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The study of mitochondrial dysfunction has become increasingly pivotal in elucidating the pathophysiology of various cerebral pathologies, particularly neurodegener-ative disorders. Mitochondria are essential for cellular energy metabolism, regulation of reactive oxygen species (ROS), calcium homeostasis, and the execution of apoptotic processes. Disruptions in mitochondrial function, driven by factors such as oxidative stress, excitotoxicity, and altered ion balance, lead to neuronal death and contribute to cognitive impairments in several brain diseases. Mitochondrial dysfunction can arise from genetic mutations, ischemic events, hypoxia, and other environmental factors. This article highlights the critical role of mitochondrial dysfunction in the progression of neurodegenera-tive diseases and discusses the need for targeted therapeutic strategies to attenuate cellular damage, restore mitochondrial function, and enhance neuroprotection.
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Academic Editor: Zhihui Feng
Received: 19 December 2024
Revised: 13 January 2025
Accepted: 16 January 2025
Published: 18 January 2025
Citation: Belenichev, I.; Popazova, O.;
Bukhtiyarova, N.; Ryzhenko, V.;
Pavlov, S.; Suprun, E.; Oksenych, V.;
Kamyshnyi, O. Targeting
Mitochondrial Dysfunction in
Cerebral Ischemia: Advances in
Pharmacological Interventions.
Antioxidants 2025,14, 108. https://
doi.org/10.3390/antiox14010108
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/
licenses/by/4.0/).
Review
Targeting Mitochondrial Dysfunction in Cerebral Ischemia:
Advances in Pharmacological Interventions
Igor Belenichev 1, Olena Popazova 2, * , Nina Bukhtiyarova 3, Victor Ryzhenko 4, Sergii Pavlov 3,
Elina Suprun 5, Valentyn Oksenych 6,* and Oleksandr Kamyshnyi 7
1Department of Pharmacology and Medical Formulation with Course of Normal Physiology, Zaporizhzhia
State Medical and Pharmaceutical University, 69000 Zaporizhzhia, Ukraine; i.belenichev1914@gmail.com
2Department of Histology, Cytology and Embryology, Zaporizhzhia State Medical and Pharmaceutical
University, 69000 Zaporizhzhia, Ukraine
3Department of Clinical Laboratory Diagnostics, Zaporizhzhia State Medical and Pharmaceutical University,
69000 Zaporizhzhia, Ukraine
4Department of Medical and Pharmaceutical Informatics and Advanced Technologies, Zaporizhzhia State
Medical University, 69000 Zaporizhzhia, Ukraine
5The State Institute of Neurology, Psychiatry and Narcology of the National Academy of Medical Sciences of
Ukraine, 46 Academician Pavlov Street, 61076 Kharkov, Ukraine
6Faculty of Medicine, University of Bergen, 5020 Bergen, Norway
7
Department of Microbiology, Virology and Immunology, I. Horbachevsky Ternopil State Medical University,
46001 Ternopil, Ukraine; kamyshnyi_om@tdmu.edu.ua
*Correspondence: popazova.o.o@zsmu.edu.ua (O.P.); valentyn.oksenych@uib.no (V.O.)
Abstract: The study of mitochondrial dysfunction has become increasingly pivotal in eluci-
dating the pathophysiology of various cerebral pathologies, particularly neurodegenerative
disorders. Mitochondria are essential for cellular energy metabolism, regulation of reactive
oxygen species (ROS), calcium homeostasis, and the execution of apoptotic processes. Dis-
ruptions in mitochondrial function, driven by factors such as oxidative stress, excitotoxicity,
and altered ion balance, lead to neuronal death and contribute to cognitive impairments
in several brain diseases. Mitochondrial dysfunction can arise from genetic mutations,
ischemic events, hypoxia, and other environmental factors. This article highlights the
critical role of mitochondrial dysfunction in the progression of neurodegenerative diseases
and discusses the need for targeted therapeutic strategies to attenuate cellular damage,
restore mitochondrial function, and enhance neuroprotection.
Keywords: mitochondrial dysfunction; ROS; cerebral ischemia; HIF-1; HSP70
1. Introduction
The relevance of the study of various types of cerebral pathology and the development
of methods for their treatment does not require detailed justification [
1
]. The World Health
Organization defines human death as the death of the brain, which in life controls all
the most important functions. In terms of prevalence and mortality, brain diseases rank
third among diseases of the population of industrialized countries, leading not only to
a decrease in life expectancy of the population but also limiting the social activity of a
person due to the development of cognitive deficit, a decrease in the individual’s ability to
think, learn, adequately perceive information, and make decisions [
2
,
3
]. In brain diseases of
destructive and degenerative genesis, mitochondrial respiratory chain, energy metabolism,
ionic homeostasis of the cell with increased content of calcium ions, development of glu-
tamate excitotoxicity and damaging effect of nitrosative and oxidative stress, initiation of
neuroapoptosis, and cell death occur [
4
]. The primary source of reactive oxygen species
Antioxidants 2025,14, 108 https://doi.org/10.3390/antiox14010108
Antioxidants 2025,14, 108 2 of 46
(ROS) is mitochondria, which play a key role in the cell’s energy supply [
5
]. Nowadays,
there is a generalized concept of “mitochondrial dysfunction”. This is a typical pathological
process that has no etiologic and nosologic specificity. The development of mitochondrial
dysfunction eventually leads to neuron death [
6
]. We can speak about mitochondrial
dysfunction as a new pathobiochemical mechanism of neurodegenerative disorders of a
wide spectrum. All of the above is the rationale for the search for highly effective cerebro-
protective drugs capable of preventing negative processes of mitochondrial dysfunction in
the cell, thereby having a cerebroprotective effect [
7
]. Currently, energy-tropic drugs such
as coenzyme Q10, carnitine, B vitamins, succinic acid derivatives, etc., are being tried to
correct mitochondrial dysfunction [
6
,
8
]. However, the rational basis for their use is poorly
developed, and effective approaches are often underutilized or ineffective ones are overesti-
mated. Medications are applied chaotically, without sufficient knowledge of their potential
and characteristics, and without planning a treatment strategy from the perspective of
appropriateness. Moreover, in cases of established mitochondrial dysfunction and the
activation of apoptotic processes, these drugs are largely ineffective as they cannot regulate
the subtle mechanisms of energy metabolism in which they act as intermediates.
Another approach to correcting mitochondrial dysfunction is also being considered:
the use of thiol antioxidants. These compete with the SH-groups of the cysteine-dependent
site of the mitochondrial inner membrane protein (ATP/ADP antiporter) for ROS and
peroxynitrite, forming stable complexes with the latter. This prevents the opening of the
mitochondrial pore under conditions of oxidative and nitrosative stress.
Particularly interesting and deserving of special attention is the use of drugs that act
as ligands for neuropeptide receptors. These can regulate apoptosis, the expression of
transcription factors, the synthesis of enzymes that regenerate mitochondrial DNA, and
enzymes that catalyze energy reactions. Recently, significant efforts have been directed
toward identifying highly effective neuroprotectors among neuropeptides.
The above highlights the scientific appeal of studying mitochondrial dysfunction in
neurons, making the development of new approaches to pharmacological correction highly
relevant and promising from the standpoint of comprehensive neuroprotection [6,9,10].
1.1. The Role of Mitochondria in the Energy Metabolism of the Brain
The relationship between the structure and function of mitochondria remains a central
focus of attention for a wide range of researchers. In recent years, revolutionary develop-
ments have taken place and are continuing in mitochondrial biology. Traditional views on
the role of mitochondria in the cell have undergone significant revisions. Until relatively re-
cently, our understanding of mitochondria was limited to their function as the cell’s energy
stations, and the role of mitochondria in the development of pathology was confined to
energy supply disruption, with all ultrastructural changes in mitochondria being viewed
exclusively from this perspective. However, many ultrastructural states of mitochondria
cannot be explained within such frameworks. Today, there is no doubt about the crucial
significance of mitochondria for the life of eukaryotic cells. The dominant role of these
organelles in ATP production, the execution of programmed cell death processes, their
involvement in the generation of ROS, and the storage of calcium ions all determine their
participation in the development of many pathological processes [6,11].
The brain has extremely high metabolic needs. It uses approximately 20% of the
body’s total oxygen and glucose consumption with only 2% of its body weight. About
70% of estimated energy expenditure is used to support neuronal signaling, including
resting potentials, action potentials, postsynaptic receptor activation, glutamate cycling,
and postsynaptic Ca
2+
signaling, while the remainder goes to nonsignaling activities such
Antioxidants 2025,14, 108 3 of 46
as biomacromolecule trafficking, axonal transport, mitochondrial proton leakage, and actin
cytoskeleton remodeling [1214].
