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antioxidants
Article
Neuroprotective and Neuromodulatory Effects
Induced by Cannabidiol and Cannabigerol in Rat
Hypo-E22 cells and Isolated Hypothalamus
Viviana di Giacomo 1, Annalisa Chiavaroli 1, Giustino Orlando 1,*, Amelia Cataldi 1,
Monica Rapino 2, Valentina Di Valerio 3, Sheila Leone 1, Luigi Brunetti 1, Luigi Menghini 1,
Lucia Recinella 1and Claudio Ferrante 1
1Department of Pharmacy, Universitàdegli Studi “Gabriele d’Annunzio”, via dei Vestini 31, 66100 Chieti,
Italy; viviana.digiacomo@unich.it (V.d.G.); annalisa.chiavaroli@unich.it (A.C.);
amelia.cataldi@unich.it (A.C.); sheila.leone@unich.it (S.L.); luigi.brunetti@unich.it (L.B.);
luigi.menghini@unich.it (L.M.); lucia.recinella@unich.it (L.R.); claudio.ferrante@unich.it (C.F.)
2Genetic Molecular Institute of CNR, Unit of Chieti, “G. d’ Annunzio” University, Via dei Vestini 31,
66100 Chieti-Pescara, Italy; m.rapino@unich.it
3Department of Medicine and Ageing Sciences, “G. d’ Annunzio” University, Via dei Vestini 31,
66100 Chieti-Pescara, Italy.; valentina.divalerio@unich.it
*Correspondence: giustino.orlando@unich.it; Tel.: +39-0871-355-4755
Received: 9 December 2019; Accepted: 10 January 2020; Published: 13 January 2020
Abstract:
Background: Cannabidiol (CBD) and cannabigerol (CBG) are non-psychotropic
terpenophenols isolated from Cannabis sativa, which, besides their anti-inflammatory/antioxidant
effects, are able to inhibit, the first, and to stimulate, the second, the appetite although there are no
studies elucidating their role in the hypothalamic appetite-regulating network. Consequently, the
aim of the present research is to investigate the role of CBD and CBG in regulating hypothalamic
neuromodulators. Comparative evaluations between oxidative stress and food intake-modulating
mediators were also performed. Methods: Rat hypothalamic Hypo-E22 cells and isolated tissues were
exposed to either CBD or CBG, and the gene expressions of neuropeptide (NP)Y, pro-opiomelanocortin
(POMC) and fatty acid amide hydrolase were assessed. In parallel, the influence of CBD on the
synthesis and release of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) was evaluated.
The 3-hydroxykinurenine/kinurenic acid (3-HK/KA) ratio was also determined. Results: Both CBD
and CBG inhibited NPY and POMC gene expression and decreased the 3-HK/KA ratio in the
hypothalamus. The same compounds also reduced hypothalamic NE synthesis and DA release,
whereas the sole CBD inhibited 5-HT synthesis. Conclusion: The CBD modulates hypothalamic
neuromodulators consistently with its anorexigenic role, whereas the CBG effect on the same mediators
suggests alternative mechanisms, possibly involving peripheral pathways.
Keywords:
cannabidiol; cannabigerol; hypothalamus; neurotransmitter; neuropeptide;
kinurenine pathway
1. Introduction
Cannabis sativa has long been considered as an efficacious pharmacological tool to treat a wide
plethora of diseases, including fever, malaria, constipation, menstrual disorders, pain, and rheumatism.
The plant, belonging to the Cannabaceae family and classified in the three recognized varieties (sativa,
indica and ruderalis), is characterized by the presence of numerous terpenophenolic compounds that
are responsible, at least partially, for the aforementioned effects. The main compounds present in the
phyto-complex are
∆
9-tetrahydrocannabinol (THC), which is also the sole psychotropic molecule, and
Antioxidants 2020,9, 71; doi:10.3390/antiox9010071 www.mdpi.com/journal/antioxidants
Antioxidants 2020,9, 71 2 of 14
cannabidiol (CBD), which is structurally related to the first but devoid of psychotropic activity [
1
].
In the last three decades, the study of the pharmacological properties of these phyto-compounds
have led to the identification and characterization of the endocannabinoid signaling, consisting in
arachinonic acid-deriving molecules including anandamide and 2-acylglycerole, and the related
metabotropic receptors, namely, the cannabinoid type 1 (CB1), expressed in prevalence at the presynaptic
level, and the CB2, which is present especially but not exclusively at the peripheral level [
2
]. The
endocannabinoid-mediated activation of CB1 and CB2 has long been implicated in the onset of
neuroprotective effects, whereas the neuroprotection occurring after phyto-cannabinoid administration
could be better explained by a multitarget mechanism involving different receptor systems, including
peroxisome proliferator-activated receptors (PPARs) and transient receptor potential (TRP) channels [
3
–
5
].
