Cannabis and Cannabinoid Research

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ª Mary Ann Liebert, Inc. scan code to access this article
DOI: 10.1089/can.2021.0240 and other resources online.

Effects of Cannabidiol Interactions with CYP2R1,

CYP27B1, CYP24A1, and Vitamin D3 Receptors
on Spatial Memory, Pain, Inflammation, and Aging
in Vitamin D3 Deficiency Diet-Induced Rats
Mahendra Kumar Trivedi,1 Sambhu Mondal,2 Mayank Gangwar,2 and Snehasis Jana2,*
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Abstract
Introduction: The study was planned to investigate memory-enhancing, anti-inflammatory, and antiaging
potential of cannabidiol (CBD) on vitamin D3 deficient diet (VDD)-induced rats.
Materials and Methods: Cytochrome P-450 enzymes were analyzed by RT-PCR method and others biomarkers
by enzyme-linked immunosorbent assay.
Results: CYP2R1 and CYP27B1-mRNA were significantly increased by 39.29 and 38.37%, respectively, while;
CYP24A1-mRNA was significantly reduced by 21.39% compared to VDD. Vitamin D3 receptor protein expression
was significantly increased by 148.3%, 60.48%, and 142.03% in liver, kidney, and brain, respectively, compared to
VDD group. Vitamin D3 metabolites and serotonin were significantly increased more than 60% and 100%, respec-
tively, compared to VDD. Spatial memory (in terms of total distance, escape latency) and pain score were im-
proved compared to VDD. Cytokines were significantly reduced than VDD. Besides, levels of superoxide
dismutase (49.61%), glutathione peroxidase (178.87%), acetylcholine (25.40%), and klotho (145.57%) were signif-
icantly increased than VDD.
Conclusions: Study findings supported that CBD interacts with CYP2R1, CYP27B1, CYP24A1, and vitamin D recep-
tors, resulting in increased vitamin D3 metabolites, which improved memory, pain tolerance, reduced inflammation,
and aging through modulating antioxidative enzymes, cytokines, and neurotransmitters in VDD-induced rats.
Keywords: cannabidiol; CYP-450-mRNA; Morris water maze; VDR; vitamin D3 deficiency diet; vitamin D3 metabolites

Introduction dementia,3 impaired motor functions,4 and aging.5

There is growing awareness of vitamin D3’s importance
in preventing disease and optimizing health worldwide.
Sufficient vitamin D3 is necessary for the optimal func-
tioning of tissues and cells. Since the mid-1980s, an
enormous body of research has accumulated about vi-
tamin D3’s role in preventing a wide range of illnesses.1
There is strong epidemiological evidence that vitamin
D deficiency strikes people of all ages, regardless of geo-
graphic location or seasonal changes, in most parts
of the world.2 The results of prospective studies and
meta-analyses demonstrate that low levels of vitamin
D3 can increase the risk of cognitive impairment and
Cannabidiol (CBD) is one of the main nonpsychotomi-
metic phytochemicals present in Cannabis sativa. CBD
plays various pharmacological roles such as analgesic
and anti-inflammatory by inhibiting the cyclooxyge-
nase and lipoxygenase enzymes in the body.6
Moreover, various scientific studies reported its prop-
erties as anxiolytic, antipsychotic, antiemetic, and neuro-
protective antioxidant that helps in elaborating its use
as a drug.7,8 In the field of mental health, the use of can-
nabis and its nonintoxicating component CBD has
been increased these days in the treatment for neurode-
velopmental disorders, such as autism spectrum

1
Trivedi Global, Inc., Henderson, Nevada, USA.
2
Trivedi Science Research Laboratory Pvt. Ltd., Thane, India.

*Address correspondence to: Snehasis Jana, PhD, Trivedi Science Research Laboratory Pvt. Ltd., Thane 400604, India, E-mail: publication@trivedisrl.com