Neurons exhibit the majority of energy consumption. They generate ATP primarily
in mitochondria via oxidative phosphorylation, with a small portion of ATP from aerobic
glycolysis in the cytoplasm. Astrocytes are highly glycolytic and convert glucose to lactate
with low oxygen consumption; lactate is then delivered to neurons for complete oxidation.
This process largely supports the energy requirements of neurons by supplying metabolic
substrates [15,16].
Oligodendrocytes also obtain ATP mainly through aerobic glycolysis. They use lactate
for their own energy needs and also supply neighboring axons with lactate. Microglia are
mainly nourished by oxidative phosphorylation but are metabolically reprogrammed to a
phenotype dominated by aerobic glycolysis under certain neurological circumstances [
17
19
].
Astrocytes have low mitochondrial activity of oxidative phosphorylation processes; this
metabolic mode of astrocytes is essential for brain lipid homeostasis. Aberrant astrocytic ox-
idative phosphorylation process can cause accumulation of lipid droplets with subsequent
development of neurodegeneration [20].
1.2. Concept of Mitochondrial Dysfunction
To date, on the basis of experimental studies, the concept of mitochondrial dysfunction, its
formation, molecular, biochemical and ultrastructural features has been formulated [
6
,
21
23
].
The development of mitochondrial dysfunction leads to the disruption of neurotrans-
mitter reuptake (catecholamines, dopamine, serotonin), ion transport, impulse generation
and conduction, de novo protein synthesis, translation and transcription processes; “para-
sitic” energy-producing reactions are activated, resulting in a significant depletion of the
neuronal cell’s energy reserves. Additionally, under the influence of ROS, particularly the
hydroxyl radical, permeability transition pores are opened, leading to the expression and
release of pro-apoptotic proteins into the cytosol. The opening of these pores occurs due to
the oxidation of thiol groups in the cysteine-dependent region of the inner mitochondrial
membrane protein (ATP/ADP antiporter), transforming it into a permeable nonspecific
channel-pore. Today, the primary mechanism for this process is recognized as the formation
of mitochondrial apoptotic pores and pores of increased permeability (PTP—permeability
transition pores) [6,2427].
The opening of the permeability pore typically allows the release of molecules up to
1500 Da. This disrupts mitochondrial metabolism, halting the synthesis of mitochondrial
proteins and the import of proteins synthesized in the cytosol. Oxidative phosphorylation
becomes uncoupled, and ATP synthesis stops. Hyperproduction of O
2
begins, and reducing
equivalents are depleted. The opening of the pore transforms the mitochondria from “power
plants into “furnaces” for oxidation substrates without ATP production [28,29].
It was found that interaction of mitochondrial structures with active derivatives of
NO and ROS, Ca
2+
“overbreathing”, reduction of intramitochondrial GSH enhances pore
opening and release of apoptogenic proteins from damaged mitochondria [
30
,
31
]. In this
context, the role of one of the neurotrophic factors, tumor necrosis factor-
α
(TNF-
α
), with
which the opening of pores in mitochondria, the subsequent disruption of their membranes
and the development of mitoptosis are associated, is significant [32].
Thus, we can speak about mitochondrial dysfunction as a new pathobiochemical
mechanism of neurodegenerative disorders of a wide spectrum. At present, two types
of mitochondrial dysfunction are distinguished—primary as a consequence of congenital
genetic defect and secondary, arising under the influence of various factors: hypoxia,
ischemia, oxidative and nitrosative stress, expression of proinflammatory cytokines. In
modern medicine, the doctrine of polysystemic disorders of cellular energy exchange, the
Antioxidants 2025,14, 108 4 of 46
so-called mitochondrial pathology, or mitochondrial dysfunction, occupies an increasingly
important position [6].
A key area of this section of medicine is hereditary syndromes based on mutations
of genes responsible for mitochondrial proteins (Kearns-Sayre, MELAS, MERRF, Pearson,
Barth, etc.) [33].
However, the class of conditions characterized by mitochondrial dysfunction is by no
means limited to these primary mitochondrial dysfunctions. A huge number of diseases
include disorders of cellular energy metabolism—secondary mitochondrial dysfunctions
as important links in pathogenesis. Among them: intracerebral hemorrhage, epilepto-
genic seizures, localized thermal brain damage, neurodegenerative disorders, transient
cerebral ischemia, chronic fatigue syndrome, migraines, cardiomyopathies, alcoholic en-
cephalopathies, senile dementia, neuroinfections, cardiomyopathies, glycogenoses, connec-
tive tissue diseases, diabetes, rickets, tubulopathies, pancytopenia, hypoparathyroidism,
liver failure, and many others (Figure 1) [6].
Antioxidants2025,14,1084of46
mitochondrialdysfunctionaredistinguished—primaryasaconsequenceofcongenitalge-
neticdefectandsecondary,arisingundertheinuenceofvariousfactors:hypoxia,ische-
mia,oxidativeandnitrosativestress,expressionofproinammatorycytokines.Inmodern
medicine,thedoctrineofpolysystemicdisordersofcellularenergyexchange,theso-called
mitochondrialpathology,ormitochondrialdysfunction,occupiesanincreasinglyim-
portantposition[6].
Akeyareaofthissectionofmedicineishereditarysyndromesbasedonmutationsof
genesresponsibleformitochondrialproteins(Kearns-Sayre,MELAS,MERRF,Pearson,
Barth,etc.)[33].
However,theclassofconditionscharacterizedbymitochondrialdysfunctionisby
nomeanslimitedtotheseprimarymitochondrialdysfunctions.Ahugenumberofdis-
easesincludedisordersofcellularenergymetabolism—secondarymitochondrialdys-
functionsasimportantlinksinpathogenesis.Amongthem:intracerebralhemorrhage,ep-
ileptogenicseizures,localizedthermalbraindamage,neurodegenerativedisorders,tran-
sientcerebralischemia,chronicfatiguesyndrome,migraines,cardiomyopathies,alcoholic
encephalopathies,seniledementia,neuroinfections,cardiomyopathies,glycogenoses,
connectivetissuediseases,diabetes,rickets,tubulopathies,pancytopenia,hypoparathy-
roidism,liverfailure,andmanyothers(Figure1)[6].
Figure1.Diseasesassociatedwithmitochondrialdysfunctionincellularenergymetabolism.The
FigurewasdesignedusingBioRender.com.Mitochondrialdysfunctionisakeyfactorinthepatho-
genesisofmanycentralnervoussystemdiseasesandcerebrovascularpathologies,includingintrac-
erebralhemorrhage,epileptogenicseizures,localizedthermalbraininjury,neurodegenerativedis-
orders,transientcerebralischemia,chronicfatiguesyndrome,migraines,alcoholicencephalopathy,
seniledementia,andneuroinfections[6].Currently,twotypesofmitochondrialdysfunctionarerec-
ognized:primary,resultingfromacongenitalgeneticdefect,andsecondary,arisingfromvarious
pathologicalfactorssuchashypoxia,ischemia,oxidativeandnitrosativestress,andtheexpression
ofpro-inammatorycytokines.Inmodernmedicine,increasingimportanceisbeingplacedonthe
Figure 1. Diseases associated with mitochondrial dysfunction in cellular energy metabolism. The Fig-
ure was designed using BioRender.com. Mitochondrial dysfunction is a key factor in the pathogenesis
of many central nervous system diseases and cerebrovascular pathologies, including intracerebral
hemorrhage, epileptogenic seizures, localized thermal brain injury, neurodegenerative disorders,
transient cerebral ischemia, chronic fatigue syndrome, migraines, alcoholic encephalopathy, senile
dementia, and neuroinfections [
6
]. Currently, two types of mitochondrial dysfunction are recog-
nized: primary, resulting from a congenital genetic defect, and secondary, arising from various
pathological factors such as hypoxia, ischemia, oxidative and nitrosative stress, and the expression
of pro-inflammatory cytokines. In modern medicine, increasing importance is being placed on the
study of systemic disturbances in cellular energy metabolism, known as mitochondrial pathology or
mitochondrial dysfunction.
The study of these disorders is of particular importance for practical medicine due to the
unavailable effective therapeutic correction methods. However, it should be noted that the
range of pathological disruptions in cellular energy metabolism is extremely wide (damages
to various links in the Krebs cycle, respiratory chain, beta-oxidation, etc.). Mitochondrial
dysfunction is closely associated with the hyperexpression of early genes, such as c-fos.
In conditions of ROS hyperproduction by the neurochemical and bioenergetic systems of
the brain during brain ischemia, as well as in several other neurodestructive pathological
Antioxidants 2025,14, 108 5 of 46
processes, there is an activation of the expression of redox-sensitive genes, many of which are
essential for protecting cells from the toxic effects of oxidative stress [3436].
