Low Doses of β-Caryophyllene Reduced Clinical and Paraclinical Parameters of an Autoimmune Animal Model of Multiple Sclerosis: Investigating the Role of CB2 Receptors in Inflammation by Lymphocytes and Microglial

Multiple Sclerosis (MS) is a prevalent inflammatory disease in which the immune system plays an essential role in the damage, inflammation, and demyelination of central nervous system neurons (CNS). The cannabinoid receptor type 2 (CB2) agonists possess anti-inflammatory effects against noxious stimuli and elevate the neuronal survival rate. We attempted to analyze the protective impact of low doses of β-Caryophyllene (BCP) in experimental autoimmune encephalomyelitis (EAE) mice as a chronic MS model. Immunization of female C57BL/6 mice was achieved through two subcutaneous injections into different areas of the hind flank with an emulsion that consisted of myelin Myelin oligodendrocyte glycoprotein (MOG)35–55 (150 µg) and complete Freund’s adjuvant (CFA) (400 µg) with an equal volume. Two intraperitoneal (i.p.) injections of pertussis toxin (300 ng) were performed on the animals on day zero (immunizations day) and 48 h (2nd day) after injection of MOG + CFA. The defensive effect of low doses of BCP (2.5 and 5 mg/kg/d) was investigated in the presence and absence of a CB2 receptor antagonist (1 mg/kg, AM630) in the EAE model. We also examined the pro/anti-inflammatory cytokine levels and the polarization of brain microglia and spleen lymphocytes in EAE animals. According to our findings, low doses of BCP offered protective impacts in the EAE mice treatment in a CB2 receptor-dependent way. In addition, according to results, BCP decreased the pathological and clinical defects in EAE mice via modulating adaptive (lymphocytes) and innate (microglia) immune systems from inflammatory phenotypes (M1/Th1/Th17) to anti-inflammatory (M2/Th2/Treg) phenotypes. Additionally, BCP elevated the anti-inflammatory cytokine IL-10 and reduced blood inflammatory cytokines. BCP almost targeted the systemic immune system more than the CNS immune system. Thus, a low dose of BCP can be suggested as a therapeutic effect on MS treatment with potent anti-inflammatory effects and possibly lower toxicity.


Introduction
Multiple Sclerosis (MS) is a prevalent inflammatory problem with special interfacing with the immune system that leads to inflammation and demyelination of neurons and axonal loss in the central nervous system (CNS) [1]. As categorized by the World Health Organization (WHO), MS is a debilitating problem that influences above one million patients globally, with a higher incidence among women (female/male ratio = 3/1) [1,2]. Despite the unclear pathophysiology mechanism of MS, some research works suggest that

Animals and Husbandry
Salari Institute of Cognitive and Behavioral Disorders (SICBD) provided female C57BL/6 mice (18-20 g, 8-10 weeks old). Afterward, the mice were maintained in the animal lab of the Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences. The room temperature where the mice were kept was controlled (22-25 • C), and 50% relative humidity and a 12/12 h light/dark cycle were applied. Animals all could freely access commercial standard rodent chow and safe and clean filtered tap water. In all procedures and experiments, the NIH's Guideline for the Care and Use of Laboratory Animals was approved by the Animal Ethics Committee of Mashhad University of Medical Sciences (No. 960037, IR.MUMS.fm.REC.1396.200).

Induction of EAE and Clinical Evaluation
Immunization of female C57BL/6 mice was achieved through two subcutaneous injections into different areas of the hind flank with an emulsion that consisted of myelin MOG  (150 µg) and CFA (400 µg) with an equal volume. Two intraperitoneal (i.p.) injections of pertussis toxin (300 ng) were performed on the animals on day zero (immunization day) and 48 h (2nd day) after injection of MOG + CFA in the Salari Institute of Cognitive and Behavioral Disorders (SICBD). The EAE induction procedure is summarized Brain Sci. 2023, 13, 1092 4 of 53 in Figure 1A. The weight of mice was obtained daily, and clinical scoring was performed from day 0 to day 37 (the experiment's last day). The procedure of clinical scoring was performed with the help of two independent researchers so that one of them was blind to the process of experiments and also grouping. The descriptions of Bittner et al. [28] were followed in the clinical scaling score of EAE, which is presented in Table 1. Also, animals were examined for the MDO (days after immunization), incidence (sick/total), MMCS (during the whole treatment course), CDI (total disease score throughout the experiment), and MCS (at the peak day).

Experiment One
The protocol was designed to evaluate the protective effects of low BCP doses (2.5, 5 mg/kg) in the EAE model (Table 2). To examine the treatment effects of BCP (2.5 and 5   [28].

Score Clinical Manifestations (Significant Comment) 0
Without clinical symptoms 1 Partly limp tail (a tip of the tail droops, normal gait) 2 Paralyzed tail (tail droops, normal gait) 3 Uncoordinated movement, hind limb paresis (uncoordinated gait, hind limbs responding to pinching, tail limps) 4 One paralyzed hind limb (uncoordinated gait with dragging one hind limb, one hind limb not responding to pinch, tail limps) 5 Two paralyzed hind limbs (uncoordinated gait with both hind limbs dragging, two hind limbs not responding to pinch, tail limps) 6 Hind limbs paralyzed, weak forelimbs (uncoordinated gait with forelimbs struggling to pull body, tail limps, forelimbs reflex following pinching) 7 Hind limbs paralyzed, one paralyzed forelimb (no movement of the mouse, one forelimb responding to a toe pinch, tail limps) 8 Hind limbs paralyzed, both forelimbs paralyzed (no movement of the mouse, both forelimbs do not respond to a toe pinch, tail limps) 9 Moribund (worsened breathing, no movement) 10 Death

