Role of Cannabidiol and Tetrahydrocannabivarin on Paclitaxel-Induced Neuropathic Pain in Rodents

Anil Kumar Kalvala FAMU: Florida Agricultural and Mechanical University Arvind Bagde FAMU: Florida Agricultural and Mechanical University Peggy Arthur FAMU: Florida Agricultural and Mechanical University Sunil Kumar Surapaneni FAMU: Florida Agricultural and Mechanical University Ramesh Nimma FAMU: Florida Agricultural and Mechanical University Aakash Nathani FAMU: Florida Agricultural and Mechanical University Mandip Singh (  mandip.sachdeva@famu.edu ) Florida A&M University: Florida Agricultural and Mechanical University https://orcid.org/0000-0001-9192-8143

Invivo Animals C57BL/6J mice (4-5 weeks old) and male Sprague Dawley rats (7-8 weeks old) were provided by Envigo (Indianapolis, IN) for the current study on PIPN. FAMU has AAALAC-accredited animal facilities, and following NIH recommendations (Guide for the care and use of laboratory animals), the current protocol was evaluated and approved by Florida Agricultural and Mechanical University's Institutional Animal Use and Care Committee (protocol numbers: 020-06 & 021-04). The animals were sacri ced using the carbon dioxide (CO2) asphyxiation procedure.

Study design
To establish peripheral neuropathy, C57BL/6J female mice (4-5 weeks old) were given PTX (8 mg/kg, i.p.) every other day for four injections. In rst set of experiments, animals were divided into three groups after they had developed neuropathy: a. untreated age-matched normal mice, b. PTX (8 mg/kg, i.p.) every other day for four injections, c. cannabidiol (CBD) group: animals were given 10 mg/kg CBD (i.p.) twice a week for six weeks following the previous PTX injection. d. Tetrahydrocannabivarin (THCV) group: animals were given 15 mg/kg of THCV (i.p.) twice a week for a total of six weeks after the last dose of PTX injection, e. Cannabidiol (CBD) + Tetrahydrocannabivarin (THCV) group: animals were given 10 mg/kg of CBD and 15 mg/kg of THCV (i.p.) and in the second set of experiments, the effects of CB1, CB2, and 5HT1A blockers on CBD and THCV-induced neurobehavioral changes in PTX-induced neuropathic mice were studied by dividing the animals into six groups, a &b: (PTX+CB1B+CBD or THCV): CB1 blocker (CB1B) 3 mg/kg/day, i.p., was given to mice for four weeks and three hours before administering CBD or THCV. c&d: (PTX+5HT1AB+CBD or THCV): 5HT1A blocker (5HT1AB), 10 mg/kg/day, i.p., was given to mice for four weeks and three hours before administering CBD or THCV e&f: (PTX+CB2B+CBD or THCV): CB2 blocker (CB2B) 1 mg/kg/day, i.p., was given to mice for four weeks and three hours before administering CBD or THCV. The PTX, CBD, THCV, AM630, Rimonabant and WAY10065 dosages were determined using existing literature reports [16][17][18][19][20][21]. After the animals were euthanized using CO2 asphyxiation and dorsal root ganglions (L1-L5) were extracted, biochemical and molecular parameters were examined.
Behavioral studies Thermal hyperalgesia Hargreaves Plantar Test: This test was carried out with some modi cations [22,23]. Brie y, the animals were housed in 12 plexiglass enclosures kept at a constant temperature (30°C) above a horizontal glass surface. With a cut-off time of the 20s, the time it took a mouse to lift its right or left paw after being exposed to a radiant heat source of infrared irradiation (40 IR units) was measured. The results are reported in seconds as paw withdrawal latency, with a 10minute time interval between each consecutive reading.
Hot and Cold plate method Thermal hyperalgesia was measured using the hot and cold plate method, as previously reported [24]. Immediately after acclimatization, the mice were placed on a hot plate (55°C) and a cold plate (10°C) where the time latency for the animal to lick its right/left foot was measured with a cut-off time of 20s and an interval of 10 minutes at each reading, and the results were reported as paw withdrawal latency in seconds.

