化合物968对类风湿性关节炎成纤维细胞样滑膜细胞的增殖和凋亡的影响及其机制

Effect of compound 968 on proliferation and apoptosis of fibroblast-like synoviocytes of rheumatoid arthritis and its mechanism

目的观察谷氨酰胺酶1(GLS1)抑制剂化合物968对类风湿性关节炎成纤维细胞样滑膜细胞(RA-FLS)的增殖和凋亡的影响,并探讨相关机制。方法不同浓度的化合物968(0,5. 0,10. 0,20. 0,40. 0,80. 0μmol/L)刺激RA-FLS细胞24,48和72 h,0μmol/L组为空白对照组。MTT法检测化合物968刺激RA-FLS细胞24,48,72 h,对细胞的增殖抑制作用。倒置显微镜观察化合物968刺激细胞48 h细胞形态的变化。DAPI染色观察化合物968刺激细胞后细胞核的变化。Annexin-Ⅴ/PI双染法检测在化合物968作用下细胞的凋亡率。Western blot检测凋亡相关蛋白Bax、Mcl-1的表达。结果 MTT法结果显示,化合物968能显著抑制RA-FLS细胞的增殖(P<0.05),呈浓度和时间依赖性,作用24,48,72 h的IC50值分别为48. 45,35. 66,20. 95μmol/L。DAPI染色显示,随着药物浓度的增加,细胞核浓染,核固缩现象显著。Annexin-Ⅴ/PI双染法结果表明,化合物968可诱导RA-FLS细胞发生凋亡,20,40,80μmol/L化合物968作用48 h时,细胞凋亡率均明显高于对照组(P<0.05)。Western blot结果显示,化合物968可下调抗凋亡蛋白Mcl-1的表达,并上调促凋亡蛋白Bax的表达。结论化合物968可以明显降低RA-FLS细胞的生存率并可以诱导细胞发生凋亡,其可能的机制是调节细胞凋亡相关蛋白Mcl-1和Bax的表达。

Objective To investigate the effect of compound 968 on the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synoviocyte(RA-FLS),and explore its mechanisms.Methods RA-FLS cells were treated with different concentrations of compound 968(0,5.0,10.0,20.0,40.0,80.0μmol/L)for 24,48 and 72 h,and 0μmol/L group served as a blank control group.The inhibitory effect of compound 968 on RA-FLS cells was detected at 24,48 and 72 h by MTT assay.The morphological changes of RA-FLS cells were observed after the stimulation of compound 968 for 48 h under inverted microscope.DAPI staining was performed to demonstrate the cell nucleus changes after stimulation of compound 968.The apoptosis rate of RA-FLS cells was analyzed by flow cytometry with Annexin-Ⅴ/PI staining.Western blotting was used to detect the expression of apoptosis-related proteins Bax and Mcl-1.Results The results of MTT assay showed that compound 968 inhibited the proliferation of RA-FLS cells in a concentration-and time-dependent manner(P<0.05).The IC 50 values of compound 968 were 48.45,35.66 and 20.95μmol/L at 24,48 and 72 h,respectively.DAPI results showed that the hyperchromatic nuclei was gradually darkened with the increase of concentration and karyopyknosis was observed.The results of Annexin-Ⅴ/PI double staining showed that compound 968 induced apoptosis of RA-FLS cells,and the apoptosis rate of compound 968 at the concentration of 20,40,80μmol/L for 48 h was significantly higher than that of 0μmol/L(P<0.05).The result of Western blot revealed that compound 968 down-regulated the expression of antiapoptotic protein Mcl-1,and up-regulated the expression of proapoptotic protein Bax.Conclusion Compound 968 can significantly reduce the survival rate of RA-FLS cells and induce the apoptosis of cells by regulating the expression of apoptosis-related proteins Mcl-1 and Bax.