First Clinical Medical College, Gannan Medical University, Ganzhou, 341000, P. R. China.
Department of Physiology, School of Basic Medicine Sciences, Gannan Medical University, Ganzhou, 341000, P. R. China.
J Neuroimmune Pharmacol. 2024 Oct 10;19(1):53. doi: 10.1007/s11481-024-10152-8.
The underlying pathogenesis of chronic inflammatory pain is greatly complex, but the relevant therapies are still unavailable. Development of effective candidates for chronic inflammatory pain is highly urgent. We previously identified that trifluoro-icaritin (ICTF) exhibited a significant therapeutic activity against complete Freund's adjuvant (CFA)-induced chronic inflammatory pain, however, the precise mechanisms remain elusive. Here, the paw withdrawal threshold (PWT), paw withdrawal latency (PWL), and CatWalk gait analysis were used to determine the pain-related behaviors. The expression and co-localization of pain-related signaling molecules were detected by Western blot and immunofluorescence staining. Our results demonstrated that ICTF (3.0 mg/kg, i.p.) effectively attenuated mechanical allodynia, thermal hyperalgesia and improved motor dysfunction induced by CFA, and the molecular docking displayed that CB2 receptor may be the therapeutic target of ICTF. Furthermore, ICTF not only up-regulated the levels of CB2 receptor, IL-10, β-endorphin and CD206, but also reduced the expression of P2Y12 receptor, NLRP3, ASC, Caspase-1, IL-1β, CD11b, and iNOS in the spinal cord of CFA rats. Additionally, the immunofluorescence staining from the spinal cord showed that ICTF significantly increased the co-expression between the microglial marker Iba-1 and CB2 receptor, IL-10, β-endorphin, respectively, but markedly decreased the co-localization between Iba-1 and P2Y12 receptor. Conversely, intrathecal administration of CB2 receptor antagonist AM630 dramatically reversed the inhibitory effects of ICTF on CFA-induced chronic inflammatory pain, leading to a promotion of pain hypersensitivity, abnormal gait parameters, microglial activation, and up-regulation of P2Y12 receptor and NLRP3 inflammasome, as well as the inhibition of CB2 receptor and IL-10/β-endorphin cascade. Taken together, these findings highlighted that ICTF alleviated CFA-induced neuroinflammation by enhancing CB2 receptor-mediated IL-10/β-endorphin signaling and suppressing microglial activation in the spinal cord, and uncovered that CB2 receptor may be exploited as a novel and promising target for ICTF treatment of chronic inflammatory pain.
慢性炎症性疼痛的潜在发病机制非常复杂，但相关治疗方法仍尚未问世。因此，开发针对慢性炎症性疼痛的有效候选药物迫在眉睫。我们之前发现，三氟地骨皮甲素（ICTF）对完全弗氏佐剂（CFA）诱导的慢性炎症性疼痛具有显著的治疗活性，然而，确切的机制仍不清楚。在此，我们采用足底缩足反射阈值（PWT）、足底缩足潜伏期（PWL）和 CatWalk 步态分析来确定与疼痛相关的行为。通过 Western blot 和免疫荧光染色检测疼痛相关信号分子的表达和共定位。结果表明，ICTF（3.0mg/kg，腹腔注射）可有效减轻 CFA 诱导的机械性痛觉过敏、热痛觉过敏和运动功能障碍，分子对接显示 CB2 受体可能是 ICTF 的治疗靶点。此外，ICTF 不仅上调了 CB2 受体、IL-10、β-内啡肽和 CD206 的水平，还降低了 CFA 大鼠脊髓中 P2Y12 受体、NLRP3、ASC、Caspase-1、IL-1β、CD11b 和 iNOS 的表达。此外，脊髓免疫荧光染色显示，ICTF 显著增加了小胶质细胞标志物 Iba-1 与 CB2 受体、IL-10、β-内啡肽的共表达，而明显减少了 Iba-1 与 P2Y12 受体的共定位。相反，鞘内给予 CB2 受体拮抗剂 AM630 则显著逆转了 ICTF 对 CFA 诱导的慢性炎症性疼痛的抑制作用，导致疼痛敏化、步态参数异常、小胶质细胞激活以及 P2Y12 受体和 NLRP3 炎性小体的上调，同时抑制了 CB2 受体和 IL-10/β-内啡肽级联反应。综上所述，这些发现表明 ICTF 通过增强 CB2 受体介导的 IL-10/β-内啡肽信号和抑制脊髓中小胶质细胞激活来缓解 CFA 诱导的神经炎症，并且揭示了 CB2 受体可能是 ICTF 治疗慢性炎症性疼痛的一个新的有前途的靶点。
