补肾强筋胶囊通过调控NLRP3炎症小体介导的细胞焦亡改善KOA滑膜炎症和纤维化的实验研究

Experimental Study of Bushen Qiangjin Capsule on Improving KOA Synovitis and Fibrosis by Regulating NLRP3 Inflammasome-Mediated Pyroptosis

目的探讨补肾强筋胶囊对膝骨关节炎(KOA)滑膜炎症和纤维化的可能效应机制。方法动物实验采用前交叉韧带离断造模法(ACLT)建立KOA动物模型,将大鼠随机分为空白组、模型组、补肾强筋胶囊组和塞来昔布胶囊组。取材时观察各组大鼠滑膜组织大体形态学变化;采用ELISA检测各组大鼠血清中白介素-1β(IL-1β)和IL-18水平;HE及天狼星红染色观察各组大鼠滑膜组织病理学变化;免疫组化染色观察各组大鼠滑膜组织NOD样受体热蛋白结构域相关蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)和消皮素D(GSDMD)、转化生长因子-β(TGF-β)和I型胶原α1重组蛋白(COL1A1)蛋白表达情况。细胞实验采用脂多糖(LPS)联合三磷酸腺苷(ATP)刺激大鼠成纤维样滑膜细胞(FLS)模拟KOA炎性环境并诱导细胞焦亡模型,使用补肾强筋胶囊或者MCC950处理细胞,采用ELISA检测各组细胞培养上清IL-1β和IL-18水平;免疫荧光染色观察NLRP3、Caspase-1和GSDMD的定位与表达;RT-qPCR和Western blot分别检测各组细胞焦亡相关分子NLRP3、Caspase-1、GSDMD和纤维化相关分子TGF-β、COL1A1、赖氨酸羟基化酶2(PLOD2)及基质金属蛋白酶抑制剂-1(TIMP1)的mRNA及蛋白表达。结果动物实验结果提示,与模型组相比,补肾强筋胶囊可明显降低KOA大鼠滑膜组织形态学评分(P<0.01);明显降低KOA大鼠血清中IL-1β和IL-18水平(P<0.01);还可降低KOA大鼠滑膜组织中NLRP3、Caspase-1、GSDMD、TGF-β及COL1A1蛋白表达(P<0.05,P<0.01)。细胞实验结果表明,与模型对照组相比,补肾强筋胶囊可显著降低细胞培养上清中IL-1β和IL-18水平(P<0.01);明显降低NLRP3、Caspase-1和GSDMD荧光相对表达量(P<0.01);显著降低NLRP3、Caspase-1、GSDMD、TGF-β、COL1A1、PLOD2和TIMP1 mRNA及蛋白表达(P<0.01)。结论补肾强筋胶囊可通过抑制NLRP3炎症小体的活化、调控FLS焦亡,从而改善KOA滑膜炎症及纤维化。

Objective To explore the possible mechanism of Bushen Qiangjin capsule on synovitis and fibrosis in knee osteoarthritis(KOA).Methods In the animal experiment,the animal model of KOA was established by anterior cruciate ligament transection(ACLT).The rats were randomly divided into blank group,model group,Bushen Qiangjin capsule group and celecoxib capsule group.The levels of interleukin-1β(IL-1β)and IL-18 in serum of rats in each group were detected by ELISA,and the histopathological changes of synovium of rats in each group were observed by HE and Sirius red staining.Immunohistochemical staining was used to observe the expression of NLRP3,Caspase-1,GSDMD,TGF-βand COL1A1 in synovium of rats in each group.In the cell experiment,Lipopolysaccharide(LPS)combined with Adenosine triphosphate(ATP)was used to stimulate rat-FLS to simulate OA inflammatory environment and induce pyroptosis model.The cells were treated with Bushen Qiangjin capsule or MCC950,and the levels of IL-1βand IL-18in the cell culture supernatant of each group were detected by ELISA;the localization and expression of NLRP3,Caspase-1 and GSDMD were observed by immunofluorescence staining;and the mRNA and protein expression of NLRP3,Caspase-1,GSDMD,TGF-β,COL1A1,PLOD2 and TIMP1 were detected by RT-qPCR and Western Blot,respectively.Results The results of animal experiment showed that compared with the model group,Bushen Qiangjin capsule could significantly reduce the score of synovial histomorphology of KOA rats(P<0.01);significantly reduce the levels of IL-1βand IL-18 in serum of KOA rats(P<0.01);also reduce the expression of NLRP3,Caspase-1,GSDMD,TGF-βand COL1A1 protein in synovium of KOA rats(P<0.05,P<0.01).The results of cell experiment showed that compared with the model control group,Bushen Qiangjin capsule could significantly reduce the levels of IL-1βand IL-18 in the supernatant of cell culture(P<0.01);significantly reduce the relative fluorescence expression of NLRP3,Caspase-1 and GSDMD(P<0.01),and significantly reduce the expression of NLRP3,Caspase-1,GSDMD,TGF-β,COL1A1,PLOD2,TIMP1 mRNA and protein(P<0.01).Conclusion Bushen Qiangjin capsule could improve KOA synovitis and fibrosis by inhibiting the activation of NLRP3 inflammasome and regulating FLS pyroptosis.