Experiment One
The protocol was designed to evaluate the protective effects of low BCP doses (2.5, 5 mg/kg) in the EAE model (Table 2). To examine the treatment effects of BCP (2.5 and 5 mg/kg doses), BCP (diffused in corn oil) was used as oral administration once per day from ten days after immunization, showing significant increases in the clinical score compared to the sham group (n = 8/each group, Figure 1A). The BCP doses were selected given our earlier in vitro tests concerning the BCP (oligodendrocytes [15] and microglia [14]) and earlier in vivo surveys concerning the BCP's protective effects (25,50 mg/kg/day [25,26]) in EAE. Accordingly, we carried out our experiments with one-tenth of the doses that were previously for BCP. DMF was regarded as a positive control group. It is a standard used orally, and the FDA has proven it for MS patients. DMF-group took 30 mg/kg DMF (diffused in 0.08% w/v methylcellulose) that was received twice daily per oral gavage (n = 8) [29,30]. In the control group, EAE mice received identical vehicle volume (n = 8). The sham group had normal mice receiving the same amount of PBS injections rather than EAE induced by MOG  .

Experiment Two
This procedure was performed to evaluate the possible role of the CB 2 receptor in the protective effects of BCP (Table 2). AM630 (1 mg/kg; i.p., [31][32][33], dissolved in 2% DMSO), as a pharmacological CB 2 receptor antagonist, was administrated 30 min before the BCP treatment (5 mg/kg; p.o., n = 8, showed the best protection in experiment one). As the control group, EAE mice took the identical volume of all vehicles, including neither BCP nor AM630 medications (n = 8). The dose of AM630 as a highly selective antagonist of the CB 2 receptor (Ki = 31.2 nM) was chosen based on previous studies indicating that the dose of 1 mg/kg of AM630 was pharmacologically optimal, devoid of any in vivo toxicity and also exerted no behavioral and molecular effects when used alone [31][32][33].

Histological Evaluation
When sacrificing (day 37), in order to evaluate the CNS inflammation level, xylazine (10 mg/kg) and ketamine (100 mg/kg) were used for the deep anaesthetization, and then they were sacrificed. Then, isolation of the spinal cord was performed, and it was kept in 4% (v/v) paraformaldehyde. Hematoxylin and Eosin (H&E) were used for staining Longitudinal sections (5 µm thick) of the spinal cords' paraffin-embedded to evaluate inflammation. Afterward, Sections were scored by light microscopy in a blinded way (by analyzing three fields) at 100× magnification (Olympus, Tokyo, Japan). Based on the previous works, the pathological scores were obtained as follows: 0-no inflammation; 1-low inflammatory cell levels; 2-perivascular infiltrates; and 3-the extended intensity of perivascular cuffing with expanding to contiguous tissue.

Serum Preparation and Inspection of the Cytokines Profile
On day 37 (the last day of the experiment), after the deep anesthesia, using cardiac puncture, 1.5 mL of blood was taken and collected in a 2 mL EDTA-coated microtube that contained aprotinin (140 µg). The samples were immediately centrifuged for 10 min at 4 • C at 2500× g. Then, we collected the plasma and kept it at −20 • C for the next experiment. The anti-inflammatory (IL-10) and inflammatory (IL-17, TNF-α, IL-6) cytokine levels were specified by the ELISA kits considering the instruction of the manufacturer, and were presented as pg per mL of plasma.

Separation of Lymphocyte from Spleen and Cell Culture
After the euthanasia of animals, we eliminated the spleen and put it into a tube (15 mL) with 5 mL ice-cold RPMI-1640/FBS (RPMI-1640, streptomycin (100 µg/mL), penicillin (100 U/mL), 2% v/v FBS). The spleen was then cut into slices by ice-cold RPMI-1640/FBS in the 75 µm cell strainer mesh to provide a high-quality cell suspension. The resulting cell suspension was centrifuged for 10 min at 4 • C at 45. Then, the supernatant was eliminated without disturbing the pellet [34]. Thereafter, sterilized PBS (15 mo) was mixed with the cell pellet, followed by pouring the suspension onto Ficoll ® (15 mo) in a 50 mL sterilized conical tube. It was then centrifuged at 400 g for 20 min at a controlled temperature (22-25 • C). After carefully removing the buffy coat layer, it was washed with buffer phosphate saline thrice for 10 min at 130 g. In order to eliminate the remaining red blood cells, we cocultured the cells with 1× diluted RBC lysis buffer (10×) based on the procedure proposed by the manufacturer, and the supernatant was aspirated without agitating the pellet. To exclude monocytes, pellets were put into a supplemented RPMI-1640 (100 U/mL penicillin, 2 mM L-glutamine, 10% FBS, 1 mM sodium pyruvate, 100 µg/mL streptomycin, HEPES buffer), followed by culturing in flasks (250 mL) for one night. Then, the supernatant, with lymphocytes, was placed into a sterilized conical tube (50 mo) [34,35]. Isolated lymphocytes were cultured in RPMI-1640 enriched with 10% v/v of heat-inactivated FBS, 1% v/v of pen/strep (100×), 2 mM L-glutamine, and 0.5 µg/mL amphotericin B. Cells were kept at 37 • C in a humidified incubator and 5% v/v CO 2 . For culturing Lymphocytes, 96-well plates were used (5 × 10 4 cells/well), and their treatment was performed with medium + MOG  peptides (30 µg/mL, [36]) or medium alone at 37 • C for 48 h and 5% v/v CO 2 . In the last 24 h, BrdU labeling solution (0.1% v/v) was mixed with the plates. Considering the manual provided by the manufacturer, a cell proliferation ELISA BrdU kit (colorimetric) was used to assess cell proliferation. The microplate reader was used for reading the absorbance at 450 nm. The mean cell proliferation level of the MOG 35-55 shame group was 100%.