CB1 and CB2 receptors were considered as promising targets for the development of anti-obesity
drugs [
6
] as well, to the point that the rimonabant, the prototype of the of CB1 blockers, was clinically
employed for a short period (more than ten years ago) for its anorexigenic effects. Nevertheless,
the rimonabant was soon after retired from the market for an increased frequency in psychiatric
disorders following the treatment [
7
]. The therapeutic failure was ascribed, at least partially, to the
specific mechanism of action of the drug, whose orthosteric inverse agonism on CB1 could lead to
supra-physiological receptor alterations [
8
]. To this regard, the CBD could represent an innovative
pharmacological approach: even though it is described as a CB1 ligand, the CBD was reported
to have a low affinity for the CB1 orthosteric site [
9
], and this could be considered as one of the
main factors influencing the lack of psychiatric symptoms following the administration
in vivo
[
10
].
Additionally, Laprairie and colleagues [
11
] suggested that the cannadidiol could act as a negative
allosteric modulator rather than an orthosteric ligand. CB1 negative allosteric modulators are molecules
devoid of intrinsic receptor activity, which depends solely on the presence of the endogenous ligands,
i.e., the endocannabinoids [
8
,
12
]. Considering that the brain level of endocannabinoids is upregulated
in obese mice [
13
], the CBD could reduce, without annulling, the endocannabinoid-stimulated CB1
signaling, thus restoring the physiological activity of the endocannabinoid system [
11
]. Another possible
anti-obesity target is the CB2 receptor. Ignatowska-Jankowska and colleagues [
14
] demonstrated
that the anorexigenic effect following CBD administration could depend on the activation of the
hypothalamic CB2 pool. Therefore, the anorexigenic effect exerted by the CBD could be the result
of a multitarget mechanism, involving the whole endocannabinoid receptor system, particularly in
the hypothalamus. Nevertheless, the potential influence of the cannabidiol on the hypothalamic
appetite-regulating network, including the involvement of neuropeptides and neurotransmitters long
considered as central transducers of peripheral appetite and satiety signals [
15
], has not been studied up
to now. Consequently, the aim of the present research is to investigate the role of the CBD in regulating
the levels of the hypothalamic peptides and neurotransmitters involved in feeding control, isolated rat
hypothalamus, and the hypothalamic Hypo-E22 cell line. Additionally, taking into consideration many
studies reporting the appetite-stimulating effects of another terpenophenol present in the C. sativa
phyto-complex, that is cannabigerol (CBG) [
16
,
17
], which was also demonstrated to act as a negative
allosteric modulator of CB1, [
11
], an evaluation of CBG effects on the experimental paradigms was
performed as well.
2. Materials and Methods
2.1. Drugs
The crystals of CBD and CBG (99% purity, 1% terpene fraction), derived from concentrated extracts
of C. sativa, were kindly provided by Enecta B.V. (Corantijnstraat 5–1, 1058DA Amsterdam, Netherlands).
The mother solutions (30 mM) were prepared in dimethylsulfoxide (DMSO) and sterilized with 0.22
µ
m
Millipore filters in sterility conditions (laminar flow hood). Afterwards, drug solutions were stepwise
diluted in Dulbecco’s modified Eagle’s medium (DMEM) for the bio-pharmacological assays, as
described below.
Antioxidants 2020,9, 71 3 of 14
2.2. In Vitro Studies
The Hypo-E22 rat hypothalamus cell line was purchased from Cedarlane Corporation (Burlington,
ON, Canada) and cultured in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum
and penicillin-streptomycin (100
µ
g/mL) (all from EuroClone SpA Life-Sciences-Division, Milano, Italy).
Cells were grown at 37
◦
C in a humified atmosphere of 5% CO
2
. When indicated, the cells were treated
with H
2
O
2
300
µ
M for 3 h and different concentrations of CBD and CBG (1, 10, 100, 1000 nM). The cell
viability was evaluated after 24 and 48 h of culture by MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) growth assay (Sigma–Aldrich, St. Louis, MO, USA), based on the capability
of viable cells to reduce MTT into a colored formazan product. The cells were seeded into 96-well
plates at 5
×
10
3
cells/well. At the established time points, the medium was replaced with a fresh one
containing 0.5 mg/mL MTT, and the cells were incubated for 3 h at 37
◦
C. After a further incubation of
the samples in DMSO for 30 min at 37
◦
C the absorbance at 570 nm was measured using a Multiscan
GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The values obtained
in the absence of cells were considered as background and subtracted from the optical density values of
the samples. Three independent experiments were performed under the same experimental conditions.