Mechanical Hyperalgesia
The Vonfrey and Randall Selitto tests were used to assess mechanical hypersensitivity in mice. The mice were poked with standard vonfrey bres of varying weights (g), and the weight at which they lifted their paws was monitored using a digital electronic readout unit and reported as paw withdrawal threshold (g).
The paw withdrawal pressure of mice was measured using Randall sellito pincture pressure on both paws. Each animal was tested ve times, with a 10-15 minute delay between each reading [25].
RNA sequencing RNA sequencing on DRG homogenates from control, PTX, CBD, and THCV-treated mice was performed by Novogene Corporation Inc (Sacramento, CA). Brie y, messenger RNA was isolated from total RNA using poly-T-oligo magnetic beads. This was followed by second strand cDNA synthesis using either dUTP or dTTP depending on the library type. Except in directed library preparation, user enzyme digestion was included after size selection. The library was quanti ed with Qubit, and the size distribution was detected with a bioanalyzer. Quanti ed libraries were pooled and sequenced on Illumina platforms. Clustering and sequencing were done following manufacturer's instructions. After generating clusters and paired-end reads, the library preparations were sequenced on Illumina. Initially, quality control of raw data in fastq format was handled using perl programs. Raw reads were cleaned by eliminating adaptor, poly-N, and low quality reads. The cleaned data Q20, Q30, and GC content were calculated and used for downstream analysis. For better mapping results, clean reads were mapped to the reference genomes using Hisat2 v2.0.5, which developed a database of splice junctions based on the gene model annotation le. Feature Counts v 1.5.0-p3 was used to count the reads mapped to each gene. In order to assess gene expression levels, FPKM (fragments per kilobase of exon per million mapped fragments) was calculated. DESeq2 R software was used to analyze differential gene expression (1.20.0). In order to reduce the false discovery rate, Benjamini and Hochberg's procedure was used. Genes with a P < 0.05 difference across groups were considered differentially expressed. The online Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) was used to examine the statistical enrichment of differential expression genes in KEGG pathways. The effective genes and critical pathways in regulating neuronal function were predicted using Reactome, disease ontology, and DisGeNET databases.

Western blotting
For this study, separated DRGs were homogenized in TPER (1:100), centrifuged at 20,000 rpm for 20 minutes, and the supernatant was collected and evaluated for total protein content using a bicinchonic acid test kit. To denature the proteins, the sample was heated to 95°C for 10 minutes with 4X laemmli buffer containing 5% mercapto-ethanol. SDS PAGE gel electrophoresis resolved 40 g protein. Semi-dry transfer of resolved gel containing segregated proteins to PVDF/nitrocellulose membranes (Transbloto, Biorad, USA). Incubation with primary antibodies (P-38 MAPkinase, p-AMPK, PI3K, p-AKT, TFAM, HO-1, Catalase, Nrf2, Bax, caspase 3, caspase 1, caspase 9 and TGF-beta of rabbit or mouse origin) at 4°C overnight followed by three washes with PBST. We next probed the membranes for 2 hours at room temperature with HRP conjugated secondary anti-rabbit and anti-mouse antibodies. The ChemiDocTMXRS+ imaging system (BIO-RAD)was used to collect the luminescence signal which was quanti ed by using image J software (version 1.48, NIH, USA) [26].