Examination of the Cytokines Profile
For providing a deeper understanding of the impact of various treatments on the Th 17 /T reg ad Th 1 /Th 2 polarization in EAE, the Th 1 (IFN-γ), Th 2 T reg (IL-10, IL-4, TGF-β 1 ), and Th 17 (IL-17) levels were evaluated related to cytokines. In addition, inflammatory cytokines TNF-α and IL-6 were also assessed in the supernatant of lymphocytes stimulated by MOG  . Thus, 12-well plates were used for culturing the isolated lymphocytes (2 × 10 6 cells/well), and they were treated with medium+MOG 35-55 peptides (30 µg/mL) or medium alone at 37 • C for 48 h and 5% v/v CO 2 . Then, after collecting the supernatants, they were maintained at −20 • C to evaluate the cytokine levels using the ELISA technique. The cytokine levels were standardized and presented as pg/mg protein. Bradford's method was used to measure each experiment's total protein content [37].

Evaluation of Intracellular Levels of Transcription Factors
In order to confirm the polarization of lymphocytes, the levels of valid and specific transcription factors for Th 1 (T-bet), Th 2 (GATA3), T reg (Foxp3), and Th 17 (ROR-γt) cells were assessed [5,38]. To this end, we cultured the separated lymphocytes (2 × 10 6 cells/well) in plates with 12-wells, followed by treatment with medium + MOG 35-55 peptide (30 µg/mL, [36]) or medium alone at 37 • C for 48 h and 5% v/v CO 2 . Then, lymphocytes were centrifuged to provide the pellets, and they were rinsed three times with PBS. The pellets were then suspended again into the cell lysis buffer (10 mM HEPES; 10 mM KCl, pH 7.5, 0.1 mM EDTA, 0.5% Nonidet-P40, 1 mM DTT, 0.5 mM PMSF with the protease inhibitor cocktail) and kept for 15-20 min with recurrent vortexing. Also, total protein contents were obtained using Bradford's method [37]. We stored separated proteins at −20 • C to evaluate the transcription factors' levels according to the ELISA procedure given in the manufacturer's guide. The levels of transcription factors were presented as pg/mg or ng protein.

Microglia Isolation
The approach provided by Lee and Tansey [39][40][41] with some modifications was used for isolating Microglial cells. After mice decapitation, mice brains were separated from meanings, undergoing enzymatic and mechanical dissociations by papain (1 mg/mL as the end concentration), dispase II (1.2 U/mL as the end concentration), and DNAse I (20 U/mL as the end concentration). The sliced brain was placed in a 15 mL tube for 20 mon, which contained the enzymes (3 mL) in the cell culture incubator. Then, the enzymes were neutralized with a complete medium (5 mL), and the cells were centrifuged at 250× g for 5 min at a controlled temperature. Following this process, the supernatants were eliminated. The pellets were suspended again into 4 mL per brain of 37% SIF (9 parts of Ficoll with 1 part 10× HBSS) and poured onto 4 mL of 70% SIF. A total of 30% SIF (4 mL) was pipetted slowly over the 37% SIF layer, and then 2 mL of HBSS was added. The mix was centrifuged at 300× g for 40 min at 18 • C with no brake. A sterilized 15-mL conical tube was used for collecting the 70-37% interphase. A total of 6 mL of HBSS per 2 mL of the obtained interphase was poured into the tube to the interphase Ficoll was thinned and centrifuged at 4 • C at 500× g for 7 min. Isolated microglia cells were cultured in the enriched DMEM (10% v/v of heat-inactivated FBS, 1% v/v of pen/strep (100×), 0.5 µg/mL amphotericin, Brain Sci. 2023, 13, 1092 8 of 53 2 mM L-glutamine). Cells were maintained in a humidified incubator at 5% v/v CO 2 and 37 • C.

Cell Proliferation Assay
Following culturing Microglia cells (8 × 10 3 cells/well) in 96-well plates, their treatment was performed with medium + MOG  peptide (30 µg/mL, [36]) or medium alone at 37 • C for 48 h at 5% v/v CO 2 . In the last 24 h, we added BrdU labeling solution (0.1% v/v) to the plates. A cell proliferation ELISA-based BrdU kit (colorimetric) was used based on the manufacturer's guide for assessing cell proliferation. The microplate reader was utilized for reading the absorbance at 450 nm. The mean cell proliferation level of the MOG 35-55 shame group was 100%.

Evaluation of the Cytokine Levels
The ELISA-based technique was used to measure levels of inflammatory and antiinflammatory (IL-10) and (PGE 2 , TNF-α cytokines. After culturing the cells in plates with 6-wells (10 6 cells/per well), they were incubated with medium + MOG 35-55 peptides (30 µg/mL, [36]) or medium alone at 37 • C for 48 h in a 5% v/v CO 2 incubator. Lastly, the levels of cytokines were measured by collecting the supernatants. The cytokine levels were presented as pg/mg protein.

Evaluation of the Intracellular Levels of Arg-1 and iNOS
Considering the manufacturer's guide, a commercial ELISA-based kit was used to assess Intra-cellular levels of Arg-1 and iNOS (as indexes of M2 and M1 cells). Following culturing the cells in a 6-well plate (2 × 10 6 cells/each well), they were incubated with medium+MOG 35-55 peptides (30 µg/mL, [36]) or medium alone at 37 • C for 48 h in a 5% v/v CO 2 incubator. The cells were collected and lysed by the use of a lysis buffer. Afterward, the cells were homogenized (DIAX 100, Germany) for 2-3 min in the cold water (0-4 • C) with vortexing (every 30 s). The cells were centrifuged for 10 min at 12,000× g at 4 • C, and 50 µL of supernatants were evaluated. The Arg-1 and iNOS levels were presented as ng/mg protein.