For the HPLC analyses, Hypo-E22 cells were seeded in 6-well plates at 10
5
cells/well. After 24 h of
exposure to CBD 1000 nM and CBG 1 nM, 300
µ
L of medium for each experimental condition were
collected and stored at −20◦C.
2.3. Ex Vivo Studies
Twenty-four male adult Sprague–Dawley rats (200–250 g) were housed in Plexiglass cages (40
×
25
×
15 cm), two rats per cage, in climatized colony rooms (22
±
1
◦
C; 60% humidity), on a 12 h/12 h
light/dark cycle (light phase: 07:00–19:00 h), with free access to tap water and food, 24 h/day throughout
the study, with no fasting periods. Rats were fed a standard laboratory diet (3.5% fat, 63% carbohydrate,
14% protein, 19.5% other components without caloric value; 13.39 kJ/g). Housing conditions and
experimentation procedures were strictly in accordance with the European Union ethical regulations
on the care of animals for scientific research. The experimental paradigm was approved by the Local
Ethical Committee (University “G. d’Annunzio” of Chieti-Pescara, Chieti-Pescara, Italy) and the Italian
Health Ministry (Authorization N. F4738.N.XTQ, delivered on 11 Novembre 2018). Specifically, rats
were sacrificed by CO
2
inhalation (100% CO
2
at a flow rate of 20% of the chamber volume per min) and
hypothalami were immediately collected and maintained in humidified incubator with 5% CO
2
at 37
◦
C
for 4 h (incubation period), in DMEM enriched with CBD (1000 nM) or CBG (1 nM). Afterwards, the
hypothalamic samples were subjected to analytical procedure for gene expression and biogenic amine
level assessment, as described in the following paragraphs. The samples (n=12) for the determination
of biogenic amines were dissected in 1 mL of a perchloric acid solution (50 mM) and filtered (PTFE
0.45
µ
m), whereas the hypothalami (n=12) intended to be used for gene expression analysis were
dissected and stored in RNAlater solution (Ambion, Austin, TX, USA) at −20 ◦C.
2.4. RNA Extraction, Reverse Transcription and Real-Time Reverse Transcription Polymerase Chain Reaction
(Real-Time RT PCR)
Total RNA was extracted from the hypothalamus using TRI Reagent (Sigma–Aldrich, St. Louis,
MO, USA), as previously reported [
18
]. Contaminating DNA was removed using 2 units of RNase-free
DNase 1 (DNA-free kit, Ambion, Austin, TX, USA). The RNA concentration was quantified at 260 nm
by spectrophotometer reading (BioPhotometer, Eppendorf, Hamburg, Germany) and its purity was
assessed by the ratio at 260 and 280 nm readings. The quality of the extracted RNA samples was
also determined by electrophoresis through agarose gels and staining with ethidium bromide, under
UV light. One microgram of total RNA extracted from each sample in a 20
µ
L reaction volume was
reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific
Inc., Monza, Italy). Reactions were incubated in a 2720 Thermal Cycler (Thermo Fisher Scientific Inc.,
Monza, Italy) initially at 25
◦
C for 10 min, then at 37
◦
C for 120 min, and finally at 85
◦
C for 5 s. Gene
Antioxidants 2020,9, 71 4 of 14
expression was determined by quantitative real-time PCR using TaqMan probe-based chemistry. PCR
primers and TaqMan probes, including
β
-actin used as the housekeeping gene, were purchased from
Thermo Fisher Scientific Inc. (Assays-on-Demand Gene Expression Products, Rn00595020_m1 for
pro-opiomelanocortin (POMC) gene, Rn00561681_m1 for neuropeptide (NP)Y gene, Rn00577086_m1
for fatty acide amide hydrolase (FAAH) gene). The real-time PCR was carried out in triplicate for each
cDNA sample in relation to each of the investigated genes. Data were elaborated with the Sequence
Detection System (SDS) software version 2.3 (Thermo Fisher Scientific Inc.). Gene expression was
relatively quantified by the comparative 2−∆∆Ct method [19].
2.5. High Performance Liquid Chromatography (HPLC) Determination of Dopamine (DA), Norepinephrine
(NE),Serotonin (5-HT), and 3-Hydroxykinurenine (3-HK)
Tissue and extracellular DA, 5-HT and NE levels were analyzed through an HPLC apparatus
consisting of a Jasco (Tokyo, Japan) PU-2080 chromatographic pump and an ESA (Chelmsford, MA,
USA) Coulochem III coulometric detector, equipped with a microdialysis cell (ESA-5014b) porous
graphite working electrode and a solid state palladium reference electrode. The analytical conditions
for biogenic amine identification and quantification were selected according to a previous study [
20
].