In-vitro DRG Primary Cultures
Primary DRG neuronal cells were generated in vitro using adult rat dorsal root ganglions from the (L1-l5) lumbar region of the spinal cord with slight modi cations as earlier reported [27]. Brie y, 9-10 week old rats were slaughtered and DRGs were separated aseptically into HAMS F12 medium with 10% FBS and 5% antibiotic/antimycotic solution. This was followed by centrifugation at 1200 rpm for 2 minutes before adding trypsin (0.25 percent) for 30 minutes and triturating with a glass pipette to dissociate into cells. This cell and tissue suspension was ltered through 70M nylon gauge. The monosuspended DRG cells were centrifuged for 3 minutes at 1200 rpm and resuspended in neurobasal media containing 10% FBS and 0.5 percent antibiotic and antimycotic solution. A 1:50 volume ratio of matrigel matrix to neurobasal media was used to grow the cells. The neurite extensions appeared two-three days after culture and were treated with various treatments at optimal concentrations. Neurite outgrowth assay Three days after DRG primary cultures in 12 well plates were treated with various concentrations of optimized drugs at speci ed intervals (48 hours), the cells were washed with fresh PBS and neurite outgrowth assay was performed using the manufacturer's kit-based protocol. Five elds were chosen at random and examined using a phase contrast microscope (Nikon ECLIPSE, Ti-U, Japan). The length of neurite outgrowths in 30 cells from each eld was measured using Image J software (NIH, USA). The number of neurite outgrowths/axon-like extensions that are twice or more than the diameter of the cell body were counted [28].
Immunocytochemistry DRG neurons were cultured on glass Coverslips in a 6-well plate at 5000 cells/well seeding density and xed with 4% paraformaldehyde solution and permeabilized with 0.5 percent Triton-X 100 for 15 minutes at room temperature, as described elsewhere. These cells were blocked for 2 hours at room temperature with a 3 percent BSA solution in PBS. After blocking, the cells were incubated overnight at 4°C with primary antibodies (p-AMPK, Complex I, and TFAM) at 1:200 dilutions in 3 percent BSA solution. The following day, cells were washed with PBST and incubated with secondary anti-rabbit and anti-mouse antibodies conjugated with rhodamine and Alexa488 (Santa Cruz Biotechnology Inc., CA, USA) at room temperature for 2 hours. Finally, the coverslips were adhered to the glass slide with DAPI mounting medium (Sigma Fluoroshield TM ). A confocal microscope was used to capture the images (Leica TCS SP8 Laser Scanning Spectral Confocal microscope, Germany) [29].
Assay for JC1 DRG primary cultures were stained with JC1 as previously described. Cultured DRG primary cells were incubated for 15 minutes with 5M JC-1 in PBS. The cells were washed with PBS to remove unbound red stain before being examined with a uorescence microscope (Nikon ECLIPSE, Ti-U, Japan) with red and green lters. The mean red and green uorescence intensity ratio was calculated using Image J software (NIH, USA), [30,31].

Mitosox Test
The Mitosox red assay was carried out in DRG primary cultures according to published protocols [32]. Brie y, the cultures were treated with various drug concentrations for 48 hours before being washed with PBS and incubated for 15 minutes at 37°C with 5M Mitosox reagent. The cells were then washed to remove any unbound Mitosox red reagent before being examined with a uorescence microscope (Nikon ECLIPSE, Ti-U, Japan) with a green lter. Image J software was used to calculate the mean red uorescence intensity (NIH, USA).

Statistical Analysis
Excel was used to calculate mean, SD, and SEM for each parameter. The results were analyzed with the newest version of graph pad prism program, with one way ANOVA to compare the groups (multiple comparison tests). When one way ANOVA demonstrated statistical signi cance, Bonferroni's multiple comparisons test was used for post hoc analysis. Statistical signi cance was de ned as P< 0.05 or less.