Evaluation of Urea and NO Levels
The supernatants of cultured microglia were used for measuring the levels of urea (generated by M 2 cells) and NO metabolites (generated by M 1 cells). The Griess technique [42] was used to examine the NO production level. Briefly, we incubated 50 µL of the supernatant with an identical volume of n-(1-naphthyl)-ethylenediamine and sulfanilamide in 2N hydrochloric acid for 10 min at the controlled temperature. A microplate reader was used for reading the absorbance at 540 nm. By the use of the sodium nitrite standard curve [43,44], the NO concentration was specified.
A commercial urea assay kit was used to determine urea levels. Shortly after, 50 µL of supernatant and equal volumes of medium (blank) and 50 µL urea-solution (5 mg/dL or 850 µM) were mixed and incubated with the 200 µL working reagent for 50 min at a controlled temperature. The Stat Fax 2100 Awareness microplate reader was utilized for reading the absorbance at 430 nm. The urea and NO levels were given as nmol/mg protein [43,44].

Statistical Analysis
GraphPad Prism ® 6 software (San Diego, CA, USA) was employed for data analysis, and data were presented in terms of their none-parametric or parametric nature as median ± range (for pathological score) or means ± SEM. For parametric data, normality tests were conducted based on Bartlett's and Kolmogorov-Smirnov tests, which examine the homogeneity of variances. In case of passing the test, two groups were compared using an ANOVA test, following Dunnett's post hoc multiple comparisons test. Following Tukey's multiple comparison tests, a two-way ANOVA test was performed for body weight and a Brain Sci. 2023, 13, 1092 9 of 53 clinical score. Moreover, Kruskal-Wallis and Dunn's post hoc multiple comparisons tests analyzed no parametric data. P-values (p) were statistically at p ≤ 0.05, 0.01, and 0.001. There was compliance between statistical analysis and data with the instructions on experimental design, analysis [45], data presentation, and sharing in preclinical pharmacology [46,47].

BCP Effect on Body Weight and the Clinical Score of EAE Mice
As illustrated in Figure 2, the clinical score level of the vehicle EAE group showed a significant elevation in comparison to the sham group (p < 0.001). DMF treatment (60 mg/kg; p.o.) notably decreased the clinical score level compared to the vehicle group (p < 0.001, Figure 2A). BCP (2.5-5 mg/kg/d; p.o.) markedly reduced the clinical score level in EAE mice compared to the vehicle group in a dose-dependent fashion (p < 0.001 for both modes, Figure 2A). The BCP (5 mg/kg) group showed a significantly lower clinical score level compared to the DMF group (60 mg/kg) ( Figure 2A). BCP effect on the clinical score level in EAE in the presence of AM6330 (1 mg/kg; i.p.) was totally abrogated (p < 0.001, Figure 2A).
As reported in Table 3, the vehicle group developed EAE, showing the initial signs of the onset of disease with a partially limp tail on day 6.72 ± 0.42, attaining an MCS of 7.75 ± 0.47 on day 17. In the BCP-treated group (2.5 and 5 mg/kg/d), the incidence of the disease was decreased to 58.3 and87.5% (p < 0.001), and disease severity was markedly attenuated, with MCS 3.75 ± 0.18 (p < 0.001) and 2.75 ± 0.43 (p < 0.001) on day 17 compared to the vehicle group (7.75 ± 0.47), respectively (Table 3). In addition, DMF (60 mg/kg/d, positive control) decreased the disease incidence to 87.5% and also significantly reduced the MCS to 3.43 ± 0.31 (p < 0.001) on day 17 in comparison with the vehicle group ( Table 3). As illustrated by our findings, the concomitant use of AM630 (1 mg/kg/d) along with BCP (5 mg/kg/day) resulted in the effects of BCP (5 mg/kg) on disease incidence and MCS on day 17 were completely reversed (p < 0.001 for both cases) and did not present a significant difference with the vehicle group (Table 3).   The body weight of vehicle-treated EAE mice reflected a significant decline in comparison with the sham group (p < 0.001, Figure 2B). Treating with BCP (2.5 and 5 mg/kg/d) or DMF (60 mg/kg/d) caused a significant elevation in the body weight of EAE mice compared to the vehicle group (p < 0.001 for all modes, Figure 2B). The body weight of the BCP group (2.5, 5 mg/kg) was significantly more significant than the DMF group in EAE mice (p < 0.001 for all modes, Figure 2B). In the AM630 presence (1 mg/kg), the BCP effect (5 mg/kg) on the body weight was considerably eliminated compared to the BCP-treated (5 mg/kg) alone group (p < 0.001, Figure 2B).