1
 2 TRIVEDI ET AL.
Graphical Abstract
Downloaded by Liz Hughston from www.liebertpub.com at 04/26/22. For personal use only.
disorder (ASD).9 The other conditions in which CBD
has already been trialed are improving spasticity,10
treatment of pain, sleep disturbances,11 and mobility12
in multiple sclerosis (MS); and anxiety symptoms in
social phobia.13 Some studies also suggest that CBD
may help in reducing seizure frequency in epileptic
syndromes associated with autistic symptoms such as
Dravet Syndrome and Lennox–Gastaut syndrome14,15;
and it was also found to improve ASD-like social defi-
cits in a mouse model of Dravet Syndrome.16 CBD
acts as an agonist at prefrontal 5-hydroxy tryptamine
1A (5-HT1A) receptors, where it suppresses glutamate
and gamma-aminobutyric acid (GABA) transmission.17
In this experiment, the vitamin D3 deficiency
diet (VDD) model was selected to induce impair-
ment of memory functions, inflammation, and aging
in Sprague– Dawley rats. Vitamin D3 is an anti-
inflammatory and neuroprotective action against neu-
rodegenerative and autoimmune diseases.18 Therefore,
we hypothesized that CBD could work on the cyto-
chrome P-450 (CYPs), CYP2R1, CYP24A1, CYP27B1,
and vitamin D3 receptors (VDRs) pathways that
might increase the levels of vitamin D3 active metabo-
lite (1,25-dihydroxy vitamin D3) responsible for the
improvement of effectors pharmacological functions.
Hence, in this experiment administered CBD in
VDD-induced rats to restore disruptive pharmacologi-
cal responses. However, there is no study to observe
the effects of CBD on VDD animals until now, and
the relationship between VDD-induced chronic pain,
inflammation, cognition impairment, and aging is
still unknown. Therefore, we hypothesized that CBD
treatment can modulate the vitamin D3 levels to im-
prove VDD-induced impairment of learning and mem-
ory, pain, inflammation, oxidative stress parameter, and
aging biomarker, the klotho protein level in the brain.
Materials and Methods
Chemicals and reagents
CBD (99.9%) was obtained from Standard Hemp Com-
pany. Standard normal chow diet (type-1324, batch/lot
no. 110118//0640) and vitamin D3 deficient diet (VDD;
type-C 1017, batch/lot no. 481/2017) were obtained
from Altromin Spezialfutter GmbH & Co. Germany.
Animals and VDD model
Adult 72 male Sprague–Dawley rats (age 10–12 weeks;
weight 250–300 g; Vivo Bio-Tech, India) were used in
this study. Experiments were conducted under the
Committee for the Purpose of Control and Supervision
 CBD IMPROVES MEMORY, REDUCES BRAIN INFLAMMATION AND AGING
of Experiment on Animals (CPCSEA) guidelines. The
test facility (Dabur Research Foundation, India) was
registered (Registration No. 64/PO/br/s/99/CPCSEA)
for the experiment of animals with the CPCSEA,
Ministry of Environment and Forest, Govt. of India.
The Institutional Animal Ethical Committee (IAEC)
approved the study protocol (IAEC/41/506) dated Jan-
uary 17, 2018. The rats were housed under environ-
mentally controlled conditions in a 12 h light/dark
cycle at 25C and were provided with food and
water. Efforts were made to reduce animal suffering
and minimize the number of animals used for these
experiments. Except for normal control (NC), all the
animals were fed with VDD for 3 weeks. Three weeks
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after initiating VDD induction, a circulatory level of
25-hydroxy vitamin D3 was estimated (data not shown).
Reduction of the level of 25-hydroxy vitamin D3 to
about 50–60% in the disease control group (VDD)
compared to NC animals was considered as VDD-
induced animals and was selected for further treatment.
Experimental
Thirty-six adult rats were randomly assigned into six
groups (n = 6) for behavioral study and 36 rats were
assigned into six groups (n = 6) for biochemical assay.
The current study design included NC with 0.5% car-
boxymethyl cellulose (CMC), disease control group
with VDD + 0.5% CMC, positive control group treat-
ment with VDD + calcitriol (0.5 lg/kg body weight
per oral), VDD + CBD (15 mg/kg body weight per