Results
CBD, THCV, and their combination on PTX-induced neurobehavioral changes PTX (8 mg/kg) treatments to the animals signi cantly (p<0.001) produced mechanical and thermal hyperalgesia in mice, and the neuropathy in animals lasted until the last day of the study (Fig.1). The neuropathic mice were given CBD (10 mg/kg) and THCV (15 mg/kg) twice a week for six weeks which reduced PTX-induced thermal hypersensitivity (p<0.01), as determined Hot and cold plate method (Fig.1). Their combination alleviated neuropathic pain by reducing PTX-induced thermal hyperalgesia. We also evaluated the role of CBD, THCV, and their combination on PTX-induced mechanical hyperalgesia. The paw withdrawal thresholds and pressures of CBD and THCV treated mice were signi cantly different from PTX treated animals in Electronic-Vonfrey and Randall selitto studies (Fig.1). Besides that, administration of 3 mg/kg rimonabant (CB1 blocker, i.p.) and 1 mg/kg AM630 (CB2 blocker) blocked the effect of CBD on mechanical and thermal hypersensitivity in mice. Further, administration of 1 mg/kg AM630 and 10 mg/kg WAY10065 (5HT1A blocker) had no effect on the neurobehavioral changes associated with THCV. However, in PTX-induced neuropathic mice, 10 mg/kg WAY10065 and 3 mg/kg rimonabant signi cantly (p<0.05 to 0.01) reduced the neurobehavioral effects of CBD and THCV, respectively (Table 1). Further, to investigate the gene regulation of CBD and THCV in DRG homogenates from PTX-treated mice, we used RNA sequencing on isolated DRG neurons from different groups of mice.

RNA seq analysis in DRG homogenates of CBD and THCV treated neuropathic mice
Primary sequencing data produced by RNA-Seq were subjected to quality control and after cleaning, the total reads and mapping ratio reads were calculated as shown in table 2 which denotes the quality of RNA seq data.
Further, we performed cluster analysis and heat map visualizations of gene expression patterns using the heatmap software. A total of 1519 genes were differentially expressed in control and PTX group, 11035 genes in CBD treatment and PTX group and 2527 genes in THCV treatment and PTX group. The number of upregulated and downregulated genes were plotted in volcano graphs as shown in Fig.2. Further, KEGG analysis unraveled several signaling pathways enriched in DRG homogenates of control and treated mice. Among these pathways, AMPK-PGC1 alpha, MAPK signalling, PI3K-AKT and NLRP3 in ammasome pathways especially attracted our attention since they have been implicated in mediating chronic pain (Fig.3). These pathways are important for regulating neuron-glial activation, cytokine production, neuroin ammation, oxidative stress, mitochondrial function, apoptosis and autophagy. Based on RNA seq analysis we further validated this pathways by performing western blotting analysis in DRG homogenates.
Effect of CBD, THCV and their combination on PI3K-AKT, P38 MAPkinase and AMPK pathway Western blotting analysis in DRG homogenates of PTX treated mice showed increased expression of PI3K (p<0.001), p-AKT (p<0.001), p-P38 MAP kinase (p<0.001), BAX (p<0.001), TGF-β (p<0.001), NLRP3 in ammasome (p<0.001) and caspase 3 (p<0.001) and decreased expression of p-AMPK (p<0.001), SIRT1 (p<0.001), NRF2 (p<0.001), HO1 (p<0.001), SOD2 (p<0.001) and catalase (p<0.001) when compared to normal control group as shown in g.4. CBD and THCV treatment signi cantly reversed the expressions of these proteins in DRG homogenates (from PTX treated mice) (Fig.4). However the combination of CBD and THCV treatment to PTX induced mice signi cantly reversed the expression of these proteins when compared to CBD and THCV alone treatment groups as indicated in Fig.4. These results suggest that the combination of these drugs would be a superior therapeutic strategy in improving neuropathy against PTX induced pain in mice. Further to understand the role of cannabinoid and non cannabinoid receptors in regulating these pathways after CBD and THCV treatment, we isolated DRGs (L1-L5 region) from rats and cultured them in our laboratory and evaluated the neuroprotective and mitoprotective effects of these drugs in presence of CB1 and 5HT1A receptor blockers. Rationality behind the usage of these blockers was based on the previous literature and on our in vivo results.

Effect of CBD, THCV and their combination on neurite outgrowths of PTX insulted DRG primary cultures
We measured the number of neurite outgrowths/ axon like extensions which were double/more than the diameter of cell body. PTX treated DRG cells signi cantly (p<0.001) reduced the neurite outgrowths and percentage of neurite bearing cells when compared to normal DRG cells (Fig.5).
