基于NLRP3信号通路探讨三痹汤对佐剂型关节炎大鼠的治疗作用及机制探讨

目的：探讨三痹汤对佐剂型关节炎（adjuvant arthritis,AA）大鼠的治疗作用以及对NLRP3信号通路的影响,明确其作用机制。方法：60只Wistar大鼠随机分为空白组（Control）、模型组（Model）、来氟米特组（来氟米特片,LeF）和三痹汤高剂量组（SBT－H）、三痹汤中剂量组（SBT－M）、三痹汤低剂量组（SBT－L）,每组各10只。采用弗氏完全佐剂制备大鼠AA模型,各组于初次免疫1周后开始灌胃相应药物。连续灌服21 d,至免疫后28 d处死大鼠。观察大鼠一般状态、足容积、关节炎指数（AI）、踝关节滑膜组织病理形态。采用ELISA法检测白介素（IL）－1β、IL－18含量。采用PCR法和免疫组化法检测NLRP3 mRNA和NLRP3、Caspase－1蛋白的表达水平。结果：与Control比较,Model大鼠踝关节肿胀和病理损伤程度严重,在第7～28天足容积显著升高（P < 0.05）,IL－1β、IL－18含量、NLRP3 mRNA蛋白表达水平及NLRP3、Caspase－1蛋白表达明显上升（P < 0.05）;与Model比较,SBT－H、SBT－M、SBT－L、LeF组踝关节肿胀和病理损伤程度均有不同程度的改善,在第7～28天足容积及关节炎指数均显著下降（P < 0.05）,IL－1β、IL－18含量、NLRP3 mRNA蛋白表达水平及NLRP3、Caspase－1蛋白表达明显下降（P < 0.05）;踝关节肿胀和病理损伤程度改善程度依次为SBT－H > SBT－M > SBT－L;与SBT－H组比较,SBT－L、IL－1β、IL－18含量上升（P < 0.05）,SBT－M、SBT－L组NLRP3 mRNA蛋白表达及NLRP3、Caspase－1蛋白水平明显升高（P < 0.05）;与LeF组比较,SBT－H踝关节肿胀和病理损伤程度改善程度基本一致,SBT－M、SBT－L组IL－1β、IL－18含量上升（P < 0.05）,SBT－H组IL－1β、IL－18含量差异无统计学意义（P > 0.05）,SBT－L组NLRP3 mRNA蛋白表达水平明显升高（P < 0.05）,SBT－M、SBT－L组NLRP3、Caspese－1蛋白表达水平升高（P < 0.05）,SBT－H组NLRP3、Caspese－1蛋白表达水平差异无统计学意义（P > 0.05）。结论：三痹汤可通过抑制NLRP3信号通路的激活,降低大鼠血清IL－1β、IL－18水平,改善佐剂型关节炎大鼠滑膜损伤的程度。

Exploration on Therapeutic Effect and Mechanism of Sanbi Decoction on Adjuvant Arthritis Rats Based on NLRP3 Signaling Pathway

Objective：To investigate the effect of Sanbi Decoction （SBD） on adjuvant arthritis （AA） rats and NLRP3 signaling pathway. Methods： Sixty Wistar rats were randomly divided into control group, model group, leflunomide group, and SBD high dose （SBD－H）, SBD medium dose （SBD－M）, and SBD low dose （SBD－L） groups, with 10 rats in each group. Rat AA model was prepared by Freund''s complete adjuvant,and corresponding drugs were administered orally to each group one week after the initial immunization. After continuous drug administration for 21 days, the rats were euthanize on 28 days after immunization. The general state, volume of foot, arthritis index （AI） and histopathologic morphology of ankle synovium were observed. The contents of IL－1β and IL－18 were detected by ELISA, and the expression levels of NLRP3 mRNA, NLRP3 and Caspase－1 protein were detected by PCR and immunohistochemistry.Results： Compared with control group,the ankle swelling and pathological injury of model group were serious,and the foot volume was significantly increased from the 7th to 28th days （P < 0.05）. The contents of IL－1βand IL－18, the expression levels of NLRP3 mRNA, NLRP3, and Caspase－1 protein were significantly increased （P < 0.05）. Compared with model group, the ankle swelling and pathological injury in SBD－H,SBD－M, SBD－L and leflunomide groups were improved to varying degrees,and the foot volume and arthritis index were significantly decreased from day 7 to 28（P < 0.05）. The contents of IL－1β and IL－18, and the expression levels of NLRP3 mRNA protein, NLRP3, and Caspase－1 protein were significantly decreased （P < 0.05）. The degree of improvement of ankle swelling and pathological injury was SBD－H > SBD－M > SBD－L. Compared with SBD－H group, the contents of IL－1β and IL－18 in SBD－L group increased （P < 0.05）, NLRP3 mRNA protein expression and NLRP3 and Caspase 1 protein levels in SBD－M and SBD－L groups were significantly increased （P < 0.05）. Compared with leflunomide group, the improvement degree of ankle swelling and pathological injury of SBD－H group was basically the same, the contents of IL－1β and IL－18 in SBD－M and SBD－L groups were increased （P < 0.05）, while the content difference of IL－1β and IL－18 in SBD－H group were not statistically significant （P > 0.05）; the NLRP3 mRNA protein expression level in SBD－L group was significantly increased （P < 0.05）, and the NLRP3, Caspase－1 protein expression levels of SBD－M and SBD－L groups were increased （P < 0.05）; however, there were no significant difference in NLRP3 and Caspase－1 protein expression levels in SBD－H group （P > 0.05）.Conclusion： Sanbi Decoction may reduce the level of IL－1β and IL－18 in serum by inhibiting the activation of NLRP3 signaling pathway,and improve the degree of synovial injury in rats with adjuvant arthritis.