oral) treatment, VDD + CBD (30 mg/kg body weight
per oral) treatment, and VDD + CBD (60 mg/kg
body weight per oral) treatment groups. The treatment
groups’ animals were treated for 8 weeks after onset
(3 weeks) of the VDD model. Five days before sacrifice,
all the animals (36) were individually subjected to be-
havioral parameters like Morris water maze (MWM)
test sessions and probe trials.
On day 55, these animals were tested for pain score
(knee joint pain measurement) using Pressure applica-
tion measurement (PAM). In the 8th week, other 36
animals were subjected to collect cerebrospinal fluid
(CSF) by a standard in-house method using a stereo-
taxic instrument for the estimation of 1,25-dihydroxy
vitamin D3, serotonin, and klotho protein by enzyme-
linked immunosorbent assay (ELISA). After CSF
collection, animals were humanely sacrificed, liver,
kidney, and brain tissues were collected, homogenized,
and stored at 80C. Kidney homogenate was used to
estimate VDR, cytochrome P450 24A1 (CYP24A1) and
3
CYP27B1, while VDR and CYP2R1 were determined
in the liver. Brain tissue was used for the estimation
of VDR, 25-hydroxy vitamin D3, acetylcholine (Ach),
proinflammatory cytokines (interleukin-1b, IL-1b and
tumor necrosis factor-alpha, TNF-a), and antioxidants
(superoxide dismutase, SOD and glutathione peroxi-
dase, GPx) by ELISA.
CYP24A1, CYP27B1, and CYP2R1-mRNA
expression by RT-PCR
RNA was extracted from frozen liver and kidney tis-
sues using TRIzol reagent (Invitrogen Corp.), as per
manufacturer’s instructions. After extraction, purity
was confirmed by spectrophotometry (UV-Vis-NIR
Spectrophotometer UV-3600 Plus; Shimadzu, Japan).
cDNA was synthesized using SuperScript II first-strand
synthesis system for PCR (Invitrogen Corp.) at 42C
for 60 min. RT-PCR was performed using TaqMan
Gene Expression Assays (Applied Biosystems), using
2 mL of cDNA as per manufacturer’s instructions. For-
ward (5¢-AGTGAGCTGAACAAGTGGTC-3¢) and re-
verse (5¢-GTCTGATTGTCAGGCAGCAC-3¢) primers
were used in PCR for CYP24A1.19 cDNA was then PCR
amplified for CYP27B1 using forward and reverse
primers (forward: 5¢-GATTGCCGCATCACCAAGG
AC-3¢; reverse: 5¢-GAGGAGACAGGTCCAGAGTCA-
3¢).20 Forward (5¢- CCA TGG ATT GGC ATC TTA
CC-3¢) and reverse (5¢- CCC AAG GTG TCC TGT
TG -3¢) primers were used in PCR for CYP2R1.21
Estimation of VDR
About 100 mg of the liver, kidney, and brain tissues
were rinsed with 1 · PBS, homogenized in 1 mL of
1 · PBS, and stored overnight at 20C. To break the
cell membrane, two freeze-thaw cycles were performed.
Then, the homogenates were centrifuged at 5000 g for
5 min at 2C–8C. The supernatant was removed and
assayed using ELISA kit (CSB-EL025832RA; CUSA-
BIO) as per manufacturer’s instruction.
MWM test
The MWM test was used to evaluate spatial mem-
ory and cognitive function in CBD-treated Sprague–
Dawley rats.22 This test was set-up on spatial cues to
locate a submerged hidden platform (10 cm diameter)
in a circular pool filled with nontransparent/cloudy
tap water with 22C – 1C. The pool was divided
into four virtual quadrants: left, right, opposite, and
target. A session trial was performed on days 51–54
to test their learning from four different starting
 4 TRIVEDI ET AL.
points in the pool. On day 55, the animals were sub-
jected to a probe trial to test their long-term spatial
memory without any platform. Total distance trav-
eled and time taken to reach the target (escape laten-
cy) were recorded and analyzed with the help of
SMART digital tracking system (Version 2.5; Panlab,
Barcelona, Spain).
Knee joint pain measurement
Static hyperalgesia or allodynia was assessed by the
application of pressure to both the knee joints of
the animal by the PAM meter (Ugo Basile, VA,
Italy; Cat. No. 38500) probe as per Jensen and Fin-
nerup, 2014 with few modifications.23 A gradual in-
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crease in pressure was applied across the knee joint
of rats using a PAM device until the animal indicated
any discomfort or pain. Animal’s limb withdrawal