CBD and THCV at 12 μM concentration signi cantly (p<0.01) improved the neurite outgrowth and percentage of neurite bearing cells when compared to PTX treated primary DRG cells. Interestingly, CBD and THCV combination improved the neurite outgrowths in PTX treated primary DRG cells two folds better than the CBD and THCV alone treatment (Fig.5). However, two hours before CBD and THCV treatment, CB1 receptors and 5HT1A receptors were blocked with WAY100135 (1 μM) (5HT1A antagonist), Rimonabant (1 μM) (CB1 antagonist). CBD and THCV failed to improve neurite outgrowths after blocking 5 HT1A receptors and CB1 receptors respectively as shown in Fig.5. These results suggest that the neuroprotective effects of CBD and THCV depend upon 5 HT1A receptors and CB1 receptors respectively.
Immunoexpression of p-AMPK, Complex I and catalase in treated DRG primary cultures After con rming neuroprotective effects of CBD and THCV in presence of 5HT1A and CB1 blockers, we sought to study the molecular effects of these drugs by blocking the same receptors. In line with the invivo study, PTX induced toxicity in DRG neuronal cells, reduced the immunoexpression of p-AMPK, complex I and Catalase as shown in Fig.6. Nevertheless, CBD and THCV treatment signi cantly increased the expression of these proteins and their combination signi cantly reversed these protein expression in PTX treated DRG neuronal cells as shown in Fig.6. However, CBD and THCV failed to increase the expression of these proteins after blocking with 5HT1A and CB1 receptors respectively. These results signify the importance of CBD and THCV combination in attenuating neuropathic pain and also suggest the mechanism of CBD and THCV in mitigating neuropathic pain against PTX induced toxicity in DRG neurons. Further, we sought to study the mitoprotective effects of these compounds in presence of same blockers.
Effect of CBD, THCV and their combination on mitochondrial membrane potentials and mitochondrial superoxide production in PTX exposed DRG cells JC1 assay explains about the mitochondrial transmembrane potentials (ΔΨm) which directly correlates the integrity and health of the mitochondria. Mitosox staining assay quanti es the mitochondrial superoxide production in cultured cells. Fluorescence imaging assay carried out in cultured DRG primary cells isolated from Rat (L1-L5 region) using mitoprobe JC1 assay kit and Mitosox staining, demonstrated mitochondrial membrane repolarization effects and reduced mitochondrial superoxides with CBD and THCV treatment respectively as shown by concentration dependent formation of red:green uorescent J1 aggregates and decreased Mitosox red uorescence in PTX treated DRG cells (Fig 7). However, CBD and THCV combination therapy have signi cantly (p<0.001) improved mitochondrial membrane potentials and reduced mitochondrial superoxides which is demonstrated by increased JC1 dimers formation (red uorescent J1 aggregates) and decreased Mitosox red uorescence respectively against PTX induced toxicity in DRG primary neuronal cells as shown in Fig 7. Interestingly, mitochondrial membrane potentials and mitochondrial superoxides were unaltered in CBD and THCV treated DRG primary cells after blocking 5HT1A and CB1 receptors respectively. These results would suggest that mitoprotective effects are prominent with CBD and THCV combination and these effects of CBD and THCV depends upon the activation of 5HT1A and CB1 receptors respectively.