The BCP Effects on the Level of the Histopathological Score in EAE Mice
The present study noted a prominent and massive leukocyte infiltration with multiple foci of inflammation in the vehicle group compared with the sham group (p < 0.001, Figure 3A,C). In DMF-treated (60 mg/kg/d) group, inflammation, and leukocyte infiltration were lower than in the vehicle group (p < 0.05, Figure 3A,D). BCP treatment (2.5 and 5 mg/kg/d) caused a significant decline in the level of the pathological score and the number of inflammatory cell infiltration into the spinal cord compared to the vehicle group (p < 0.05 and p < 0.001, Figure 3A,E,F). Nevertheless, the two doses of BCP did not show a significant difference. The addition of AM630 (1 mg/kg/d) before BCP (5 mg/kg/d) caused a considerable leukocyte infiltration into the spinal cords with multiple foci of inflammation compared to the BCP-treated alone group(5 mg/kg/d) (p < 0.001, Figure 3A,G), and no difference was noted compared to the vehicle-treated group.
Furthermore, our findings indicated that BCP (2.5, 5 mg/kg) generated a significant decline in the TNF-α levels (p < 0.001 for both modes, Figure 4A), IL-6 (p < 0.001 for both modes, Figure 4B), and IL-17 (p < 0.001 for both modes, Figure 4C), and IL-17/IL-10 ratio (p < 0.001 for both modes, Figure 4E) compared with the vehicle group. The IL-10 level in the BCP-treated group (2.5, 5 mg/kg/d) was meaningfully improved compared with the vehicle group (p < 0.01 for BCP 2.5 mg/kg, and p < 0.001 for BCP 5 mg/kg, Figure 4D). We observed that there is a meaningful difference between the effects of 2.5 and 5 mg/kg of BCP in EAE mice (p < 0.001 for TNF-α, IL-6, and IL-17, and p < 0.05 for IL-10, Figure 4A-D). In the presence of AM630 (1 mg/kg), the effects of BCP (5 mg/kg) on all measured parameters were utterly reversed compared with the BCP-treated alone group (5 mg/kg) (p < 0.001 for all modes, Figure 4A-E), and did not differ to vehicle group. tion were lower than in the vehicle group (p < 0.05, Figure 3A,D). BCP treatment (2.5 and 5 mg/kg/d) caused a significant decline in the level of the pathological score and the number of inflammatory cell infiltration into the spinal cord compared to the vehicle group (p < 0.05 and p < 0.001, Figure 3A,E,F). Nevertheless, the two doses of BCP did not show a significant difference. The addition of AM630 (1 mg/kg/d) before BCP (5 mg/kg/d) caused a considerable leukocyte infiltration into the spinal cords with multiple foci of inflammation compared to the BCP-treated alone group(5 mg/kg/d) (p < 0.001, Figure 3A,G), and no difference was noted compared to the vehicle-treated group.  ; 0-without inflammation; 1-low inflammatory cell levels, 2-perivascular infiltrates; and 3-the extended intensity of perivascular cuffing with extension into contiguous tissue. Data were presented as mean ± range, n = seven animals per group, three to four fields of view per animal for each experiment protocol. Considering that data has non-parametric nature, data analysis was performed using the Kruskal-Wallis test and Dunn's post hoc multiple comparisons test. (*) indicates the comparison between varying doses of BCP and DMF groups and the vehicle group, *: p < 0.05, and ***: p < 0.001.; ( + ) makes a comparison between the vehicle and sham groups, +++ : p < 0.001.; # makes comparisons between BCP to DMF group in each graph, # : p < 0.05.

The BCP Effects on Serum Levels of Cytokines in EAE Mice
According to our findings, the inflammatory cytokine levels, including TNF-α (p < 0.001, Figure 4A), IL-6 (p < 0.001, Figure 4B), IL-17 (p < 0.001, Figure 4C), and IL-17/IL-10 ratio (p < 0.001, Figure 4E) were significantly higher in the vehicle-treated group compared to the sham group. On the contrary, the IL-10 level (p < 0.001, Figure 4D) was significantly ; 0-without inflammation; 1-low inflammatory cell levels, 2-perivascular infiltrates; and 3-the extended intensity of perivascular cuffing with extension into contiguous tissue. Data were presented as mean ± range, n = seven animals per group, three to four fields of view per animal for each experiment protocol. Considering that data has non-parametric nature, data analysis was performed using the Kruskal-Wallis test and Dunn's post hoc multiple comparisons test. (*) indicates the comparison between varying doses of BCP and DMF groups and the vehicle group, *: p < 0.05, and ***: p < 0.001.; ( + ) makes a comparison between the vehicle and sham groups, +++ : p < 0.001.; # makes comparisons between BCP to DMF group in each graph, # : p < 0.05.

The Effects of BCP on the Levels of Transcription Factors T-Bet (Th 1 ), ROR-γt (Th 17 ), Foxp3 (T reg ), and GATA3 (Th 2 ) of EAE Mice Lymphocytes with and without MOG Stimulation
Without MOG stimulation, spleen lymphocytes of the vehicle group significantly expressed higher levels of ROR-γt (Th 17 , p < 0.001, Figure 6C), T-bet (Th 1 , p < 0.001, Figure 6A), and the ratios of ROR-γt/Foxp3 (Th 17 /T reg , p < 0.001, Figure 6F) and Tbet/GATA3 (Th 1 /Th 2 , p < 0.001, Figure 6E) compared to spleen lymphocytes of the sham group. However, the levels of GATA3 (Th 2 , p < 0.001, Figure 6B) and Foxp3 (T reg , p < 0.001, Figure 6D) in the spleen lymphocytes of the vehicle group showed a markedly lower level compared to the sham group. In addition, according to our results, treatment with BCP (2.5 and 5 mg/kg) and DMF (60 mg/kg) showed a reduction in the levels of ROR-γt (p < 0.001 for all modes, Figure 6C) and T-bet (p < 0.01 for all modes, Figure 6A), and the ratios of ROR-γt/Foxp3 (p < 0.001 for all modes, Figure 6F) and T-bet/GATA3 (p < 0.001 for all modes, Figure 6E) in spleen lymphocytes compared to vehicle-treated group. Additionally, it was found that levels of Foxp3 (p < 0.001 for both cases, Figure 6D) and GATA3 (p < 0.001 for both cases, Figure 6B) were markedly increased by BCP (2.5 and 5 mg/kg) in comparison with the vehicle-treated group, while DMF (60 mg/kg) caused a decline in the Foxp3 level (p < 0.001, Figure 6D) in comparison to the vehicle group. Our findings also showed that the combination therapy of AM630 (1 mg/kg) with BCP (5 mg/kg) led to a complete blockade of the effects of BCP on transcription factors and their ratios (p < 0.001 for all modes, Figure 6A-F).

