Celastrol attenuates inflammatory and neuropathic pain mediated by cannabinoid receptor type 2

Abstract

Celastrol, a major active ingredient of Chinese herb Tripterygium wilfordii Hook. f. (thunder god vine), has exhibited a broad spectrum of pharmacological activities, including anti-inflammation, anti-cancer and immunosuppression. In the present study, we used animal models of inflammatory pain and neuropathic pain, generated by carrageenan injection and spared nerve injury (SNI), respectively, to evaluate the effect of celastrol and to address the mechanisms underlying pain processing. Intraperitoneal (i.p.) injection of celastrol produced a dose-dependent inhibition of carrageenan-induced edema and allodynia. Real-time PCR analysis showed that celastrol (0.3 mg/kg, i.p.) significantly reduced mRNA expressions of inflammatory cytokines, TNF-α, IL-6, IL-1β, in carrageenan-injected mice. In SNI mice, pain behavior studies showed that celastrol (1 mg/kg, i.p.) effectively prevented the hypersensitivity of mechanical nociceptive response on the third day post-surgery and the seventh day post-surgery. Furthermore, the anti-hyperalgesic effects of celastrol in carrageenan-injected mice and SNI mice were reversed by SR144528 (1 mg/kg, i.p.), a specific cannabinoid receptor-2 (CB2) receptor antagonist, but not by SR141716 (1 mg/kg, i.p.), a specific cannabinoid receptor-1 (CB1) receptor antagonist. Taken together, our results demonstrate the analgesia effects of celastrol through CB2 signaling and propose the potential of exploiting celastrol as a novel candidate for pain relief.

Figure 1

Celastrol reduced edema and hyperalgesia in the carrageenan-induced inflammatory pain model. The effect of vehicle (VEH, 5% PEG/5% Tween-80 in saline, 10 mL/kg, i.p.) and celastrol (CEL, 0.3 mg/kg, i.p.) on carrageenan-induced edema (A) and pain hypersensitivity (B). *** p< 0.001 vs. non-carrageenan injection (CT), ## p < 0.01 vs. VEH; one-way ANOVA followed by Bonferroni’s multiple comparison test, n = 5–6. The time- and dose-dependent effect of celastrol on carrageenan-induced edema (C) and pain hypersensitivity (D). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle, two-way ANOVA with Bonferroni’s post-tests, n = 5–6/group.

Figure 2

Analgesia effect of celastrol in carrageenan-induced inflammatory pain was mediated by cannabinoid receptor-2 (CB₂) signaling. The effects of celastrol, SR141716, SR144528, a combination of celastrol and SR141716 and a combination of celastrol and SR144528 on mechanical hyperalgesia, assessed by the allodynia test, in carrageenan-injected mice (closed bar). Open bar, non-carrageenan injected mice; vehicle, 5% PEG/5% Tween-80 in saline, 10 mL/kg, i.p; celastrol, 0.3 mg/kg, i.p.; SR141716, 1 mg/kg, i.p.; SR144528, 1 mg/kg, i.p. *** p < 0.001; N.S., not significant; one-way ANOVA followed by Bonferroni’s multiple comparison test, n = 8.

Figure 3

Celastrol suppressed carrageenan-induced inflammation. The effect of vehicle (VEH, 5% PEG/5% Tween-80 in saline, 10 mL/kg, i.p.) and celastrol (CEL, 0.3 mg/kg, 10 mL/kg, i.p.) on the mRNA expressions of (A) TNF-α; (B) IL-6; and (C) IL-1β in the paw tissues of carrageenan injected mice (close bars). *** p < 0.001 vs. non-carrageenan injection (CT, open bars); # p < 0.05, ## p < 0.01, ### p < 0.001 vs. VEH, one-way ANOVA with Bonferroni’s multiple comparison test. n = 6.

Figure 4

Celastrol reduced hyperalgesia in SNI mice. The time-course effect of vehicle (5% PEG/5% Tween-80 in saline, 10 mL/kg, i.p.) and celastrol (1 mg/kg, 10 mL/kg, i.p.) on mechanical allodynia in SNI mice (closed bars) on the third day post-surgery (A); and the seventh day post-surgery (B). Sham, sham-operated mice (open bars); 1, 1 h after drug administration; 4, 4 h after drug administration; 8, 8 h after drug administration. *** p < 0.001 vs. Sham-operated mice; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. vehicle, one-way ANOVA with Bonferroni’s multiple comparison test, n = 7–8.

Figure 5

Analgesia effect of celastrol in SNI mice was blocked by the CB₂ antagonist. The effects of vehicle (5% PEG/5% Tween-80 in saline, 10 mL/kg, i.p.), SR141716 (1 mg/kg, i.p.), SR144528 (1 mg/kg, i.p.) and celastrol (0.3 mg/kg, i.p.) on the mechanical allodynia test in the seventh day post-surgery SNI mice (closed bars). Open bars, sham-operated mice. Pain withdraw thresholds were measured two hours after drug administration. N.S., not significant; *** p < 0.001, ** p < 0.01, one-way ANOVA with Bonferroni’s multiple comparison test, n = 5–6.