Effects of gonadal hormones on the peripheral cannabinoid receptor 1 (CB1R) system under a myositis condition in rats

Abstract

In this study, we assessed the effects of peripherally administered cannabinoids in an orofacial myositis model, and the role of sex hormones in cannabinoid receptor (CBR) expression in trigeminal ganglia (TG). Peripherally administered arachidonylcyclopropylamide (ACPA), a specific CB1R agonist, significantly attenuated complete Freund's adjuvant (CFA)-induced mechanical hypersensitivity in the masseter muscle in male rats. The ACPA effect was blocked by a local administration of AM251, a specific CB1R antagonist, but not by AM630, a specific CB2R antagonist. In female rats, a 30-fold higher dose of ACPA was required to produce a moderate reduction in mechanical hypersensitivity. CFA injected in masseter muscle significantly upregulated CB1R mRNA expression in TG in male, but not in female, rats. There was a close correlation between the CB1R mRNA levels in TG and the antihyperalgesic effect of ACPA. Interleukin (IL)-1β and IL-6, which are elevated in the muscle tissue following CFA treatment, induced a significant upregulation of CB1R mRNA expression in TG from male rats. The upregulation of CB1R was prevented in TG cultures from orchidectomized male rats, which was restored by the application of testosterone. The cytokines did not alter the CB1R mRNA level in TG from intact as well as ovariectomized female rats. Neither estradiol supplement nor estrogen receptor blockade had any effects on CB1R expression. These data indicate that testosterone, but not estradiol, is required for the regulation of CB1Rs in TG under inflammatory conditions, which provide explanations for the sex differences in the antihyperalgesic effects of peripherally administered cannabinoids.

Fig. 1

Effects of arachidonylcyclopropylamide (ACPA) on complete Freund’s adjuvant (CFA)-induced mechanical hypersensitivity. (A) CFA-induced mechanical hypersensitivity measured in the masseter muscle of male and normally cycling female rats. Mechanical force (g) that produced the head withdrawal responses 50% of the trials is plotted for pre- and 1, 2, 3, 7, 10, and 14 days post-CFA injection. ⁺denotes significant time effects at P < 0.05 compared to the pre-CFA values. (B, C) Effects of intramuscular ACPA on mechanical sensitivity 3 days after CFA treatment in male and female rats. Bar graphs show mean % change in EF₅₀ values in vehicle (phosphate-buffered saline [PBS])- and ACPA-treated rats. *denotes significant differences at P < 0.05 compared to the vehicle group. Contra-ACPA injected in the masseter muscle contralateral to the mechanical sensitivity testing. (D). Effects of ACPA on mechanical sensitivity of naïve male and female rats. (E, F). Effects of a CB1R antagonist (AM251) and a CB2R antagonist (AM630) on ACPA-mediated antihyperalgesic responses 3 days after CFA treatment in male rats. *denotes significant differences at P < 0.05 compared to the vehicle group. All data are shown as mean ± SE and each group consisted of 6–7 animals.

Fig. 2

(A) Real-time polymerase chain reaction data showing complete Freund’s adjuvant (CFA)-induced changes in CB1R mRNA levels in intact trigeminal ganglia of male and female rats. All data are normalized to naïve male rats and presented as mean percent change. ⁺denotes significant effects with respect to naïve condition at P < 0.05. (B) Effects of arachidonylcyclopropylamide (ACPA) on masseter hypersensitivity 1 day after CFA treatment in male rats. Bar graphs show mean % change in EF₅₀ values in vehicle (phosphate-buffered saline [PBS])- and ACPA-treated rats. Data are shown as mean ± SE and each group consisted of 6 animals.

Fig. 3

Effects of inflammatory cytokines on CB1R mRNA expression in trigeminal ganglia primary cultures. Real-time polymerase chain reaction data following individual or combination of cytokines applications were compared to those of phosphate-buffered saline (PBS) treatment in cultures prepared from male (A,B) and female (C,D) rats. All data are normalized to untreated samples and presented as mean percent change in CB1R mRNA transcripts. Data are shown as mean ± SE and each group consisted of 5–6 replicates. *denotes significant differences compared to PBS group at P < 0.05. IL, interleukin; TNF, tumor necrosis factor.

Fig. 4

(A) Role of inflammatory cytokines in complete Freund’s adjuvant (CFA)-induced upregulation of CB1R mRNA in intact trigeminal ganglia (TG) of male rats. CFA-induced changes in CB1R mRNA levels were assessed in intact TG of male rats treated with interleukin (IL)-1 receptor antagonist (IL-1RA), IL-6 inhibitor, gp130, and vehicle. Data are normalized to that of TG obtained from naïve rats. Each group consisted of 6 rats. (B) Effects of arachidonylcyclopropylamide (ACPA) on CFA-induced masseter hypersensitivity were assessed under the same experimental conditions as in A. Each group consisted of 6–8 rats. (C) CB1R mRNA in intact TG from male rats treated with direct masseteric injections of IL-1β, IL-6 with its receptor (IL-6R), and vehicle control. Data are normalized to that of naïve male rats and each group consisted of 6 rats *denotes significant group effects at P < 0.05. PBS, phosphate-buffered saline.

Fig. 5

Role of estrogen in CB1R expression in trigeminal ganglia (TG) from female rats. (A) CB1R mRNA levels were assessed from TG of ovariectomized (OVX) rats treated with cytokines. (B) Estradiol benzoate (ES) alone or ES combined with cytokines were applied to TG cultures from intact female rats and CB1R mRNA levels assessed. (C) An estrogen receptor antagonist, ICI-182,780 (ICI) alone or ICI combined with cytokines were applied to TG cultures from intact female rats and CB1R mRNA assessed. All data are normalized to untreated samples and presented as mean percent change in CB1R mRNA transcripts. Data are shown as mean ± SE and each group consisted of 5–6 replicates. *denotes significant differences compared to PBS group at P < 0.05. PBS, phosphate-buffered saline; IL, interleukin; TNF, tumor necrosis factor.

Fig. 6

Role of testosterone in CB1R mRNA expression in trigeminal ganglia (TG) from male rats. (A) Real-time polymerase chain reaction data following individual or combination of cytokines applications were compared to those of PBS treatment in orchidectomized (GDX) rats. (B) Testosterone (TS) alone or TS combined with cytokines were applied to TG cultures from GDX rats and CB1R mRNA levels assessed. All data are normalized to untreated samples and presented as mean percent change in CB1R mRNA transcripts. Data are shown as mean ± SE and each group consisted of 5–6 replicates. *denotes significant differences compared to PBS group at P < 0.05. (C) Effects of arachidonylcyclopropylamide (ACPA) on masseter hypersensitivity 3 days after complete Freund’s adjuvant treatment in GDX rats. Bar graphs show mean % change in EF₅₀ values in vehicle (phosphate-buffered saline [PBS])- and ACPA-treated rats. Data are shown as mean ± SE and each group consisted of 6 animals.

Fig. 7

Role of testosterone (TS) in CB1R mRNA expression in trigeminal ganglia (TG) from female rats. TS alone or TS combined with cytokines were applied to TG cultures from intact female rats and CB1R mRNA levels assessed. All data are normalized to untreated samples and presented as mean percent change in CB1R mRNA transcripts. Data are shown as mean ± SE and each group consisted of 5–6 replicates. OVX, ovariectomized; PBS, phosphate-buffered saline; IL, interleukin; TNF, tumor necrosis factor.