The Relative Effects of BCP on the CNS Immunity and Systemic Immunity Levels in EAE Mice
As illustrated in Figure 11, the relative effects of BCP, DMF, and the AM630-BCP combination on the levels of systemic immunity (spleen lymphocytes and blood) and CNS immunity (microglia) were measured as a percentage of reduction of inflammatory/antiinflammatory ratios in the blood (IL-10/ IL-17) spleen lymphocytes (Th 17 /T reg and Th 1 /Th 2 ) and microglia (M 1 /M 2 ) compared with the vehicle group in EAE mice. The results showed that BCP (2.5 mg/kg) and DMF (60 mg/kg) declined the Th 17 /T reg levels and blood IL-17/IL-10 ratios more than the Th 1 /Th 2 levels and M 1 /M 2 ratios ( Figure 11). However, BCP (5 mg/kg) simultaneously targeted both CNS immune system and the systemic system ( Figure 11). In contrast, the addition of AM630 before BCP (5 mg/kg) reduced all effects of BCP (5 mg/kg) on the systemic system and CNS immune system ( Figure 11). inflammatory ratios in the blood (IL-10/ IL-17) spleen lymphocytes (Th17/Treg and Th1/Th2) and microglia (M1/M2) compared with the vehicle group in EAE mice. The results showed that BCP (2.5 mg/kg) and DMF (60 mg/kg) declined the Th17/Treg levels and blood IL-17/IL-10 ratios more than the Th1/Th2 levels and M1/M2 ratios ( Figure 9). However, BCP (5 mg/kg) simultaneously targeted both CNS immune system and the systemic system (Figure 9). In contrast, the addition of AM630 before BCP (5 mg/kg) reduced all effects of BCP (5 mg/kg) on the systemic system and CNS immune system (Figure 9).