Briefly, the analytical cell was set at
−
0.150 V for detector 1 and at +0.300 V for detector 2, with a
range of 100 nA. The chromatograms were monitored at the analytical detector 2. Integration was
performed by Jasco Borwin Chromatography software version 1.5. The chromatographic separation
was performed by isocratic elution on a Phenomenex Kinetex reverse phase column (C18, 150
×
4.6 mm i.d., 2.6
µ
m). As regards the separation of DA, NE and 5-HT, the mobile phase was (10:90, v/v)
acetonitrile and 75 mM pH 3.00 phosphate buffer containing octanesulfonic acid 1.8 mM, EDTA 30
µ
M
and triethylamine 0.015% v/v. The mobile phase for 3-HK analysis consisted of 1.5% acetonitrile, 0.9%
triethylamine, 0.59% phosphoric acid, 0.27 mM EDTA, and 8.9 mM octanesulfonic acid. Flow rate was
0.6 mL/min and the samples were manually injected through a 20
µ
l loop. Neurotransmitter peaks were
identified by comparison with the retention time of pure standard. Neurotransmitter concentrations in
the samples were calculated by linear regression curve (y=bx+m) obtained with standard. Neither
internal nor external standard were necessary for neurotransmitter quantification in the hypothalamus
homogenate, and all tests performed for method validation yielded results in accordance with limits
indicated in official guidelines for applicability in laboratory trials. The standard stock solutions of
DA, NE, and 5-HT at 2 mg/mL were prepared in bidistilled water containing 0.004% EDTA and 0.010%
sodium bisulfite. The stock solutions were stored at 4
◦
C. Work solutions (1.25–20.00 ng/mL) were
obtained daily by progressively diluting the stock solutions in the mobile phase.
2.6. HPLC-Fluorimetric Determination of Kinurenic Acid (KA)
The KA quantitative determination in the cell medium was carried out on a reversed phase
HPLC-fluorimeter in agreement with the method employed by Pocivavsek and colleagues [
21
]. Analyses
were performed by using a liquid chromatograph (MOD. 1525, Waters Corporation, Milford, MA,
USA) equipped with a fluorimetric detector (MOD. 2475, Waters Corporation), a C18 reversed-phase
column (AcclaimTM 120, 3
µ
m, 2.1
×
100 mm, Dionex Corporation, Sunnyvale, CA, USA), and an
on-line degasser (Biotech 4-CH degasi compact, LabService, Anzola Emilia, Italy). The separation
was conducted in isocratic conditions and the mobile phase consisted of 250 mM zinc acetate, 50 mM
sodium acetate, and 3% acetonitrile (pH adjusted to 6.2 with glacial acetic acid), using a flow rate of
1.0 mL/min. In the eluate, the KA was identified and measured fluorimetrically (excitation: 344 nm;
emission: 398 nm).
2.7. Statistical Analysis
Statistical analysis was carried out through GraphPad Prism version 5.01 for Windows (GraphPad
Software, San Diego, CA, USA). Means
±
S.D. were determined for each experimental group and
analyzed by one-way analysis of variance (ANOVA), followed by Newman–Keuls comparison multiple
Antioxidants 2020,9, 71 5 of 14
test. Statistical significance was set at p<0.05. As regards the animals employed for the experiments,
their number per condition (n=4) was calculated with the software G*Power (v3.1.9.4, UCLA,
Los Angeles, CA, USA). The values of study potency (1-
β
) and significance level (
α
) were 0.8 and
0.05, respectively.
3. Results
Cannabidiol and cannabigerol show no significant effect on Hypo-E22 proliferation when they are
added to the cell medium at different concentrations for 24 h (Figure 1A). On the contrary, the MTT
assay at 48 h shows that, whereas the CBD 1000 nM has an OD value similar to the control, the lower
CBD concentrations (1, 10, 100 nM) and all the CBG concentrations appear to induce a slight decrease
in cell viability, even though it is not always significant. On the other hand, when the hypothalamic
cell line is challenged with hydrogen peroxide (Figure 1B), its viability is markedly reduced (0.32
±
0.01
H
2
O
2
vs. 0.93
±
0.06 ctrl 24 h and 1.00
±
0.04 H
2
O
2
vs. 1.56
±
0.05 ctrl 48 h). The addition of the two
substances to the cell culture results in an improved viability for all the experimental points. Both after
24 and 48 h of exposure, CBD and CBG are able to protect the cells from the oxidative stress induced by
the hydrogen peroxide. For the subsequent analyses, two concentrations were chosen, namely, 1000 nM
for CBD, having shown the best recover from the H
2
O
2
damage at both 24 h (0.76
±
0.10 vs. 0.32
±
0.01
of the H
2
O
2
sample) and 48 h (1.32
±
0.01 vs. 1.00
±
0.04 of the H
2
O
2
sample) and 1 nM for CBG, being
the lowest concentration to give the highest cell viability (0.73 ±0.02 at 24 h and 1.21 ±0.08 at 48 h).