Discussion
Chemotherapy-induced peripheral neuropathy (CIPN) is a critical challenge for most cancer patients. For many solid organ cancers, PTX is a mainstay chemotherapeutic drug and it triggers irreversible neurotoxicity, rendering it inappropriate for long-term usage. Currently no speci c treatment is available in clinical settings to reduce the severity of this disease [2]. In this study, we investigated for the rst time, the neuroprotective effects of synthetic CBD, THCV, and their combination in PTX-induced neuropathic mice. Although there have been reports of CBD's impact on PTX-induced neuropathic pain, the molecular mechanism behind their neuroprotective bene ts still remains to be investigated Nonpsychoactive phytocannabinoids constitute a viable therapeutic for treatment of neuropathic pain [33]. CBD-containing gelatin given ad libitum orally for three weeks after surgery dramatically reduced (p<0.01) allodynia in a sciatic nerve damage mouse model [34]. Also, CBD and its modi ed derivatives reduced (p<0.001) thermal and mechanical hyperalgesia in mice and rats after intraplantar injection of 10 µl complete Freund's adjuvant and ligation of the L5 spinal nerve respectively [35]. Further, CBD also inhibited mechanical and thermal allodynia in mice and prevented mechanical sensitivity in investigating PTXinduced neuropathic pain [36]. Despite CBD's importance in the treatment of neuropathic pain, THCV has also been shown to have similar effects. For example, in mice, THCV reduced the thermal and mechanical hyperalgesia generated by formalin and carrageenan [37]. In line with earlier studies, current study also demonstrated that synthetic CBD and THCV treatment reduced thermal and mechanical hyperalgesia against PTX induced neuropathic mice. Interestingly, combining these two drugs proved to be more effective in providing neuroprotection against PTX-induced neuropathy in mice (P<0.01).
PTX has been shown to damage peripheral sensory neurons, including dorsal root ganglions, and dorsal root ganglions axons, as shown by a number of studies in PTX-induced peripheral neuropathy [38,39]. In the current study, PTX treatment induced damage in isolated DRGs from mice, as demonstrated by decreased neurite outgrowths and the quantity of neurites carrying cells, as well as a change in neuron morphology. On the other hand, CBD and THCV therapy restored all of the PTX-induced alterations in the neurons with higher e cacy when used together.
RNA sequencing (KEGG analysis) in DRG homogenates of neuropathic mice treated with CBD and THCV revealed differences in gene expression levels, indicating the involvement of p38 MAPkinase, AMPK, PI3-AKT, autophagy, oxidative phosphorylation, retrograde endocannabinoid signaling, GABAmergic, glutamergic and dopamergic synapse, axon guidance and in ammatory pathways. These pathways are linked to mitochondrial biogenesis and function, oxidative stress, autophagy, apoptosis, ER stress, and neuroin ammation [40,41]. Although similar transcriptome ndings with PTX injury in DRG neurons have previously been published [42], the RNA seq data with CBD and THCV therapy in neuropathic mice is unique and is being reported for the rst time.
The modulation of nociceptive information induced by PTX exposure in DRGs is well established to involve MAPK activation pathways [43]. MAPKs are a family of serine/threonine protein kinases that have been implicated in different aspects of cell communication and gene expression in the peripheral nervous system [44,45]. In ammation-induced pain hyperalgesia is considered to be modulated by MAPKs in DRGs and the spinal cord. P38 MAPK is activated, and expression levels in the spinal dorsal horns are increased following peripheral nerve damage [46]. P38 MAPK has been shown to stimulate several in ammatory pathways, including the generation of in ammasomes, and initiate apoptosis in the cellular milieu by activating Bax and caspases [47]. In the current study, we observed an increase in p-p38 MAPKinase expression, NLRP3 in ammasome formation, TGF beta expression, and BAX expression following PTX assault, which is consistent with earlier results. Surprisingly, CBD and THCV therapy lowered the expressions of these proteins, but the reduction in protein expressions with their combination was two times greater than with each treatment alone. In another study, CBD delivery intranasally and intravenously reduced type 1 diabetic neuropathic pain and suppressed microglial density and phosphorylation of p38 mitogen-activated protein kinases [48], thus corroborating our ndings. Moreover, THCV has been shown to diminish in ammation caused by formalin and carrageenan in rodents, and a number of studies have observed that p-p38 MAPKinase is important in regulating in ammation caused by formalin and carrageenan in rats [49]. These studies and our ndings suggest that phytocannabinoids' suppression of p-p38 MAPkinase is important in reducing PTX-induced neuropathic pain in mice.
