摘要
目的观察新风胶囊(XFC)含药血清对脂多糖(LPS)诱导的类风湿关节炎滑膜成纤维细胞(RA-FLS)焦亡的影响及其机制。方法将20只SD大鼠利用随机数字表法分为2组:空白组和XFC组,XFC组予0.324 mg/g灌胃7 d后制备含药血清。CCK-8法检测XFC含药血清作用于RA-FLS最佳干预浓度和时间。将RA-FLS分为空白组(RA-FLS)、模型组(RA-FLS+5μg/mL LPS)、MCC950组(RA-FLS+LPS+MCC950)、XFC组(RA-FLS+LPS+XFC含药血清)。CCK-8法检测细胞活性,电镜观察RAFLS焦亡形态,ELISA检测IL-1β、IL-18浓度,Western blotting和q RT-PCR检测NLRP3、caspase-1、GSDMD的蛋白和mRNA的表达。结果XFC作用于RA-FLS最佳干预浓度和时间分别为20%和24 h。与空白组相比,模型组细胞活性显著上升(P<0.05),电镜下可见RA-FLS内形成大量小泡,膜上形成孔隙,胞膜破裂,细胞内容物流出,IL-1β、IL-18浓度和NLRP3、GSDMD、caspase-1mRNA及蛋白表达显著升高(P<0.05或P<0.01);与模型组相比,XFC含药血清组、MCC950组细胞活性显著下降(P<0.01),细胞焦亡情况改善,IL-1β、IL-18浓度和NLRP3、GSDMD、caspase-1蛋白及mRNA表达明显降低(P<0.05、P<0.01)。结论XFC可能通过NLRP3/GSDMD通路抑制FLS细胞焦亡,下调炎症细胞因子的分泌,减轻关节局部炎症反应而发挥治疗作用。
Objective To observe the effect of serum from rats treated with Xinfeng Capsule(XFC)on lipopolysaccharide(LPS)-induced pyroptosis of rheumatoid arthritis synovial fibroblasts(RA-FLS)and explore the possible mechanism.Methods Twenty SD rats were divided into blank control group and XFC group.The rats in XFC group was given 0.324 mg/g XFC by gavage for 7 days to prepare the drug-containing serum.CCK-8 assay was used to determine the optimal concentration and duration of the serum for cell treatment.The effect of the drug-containing serum or MCC950 on viability of RA-FLS stimulated with 5μg/mL LPS was assessed with CCK-8 assay,and pyroptosis of the cells was observed using electron microscope;the levels of IL-1βand IL-18 in the cell culture supernatant were detected by ELISA,and the protein and mRNA expressions of NLRP3,caspase-1 and GSDMD were detected using Western blotting and qRT-PCR.Results The optimal concentration and duration of XFC for RA-FLS treatment were 20%and 24 h,respectively.Compared with the blank control cells,the cells with LPS stimulation showed significantly increased cell viability(P<0.05)and electron microscopy revealed a large number of vesicles in the cells with formation of membrane pores,cell membrane rupture,and leakage of cell contents.LPS stimulation significantly increased IL-1βand IL-18 levels and expressions of NLRP3,GSDMD,and caspase-1 in the cells(P<0.05 or 0.01).Treatment with the drug-containing serum or MCC950 significantly decreased the viability of LPS-stimulated RA-FLS(P<0.01),reduced cell pyroptosis,and lowered the concentrations of IL-1βand IL-18 and expressions of NLRP3,GSDMD,and caspase-1(P<0.05 or 0.01).Conclusion XFC alleviates local inflammatory response of joints in RA possibly by inhibiting pyroptosis of the FLS through inhibition of the NLRP3/GSDMD pathway,which results in reduced secretion of inflammatory cytokines.
作者
王露琳
汪元
刘健
黄传兵
张皖东
万磊
孙玥
黄旦
WANG Lulin;WANG Yuan;LIU Jian;HUANG Chuanbing;ZHANG Wandong;WAN Lei;SUN Yue;HUANG Dan(Anhui University of Traditional Chinese Medicine,Hefei 230038,China;Department of Rheumatology,First Affiliated Hospital of Anhui University of Traditional Chinese Medicine,Hefei 230031,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2022年第12期1846-1851,共6页
Journal of Southern Medical University
基金
安徽省青年领军后备人才支持项目(中发展[2022]4号)
新安医学教育部重点实验室开放基金项目(2020xayx01)
安徽省高校优秀青年人才支持计划项目(gxyq2019031)
国家临床重点专科中医风湿病科建设项目(财社[2013]239号)
安徽省重点实验室建设项目(1306c083035)
安徽省名中医刘健工作室建设项目(中医药发展秘[2018]11号)
安徽省第12批“115”创新团队项目(皖人才办[2019]1号)。