Discussion
As far as we know, it is the first work regarding the protective effects of low doses of BCP in treating EAE as a well-established model of chronic MS. In our work, the impacts of low doses of BCP were examined in the presence and absence of a selective pharmacological CB 2 antagonist (AM630) on the blood cytokine levels, microglia (M 1 /M 2 ) polarizations, and lymphocytes (Th 1 /Th 2 and Th 17 /T reg ) in EAE mice. As a result, we indicated that low BCP doses significantly decreased clinical and pathological score levels in a dosedependent fashion, and a selective CB2 antagonist AM630, completely abolished their effects. Indeed, BCP effectively regulated the Th1/Th2 ratios, M1/M2 and Th17/Treg from inflammatory states Th1, Th17, and M1 towards anti-inflammatory conditions Th2, Treg and M2, respectively. Also, according to the results, the BCP effects on the polarizations of lymphocytes and microglia were also strikingly associated with the activation of the CB 2 receptor since the BCP effects were reversed entirely using CB 2 antagonist AM630.
Several evidence lines indicate that the EAE model provides inability and inflammation in mice similar to those observed in chronic MS patients. In our study, we demonstrated that EAE mice receiving vehicles represent worsened signatures in the clinical score, body weight, histopathology (H&E staining) and CNS (microglia) and systemic (lymphocytes and blood) inflammations, which are in accordance with the previous studies [48][49][50]. Furthermore, many studies indicated that Th 17 (expressing ROR-γt and producing IL-17) and Th 1 (representing T-bet and producing IFN-γ) cells are the primary culprits in the pathogenesis of EAE and MS [5]. In contrast, the anti-inflammatory population of T cells, such as T reg (expressing Foxp3, producing IL-10 and TGF-β) and Th 2 (expressing GATA3, producing IL-4), results in inflammation resolution [5,51]. In fact, it is worth mentioning that disturbance in ratios of T cells sub-population including Th 17 /T reg and Th 1 /Th 2 with the propagation of Th 17 and Th 1 cells, and their cytokines and transcription factors are recently paid more attention in the pathogenesis of both MS and EAE [51]. Also, infiltration of T cells into the CNS leads to the activation and polarization of microglia toward the inflammatory M 1 phenotype (producing high levels of inflammatory cytokines and iNOS) [5,52]. Our results also showed that MOG 35-55 -induced EAE markedly increased the inflammatory cytokine levels in the blood and secreted from spleen lymphocytes and microglial cells. Also, based on the results, the anti-inflammatory cytokine levels secreted from microglia and spleen lymphocytes were notably reduced in this model. Accordingly, the levels of Th 1 /Th 2 , Th 17 /T reg , M 1 /M 2 , and blood IL-17/IL-10 ratios shifted towards Th 1 , Th 17 , M 1 and IL-17 as inflammatory states. In agreement with our findings, several studies also indicated that Th 1 , M 1 , and Th 17 cells show dominance in the EAE model [53]. In this case, it was demonstrated that MOG 35-55 -induced EAE provided both systemic and CNS inflammation by elevating the inflammatory cell levels, such as lymphocytes and microglia, respectively [54]. Our results also showed the high sensitivity and specificity of lymphocytes and microglia to MOG  peptide stimulation because their proliferation and levels of cytokine were dramatically increased in the MOG stimulation presence.
In the present study, DMF was considered the positive control as an oral medicine approved by FDA for MS treatment. According to findings, DMF (60 mg/kg; p.o.) attenuated the severity of clinical scores and pathological signatures in EAE mice. In addition, the group receiving DMF significantly expressed lower levels of inflammatory cytokines and related transcription factors and higher anti-inflammatory cytokines and related transcription factors. Collectively, DMF caused the shifting of M 1 /M 2, Th 1 /Th 2 , and Th 17 /T reg ratios from inflammatory phenotypes toward anti-inflammatory phenotypes T reg , M 2 , and Th 2 . Consistent with the present work, various research works described that oral administration of DMF is an effective treatment in different inflammatory and oxidative stress models. In this regard, it has been shown that DMF suppressed Th 1 and Th 17 cells differentiation by decreasing the secretion and expression of their pro-inflammatory cytokines, such as TNF-α, IL-17, and IFN-γ [55,56], and expanding the population of the levels of IL-10, IL-5, and IL-4 cytokines and Th 2 cells [56,57].
Moreover, we demonstrated that DMF treatment (60 mg/kg) mainly targeted blood cytokines and systemic immunity and affected the Th 17 /T reg ratio more than the Th 1 /Th 2 ratio. Its effects may provide the protective effects of DMF in the EAE model on microglia cells as CNS immunity and systemic immunity. Additionally, we showed that microglia and lymphocytes isolated from EAE mice receiving DMF possessed lower inflammatory cytokine levels and transcription factors in the absence of MOG stimulation than the vehicle group. In agreement with our results, it has been recently reported that patients with MS receiving DMF medication had a persistent change in innate and adaptive immune systems [58]. In this regard, it has been explained that long-term therapy with DMF changes expression patterns of CD4 + T cell cytokines by reducing the pro-inflammatory (IFN-γ, GM-CSF, IL-17) and boosting the anti-inflammatory cytokine levels (IL-4, IL-10) [58]. In another study, Parasio and coworkers described the protective effects of DMF on lipopolysaccharide (LPS)-stimulated primary murine microglial cells. Contextually, DMF suppressed the activation of NF-κB and protected neurons against activated microglial cells through both Nrf2-dependent and independent pathways [59]. Peng and colleagues also indicated DMF's ability to attenuate the levels of inflammatory cytokines, chemokines, and nitric oxide in LPS/IFN-γ-activated microglia and provide healing and anti-inflammatory phenotype M 2 [60].
Interestingly, we indicated that the low doses of BCP (2.5, 5 mg/kg; p.o.) ameliorated the clinical score, body weight, histopathology (H&E staining), and both CNS (microglia) and systemic (lymphocytes and blood) inflammations, and elevated the levels of antiinflammatory cytokine IL-10 in EAE mice. Previous works have reported the critical role of microglia and lymphocytes during the neuroinflammatory and neurodegenerative processes in EAE and patients with MS [7,14,15,61], which the regulation of these cells is considered an essential target for the resolution of leukocyte infiltration, inflammation, and disease progression in EAE. We revealed that BCP (2.5, 5 mg/kg) reduced the inflammatory cytokine levels and transcription factors and shifted the imbalanced Th 1 /Th 2 (IFN-γ/IL-4, T-bet/GATA3), Th 17 /T reg (IL-17/IL-10 and TGF-β 1 , ROR-γt/Foxp3) and M 1 /M 2 (iNOS/Arg-1, NO/urea) ratios from the inflammatory state to anti-inflammatory Th 2 (IL-4, GATA3), T reg (IL-10, TGF-β 1 , Foxp3) and M 2 (Arg-1, urea) state. In fact, BCP exerts its protective effects by reducing the primary culprit cells (Th17 and Th1) involved in the EAE pathogenesis and reactivation of anti-inflammatory cells (M2 and Treg), resulting in the resolution of inflammatory responses. In accordance with our findings, various in vivo and in vitro reported the protective effects of BCP against inflammation.