Thus, under normal oxygen concentration in the surrounding cell environment (nor-
moxia), the activation of JunB and ATF-2 transcription factors mainly occurs under the
action of ROS, while under oxidative stress, the activation of c-Jun and c-fos factors pre-
dominates. The activation of these specific transcription factors under conditions of ROS
hyperproduction is explained by the fact that JunB and c-fos contain cysteine residues
(Cys252, Cys54, Cys61) in their DNA-binding domains that are highly sensitive to ROS.
Oxidation of their SH-groups leads to the reverse inactivation of AR-1 and NF-kB [
6
,
37
,
38
].
In addition, c-fos protein is directly involved in the process of mitochondrial DNA
fragmentation and initiation of apoptotic neuronal cell death. c-fos is responsible for NO
hyperproduction in neurodegenerative diseases through iNOS activation. c-fos is one
of the main nuclear targets for signaling regulation of cell growth and transformation
and is involved in many cellular functions, including cell proliferation and differentiation
processes [3941].
It is currently known that the main manifestations of mitochondrial dysfunction are a
decrease in ATP levels in the cell, activation of cell death mechanisms, and the production
of ROS by the mitochondria. It is known that during the functioning of the mitochondrial
respiratory chain, small amounts of superoxide radical (O
•−2
) are formed as a byproduct
of the respiratory complexes [42,43].
2. Molecular Mechanisms Behind Mitochondrial Dysfunction
2.1. Markers of Mitochondrial Dysfunction
One important mitochondrial control system that has recently been recognized is the
sirtuins. These conserved class III histone deacetylases possess a catalytic nicotine-adenine
dinucleotide + (NAD+) binding domain and play an important role in the regulation of
both inflammation and metabolism [44,45].
In mitochondria, sirtuins utilize NAD+ as a sensor of mitochondrial energy status,
with SIRT3 enhancing oxidative stress control by increasing SOD levels, SIRT 4 enhancing
the ATP/ADP transporter, and SIRT 5 enhancing urea cycle function [46,47].
As a marker of mitochondrial dysfunction we use AMA-M2—antimitochondrial
antibodies directed to different proteins of the inner membrane of mitochondria. M2 is
directed to the pyruvate dehydrogenase complex, and M3 to the malate dehydrogenase
complex [4852].
Also used as a marker of mitochondrial dysfunction, is Cytochrome C, which is
synthesized as apocytochrome C and enters the mitochondrion where it binds to the inner
surface of the membrane and then exits into the cytoplasm through channels that are
opened for it by Bcl-2 family proteins [5355].
Biochemical markers of mitochondrial dysfunction such as lactate and pyruvate are
widely recognized. Pyruvate is a product of glycolysis, the pathway that metabolizes glu-
cose for fuel, and is the first step in mitochondrial metabolism of carbohydrates. Pyruvate
enters the tricarboxylic acid cycle, one of the main mitochondrial metabolic pathways. If
there is mitochondrial dysfunction, the tricarboxylic acid cycle slows down, leading to
pyruvate accumulation. As pyruvate accumulates, it is diverted to other pathways for
metabolism, specifically lactate and alanine. Thus, when mitochondria become dysfunc-
tional, three metabolic biomarkers can rise in the blood: pyruvate, lactate, and alanine. In
fact, lactate and pyruvate are used in the diagnostic criteria for mitochondrial disease. The
two ratios are also used to monitor mitochondrial function. The lactate to pyruvate ratio is
elevated when lactate is overproduced from pyruvate. Another lesser-known ratio is the
alanine-to-lysine ratio. Lysine is produced as a product of acetyl-CoA, the first step of the
Antioxidants 2025,14, 108 6 of 46
tricarboxylic acid cycle. Thus, when the tricarboxylic acid cycle slows down, less lysine
and more alanine is produced, raising the alanine-to-lysine ratio to over 2.5 [5659].
Carnitine is used as a biochemical marker of mitochondrial dysfunction. Low levels of
total or free carnitine are markers of several diseases associated with primary mitochondrial
dysfunction. Carnitine plays an important role in fatty acid metabolism but also serves
another purpose. Carnitine can bind to fatty acids in the blood as acylcarnitines and
can also bind to excess organic acids. This increases the amount of carnitine esters and
decreases free carnitine. Carnitine can then be excreted in the urine along with these fatty
acids or organic acids if in excess, resulting in a loss of carnitine in the body, reducing
the total amount of carnitine. Short and medium-chain fatty acids can directly enter the
mitochondria, while long-chain fatty acids are bound to carnitine and transported through
the carnitine shuttle. Fatty acids are metabolized by
β
-oxidation, which is a cycle that
shortens a fatty acid by two carbons with each turn, producing acetyl-CoA, which directly
enters the first step of the tricarboxylic acid cycle, and FADH 2, which directly contributes
to the electron transport chain as a substrate for complex II. Elevated levels of Acyl-CoA are
markers of diseases associated with primary mitochondrial dysfunction (Figure 2) [
60
64
].
Antioxidants2025,14,1087of46
Figure2.Diseasesassociatedwithprimarymitochondrialdysfunction.TheFigurewasdesigned
usingBioRender.com.Cerebralischemia,resultingfromasharpdecreaseinoxygenpartialpressure
(pO2),leadstodiscoordinationintheKrebscycle,inhibitionofcompensatoryenergyshunts(such
astheRobertsshuntandthemalate-aspartateshule),activationofglycolysis,lacticacidosis,phos-
pholipaseactivation,anddisruptionofcalcium(Ca
++
)transport.ATPproductionimpairmentand
energydecitcontributetoglutamateexcitotoxicity,Ca
++
overload,activationofneuronalnitricox-
idesynthase(nNOS),andexcessivenitricoxide(NO)production.IncreasedROSproductioninmi-
tochondriathroughNAD(P)H-dependentreactions,combinedwithexcessNO,triggersburstsof
freeradicalreactions.Inthecontextofendogenousantioxidantdeciency,thisleadstooxidative
andnitrosativestressandreducedexpressionofHSP70andHIFproteins,impairingmitochondrial
functionalactivity.Energyproductioninmitochondriaduringischemiareliesonthefunctioningof
themalate-aspartateshule,regulatedbyHSPandHIFproteins.Oxidativeandnitrosativestress,
drivenbyasignicantshiftinthethiol-disuldebalanceandaccumulationofcytotoxicnitricoxide
derivatives(peroxynitrite,nitroxyl,nitrosonium),resultsinoxidativeinhibitionofmitochondrial
respiratorychainenzymesanddirectcytotoxicmodicationsofmitochondrialproteinsandmem-
branelipids.Theenhancementoffreeradicalproductionunderacidosisislinkedtotheincreased
releaseofiron,atriggerforoxidativemechanisms,fromtransferrin-likeproteinsinanacidicenvi-
ronment,intensifyingHaber-Weissreactions.Disruptionofelectrontransportinmitochondria
leadstosecondaryROSgeneration,furtheramplifyingoxidativeandnitrosativestresses.ROSand
freeradicalscauseoxidativemodicationofmitochondrialproteinstructures,particularlyporepro-
teins,increasemembranepermeability,andimpairtranslationandtranscriptionprocesses,aswell
asproteinsynthesisandimport.Ultimately,damagedmitochondriainitiatecelldeathprogramsvia
apoptosisornecrosis.Arrowsindicateincreaseanddecrease
Figure 2. Diseases associated with primary mitochondrial dysfunction. The Figure was designed
using BioRender.com. Cerebral ischemia, resulting from a sharp decrease in oxygen partial pres-
sure (pO2), leads to discoordination in the Krebs cycle, inhibition of compensatory energy shunts
(such as the Roberts shunt and the malate-aspartate shuttle), activation of glycolysis, lactic acidosis,
phospholipase activation, and disruption of calcium (Ca
++
) transport. ATP production impairment
and energy deficit contribute to glutamate excitotoxicity, Ca
++
overload, activation of neuronal nitric
oxide synthase (nNOS), and excessive nitric oxide (NO) production. Increased ROS production in
mitochondria through NAD(P)H-dependent reactions, combined with excess NO, triggers bursts of
Antioxidants 2025,14, 108 7 of 46
free radical reactions. In the context of endogenous antioxidant deficiency, this leads to oxidative
and nitrosative stress and reduced expression of HSP70 and HIF proteins, impairing mitochondrial
functional activity. Energy production in mitochondria during ischemia relies on the functioning
of the malate-aspartate shuttle, regulated by HSP and HIF proteins. Oxidative and nitrosative
stress, driven by a significant shift in the thiol-disulfide balance and accumulation of cytotoxic
nitric oxide derivatives (peroxynitrite, nitroxyl, nitrosonium), results in oxidative inhibition of
mitochondrial respiratory chain enzymes and direct cytotoxic modifications of mitochondrial proteins
and membrane lipids. The enhancement of free radical production under acidosis is linked to the
increased release of iron, a trigger for oxidative mechanisms, from transferrin-like proteins in an acidic
environment, intensifying Haber-Weiss reactions. Disruption of electron transport in mitochondria
leads to secondary ROS generation, further amplifying oxidative and nitrosative stresses. ROS and
free radicals cause oxidative modification of mitochondrial protein structures, particularly pore
proteins, increase membrane permeability, and impair translation and transcription processes, as well
as protein synthesis and import. Ultimately, damaged mitochondria initiate cell death programs via
apoptosis or necrosis. Arrows indicate increase and decrease.
