ArticlePDF AvailableLiterature Review

Royal Jelly as an Intelligent Anti-Aging Agent-A Focus on Cognitive Aging and Alzheimer's Disease: A Review

Authors:

Abstract and Figures

The astronomical increase of the world's aged population is associated with the increased prevalence of neurodegenerative diseases, heightened disability, and extremely high costs of care. Alzheimer's Disease (AD) is a widespread, age-related, multifactorial neurodegenerative disease that has enormous social and financial drawbacks worldwide. The unsatisfactory outcomes of available AD pharmacotherapy necessitate the search for alternative natural resources that can target the various underlying mechanisms of AD pathology and reduce disease occurrence and/or progression. Royal jelly (RJ) is the main food of bee queens; it contributes to their fertility, long lifespan, and memory performance. It represents a potent nutraceutical with various pharmacological properties, and has been used in a number of preclinical studies to target AD and age-related cognitive deterioration. To understand the mechanisms through which RJ affects cognitive performance both in natural aging and AD, we reviewed the literature, elaborating on the metabolic, molecular, and cellular mechanisms that mediate its anti-AD effects. Preclinical findings revealed that RJ acts as a multidomain cognitive enhancer that can restore cognitive performance in aged and AD models. It promotes brain cell survival and function by targeting multiple adversities in the neuronal microenvironment such as inflammation, oxidative stress, mitochondrial alterations, impaired proteostasis, amyloid-β toxicity, Ca excitotoxicity, and bioenergetic challenges. Human trials using RJ in AD are limited in quantity and quality. Here, the limitations of RJ-based treatment strategies are discussed, and directions for future studies examining the effect of RJ in cognitively impaired subjects are noted.
Content may be subject to copyright.
antioxidants
Review
Royal Jelly as an Intelligent Anti-Aging Agent—A
Focus on Cognitive Aging and Alzheimers Disease:
A Review
Amira Mohammed Ali 1, 2, * and Hiroshi Kunugi 1,3
1
Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology
and Psychiatry, Tokyo 187-0031, Japan; hkunugi@ncnp.go.jp
2Department of Psychiatric Nursing and Mental Health, Faculty of Nursing, Alexandria University,
Alexandria 21527, Egypt
3Department of Psychiatry, Teikyo University School of Medicine, Tokyo 173-8605, Japan
*Correspondence: mercy.ofheaven2000@gmail.com; Tel.: +81-042-346-1714
Received: 30 August 2020; Accepted: 24 September 2020; Published: 29 September 2020


Abstract:
The astronomical increase of the world’s aged population is associated with the increased
prevalence of neurodegenerative diseases, heightened disability, and extremely high costs of care.
Alzheimer’s Disease (AD) is a widespread, age-related, multifactorial neurodegenerative disease that
has enormous social and financial drawbacks worldwide. The unsatisfactory outcomes of available
AD pharmacotherapy necessitate the search for alternative natural resources that can target the
various underlying mechanisms of AD pathology and reduce disease occurrence and/or progression.
Royal jelly (RJ) is the main food of bee queens; it contributes to their fertility, long lifespan, and memory
performance. It represents a potent nutraceutical with various pharmacological properties, and has
been used in a number of preclinical studies to target AD and age-related cognitive deterioration.
To understand the mechanisms through which RJ aects cognitive performance both in natural aging
and AD, we reviewed the literature, elaborating on the metabolic, molecular, and cellular mechanisms
that mediate its anti-AD eects. Preclinical findings revealed that RJ acts as a multidomain cognitive
enhancer that can restore cognitive performance in aged and AD models. It promotes brain cell
survival and function by targeting multiple adversities in the neuronal microenvironment such as
inflammation, oxidative stress, mitochondrial alterations, impaired proteostasis, amyloid-βtoxicity,
Ca excitotoxicity, and bioenergetic challenges. Human trials using RJ in AD are limited in quantity
and quality. Here, the limitations of RJ-based treatment strategies are discussed, and directions for
future studies examining the eect of RJ in cognitively impaired subjects are noted.
Keywords:
Alzheimer’s disease; neurodegenerative disorders; aging; alternative therapy;
apitherapy; amyloid-
β
; cognitive impairment; dementia; mitochondrial dysfunction; oxidative
stress; neuroinflammation; gut-brain axis; royal jelly
1. An Overview of Cognitive Aging
The world has witnessed an astronomical increase in the aged population, both in developed
and developing countries. Unfortunately, longevity comes at a high price. Aging is associated
with progressive deterioration of overall homeostasis and a decline of cognitive, visual, hearing,
and muscular functions, as well as sleep. These functional alterations lead to a trail of burdensome
problems including fatigue, mood dysregulation, overall physical dysfunction, low quality of life,
low life-expectancy, high disability and dependency, and eventually institutionalization [16].
Evidence indicates that aged brains undergo several pathological changes: higher metabolic
stress, reduced neurogenesis, increased synaptic aberrations, immune dysregulation (high expression
Antioxidants 2020,9, 937; doi:10.3390/antiox9100937 www.mdpi.com/journal/antioxidants
Antioxidants 2020,9, 937 2 of 47
of inflammatory markers), and low expression of neuroprotective factors. Altered brain physiology,
along with disturbances in the function and synchronization of the circadian system, significantly
increase the prevalence of neurodegeneration, neurobehavioral deficits, and cognitive aging [
7
,
8
].
Research documents that normal cognitive aging involves synaptic changes and decreased neuronal
plasticity of the cortex and hippocampus, which directly accelerate declines in memory, reasoning,
and speed; these alterations may start from early adulthood [9,10].
Chronic neuroinflammation is one of the main pathologies that contribute to age-related cognitive
decline. In particular, microglia (the resident immune cells of the brain) are persistently activated in
response to neuroinflammation, leading to cytotoxic eects and pathologic changes in cortical white
matter [
11
], which involve protein nitration, white mater lesions (signal abnormalities), and myelin
loss. White matter tracts play a major role in learning and information processing, whereas age-related
alterations of white matter represent a main source of cognitive deterioration among the elderly. Indeed,
the acceleration of age-dependent microglial activation and proliferation in human white matter and
its associated disruption of white matter integrity can be detected early in middle-age (around the
age 50 years) [
12
]. In addition, cerebral arterial stiness increases with age, and is associated with the
formation of white matter lesions and cognitive decline in normal aging [
13
]. Interestingly, the term
“normal cognitive aging” can be misleading, given that researchers have identified discrete cognitive
phenotypes that reflect nonoverlapping vulnerabilities for cognitive decline (e.g., speed and memory)
in high-functioning adults. Therapeutic targets within the “normal” spectrum can be identified early
through suitable biomarkers [
14
]. Altogether, increased aging remains the main underlying etiology of
cognitive deterioration in the elderly population [14,15].
2. Overview of Alzheimer’s Disease
Dementia is a clinical syndrome that manifests mainly by progressive and irreversible deficits in
cognitive performance, which lead to complete dependence for activities of daily living (ADL) [
5
,
16
].
Worldwide, dementia represents the second most prevalent neurological disorder (around 50 million
cases in 2015) after dierent types of headache [17]. Alzheimer’s disease (AD) accounts for 70 to 80%
of dementia cases. Currently, 36 million people suer from AD. Around 5 million new cases develop
every year, and it is estimated that the AD population will reach 115 million cases by 2050, given the
continuous increases in life expectancy [
5
,
18
]. AD is the sixth leading cause of death in the USA, and its
annual cost of care exceeds $232 billion, making it the third most burdensome disease after cancer and
coronary heart disease. It represents a massive form of disability that significantly impairs all aspects
of life, including lower life expectancy, poor quality of life, and high levels of institutionalization.
In addition, family caregivers of AD patients are vulnerable to serious mental and physical health
problems due to burnout and distress [5,19].
Few people get diagnosed and treated in the early stages of AD, despite the fact that the disease
can be clinically diagnosed by physical and neurological examination, and its symptoms may be
eectively managed in the early stages. This is because the brain pathology of AD may precede the
clinical diagnosis of dementia by up to 20 years [
20
22
]. In addition, the course of the disease is
highly diverse. AD comprises three stages based on the level of cognitive and functional impairments:
(1) a preclinical
stage characterized by normal cognitive performance, which can last up to 10 years,
(2) a prodromal stage characterized by mild cognitive impairment (MCI), which can last up to 4 years,
and (3) dementia, which is characterized by manifest functional impairments, which may last longer
than 6 years [
14
,
22
]. Moreover, a lack of agreement among AD specialists on the terminology related
to clinically “normal” cognitive aging and dementia may cause patients with preclinical AD to miss
the chance of getting promptly diagnosed and treated [23,24].
In addition to cognitive deterioration, the main characteristic symptom of AD, numerous other
symptoms develop such as language disturbances, functional impairments in ADL, and a wide range
of neuropsychiatric symptoms, e.g., depression, irritability, anxiety, agitation, apathy/indierence,
delusions, and hallucinations. Evidence indicates that depression and irritability represent early AD
Antioxidants 2020,9, 937 3 of 47
symptoms that exist alongside MCI before AD diagnosis [
25
]. The course of the disease begins with
mild symptoms that gradually get more severe [
26
]. Symptoms associated with AD heighten caregivers’
burden and increase rates of institutionalization and mortality [25].
3. The Mechanism Underlying AD Development
Understanding the pathophysiology of AD is necessary for early detection and the development
of specific eective treatments. Despite extensive investigations of AD, the exact cause remains
unclear. However, AD researchers came to a consensus on the main features of AD pathogenesis:
progressive buildup of the neurotoxic oligomers of beta-amyloid (A
β
) protein fragments to form
insoluble senile plaques (SPs) outside neurons. The amyloidogenic pathway is considered the main
molecular event contributing to the accumulation of A
β
. The rate-limiting step in the process of A
β
production involves proteolytic cleavage of amyloid precursor protein (APP) by
β
-secretase (beta-site
APP cleaving enzyme 1, BACE1), which results in the production of part-soluble APP peptide-b and
C-terminal APP fragment-b, which then get further cleaved by
γ
-secretase to produce hydrophobic A
β
polypeptides [27,28].
3.1. Role of the Immune System in Alzheimer’s Disease
AD pathology in 99% of cases involves multiple interactions between environmental, lifestyle,
and genetic factors. Genetics constitute 53% of total phenotypic variance [
29
]. A current wide-scale
meta-analysis of genome-wide association studies has identified 215 risk-increasing genes of AD. Most
of these genes are related to body tissues involved in the immune system: whole blood, spleen, liver,
and microglia [
30
]. Accumulating evidence confirms the core casual involvement of the immune system
and chronic neuroinflammation in the pathology of age-related neurodegenerative diseases, such as
AD [
11
,
31
]. Aged brains that undergo inflammation develop a ‘sensitized’ or ‘primed’ phenotype,
mainly because microglia exhibit dystrophic morphology, increased production of pro-inflammatory
molecules, and diminution of neuroprotective factors [
32
]. A sensitized phenotype of the aged brain
is highly vulnerable to secondary insults such as infections and psychological stress. Inflammatory
cytokines foster the transcriptional upregulation of
β
-secretase and APP and increase A
β
aggregation,
which contribute to the characteristic neuropathologic substrate of AD—Aβplaques [7,11,33].
Neuroinflammation synergizes the expression of the genes involved in AD development in aged
brains. In this respect, astrocytes play a major role in the neuroinflammatory and neurodegenerative
processes underlying AD. They represent the main site where genes associated with AD are expressed,
such as the apolipoprotein E (APOE) gene [
34
]. APOE-
ε
4, the main APOE isomer, is strongly
associated with the onset of late-onset familial and sporadic AD [
35
]. In mice, APOE-
ε
4 is associated
with low spontaneous excitatory postsynaptic currents in the amygdala in middle age and high
excitatory activity in old age, with multiple long-term hippocampal alterations related to metabotropic
glutamatergic receptors, i.e., the extrasynaptic N-methyl-d-aspartate receptor (NMDAR)-dependent
signaling pathway. Inheritance of APOE-
ε
4 in humans is associated with alterations of the brain
structure and function, which occur very early before the onset of AD, e.g., behavioral deficits and
reduced glucose metabolism in the temporal cortex and parahippocampal gyrus occur in young and
middle-age individuals. In old age, APOE-
ε
4 is associated with heightened brain atrophy in the medial
temporal lobe and accelerated A
β
deposition [
21
]. Moreover, carriers of APOE-
ε
4, individuals in
early stages of AD, and cognitively normal people with A
β
aggregation demonstrate hyperactivity
or dysregulated activation of the default-mode network during hippocampal memory-encoding.
Experimentally, these dysregulations occur even before the formation of Aβlesions [36]. Though the
exact mechanism through which APOE contributes to neurodegeneration is not well-understood,
current research indicates that APOE-lipoproteins bind to various cell-surface receptors and interfere
with processes essential for the generation and clearance of A
β
and tau, such as the transport of
brain lipid (e.g., lipophilic A
β
peptide), neuronal signaling, mitochondrial function, and glucose
metabolism [
37
]. Furthermore, overproduction of APOE by activated microglia might exacerbate
Antioxidants 2020,9, 937 4 of 47
neuroinflammation, leading to sporadic brain neurodegeneration [
31
]. In line with this, our research
group demonstrated increased APOE protein levels in the cerebrospinal fluid of people with APOE-
ε
4
allele, compared to those without the allele [38].
3.2. Role of the Gut-Brain Axis in AD Pathogenesis
From another angle, emerging knowledge attributes the origin of the inflammatory processes
underlying AD to microbiome alterations [
39
]. Various external factors such as imbalanced diet
(high in fat and low in fiber) and the ingestion of toxins and pathogens cause propagation of
enterotoxigenic bacterial strains such as Gram-negative bacilli Bacteroides fragilis and Escherichia (E.)coli.
This imbalance is associated with a reduction of beneficial bacteria such as Lactobacillus johnsonii [
39
41
].
Under imbalanced gut conditions, the symbiotic interactions of the gut microbiome that are essential
for human physiological functions get disrupted, allowing enterotoxigenic bacteria to cause virulent
aberrations and inflammation of the gastrointestinal (GI) wall [
42
]. Consequently, the production and
availability of neuroactive molecules produced by enteric neurons (e.g., serotonin) deteriorate [
39
,
41
,
43
].
Furthermore, inflamed gut undergoes local activation of inducible nitric oxide synthase (iNOS, also
known as NOS2) signaling and increased expression of gut nitric oxide (NO). Local activation
of inflammatory pathways follows iNOS activation, leading to increased production of cytokines,
which further accelerates GI inflammation and dysbiosis/leaky gut. GI dysbiosis enables endobacteria
and their toxins, i.e., potent pro-inflammatory neurotoxins (e.g., lipopolysaccharide (LPS)), to get into
the blood stream and induce systemic inflammation and oxidative stress. All these events lead to
pathological clotting, leakage of the blood brain barrier (BBB), suppression of neurotrophins, and the
initiation of neuroinflammatory processes that lead to the accumulation of Aβ[3941,43] (Figure 1).
Antioxidants 2020, 9, x 4 of 46
neurodegeneration [31]. In line with this, our research group demonstrated increased APOE protein
levels in the cerebrospinal fluid of people with APOE-ε4 allele, compared to those without the allele
[38].
3.2. Role of the Gut-Brain Axis in AD Pathogenesis
From another angle, emerging knowledge attributes the origin of the inflammatory processes
underlying AD to microbiome alterations [39]. Various external factors such as imbalanced diet (high
in fat and low in fiber) and the ingestion of toxins and pathogens cause propagation of
enterotoxigenic bacterial strains such as Gram-negative bacilli Bacteroides fragilis and Escherichia (E.)
coli. This imbalance is associated with a reduction of beneficial bacteria such as Lactobacillus johnsonii
[39–41]. Under imbalanced gut conditions, the symbiotic interactions of the gut microbiome that are
essential for human physiological functions get disrupted, allowing enterotoxigenic bacteria to cause
virulent aberrations and inflammation of the gastrointestinal (GI) wall [42]. Consequently, the
production and availability of neuroactive molecules produced by enteric neurons (e.g., serotonin)
deteriorate [39,41,43]. Furthermore, inflamed gut undergoes local activation of inducible nitric oxide
synthase (iNOS, also known as NOS2) signaling and increased expression of gut nitric oxide (NO).
Local activation of inflammatory pathways follows iNOS activation, leading to increased production
of cytokines, which further accelerates GI inflammation and dysbiosis/leaky gut. GI dysbiosis enables
endobacteria and their toxins, i.e., potent pro-inflammatory neurotoxins (e.g., lipopolysaccharide
(LPS)), to get into the blood stream and induce systemic inflammation and oxidative stress. All these
events lead to pathological clotting, leakage of the blood brain barrier (BBB), suppression of
neurotrophins, and the initiation of neuroinflammatory processes that lead to the accumulation of
Aβ[39–41,43] (Figure 1).
