摘要
目的 观察程氏蠲痹汤(JBT)对TNF-α诱导的滑膜成纤维细胞(FLS)焦亡的抑制作用及其分子机制。方法 以FLS为研究对象,按CCK8实验结果将其分为A:FLS组;B:TNF-α+FLS组;C:TNF-α+FLS+JBT组(最佳程氏蠲痹汤剂量)。电镜观察细胞形态,使用乳酸脱氢酶(LDH)释放测定各组质膜孔隙形成的情况,采用RT-qPCR、Western Blot(WB)实验观察各组细胞中细胞标志基因、蛋白的表达,CO-IP检测NLRP3和Caspase-1相互作用。结果 CCK8实验结果发现JBT冻干粉浓度在0.5 mg/mL,培养48 h效果最佳。透射电镜显示总体病变程度B组<C组<A组。与B组相比,C组的LDH漏出率显著降低(P<0.01),C组和A组相比,LDH漏出率差异无统计学意义(P>0.05)。RT-qPCR及WB结果显示,A组和C组、B组相比,NLRP3、Caspase-1、IL-18、IL-1β mRNA及蛋白表达上调(P<0.01)与B组相比,C组的NLRP3、Caspase-1、IL-18、IL-1βmRNA及蛋白的表达下调(P<0.01)。根据灰度值的读取结果IP组模型与药物的差异程度与Input组内模型与药物的差异程度存在差异性表明JBT可在一定程度上影响NLRP3和Caspase-1的结合。结论 程氏蠲痹汤通过下调NLRP3/Caspase-1细胞焦亡信号通路而抑制TNF-α诱导的滑膜成纤维细胞焦亡。
Objective To investigate the inhibitory effect of Cheng's Juanbi Decoction(JBT)on TNF-α-induced pyroptosis of fibroblast-like synoviocyte(FLS)and its molecular mechanism.Methods FLS were taken as the research objects,and they were divided into A:FLS group,B:TNF-α+FLS group,C:TNF-α+FLS+JBT group(the optimal dose of Cheng's Juanbi Decoction)according to the CCK 8 test results.The cell morphology was observed by electron microscope,the formation of plasma membrane pores in each group was determined by the release of lactate dehydrogenase(LDH),and the expression of pyroptosis signaling pathway marker genes and proteins in the cells of each group were observed by RT-qPCR and Western Blot(WB),CO-IP Detection of NLRP3 and Caspase-1 interaction.Results The results of CCK 8 experiment showed that the concentration of JBT freeze-dried powder was 0.5 mg/mL,and the effect of culturing for 48 h was the best.Transmission electron microscopy showed that the overall degree of lesions was ranked as follows:B group<C groupA group.Compared with B group,the LDH leakage rate in C group was significantly down(P<0.01),and there was no statistical difference in the LDH leakage rate between C group and A group(P>0.05).RT-qPCR and WB results showed that compared with C group and B group,the mRNA and protein expressions of NLRP3,Caspase-1,IL-18,IL-1βwere up-regulated in FLS group(P<0.01),the mRNA and protein expressions of NLRP3,Caspase-1,IL-18 and IL-1βwere down-regulated in C group and B group(P<0.01).According to the results of Input,it can be seen that the expression of Caspase-1 and NLRP3 in the target cells is normal,and the balance can detect strong protein expression.Non-specific IgG was used as a negative control group and failed to precipitate the target protein.The co-precipitation experiments of NLRP3 and Caspase-1 were used,and it was found that the target protein could be precipitated,indicating that NLRP3 and Caspase-1 were bound in both forward and reverse directions.Moreover,it can be seen from the results that TNF-α
作者
孙广瀚
朱俊
许霞
万磊
南淑玲
王玉凤
赵黎
程卉
王坤
方妍妍
孙朗
SUN Guang-han;ZHU Jun;XU Xia;WAN Lei;NAN Shu-ling;WANG Yu-feng;ZHAO Li;CHENG Hui;WANG Kun;FANG Yan-yan;SUN Lang(School of Chinese Medicine,Anhui University of Chinese Medicine,Hefei 230012;The First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei 230031;Scientific Research and Technology Center,Hefei 230021)
出处
《湖北中医药大学学报》
2023年第4期10-14,共5页
Journal of Hubei University of Chinese Medicine
基金
2020年度国家中医药管理局“中医药古籍文献和特色技术传承专项”(项目编号:GZY-KJS-2020-044)
2020年安徽省高等学校自然科学研究项目(项目编号:KJ2020A0378)
2019年度安徽省社科规划项目(项目编号:AHSKY2019D070)
全国中医药创新骨干人才培训项目(项目编号:国中医药人教函[2019]128号)
安徽省省级质量工程教学研究项目(项目编号:2018jyxm1068)
2019年安徽省高校优秀青年骨干人才项目(项目编号:gxgwfx201904)。