2.2. Mitochondrial Antioxidant System
In functionally complete mitochondria, the action of the mitochondrial antioxi-
dant system, including glutathione, thioredoxin-2, glutathione peroxidase, phospholipid-
hydroperoxide-glutathione peroxidase, and Mn-superoxide dismutase, prevents damage
to mitochondrial structures by reactive oxygen species [65,66].
GSH is the most important antioxidant in the cell, and the ratio of GSH to GSSH is the
most important biomarker of the redox state of the cell. GSH cannot be produced within
mitochondria, so it must be imported. A pathway that produces more GSH requires ATP,
an energy molecule produced by the mitochondria, so poor mitochondrial function will
result in decreased GSH production and reduced ability of the mitochondria to produce
cellular energy. ROS is controlled at the inner mitochondrial membrane by leakage of
protons back across the membrane, a process that makes mitochondria less efficient. This is
accomplished by several proton channels such as uncoupling protein (UCP) [6770].
We found that the introduction of chloro-2,4-dinitrobenzene (CDNB, 1 mM) into the
suspension of mitochondria isolated from the brain of white rats leads to a 62% decrease
in the amount of GSH and more than 47% inhibition of Mn-SOD activity. A decrease in
mitochondrial membrane potential and an increase in mitochondrial pore opening rate
were also detected. Deprivation of the mitochondrial glutathione system leads to a decrease
in other antioxidant systems as well and shapes the development of mitochondrial dysfunc-
tion. GSH deficiency in mitochondria leads to increased formation of ROS and nitrogen
and oxidation of cysteine-dependent sites of proteins forming the mitochondrial pore.
Excess of active nitrogen forms (peroxynitrite, nitrosonium ion) formed at GSH deficiency
in mitochondria leads to oxidative modification of Mn-SOD and reduction of its activ-
ity. Reduction of Mn-SOD activity promotes a secondary “burst” of free-radical reactions
and intensification of oxidative destruction of Red-Oxi—sensitive sites of mitochondrial
membrane and formation of persistent mitochondrial dysfunction [71].
2.3. The Production of ROS by Mitochondria and the Lack of Mitochondria
It is believed that a key event in the development of mitochondrial dysfunction after
hypoxia/reoxygenation is an increase in the production of ROS by mitochondria. The main
causes of mitochondrial dysfunction include electron overload in the respiratory chain
under hypoxic conditions, as well as a decrease in the activity of cytochrome c oxidase
and Mn-superoxide dismutase (Mn-SOD). Inhibition of cytochrome c oxidase during
subsequent reperfusion contributes to a disruption in the process of electron transfer to
the final acceptor in the respiratory chain—oxygen—which leads to an increase in the
production of superoxide radicals by the respiratory complexes [72].
Antioxidants 2025,14, 108 8 of 46
Superoxide is formed in the so-called “parasitic” reactions in the initial part of the
mitochondrial respiratory chain (CoQH2-NAD+) with the participation of NADH-CoQH2-
reductase, the activity of which is increased by blockade of cytochrome-C-dependent
receptor on the outer surface of the mitochondrial membrane against the background of
increased reduced flavins. In addition to superoxide, the key role in the development of
mitochondrial disorders and the development of apoptosis/necrosis belongs to NO and its
more aggressive form—peroxynitrite [73,74].
Neuronal mitochondria are an important source of NO. The presence of a constitutive
form of NOS localized in the inner membrane and NO production in mitochondria of
hippocampal neurons has been shown. Mitochondrial NOS at suboptimal concentrations
of L-arginine is able to produce superoxide. Mitochondrial NOS (mtNOS) is significantly
activated in response to the development of glutamate “excitotoxicity” and calcium uptake
by mitochondria [75,76].
It should be noted that IL-1b and TNF-a play a certain role in mtNOS activation.
mtNOS through the production of a dosed level of NO, is able to regulate mitochondrial
respiration in normal conditions and at the initial, compensated stages of ischemia, modu-
lating the activity of cytochrome-C-oxidase, complexes I and II of the electron-transport
chain and the level of NADPH, FAD, and coenzyme Q10, as well as changing the availability
of O
2
for electron acceptance. Further, the role of mtNOS changes to the cardinally opposite
one—it participates in activation of “parasitic” reactions of ROS formation by mitochondria.
The thiol-disulfide system deserves special attention in expanding the understanding of
the mechanisms of NO cytotoxicity and neuronal death [7779].
Intermediates of the thiol-disulfide system have transport properties with respect
to NO, thereby increasing its bioavailability; moreover, many thiols such as glutathione,
cysteine, and methionine can significantly limit the cytotoxicity of NO and its derivatives,
increasing the chance of neuronal mitochondria to survive ischemia [26,80,81].
2.4. Interaction of ROS and NO with the Mitochondria
When electron transfer between the components of the respiratory chain is disrupted,
the generation of superoxide anion (O
•−2
) by mitochondria is significantly enhanced. A
deficiency in the functions of the mitochondrial antioxidant system will contribute to the
development of oxidative stress, activation of self-sustaining processes of lipid peroxidation,
and oxidative damage to proteins and nucleic acids within the mitochondria [82].
The increase in NO concentration in mitochondria observed in the post-ischemic
period leads to the interaction of NO with heme iron and paired thiol groups, forming
dinitrosol iron complex (DNIC). DNIC, in contrast to NO, is a stronger nitrosylating
agent, interacting with protein thiols, histidine, aspartate, glutamine, methionine, cysteine,
glutathione and forming N- and S-nitrosothiols [83].
Cerebral ischemia is accompanied by a sharp shift of thiol-disulfide equilibrium to-
wards oxidized thiols, a drop in the activity of enzymes of the thiol-disulfide system
(glutathione reductase, glutathione-S-transferase). When glutathione-dependent enzymes
are inhibited under ischemia conditions, even greater oxidative modification of low molec-
ular weight thiols occurs, homocysteine formation and, as a consequence, NO transport
is impaired with the formation of its cytotoxic derivatives that further enhance thiol oxi-
dation. The presence in a neuron of a sufficiently active thiol antioxidant system capable
of regulating NO transport provides cell resistance to nitrosative stress, the earliest neu-
rodegenerative mechanism under ischemia conditions [
84
86
]. It is known that in the first
minutes of brain ischemia, NO (macrophagal or exogenous) inhibits oxidative phosphoryla-
tion in mitochondria of target cells due to reversible binding to mitochondrial cytochrome
Antioxidants 2025,14, 108 9 of 46
C-oxidase. Suppression of electron transport in mitochondria, as shown above, leads to
generation of superoxide and, as a consequence, to the formation of ONOO- [26].
Excess superoxide radical, peroxynitrite further oxidizes the thiol groups of the
cysteine-dependent site of the mitochondrial inner membrane protein (ATP/ADP-
antiporter), which turns it into a permeable nonspecific channel pore. The mitochondrial
pore is a supramolecular channel connecting the cytosolic and intramitochondrial spaces,
composed of a complex of proteins including the adenine nucleotide translocator, the
benzodiazepine receptor (translocator protein), and the voltage-dependent anion channel.
Cyclophilin D, ATPase, and the mitochondrial inorganic phosphate carrier are not part of
the pore structure but act as regulatory factors. The anti-apoptotic bcl-2 and pro-apoptotic
Bax proteins are associated with the benzodiazepine receptor. These proteins are compo-
nents of the outer membrane and cytosol and participate in apoptosis by controlling the
release of cytochrome C [87,88].
Under conditions of oxidative stress, the mitochondrial pore binds to Ca
2+
and sub-
stances with large molecular weight can pass through the membrane pore. This leads to
a drop in membrane potential and matrix swelling, the integrity of the outer membrane
is inevitably compromised, and apoptosis proteins are released from the intermembrane
space into the cytoplasm [89].