Figure 1. Contribution of disrupted microbiota to the permeability of the blood brain barrier and
neuroinflammation in Alzheimer’s disease. Abbreviations: denotes increase, BBB: blood brain
barrier, RAGE: receptor for advanced glycation end products, NF-kB: nuclear factor-kappa B, iNOS:
inducible nitric oxide synthase, Aβ: beta-amyloid protein fragments, ROS: reactive oxygen species,
NOS: nitric oxide species.
As illustrated by the yellow arrows, the ingestion of pathogens, toxic substances, and improper
diet (low fiber and high fat) disrupt the gut microbiota. Microbiota imbalance causes the propagation
of highly toxic endobacteria such as Gram-negative bacteria and a reduction in numbers of beneficial
bacteria such as lactic acid bacteria and bifidobacteria. Endobacteria toxins cause local inflammation
and oxidative stress, resulting in aberrations and injuries of the gastrointestinal wall, which allow the
passage of endobacteria and their toxins into the blood stream, as illustrated by the red arrows. The
persistent gastrointestinal leak causes continuous activation of the immune system. The activation of
pathways of inflammation and oxidative stress such as NF-kB and iNOS results in high production
of inflammatory cytokines, free radicals, adhesion factors, and advanced glycation end-products. The
latter form as a result of nonenzymatic glycation and the oxidation of lipids, long-lived proteins, and
nucleic acids. They bind to RAGE and other cell-surface receptors or cross-link with other proteins to
Figure 1.
Contribution of disrupted microbiota to the permeability of the blood brain barrier and
neuroinflammation in Alzheimer’s disease. Abbreviations:
denotes increase, BBB: blood brain barrier,
RAGE: receptor for advanced glycation end products, NF-
κ
B: nuclear factor-kappa B, iNOS: inducible
nitric oxide synthase, A
β
: beta-amyloid protein fragments, ROS: reactive oxygen species, NOS: nitric
oxide species.
As illustrated by the yellow arrows, the ingestion of pathogens, toxic substances, and improper
diet (low fiber and high fat) disrupt the gut microbiota. Microbiota imbalance causes the propagation
of highly toxic endobacteria such as Gram-negative bacteria and a reduction in numbers of beneficial
bacteria such as lactic acid bacteria and bifidobacteria. Endobacteria toxins cause local inflammation
and oxidative stress, resulting in aberrations and injuries of the gastrointestinal wall, which allow
the passage of endobacteria and their toxins into the blood stream, as illustrated by the red arrows.
The persistent gastrointestinal leak causes continuous activation of the immune system. The activation
of pathways of inflammation and oxidative stress such as NF-
κ
B and iNOS results in high production
of inflammatory cytokines, free radicals, adhesion factors, and advanced glycation end-products.
The latter form as a result of nonenzymatic glycation and the oxidation of lipids, long-lived proteins,
and nucleic acids. They bind to RAGE and other cell-surface receptors or cross-link with other proteins
Antioxidants 2020,9, 937 5 of 47
to induce a vicious cycle of oxidative stress and inflammation. Bacterial toxins, along with cytokines,
ROS, and activated adhesion factors attack the tight junction proteins of the BBB, leading to their
degradation and permeability of the BBB, which allows the passage of toxic solutes (e.g., A
β
) from the
blood stream to the brain tissue. Glial cells (microglia, astrocytes, and oligodendrocytes—star-shape-like
cells colored in brown on Figure 1) get activated in order to protect neurons against toxic A
β
and
other products of inflammatory and oxidative responses. Glial activation phenotype develops as a
result of the depletion of resources of these cells and their failure to counteract constant inflammation,
which results in a cycle of self-perpetuating neuroinflammation and neuronal toxicity.
Of interest, rodents fed fecal E. coli from rats with colitis exhibited significant memory and learning
impairment, along with changes in the composition of gut microbiota and elevation of LPS levels
in the feces, blood, and brain [
40
]. Evidence reveals that E. coli can form extracellular functional
amyloid or amyloid-like structures [
44
,
45
]. Evolving knowledge indicates that all human brains and the
erythrocytes of AD patients contain bacterial-encoded 16S rRNA, which is largely (i.e., more than 70%)
Gram-negative bacteria [
41
,
46
]. In the meantime, iron dysregulation—as indicated by exceptionally
high serum ferritin levels—is common in AD patients; it contributes to oxidative stress and causes
virulent reactivation of dormant blood and tissue microbiome [
39
]. E. coli bacterial lipoproteins act as
agonists of toll-like receptor (TLR) 2 and nucleotide-binding domain and leucine-rich repeat containing
protein 3 (NLRP3); they induce inflammation and increase cytokine production through their interaction
with recombinant human Serum Amyloid A1 (hSAA1) [
47
]. In addition, various levels of bacterial LPS
have been detected in blood samples and brain lysates from the superior temporal lobe neocortex and
hippocampus of AD patients. Meanwhile, levels of hippocampal LPS in advanced AD cases can be up
to 26-fold higher than in cognitively normal age-matched controls [
41
,
48
]. LPS localizes in high levels
with A
β
1-40/42 in A
β
lesions, around cerebral vessels, and inside oligodendrocytes and neurons in the
brain of AD patients [
49
]. LPS contributes to myelin injury and white matter hyperintensities, a main
pathophysiological feature that is associated with the severity of neurodegeneration and cognitive
decline in AD. In addition, AD patients exhibit extremely high blood titer of autoantibodies against
various myelin proteins, and myelin damage precedes tau pathology and amyloid aggregation in
experimental models of AD. Meanwhile, focal loss of myelin and oligodendrocytes occurs within
and around A
β
lesions in familial and sporadic AD [
50
]. Mechanistically, once bacterial LPS crosses
the BBB, it targets glial cells (oligodendrocytes) in the white and grey matter by activating leukocyte
and glial TLR4-CD14/TLR2 and low-density lipoprotein receptor-related protein 1 (LRP-1). The latter
regulates lipid metabolism and transports A
β
out of the brain. LPS binding to these receptors
initiates intracellular signaling cascades associated with mitochondrial oxidative stress (e.g., iNOS) and
inflammation (e.g., nuclear factor-kappa B; NF-
κ
B) [
51
,
52
]. Cellular failure to adapt to the continuously
increased production of ROS and proinflammatory cytokines eventually causes serious injuries to
oligodendrocytes, degrades myelin proteins, e.g., myelin basic protein (MBP), and stimulates the
aggregation of A
β
, because A
β
clearance eciency of LRP-1 diminishes. Moreover, A
β
1–42 directly
associates with LRP-1 and binds to degraded MBP, which accelerates plaque formation. In the meantime,
leaky gut and leaky BBB promote chronic LPS intrusion, cellular damage, and A
β
1–42-induced agonism
of TLR4 receptors. These events generate a vicious cycle of incorrectly modulated glial activation
phenotype that supports progressive neurotoxicity [51,53].
3.3. Multiple Medical Conditions Contribute to AD by Enhancing Neuroinflammation
The diminution of sex hormones that occurs with aging is associated with memory and
cognitive changes. In particular, the drop in estrogen levels that occurs after menopause aggravates
neuroinflammation through the activation of inflammatory pathways such as NF-
κ
B. Accordingly,
estrogen deficiency stimulates multiple autonomic nervous changes including A
β
accumulation,
resulting in memory impairment and increased susceptibility to AD [24,54]. In addition, loss of bone
mass is common in AD patients; it usually occurs as a direct eect of estrogen deficiency [
55
].
The literature documents sex dierences in the prevalence, cerebral pathology, and molecular
Antioxidants 2020,9, 937 6 of 47
mechanisms of AD. Compared with male APP/PS1/tau triple-transgenic AD mice, AD female mice
exhibit more prominent A
β
plaques, neurofibrillary tangles, neuroinflammation, spatial cognitive
deficits, and dysregulation of hippocampal cyclic adenosine monophosphate (cAMP)-response element
(CRE)-binding protein (CREB) signaling. These dierences are likely to be induced by estrogen
deficiency in female mice [56].
Advanced aging is associated with multiple systemic failures such as reduced vascular elasticity,
including cerebral blood vessels. Emerging knowledge emphasizes the fact that APOE and age-related
vascular pathologies such as arteriosclerosis lead to failure of drainage of A
β
and soluble proteins
from perivenous spaces [
57
]. Cerebral artery pathology unrelated to A
β
, which occurs prior to AD,
contributes to the impairment of downstream arterioles in AD. Cerebral amyloid angiopathy develops
as a result of A
β
deposition on the inner walls of blood vessel, resulting in poorer A
β
drainage [
58
],
the formation of lesions in white matter, and greater a degree of cognitive decline [13,58].
Several lines of evidence suggest that the pathogenetic processes of AD, mainly neuroinflammation,
are initiated by various metabolic dysfunctions, e.g., insulin resistance, nutritional problems
(e.g., vitamin D deficiency), and hormonal abnormalities e.g., thyroid dysfunction [
5
,
59
,
60
].
Investigations of neural exosomes and nanosomes revealed that AD patients exhibit metabolic
disturbances many years before they develop AD [
61
]. Dysregulated insulin signaling is one of the
main metabolic dysfunction that contributes to AD pathology [
62
]. Insulin is one of the hormones
that aect every single cell in the body, and the insulin/insulin-like growth factor (IIS) signaling
pathway interacts with other pathways and aects their functioning [
63
]. It plays a major role in
cellular growth, autophagy, energy utilization, mitochondrial function, management of oxidative stress,
synaptic plasticity, and cognitive function [
62
]. Longitudinal and autopsy studies suggest an important
contribution of vascular mechanisms (small vessel disease) to cognitive decline in AD associated
with type 2 diabetes mellitus [
60
]. Hyperglycemia induces vascular permeability by upregulating
hypoxia inducible factor-1
α
(HIF-1
α
), which subsequently activates vascular endothelial growth factor
(VEGF) [
64
]. Hyperglycemia also amplifies inflammatory and oxidative processes by contributing to the
production of advanced glycation end-products (AGEs) through nonenzymatic glycation and oxidation
of lipids, long-lived proteins, and nucleic acids. AGEs are heterogenous compounds that bind to the
receptor for advanced glycation end products (RAGE) and other cell-surface receptors, or cross-link
with other proteins to induce oxidative stress and inflammation [
33
,
64
]. Meanwhile treatments that
resensitize brain insulin signaling have been shown to improve cognitive function in AD patients [
62
].
In the same way, hypertension reduces brain reserve and contributes to amyloid-dependent pathway
signaling, which facilitates A
β
and tau deposition and related neuronal injury, especially in APOE-
ε
4
carriers, leading to AD development [
65
]. A recent systematic review shows that the use of any eective
antihypertensive medication decreases the incidence of AD in hypertensive patients [66].
Hypercholesterolemia represents another common deleterious age-related health problem which is
strongly linked to hypertension [
67
]. Biophysical properties and signaling of the biomembrane in both
physiological and pathological processes are aected by asymmetries of lipid distribution: condensed
cholesterol localizes mostly in the external hemilayers in raft domains/lipid-ordered microdomains
along with saturated phosphatidyl lipids and sphingolipids [
68
]. A considerable number of AD
genes are related to liver function, denoting a prominent role of lipids in the pathogenesis of AD [
30
].
High cholesterol levels and dysfunctional lipid metabolism in the brain, which is regulated by APOE,
promote the pathogenesis of AD by altering the vascular integrity of the brain and the immune responses
of glial cells [
30
,
69
,
70
]. The accumulation of A
β
particles in the cell membrane mostly occurs in raft
domains, the cleavage location of the precursor APP by
β
- and
γ
- secretase [
68
]. Human extracellular
A
β
fibrils contain large amounts of cholesterol rich lipids. The region of residues 22–35 in the A
β
peptide has been identified as a potential cholesterol binding site where cholesterol vesicles accelerate
the primary nucleation of Aβ42 by up to 20-fold via a heterogeneous nucleation pathway [69,70].
AD is a multifactorial disease; several other risk factors with detrimental eects have been
proposed such as hypothyroidism, chronic stress (e.g., income inequality), improper diet (e.g., high fat
Antioxidants 2020,9, 937 7 of 47
and low fiber), and pollution (e.g., heavy metals) [
5
,
15
,
41
]. These factors aect the homeostasis of
the whole body, including the gut-brain axis, leading to aging-like sensitization of microglia and
heightened reactivity to secondary insults [
7
]. As a result, most physiological processes in aging
deteriorate, which synergizes AD-related genetic interactions [
5
,
15
]. The resulting alterations entail
the activation of various pathological pathways that involve the depletion of endogenous antioxidants,
oxidative stress, neuroinflammation, dysfunctional proteostasis, expression of proapoptotic proteins
leading to mitochondrial breakdown, depletion of neurotrophins, glutamatergic neurotoxicity, synaptic
dysfunction, and neuron loss [7173].
3.4. Amyloid Pathology Promotes Neurodegeneration by Altering Cellular Structure and Function
Monomers and oligomers of the A
β
peptide are highly disordered, and they disrupt the
homeostasis of cellular elements including bio-metals [
71
]. The mechanism and kinetics through which
A
β
aggregates depend on its interaction with transition metal ions such as copper, iron, and zinc [
74
].
A
β
interacts with zinc (Zn) ions, which exist in large amounts in SPs in AD patients. ZnA
β
oligomers
adopt the same
β
-sheet structure as in A
β
fibrils. However, they exhibit a greater potential to accelerate
hippocampal microglia activation. Cell viability and cytotoxicity assays show that ZnA
β
oligomers
are more cytotoxic than A
β
oligomers [
75
]. Zn is involved in the regulation of multiple AD-related
metabolic processes such as hormonal signaling, insulin desensitization, and proteolytic activities.
Being trapped inside A
β
plaques, a sort of Zn deficiency occurs, which hastens metabolic, genetic,
and epigenetic alterations that contribute to a widespread pathology in the cortex. Thus, this pathology
results in early nonamnestic features such as dyscalculia and aphasia [
61
]. Copper and iron are
core redox active transition metals that interact with A
β
to facilitate the electron transfer necessary
for the generation of ROS and reactive nitrogen species (RNS) in a reaction that requires tyrosine
10 [
76
]. Hydrogen peroxide (H
2
O
2
), one of the most prominent free radicals produced in AD
brains due to oxidative stress, is an uncharged, stable, and freely diusible ROS. H
2
O
2
amplifies A
β
neurotoxicity by interacting with iron and copper to generate highly toxic ROS that accelerate the
buildup of inflammatory cytokines and attract activated microglia to encircle A
β
plaques, causing
more ROS/H
2
O
2
production and leading to drastic damages of cellular proteins, lipids, and DNA,
and eventually neuronal loss [77,78] (Figure 2).
Antioxidants 2020, 9, x 7 of 46
fat and low fiber), and pollution (e.g., heavy metals) [5,15,41]. These factors affect the homeostasis of
the whole body, including the gut-brain axis, leading to aging-like sensitization of microglia and
heightened reactivity to secondary insults [7]. As a result, most physiological processes in aging
deteriorate, which synergizes AD-related genetic interactions [5,15]. The resulting alterations entail
the activation of various pathological pathways that involve the depletion of endogenous
antioxidants, oxidative stress, neuroinflammation, dysfunctional proteostasis, expression of
proapoptotic proteins leading to mitochondrial breakdown, depletion of neurotrophins,
glutamatergic neurotoxicity, synaptic dysfunction, and neuron loss [71–73].
3.4. Amyloid Pathology Promotes Neurodegeneration by Altering Cellular Structure and Function
Monomers and oligomers of the Aβ peptide are highly disordered, and they disrupt the
homeostasis of cellular elements including bio-metals [71]. The mechanism and kinetics through
which Aβ aggregates depend on its interaction with transition metal ions such as copper, iron, and
zinc [74]. Aβ interacts with zinc (Zn) ions, which exist in large amounts in SPs in AD patients. ZnAβ
oligomers adopt the same β-sheet structure as in Aβ fibrils. However, they exhibit a greater potential
to accelerate hippocampal microglia activation. Cell viability and cytotoxicity assays show that ZnAβ
oligomers are more cytotoxic than Aβ oligomers [75]. Zn is involved in the regulation of multiple
AD-related metabolic processes such as hormonal signaling, insulin desensitization, and proteolytic
activities. Being trapped inside Aβ plaques, a sort of Zn deficiency occurs, which hastens metabolic,
genetic, and epigenetic alterations that contribute to a widespread pathology in the cortex. Thus, this
pathology results in early nonamnestic features such as dyscalculia and aphasia [61]. Copper and
iron are core redox active transition metals that interact with Aβ to facilitate the electron transfer
necessary for the generation of ROS and reactive nitrogen species (RNS) in a reaction that requires
tyrosine 10 [76]. Hydrogen peroxide (H
2
O
2
), one of the most prominent free radicals produced in AD
brains due to oxidative stress, is an uncharged, stable, and freely diffusible ROS. H
2
O
2
amplifies Aβ
neurotoxicity by interacting with iron and copper to generate highly toxic ROS that accelerate the
buildup of inflammatory cytokines and attract activated microglia to encircle Aβ plaques, causing
more ROS/H
2
O
2
production and leading to drastic damages of cellular proteins, lipids, and DNA,
and eventually neuronal loss [77,78] (Figure 2).