To better evaluate the effect of cannibidiol and cannabigerol on the Hypo-E22 cells in their basal
state, the detection of the extracellular release of 3-HK and KA was performed. The ratio 3-HK/KA,
a well-known index of neurotoxicity, was considerably reduced following CBD and CBG treatment
(Figure 2), with a higher efficacy found when the cells were exposed to CBD (0.08
±
0.014 vs. 0.17
±
0.015
of the CBG group). The stimulation of isolated rat hypothalamus with CBD 1000 nM and CBG 1 nM
led to significant alterations in the expression pattern of the genes NPY and POMC, with a significant
reduction (p<0.0001) of their mRNA level (Figure 3) compared to control-treated group. On the other
hand, both compounds revealed ineffective in altering the gene expression of FAAH (Figure 3). In
parallel, a significant inhibition (p<0.0001) of NE steady state level was observed (Figure 4A) when
the hypothalami were exposed both to CBD and CBG. Conversely, a null effect was observed on DA
concentration (Figure 4B), whereas only CBD proved able to increase the hypothalamic level of 5-HT,
following the 4 h treatment (Figure 4C; p<0.001). As for the effects of the two compounds on the
modulation of the biogenic amine release from Hypo-E22 cells, both CBD and CBG decreased the DA
level (p<0.001) without affecting NE and 5-HT extracellular concentrations (Figure 5).
Antioxidants 2020,9, 71 6 of 14
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Figure 1. MTT assay of hypothalamic Hypo-E22 cells exposed to different concentrations (1–1000 nM)
of either cannabidiol (CBD) or cannabigerol (CBG) for 24 and 48 h. (A) Cells in basal conditions. (B)
Cells challenged with 300 µM H
2
O
2
. ANOVA, p < 0.001; ** p < 0.01, * p < 0.05 vs. control group.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
24 h
48h
MTT
* * *
CB
D
CB
G
CB
G
CB
D
A
ABS
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
24h
48h
CB
D
CB
G
CB
G
CB
D
**
** *
B
**
ABS
Figure 1.
MTT assay of hypothalamic Hypo-E22 cells exposed to different concentrations (1–1000 nM)
of either cannabidiol (CBD) or cannabigerol (CBG) for 24 and 48 h. (
A
) Cells in basal conditions. (
B
)
Cells challenged with 300 µM H2O2. ANOVA, p<0.001; ** p<0.01, * p<0.05 vs. control group.
Antioxidants 2020,9, 71 7 of 14
Antioxidants 2019, 8, x FOR PEER REVIEW 7 of 14
Figure 2. Inhibitory effects induced by cannabidiol (CBD) 1000 nM and cannabigerol (CBG) 1 nM on
extracellular 3-hydroxykinurenine/kynurenic acid (3-HK/KA) ratio in hypothalamic Hypo-E22 cells.
ANOVA, p < 0.001; ** p < 0.01, * p < 0.05 vs. control group.
Figure 3. Inhibitory effects induced by cannabidiol (CBD) 1000 nM and cannabigerol (CBG) 1 nM on
neuropeptide (NP)Y and pro-opimelanocortin (POMC) gene expression (Relative Quantification:
R.Q.), in isolated rat hypothalamus. ANOVA, p < 0.0001; *** p < 0.001, ** p < 0.01 vs. control group.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Control CBD 1000 nM CBG 1 nM
3-HK/KA ratio
0
0.2
0.4
0.6
0.8
1
1.2
Control CBD 1000 nM CBG 1 nM
Gene Expression (R.Q.)
NPY POMC FAAH
***
**
**
*
Figure 2.
Inhibitory effects induced by cannabidiol (CBD) 1000 nM and cannabigerol (CBG) 1 nM on
extracellular 3-hydroxykinurenine/kynurenic acid (3-HK/KA) ratio in hypothalamic Hypo-E22 cells.
ANOVA, p<0.001; ** p<0.01, * p<0.05 vs. control group.
Antioxidants 2019, 8, x FOR PEER REVIEW 7 of 14
Figure 2. Inhibitory effects induced by cannabidiol (CBD) 1000 nM and cannabigerol (CBG) 1 nM on
extracellular 3-hydroxykinurenine/kynurenic acid (3-HK/KA) ratio in hypothalamic Hypo-E22 cells.
ANOVA, p < 0.001; ** p < 0.01, * p < 0.05 vs. control group.