There are several pro-apoptotic proteins: apoptosis-inducing factor (AIF), second
mitochondria-derived activator of caspases (Smac), and some procaspases. The inducing
factor goes directly to the nucleus, where it causes DNA degradation. Along with specific
apoptosis proteins, cytochrome C, which normally serves as the final link in the electro-
transport chain, leaves the mitochondrion through the open pore. In the cytoplasm, this
protein binds to the protein Apaf-1 (apoptotic protease activating factor-1) and forms an
apoptosome complex. With the help of Smac and another factor (Omi/HtrA2), it acti-
vates procaspase-9, which, becoming caspase-9, transforms two other proenzymes into
caspases-3 and -7; and they already cleave structural proteins, leading to the appearance of
biochemical and morphological signs of apoptosis in the neuronal cell [90,91].
2.5. Disorder in the Lipid Layer of Mitochondrial Membranes
In parallel with the above-mentioned destructive processes in the mitochondrial
matrix, oxidative changes in the lipid layer of mitochondrial membranes are observed: the
level of phospholipids decreases and free fatty acids and lysophosphatides accumulate.
Depletion and oxidative modification of mitochondrial pool of membrane phospholipids
promote mobilization of cytochrome C in the intermembrane space, which significantly
facilitates its release into the cytoplasm after opening of mitochondrial pores [92,93].
It was found that changes in the lipid composition of mitochondrial membranes lead
to impaired functioning of mitochondrial membrane enzymes. The increase in the content
of free fatty acids in the inner mitochondrial membrane promotes dissociation of oxidation
and phosphorylation processes, which leads to suppression of ATP synthesis [94,95].
ROS are produced through metabolic processes, particularly by Complexes I and III
of the electron transport chain (ETC). ROS can be destructive to many vulnerable parts
of the mitochondria. Lipid membranes are especially vulnerable to ROS. The integrity of
lipid membranes is crucial for key mitochondrial structures, such as the ETC. If the lipid
membrane is damaged, protons may leak through the inner mitochondrial membrane,
which can reduce the proton gradient responsible for driving Complex V of the ETC
to produce ATP. This will decrease ATP production and lower the inner mitochondrial
membrane potential [42,96].
Antioxidants 2025,14, 108 10 of 46
3. Genetic and Cellular Factors Involved in Mitochondrial Dysfunction
3.1. Mitochondrial DNA Damage in Mitochondrial Dysfunction
Another mechanism of mitochondrial dysfunction is the accumulation of damage in
the mitochondrial genome and the depletion of the mitochondrial DNA pool. ROS can
also damage mitochondrial DNA (mtDNA), as mtDNA is not protected like nuclear DNA,
and mitochondria are inefficient in repairing mtDNA. This can lead to harmful mutations
in mtDNA if the damage occurs in a critical genetic region of the mtDNA. Ultimately,
these events lead to a decrease in cell function and the accumulation of mutations in both
mitochondrial and nuclear DNA. In turn, damage to mitochondrial genes contributes to
the disruption of the electron transfer process in the respiratory chain, resulting in further
increased production of free radicals in the mitochondria [97,98].
ROS, interacting with nucleic acids, modify bases, deoxyriboses, and also form new
covalent bonds. The most significant modification occurs at the bases [
99
]. When ROS
and free radicals interact with thymine, 5,6-dihydroxy-5,6-dihydrothymine isomers are
formed. Cytosine, when interacting with ROS and hydroperoxides, forms hydroxylated
cytosine. The interaction of ROS with purines leads to the breakage of the imidazole ring of
the molecule fragment, resulting in the formation of formamidopyrimidine residues [
100
].
Among the products of oxidative modification of purines, the most notable are 8-
oxoguanine (8-OG) and its tautomer 8-hydroxyguanine (8-OHG). The formation of these
products occurs continuously, but it significantly increases under various pathological
conditions and can be considered a marker of oxidative stress. Currently, the determination
of 8-OHG is a popular, non-invasive method for assessing oxidative stress levels. Under the
influence of ROS, deamination of guanine and adenine may occur, leading to the formation
of xanthine and hypoxanthine in the DNA molecule, which have mutagenic effects. The
consequences of various types of oxidative damage are not the same. For example, thymine
glycols and formamidopyrimidines block replication, are cytotoxic, but their mutagenic
potential is limited. The most mutagenic is 8-OHG, and if it is present in the template, all
replicative DNA polymerases insert dAMP opposite it and carry out a guanine-cytosine to
thymine-adenine substitution. In addition, ROS can directly induce DNA strand breaks
by cleaving the sugar-phosphate base [
101
,
102
]. DNA aberration induces an enhanced
stress response in the mitochondria, which ultimately leads to the formation of defective
mitochondrial proteins. ROS are capable of causing indirect damage to nucleic acids. ROS
trigger the release of calcium from the mitochondria, which subsequently leads to an
increase in nuclease activity. Nitric oxide (NO) plays a significant role in DNA damage by
causing the deamination of nucleic acids, followed by the activation of NAD-dependent
polymerase (PARP), which catalyzes the attachment of ADP-ribose to histone proteins and
DNA [103,104].
Additionally, it should be noted that transporting 1 mole of ADP-ribose requires 1 mole
of NAD+ and 4 moles of ATP, which, with significant mobilization of PARP following
extensive DNA damage, rapidly depletes the cell’s energy reserves. ROS also stimulate
ADP-dependent ribosylation of glyceraldehyde-3-phosphate dehydrogenase, leading to
the subsequent inactivation of the enzyme and disruption of glycolysis reactions. The
impairment of the mitochondria’s energy-producing function is also associated with the
dysfunction of the respiratory chain, caused by mutations in mitochondrial DNA (mtDNA).
Such disruptions affect various biochemical functions of the mitochondria, such as the
mitochondrial membrane potential, ATP synthesis, the ATP/ADP ratio (which indicates
the state of the oxidative phosphorylation system), ROS generation, and mitochondrial
turnover. It has been established that once the proportion of ROS-mediated mtDNA
deletions exceeds a sensitivity threshold, the mitochondrial membrane potential, ATP
synthesis rate, and ATP/ADP ratio sharply decrease [105107].
Antioxidants 2025,14, 108 11 of 46
The main consequences of the aforementioned processes are the disruption of the res-
piratory chain function, weakening of the mitochondrial antioxidant defense, accumulation
of cytotoxic oxidatively damaged proteins and nucleic acids; mobilization of cytochrome
C in the intermembrane space, which, after reoxygenation, transforms the mitochondria
into a source of free radicals, reduces their ability to synthesize ATP, and increases the
mitochondria’s sensitivity to thanatogenic signals [108,109]
3.2. Ca2+ and Mitochondrial Dysfunction
A significant impact on the mitochondria is exerted by the increase in the cytoplasmic
level of calcium ions due to the disruption of the cell’s ionic homeostasis. It has been shown
that the increase in calcium ion concentration in the cytoplasm promotes the induction of
the release of thanatogenic factors from the mitochondria, initiates lipolysis processes in the
mitochondrial membranes, and disrupts the function of respiratory complexes. It has been
established that Ca
2+
ions activate proteins that facilitate the formation of mitochondrial
channels: Bax and Bid, by activating calpains, as well as Bad and Bik, through the activation
of calcineurin [110112].
Movement of excessive amounts of calcium ions into the mitochondrial matrix of Ca
2+
ions can lead to additional opening of permeability transition pores. Another of the known
mechanisms of the effect of excessive concentration of Ca
2+
ions on mitochondrial structures
is damage to the mitochondrial membrane due to activation of phospholipase A2 [
113
].
The possibility of stimulation of oxidative stress in the cell by Ca
2+
ions through activation
of calcium-sensitive isoform of NOS has been shown [
114
]. It is known that uncontrolled
increase in the concentration of neurotransmitters such as glutamate and dopamine in the
extracellular space contributes to the development of neurotoxic processes in the affected
area of the brain during stroke. It has been established that excessive glutamate induces
unregulated Ca
2+
ion influx into the cytosol, further impairing the functional activity
of mitochondria [
115
]. Glutamate is the main excitatory neurotransmitter of the CNS, is
involved in cognitive functions, along with acetylcholine maintains the level of wakefulness,
but in high concentrations is a neurotoxin [116].
Glutamate realizes its effects through a group of ionotropic membrane receptor chan-
nels: NMDA, AMPA, and kainate receptors. Excitation of glutamate NMDA receptors,
which regulate the content of K
+
, Na
+
, Ca
++
, Cl
in the extra- and intracellular space, acti-
vates Ca
++
-channels, which leads to an increase in the flow of extracellular Ca
++
into the cell
and release of intracellular Ca++ from the depot, activating various enzyme systems [117].