Figure 2. Beta-amyloid protein fragments induce multiple molecular and cellular changes that
promote neurodegeneration. Abbreviations: denotes increase, Aβ: beta-amyloid protein
fragments, ROS: reactive oxygen species, ER: endoplasmic reticulum, UPR: unfolded protein
response, MAP-2: microtubule-associated protein 2. Aβ pathology contributes to various molecular
and cellular changes that induce neuronal death.
Figure 2.
Beta-amyloid protein fragments induce multiple molecular and cellular changes that
promote neurodegeneration. Abbreviations:
denotes increase, A
β
: beta-amyloid protein fragments,
ROS: reactive oxygen species, ER: endoplasmic reticulum, UPR: unfolded protein response, MAP-2:
microtubule-associated protein 2. A
β
pathology contributes to various molecular and cellular changes
that induce neuronal death.
Antioxidants 2020,9, 937 8 of 47
On one side, A
β
interacts with transition metal ions such as copper, iron, and zinc, leading to the
formation of highly toxic A
β
oligomers and high production of hydrogen peroxide (H
2
O
2
), a highly
toxic ROS. H
2
O
2
amplifies A
β
neurotoxicity by interacting with iron and copper to generate highly toxic
ROS that accelerate the buildup of inflammatory cytokines and attract activated microglia to encircle
A
β
plaques, causing more ROS/H
2
O
2
production. These events ultimately lead to drastic damages of
cellular proteins, lipids, and DNA. On the other side, A
β
activates the phosphorylation of various
tau proteins via a mechanism that involves alteration of the ER by oxidative stress. ER is the main
region involved in protein folding and secretion. ER stress activates UPR signaling. UPR contextually
regulates protein misfolding and neurodegeneration in Alzheimer’s disease. MAP-2, the main protein
in neuronal axon, is a key tau protein that is aected by A
β
activity. Hyperphosphorylation of
MAP-2 leads to axonal collapse, dendrite instability, degeneration of synaptic spines, and disrupted
signal transduction.
Oxidative stress alters endoplasmic reticulum (ER), the main region involved in protein folding
and secretion. ER stress activates unfolded protein response (UPR) signaling pathway, which regulates
protein misfolding and neurodegeneration. UPR contextually regulates protein misfolding and
neurodegeneration in AD [
72
]. In particular, the phosphorylation of protein kinase RNA like ER
kinase (PERK), a stress-responsive transmembrane protein embodied in UPR, leads to the activation
of several signaling cascades that regulate redox proteins, cholesterol metabolism, and genes of
foldases and chaperones [
79
]. In this respect, the formation of A
β
plaques is significantly associated
with proteotoxicity and tauopathy, i.e., protein misfolding, hyperphosphorylation and nucleation
of axonal tau proteins, resulting in a wide spread of twisted strands of the tau protein and the
formation of neurofibrillary tangles (NFTs) inside neurons, particularly in neocortical regions [
20
,
23
].
However, soluble tau can also be neurotoxic. In addition, the hyperphosphorylation of some tau
proteins such as microtubule-associated protein 2 (MAP-2) contributes to neuronal death. MAP-2
is a cytoskeleton protein that regulates dendrite branching, microtubule assembly, and synaptic
signal transduction. The hyperphosphorylation of MAP-2 leads to microtubule collapse, dendrite
instability, axon degeneration, and dysfunctional axoplasmic transport. As a result, the synthesis,
transport, release, and uptake of neurotransmitters gets disrupted. Such interactions promote
neurotoxicity, aggravate tau and A
β
deposition, and ultimately lead to more cellular lesions and sporadic
neurodegeneration [
73
,
76
,
80
]. Finally, progressive damage and death of neurons in several areas of
the brain, expressly the hippocampus—an important region for learning and memory—represent the
direct cause of clinical manifestations in AD [16,23,28].
Accumulating A
β
lesions are involved in complex interactions that greatly aect the neurochemical
environment and the morphology of the surrounding brain tissues, resulting in the cognitive and
behavioral features of AD [
81
] (Figure 3). Synaptic dysfunction in AD originates from the high
sensitivity of axons and presynaptic oligomers, particularly long-range glutamatergic projecting
axons of pyramidal neurons, to insoluble and soluble oligomeric species of A
β
. Both A
β
plaques
and soluble A
β
oligomers induce synaptic swelling and dystrophic changes, making these sites
favorable for the ectopic release of glutamate, which aggregates in the extracellular space [
68
,
81
].
On the other hand, presenilin 1 (PS1)—the catalytic component of
γ
-secretase, the enzyme responsible
for the final processing of APP to produce A
β
—directly interacts with ryanodine receptors and
synaptic vesicle release machinery protein (synaptotagmin 1) in neurons to modulate the release
of neurotransmitters at the synapse. PS1 also interacts with the major glutamate transporter in the
central nervous system (CNS), glutamate transporter 1 (GLT-1), also known as excitatory amino acid
transporter (EAAT), at the PS1 large cytosolic loop located between the 6th and 7th transmembrane
domains [
36
]. This interaction occurs both in neurons and astrocytes, resulting in altered expression
and function of GLT-1, which drastically aects brain metabolism and synaptic signaling in a fashion
that contributes to excitotoxicity and neurodegeneration [
36
,
81
]. In particular, downregulation
of GLT-1 entails enhancement of glutamate signaling through the activation of NMDA receptors.
The latter is a principal element of the memory formation system in the brain that functions through
Antioxidants 2020,9, 937 9 of 47
glutamate-mediated neurotransmission. Glutamate is a major excitatory neurotransmitter, and its
transport from the extracellular space into the cytoplasm induces neuronal hyperactivity at the initial
stages of AD pathogenesis [36,81,82].
Antioxidants 2020, 9, x 9 of 46
neurotransmitter, and its transport from the extracellular space into the cytoplasm induces neuronal
hyperactivity at the initial stages of AD pathogenesis [36,81,82].
Aβ-related alterations of glutamate signaling limit synaptic plasticity and stimulate pathological
synaptic transmission through disruption of calcium homeostasis [81]. Calcium (Ca
2+
) is a second
messenger that plays a core role in the regulation of various basic neuronal processes, e.g., cellular
differentiation, proliferation, growth, survival, apoptosis, gene transcription, synaptic plasticity, and
membrane excitability [83]. The homeostasis of extracellular free Ca
2+
, as well as axon and dendrite
development, are regulated by Ca
2+
-sensing receptor (CaSR) [84]. Neurons are highly sensitive to any
change in Ca
2+
levels, and even minor changes can destructively alter neuronal activity [83]. Amyloid
pathology in AD contributes to the dysregulation of neuronal Ca
2+
signaling [81,85]. On the other
side, the binding of both exogenous and accumulating Aβ42 oligomers to CaSR in neurons and
astrocytes at the plasmalemma activates a set of intracellular signaling cascades such as iNOS,
vascular endothelial growth factor-A (VEGF-A), and GTP cyclohydrolase 1. As a result, many
cytotoxic effects ensue, including increased production of NO, increased vascular permeability,
aggravated intracellular influx and aggregation of Aβ, and diminution of Aβ proteolysis [84].
Moreover, distorted Ca
2+
channels contribute to neuroinflammation and neurodegeneration by
causing neuronal membrane disruption, which allows irregular transfer of Ca
2+
to occur and alters
the release of neurotransmitters, ending with the activation of apoptotic signaling cascades [81,85].
Polymerization of the dimers of CaSR occurs at the ER [84], whereas synaptic loss in AD embraces
increased Ca
2+
levels in the ER and decreased neuronal store-operated Ca
2+
entry [83].
Figure 3. Schematic illustration of the mechanism underlying synaptic destruction by beta-amyloid
peptide (Aβ). Abbreviations: denotes increase, denotes decrease, Aβ: beta-amyloid protein
fragments, MAP-2: microtubule-associated protein 2, PS1: presenilin 1, nAChRs: nicotinic
acetylcholine receptors, GLT-1: glutamate transporter 1, ROS: reactive oxygen species. Aβ pathology
contributes to synaptic destruction, which promotes neuronal apoptosis through various
mechanisms.
On one hand, Aβ activates the phosphorylation of MAP-2, a protein that is abundant in neuronal
axon terminals, leading to axon collapse and dendrite instability. Aβ oligomers induce local
production of cytokines and free radicals, which directly cause synaptic swelling. PS1, the catalytic
component of γ-secretase, modulates the release of neurotransmitters by directly interacting with
ryanodine receptors and synaptotagmin 1 in neurons. PS1 also stimulates the ectopic release of
glutamate by interacting with GLT-1, the major glutamate transporter in the central nervous system.
On the other hand, Aβ pathology triggers locational and functional alterations in nAChRs by causing
perturbations of the cholesterol and phospholipid components of the biomembrane, where nAChRs
Figure 3.
Schematic illustration of the mechanism underlying synaptic destruction by beta-amyloid
peptide (A
β
).
Abbreviations
:
denotes increase,
denotes decrease, A
β
: beta-amyloid protein
fragments, MAP-2: microtubule-associated protein 2, PS1: presenilin 1, nAChRs: nicotinic acetylcholine
receptors, GLT-1: glutamate transporter 1, ROS: reactive oxygen species. A
β
pathology contributes to
synaptic destruction, which promotes neuronal apoptosis through various mechanisms.
A
β
-related alterations of glutamate signaling limit synaptic plasticity and stimulate pathological
synaptic transmission through disruption of calcium homeostasis [
81
]. Calcium (Ca
2+
) is a second
messenger that plays a core role in the regulation of various basic neuronal processes, e.g., cellular
dierentiation, proliferation, growth, survival, apoptosis, gene transcription, synaptic plasticity,
and membrane excitability [
83
]. The homeostasis of extracellular free Ca
2+
, as well as axon and dendrite
development, are regulated by Ca
2+
-sensing receptor (CaSR) [
84
]. Neurons are highly sensitive to any
change in Ca
2+
levels, and even minor changes can destructively alter neuronal activity [
83
]. Amyloid
pathology in AD contributes to the dysregulation of neuronal Ca
2+
signaling [
81
,
85
]. On the other side,
the binding of both exogenous and accumulating A
β
42 oligomers to CaSR in neurons and astrocytes at
the plasmalemma activates a set of intracellular signaling cascades such as iNOS, vascular endothelial
growth factor-A (VEGF-A), and GTP cyclohydrolase 1. As a result, many cytotoxic eects ensue,
including increased production of NO, increased vascular permeability, aggravated intracellular influx
and aggregation of A
β
, and diminution of A
β
proteolysis [
84
]. Moreover, distorted Ca
2+
channels
contribute to neuroinflammation and neurodegeneration by causing neuronal membrane disruption,
which allows irregular transfer of Ca
2+
to occur and alters the release of neurotransmitters, ending with
the activation of apoptotic signaling cascades [
81
,
85
]. Polymerization of the dimers of CaSR occurs
at the ER [
84
], whereas synaptic loss in AD embraces increased Ca
2+
levels in the ER and decreased
neuronal store-operated Ca2+entry [83].
On one hand, A
β
activates the phosphorylation of MAP-2, a protein that is abundant in neuronal
axon terminals, leading to axon collapse and dendrite instability. A
β
oligomers induce local production
of cytokines and free radicals, which directly cause synaptic swelling. PS1, the catalytic component
of
γ
-secretase, modulates the release of neurotransmitters by directly interacting with ryanodine
receptors and synaptotagmin 1 in neurons. PS1 also stimulates the ectopic release of glutamate by
interacting with GLT-1, the major glutamate transporter in the central nervous system. On the other
Antioxidants 2020,9, 937 10 of 47
hand, A
β
pathology triggers locational and functional alterations in nAChRs by causing perturbations
of the cholesterol and phospholipid components of the biomembrane, where nAChRs are located,
which result in oectopic glutamate release and Ca
2+
influx. These events lead to synaptic destruction
and impair signal transduction in a manner that promotes ROS production, tauopathy, amyloidogenesis,
and apoptosis.
Cholinergic transmission is modulated by acetylcholine (ACh). ACh plays a major role in cortical
activation and arousal, which are involved in higher brain functions such as memory, learning,
and attention, as well as in overall brain homeostasis and plasticity [
86
]. The balance of ACh is
regulated by its synthesis through the activities of choline acetyltransferase (ChAT) and degradation
by acetylcholinesterase (AChE) [
87
]. Nicotinic acetylcholine receptors (nAChRs) are located in the
lipid-ordered domains of the biomembrane, where A
β
accumulation takes place. A
β
pathology causes
perturbations of the cholesterol and phospholipid components of the membrane, leading to locational
and functional alterations of nAChRs [
68
]. In the same way, degeneration associated with NFTs causes
progressive death and dysfunction of cholinergic neurons in the forebrain and neocortex, leading
to a widespread presynaptic cholinergic denervation [
86
]. Therefore, the characteristic cognitive
dysfunction in AD is mostly attributed to dysregulation of the cortical cholinergic system caused by the
deficiency of its main neurotransmitter [
88
]. A group of the few approved medications of AD is based
on increasing ACh levels [
86
]. ACh has a neuroprotective eect against A
β
toxicity, both in cholinergic
and noncholinergic cells, by enhancing the soluble A
β
peptide conformation and discouraging the
aggregation-prone
β
-sheet conformation [
89
]. The homomeric
α
7- nAChR is the most widely used
form of nAChRs, and has high permeability to Ca
2+
and high anity for A
β
. Elevated astrocytic
α
7-nAChR in the hippocampus, entorhinal cortex, and temporal cortex of sporadic AD patients and
carriers of the Swedish APP 670/671 mutation suggest its involvement in AD pathology. Nonetheless,
evidence denotes that it could be either neuroprotective or neurodegenerative, depending on A
β
levels,
which vary according to disease stage. Experimentally, activation of
α
7-nAChRs by picomolar levels
of A
β
42 promotes long-term potentiation and memory via spontaneous intracellular Ca
2+
and NMDA
signaling. On the other hand, inhibition of
α
7-nAChRs occurs by low micromolar levels of A
β
42 and
A
β
25-35, which is followed by intercellular Ca
2+
influx and ectopic release of glutamate, resulting
in the destruction of synaptic spines out of accelerated production of NO, p-tau oligomers, and the
activity of caspase-3 [
84
], a protein that belongs to the cysteine-aspartic acid protease (caspase) family,
which, when activated, interacts with caspase-8 and caspase-9 to contribute to the execution-phase of
cell apoptosis [28].
4. Current Treatments of AD
AD is not currently curable [
15
,
90
], and no new AD drugs have been approved in the last
16 years. Meanwhile, disease-modifying treatments, which aim to prevent neurodegenerative
processes at early stages before the development of clinical manifestations, are lacking [
91
].
The available pharmacotherapy consists of two classes: cholinesterase inhibitors (donepezil,
galantamine, rivastigmine, tacrine) that increase brain levels of ACh, and glutamatergic antagonist
(memantine) that protects neurons against glutamate mediated excitotoxicity. Nonetheless, these drugs
fail to treat the underlying causes of AD and only provide a symptomatic relief [18,26].
Researchers have desperately attempted to develop alternative pharmacologic treatments of
AD. However, failure rates of drug development programs are exceptionally high. A current review
that examined clinicaltrials.gov for all pharmacologic AD trials in 2019 revealed that 132 agents are
being tested in trials that mostly include preclinical and prodromal AD. Out of the tested compounds,
19 agents target cognitive enhancement, 14 target neuropsychiatric and behavioral symptoms associated
with AD, 38 agents target A
β
, and 17 agents target tau; agents targeting A
β
and tau include small
molecules and monoclonal antibodies or biological therapies [
91
]. In other words, not a single drug
among the available AD treatments is able to address the disease in its active phase or from its dierent
pathological dimensions. It is worth noting that treatments that enhance brain A
β
clearance activity
Antioxidants 2020,9, 937 11 of 47
(e.g., A
β
immunotherapy) have serious adverse eects. For instance, therapeutic antibodies may
cause vasogenic edema, microhemorrhage, and neuronal hyperactivity [
28
]. Therefore, attention has
been recently directed toward natural resources to treat the underlying causes of AD and prevent its
development in vulnerable people [
18
]. Research has recently shown that dietary intervention may
promote cognitive health and prevent AD [92].
5. Royal Jelly, Its Ingredients and Pharmacological Properties
Royal jelly (RJ) is a white or yellowish gelatinous substance secreted from the mandibular
and hypopharyngeal glands of young nurse worker bees (Apis mellifera). It has a pungent smell,
a distinct sweet-sour taste, and an acidic pH (3.4–4.5) [
93
,
94
]. RJ possesses a wide range of
health-promoting activities: antioxidant, anti-inflammatory, neurotrophic, hypotensive, antidiabetic,
antihypercholesterolemic, antirheumatic, antitumor, antifatigue, antimicrobial, nematocidal,
and anti-aging [
95
,
96
]. Consequently, RJ and its major active compounds have been used to attain
therapeutic benefits in cancer, diabetes, hypertension, hyperlipidemia, skin diseases, etc. [63,97].