Figure 3. Inhibitory effects induced by cannabidiol (CBD) 1000 nM and cannabigerol (CBG) 1 nM on
neuropeptide (NP)Y and pro-opimelanocortin (POMC) gene expression (Relative Quantification:
R.Q.), in isolated rat hypothalamus. ANOVA, p < 0.0001; *** p < 0.001, ** p < 0.01 vs. control group.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Control CBD 1000 nM CBG 1 nM
3-HK/KA ratio
0
0.2
0.4
0.6
0.8
1
1.2
Control CBD 1000 nM CBG 1 nM
Gene Expression (R.Q.)
NPY POMC FAAH
***
**
**
*
Figure 3.
Inhibitory effects induced by cannabidiol (CBD) 1000 nM and cannabigerol (CBG) 1 nM
on neuropeptide (NP)Y and pro-opimelanocortin (POMC) gene expression (Relative Quantification:
R.Q.), in isolated rat hypothalamus. ANOVA, p<0.0001; *** p<0.001, ** p<0.01 vs. control group.
Conversely, CBD and CBG exerted a null effect on fatty acid amide hydrolase (FAAH) gene expression.
Antioxidants 2020,9, 71 8 of 14
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Conversely, CBD and CBG exerted a null effect on fatty acid amide hydrolase (FAAH) gene
expression.
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
Control CBD 1000 nM CBG 1 nM
NE (ng/mg wet tissue)
**
*
A
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Control CBD 1000 nM CBG 1 nM
DA (ng/mg wet tissue)
B
Figure 4. Cont.
Antioxidants 2020,9, 71 9 of 14
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Figure 4. Effects of cannabidiol (CBD) 1000 nM and cannabigerol (CBG) 1 nM on norepinephrine (NE),
dopamine (DA), and serotonin (5-HT) levels (ng/mg wet tissue) in isolated rat hypothalamus. (A)
CBD and CBG inhibited NE level in the hypothalamus. ANOVA, p < 0.0001; *** p < 0.001, ** p < 0.01
vs. control group. (B) Neither CBD nor CBG influenced DA level, in the hypothalamus. (C)
Conversely, CBD and CBG stimulated hypothalamic 5-HT level. ANOVA, p < 0.001; ** p < 0.01 vs.
control group.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
Vehicle CBD 1000 nM CBG 1 nM
5-HT (ng/mg wet tissue)
** C
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control CBD 1000 nM CBG 1 nM
Extracellular neurotransmitter level (ng/mL)
NE DA 5-HT
** **
Figure 4.
Effects of cannabidiol (CBD) 1000 nM and cannabigerol (CBG) 1 nM on norepinephrine (NE),
dopamine (DA), and serotonin (5-HT) levels (ng/mg wet tissue) in isolated rat hypothalamus. (
A
) CBD
and CBG inhibited NE level in the hypothalamus. ANOVA, p<0.0001; *** p<0.001, ** p<0.01 vs.
control group. (
B
) Neither CBD nor CBG influenced DA level, in the hypothalamus. (
C
) Conversely,
CBD and CBG stimulated hypothalamic 5-HT level. ANOVA, p<0.001; ** p<0.01 vs. control group.
Antioxidants 2019, 8, x FOR PEER REVIEW 9 of 14
Figure 4. Effects of cannabidiol (CBD) 1000 nM and cannabigerol (CBG) 1 nM on norepinephrine (NE),
dopamine (DA), and serotonin (5-HT) levels (ng/mg wet tissue) in isolated rat hypothalamus. (A)
CBD and CBG inhibited NE level in the hypothalamus. ANOVA, p < 0.0001; *** p < 0.001, ** p < 0.01
vs. control group. (B) Neither CBD nor CBG influenced DA level, in the hypothalamus. (C)
Conversely, CBD and CBG stimulated hypothalamic 5-HT level. ANOVA, p < 0.001; ** p < 0.01 vs.
control group.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
Vehicle CBD 1000 nM CBG 1 nM
5-HT (ng/mg wet tissue)
** C
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control CBD 1000 nM CBG 1 nM
Extracellular neurotransmitter level (ng/mL)
NE DA 5-HT
** **
Figure 5.
Inhibitory effects induced by cannabidiol (CBD) 1000 nM and cannabigerol (CBG) 1 nM on
extracellular dopamine (DA) levels (ng/mL), in hypothalamic Hypo-E22 cells. ANOVA, p<0.001;
** p<0.01 vs. control group. Conversely, CBD and CBG exerted a null effect on extracellular
norepinephrine (NE) and serotonin (5-HT) levels.