This leads to impaired phosphorylation of proteins, cleavage of phospholipids and re-
lease of arachidonic acid, formation of toxic products, free radicals that have cytotoxic,
immunogenic and mutagenic effects, damaging cellular DNA and mRNA [118].
Along with the swelling of mitochondria caused by the influx of calcium, the process
shifts into the cytoplasm of the cell and extends to the intercellular level, making the
hypoxia tissue-wide. This stage of the ischemic cascade can no longer be reversed by
restoring oxygen supply or reperfusion, as the deeply damaged mitochondria stop utilizing
oxygen and substrates. They combine with sodium and calcium in the cytoplasm to form
endogenous soaps, which literally dissolve (wash away) lipid membranes [119122].
3.3. Disruption of Native Protein Structure (Unfolded Protein Response—UPR) and
Mitochondrial Dysfunction
An additional factor contributing to mitochondrial damage is the “unfolded protein
response” (UPR), which is activated under hypoxic conditions. It has now been shown
that the endoplasmic reticulum (ER), when under stress mediated by UPR, can promote
the development of degenerative changes in mitochondria by releasing calcium ions into
the cytoplasm from the ER, along with the membrane-associated ER protein inositol-
Antioxidants 2025,14, 108 12 of 46
requiring enzyme 1 (IRE1). The ER stress sensor IRE1 interacts with STIM1, facilitating
the influx of Ca
++
and initiating calcium-dependent responses, including the activation of
NOS-regulated proteins [123125].
UPR normally mediates cell death by activation of the intrinsic apoptotic pathway,
but recent studies have shown that in mitochondrial dysfunction there is a strong acti-
vation of UPR, which may lead to activation of programmed necrosis pathways such as
necroptosis [
126
]. To date, it is known that mitochondrial dysfunction is possible through
inactivation of the hypoxia-inducible transcription factor HIF-1 [127].
Recent studies have established that adaptation to hypoxia at the cellular and subcel-
lular levels is closely associated with the transcriptional expression of late-acting hypoxia-
inducible genes, which are involved in regulating multiple cellular and systemic functions
and are necessary for the formation of adaptive traits. It is known that at low oxygen
concentrations, this process is primarily controlled by the specific transcription factor HIF-1,
which is induced by hypoxia in all tissues. Disruption of energy metabolism due to ischemia
or hypoxia, and a decrease in the concentration of mitochondrial ATP, leads to the activation
of mitochondrial UPR (UPRmt). In the early stages of ischemia, UPRmt “attempts” to
restore mitochondrial energy homeostasis, and if unsuccessful, it suppresses the expression
of protective proteins, including HIF-1, and initiates cell death mechanisms [
128
130
]. The
use of novel specific UPR inhibitors as remedies for mitochondrial dysfunction is of interest.
3.4. HIF-1 and Mitochondrial Dysfunction
Hypoxia-induced factor, discovered in the early 90s, functions as a master regulator of
oxygen homeostasis and is the mechanism by which the organism, responding to tissue
hypoxia, controls the expression of proteins responsible for the mechanism of oxygen
delivery to the cell, i.e., regulates the adaptive responses of the cell to changes in tissue
oxygenation [131133].
Currently, more than 60 direct target genes have been identified for it. All of them
contribute to the improvement of oxygen delivery (erythropoiesis, angiogenesis), metabolic
adaptation (glucose transport, enhanced glycolytic ATP production, ion transport) and cell
proliferation. HIF-1 regulated products act at different functional levels. The end result of
such activation is an increase in O2supply to the cell [119,134].
The identification and cloning of HIF-1 have revealed that it is a heterodimeric redox-
sensitive protein composed of two subunits: the inducibly expressed oxygen-sensitive
subunit HIF-1
α
and the constitutively expressed subunit HIF-1
β
(aryl hydrocarbon receptor
nuclear translocator—ARNT). By heterodimerizing with the aryl hydrocarbon receptor
(AHR), it forms a functional dioxin receptor. Other proteins of the HIF-1
α
family are also
known, including HIF-2
α
and HIF-3
α
. All of them belong to a family of basic proteins,
each containing a basic helix-loop-helix (bHLH) domain at the amino-terminal end of
each subunit, which is characteristic of various transcription factors and essential for
dimerization and DNA binding [135137].
HIF-1
α
consists of 826 amino acid residues (120 kD) and contains two transcriptional
domains at the C-terminal end. Under normoxic conditions, its synthesis occurs at a low
rate and its content is minimal because it undergoes rapid ubiquitination and degradation
by proteasomes. This process depends on the interaction of the primary structure of HIF-
1
α
and its specific oxygen dependent degradation domain (ODDD—oxygen dependent
domain degradation) with von Hippel Lindau (VHL), a tumor growth suppressor, which
acts as a protein ligase, widely distributed in tissues [138140].
The molecular basis for this regulation is the O
2
-dependent hydroxylation of its two
proline residues P402 and P564, part of the HIF-1
α
structure, by one of three enzymes
collectively known as “prolyl hydroxylase domain (PHD) proteins, or HIF-1
α
prolyl hy-
Antioxidants 2025,14, 108 13 of 46
droxylases,” which is required for binding of HIF-1
α
to the VHL protein.
α
-Ketoglutarate,
vitamin C and iron are also obligatory components of the process. Along with this, hy-
droxylation of an asparagine residue in the C-terminal transactivation domain (C-TAD)
occurs, resulting in suppression of HIF-1
α
transcriptional activity. After hydroxylation of
the proline residue in the ODDD and the asparagine residue, HIF-1
α
binds to the VHL
protein, which makes this subunit available for proteasomal degradation [141,142].
Under conditions of severe oxygen deficiency, the oxygen-dependent process of hy-
droxylation of proline residues, which is characteristic of normoxia, is suppressed. As a
result, VHL (von Hippel-Lindau protein) cannot bind to HIF-1
α
, and its degradation by the
proteasome is limited, allowing for its accumulation. In contrast, p300 and CREB binding
protein (CBP) can bind to HIF-1
α
, as this process does not depend on asparaginyl hydroxy-
lation. This enables the activation of HIF-1
α
, its translocation to the nucleus, dimerization
with HIF-1
β
, leading to conformational changes and the formation of a transcriptionally
active complex (HRE), which triggers the activation of a broad range of HIF-1-dependent
adaptive processes aimed at enhancing the synthesis of endogenous cytoprotective proteins.
Among the most important proteins in this group are the so-called heat shock proteins,
such as HSP70 (heat shock proteins) [143,144].
HIF-1 mediates mitochondrial biogenesis, mitophagy, and mitochondrial dynamics
to regulate mitochondrial population. HIF-1 activation induced mitochondrial fission in
human models of pulmonary arterial hypertension (PAH) by phosphorylation of DRP1 by
serine 616 [145].
HIF-1 can alter the intracellular distribution of mitochondria by regulating their
mobility. The mitochondrial movement regulator (HUMMR) is activated by HIF-1
α
, and
mitochondrial transport shifts in the anterograde direction for efficient distribution through-
out the neuron. Additionally, HUMMR may help preserve mitochondrial content in axons
dependent on HIF-1
α
. HIF-1 regulates mitochondrial morphology, such as size, shape, and
structure, which could underlie functional changes or be secondary to functional regula-
tion. In mitochondria with stimulated HIF-1 expression, there was an increase in energy
metabolism reactions and regulation of ROS production by mitochondria. HIF-1 positively
regulates the expression of lactate dehydrogenase A (LDHA) and promotes the conver-
sion of pyruvate to lactic acid under hypoxic conditions. HIF-1 serves as a sensor for ROS,
limiting excessive mitochondrial ROS production in response to cytokine stimulation. Over-
expression of HIF-1
α
blocks the reduction of mitochondrial membrane potential (
∆Ψ
m)
under hypoxia and inhibits mitochondrial mechanisms that initiate apoptosis [
127
,
146
,
147
].
There is evidence for a relationship between HIF-1
α
and iron homeostasis, especially
at the mitochondrial level, because Fe
2+
is mobilized in the Fenton reaction, which produces
hydroxyl radical (
OH) from H
2
O
2
and lipid alkoxyl radicals from lipid peroxides. Notably,
the mitochondrial antioxidant apparatus allows iron homeostasis to be maintained by
limiting H
2
O
2
production and converting lipid peroxides to lipid alcohol using SOD and
GPX4, respectively [127,148].
3.5. HSP70 and Mitochondrial Dysfunction
Enhanced expression of genes encoding HSP is triggered not only by heat stress but
also by a number of different factors and pathogens. Evolutionarily, HSP70 is classified
as a highly conserved protein, indicating that it performs fundamental cellular functions.