threshold as a result of pain in gram force (GF) was
digitally recorded in the PAM instrument with the
help of DCA software.24
Biomarkers estimation in brain and CSF
About 100 mg of the brain tissue was rinsed with
1 · PBS, homogenized in 1 mL of 1 · PBS, and stored
overnight at 20C. To break the cell membranes,
two freeze-thaw cycles were performed. The homoge-
nates were centrifuged for 5 min at 5000 g, at 2C–
8C. The supernatant was removed for the estimation
of 25-hydroxy vitamin D3 (CSB-E08098r; CUSABIO),
Ach (MBS282680, My Bio Source), IL-1b (CSB-
E08055r; CUSABIO), TNF-a (CSB-E08364r; CUSA-
BIO), SOD (706002; Cayman Chemical), and GPx
(703102; Cayman Chemical) using ELISA method as
per manufacturer’s instruction. However, the CSF
was collected by a standard in-house method using
the stereotaxic instrument for the estimation of 1,25-
dihydroxy vitamin D3 (MBS2601701; My Bio Source),
serotonin (CSB-E08364r; CUSABIO), and klotho (CSB-
E10958r; CUSABIO) using ELISA method as per man-
ufacturer’s instruction.
Statistical analysis
The obtained data are shown (mean – standard error
of the mean) and subjected to statistical analysis
using Sigma-Plot (V11.0). For multiple comparison,
One-way analysis of variance (ANOVA) followed
by post hoc analysis by Tukey’s test and for between
two groups comparison Student’s t-test was per-
formed. F values p < 0.05 were regarded as statistically
significant.
Results
RT-PCR analysis of CYP24A1, CYP27B1,
and CYP2R1-mRNA expression
The real-time PCR analysis revealed that pair t-test
analysis showed that CYP2R1-mRNA level in liver was
significantly (95% CI = 24.434 to 5.886; t = 3.642,
p = 0.005) increased by 38.37% in CBD-60 mg/kg
group compared to disease control (VDD) group.
Additionally, CYP27B1-mRNA expression in kidney
tissue was significantly (95% CI = 21.514 to 0.486;
t = 2.331, p = 0.042) increased by 39.29% in CBD-
60 mg/kg group compared to VDD group. Further,
pair t-test analysis showed that CYP24A1-mRNA tran-
scripts in kidney tissues were significantly (95% CI =
1.227–10.230; t = 2.835, p = 0.018) reduced by 21.39%
in CBD-60 mg/kg group compared to VDD (Fig. 1).
VDR expression
VDR protein expression in liver tissue was significantly
(95% CI = 41.313–99.599; t = 5.387, p £ 0.001) decreased
by 53.08% in VDD than NC group. Pair t-test analysis
showed that it was significantly (95% CI = 148.560 to
36.152; t = 3.661, p = 0.004) increased by 687.40%,
101.20%, 118.20%, and 148.30% in calcitriol, CBD-15,
CBD-30, and CBD-60 groups, respectively than VDD
group (Fig. 2A). Besides, VDR protein expression in
kidney tissue was significantly (95% CI = 5.633–
18.765; t = 4.139, p = 0.002) decreased by 50.95% in
VDD than NC. Pair t-test analysis revealed that it
was significantly (95% CI = 12.107 to 2.087;
t = 3.156, p = 0.010) increased by 60.48% in CBD-
60 mg/kg group than VDD. Pair t-test analysis revealed
that VDR protein expression in brain tissue was signifi-
cantly (95% CI = 8.795 to 1.005; t = 2.803,
p = 0.019) increased by 142.03% in CBD-60 mg/kg
group than VDD group (Fig. 2B).
Vitamin D3 metabolites
The level of first metabolite like, 25-hydroxy vita-
min D3 in brain homogenate was nonsignificantly
decreased by 22.49% in the VDD group than NC.
Tukey’s post hoc analysis revealed that it was significantly
(F(4,25) = 15.054, p £ 0.001) increased by 59.84%, 65.02%,
75.19%, and 92.79% in calcitriol, CBD-15, CBD-30,
and CBD-60 groups, respectively compared to VDD
(Fig. 3A). Moreover, the level of second active metabo-
lite like, 1,25-dihydroxy vitamin D3 in CSF was signif-
icantly (95% CI = 0.133–1.344; t = 2.718, p = 0.022)
decreased by 46.78% in the VDD group than NC.
Pair t-test analysis revealed that it was significantly
 FIG. 1. Response to mRNA expression of CYP24A1 and CYP27B1 in kidney tissues; while CYP2R1 in the liver
after treatment with CBD for 56 days in VDD-induced Sprague–Dawley rat model by RT-PCR. CBD@ 15, 30,
Downloaded by Liz Hughston from www.liebertpub.com at 04/26/22. For personal use only.