Antioxidants 2020,9, 71 10 of 14
4. Discussion
The protective effects exerted by the two metabolites of C. sativa (CBD and CBG) on hypothalamic
cells challenged with H
2
O
2
, as demonstrated by the MTT assay results, are confirmed by the modulation
of the release of kynurenine metabolites by the same cells. The 3-HK and KA are key products of the
kynurenine pathway, which represents, together with the 5-HT pathway, the two main tryptophan
degradative systems [
22
]. Additionally, tissue and plasma levels of these two molecules are well
known to be related to inflammatory and oxidative stress conditions in both peripheral and central
tissues [
23
–
25
]. Specifically, the 3-HK/KA is a reliable marker of neurotoxicity [
26
], and our findings
of reduction of this ratio from Hypo-E22 cells after pharmacological treatment further support the
neuroprotective role exerted by both CBD and CBG. Feeding behavior and energy balance are finely
modulated in the hypothalamus, which has long been considered a cornerstone in this process [
27
].
In this region of the brain, neuropeptides, including NPY and POMC, and biogenic amines, namely
DA, NE, and 5-HT, act as central transducers of peripheral short- and long-term satiety signals [
15
].
Although the role of NPY as central appetite stimulant is well-established [
15
], the involvement of
POMC is still controversial, having this neuropeptide demonstrated both stimulatory and inhibitory
effects, depending on the post-transcriptional pathway activation, that could lead to the production of
both the anorexigenic
α
-melanocyte stimulating hormone (
α
-MSH) and the orexigenic
β
-endorphin
(
β
-END) [
28
]. In this regard, Koch and colleagues [
29
] suggested that cannabinoid-induced feeding
could be mediated by increased levels of the POMC-derived peptide
β
-endorphin. Actually, the
inhibition of the POMC gene expression following CBD and CBG treatment is consistent with their
putative role as negative allosteric modulators of CB1 receptor [
30
]. In contrast, the inhibition of
the expression of the NPY gene registered after the treatment, although being consistent with the
anorexigenic effects ascribed to CBD [
6
], appears to be discrepant with the orexigenic role of CBG [
16
,
17
].
This result is, however, in agreement with the expression of the CB1 receptor on synaptic endings
innervating both NPY and POMC first order neurons in the hypothalamus [
29
,
31
]. Interestingly, the
anorexigenic effects induced by bisphenol A in mice were followed by the concomitant reduction and
stimulation of CB1 and cocaine and amphetamine-regulating transcript (CART) peptide gene expression,
respectively; thus, further highlighting the importance of hypothalamic arcuate nucleus first order
neurons as key targets of the anti-obesity effects of CB1-modulating compounds [
32
]. Nevertheless,
the study performed by Merroun and colleagues [
33
] suggested the lateral hypothalamus-derived
orexin A as a mediator of the anorexigenic effects induced by CB1 antagonist AM251 as well [
33
]. The
anorexigenic effects of CBD were also related to CB2 receptor activation [
14
], whilst the cannabigerol
proved to challenge brain
α
2-adrenoceptor [
34
], whose activation is well known to be related to a
feeding stimulating effect [
35
]. Another parameter investigated after CBD and CBG treatment was the
gene expression of FAAH, a key enzyme known for being able to stimulate food intake and notoriously
involved in the degradation of endocannabinoids such as anandamide [
7
]. The null effect on FAAH gene
expression registered after the exposure to CBD and CBG is consistent with their low potency as FAAH
inhibitors [
36
], thus excluding, in this experimental system, the direct involvement of endocannabinoid
levels in mediating the observed modulatory effects in the hypothalamic appetite-regulating network.
The steady state levels of DA, NE, and 5-HT in isolated hypothalamus, were assayed after the
challenging with CBD and CBG. The role of DA on neuroendocrine control of food intake is still
a matter of debate, being dependent on the hypothalamic site of administration for the result in
stimulation or inhibition [
37
]. However, mesolimbic dopaminergic pathways seem to be involved
in the reward underlying the ingestion of palatable foods, thus suggesting a stimulating effect on
appetite [
38
]. Hypothalamic NE is involved in feeding regulation as well, with a role in inhibiting
or stimulating the food intake mediated by
α
1- or
α
2-adrenoceptors, respectively, depending on the
circadian alteration in the
α
1/
α
2 ratio [
35
]. Although multiple studies suggest the inhibition of the
monoamine release as a possible mechanism of action of peripheral anorexigenic hormones [
18
,
39
,
40
],
central 5-HT is well known to reduce appetite and increase energy expenditure [
41
], while its release in
the hypothalamus could be increased by peripheral anorexigenic hormones [
42
]. Having both CBD and
Antioxidants 2020,9, 71 11 of 14
CBG demonstrated their effectiveness in reducing NE steady state level in isolated rat hypothalamus,
this monoamine can be suggested as a possible mediator of the effects of the two terpenophenols
on food intake. In a previous study, the RVD-hemopressin-
α
, an endogenous anorexigenic peptide,
proved to be a negative allosteric modulator of CB1 [
43
] and to inhibit hypothalamic NE levels
following peripheral administration despite being ineffective against DA and 5-HT levels [
30
]. The
two terpenophenols objects of this study were also ineffective against the DA level, whereas the sole
CBD stimulated 5-HT levels, and this could explain, albeit partially, the aforementioned anorexigenic
effects [
14
]. The evaluation of the biogenic amine steady state level is currently considered a useful
tool to predict the effect of a drug on the activity in the brain, particularly in
in vivo
studies [
44
,
45
].