Water comprises the greatest part of crude RJ (50–70% w/w) [
96
,
98
,
99
]. Proteins represent the main
active ingredient of RJ, accounting for 50% of its dry matter weight. Nine water-insoluble proteins
constitute the majority of RJ protein content, which are known as major royal jelly proteins (MRJPs) [
96
].
Small amounts of other proteins exist in RJ such as royalisin, jelleines, and aspimin [
97
]. Novel proteins
that do not belong to the MRJPs group have been newly discovered [
100
]. The antioxidant eect of
RJ is attributed to its protein fraction—up to 29 antioxidative peptides have been identified in RJ
hydrolysates. They contain a phenolic hydroxyl group, which possesses the ability to scavenge free
radicals by donating a hydrogen atom [
97
]. The lipid fraction of RJ accounts for 3–6% and 7–18% of
its wet and dry weights, respectively [
99
,
101
]. Short hydroxyl fatty acids constitute 80–85% of this
fraction, while the rest consists of phenols (4–10%), waxes (5–6%), steroids (3–4%), and phospholipids
(0.4–0.8%) [
102
]. Trans-10-hydroxy-2-decenoic acid (10-HDA) is the main fatty acid in RJ; it exhibits
a plethora of biological properties, e.g., anti-aging, neurogenic, anticancer, antiobesity, antibacterial,
and many others [
63
,
98
]. In addition, fructose and glucose constitute 90% of the carbohydrates
fraction of RJ (7.5–16%) [
63
,
97
,
102
]. RJ contains small amounts of vitamins, minerals, phenols, esters,
aldehydes, ketones, and alcohol. Numerous bioactive substances are available in RJ such as ACh and
nucleotides. Nucleotides exist either as free bases such as adenosine and guanosine, or phosphates
such as adenosine diphosphate and adenosine monophosphate [
63
,
101
,
102
]. Additionally, RJ contains
currently undefined ingredients [100]. Table 1illustrates the structure of RJ in detail.
Various factors influence the quality of RJ by aecting its ingredients and their activity. For
instance, feeding bees with sugars causes significant alterations in the amounts and structure of
vital constituents of RJ such as amino acids (e.g., tryptophan and lysine), derivatives of amino acids
(e.g., pyroglutamic acid), amines (e.g., cadaverine), carbohydrates, and vitamins [
103
]. Of importance,
RJ should be stored in a frozen state in order to retain its biological properties. Storage at 5
C or
above markedly reduces its soluble nitrogen and free amino acids due to activation of enzymatic
degradation and interactions between its lipid and protein fractions, eventually ending with a darker,
rancid, and more viscous substance that consists of water insoluble nitrogenous compounds [94].
RJ is the only food consumed by bee queens throughout their entire life [
63
]. It is suggested that RJ
contributes to the unique qualities of bee queens: long lifespan, high fertility, and excellent learning and
memory ability [
104
]. Hence, this review aims to investigate the anti-aging properties of RJ with a focus
on cognitive function in advanced aging and AD. In this respect, it reviews, synthesizes, and discusses
the most relevant studies that examine the eect of RJ on cognitive aging and AD pathology, both
in cell cultures and animal models, as well as in humans when possible. It also elaborates on the
molecular changes that lie behind these eects.
Antioxidants 2020,9, 937 12 of 47
Table 1. Composition of royal jelly.
Compounds Percentage Examples References
Moisture 50–70% Mainly water. [96,98,99]
Carbohydrates 7.5–16% Sugars such as fructose, glucose, maltose, melibiose,
and ribose. [98,99]
Proteins 9–18%
MRJPs (80% of protein content), minor proteins
(e.g., aspimin, royalisin and jelleines), peptides (in the
form of dipeptides or tripeptides, e.g., alanine-leucine,
leucine- aspartic acid-arginine), and free amino acids
(e.g., threonine, valine, glycine, isoleucine, leucine,
proline, serine, methionine, and tryptophan).
[63,99,102]
Lipidsa 3–6% 10-HDA, sebacic acid, phenols (4–10%), waxes (5–6%),
steroids (3–4%), and phospholipids (0.4–0.8%). [63,98,99,101,102]
Vitamins ?
B5 (52.8 mg/100 g), B6 (42.42 mg/100 g), niacin
(42.42 mg/100 g), and traces of B1, B2, B8, B9, B12,
ascorbic acid (vitamin C), vitamin E and A.
[63,102]
Minerals ?
Potassium, sodium, magnesium, calcium, phosphor,
sulfur, cupper, iron, zinc, selenium, barium, cobalt,
manganese, etc.
[42,63,98]
Bioactive
compounds ?
ACh and nucleotides both as free bases (e.g., adenosine,
guanosine, iridin, and cytidine) and as phosphates
(e.g., adenosine 50-monophosphate, adenosine
50-diphosphate, and adenosine 50-triphosphate).
[63,102]
Others ?
Volatile organic compounds (e.g., esters, aldehydes,
ketones, and alcohols), and minor heterocyclic
compounds.
[63,98,102]
Abbreviations
: MRJPs: major royal jelly proteins, 10-HDA: Trans-10-hydroxy-2-decenoic acid, ACh: acetylcholine.
N.B. percentages of constituents are reported in fresh RJ, and data on the percentages of RJ constituents denoted by
“?” are not clearly available.
6. Eect of Royal Jelly on Cognitive Performance and Related Biological Markers
6.1. Evidence from Preclinical Studies
Research denotes that queen bees, which feast on RJ throughout their entire lives, demonstrate up
to 5-fold higher learning and memory abilities than worker bees, which feast on honey and pollens.
The exceptional cognitive abilities of queen bees are associated with persistent expression of Dnmt3
gene encoding DNA methyltransferase-enzyme catalyzing DNA methylation, which plays a principle
role in the formation of long-term memory [
104
]. Interestingly, supplementing the diet of bee workers
with RJ significantly increased olfactory learning and memory [
105
,
106
], and accelerated the expression
of learning and memory-related genes (GluRA and Nmdar1) compared with control bees. Higher
concentrations of RJ (20%) produced better eects than lower ones [
106
]. A number of studies have
utilized various animal species as models of natural aging and AD (Figure 4) in order to test the eect
of RJ and its elements on cognitive function. The findings of these investigations highlight the ability
of RJ to enhance learning and memory retention, as well as to prevent and treat cognitive behavioral
deficits. Rodents have been widely used as models of natural aging and AD.
RJ significantly improved spatial learning and enhanced memory retention by up to 48.5%
in normally aged rats [
107
110
]. It also corrected cognitive deficits in dierent AD models
such as copper and cholesterol-fed rabbits [
70
], streptozotocin-induced hippocampal neuronal
death [
111
], trimethyltin-induced hippocampal neuronal death [
112
], and double transgenic APP/PS1
mice, which express two mutations associated with early-onset AD—chimeric mouse/human APP
(Mo/HuAPP695swe) and human PS1 (PS1-dE9) [
28
]. Cognitive enhancement/recovery manifested
in the improvement of the spontaneous alternation rate in the Y-maze [
112
], shorter time and path
Antioxidants 2020,9, 937 13 of 47
lengths to find an underwater escape plate in the Morris Water Maze test, increased time spent in the
target quadrant and increased time of the first entrance to the dark room one week after receiving an
electrical shock on the step-down passive avoidance test (the probe trial), increased crossings by up
to 177.4% [
28
,
108
,
109
], as well as decreased time searching for food, and improved response rate to
sudden sound on open field test [95] (Table 2).
Antioxidants 2020, 9, x 13 of 46
Figure 4. Royal jelly improves cognitive function-related parameters in various animal models and in
humans. Abbreviations: AD: Alzheimer’s disease, PQ: paraquat, H
2
O
2
: hydrogen peroxide, Aβ: beta-
amyloid peptide, APP/PS1 mice express 2 mutations associated with early-onset AD—chimeric
mouse/human APP (Mo/HuAPP695swe) and human PS1 (PS1-dE9), LPS: lipopolysaccharide.
RJ significantly improved spatial learning and enhanced memory retention by up to 48.5% in
normally aged rats [107–110]. It also corrected cognitive deficits in different AD models such as
copper and cholesterol-fed rabbits [70], streptozotocin-induced hippocampal neuronal death [111],
trimethyltin-induced hippocampal neuronal death [112], and double transgenic APP/PS1 mice,
which express two mutations associated with early-onset AD—chimeric mouse/human APP
(Mo/HuAPP695swe) and human PS1 (PS1-dE9) [28]. Cognitive enhancement/recovery manifested in
the improvement of the spontaneous alternation rate in the Y-maze [112], shorter time and path
lengths to find an underwater escape plate in the Morris Water Maze test, increased time spent in the
target quadrant and increased time of the first entrance to the dark room one week after receiving an
electrical shock on the step-down passive avoidance test (the probe trial), increased crossings by up
to 177.4% [28,108,109], as well as decreased time searching for food, and improved response rate to
sudden sound on open field test [95] (Table 2).
The improvement of cognitive function associated with RJ treatment results from the
amelioration of oxidative damage and increase of antioxidant capacity [87,106,111]. In vivo studies
involving AD models show that RJ enhanced the endogenous production of antioxidants, increasing
superoxide dismutase (SOD) by up to 27% and reducing free radicals such as ROS, NOS, and
malonaldehyde (MDA) in the cortex and hippocampus [70,95]. A limited number of in vitro studies
examined the mechanistic effect of RJ on AD pathology. The results go in agreement with those
reported by in vivo experiments. RJ protected LPS-stimulated BV-2 microglia against oxidative stress
and attenuated microglial inflammatory processes that contribute to amyloidgenesis. It significantly
decreased the production of ROS, NOS, IL-6, IL-1β, and TNF-α and increased the mRNA expression
of antioxidants such as heme oxygenase-1 (HO-1) and glutathione peroxidase (GSH-Px) [113,114].
Figure 4.
Royal jelly improves cognitive function-related parameters in various animal models and
in humans. Abbreviations: AD: Alzheimer’s disease, PQ: paraquat, H
2
O
2
: hydrogen peroxide, A
β
:
beta-amyloid peptide, APP/PS1 mice express 2 mutations associated with early-onset AD—chimeric
mouse/human APP (Mo/HuAPP695swe) and human PS1 (PS1-dE9), LPS: lipopolysaccharide.
The improvement of cognitive function associated with RJ treatment results from the amelioration
of oxidative damage and increase of antioxidant capacity [
87
,
106
,
111
].
In vivo
studies involving AD
models show that RJ enhanced the endogenous production of antioxidants, increasing superoxide
dismutase (SOD) by up to 27% and reducing free radicals such as ROS, NOS, and malonaldehyde
(MDA) in the cortex and hippocampus [
70
,
95
]. A limited number of
in vitro
studies examined
the mechanistic eect of RJ on AD pathology. The results go in agreement with those reported
by
in vivo
experiments. RJ protected LPS-stimulated BV-2 microglia against oxidative stress and
attenuated microglial inflammatory processes that contribute to amyloidgenesis. It significantly
decreased the production of ROS, NOS, IL-6, IL-1
β
, and TNF-
α
and increased the mRNA expression
of antioxidants such as heme oxygenase-1 (HO-1) and glutathione peroxidase (GSH-Px) [
113
,
114
].
The major fatty acid in RJ, 10-HDA, protected the BBB against LPS-induced leakage by inhibiting the
degradation of tight junction proteins (occludin, claudin-5 and ZO-1) in LPS-stimulated C57BL/6 mice,
as well as by increasing the expression of tight junction proteins, and decreasing the expression of
chemokines (CCL-2 and CCL-3), adhesion molecules (e.g., intercellular adhesion molecule-1 (ICAM-1)
Antioxidants 2020,9, 937 14 of 47
and vascular cell adhesion molecule-1 (VCAM-1)), and matrix metalloproteinases (MMP-2 and MMP-9)
in LPS-stimulated human brain microvascular endothelial cells (HBMECs) [114].
RJ decreased A
β
synthesis and enhanced its clearance in AD rat models, which is associated with
a reduction in the total size and number of cortical and hippocampal SPs [
28
,
95
]. In a unique study,
synchronous CL2006 worms were used as a nematode AD model. Supplementing aged worms with
RJ/enzyme-treated RJ (eRJ) (2 mg/mL and 1 mg/mL) for 10 days at 20
C decreased the total amount of
A
β
species by 13.61% and 21.90%, respectively. Both RJ and eRJ reduced paralysis induced by A
β
toxicity and increased levels of soluble proteins by 27.13% and 21.27%, respectively [
92
]. Similarly,
an
in vitro
study showed that purified RJ peptides (RJPs) (1–9
µ
g/mL) can interfere with the process
of A
β
formation and prevent the external production of A
β
1-40 and A
β
1-42 in N2a/APP695swe cell
cultures (which produce high levels of APP in AD, because they are stably transfected with the human
APP gene) via down-regulation of
β
-secretase [
115
]. Another study reported increased the clearance
of soluble A
β
by a DMSO-soluble fraction of RJ [
116
]. In addition, RJ corrected pathologies that
promote amyloid cleavage, e.g., it decreased cholesterol levels [
70
] and increased free thyroxine (fT4) in
hypothyroidism rat models [
73
]. MRJPs contributed to DNA repair in aged rats by increasing levels of
xanthosine, which supports the anabolism of nucleic acid [
109
]. RJ activated autophagy genes [
117
,
118
]
and protected against neuronal apoptosis [
28
] (the mechanisms underlying these eects are described
in detail in Section 7).
Antioxidants 2020,9, 937 15 of 47
Table 2. Eects of royal jelly on cognition and related molecular-level changes in experimental models and humans (N of reviewed studies =30).
Animal/Cell Line Model RJ Treatment Summary of Eects and Mechanism Reference
Hippocampal SST and NEP positive neurons DRJ (100 mg/mL) SST and NEP gene expression and CREB-binding to CRE at the
promoter region of SST. [116]
N2a/APP695 cells RJPs (1–9 µg/mL) Aβ1-40, Aβ1-42, and BACE1. [115]
LPS-stimulated BV-2 microglia RJ (0.3–3 mg/mL) IL-6, IL-1β, TNF-α, iNOS, and COX-2. [113]
Apis mellifera workers as a model of learning RJ (10% and 20%) in 50% sucrose solution
Olfactory learning, memory, and expression of memory-related genes
(GluRA and Nmdar1). [105,106]
Naturally aged Drosophila and Drosophila treated
with H2O2and paraquat eRJ (1–5 mg/mL) plus CP at a ratio of 2:3
T-SOD, GSH-Px, CAT, average life span, food consumption, weight
gain, and exercise capacity.
MDA and protein carbonyl.
[96]
Aβtoxicity in CL2006 worm model of AD RJ (2 mg/mL) and eRJ 1 mg/mL)/day/10 days at
20 C
Aβspecies, Aβ-induced body paralysis, and IIS signaling.
Soluble proteins. [92]
LPS-stimulated C57BL/6J mice and microglial
BV-2 cells Oral 10-HAD (100 mg/kg/day for 1 month)
TNF-
α
, Tnfrsf8, Traf1, IL-1
β
, NF-
κ
B and NLRP3 inflammasome-IL-1
β
signaling, and SQSTM1.
FOXO1-mediated autophagy, ULK, and LC3-II.
[117]
LPS-stimulated C57BL/6 mice and HBMECs Oral 10-HAD (100 mg/kg/day for 1 month)
ROS, CCL-2, CCL-3, ICAM-1, VCAM-1, MMP-2, and MMP-9, BBB
permeability, and tight junction proteins degradation.
Expression of tight junction proteins, and AMPK/PI3K/AKT signaling.
[114]
OVX cholesterol-fed rabbit model of AD Oral RJ (400 mg/kg/day/12 weeks)
Behavioral cognitive deficits, body weight, blood lipid, BBB
permeability, brain levels of MDA, Aβ, AchE, BACE1, and RAGE.
ChAT, SOD, LRP-1, heart rate variability, and Baroreflex sensitivity.
[95]
OVX rat model of aging Oral eRJ (250 mg/mL tap water: 10 mL/kg/day/
82 days)
Cognitive and depressive-like behavioral deficits.
Brain weight and myelin galactolipids. [87]
A rat model of AD induced by streptozotocin (icv)
Oral RJ (200 mg/kg/day/14 days)
O
2-
(in the DG and hilus regions) and neurodegeneration (in the DG).
Working memory and neurogenesis in the DG. [111]
Hypothyroidism rat model of cognitive
impairment Intragastric RJ (100 mg/kg/day/20 days) Brain vascular dilation, edema, and degeneration.
MAP-2 and fT4. [73]
APP/PS1 transgenic mice model of AD Intragastric RJ (300 mg/kg/day/3 months)
Spatial learning and memory.
MDA, p-JNK and bax/bcl-2 ratio, caspase-3, BACE1, A
β
40 and A
β
42,
and the total area and number of senile plaques in the cortex and
hippocampus.
cAMP, p-PKA, p-CREB, BDNF, IDE, and LRP-1.
[28]
Antioxidants 2020,9, 937 16 of 47
Table 2. Cont.
