益肾蠲痹丸干预相关通路介导细胞焦亡对膝骨性关节炎大鼠的影响

The effect of Yishen Juanbi pill on knee osteoarthritis through intervention of cell pyroptosis mediated by NLRP3/NF-κB/Caspase-1 signaling in rats

目的探讨益肾蠲痹丸干预NLRP3/NF-κB/Caspase-1信号通路对膝骨性关节炎(knee osteoarthritis,KOA)大鼠的保护作用,并分析其对细胞焦亡相关蛋白的影响。方法随机将90只SPF级SD大鼠分为空白组、模型组、阳性对照组和益肾蠲痹丸低、中、高剂量组,每组15只。空白组不做处理,其他组制备KOA大鼠模型后进行干预治疗。比较6组大鼠干预前后膝关节直径及足趾容积情况;ELISA法检测血清中IL-1β、IL-18及血管内皮生长因子(vascular endothelial growth factor,VEGF)含量变化;HE染色观察关节软骨组织病理学改变情况;TUNEL法检测细胞凋亡情况;免疫组化法分析Caspase-1相关蛋白表达;Western blot及qRT-PCR法检测NLRP3/NF-κB/Caspase-1相关蛋白及mRNA表达水平。结果KOA造模后,与空白组比较,模型组大鼠膝关节直径与足趾容积明显增大,血清中IL-1β、IL-18及VEGF含量明显升高,软骨组织细胞排列紊乱,出现炎性浸润,细胞凋亡程度增高,NLRP3/NF-κB/Caspase-1相关蛋白及mRNA表达水平明显升高(P均<0.05);与模型组比较,阳性对照组及益肾蠲痹丸治疗组大鼠膝关节直径与足趾容积明显减轻,血清中IL-1β、IL-18及VEGF含量下降,软骨组织细胞炎性浸润改善,细胞凋亡程度减轻,NLRP3/NF-κB/Caspase-1相关蛋白及mRNA表达水平明显下降(P均<0.05),其中益肾蠲痹丸高剂量组显著优于益肾蠲痹丸低剂量组(P<0.01)。结论益肾蠲痹丸能够改善KOA大鼠关节软骨组织损伤,降低炎症因子表达和细胞凋亡程度,其机制可能与调控NLRP3/NF-κB/Caspase-1信号通路减轻细胞焦亡有关。

Objective To investigate the protective effect of Yishen Juanbi Pill on knee osteoarthritis(KOA)rats by interfering with NLRP3/NF-κB/Caspase-1 signal pathway,and to analyze its effect on pyroptosis related proteins.Methods Ninety SPF SD rats were randomly divided into blank group,model group,positive control group,and low,middle and high dose of Yishen Juanbi Pill groups,with 15 rats in each group.Rats in the blank group was not treated.Rats in the other groups were intervened after preparing the rat model of knee osteoarthritis.The knee joint diameter and toe volume of 6 groups were compared before and after the intervention.The contents of IL-1β,IL-18,and VEGF in rat serum were detected with ELISA method.Histopathological changes of articular cartilage were observed with HE staining.TUNEL method was used to detect the apoptosis of articular chondrocytes in each group.The expressions of caspase-1 related proteins were analyzed with immunohistochemistry.The expression levels of NLRP3/NF-κB/Caspase-1 related proteins and mRNAs in each group were detected using Western blotting and qRT-PCR.Results After modeling,compared to those in the blank group,the diameter of knee joint and the volume of toe in the model group increased significantly,and the contents of IL-1β,IL-18 and VEGF in serum increased significantly.The cells in cartilage tissue were in disorder,inflammatory infiltration appeared,and the degree of apoptosis increased.The expression levels of NLRP3/NF-κB/Caspase-1 related proteins and mRNAs increased significantly(all P<0.05).Compared to those in the model group,the diameter of knee joint and toe volume of rats in the positive control group and the Yishen Juanbi Pill treatment group reduced significantly,the contents of IL-1β,IL-18,and VEGF in serum decreased,the inflammatory infiltration of chondrocytes improved,the degree of apoptosis reduced,and the expression levels of NLRP3/NF-κB/Casase-1 related proteins and mRNAs decreased significantly(all P<0.05).Among them,the high dose group of Yishen Juanbi Pill was significantly better than the low dose group(P<0.01).Conclusion Yishen Juanbi Pill effectively relieves the articular cartilage tissue damage of KOA rats,reduces the expression of inflammatory factors and the degree of cell apoptosis..Its mechanism may be related to the regulation of the NLRP3/NF-κB/Caspase-1 signal pathway to reduce pyroptosis.