【摘要】 目的：探讨甘草附子汤加减方（GCFZD）抑制gasdermin D (GSDMD)介导细胞焦亡途径对胶原诱导性关节炎（CIA）大鼠的治疗效果。方法：SD大鼠尾跟部注射由牛Ⅱ型胶原与弗氏佐剂混合形成的乳化剂，构建CIA大鼠模型并对其关节炎指数进行评估，将成功诱发关节炎的30只雄性SD大鼠随机分为模型组、甘草附子汤低（4 g/kg）、中（8 g/kg）和高（16 g/kg）剂量组及甲氨蝶呤（1 mg/kg）组，每组6只，连续给药30 d。测定大鼠的体重、关节炎指数和足爪肿胀指数；X线影像学观察骨质破坏和软组织厚度改变；HE染色观察脾脏和关节组织病理学改变；番红O-固绿染色观察关节软骨改变；TUNEL染色观察关节内细胞焦亡发生情况；Western blot测定TLR4、caspase-1、NLRP3和GSDMD蛋白表达；real-time PCR和ELISA测定IL-1β、IL-6和IL-10细胞因子表达。结果：与模型组相比，GCFZD治疗显著改善CIA大鼠软组织肿胀和骨质破坏，降低脾脏和胸腺指数；HE染色结果显示，GCFZD治疗减轻CIA大鼠脾脏和踝关节组织病理学改变；番红O-固绿染色结果显示，GCFZD治疗减少软骨组织破坏，修复软骨结构；TUNEL染色结果显示，GCFZD显著抑制关节组织内的细胞焦亡；Western blot结果表明，GCFZD降低CIA大鼠脾脏中TLR4、cleaved caspase-1、NLRP3和GSDMD-N的蛋白表达；real-time PCR和ELISA结果显示，GCFZD减少CIA大鼠IL-1β和IL-6的mRNA和蛋白水平的表达，上调IL-10的mRNA和蛋白水平的表达。结论：GCFZD能够抑制GSDMD介导的细胞焦亡，减少IL-1β和IL-6分泌，促进IL-10分泌，有效减轻CIA大鼠的类风湿关节炎症状。更多还原

【Abstract】 AIM: To explore the therapeutic effect of Gancao-Fuzi Decoction(GCFZD) on collagen-induced arthritis(CIA) in rats by inhibiting the GSDMD-mediated pyroptosis. METHODS: The tail heels of rats were injected with an emulsifier formed by mixing the bovine type Ⅱ collagen with Freund’s adjuvant to construct the CIA rat models and evaluate its arthritis indexes. The 30 male CIA rats were randomly divided into the model group, the low-(4 g/kg), medium-(8 g/kg) and high-dose(16 g/kg) groups of GCFZD and the methotrexate group(1 mg/kg), 6 rats per group for 30days. The body weight, arthritis index and paw swelling index were recorded in each group of rats. X-ray imaging to observe the changes in bone destruction and soft tissue thickness, and the spleen and joints were detected by HE staining to observe the histopathological changes. The safranine o-fast green staining was used to observe articular cartilage changes.TUNEL staining was performed to evaluate cellular pyroptosis. Western blot for TLR4, caspase-1, NLRP3 and GSDMD protein expression. Real-time PCR and ELISA for IL-1β, IL-6 and IL-10 cytokine expression. RESULTS: Compared with the model group, there were significant improvements in soft tissue swelling and bone destruction, as well as reduced spleen and thymus indices in CIA rats with GCFZD treatment. HE staining results showed that the histopathological changes in the spleen and ankle joint of CIA rats were significantly improved with GCFZD treated. The safranine o-fast green staining showed that GCFZD could reduce the cartilage tissue destruction and repaired cartilage structure. TUNEL staining results indicated that GCFZD could significantly inhibit joint tissue pyroptosis. Western blot showed that TLR4, cleaved caspase-1, NLRP3 and GSDMD-N were decreased in spleen of CIA rats in GCFZD treatment groups. Real-time PCR and ELISA results showed that the GCFZD decreased the mRNA and protein level expression of IL-1β and IL-6 while upregulating the mRNA and protein level expression of IL-10 in blood serum of CIA rats. CONCLUSION: GCFZD may inhibit GSDMD-mediated pyroptosis and decrease IL-1β and IL-6 secretion and promote IL-10 secretion, resulting in an effective reduction of rheumatoid arthritis symptoms in CIA rats.更多还原