and 60 mg/kg body weight per oral (p.o.); calcitriol (0.5 lg/kg body weight as positive control, p.o.). Data
show mean – standard error of the mean (n = 6). *p £ 0.05 and **p £ 0.01 versus disease control (VDD). CBD,
cannabidiol; VDD, vitamin D3 deficiency diet; NC, normal control; RT-PCR, real-time polymerase chain
reaction.

FIG. 2. Response to VDR protein expression (A) in kidney and brain tissues; and (B) in the liver after
treatment with CBD for 56 days in VDD-induced Sprague–Dawley rat model by ELISA. CBD@ 15, 30, and
60 mg/kg body weight per oral (p.o.); calcitriol (0.5 lg/kg body weight as positive control, p.o.). Data show
mean – standard error of the mean (n = 6). $$p £ 0.01 and $$$p £ 0.001 versus NC; *p £ 0.05 and **p £ 0.01
versus disease control (VDD). ELISA, enzyme-linked immunosorbent assay; VDR, vitamin D receptor.

5
 6 TRIVEDI ET AL.
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FIG. 3. Response to vitamin D3 metabolites (A) 25-hydroxy-vitamin D3 and (B) 1,25-dihydroxy-vitamin D3

in brain homogenate and CSF, respectively after treatment with CBD for 56 days in VDD-induced Sprague–
Dawley rat model. CBD@ 15, 30, and 60 mg/kg body weight per oral (p.o.); calcitriol (0.5 lg/kg as positive
control p.o.). Data show mean – standard error of mean (n = 6). $p £ 0.05 versus NC; *p £ 0.05 and ***p £ 0.001
versus disease control (VDD).

(95% CI = 1.367 to 0.180; t = 2.904, p = 0.016) in-
creased by 92.10% in CBD-60 group compared to
VDD (Fig. 3B).
MWM test for spatial memory
Total distance traveled was significantly (95% CI =
226.219 to 97.774; t = 5.620, p £ 0.001) increased
by 135.22% in VDD as compared to NC. Pair t-test
analysis showed it was significantly (95% CI = 2.498–
120.209; t = 2.323, p = 0.043) decreased by 21.77% in
CBD-60 group compared to VDD group (Fig. 4A).
Further, escape latency was significantly (95% CI =
26.257 to 9.654; t = 4.819, p £ 0.001) increased
by 269.76% in VDD than NC. Tukey’s post hoc analysis
identified a significant reduction in escape latency
(F(2,15) = 3.853, p = 0.045) by 37.04% and 35.87% in
the calcitriol and CBD-60 groups, respectively com-
pared to VDD group (Fig. 4B).
Knee joint pain measurement
Assessment of pain in terms of GF was significantly
(95% CI = 94.730 to 16.410; t = 3.162, p = 0.010)
increased by 9.64% in the CBD-60 compared to VDD
using paired t-test analysis (Fig. 5).
Neurotransmitters (Ach and serotonin) in brain
The level of neurotransmitter, Ach in brain ho-
mogenate was significantly (95% CI = 0.775–45.011;
t = 2.306, p = 0.044) decreased by 16.87% in the disease
control (VDD) group than NC. Tukey’s post hoc anal-
ysis showed that Ach was significantly (F(4,25) = 4.187,
p = 0.010) increased by 25.40% in CBD-60 compared to
VDD (Fig. 6A). The level of serotonin in CSF was sig-
nificantly (95% CI = 0.691–4.832; t = 2.972, p = 0.014)
decreased by 46.67% in VDD group than NC. Tukey’s
post hoc analysis showed that it was significantly
(F(4,25) = 6.073, p = 0.001) increased by 125.05%, 107.81%,
Downloaded by Liz Hughston from www.liebertpub.com at 04/26/22. For personal use only.

FIG. 6. Response to neurotransmitters (A) Ach

FIG. 4. Response to learning, spatial memory,
and cognitive behavior by Morris water maze
task (A) total distance (centimeter) and
(B) escape latency (seconds) after treatment
with CBD for 56 days in VDD-induced Sprague–
Dawley rat model. CBD@ 15, 30, and 60 mg/kg
body weight per oral (p.o.); calcitriol (0.5 lg/kg
as positive control p.o.). Data show
mean – standard error of the mean (n = 6).
$$$
p £ 0.001 versus NC; *p £ 0.05 versus disease
control (VDD).
and (B) serotonin in brain homogenate and CSF,
respectively after treatment with CBD for 56
days in VDD-induced Sprague–Dawley rat
model. CBD@ 15, 30, and 60 mg/kg body weight
per oral (p.o.); calcitriol (0.5 lg/kg as positive
control p.o.). Data show mean – standard error
of the mean (n = 6). $p £ 0.05 versus NC;
**p £ 0.01 and ***p £ 0.001 versus disease control
(VDD). Ach, acetylcholine.