Animal/Cell Line Model RJ Treatment Summary of Eects and Mechanism Reference
A rabbit model of AD induced by cholesterol diet
and copper sulfate Oral RJ (400 mg/kg/day/12 weeks)
TC, LDL-C, MDA, ROS, RNS, Cho/Cr, mI/Cr, caspase-3, BACE1,
Aβ1-40, Aβ1-42, Aβplaque, and neuronal loss.
SOD, LRP-1, IDE, NAA/Cr, and glutamate/Cr.
[70]
A mouse model of streptozotocin-induced
cognitive impairment Dietary RJ (3% w/w/day/10 days) Streptozotocin-induced defects in learning and memory. [16]
A mouse model of trimethyltin-induced
hippocampal DG damage Dietary RJ (1% or 5% w/w/day/6 days) Cognitive impairment and neuronal cell loss.
Number of hippocampal DG granule cells. [112]
A mouse model of cadmium-induced cortical
damage Intragastric RJ (85 mg/kg/day/7 days)
NRF2, GSH-Px, GSH-R, SOD, CAT, Bcl-2, norepinephrine, dopamine,
and serotonin.
iNOS, ROS, NOS, TNF-α, IL-1β, Bax, caspase-3, and cadmium level
in cortical neurons.
[119]
A mouse model of tartrazine-induced cortical
damage Ora RJ (300 mg/kg/day/30 days) CAT, SOD, GSH, and brain levels of GABA, dopamine, and 5HT.
MDA, cortical pyknotic nuclei, and ssDNA positive apoptotic cells. [120]
Naturally aged rats Oral RJ (50 and 100 mg/kg/day/8 weeks) Memory and learning. [107]
Naturally aged rats Dietary RJ (3% w/w/day/10 days) Memory and learning. [108]
Naturally aged rats Intragastric MRJPs (125 mg/kg/day/14 weeks)
Learning, memory, gluconeogenesis, brain glucose supply and ATP
level, nicotinate and nicotinamide metabolism—NaMN,
and cysteine-taurine metabolism.
ROS, AKT, and GABA.
[109]
Naturally aged rats Oral/intragastric RJ (50 and 100 mg/kg/day/
8 weeks)
Learning, spatial memory, and motor performance.
5-HT, dopamine, MHPG and its turnover.
5HIAA, DOPAC and their turnover in the prefrontal cortex.
DOPAC and 5HIAA in the striatum.
[107,110]
Naturally aged rats Subcutaneous RJ (100 and 500 mg/kg/day/6 days) Serotonin activity in the hippocampus and prefrontal cortex. [121]
Naturally aged rats Intragastric RJ (50 and 100 mg/kg/day/8 weeks) GABA in the striatum and hypothalamus. [122]
d-galactose induced mouse model of aging Intragastric RJ and eRJ (0.7 and 1.4 mg/kg/day/
90 days)
ROS and body weight loss.
Memory, learning, muscular performance, and levels of internal
antioxidant enzymes.
[123]
d-galactose induced mouse model of aging Intragastric RJ (0.7 and 1.4 mg/kg/day/90 days)
Spatial learning, memory, brain levels of norepinephrine, dopamine,
and SOD.
MDA.
[124]
Antioxidants 2020,9, 937 17 of 47
Table 2. Cont.
Animal/Cell Line Model RJ Treatment Summary of Eects and Mechanism Reference
Elderly with MCI RJ plus herbal extracts Scores of the Mini-Mental State Scale. [125]
Postmenopausal women with menopausal
complaints RJ plus flower pollen Problem-solving ability, HDL, and TG.
Depression, menopausal symptoms, TC, and LDL. [126]
Abbreviations:
denotes increase,
denotes decrease, RJ: Royal jelly, eRJ: enzyme-treated RJ, MRJPs: major RJ proteins, RJPs: purified RJ peptides, SST: somatostatin, NEP: neprilysin, cAMP:
cyclic adenosine monophosphate, CREB: cAMP-response element (CRE)-binding protein, DRJ: DMSO-soluble fraction of RJ, A
β
: amyloid-
β
peptide, APP: amyloid precursor protein,
N2a/APP695 cells: an
in vitro
model of AD pathology being stably transfected with the human APP gene to produce high levels of A
β
, DG: dentate gyrus granule, BACE1/
β
-secretase:
beta-site APP cleaving enzyme 1, IL-6: interleukin-6, IL-1
β
: interleukin-1
β
, TNF-
α
: tumor necrosis factor alpha, icv: intracerebroventricular injection, iNOS: inducible nitric oxide synthase,
COX-2: cyclooxygenase-2, NF-
κ
B: nuclear factor-kB, CP: collagen peptide, T-SOD: total superoxide dismutase, GSH-Px: glutathione peroxidase, CAT: catalase, IIS: insulin/insulin-like
growth factor, NLRP3: nucleotide-binding domain and leucine-rich repeat containing protein 3, SQSTM1: Sequestosome 1, ULK: Unc-51-like autophagy activating kinase, LC3-II:
Microtubule-associated protein 1 light chain 3-II, TNFRSF8: tumor necrosis factor receptor superfamily, member 8, TRAF1: TNF receptor-associated factor 1, 10-HDA: 10-hydroxy-decanoic
acid, HBMECs: human brain microvascular endothelial cells, ROS: reactive oxygen species, RNS: reactive nitrogen species, CCL-2: C-C motif ligand 2, CCL-3: C-C motif ligand
3, ICAM-1: intercellular adhesion molecule 1, VCAM-1: vascular cell adhesion molecule-1, MMP: matrix metalloproteinase, BBB: blood brain barrier, AMPK: 5
0
-AMP-activated
protein kinase, PI3k: phosphoinositide-3 kinase, ATP: Adenosine triphosphate, AKT: a serine/threonine nutrient sensing protein kinase of the PI3k family, OVX: ovariectomized, AchE:
acetylcholinesterase, RAGE: receptor for advanced glycation end products, LRP-1: low density lipoprotein receptor-related protein 1, MDA: Malonaldehyde, MAP-2: microtubule-associated
protein 2, fT4: free thyroxine, p-JNK: Phosphorylated p-Jun N-terminal kinase, NRF2: nuclear factor erythroid 2, Bcl-2: B-cell lymphoma 2, Bax: Bcl-2-associated X protein, PKA:
adenosine A2A receptor-mediated protein kinase A, BDNF: brain-derived nerve factor, IDE: insulin-degrading enzyme, TC: total cholesterol, LDL-C: low density lipoprotein C, HDL:
high-density lipoproteins, TG: triglycerides, NAA: N-acetyl aspartate, Cho: choline, mI: myo-inositol, Cr: creatine, GABA: gamma-aminobutyric acid, 5HT: serotonin transporter, 5HIAA:
5-hydroxyindoleacetic acid, DOPAC: 3,4-dihydroxyphenylacetic acid, MHPG: 3-methoxy-4-hydroxyphenylglycol, NaMN: nicotinic acid mononucleotide.
Antioxidants 2020,9, 937 18 of 47
6.2. Evidence from Clinical Trials
Preclinical trials show that RJ/eRJ, 10-HDA, RJ peptides, and MRJPs exhibit significant eects
against AD pathology in animal models and in cell lines by interfering with amyloid synthesis
and protein misfolding, enhancing amyloid clearance, and correcting pathologies that contribute
to amyloidogenesis, such as inflammation and oxidative stress (Table 2). However, concerns have
arisen about the notion that similar eects can be obtained in humans. Unfortunately, research using
RJ to treat cognitive impairment in humans is scarce. In a single randomized clinical trial (RCT),
66 patients (50-80 years old) with MCI received a daily capsule of Memo
®
, a triple combination
of 750 mg of lyophilized RJ with standardized extracts of Ginkgo biloba (120 mg) and Panax ginseng
(150 mg), for 4 weeks. Memo
®
significantly improved scores of the Mini-Mental State Examination
compared with placebo treatment [
125
]. Similarly, daily consumption of 2 capsules of Lady 4—a
combination of 200 mg of lyophilized RJ, 250 mg of evening primrose oil, 100 mg of Turnera diusa,
and 50 mg of Panax ginseng—for 4 weeks significantly reduced total scores of the Menopause Rating
Scale. This scale measures menopausal symptoms including impaired memory, poor concentration,
nervousness, depression, and insomnia. However, the authors only reported the total score of
the scale, and it is unclear if Lady 4 had any eect on cognitive and psychological symptoms of
menopause [
127
]. Another uncontrolled, open-label trial treated 55 postmenopausal women (having
menopausal complaints) with Melbrosia, a dietary supplement that combines RJ with flower pollen
and fermented flower pollen, for 3 months. Melbrosia significantly improved participants’ scores on
the problem-solving subscale of the Frankfurt Self-concept Scale, relieved depressive and menopausal
symptoms, decreased total cholesterol and low-density lipoproteins, and increased high-density
lipoproteins and triglycerides, whereas VCAM-1 and C-reactive protein levels were not aected [
126
].
In contrast, a former RCT reported no eect of Melbrosia on biochemical parameters in women with
severe menopausal symptoms, although it significantly improved vitality and relieved symptoms
of headache, urinary incontinence, and vaginal dryness [
128
]. Minor adverse eects were reported
including weight gain (a Melbrosia tablet contains 307 kcal), brief GI discomfort, transient facial flush,
mild nausea, and mild transient headache in 6 subjects [
125
,
126
]. Nonetheless, it is impossible to
confirm either the eectiveness of RJ in AD patients or its side eects from the data reported in these
studies. In all trials, RJ was combined with other elements, biomarkers were not assessed in patients
with cognitive impairment, and various methodological flaws were incorporated, e.g., small sample
sizes, lack of blinding, and absence of a control group [125,126].
7. Mechanisms Underlying Eects of RJ on Cognition and AD-Related Pathology
The findings from the studies examined in this review indicate that RJ targets a variety
of pathophysiological mechanisms of cognitive aging and AD. The neurotrophic, antioxidative,
anti-inflammatory, anti-apoptotic, and anti-amyloidogenic properties of RJ allow it to act as a
multidomain cognitive enhancer, which may delay the onset of AD, slow its progression, and foster
recovery. Figure 5summarizes the anti-AD and cognitive promoting activities of RJ. This section
describes the mechanisms underlying these activities in detail.
RJ improves cognition via a network of inter-related mechanisms. It alleviates A
β
pathology by
(1) decreasing its influx through the BBB via inhibition of RAGE, (2) preventing the cleavage of APP
into A
β
via inhibition of BACE1, and (3) facilitating the degradation and clearance of A
β
by IDE, NEP,
and LRP1. From another perspective, RJ activates AMPK, a master signaling pathway that activates
various signaling pathways (mainly through an indirect pathway mediated by phosphorylation
of FOXO, which inhibits mTOR). AMPK activity promotes autophagy and antioxidant production,
and suppresses microglial inflammation via inhibition of various oxidative, inflammatory, and apoptotic
pathways, e.g., iNOS and NF-
κ
B. RJ and its lipids bind to estrogen receptors
β
and
α
to enhance the
production of neurotrophins such as NGF and BDNF, which promote ACh production, neurogenesis,
and synaptogenesis, and counteract amyloidogenesis, etc.
Antioxidants 2020,9, 937 19 of 47
Antioxidants 2020, 9, x 17 of 46
(50-80 years old) with MCI received a daily capsule of Memo
®
, a triple combination of 750 mg of
lyophilized RJ with standardized extracts of Ginkgo biloba (120 mg) and Panax ginseng (150 mg), for 4
weeks. Memo
®
significantly improved scores of the Mini-Mental State Examination compared with
placebo treatment [125]. Similarly, daily consumption of 2 capsules of Lady 4—a combination of 200
mg of lyophilized RJ, 250 mg of evening primrose oil, 100 mg of Turnera diffusa, and 50 mg of Panax
ginseng—for 4 weeks significantly reduced total scores of the Menopause Rating Scale. This scale
measures menopausal symptoms including impaired memory, poor concentration, nervousness,
depression, and insomnia. However, the authors only reported the total score of the scale, and it is
unclear if Lady 4 had any effect on cognitive and psychological symptoms of menopause [127].
Another uncontrolled, open-label trial treated 55 postmenopausal women (having menopausal
complaints) with Melbrosia, a dietary supplement that combines RJ with flower pollen and fermented
flower pollen, for 3 months. Melbrosia significantly improved participants’ scores on the problem-
solving subscale of the Frankfurt Self-concept Scale, relieved depressive and menopausal symptoms,
decreased total cholesterol and low-density lipoproteins, and increased high-density lipoproteins
and triglycerides, whereas VCAM-1 and C-reactive protein levels were not affected [126]. In contrast,
a former RCT reported no effect of Melbrosia on biochemical parameters in women with severe
menopausal symptoms, although it significantly improved vitality and relieved symptoms of
headache, urinary incontinence, and vaginal dryness [128]. Minor adverse effects were reported
including weight gain (a Melbrosia tablet contains 307 kcal), brief GI discomfort, transient facial flush,
mild nausea, and mild transient headache in 6 subjects [125,126]. Nonetheless, it is impossible to
confirm either the effectiveness of RJ in AD patients or its side effects from the data reported in these
studies. In all trials, RJ was combined with other elements, biomarkers were not assessed in patients
with cognitive impairment, and various methodological flaws were incorporated, e.g., small sample
sizes, lack of blinding, and absence of a control group [125,126].
7. Mechanisms Underlying Effects of RJ on Cognition and AD-Related Pathology
The findings from the studies examined in this review indicate that RJ targets a variety of
pathophysiological mechanisms of cognitive aging and AD. The neurotrophic, antioxidative, anti-
inflammatory, anti-apoptotic, and anti-amyloidogenic properties of RJ allow it to act as a
multidomain cognitive enhancer, which may delay the onset of AD, slow its progression, and foster
recovery. Figure 5 summarizes the anti-AD and cognitive promoting activities of RJ. This section
describes the mechanisms underlying these activities in detail.
Figure 5. Possible mechanisms of action behind royal jelly-related effects in cognitive aging and
Alzheimer’s disease. Abbreviations: denotes increase, denotes decrease, Aβ: beta-amyloid
protein fragments, RAGE: receptor for advanced glycation end products, BBB: blood brain barrier,
ROS: reactive oxygen species, APP: amyloid precursor protein, BACE1: beta-site APP cleaving
enzyme 1, IDE: insulin-degrading enzyme, NEP: neprilysin, SST: somatostatin, LRP-1: low density
Figure 5.
Possible mechanisms of action behind royal jelly-related eects in cognitive aging and
Alzheimer’s disease. Abbreviations:
denotes increase,
denotes decrease, A
β
: beta-amyloid
protein fragments, RAGE: receptor for advanced glycation end products, BBB: blood brain barrier,
ROS: reactive oxygen species, APP: amyloid precursor protein, BACE1: beta-site APP cleaving
enzyme 1, IDE: insulin-degrading enzyme, NEP: neprilysin, SST: somatostatin, LRP-1: low density
lipoprotein) receptor-related protein 1, iNOS: inducible nitric oxide synthase, NRF2: nuclear
factor-erythroid 2-related factor 2, AMPK: AMP-activated protein kinase, FOXO: Forkhead Box O
transcription factor, HSF-1: heat shock transcription factor 1, PI3K: phosphatidylinositol 3-kinase, PKA:
cAMP-dependent protein kinase, cAMP: cyclic adenosine mono phosphate, CREB: cAMP-response
element (CRE)-binding protein, mTOR: mammalian target of rapamycin, ULK: Unc-51-like kinase,
LC3-II: microtubule-associated protein 1 light chain 3-II, SQSTM1: sequestosome 1, Bcl-2: B-cell
lymphoma-2, Bax: Bcl-2-associated X protein, NF-
κ
B: nuclear factor-kappa B, JNK: c-Jun NH2-terminal
kinases, NLRP3: nucleotide-binding domain and leucine-rich repeat containing protein 3, GSK-3
β
:
glycogen synthase kinase-3
β
, IIS: insulin/insulin-like growth factor, ER
β
and
α
: estrogen receptors
β
and
α
, MAPK: mitogen-activated protein kinase, ERK1/2: extracellular signal-regulated kinase 1
or 2, NGF: nerve growth factor, ACh: acetylcholine, ChAT: choline acetyltransferase, BDNF: brain
derived neurotrophic factor, p90RSK: pp90 ribosomal S6 kinase, MSK1/2 and MAPKAP: mitogen- and
stress-activated protein kinase and kinase 2, PC12: progenitor stem cells.