Additionally, the current work showed that BCP was effective on both CNS immune system (microglia) and systemic system (lymphocytes and blood). However, it seems that BCP simultaneously targets both CNS (microglia) immune systems and systemic (lymphocytes and blood), but we assessed the relative effects of BCP on microglia (M 1 /M 2 ) and blood (IL-17/IL-10) lymphocytes (Th 1 /Th 2 and Th 17 /T reg ) in the presence of MOG stimulation. Intriguingly, it was illustrated that the effects of BCP at 2.5 mg/kg were different from BCP at 5 mg/kg. Essentially, BCP at 5 mg/kg simultaneously targeted both systemic and CNS immune systems, but BCP at 2.5 mg/kg mainly affected blood cytokines and Th 17 /T reg ratio rather than Th 1 /Th 2 and M 1 /M 2 ratios. However, this finding shows that BCP specifically targets Th 17 and T reg cells, but its effects on CNS immunity cannot be excluded. Recently, the protective effects of BCP on both oligodendrocytes and microglial cells against LPS-induced inflammatory states [14,15] were demonstrated. In this context, we showed that BCP at both high and low concentrations significantly exerted its protective effects via reducing the inflammatory mediator levels (TNF-α, IL-1β, PGE 2 , NO, ROS, iNOS), increasing the anti-inflammatory parameters (Arg-1, IL-10, urea), and polarization of microglial cells to M 2 anti-inflammatory cells [14], which all these effects were associated in a dose-dependent manner. As determined in our previous works, low BCP concentrations are an efficient therapy against inflammatory and oxidative stress and are also more potent than high concentrations. In agreement with our results, low doses of BCP (1, 5, and 10 mg/kg) impose considerable analgesic effects on pain types in different animal models [62].
Moreover, they showed that BCP (10 mg/kg) attenuates alcoholic steatohepatitis by decreasing inflammation and metabolic dysregulation in mice [63]. These works could support our results about the protective effects of low doses of BCP in EAE as an MS chronic model. Nonetheless, two separate works have reported the efficacy of BCP's high doses in EAE [25,26]. Contextually, they explained that BCP (25 and 50 mg/kg once a day; p.o. [26], and 25 and 50 mg/kg twice a day; p.o. [25]) had several protective effects on pathological and clinical defects in the EAE model. Additionally, these authors have not tried to show the role of CB 2 receptors in the protective effects of BCP as a known CB 2 receptor agonist. As revealed by our previous works, both high and low BCP concentrations exert protective effects via PPAR-γ and Nrf2 receptor signaling pathways depending on the activation of the CB 2 receptor [14,15]. However, it should take into account that high doses of BCP could provide further nonspecific and adverse effects in the long term of usage, likely through CB 1 or other involved mechanisms.
Furthermore, another set of experiments was conducted to clarify the role of CB2 receptors in the protective effects of BCP. Interestingly, we demonstrated that BCP results were CB 2 receptor-mediated since the use of a selective CB 2 receptor antagonist AM630 entirely disappeared from all protective effects of BCP. Available evidence demonstrated that low concentrations/doses of BCP can exert its protective effects exclusively via the CB 2 receptor but not CB 1 [19,62,64,65]. BCP (1, 5, and 10 mg/kg) experimentally provides important relieving effects in animal models of neuropathic and inflammatory pain via CB 2 receptor due to using a CB2 receptor antagonist or genetical elimination of CB 2 decreased the protective responses of BCP [62]. Moreover, it was described that using BCP reduces the infiltration and activity of astrocytes and macrophages in the dorsal horn of the spinal cord. Therefore, these effects were all mediated by the CB 2 receptor [62].
Furthermore, as observed, BCP (10 mg/kg) presents protection for the liver and hinders the over-activation of hepatic macrophage Kupffer cells through their switching toward M 2 cells. These were the CB 2 receptor-mediated effects since the CB 2 receptor ablation entirely inverted the BCP protective effects against binge chronic ethanol-induced inflammation and liver injury [63]. Overall, these research works can approve the efficacy of a low BCP dose in reducing inflammation and demyelination in the spinal cord by hindering the inflammatory cells' infiltration into the area and polarization of inflammatory macrophages in a CB 2 receptor-dependent manner.
There are also many studies regarding the protective effects of CB 2 receptor agonists in preventing and treating EAE. In one study, it has been reported that the CB 2 receptor activation, but not CB 1 , is mainly required for the therapeutic effect of alpha/beta-hydrolase domain-6 (ABHD6) inhibitor WWL70 in the EAE model because WWL70 did not exert any protective effects in CB 2 receptor knockout mice or in the presence of a CB 2 antagonist [66]. Contextually, they found that WWL70 strikingly inhibits the COX-2, iNOS, IL-1β, and TNFα production and the NF-κB phosphorylation, as well as demyelination and axonal loss. Moreover, amidoalkylindoles derivatives as selective CB 2 agonists remarkably improve clinical symptoms and protect the CNS against EAE [67]. In another study, a highly selective CB 2 agonist, Gp1a, ameliorates the clinical scores and reduces the recovery period in EAE, along with the prolonged decrease in demyelination and axonal loss [68]. Our results indicated that Gp1a exerts its protective effects by decreasing pathogenic T cells in the CNS and reducing Th1/Th17 differentiation in peripheral immune organs. On the other hand, CB 2 receptor agonists provide their protection by decreasing the inflammatory cytokine levels and polarization of M1/Th1/Th17 and increasing the anti-inflammatory cytokine levels and M 2 /Th 2 /T reg polarization [38,68,69]. Also, it has been explained that CB 2 agonist decreases the level of IL-6 as Th 17 polarizing cytokine [68,70,71]. Accordingly, we observed that BCP caused a decline in the inflammatory cytokines, such as IL-6, TNF-α, IL-17, IFN-γ and the transcriptional factors of Th 17 (ROR-γt) and Th 1 (T-bet), and a significant elevation in the anti-inflammatory cytokines, including TGF-β 1 , IL-10, IL-4, and the transcriptional factors of Th 2 (GATA3) and T reg (Foxp3), in a dose-dependent fashion. Indeed, the findings exhibited that BCP modulates M 1 /Th 1 /Th 17 cells towards M 2 /Th 2 /T reg , and these effects were tightly associated with activating the CB 2 receptor since the use of AM630 thoroughly blocked the BCP effects. Moreover, recently, we have shown the functional selectivity of BCP (10 −11 to 10 −4 M) by assessing the level of intracellular cAMP in the absence or presence of CB 1 and CB 2 receptor antagonists in both oligodendrocytes and microglia models of inflammation [14,15]. Accordingly, BCP dose-dependently reduces the cAMP level and produces a rightward shift by a CB 2 antagonist. Undoubtedly, it implies that the effects of high and low doses of BCP are both CB 2 receptor-mediated. The mechanism of protective effects of selective CB 2 agonists at high doses is different from the signaling mentioned above pathways and depends on the activation of PPAR-γ. Several studies indicated that the ceramide level and PPAR-γ activation signaling pathway are responsible for the effects of higher concentrations/doses of CB 2 receptor ligands [16,72,73]. Similarly, it has been demonstrated that the protective effects of high concentrations and amounts of BCP (>5 µM and (>25 mg/kg) were mediated via the CB 2 receptor-PPAR-γ pathway, since the use of PPAR-γ and CB 2 antagonists totally annihilated the protective effects of BCP [14,15,27,32,74].
In conclusion, according to the present study's findings, low BCP doses also effectively treated EAE as a chronic MS model. In addition, it was found that the CB2 receptor mediated the protective effects of BCP. Furthermore, our ex vivo and in vivo surveys revealed that BCP decreases the pathological and clinical score in EAE by modulating both adaptive (lymphocytes) and innate (microglia) immune systems from the inflammatory (Th 1 /Th 17 /M 1 ) to anti-inflammatory state (Th 2 /T reg /M 2 ).