FIG. 5. Assessment of pain in terms of GF after chronic administration of CBD at different doses in VDD-
induced Sprague–Dawley rat using PAM meter. CBD@ 15, 30, and 60 mg/kg body weight per oral (p.o.);
calcitriol (0.5 lg/kg as positive control p.o.). Data shows mean – standard error of mean (n = 6). **p £ 0.01
versus disease control (VDD). PAM, pain application measurement; GF, gram force.

7
 8 TRIVEDI ET AL.
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FIG. 7. Response to proinflammatory cytokines
(A) IL-1b and (B) TNF-a in brain homogenate
after treatment with CBD for 56 days in VDD-
induced Sprague–Dawley rat model. CBD@ 15,
30, and 60 mg/kg body weight per oral (p.o.);
calcitriol (0.5 lg/kg as positive control p.o.). Data
show mean – standard error of mean (n = 6).
$
p £ 0.05 and $$p £ 0.01 versus NC; *p £ 0.05 and
**p £ 0.01 versus disease control (VDD). TNF-a,
tumor necrosis factor alpha; IL-1b, interleukin-
1b.
160.53%, and 205.75% in calcitriol-0.5, CBD-15, CBD-30,
and CBD-60 groups, respectively compared to VDD
(Fig. 6B).
Proinflammatory cytokines (IL-1b and TNF-a)
in brain
The level of proinflammatory cytokine, IL-1b in brain
homogenate was significantly (95% CI = 155.155 to
3.445; t = 2.329, p = 0.042) increased by 39.39% in
the disease control (VDD) group than NC. Pair t-test
revealed that IL-1b was significantly (95% CI = 0.327–
135.673; t = 2.239, p = 0.049) decreased by 21.43% in
CBD-60 compared to VDD (Fig. 7A). As well, level
of TNF-a in brain homogenate was significantly (95%
CI = 15.465 to 3.745; t = 3.652, p = 0.004) in-
creased by 46.70% in VDD group than NC. Tukey’s
post hoc analysis showed that it was significantly
(F(4,25) = 4.576, p = 0.007) decreased by 28.34 and 29.60%
in calcitriol-0.5 and CBD-60, respectively compared
to VDD (Fig. 7B).
FIG. 8. Response to antioxidative enzymes
(A) SOD and (B) GPx in brain homogenate;
while (C) klotho protein in CSF after treatment
with CBD for 56 days in VDD-induced Sprague–
Dawley rat model. CBD@ 15, 30, and 60 mg/kg
body weight per oral (p.o.); calcitriol (0.5 lg/kg
body weight as positive control, p.o.). Data show
mean – standard error of mean (n = 6). $p £ 0.05
versus NC; *p £ 0.05, **p £ 0.01, and ***p £ 0.001
versus disease control (VDD). SOD, superoxide
dismutase; GPx, glutathione peroxidase; CSF,
cerebrospinal fluid.
Antioxidant and klotho in brain
Expression of SOD in brain homogenate was signifi-
cantly (95% CI = 0.0216–1.041; t = 2.323, p = 0.043) de-
creased by 11.08% in disease control (VDD) group
than NC. Tukey’s post hoc test revealed that SOD was
significantly (F(4,25) = 20.526, p £ 0.001) increased by
49.61% in CBD-60 compared to VDD (Fig. 8A). Addi-
tionally, level of GPx in brain homogenate was de-
creased by 36.23% in VDD group than NC. Pair
t-test analysis showed that it was significantly (95%
CI = 6.120 to 1.300; t = 3.430, p = 0.006) increased
by 178.87% in CBD-60 compared to VDD (Fig. 8B).
 CBD IMPROVES MEMORY, REDUCES BRAIN INFLAMMATION AND AGING
Further, expression of klotho protein in CSF was signif-
icantly (95% CI = 17.684–377.899; t = 2.447, p = 0.034)
decreased by 50.73% in disease control (VDD) group
than NC. Tukey’s post hoc analysis showed that klotho
level was significantly (F(4,25) = 6.104, p = 0.001) in-
creased by 110.80%, 102.77%, 103.76%, and 145.57%
in calcitriol, CBD-15, 30, and 60 mg/kg, respectively
compared to VDD (Fig. 8C).
Discussion
VDD possesses more abundance of bioactive lipids like
endocannabinoids (ECs). Gene expression study ex-
plored that increased ECs tone demonstrated increased
synthesis and metabolism of anandamide (a fatty acid
Downloaded by Liz Hughston from www.liebertpub.com at 04/26/22. For personal use only.
neurotransmitter and first EC that bind to the cannabi-
noid receptors) compared with vitamin D3 sufficient
diet.25 Cheng Fang’s 2014 docking study reported
that CB2 inverse agonist modulates VDR.26 Besides,
it has been reported that CBD can behave as a CB2 re-
ceptor inverse agonist for its well-documented anti-