7.1. Royal Jelly-Related Neuroprotection Is Mediated by Regulating the Production of Neurotrophins
The depletion of neurotrophins such as brain derived neurotrophic factor (BDNF), glial cell
line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF) significantly aects neuronal
and nonneuronal responses to AD and accelerates disease progression [
9
,
15
]. On the other hand,
the enhancement of the production of endogenous neurotrophins limits neurodegeneration. NGF, a key
member of the neurotrophin family, promotes the survival and function of cholinergic neurons of the
basal forebrain, which undergo massive degeneration in AD [
129
]. NGF boosts the production of ACh
in cholinergic neurons by enhancing the release and activity of ChAT [
85
]. In fact, RJ (which stimulates
NGF production [
130
,
131
]) was shown to increase the activities of ChAT and decrease AChE in
the cortex of a rabbit model of AD induced by ovariectomy and cholesterol diet [
95
]. NGF also
enhances healing of injured astrocytes in dierent brain areas [
132
]. The highest levels of NGF
originate from brain regions that are most insulted in AD: the cortex, hippocampus, and the basal
ganglia [
133
,
134
]. External NGF replacement has been used as a potential treatment to promote neural
regeneration in neurodegenerative disease such as AD [
133
,
134
]. However, the passage of NGF across
the BBB represents a major challenge for nonviral gene delivery via a systematic transvascular route.
Therefore, the delivery of this therapy is limited to invasive intracerebral injection [
135
]. Nonetheless,
its eciency is also questionable. For example, a two-year RCT examined the eect of intracerebral
injections of adeno-associated viral vector (serotype 2)-NGF in AD patients using magnetic resonance
Antioxidants 2020,9, 937 20 of 47
imaging, fludeoxyglucose F18-labeled positron emission tomography imaging, and neuropsychological
testing. The results revealed that NGF was safe and well-tolerated, but there was no evidence of
ecacy [
133
]. Therefore, the use of natural agents that are capable of stimulating internal production
of NGF may support healing of injured brain tissues in a more cost-eective manner than external
NGF administration.
AMP N1-oxide is a chief RJ derivative that can modulate neuronal function and stimulate
nitrite outgrowth and processes formation via adenosine A2A receptors [
130
,
131
]. Targeting glial A2A
receptors, in particular, is a promising target for AD treatment. A2A receptors are expressed in the frontal
cortex and hippocampus, and they play a major role in neuroinflammation and neurodegeneration
by controlling synaptic plasticity, NMDAR activity, and glutamate uptake by astrocytes [
84
]. Strong
activation of A2A receptor genes by eRJ and AMP N1-oxide highlights their role in the regulation of
synaptic plasticity, which is crucial for early neuronal development [
130
,
136
]. The eects demonstrated
by AMP N1-oxide at concentrations of 20 or 40 mM are similar to the eect of 10 or 50 ng/mL of NGF.
Meanwhile, combining NGF and eRJ had no eect on the percentage of process-bearing cells compared
with solo NGF treatment, but a marked increase in the percentage of cells with processes longer than
two times the cell diameter was recorded [
131
]. Evidence denotes that AMP N1-oxide mimics the
regenerative eect of NGF [
130
]. NGF stimulates neurite outgrowth in neuronal progenitor stem cells
(PC12) and contributes to synaptic plasticity through binding receptors p75 and tropomyosine-related
kinase A (TrkA), a member of the tyrosine kinases family [
130
,
137
]. This process entails increasing the
expression of a protein of mature neurons known as neurofilament M and enhancing the dierentiation
of pheochromocytoma PC12 into neurons similar to sympathetic neurons. Eects of AMP N1-oxide
on cell outgrowth and dierentiation involve the activation of two main cellular signaling cascades:
adenosine A2A receptor-mediated phosphatidylinositol 3-kinase (PI3K)/Akt/cAMP-dependent protein
kinase (PKA)/CREB and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated
kinase 1 or 2 (ERK1/2) signaling pathways [
130
]. Phosphorylation of ERK1/2 or the p38 MAPK pathway
for CREB takes place via an indirect pathway mediated by the phosphorylation of pp90 ribosomal
S6 kinase (p90RSK), mitogen- and stress-activated protein kinase (MSK)1/2, MAPKAP kinase 2 [
138
].
Integrin receptor signaling is necessary for the activation of ERK1/2, because it stimulates Mn
2+
to
accelerate neurite outgrowth from PC12 cells treated with eRJ or AMP N1-oxide [
136
]. In addition,
AMP N1-related activation of PI3K/Akt upregulates phosphorylation of glycogen synthase kinase-3
β
(GSK-3β) to enhance cellular survival by abrogating inflammatory signaling [139].
BDNF, especially in the hippocampus, plays a major role in the regulation of processes
involved in learning and memory, such as long-term potentiation, synaptic plasticity, axonal
sprouting, and proliferation of dendritic arbor, mainly through its interaction with Trk B receptor [
9
].
Polymorphisms of BDNF have been reported to be associated with AD [
140
,
141
]. RJ, 10-HDA,
and 10-HDA-related esters (e.g., Trans-2-decenoic acid ethyl ester (DAEE) and 4-Hydroperoxy-2-
decenoic acid ethyl ester (HPO-DAEE)) are reported to generate intracellular signals like BDNF,
which grants them the potential of neuronal regeneration. In addition to stimulating neurogenesis and
neurite outgrowth, these compounds promote neuronal survival and demonstrate neuroprotection
against physical injury and neurotoxicity [
112
,
136
,
142
,
143
]. A comparison of signaling induced
by BDNF and DAEE in cultured rat embryonic neurons revealed that DAEE increased the mRNA
expression of BDNF and neurotrophin-3 and enhanced synaptogenesis as portrayed by increased
synapse-specific proteins such as synaptophysin, synapsin-1, and syntaxin [
144
]. Evolving evidence
indicates that RJ-related induction of BDNF signaling may alleviate AD symptoms in APP/PS1 mice by
reducing cortical and hippocampal levels of BACE1, soluble and insoluble A
β
40 and A
β
42, as well as
the number and size of Aβplaques [28].
GDNF is a diusible peptide, which is centrally involved in neuronal dierentiation and survival;
it has been suggested that it regulates processes implicated in the repair of damaged brain tissues [
145
].
Research shows that oral administration of RJ enhances the production of GDNF and neurofilament H
in the hippocampus of adult mice [
146
]. Interestingly, the expression of neurofilament M was associated
Antioxidants 2020,9, 937 21 of 47
with increased dierentiation of neural stem/progenitor cells (NS/NPCs) into mature neurons [
130
].
NS/NPCs exist in the adult hippocampal dentate gyrus (DG), and can dierentiate into neurons that
regulate memory and learning [
112
]. Injecting umbilical cord blood mononuclear cells (UCBMCs)
transduced with adenoviral vectors expressing GDNF to the sites of neurodegeneration into AD
transgenic mice resulted in the presence of UCBMCs in the hippocampus and cortex several weeks after
transplantation. This treatment induced long-lasting neuroprotection and stimulated synaptogenesis,
delineated by the restoration of postsynaptic density protein 95 and synaptophysin levels in the
hippocampus, which was associated with improvement of spatial memory [145].
7.2. Royal Jelly Regulates Neurotransmission in Models of Advanced Aging and Alzheimer’s Disease
Gamma-aminobutyric acid (GABA) is a major brain neurotransmitter that is synthesized inside
neurons by glutamate decarboxylase (GAD) and metabolized by GABA-transaminase (GABA-T) [
122
].
Current research highlights the role of GABA in memory and spatial learning. RJ treatment increased
GABA levels in the cortex of rats undergoing cortical damage induced by tartrazine [
120
]. In contrast,
RJ supplementation to naturally aged rats decreased GABA in the striatum and hypothalamus.
This eect was not associated with a drop in glutamate, the precursor of GABA [
122
]. It seems
that minor changes in GABA neurotransmission aect glutamate-mediated neurotransmission via
NMDAR activity [
82
]. Dierent GABA-ergic receptors work in a fashion that sustains the balance
between the excitatory glutamatergic and the inhibitory GABAergic neurotransmitters [
122
]. In this
regard, antagonism of GABA A receptor by injecting bicuculline into CA1 altered spatial memory
and maintained nonspatial memory under conditions of NMDAR antagonism by ketamine. GABA
A receptor agonism, under the same conditions, did not alter nonspatial change detection ability
but altered spatial novelty [
82
]. In another study, agonism of GABA A and GABA B receptors by
intra-CA1 microinjections of muscimol and baclofen impaired memory retention in the same way as
D-AP5 (a NMDAR antagonist). Meanwhile, agonism of GABA A receptor accelerated the memory
impairment eect of D-AP5, whereas its antagonism had no eect. The activation and blockade of
GABA B receptor by baclofen and phaclofen at a low dose (0.1
µ
g/rat) potentiated memory impairment
induced by simultaneous administration of low doses of D-AP5 (0.0625 and 0.125
µ
g/rat); however,
they abolished the memory impairment eect of higher D-AP5 doses (0.25
µ
g/rat). Thus, the agonism
or antagonism of dierent GABAergic receptors may aect memory in a dual manner, depending
on the level of NMDAR neurotransmission [
147
]. It is possible that RJ modulation of GABA in the
hypothalamus is associated with increased activity of the hypothalamus and enhanced production
of gonadotropin-releasing hormone and sex steroids, which express memory promoting activities
(detailed in Section 7.8.) [122].
The destruction of synaptic spines in AD brains results from increased activity of free radicals,
phosphorylated tau oligomers, and caspase-3, which follow excessive intercellular Ca
2+
influx.
Synaptic loss triggers dysregulation of synaptic signal transduction, i.e., the synthesis, transport,
release, and uptake of neurotransmitters, which accelerates neurodegeneration [
73
,
80
,
84
]. Evidence
shows that serotonergic transmission, which plays a role in the pathogenesis of depression [
148
],
influences processes of learning and memory. Serotonin receptors exist in the prefrontal cortex,
amygdala, and hippocampus—the main brain structures involved in high cognitive functions [
121
,
149
].
Accumulating evidence suggests that agonism of 5-hydroxytryptamine (5-HT)2A/2C and 5-HT4
receptors or antagonism of 5-HT1A, 5HT3, and 5-HT1B receptors protects against memory impairment
and promotes learning under conditions of high cognitive demand [
149
]. Meanwhile, 90% of the
metabolism of tryptophan, the precursor of serotonin, is directed toward the synthesis of kynurenine.
Kynurenine is a precursor of kynurenic acid, which aects cognition by antagonizing glutamate
ionotropic receptors. In this light, tryptophan deficiency is associated with increased memory
impairment [
149
]. Glial activation and high levels of cytokines contribute to the upregulation of
astrocytic serotonin transporter (5-HTT) and the reduction of extracellular serotonin/5-HT levels [
148
].
From another standpoint, evolving knowledge highlights an important role of dopamine and its
Antioxidants 2020,9, 937 22 of 47
D2-like dopamine receptor gene (AmDOP3) in learning and memory via the homovanillyl alcohol
target dopamine pathways in the brain [
150
]. RJ contains tryptophan and tyrosine, the precursors of
serotonin and dopamine [
63
,
103
,
151
]. Treating experimental models with RJ and tyrosine significantly
increases brain levels of dopamine and its metabolites [
152
,
153
]. Therefore, it is possible that enhancing
brain neurotransmission is one of the molecular changes that lie behind the cognition-agonizing
eects of RJ. In two studies, both short- and long-term application of RJ to naturally aged rats
significantly improved learning, spatial memory, and motor performance. Such eects were associated
with significant modifications in brain content of various neurotransmitters and their metabolites;
the serotonergic and dopaminergic activity increased in the prefrontal cortex, as denoted by a decrease
of 5-HT and dopamine and an increase of their metabolites—5-hydroxyindoleacetic acid (5HIAA)
and 3,4-dihydroxyphenylacetic acid (DOPAC)—and increased turnover of these metabolites. RJ also
decreased cortical levels of 3-methoxy-4-hydroxyphenylglycol, a metabolite of noradrenaline, and its
turnover. Striatal DOPAC increased, which is in line with the improvement of motor activity by RJ
treatment. Short-term RJ treatment increased hippocampal 5-HT content and decreased serotonin
turnover but had no eect on levels of dopamine or noradrenaline though their metabolism in the
prefrontal cortex, hippocampus, and striatum changed [
107
,
121
]. In another study, improvements in
learning and memory following RJ treatment were associated restoration of brain levels of noradrenaline
and dopamine in a mouse model of aging induced by intraperitoneal injection of d-galactose [
124
].
In two other studies, RJ restored cortical levels of serotonin, dopamine, and noradrenaline and
attenuated cortical neuron death induced by tartrazine and cadmium [
119
,
120
]. Overall, these reports
suggest that RJ treatment of cognitively impaired animals was associated with less synaptic destruction
and enhancement of synaptogenesis—a finding reported in several previous studies [
130
,
136
,
144
,
154
].
7.3. Royal Jelly Regulates Energy Metabolism in the Brain
Glucose is necessary for neuronal functioning, and glucose transporters (mainly GLUT1, and to a
limited extent, GLUT2, GLUT 3, GLUT4, GLUT8) mediate glucose transport across the BBB and glucose
uptake into neurons and astrocytes [
64
,
155
,
156
]. Higher metabolic rates and higher predisposition
to deposit fat along with changes in the allocation of energy supplies are essential for the evolution
of brain size and complexity [
157
]. Human carriers of APOE-
ε
4 experience decreased glucose
metabolism in the temporal cortex and parahippocampal gyrus early, i.e., during young adulthood
and middle-age [
21
]. Meanwhile, glucose and oxygen metabolic rates in brain cells decrease with
normal aging and markedly diminish in AD [
157
,
158
]. On one hand, aging entails increased expression
of RAGE, which aects various major metabolic functions (e.g., glucose tolerance) in conditions
predisposing to AD such as obesity, metabolic syndrome, and type 2 diabetes [
64
,
159
]. In fact, diabetes
mellitus is associated with alterations in the brain expression levels of dierent glucose transporters,
which contribute to poor glucose supply to the brain [
64
,
155
]. On the other hand, AD embraces
deficiency of adiponectin, an adipose tissue-derived adipokine that plays major roles in inflammation,
oxidative stress, lipid and glucose metabolism, and neurogenesis. Moreover, it activates peroxisome
proliferator-activated receptor-
α
(PPAR-
α
) signaling in microglia exposed to toxic A
β
oligomers.
Reduced levels of adiponectin, such as in obesity and type 2 diabetes, worsen cognitive impairments
through inhibition of AMP-activated protein kinase (AMPK), which is associated with the development
of cerebral insulin resistance [33].
In light of these reports, processes of glycolysis and gluconeogenesis evidently aect higher
brain functions, e.g., learning and memory [
109
]. Evidence has revealed that a slight increase of
brain glucose supply, indicated by elevated energy consumption in neurons of the mushroom body
(i.e., the main memory center in fruit flies), is necessary for long-term memory formation. This eect is
mediated by dopamine signaling [
160
]. RJ reduced amyloidogenesis in aged Caenorhabditis (C.)elegans
by inhibiting IIS signaling [
92
]. Downregulation of IIS improves insulin sensitivity [
63
] and triggers
local transfer of Ca
2+
from the ER to mitochondria to stimulate GLUT4 translocation to the cell surface
to increase glucose uptake [
161
]. Supplementing aged rats with MRJPs protected against memory
Antioxidants 2020,9, 937 23 of 47
impairment through restoration of brain levels of adenosine triphosphate (ATP) and improvement of
brain metabolism. Mechanistically, MRJPs modestly enhanced glucose supply to the brain, resulting in
enhanced brain levels of phosphoenolpyruvic acid, which stimulates gluconeogenesis to modulate its
level. Metabolomics analysis revealed that urine metabolites of MRJPs-treated rats were similar to those
of young rats, which significantly diered from the untreated control aged rats [
109
]. Experimental
trials showed that RJ can support neuronal metabolic activities in AD brains. In an AD rabbit model
induced by copper and a high cholesterol diet, N-acetyl aspartate and glutamate markedly dropped
and choline and myo-inositol increased compared with the normal control group. On the other hand,
RJ treatment significantly improved neuronal metabolic activities, as shown by increased levels of
N-acetyl aspartate and glutamate and their ratio to creatine, whereas levels of choline and myo-inositol
and their ratio to creatine decreased compared with untreated AD rabbits [
70
]. Furthermore, cumulative
knowledge denotes the contribution of royal jelly to the production of ketone bodies [
162
]. AD brains
utilize ketones, such as acetoacetate and
β
-hydroxybutyrate, as major alternative energy substrates
to glucose [
158
]. Empirical evidence shows that consumption of a ketogenic diet improves verbal
memory and processing speed in patients with cognitive impairments [163,164].
One of the mechanisms through which RJ controls energy expenditure involves the modulation
of eat-2 mutant, which activates multiple dietary restriction-related signaling cascades [
63
,
165
].
Brain cells adaptively respond to bioenergetic challenges such as stress induced by food deprivation
through activation of signaling pathways that enhance synaptic structure and function, stimulate
synaptic formation, increase the production of new neurons from stem cells, and promote neuronal
resistance to metabolic, oxidative, excitotoxic, and proteotoxic stresses which are in the pathogenesis of
disorders such as AD [
157
]. Experimentally, dietary restriction in aged rats was shown to increase
cortical expression of GLUT1, GLUT3, and GLUT4, while intermittent feeding increased AMPK
phosphorylation in the hippocampus [
156
]. It seems that RJ contributes to the regulation of neurons
under conditions of low energy supply. In this context, positive eects of RJ (e.g., suppression of
inflammation and oxidative stress, increased autophagy, etc.) have been associated with the activation
of AMPK [114]. AMPK is an energy sensing pathway that gets activated under conditions that entail
deficiency of cellular nutrients. It is a downstream eector of IIS [
63
]. AMPK phosphorylates Forkhead
Box O transcription factor (FOXO) to induce a catabolic response for energy supply in cells low in
nutrients [63,166].