The native HSP70 molecule is a dimer with the ability to form highly oligomeric complexes
with many structures in the cell, as well as cytosolic and mitochondrial proteins, and has at
least 8 isoforms, the exact number and concentration of which depends on the cell type and
is controlled by the type of stressor. HSP70 proteins belong to a class of cellular proteins
referred to as “molecular chaperones” (chaperone-mediator). Chaperone activity refers
Antioxidants 2025,14, 108 14 of 46
to the ability of HSP70 to recognize and bind exposed hydrophobic surfaces of native
polypeptide chains, denatured and oxidatively damaged polypeptides. HSP70 are the main
participants in the process of folding of newly synthesized polypeptide chains [149].
The ability of HSP70 to fold polypeptides in a specific conformation is used in normal
cellular processes to regulate key signaling molecules such as cell cycle kinases, caspases,
steroid hormone receptors, and vitamin D receptors. HSP70 are also involved in the pro-
cesses of polypeptide translocation into mitochondria, in restoring the structure of damaged
and denatured proteins, and in the formation of oligomeric protein complexes. In addition
to chaperone function, HSP70 function as regulators/modulators of protein proteolysis
(ubiquitins, redox reactions, synthesis of proinflammatory cytokines, synaptic transmission,
and Ca
++
-dependent K
+
-channels). In addition, the ability of HSP70 to stabilize under
ischemia conditions the factor HIF-1 was investigated. Thus, under normoxia conditions,
HSP-70 is in complex with HIF-1 [150,151].
Under hypoxia conditions, HSP70 is displaced from the complex with HIF-1 by ARNT
protein, with further exercise of its chaperone function against HIF-1, in addition, these
proteins exert unidirectional action with respect to cell protection against oxidative stress
during ischemia [149,152,153].
Recent studies have established the neuroprotective activity of HSP70 and HIF-1 aimed
at reducing the phenomena of mitochondrial dysfunction and related oxidative stress. We
found that HSP70 and HIF-1 in brain ischemia increase the activity of mitochondrial
antioxidant enzymes protecting them from oxidative destruction, restore thiol-disulfide
equilibrium, normalize the processes of energy metabolism due to folding of respiratory
chain proteins, as well as increase the functional activity of mitochondria, eliminating
damaged and denatured proteins. Start and regulate the activity of the compensatory
malate-aspartate shuttle mechanism of energy production [153,154].
This statement is confirmed by the works of some authors. The role of increased
expression of HSP70 in brain cells (astrocytes) in protecting them from death caused by
oxygen starvation has been shown [155,156].
In addition, the ability of a purified HSP70 preparation to enhance the survival of
neurons involved in glutamatergic synaptic transmission in the olfactory cortex of rat brain
was demonstrated against the damaging effects of severe anoxia [
157
,
158
]. Nevertheless,
the mechanism of the protective effect of HSP70 is still unclear. Taking into account the
data on the ability of HSP70 to enhance neuronal cell viability under hypoxia conditions
and the fact of interaction between HSP70 and HIF-1, which plays a primary role in the
cellular response to hypoxia, we can assume that HSP70 is involved in the regulation of
signaling pathways of the cell response to hypoxic stress at the level of regulation of HIF-1
stability [149].
In addition, the neuroprotective effect of HSP70 in ischemia is also explained by its
antiapoptotic and “mitoprotective” action. Currently, three main pathways of HSP70
influence on apoptosis processes are postulated in the literature. Firstly, HSP70 may affect
the functioning and signaling of the Fas/Apo1 receptor inside the cell; secondly, HSP70
may in one way or another affect the release of cytochrome C from mitochondria; and
finally, thirdly, HSP70 may affect the formation of apoptosomes and the activation of the
caspase cascade. HSP70 blocks apoptosis induced by activation of the Fas/Apo1 receptor.
After binding to the ligand, the receptor interacts with adaptor proteins, one of which may
be the FADD protein [159162].
This adaptor protein FADD binds inactive procaspase 8 and promotes its activation
upon receptor-ligand binding. Caspase 8 activates caspases 3, 6, and 7 and thereby initiates
proteolysis of target proteins, ultimately leading to apoptosis. In addition, caspase 8 can activate
the Bid protein, which induces the release of cytochrome c from mitochondria [163,164].
Antioxidants 2025,14, 108 15 of 46
The site of action of HSP70 in this complex chain of reactions has not yet been precisely
determined. An alternative pathway for triggering apoptosis via Fas/Apo1 involves the
Daxx protein. The mechanism of action of this protein is not well understood. Normally,
Daxx is localized in the nucleus, where it is bound to certain proteins, but it can move to
the cytoplasm and play the role of an adaptor protein responsible for triggering the cascade
of JNK-kinases by activating Fas/Apo [165,166].
It is assumed that HSP70 is able to move to the nucleus, where it interacts with Daxx,
preventing its release into the cytoplasm and activation of the receptor. Previously, it was
noted that HSP70 may participate in the regulation of apoptosis not only at the level of the
receptor Fas/Apo1, but also at the level of certain intracellular target proteins [167,168].
Indeed, HSP70 has been shown to prevent mitochondria-initiated apoptosis, and
different mechanisms of action of heat shock proteins are possible. The drop in membrane
potential induced by cerebral ischemia is known to result in the release of cytochrome c
from mitochondria. In the cytoplasm, cytochrome C binds to Apaf1 protein, deoxy ATP,
and procaspase 9, forming the so-called apoptosome [169,170].
Apoptosome formation is accompanied by autocatalytic activation of procaspase 9
and its transfer into the active form of caspase 9. This enzyme activates procaspase 3 and
the following caspases involved in the process of apoptosis. HSP70 inhibits apoptosis in
the step between cytochrome c release and cleavage of procaspase 9 in the apoptosome.
Recently, the literature has provided evidence that HSP70 is able to interact with cytochrome
C [
171
,
172
]. The question of what part of cytochrome C released from mitochondria binds
to HSP70 remains open. A number of studies have shown that HSP70 binds only a very
small fraction of cytochrome C released from mitochondria and, therefore, cannot play a
significant role in apoptosome formation [
173
,
174
]. HSP70 is known to prevent the decrease
in membrane potential induced by Bax protein, but does not interact with this protein.
There is a hypothesis that in the mitochondrial pathway of apoptosis HSP70 acts at earlier
stages of this complex process and prevents disruption of the actin filament structure [
154
].
Our works established that modeling of chronic cerebral circulatory disturbance by
disruption of cerebral blood circulation led to persistent neurological disorders in surviving
animals by the 18th day of the experiment, as well as to the disruption of mitochondria
ultrastructure of hippocampal CA1 neurons, which was characterized by an increase in the
absolute number of damaged mitochondria, more than 11 times in relation to the intact
group of animals, as well as a decrease in intramitochondrial HSP70 3,4 in comparison with
similar indicators of the group of falsely operated animals [25].
Thus, summarizing the above, we can conclude that HSP70 and Hif1b proteins are
inevitable companions of pathobiochemical reactions that develop during ischemic brain
damage and perform a protective function under these conditions, which is realized by
means of enhancing the synthesis of antioxidant enzymes, stabilization of oxidatively
damaged macromolecules, direct antiapoptotic and mitoprotective action. Such a role of
these proteins in cellular reactions during ischemia raises the question of the development
of new neuroprotective agents capable of modulating the expression and synthesis of HSP-
and HIF-proteins [6].
4. Mitochondrial Dysfunction and Apoptosis
The activation of neuroapoptosis, according to many researchers, is the primary cause
of persistent cognitive and mnemonic dysfunctions in the central nervous system (CNS).
Neuroapoptosis develops as a cascade process accompanied by the activation (induction
of formation) of specific pro- or anti-apoptotic proteins, as well as specialized proteolytic
enzymes—caspases. Among the factors triggering apoptosis, the formation of ROS during
the “distorted” pathway of oxidative metabolism in the cell should be noted. Convincing
Antioxidants 2025,14, 108 16 of 46
evidence exists that mitochondria play a central role in ROS production and the subsequent
development of apoptosis and necrosis. This involves changes in the permeability of
mitochondrial membranes due to the formation of specific mitochondrial pore complexes
and the initiation of mitoptosis (Figure 3) [175,176].
Antioxidants2025,14,10817of46
Figure3.Disruptionsinmitochondrialfunction.TheFigurewasdesignedusingBioRender.com.
Mitochondrialdysfunctionisatypicalpathologicalprocesslackingetiologicalandnosologicalspec-
icity,ultimatelyleadingtocelldeath.Itresultsinenergydecit,impairedneurotransmier
reuptake,neurotransmierautocoidosis,disruptedCa
++
transport,impairednerveimpulseconduc-
tion,andhyperproductionofROSbybioenergeticsystems.ROSoxidizethethiolgroupsoftheCys-
dependentregionoftheinnermitochondrialmembraneprotein(ATP/ADPantiporter),causingthe
massivereleaseofpro-inammatoryandpro-apoptoticfactors.Inthecontextofdepletedmitochon-
drialantioxidantdefenses(MnSOD,GSH),ROSinduceoxidativemodicationofproteins,nucleic
acids,andlipids.