inflammatory properties by inhibiting immune cell mi-
gration and reducing clinical signs of inflammation.27
The CYP27B1 gene influences 1-alpha-hydroxylase
(1a-hydroxylase) to convert vitamin D to its active
form, 1,25-dihydroxyvitamin D3.28 The CYP24A1 is
a mitochondrion enzyme that catalyzes hydroxyl-
ation reactions, leading to the degradation of 1,25-
dihydroxyvitamin D3. Thus, this 1,25(OH)2D regulates
its own feedback metabolism via upregulation of
CYP24A1 and downregulation of CYP27B1 expression.
This process ultimately reduced the levels of both
25(OH)D and 1,25(OH)2D.29 Besides, the CYP2R1
gene encodes 25-hydroxylase that is responsible for
conversion from vitamin D to its active form, 1,25-
dihydroxyvitamin D3, also known as calcitriol.30 In
this experiment, authors observed that CBD treatment
significantly inhibits the CYP24A1 (kidney) and simul-
taneously enhanced CYP27B1 (kidney) and CYP2R1
(liver) expression. Therefore, CBD will be able to op-
pose the degradation of 1,25-dihydroxyvitamin D3
and consequently increased more formation of 1,25-
dihydroxyvitamin D3 to maintain an adequate level
of active metabolites in circulation that are required
for normal physiological functions.
Treatments with CBD and vitamin D3 improve bone
cells differentiation and mineralization in isolated stem
cells.31 The behavioral profile of CBD was investigated
with MWM test after prolonged CBD treatment in
VDD-induced rats. Recently, Amini and Abdolmaleki
2021 reported CBD significantly reduces escape latency
9
and traveled distance; simultaneously increased protein
expression of both the cannabinoids type 1 (CB1) and
CB2 receptors in rat brain, and improved learning and
memory function.32 Others findings showed that CBD
improves memory and cognition in multiple preclinical
cognitive impairment models.33 It also evidenced that
CBD can oppose the cognitive-impairing effects of
D9-tetrahydrocannabidiol, when co-administered with
1:1.34 In this experiment, chronic oral administration
of CBD at 60 mg/kg body weight significantly reduced
both total distance traveled and escape latency, suggest-
ing an improved learning and memory function in the
VDD-induced rat model.
Pressure application measurement (PAM) has been
used as a behavioral tool to determine primary mechan-
ical hypersensitivity in rats with the inflammatory joint
pain model.24 In this experiment, PAM applies a quan-
tifiable force for direct stimulation of the joint and au-
tomatic recorded the animal response. Lack of vitamin
D can lead to allodynia, causes muscle weakness and
pain in children and adults.35 CBD possess an anti-
inflammatory and analgesic activity on CB1 receptor an-
tagonist was approved in Canada in 2005 to treat central
neuropathic pain in MS and in 2007 for intractable can-
cer pain.36 In this experiment, authors have found signif-
icant improvement of pain score measured by PAM
after administration of CBD in VDD-induced rat model.
Emerging evidence suggests that CBD increases Ach
levels in a brain region as wakefulness modulation.37
Recent studies have found that CBD inhibits acetylcho-
linesterase (AChE) enzyme, thus retained levels of
Ach.38 Besides, CBD escalates 5-hydroxy-tryptamine
(5-HT) or serotonin and glutamate levels in the pre-
frontal cortex through 5-HT1A receptor-mediated acti-
vation.39 Apart from this, CBD acts as an agonist of the
human 5-HT1a receptor by increasing [35S]GTPcS
binding in G protein-coupled receptor system and re-
ducing cyclic adenosine monophosphate concentra-
tion.17 Based on the above research findings, our data
support the improvement in neurotransmitters in the
brain, but the most fascinating intervention was in
the VDD model.
From the literature point of view, CBD mini-
mizes inflammation in the brain by reducing the lev-
els of proinflammatory cytokines like TNF-a in the
hippocampus and IL-6 in the prefrontal cortex by
binding subtypes of CB1 and CB2 receptors within
the brain.40 Based on the human cell-lines study,
CBD reduced inflammation by reducing the levels
