摘要
目的:观察乌头碱对类风湿性关节炎成纤维细胞样滑膜细胞(RA-FLS)增殖、凋亡及自噬的影响,探讨其在抗类风湿关节炎疾病中的作用机制。方法:对类风湿关节炎(RA)患者的膝关节滑膜组织进行原代细胞培养RA-FLS,采用不同浓度的乌头碱对RA-FLS进行处理,运用相差显微镜观察RA-FLS的形态结构,运用MTT法测定RA-FLS的增殖活性,流式细胞仪测定RA-FLS的细胞凋亡率,采用定量RT-qPCR、Western blot、免疫荧光法测定LC3基因和蛋白的表达。结果:乌头碱能有效抑制RA-FLS细胞增殖,倒置显微镜下发现经乌头碱处理后RA-FLS细胞逐渐变圆、间隙增加;流式检测乌头碱能促进RA-FLS凋亡;定量RT-qPCR、Western blot及免疫荧光检测LC3基因及蛋白的水平均随乌头碱浓度的增加而增加,LC3表达水平明显增强。结论:乌头碱可抑制RA-FLS增殖并诱导凋亡,且具有浓度依赖性,这可能与其过度激活RA-FLS中的自噬水平有关。
Objective:To observe the effects of aconitine on proliferation,apoptosis and autophagy of rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS),and to explore its mechanism of action in rheumatoid arthritis.Methods:Primary cultured RA-FLS was performed on the synovial tissue of patients with rheumatoid arthritis(RA).RA-FLS was treated with different concentrations of aconitine.The morphology of RA-FLS was observed by phase contrast microscope.The proliferation activity of RA-FLS was determined by MTT assay.The apoptosis rate of RA-FLS was determined by flow cytometry.The expression of LC3 gene and protein was determined by quantitative RT-qPCR,Western blot and immunofluorescence.Results:Aconitine could effectively inhibit the proliferation of RA-FLS cells.It was observed by inverted microscope that RA-FLS cells gradually became round and the gap increased after aconitine treatment.Flow-through detection of aconitine promoted RA-FLS apoptosis.The levels of LC3 gene and protein were increased by quantitative RT-qPCR,Western blot and immunofluorescence with the increase of aconitine concentration,and the expression level of LC3 was significantly enhanced.Conclusion:Aconitine can inhibit the proliferation of RA-FLS and induce apoptosis in a concentration-dependent manner,which may be related to the level of autophagy in over-activation of RA-FLS.
作者
邵鑫
袁叶双
蒋先虹
贾艾敏
刘静
陈勇
刘福
SHAO Xin;YUAN Ye-Shuang;JIANG Xian-Hong;JIA Ai-Min;LIU Jing;CHEN Yong;LIU Fu(School of Pharmacy,North Sichuan Medical College,Nanchong 637000,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2019年第24期3066-3070,F0004,共6页
Chinese Journal of Immunology
基金
四川省卫计委项目基金(18ZD009)资助