Undertheinuenceofhydroxylradicals,mitochondrialporesopen,leadingtothe
expressionandreleaseofpro-apoptoticproteinsintothecytosol.Theopeningofthese
porestransformsmitochondriafrom“powerplantsinto“furnaces”foroxidationsub-
strateswithoutATPproduction.Precisebiochemicalstudieshaveestablishedthatdisrup-
tionsintissueoxygenation,hyperproductionofexcitotoxicaminoacids,adecreasein
“normalCa
2
⁺accumulationbymitochondria,andoxidativedamagetomitochondrial
membranesbyROSexacerbateporeopeningandthereleaseofapoptogenicproteinsfrom
damagedmitochondria.
Inthiscontext,theroleofoneneurotrophicfactor—tumornecrosisfactor-α(TNF-
α)—issignicant.TNF-αisassociatedwiththeopeningofmitochondrialpores,subse-
quentmembranedamage,andtheprogressionofmitoptosis.Themitochondrialporeisa
channeltraversingbothmitochondrialmembranesandcomprisesthreeproteins:thead-
eninenucleotidetranslocator,thevoltage-dependentanionchannel(porin),andtheben-
zodiazepinereceptor.WhenthiscomplexbindstoCa
2
,substanceswithsmallmolecular
Figure 3. Disruptions in mitochondrial function. The Figure was designed using BioRender.com.
Mitochondrial dysfunction is a typical pathological process lacking etiological and nosological speci-
ficity, ultimately leading to cell death. It results in energy deficit, impaired neurotransmitter reuptake,
neurotransmitter autocoidosis, disrupted Ca
++
transport, impaired nerve impulse conduction, and
hyperproduction of ROS by bioenergetic systems. ROS oxidize the thiol groups of the Cys-dependent
region of the inner mitochondrial membrane protein (ATP/ADP antiporter), causing the massive
release of pro-inflammatory and pro-apoptotic factors. In the context of depleted mitochondrial
antioxidant defenses (MnSOD, GSH), ROS induce oxidative modification of proteins, nucleic acids,
and lipids.
Under the influence of hydroxyl radicals, mitochondrial pores open, leading to the
expression and release of pro-apoptotic proteins into the cytosol. The opening of these
pores transforms mitochondria from “power plants” into “furnaces for oxidation substrates
without ATP production. Precise biochemical studies have established that disruptions in
tissue oxygenation, hyperproduction of excitotoxic amino acids, a decrease in “normal” Ca
2+
accumulation by mitochondria, and oxidative damage to mitochondrial membranes by ROS
exacerbate pore opening and the release of apoptogenic proteins from damaged mitochondria.
In this context, the role of one neurotrophic factor—tumor necrosis factor-
α
(TNF-
α
)—is
significant. TNF-
α
is associated with the opening of mitochondrial pores, subsequent
membrane damage, and the progression of mitoptosis. The mitochondrial pore is a channel
traversing both mitochondrial membranes and comprises three proteins: the adenine nu-
cleotide translocator, the voltage-dependent anion channel (porin), and the benzodiazepine
receptor. When this complex binds to Ca
2+
, substances with small molecular masses can
Antioxidants 2025,14, 108 17 of 46
pass through the membrane pore. This process leads to a decrease in membrane potential
and matrix swelling, which inevitably compromises the integrity of the outer membrane.
Consequently, apoptosis-related proteins are released from the intermembrane space into
the cytoplasm [89,177].
There are several of them: apoptosis-inducing factor, secondary mitochondria-derived
activator of caspases (Smac), and some procaspases. The inducing factor goes directly to
the nucleus, where it causes DNA degradation. Along with specific apoptosis proteins,
cytochrome C, which normally serves as the final link in the electrotransport chain, leaves
the mitochondrion through the open pore. In the cytoplasm, this protein binds to the protein
Apaf-1 (apoptotic protease activating factor-1) and forms an apoptosome complex. It, with
the help of Smac and another factor (Omi/HtrA2), activates procaspase-9, which, becoming
caspase-9, transforms two other proenzymes into caspases-3 and 7; and they already cleave
structural proteins, leading to the appearance of biochemical and morphological signs
of apoptosis [
178
180
]. Among the earliest events, in particular, are the translocation
of phosphatidylserine to the outer membrane layer and DNA fragmentation under the
influence of ROS and NO [181].
Among the secondary signs, the most characteristic are the “shedding” of the cell
from the matrix, membrane wrinkling, nuclear condensation, and the formation of vesicles
containing cellular contents—apoptotic bodies [
182
]. The release of cytochrome c into the
cytoplasm is facilitated by a decrease in pH during the development of lactate acidosis
and an increase in oxidative modification of mitochondrial proteins and lipids. This latter
reaction is directly triggered by ROS, which are inevitably formed as a result of “parasitic”
energy reactions [
183
]. Cytochrome C can be released in response to an increase in Ca
2+
ion concentration, which triggers pore opening, and its release is also regulated by proteins
of the Bcl-2 family [184,185].
They are the ones that regulate apoptosis at the mitochondrial level. Some of the
proteins of this large family (Bcl-2, as well as Bcl-xL, Bcl-w, Mcl-1, Al, and Boo) prevent
apoptosis; others (Bax, Bad, Bok, Bcl-xS, Bak, Bid, Bik, Bim, Krk, and Mtd) promote its
initiation [186,187].
The entire superfamily of Bcl-2-related proteins is considered to be one of the most
important classes of apoptosis-regulating gene products. Their ever-expanding list includes
both cell death antagonists and apoptosis-inducing proteins. Due to the large number
of representatives of this family described to date, they are usually divided into three
groups [188,189].
1.
Anti-apoptotic proteins Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Bfl-1 and Boo with homology in
the BH1, 2, 3 and 4 regions;
2.
Pro-apoptotic proteins Bax, Bak, Bad, Bok and Diva with homology in regions BH1,
2 and 3 but not BH4;
3.
“BH3-proteins” are pro-apoptotic proteins such as Bik, Bid, Bim, Hrk, and Blk, which
share homology exclusively in the BH3 domain.
The combined action of related cell death agonists and antagonists from the Bcl-2
family represents a regulatory switch whose function is determined, at least in part, by
selective protein-protein interactions. These proteins are characterized by the ability to
form heterodimers in which partners repress each other [190].
Proapoptotic proteins are mostly localized in the cytosol and translocate to the mito-
chondrial membrane in response to certain stimuli. Some studies claim that Bach proteins
translocate from the cytosol to the mitochondria, while others suggest that they undergo
conformational changes enhanced by interaction with Bid proteins [191].
Bid proteins are known to be hydrolyzed by caspase-8 and their C-terminal part
interacts with mitochondria. Several models have been proposed for the participation of
Antioxidants 2025,14, 108 18 of 46
Bcl-2 family proteins in the regulation of protein transfer from the mitochondrion to the
cytosol [192194].
1.
Since Bax (like other Bcl-2 family proteins) can form pores in the outer mitochondrial
membrane, it has been hypothesized that these pores might be large enough to allow
cytochrome c to exit. However, this has not yet been confirmed;
2.
There is a hypothesis suggesting that Bax interacts with VDAC, resulting in the
formation of an even larger channel capable of accommodating cytochrome C. Notably,
the conductivity of this channel is blocked by Bcl-xL [195];
3.
Bcl-2 can also form channels in the outer mitochondrial membrane that allow adenine
nucleotides to pass through. It is hypothesized that bach closes VDAC, ATP/ADP
exchange between mitochondrion and cytoplasm is disrupted, resulting in the opening
of RTR. Moreover, all these phenomena are prevented by Bcl-2 [196198];
4.
There is also a hypothesis that suggests that the Bcl-2 family protein complex inter-
acts with the giant pore complex in the inner mitochondrial membrane and leads to
membrane depolarization, mitochondrial swelling, and cytochrome C release [
199
,
200
].
The Bcl-2 protein has a direct inhibitory effect on pore opening, but it does not protect
against all permeability change inducers. Because the mitochondrial pore is regulated by
a complex that includes Bcl-2 and Bax antagonists, changes in its stoichiometry (e.g., in-
creased Bax synthesis or Bcl-2 modification) may contribute to permeability changes. It is
hypothesized that the ratio between Bcl-2 and Bax proteins, and their phosphorylation, pro-
motes either cell survival (excess