On the other hand, we observed an increase in PI3K and AKT protein expression in PTX-treated DRGs isolated from rodents, which has been previously documented [50]. These protein expressions were reduced in CBD and THCV-treated mice, and the decrease was even greater (p<0.001) in their combined treatment. According to a growing body of evidence, blocking the PI3K/Akt signaling pathway has been shown to be analgesic in neuropathic pain models [51]. According to the ndings, activation of PI3K and PI3K/Akt appears to be implicated in the progression of PTX-induced neuropathic pain. Previous research has also documented that the PI3K and PI3K/Akt signaling pathways are critical in modulating the actions of in ammatory markers, including NLRP3 in ammasome, IL-1, and TNF-α, which are signi cant in neuropathic pain [52]. Similarly, the current study observed increased NLRP3 in ammasome production in DRG homogenates of PTX-treated mice, which was reduced by CBD, THCV, and their combination treatment; however the combination showed higher e cacy. With these ndings, we can deduce that PTX-induced activation of the P-38 MAPKinase, PI3K, and PI3K/Akt signaling pathways may cause changes in in ammatory markers and contribute to the development of neuropathic pain, while CBD, THCV, and their combination treatments reduce neuropathic pain by regulating these signaling pathways (Fig. 8).
AMP-activated protein kinase (AMPK) is an energy-sensing kinase that can block mitogen-activated protein kinase (MAPK) signaling, which has been associated to pain enhancement following injury and the development of hyperalgesic priming [53]. Of note, Inyang et al., observed that PTX administration induced mechanical hypersensitivity in both male and female mice, which was counteracted by administering AMPK activators [53]. In agreement with previous studies, current study revealed that AMPK was signi cantly downregulated in PTX-treated DRGs when compared to the normal control group. Despite this, phytocannabinoids can boost AMPK activation through a variety of signaling pathways, which can help with appetite and heart function [54]. Similarly, we observed that CBD and THCV treatment elevated p-AMPK expression, and that their combination was superior in this effect, suggesting that combination therapy may provide additive to synergistic neuroprotection against PTX-induced neuropathy in mice which however has to be further investigated. Nrf2 is known to be activated by p-AMPK and plays a key role in regulating endogenous antioxidant defense by regulating the transcription of downstream target genes such as heme oxygenase-1 (HO-1), superoxide dismutase, glutathione reductase, and NAD(P)H: quinone oxidoreductase 1 [55]. As a result, Nrf2 activators reduced PTX-induced neuropathic pain in experimental animals [56]. Moreover, CBD's antioxidant potential has been established via its effect on the activity of the Nrf2 transcription factor, which is involved in the development of cytoprotective proteins such as antioxidant enzymes [57]. In this work, we also discovered that neuropathic mice with CBD and THCV treatment had elevated expressions of Nrf2, HO1, and catalase in DRGs, and that these protein expressions were considerably higher (p<0.001) in the combination treatment. This data implies that when these phytocannabinoids are combined, their antioxidant capacity increases in reducing the neuropathic pain (Fig. 8).
Further, by activating TFAM, p-AMPK reduced mitochondrial functional de ciencies in various animal and cell culture models of diseases by increasing mitochondriogenesis and respiratory capacity [58,59]. TFAM is a nuclear protein that binds to the mitochondrial genome and controls the transcription of subunits of mitochondrial complexes [60]. Overexpression of TFAM has also been shown to have protective effects in neuropathological disorders such as age related hearing loss, amyotrophic lateral sclerosis, alzheimer disease, and memory loss in animal systems. CBD, THCV, and their combination treatment improved TFAM levels(p<0.01 to 0.001) in DRG homogenates of PTX-induced neuropathic mice in the current investigation [61]. Immunocytochemistry investigations in DRG primary cultures demonstrated a rise in mitochondrial complex I subunits and JC1 labeling suggesting mitochondrial repolarisation or restoration of mitochondrial membrane potentials. These ndings imply that CBD and THCV increase mitochondrial function via activating the AMPK-Nrf2-TFAM axis, and that their combination is more effective than either medication alone.
THCV is a non-psychoactive, neutral CB1 antagonist/reverse agonist that can act as an agonist or antagonist at CB2 receptors depending on the dose [18].