7.4. Royal Jelly Protects Against Neuroinflammation
Accumulating evidence shows that the microglia of senescent brains are most aected by
age-related activation of inflammatory signaling. High expression of microglial inflammation markers
leads to dystrophic morphology (e.g., deramification, cytoplasmic fragmentation, and shortening
of cellular processes). Such manifestation of microglial senescence alters their ability to
promote proper neuronal function or protect neurons against the accumulation of NFT and A
β
oligomers [
7
,
167
]. Neuroinflammation broadens neuron loss by accelerating oxidative stress.
A number of investigations included in this article challenged microglia or rodent models with
LPS to induce a neuroinflammatory phenotype that was parallel to that in AD [
113
,
114
,
117
].
LPS induces inflammation by regulating the activity of NF-
κ
B, MAPK, signal transducer and activator
of transcription 1 (STAT1), and activator protein (AP-1), and c-Jun NH2-terminal kinases (JNK) [
113
].
Furthermore, markers of neuroinflammation are evidently high in AD models produced by toxic
treatments [
113
,
114
,
117
,
119
]. In these experiments, RJ demonstrated anti-inflammatory eects by
inhibiting the expression of proinflammatory cytokines (IL-6, IL-1
β
, and TNF-
α
) [
113
,
114
,
117
,
119
],
chemokines (CCL-2, CCL-3) [
114
], and related genes of iNOS and cyclooxygenase-2 (COX-2) [
113
].
NF-
κ
B and cytokines stimulate the slong-term expression of adhesion molecules such as ICAM-1
and VCAM-1 in the endothelium. ICAM-1 and VCAM-1 bind actin cytoskeleton to activate several
intracellular signaling pathways that influence immunological synapse formation and cellular immune
responses such as cytokine production and microvascular permeability. The latter facilitates the transfer
Antioxidants 2020,9, 937 24 of 47
of solutes (e.g., A
β
) and leukocytes into peripheral tissues [
168
,
169
]. Downregulation of inflammatory
cytokines by RJ was associated with inhibition of the gene expression of ICAM-1, VCAM-1, MMP-2,
and MMP-9, as well as with less degradation of tight junction proteins and an increase of their mRNA
expression. These eects resulted in less BBB permeability [114].
RJ demonstrates an immunomodulatory eect, and its glycoproteins are reported to contain
a T-antigen unit [
170
]. However, the anti-inflammatory eects of RJ in AD-related models are
complex. On one hand, RJ suppressed the phosphorylation of some of the main inflammatory
pathways such as NF-
κ
B, p38, and JNK [
28
,
113
,
117
]. In particular, 10-HDA has the ability to positively
aect TLR signaling by inhibiting the gene expression of TNF receptor-associated factor 1 (TRAF1)
in LPS-stimulated microglia [
117
]. TRAF1 activates NF-
κ
B and JNK signaling, and it negatively
regulates TLR and Nod-like receptor signaling. It produces these actions by interacting with TNFR2
to form a heterodimeric signaling complex. This complex recruits cIAPs, which possess E3 ligase
activity that promotes the addition of K63-linked polyubiquitin (K63-Ub) to receptor-interacting
serine/threonine-protein kinase 1 (RIPK1). K63-Ub activates Inhibitor of kappa B kinase (IKK) and
MAPK by recruiting transforming growth factor-
β
(TGF-
β
)-associated kinase (TAK1), TAK binding
protein (TAB), and the linear ubiquitin assembly complex (LUBAC). LUBAC modifies K63-Ub to
hybrid molecules and catalyzes the addition of polyubiquitin polymerized through the M1 position
(M1-Ub) [
171
]. M1-Ub facilitates the phosphorylation of Inhibitor of
κ
B (I
κ
B) through the activity of
IKK, which modifies it to K48-Ub. Degradation of K48-Ub stimulates the nuclear translocation of
NF-
κ
B [
117
,
171
]. Western blotting shows that 10-HDA inhibits the nuclear translocation of NF-
κ
B p65
and NF-
κ
B p50 subunits in activated microglia by impeding the phosphorylation and degradation
of I
κ
B
α
[
117
]. On the other hand, 10-HDA can suppress the activity of NLRP3 inflammasome-IL-1
β
signaling—a cytosolic protein oligomer that activates inflammatory responses by promoting the
proteolytic cleavage, maturation, and secretion of inflammatory cytokines [117].
Autophagosomes initiate innate and adaptive immune responses by supplying major
histocompatibility complex-loading compartments and endosomal pattern recognition receptors
with intracellular pathogen-associated molecular patterns [
172
]. Research reports impaired autophagic
influx under inflammatory conditions in AD mice [
173
]; 10-HDA was reported to promote autophagic
immune regulation and evoke autophagy in activated microglia and LPS-treated mice by upregulating
both Unc-51-like autophagy activating kinase (ULK) and microtubule-associated protein 1 light chain
3-II (LC3-II) and inhibiting sequestosome 1 (SQSTM1/p62). ULK and LC3-II are proteins that reflect
activation of autophagy [117]. The former gets activated through the inhibition of mammalian target
of rapamycin (mTOR) under nutrient-starving contexts [
174
]. SQSTM1 is a key autophagy gene that
regulates the activity of many signaling cascades including cytokine signaling. It directs ubiquinated
cargoes to autophagosomes for degradation. Though clearing dysfunctional proteins and organelles is
important for cellular function, the removal of basic cellular protein structures represents a prodeath
factor. Research shows that reduction of SQSTM1 delays brain damage induced by injury [
175
]. In total,
these reports indicate that RJ and its constituents promote qualified cellular housekeeping under
inflammatory conditions.
Evidence shows that the anti-inflammatory eects of RJ originate from compensatory changes
in energy sensing pathways. For example, 10-HDA treatment of LPS-stimulated mice and HBMECs
alleviated inflammation through activation of the energy sensing AMPK/PI3K/AKT signaling
pathways [
114
]. Inactivation of AMPK in AD is associated with the severity of cognitive impairment [
33
].
Meanwhile, activation of AMPK improves immunometabolism and the function of immune cells.
AMPK inhibits the activity of NF-
κ
B and suppresses its related pro-inflammatory responses [
176
] via
the activation of multiple signaling pathways including FOXO [
177
]. Interestingly, the eect of 10-HDA
on neuroinflammation and autophagy in LPS-treated mice and microglial BV-2 cells was mediated
by the upregulation of FOXO1. Chemical inhibition of FOXO1 dampened the eect of 10-HDA on
NF-
κ
B and NLRP3 inflammasome-IL-1
β
signaling [
117
]. FOXO downregulates AKT (a substrate
of mTOR) through PHA-4/FOXA transcription factor and S6K, which stimulate the expression of
Antioxidants 2020,9, 937 25 of 47
various autophagy genes and the translation of specific mRNAs that face inflammation and preserve
homeostasis by facilitating the repair or degradation of endogenous proteotoxic stress that occurs with
aging [178181].
7.5. Royal Jelly Protects Against Oxidative Stress
Reduced levels of endogenous antioxidants contribute to memory alteration [
87
]. Likewise,
increased production of free radicals and oxidative stress destroy biological infrastructures (e.g., lipids,
proteins, and DNA) in the CNS of AD patients [
28
]. Most studies in this review reported high
ROS/NOS production in AD models (Table 2). A recent study treated Drosophila Canton-S with
H
2
O
2
or paraquat (N,N
0
-dimethyl-4,4
0
-bipyridinium dichloride, a toxic substance widely used as
a herbicide) to induce an oxidative stress-model of aging similar to AD [
96
]. Studies involving
AD models show that RJ can suppress oxidative stress as indicated by a reduction of oxidative
biomarkers such as MDA, ROS, and NOS, both in the plasma and the brain, particularly in the
hippocampus and the cortex [
28
,
70
,
95
,
96
,
113
,
114
,
119
,
120
,
124
]. RJ can also enhance the internal
antioxidant capacity by stimulating the production of antioxidants such as HO-1, GSH-Px, SOD,
and catalase [
70
,
95
,
96
,
113
,
119
,
120
,
124
]. The antioxidant activity of RJ is a principal contributor to the
reported amelioration of apoptosis of cortical and hippocampal neurons in AD models [
28
,
70
,
119
,
120
].
It has been suggested that ACh and the enzyme ingredients (e.g., lipase and SOD) of RJ ameliorate
oxidative damage and improve memory [
106
]. In addition, existing knowledge emphasizes the
antioxidant properties of amino acids, peptides, and proteins in RJ [63].
It appears that RJ produces its antioxidant and anti-inflammatory eects by influencing an
integrated network of dierent signaling pathways, which have positive feedback eects on the activity
of each other. In this context, 10-HDA treatment of LPS-stimulated mice and HBMECs alleviated
inflammation and resulted in numerous positive eects, including reduction of ROS emission through
activation of AMPK/PI3K/AKT signaling [
114
]. The activation of AMPK regulates the function of nuclear
factor-erythroid 2-related factor 2 (NRF2), the principal antioxidant pathway [
177
]. NRF2 stimulates the
expression of antioxidant genes such as HO-1 and SOD [
154
]. In fact, RJ treatment of experimental AD
models resulted in increased activity of NRF2 [
119
]. In addition to targeting the regulation of oxidative
stress, NRF2 and related antioxidants (e.g., HO-1) directly silenced neuroinflammation by inhibiting
the transcription of cytokines such as IL-6 and IL-1
β
[
154
,
182
]. From another aspect, RJ significantly
downregulated the signaling of iNOS as a result of inhibition of inflammatory pathways [
113
,
119
].
The activation of iNOS occurs in immune activated cells, and it results in persistent emission of NO,
leading to cytotoxic eects. In contrast, the activation of endothelial NO synthase (eNOS) or neuronal
NO synthase (nNOS) stimulates a brief production of NO (for seconds or few minutes), which acts as a
signal molecule that regulates innate immune response [183,184].
Pyridine nucleotide nicotinamide adenine dinucleotide (NAD+) is a chief molecule that contributes
to health and lifespan via the regulation of normal cellular bioenergetics, scavenging of free radicals,
autophagy, DNA repair, and genome stability [
185
]. Cognitive decline that occurs with advanced aging
is associated with diminution of NAD+and its downstream eector, calcium/calmodulin-dependent
serine protein kinase in the CA1 hippocampal region [
186
]. Recently, supplementation of NAD+
precursors has been adopted to therapeutically increase NAD+levels, and consequently, counteract
oxidative stress [
185
]. Aged rats receiving MRJPs demonstrated increased levels of nicotinic acid
mononucleotide (NaMN), a metabolite of nicotinic acid (NA) and a precursor of NAD+[
109
].
High intracellular NA levels are cytotoxic, and its metabolism is necessary for therapeutic actions
to occur [
187
]. It has been suggested that all anabolism of NAD+biosynthesis goes through NaMN,
which maintains intracellular redox status when catalyzed to [NAD+(H)] by keeping a balanced
NADH/NAD+ratio [
109
]. The molecular mechanisms employed by NaMN to ameliorate the cytotoxic
eects of oxidative stress involve the inhibition of AKT [187].
Downregulation of AKT by RJ is associated with the activation of FOXO and inhibition of
the nutrient sensing mTOR pathway, a master regulator of metabolism and autophagy [
63
,
166
].
Antioxidants 2020,9, 937 26 of 47
mTOR has two distinct forms: mTORC1 and mTORC2. The latter has four main proteins, namely
mTOR, rapamycin-insensitive companion of target of rapamycin, mammalian stress-activated protein,
and kinase interacting protein. All these proteins are located at mitochondria-associated ER
membranes [
166
]. UPR signaling is aected by stress of the ER while experimental maneuvering
of this pathway alleviates neurodegeneration in preclinical models of AD [
72
]. In detail, PERK
homodimerizes after its activation, leading to the phosphorylation of serine residues on cytoplasmic
eukaryotic initiation factor 2 alpha (eIF2
α
) and the phosphorylation of the activating transcription
factor 4 (ATF4). ATF4 controls apoptosis by targeting CCAAT enhancer-binding (C/EBP) protein
homologous protein (CHOP) and triggers adaptive programs that maintain homeostasis via regulation
of redox proteins. Independent of eIF2
α
, PERK also activates NRF2, which modulates inflammation
and oxidative stress [
79
]. The organized interaction of these signaling pathways further regulates
the activity of AMPK to promote a cycle of activation of the basic processes involved in cellular
homeostasis, such as stress resistance, antioxidative capacity, autophagy, and DNA repair [176,177].
The reduction of AGEs by RJ may be another molecular mechanism for counteracting oxidative
damage. RJ treatment decreased the expression of RAGE (the main receptor for AGEs) in an AD
model [
70
]. RAGE increases with aging, and is highly expressed in AD brains. When it binds to AGE,
it activates the intracellular signaling involved in oxidative stress and inflammation. Thus, it floods
the body with free radicals and inflammatory mediators like TNF-
α
, IL-6, and C-reactive protein,
which induce microglial activation and disrupt cellular structure and A
β
metabolism [
33
,
64
]. Treating
Drosophila intoxicated by H
2
O
2
or paraquat with a combination of peptides of eRJ and collagen peptides
from the skin of Carcharhinus falciformis fish reduced carbonyl proteins [
96
]. This finding may represent
a read out of reduced activity of RAGE. RAGE binds to several other ligands such as oligomeric forms
of A
β
and ligands involved in inflammatory responses, e.g., S100/calgranulins and high mobility
group box 1 [
64
,
159
]. In addition, the activation of RAGE causes generalized endothelial dysfunction,
which increases vascular disorders (a core AD risk factor), decreases cerebral blood flow, increases
the permeability of the BBB, and facilitates the transfer of excessive amounts of A
β
from the blood to
the brain [
33
,
64
]. Evidence indicates that RJ can protect against endothelial insult and restore normal
endothelial function during chronic exposure to hyperglycemic conditions, which stimulate RAGE and
increase the production of AGEs [
33
,
64
]. A recent study found that RJ treatment of human endothelial
cells exposed to 30 mM glucose over 72 h increased the expression of LC3-II autophagy-related factor,
and decreased the activity of MPP-2 and MPP-9 [
118
]. Altogether, these reports suggest that the
inhibition of RAGE by RJ represents a multidimensional mode of cognitive enhancement: controlling
oxidative stress, inflammation, glial activation, and A
β
metabolism, in addition to enhancing vascular
integrity and protecting against BBB leakage under conditions of metabolic dysfunction.
7.6. Royal Jelly Promotes Neuronal Regeneration and Attenuates Apoptosis
The pathologies contributing to cognitive deterioration and AD development cause serious damage
to the cortical and hippocampal neurons, especially in the DG [
112
]. The neuroprotective eects of RJ
in AD models comprise improved neuronal cell structures, increased cellular survival, and reduced
neuronal loss [
70
]. Evidence documents that RJ, AMP N1-oxide, and 10-HDA promote neurogenesis and
gliogenesis by triggering the proliferation of NS/NPCs
in vitro
[
143
,
188
]. Experimentally, RJ enhances
cognitive ability by promoting neuronal regeneration
in vivo
. In one experiment, mice intraperitoneally
injected with trimethyltin, a toxic organotin substance that induces selective acute neuronal death
in hippocampal DG, were treated with dietary RJ for 6 days. Trimethyltin decreased the number
of DG neurons by 50% and caused intense cognitive dysfunction. Hippocampal DG contains
NS/NPCs, which generate the neurons responsible for cognitive function. RJ treatment reversed
trimethyltin-induced DG neurotoxicity by enhancing neurogenesis, resulting in functional neurons
that were capable of ameliorating cognitive impairment [
112
]. In another study, a cholesterol diet and
copper intoxication induced AD in ovariectomized rabbits, which manifested by nucleus shrinkage,
neuronal loss, and the disappearance of Nissl bodies in the cortex and hippocampus. RJ treatment
Antioxidants 2020,9, 937 27 of 47
significantly increased the number of neurons in the hippocampal CA1, CA3, and DG regions and
cortical PCL and MCL areas by 40%, 56%, 34%, 23%, and 34% respectively [70].