Nevertheless, other experimental paradigms, such as microdialysis/push-pull, synaptosome perfusion,
and cell cultures can give a more detailed assessment with regard to the effects of drugs on brain
neurotransmitter release. In order to better elucidate a possible direct effect on aminergic signaling, the
hypothalamic cell line Hypo-E22 was exposed to CBD and CBG to evaluate their influence on DA,
NE, and 5-HT release. Since both CBD and CBG were able to reduce extracellular DA level in the cell
line while there was no change in isolated rat hypothalamus, an inhibitory effect on neurotransmitter
release, possibly from the ready-releasable vesicles, can be hypothesized. Conversely, the null effect of
CBD on 5-HT release in the Hypo-E22 indicates that the increased 5-HT level in isolated hypothalamus,
after CBD treatment, could happen through a stimulating effect on neurotransmitter synthesis. It
should also be highlighted that the hypothalamic 5-HT level tended to increase, without resulting in
significant, after CBG exposure. Intriguingly, both stimulating effects on 5-HT level were paralleled by a
significant decrease in the 3-HK/KA ratio. Considering that pro-inflammatory conditions could activate
the kinurenine pathway, thus leading to increased 3-HK/KA and 5-HT turnover [
46
], the inverse trend
observed in the rat hypothalamus following CBD and CBG treatment suggests tryptophan degradative
pathways as potential targets underlying the pharmacological effects of these compounds. In order
to validate this hypothesis, a deepening further study is required to investigate the effects of CBD
and CBG on kinurenine and tryptophan levels and the expression of the enzymes involved in the
respective biochemical pathways.
5. Conclusions
Collectively, the present results indicate a modulation induced by both CBD and CBG on the
hypothalamic neuropeptides and neurotransmitters playing a master role in feeding behavior. The
increased 5-HT level, the reduced NPY and POMC gene expression, the NE tissue level, and the DA
release are consistent with the anorexigenic role of CBD, after peripheral administration [
14
]. The
CBG, on the contrary, besides being ineffective in modulating the hypothalamic 5-HT level, showed a
pharmacological profile against the other tested neuromodulators that is very close to that of CBD.
In this context, the involvement of other mechanisms and mediators not included in the present
research could also explain the observed effects of CBG on orexigenic molecule balance following
peripheral administration. Therefore, further investigations are needed to elucidate the effects of these
compounds in the neuroendocrine mechanisms of feeding behavior, possibly taking into consideration
the involvement of peripheral signals as well.
Author Contributions:
Conceptualization, G.O., A.C. (Amelia Cataldi) and L.B.; methodology, C.F., V.d.G.;
software, L.M.; validation, C.F., L.M.; formal analysis, V.d.G., C.F.; investigation, A.C. (Annalisa Chiavaroli),
M.R., V.d.V., L.R., S.L.; resources, G.O.; data curation, V.d.G., C.F., G.O.; writing—original draft preparation, C.F.,
V.d.G.; writing—review and editing, A.C. (Amelia Cataldi), V.d.G., C.F., G.O.; visualization, L.B.; supervision, A.C.
(Amelia Cataldi), L.B.; project administration, V.d.G., G.O., C.F.; funding acquisition, V.d.G., A.C. (Amelia Cataldi),
A.C. (Annalisa Chiavaroli), G.O., C.F. All authors have read and agreed to the published version of the manuscript.
Funding:
This study was entrusted by Enecta B.V. (Corantijnstraat 5–1, 1058DA Amsterdam, The Netherlands)
within the project entitled “Effects of cannabidiol and cannabigerol on the neuroendocrine mechanisms underlying
feeding behavior and energy balance” (Coordinators: Claudio Ferrante, Giustino Orlando and Luigi Menghini).
This study was also supported by the following funds: (1) “G. d’Annunzio” Foundation fund granted to Claudio
Ferrante. (2) Italian Ministry of University funds (FAR 2019) granted to Viviana di Giacomo, Amelia Cataldi,
Annalisa Chiavaroli and Giustino Orlando.
Antioxidants 2020,9, 71 12 of 14
Conflicts of Interest: The authors declare no conflict of interest.
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