of TNF-a.41,42
 10
This study data also found that there was a signifi-
cant reduction of proinflammatory cytokines like
TNF-a and IL-1b in brain tissue after treatment with
CBD at various doses. Oxidative stress plays a vital
role in the pathogenesis of brain disorders such as Alz-
heimer’s disease, Parkinson’s disease (PD), depression,
anxiety, epilepsy, and other seizure disorders. However,
SOD and GPx are the first-line defense mechanism43 in
the entire defense grid. CBD showed antioxidative and
anti-inflammatory potential.44 The experimental data
suggested that CBD significantly improved brain anti-
oxidant capacity by improving levels of SOD and
GPx. Cognitive decline, aging, and cannabis use are
closely associated with each other.45
Downloaded by Liz Hughston from www.liebertpub.com at 04/26/22. For personal use only.
For mechanistic perspective, we can see that CBD can
enhance the activity of cytochrome P-450 (CYPs),
CYP2R1 and CYP27B1; while inhibition of CYP24A1 is
based on our findings. That leads to abundance of active
metabolites. As per literature, deficiency of vitamin D3
containing diet leads to more production of ECs. Further,
gene expression study indicated that an increased ECs
tone demonstrated an increased synthesis and metabo-
lism of anandamide as compared with diet with sufficient
vitamin D3.25 There is a co-relation between vitamin D3
and ECs. Besides, from existing literature it has also been
reported that CB2 inverse agonist modulates VDRs, and
CBD specifically bind with CB2 receptor rather than
CB1.26 Based on the above facts and current study find-
ings, authors conclude that CBD can modulate the VDRs
through activation and/or inhibition of CYPs mixed
function oxidases and consequently increased the levels
of vitamin D3 active metabolites in the systemic circula-
tion, those are responsible for pharmacological re-
sponses. However, for more mechanistic perspective
detailed molecular studies are required.
Based on the study outcomes, CBD improved vita-
min D3 metabolites in brain and VDRs in brain and
kidney via modulation of CYPs and justified our hy-
pothesis predominately.
Conclusions
In conclusion, the present study demonstrates that
treatment with CBD improved cognitive impair-
ment, pain tolerance, and aging caused by consuming a
VDD. Besides, it raised vitamin D3 metabolites by mod-
ulating the expression of CYP-450-mRNAs (CYP2R1,
CYP27B1, and CYP24A1) and VDRs in the VDD-
induced rat model. These effects are supported through
the antioxidant, anti-inflammatory, and neuroprotec-
tive properties of CBD.
TRIVEDI ET AL.
Acknowledgment
The authors extend their sincere thanks and gratitude
to Dabur Research Foundation, India, for providing
the facilities and support that enabled the successful
completion of the work.
Author Disclosure Statement
All authors claim that there are no conflicts of interest
with respect to personal financial interests, funding,
employment, and other competing interests.
Funding Information
No funding was received for this article.
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Cite this article as: Trivedi MK, Mondal S, Gangwar M, Jana S (2022)
Effects of cannabidiol interactions with CYP2R1, CYP27B1, CYP24A1,
and vitamin D3 receptors on spatial memory, pain, inflammation, and
aging in vitamin D3 deficiency diet-induced rats, Cannabis and Can-
nabinoid Research X:X, 1–11, DOI: 10.1089/can.2021.0240.
Abbreviations Used
Ach ¼ acetylcholine
ASD ¼ autism spectrum disorder
CBD ¼ cannabidiol
CMC ¼ carboxymethyl cellulose
CPCSEA ¼ committee for the purpose of control and supervision
of experiment on animals
CSF ¼ cerebrospinal fluid
EC ¼ endocannabinoid
ELISA ¼ enzyme-linked immunosorbent assay
GF ¼ gram force
GPx ¼ glutathione peroxidase
IAEC ¼ institutional animal ethical committee
IL-1b ¼ interleukin-1b
MS ¼ multiple sclerosis
MWM ¼ Morris water maze
PAM ¼ pressure application measurement
SOD ¼ superoxide dismutase
TNF-a ¼ tumor necrosis factor-alpha
VDD ¼ vitamin D3 deficient diet
VDR ¼ vitamin D receptor