When THCV was given to genetically obese mice at doses of 0.1-12.5 mg/kg once daily for 45 days, the total fat content was reduced by 31.1 percent when compared to the obese control group [12]. It was observed that THCV had a high a nity for CB1 receptors and high brain penetration, resulting in metabolically favorable effects that are typical of CB1 receptor inverse agonists. Further, THCV has been demonstrated to improve insulin sensitivity and reduce obesity in diet-induced obese mice models via altering metabolic processes through interacting with TRPV1 ion channels [62]. However, in the current investigation, we discovered that administering a selective CB1 blocker (rimonabant) to PTX-induced neuropathic mice prevented the antinociceptive effects of THCV. Furthermore, we observed that inhibiting CB1 receptors with Rimonabant in DRG primary cultures also prevented THCV-induced increases in AMPK, complex I, and catalase expression. These ndings suggest that THCV modulates mitochondrial bioenergetics via the CB1/AMPK/nrf2/TFAM axis and the exact process by which THCV interacts with CB1 receptors still needs more investigations.
Our research also demonstrated that CBD treatment, when combined with a 5HT1A receptor blocker, it had no effect on nociception caused by PTX injection in mice. CBD provided protection against STZ-induced diabetes pain by speci cally activating 5HT1A receptors. Pascual et al. observed that the CB1/CB2 dual agonist WIN55,212-2 decreased the thermal hyperalgesia and tactile allodynia elicited by PTX in rats, and that this action was prevented by the CB1 antagonist SR141716, implying that the CB1 receptor is involved [63,64]. Another study suggested that injecting CBD into the infralimbic cortex of the rat brain reduced fear by activating CB1 receptors, as determined by lower levels of freezing during an extinction test [65]. However, neither CB1 nor CB2 receptors are involved in CBD's ability to reduce neuropathic pain, according to Segrado et al. and Sara et al [66,67]. Furthermore, despite its indirect activation of CB1/CB2 receptors via increasing endocannabinoid levels, very few studies have shown that CBD has a lower binding a nity for CB1 or CB2 receptors. In this study, we also observed that CBD's anti-nociceptive effects are not dependent on CB1 activation, but rather on 5HT1A receptor activation, which is consistent with earlier results. Additionally, 5HT1A receptors have been shown to be important in the phosphorylation of CaMKII, an upstream regulator of the AMPK pathway [68]. As a result of the activation of 5HT1A receptors, CBD may have the capacity to regulate mitochondrial bioenergetics, redox, and in ammatory homeostasis in neuronal cells by regulating AMPK pathway. Because CBD and THCV work in distinct ways, our study demonstrates that combining them results in entourage neuroprotective advantages.

Conclusion
Synthetic CBD and THCV showed neuroprotective bene ts in PTX-induced neuropathic mice, as measured by a variety of neurobehavioral characteristics. MAPkinase, PI3K-AKT, AMPK, and in ammasome pathways, along with mitochondrial function-related genes, were observed to be involved in the pathogenesis of PTX-induced neuropathy by transcriptome analysis of DRG homogenates from diverse treatment groups of mice. The neuroprotective effects of CBD and THCV in mouse/rat DRGs are dependent on the modulation of 5HT1A and CB1 receptors, respectively, in reducing PTX-induced neuropathic pain. The combination of CBD and THCV has been shown to provide better neuro and mitoprotection against PTX-induced insult than either treatment alone.    The effects of CBD, THCV, and its combination on the p38 MAPKinase, AMPK, and PI3-AKT pathways.
(a) Western blots of DRG homogenates from PTX-treated mice demonstrate CBD, THCV, and their combination therapy for six weeks after the nal PTX dose.   Respective quanti cation of red: green uorescence ratio indicating depolarized mitochondria and (c) respective mitosox staining red orescence quanti cation indicating the mitochondrial superoxides production in different groups (n=3). ***p<0.001 Vs normal control, ^p<0.05, ^^p<0.01 and ^^^p<0.001 Vs PTX. Transforming growth factor beta.