甘草附子汤加减方通过抑制G...疗CIA大鼠的作用机制研究_张铭.pdf

中国病理生理杂志 Chinese Journal of Pathophysiology 2023，39（2）：325-334杂志网址：http:/甘草附子汤加减方通过抑制GSDMD介导的焦亡治疗CIA大鼠的作用机制研究*张铭1，2，吴天宇3，刘小碣1，张妍4，何晓宇1，刘赛赛5，赵士弟1，5，陈莉秋6，陈传好2，张小楠1，5（1蚌埠医学院心脑血管疾病基础与临床重点实验室，2蚌埠医学院人体解剖学教研室，3蚌埠医学院公共卫生学院，4蚌埠医学院临床医学院，5蚌埠医学院病理生理学教研室，6蚌埠医学院第二附属医院中医科，安徽 蚌埠 233030）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-1、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-1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-1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。关键词 甘草附子汤；类风湿关节炎；gasdermin D；细胞焦亡中图分类号 R593.21；R363.2 文献标志码 A doi：10.3969/j.issn.1000-4718.2023.02.015Mechanism of Gancao-Fuzi Decoction in treatment of collagen-induced arthritis rats via inhibiting GSDMD-mediated pyroptosisZHANG Ming1，2，WU Tianyu3，LIU Xiaojie1，ZHANG Yan4，HE Xiaoyu1，LIU Saisai5，ZHAO Shidi1，5，CHEN Liqiu6，CHEN Chuanhao2，ZHANG Xiaonan1，5（1Key Laboratory of Cardiovascular and Cerebrovascular Diseases，Bengbu Medical College，2Department of Human Anatomy，Bengbu Medical College，3School of Public Health，Bengbu Medical College，4Clinical Medicine Department of Bengbu Medical College，5Department of Pathophysiology of Bengbu Medical College，6Department of Traditional Chinese Medicine，the Second Affiliated Hospital of Bengbu Medical College，Bengbu 233030，China.E-mail：；）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 Freunds 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 30 文章编号 1000-4718（2023）02-0325-10 收稿日期 2022-09-07 修回日期 2022-12-14*基金项目 安徽省高校自然科学研究项目重点项目（No.KJ2020A0588）；安徽省重点科研平台开放课题基金项目（No.KLICD-2022-Z5）；蚌埠医学院研究生创新项目（No.Byycx21012、21058）；安徽省大学生创新项目（No.S202110367076）通讯作者 张小楠 Tel：13609827842；E-mail：；陈传好 Tel：15212108484；E-mail：325days.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-1，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-1 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-1 and IL-6 secretion and promote IL-10 secretion，resulting in an effective reduction of rheumatoid arthritis symptoms in CIA rats.KEY WORDS Gancao-Fuzi Decoction；rheumatoid arthritis；gasdermin D；pyroptosis类风湿关节炎（rheumatoid arthritis，RA）是一种慢性自身免疫性疾病，以关节骨质破坏和侵蚀性滑膜炎症为主要病理特征，致残率高且预后较差，尚缺乏有效防治措施1。RA临床药物治疗常通过免疫抑制剂与非甾体药物的联合应用缓解患者病情，但常规药物治疗导致的治疗耐受和毒副作用致使其临床应用受限2。因此，探索RA治疗新途径尤为重要。甘草附子汤源于张仲景的 伤寒论，包含甘草、附子、白术和桂枝四种中药，制附子补火助阳、逐风驱湿；桂枝助阳化气、温经散寒；白术除湿益燥、健脾益气；甘草通血利痹、调和诸药，全方共奏除湿祛风、温经散寒之效，能够有效缓解关节肿胀、疼痛、僵直等症状，临床已有报道其对 RA 具有较好治疗效果 3-4，本研究前期根据临床患者实际症状和兼症，在原方剂的基础上加入了知母、白芍、威灵仙、鸡血藤、木瓜和伸筋草等六味中药，临床治疗效果较好，但其具体机制不明。细胞焦亡是一种由Gasdermin D（GSDMD）蛋白介导的溶解性细胞程序性死亡，研究表明，依赖GSDMD诱导的细胞焦亡途径与RA滑膜炎症的发生密切相关，通过抑制GSDMD的表达有助于缓解RA5-6。已有研究证明甘草附子汤能够通过抑制滑膜成纤维细胞增殖、炎症因子分泌等减轻RA疾病活动度7-8，但从细胞焦亡层面探究GCFZD的药效学机制尚缺乏文献报道。因此，本研究对CIA模型大鼠给予不同剂量的 GCFZD 处理后，通