Immunofluorescence staining and Western blotting revealed that RJ inhibited apoptosis in the
brains of AD rabbits and APP/PS1 transgenic mice; the number of activated caspase-3 immunolabeled
cells, as well as the covered areas of activated caspase-3, decreased by up to 57% in the cortex and
hippocampus of RJ-treated AD models compared with untreated animals [
28
,
70
]. Mechanistically,
RJ amelioration of neuronal apoptosis resulted from the inhibition of the phosphorylation of I
κ
B
α
, p38,
and p- JNK via inhibition of the translocation of NF-
κ
B p65 into the nucleus [
28
,
113
]. As a result, levels
of the mitochondrial anti-apoptotic signaling molecule B-cell lymphoma-2 (Bcl-2) increased, and levels
of pro-apoptotic signaling molecule Bcl-2-associated X protein (Bax) decreased [
119
]. Downregulation
of the Bax/Bcl-2 ratio by RJ was associated with an evident decrease of the expression of cleaved
caspase-3 in the hippocampus and cortex [
28
,
119
]. Bcl-2 promotes mitochondrial integrity, while
Bax is a chief eector of the mitochondrial apoptosis pathway. Emerging knowledge signifies that
AD neurons exhibit drastic ER stress, which stimulates mitochondrion Ca
2+
fluxes and apoptotic
responses by promoting Bax phosphorylation through direct conformational changes, or through
the displacement of anti-apoptotic Bcl-2 proteins. Activated Bax translocates into the mitochondria
to form protein-permeable channels/pores within the outer mitochondrial membrane in a process
known as mitochondrial outer membrane permeabilization (MOMP). MOMP stimulates the release
of pro-apoptotic factors (e.g., AIF, cyt-c, EndoG) from the mitochondrial intermembrane space
into the cytoplasm to activate a caspase-dependent neuronal apoptosis in AD brains [
189
,
190
].
The investigations addressed by this review denote that the amelioration of apoptosis of cortical
and hippocampal neurons in AD models is closely linked to the antioxidant and anti-inflammatory
activities of RJ [28,70,119,120].
On the other hand, RJ promotes the production of neuroprotective molecules. In this regard,
MRJPs increased cysteic acid in aged rats, indicating an involvement of the cysteine-taurine metabolism
pathways in memory enhancement by RJ. MRJPs are rich in cystine, which can be converted into
cysteine and then into cysteic acid by cysteine lyase [
109
]. Cysteine is used as a rate-limiting substrate
for the production of the neuroprotective gaseous physiological modulator hydrogen sulfide (H
2
S) by
cystathionine beta-synthase in the parenchyma of the brain [
191
]. In addition, cysteic acid is a precursor
for taurine, a sulfur-containing, free amino acid that is abundant in excitable tissues such as the brain
and the heart. Taurine deficiency is associated with multisystem failure and memory impairment [
192
].
Meanwhile, diets rich in taurine and cysteine enhance brain production of H
2
S [
191
]. Indeed,
RJ treatment of ovariectomized rats increased myelin galactolipids including galactosylceramide and
sulfatide—an eect that was associated with a slight increase in brain weight [
87
]. Taurine acts as a
neuromodulator that regulates intracellular Ca
2+
homeostasis, prevents hippocampal neuron loss,
guards synaptic plasticity against excitotoxicity, decreases A
β
accumulation in the hippocampus,
and enhances memory in AD experimental models through the modulation of the GABAergic system
(Section 7.2) [192,193].
7.7. Royal Jelly Mitigates Amyloid-Related Neurotoxicity
Both
in vivo
and
in vitro
studies showed that RJ significantly decreased blood and brain levels of
A
β
[
28
,
95
] and reduced the deposition of A
β
, both in advanced aging and AD models [
28
,
92
,
95
,
115
,
116
].
Imbalance between the formation and degradation of A
β
plays a major role in its excessive deposition
in AD. RJ abrogates A
β
pathology by regulating processes essential for its production, degradation,
and clearance [
28
]. In one study, RJ reduced levels of soluble A
β
40 and A
β
42 by 24% and 40%,
respectively. However, the eciency of removal of insoluble A
β
40 and A
β
42 was even much higher
(60%). On one hand, RJ interferes with A
β
synthesis by decreasing the expression of BACE1
in vitro
[
115
]
and
in vivo
—in cortical and hippocampal neurons by up to 44% and 24%, respectively [
28
,
70
,
95
].
BACE1 is the rate-limiting enzyme that catalyzes the proteolytic cleavage of APP into A
β
[
27
].
Thus, RJ inhibition of BACE1 indicates its ability to block the initial pathogenic steps underling the
Antioxidants 2020,9, 937 28 of 47
formation of A
β
plaques, the main pathological structure of AD. On the other hand, RJ significantly
increased the expression of some A
β
-degrading enzymes such as insulin-degrading enzyme (IDE) [
28
]
and neprilysin (NEP) [
116
]. These enzymes convert A
β
polypeptide into benign forms [
28
]. RJ also
promoted A
β
clearance by upregulating the expression of LRP-1, which facilitates the removal of
degraded Aβout of brain cells [28,95].
RJ has been used to activate cAMP/PKA and CREB-dependent signaling linked to CRE-mediated
transcription in PC12 cells via the ERK/MAPK signaling cascade [
93
,
130
]. CRE-mediated transcription
is a principal signaling pathway that promotes learning and memory through long-term hippocampal
potentiation. Dysregulation of this pathway influences A
β
metabolism and stimulates tau
phosphorylation, which contributes to the development of cognitive impairment in neurodegenerative
diseases such as AD [
80
,
93
]. Several lines of evidence note that RJ repairs dysregulated neurons
encompassing A
β
pathology via activation of CREB signaling [
28
,
116
]. In one study, intragastric
administration of RJ (300 mg/kg/day) to ten-month-old APP/PS1 transgenic mice for three months
markedly ameliorated cognitive deficits and decreased soluble and insoluble A
β
40 and A
β
42 (25%,
40%, and 60%), as well as the size and number of SPs, both in the cortex and hippocampus, through
the activation of cAMP, p-PKA, p-CREB and BDNF [
28
]. Likewise, another
in vitro
study reported
that a DMSO-soluble fraction of RJ increased the clearance of soluble A
β
through the activation
of the NEP-somatostatin system in hippocampal neurons. Chromatin immunoprecipitation-qPCR
assays revealed that RJ enhanced the gene expression of NEP and somatostatin in hippocampal
neurons. The former is a basic degrading enzyme of A
β
oligomers, while the latter facilitates
NEP-mediated proteolytic degradation in the hippocampus. RJ facilitated CREB-binding to the
prototypical functional CRE at the promoter region of somatostatin. The activation of the hippocampal
NEP-somatostatin system plays an important role in memory formation, synaptic plasticity, neural
development, and neuronal circuit homeostasis [116].
Downregulation of IIS accounts for another mechanism employed by RJ to reduce amyloidogenesis
and proteotoxicity [
92
]. In aged C. elegans, RJ downregulated daf-2, a key upstream component of IIS,
resulting in phosphorylation of three core downstream transcription factors of IIS: DAF-16—the
C. elegans counterpart to the mammalian FOXO, heat shock transcription factor 1 (HSF-1),
and SKN-1/NRF2 [
92
]. These transcription factors represent specific longevity pathways that have
the potential to activate multiple cascades essential for antioxidant and anti-inflammatory activities,
DNA repair, autophagy, stress resistance, and cell proliferation [
63
]. On the other hand, cumulating
knowledge indicates that aging-related physiological alterations increase the production of highly
toxic A
β
fibrils via attenuation of HSF1 signaling [
194
]. Knock down of DAF-16, SKN-1, and HSF-1
genes via RNA interference abolished the eect of RJ/eRJ on paralysis induced by A
β
toxicity in C.
elegans, which confirms the main role of these genes in RJ protection against neurodegeneration [
92
].
Emerging knowledge highlights the role of interventions that foster the activity of FOXO and HSF-1 in
hindering or possibly preventing the onset of neurodegeneration [194].
7.8. Royal Jelly Alleviates Hormonal and Metabolic Abnormalities Underlying Cognitive Impairment
Accumulating knowledge shows that sex steroids (estrogens, androgens, and luteinizing hormone)
possess strong anti-inflammatory and neuroprotective eects. They interact with many pathways
involved in AD pathology to improve insulin resistance and enhance adaptive programs that promote
DNA repair capacity of the CNS. Age-related declines in these hormones contribute to the development
of cognitive impairments and even AD in people with genetic and environmental vulnerabilities [
24
,
195
,
196
]. Estrogen supplementation has been used to improve cognitive performance and alleviate
pathological damages embedded in various physical disorders (e.g., cardiovascular diseases) in
reproductively-senescent women. However, estrogen as a supplement may cause devastating eects
such as increased risk of cancer [
63
,
87
]. Therefore, research has been focused on the use of natural
agents such as foods rich in phytoestrogens as safe substitutes for estrogen. Phytoestrogens are similar
to estrogen chemically, structurally, and functionally [85,87].
Antioxidants 2020,9, 937 29 of 47
RJ is reported to improve testosterone, dehydroepiandrosterone sulfate, and estradiol in healthy
adults [
197
,
198
]. It is widely used to treat infertility because of its phytoestrogen content [
87
,
199
].
Thus, it is possible that RJ-related improvement of gonadal function may represent an endocrine
modulation mechanism for enhancing cognitive function in old age. In fact, fatty acids and sterols in RJ,
e.g., 10-HDA and 24-methylenecholesterol, exert neurogenic eects and improve brain function through
estrogen signaling [
95
,
143
]. Estrogen receptor 1 represents a common single nucleotide polymorphism
that contributes to the onset of AD, as indicated by genome-wide association studies [
200
]. The binding
of RJ fatty acids to estrogen receptors
β
and
α
modulates cell proliferation, increases the production
of neurotrophins such as BDNF and NGF, regulates the expression of various genes that counteract
inflammation and oxidative stress in cholinergic neurons, promotes Ca
2+
outflow and ACh release,
reduces tau protein phosphorylation, decreases A
β
production, and inhibits A
β
-related destructive
eects on cellular homeostasis [
85
,
87
,
201
]. These eects result from multiple distinct mechanisms:
(1) estrogen translocation into the nucleus to bind estrogen response elements (EREs) located in
the promoters of target genes, (2) facilitating protein–protein interactions with other DNA-binding
transcription factors in the nucleus, and (3) nongenomic actions of estrogen that involve modifying
functions of cytoplasmic proteins leading to the regulation of gene expression [
143
,
202
]. Experimental
modulation of estrogen by supplementing ovariectomized animals with RJ was shown to significantly
enhance cognitive behaviors, improve autonomic and cardiovascular conditions, and be capable of
treating pathologies associated with AD such as Aβdeposition and hypercholesterolemia [87,95].
A strong correlation between thyroid dysfunction and AD was demonstrated by 14 out of
23 studies [
203
]. Research documents an association of thyroid stimulating hormone and free
triiodothyronine (T3) with regional cerebral blood flow in patients with MCI and AD [
204
]. Deficiency
of T3 and thyroxine (T4) promotes oxidative stress and alters cellular metabolism, which impair the
function of all organs and contribute to metabolic and cardiovascular disorders, increasing the risk of AD.
On the other hand, hypothyroidism impairs neurotransmitter synthesis, induces hyperphosphorylation
of tau proteins, promotes glutamate excitotoxicity, reduces synaptic plasticity, inhibits hippocampal
neurogenesis, and promotes hippocampal neuron death, which result in symptoms of poor memory
and concentration [
5
,
73
]. RJ supplementation (100 mg/kg/day/20 days) to rat models of hypothyroidism
(induced by a daily intraperitoneal injection of 10 mg/kg propylthiouracil) increased serum T4, reduced
vascular dilation and neurodegeneration in CA3 and CA1 hippocampal regions, and increased
hippocampal MAP-2 levels [
73
]. These findings suggest that RJ prevents neurodegeneration by
maintaining microtubule and axon stability and decreasing tau phosphorylation through microtubule
anity regulating kinase signaling [73,80].
Cholesterol promotes the formation of A
β
plaque and neuronal loss by accelerating the cleavage
of APP [
95
]. Research also indicates that high fat diets impair memory by inhibiting the signaling
associated with the expression of genes involved in memory, as well as by evoking several other
drastic disturbances: increased triglyceride stores, impaired metabolism, hypertension and other
cardiovascular disorders, obesity, and shortened life span [
205
]. Two studies included in this review
treated ovariectomized animals with a high cholesterol diet to induce cognitive impairment [
70
,
95
].
RJ and peptides in pRJ exhibit antihypertensive and anticholesterol eects in rabbit models of AD and
in rats with high spontaneous blood pressure, such as: inducing aortic relaxation; improving heart
rate variability and baroreceptor sensitivity; and reducing blood pressure, plasma lipids (e.g., TC and
LDL-C), body weight, and brain levels of A
β
, AchE, and MDA [
70
,
206
,
207
]. Such eects were associated
with increased antioxidative capacities, amelioration of A
β
pathology, and protection against neuronal
damage [
70
]. The suggested mechanism entails agonism of muscarinic receptors by ACh in RJ, leading
to a brief increase in the levels of NO and cyclic guanosine monophosphate, as well as suppression of
norepinephrine-related intracellular Ca2+release and K+-related extracellular Ca2+influx [206].
Antioxidants 2020,9, 937 30 of 47
8. Discussion
The results of this review indicate that RJ can enhance cognitive performance and boost learning
and memory in old age and in AD models. Most studies promisingly signify a general trend of positive
dose-dependent eects of RJ on the pathologies underlying cognitive impairments (e.g., oxidative stress,
neuroinflammation, and A
β
and tau pathology) [
28
,
70
,
92
,
95
,
115
,
116
,
119
]
in vitro
and
in vivo
, despite
the large heterogeneity in cell lines and species that were used as models of AD or cognitive aging,
as well as variations in RJ preparation (e.g., enzyme treatment, lyophilization, etc.), dose, and route
of administration (oral, gavage, or subcutaneous) across the available literature. Additionally, RJ as
a monotherapy for AD has not been tested in a single human trial until now. Nonetheless, a few
studies have investigated the eect of herbal mixtures containing RJ on cognitive function in humans.
The methodological flaws in these studies give rise to serious concerns, e.g., small sample size, lack of
assessment of biological markers, lack of blinding, and absence of a comparison group, to name a
few [125,126].
The eects of RJ on cognition are closely associated with the modulation of various biological
activities that boost the structure and operative performance of both neurons and non-neuronal
brain cells (e.g., glial cell and HBMECs), and eventually enhance cell survival and prevent
neurodegeneration [
28
,
112
114
]. Among these pharmacological activities, RJ regulated the production
of neurotransmitters such as serotonin [
107
,
119
,
121
] and neurotrophins such as BDNF and NGF,
which protect against synaptic loss in the cortex and hippocampus [
28
]. It was also shown to deactivate
cellular-stress signaling pathways engaged in inflammation, oxidative stress, and mitochondrial-related
apoptosis [
28
,
70
,
119
,
208
]. In addition, it ameliorated the detrimental eects of A
β
by decreasing its
production, sequestering its insoluble form, and enhancing its removal out of the brain [
28
,
92
,
95
,
115
,
116
].
Additionally, RJ improved cognitive behavioral deficits in AD by correcting systemic pathologies
contributing to neurodegeneration, e.g., enhancing estrogen levels, improving autonomic, metabolic,
and cardiovascular conditions in ovariectomized animals [
95
], and increasing fT4 levels in
hypothyroidism rodents model of cognitive dysfunction [
73
]. Therefore, future RCTs should explore
if RJ can comprehensively optimize metabolic and endocrine parameters in individuals enduring
age-related cognitive impairments by expanding standard laboratory evaluations.
8.1. Royal Jelly May Improve Health and Extend Lifespan in Cognitively Impaired Subjects
Aging is a key risk factor for AD. Despite the long lifespan of people with AD, they suer
prolonged disability out of the dramatic progress of the disease and a variety of other aging-related
comorbidities [
25
,
60
,
65
]. As illustrated before, AD pathology may result from other existing metabolic
and hormonal dysregulations such as hyperlipidemia, obesity, type 2 diabetes mellitus, hypertension
and other cardiovascular disorders, as well as severely decreased levels of sex steroids [
5
,
13
,
54
,
58
].
Research shows that A
β
, tauopathy, and mutations of genes contributing to AD, e.g., Ankyrin 1
gene increase neurodegeneration, disrupt memory, decrease locomotion, and shorten lifespan in
Drosophila [
209
]. We have previously shown that RJ modulates several aging pathways to prolong
lifespan in various model organisms and improve health both in humans and animal models [
63
].
The literature examined in the current review indicates that RJ brings about other benefits along with
improved cognitive function. In this regard, RJ positively aected lipid profiles in postmenopausal
women (who are at high risk for AD) [
126
] and in AD models induced by high cholesterol diet on top of
ovariectomy or copper intoxication [
70
,
95
]. It also improved estrogen levels; estrogen expresses a range
of health promoting activities, e.g., promoting cardiovascular function and bone density [
210
,
211
].
More, cognitive recovery resulting from RJ treatment in hypothyroidism models was associated with
improvement of plasma levels of fT4 [
73
]. Evidence from a current Drosophila model shows that RJ
improved food intake, body weight, exercise capacity, and mean lifespan in flies treated with H
2
O
2
or paraquat, as well as in naturally aged flies [
96
]. Taken together, these reports suggest that RJ
might improve health, optimize lifespan, and promote overall wellness in cognitively impaired people
by targeting age-related physiological declines. Nonetheless, studies addressing the eect of RJ on