雷公藤甲素抑制类风湿性关节炎成纤维细胞样滑膜细胞的自噬与存活

Triptolide suppresses autophagy and survival in rheumatoid arthritis fibroblast-like synoviocytes

目的探讨雷公藤甲素抑制类风湿性关节炎成纤维细胞样滑膜细胞(rheumatoid arthritis fibroblast-like synoviocytes, RA-FLSs)的自噬与存活。方法体外培养人RA-FLSs细胞,使用不同浓度的雷公藤甲素(0、10、20、30、40、50 ng/mL)处理RA-FLSs细胞,处理24、48、72 h后,采用CCK-8试剂盒测定RA-FLSs的增殖能力,确定对细胞增殖能力抑制50%时所需的雷公藤甲素浓度(半数最大抑制浓度,IC_(50))。基于RA-FLSs增殖能力测定的实验结果,选取最佳雷公藤甲素作用浓度处理RA-FLSs,处理24、48、72 h后,检测RA-FLSs自噬荧光强度;采用Western blot检测LC3Ⅱ/LC3Ⅰ、Beclin1、Bax和caspase-3蛋白表达水平。结果雷公藤甲素呈剂量依赖性的抑制RA-FLSs细胞的增殖,雷公藤甲素浓度为40 ng/mL时,细胞增殖活力显著下降(P<0.001),24、48、72 h的IC_(50)分别是42.31、40.71、42.71 ng/mL。因此后续的实验选用雷公藤甲素的上药浓度为40 ng/mL。与空白组相比,40 ng/mL雷公藤甲素作用RA-FLSs 24、48、72 h后,均能减弱LC3Ⅱ自噬荧光、显著降低自噬蛋白LC3Ⅱ/LC3Ⅰ、Beclin1表达水平(P<0.05),显著增加凋亡蛋白Bax、caspase-3表达水平(P<0.05);且在处理48 h时,40 ng/mL雷公藤甲素抑制自噬及促进凋亡的作用较处理24 h及72 h强。结论雷公藤甲素治疗类风湿性关节炎的作用机制可能与通过抑制RA-FLSs自噬,从而对RA-FLSs产生抑制增殖和促进凋亡作用。这一发现表明雷公藤甲素是一种治疗类风湿性关节炎的潜在药物。

Objective To evaluate the effects of triptolide on the survival and autophagy of RA-fibroblast-like synoviocytes(FLSs).Methods Human RA-FLSs cells were cultured in vitro. RA-FLSs cells were treated with different concentrations of triptolide(0, 10 20, 30, 40, 50 ng/mL), respectively. After 24, 48, 72 h treatment, the proliferation ability of RA-FLSs was determined by CCK-8 kit to determine IC_(50). Based on the results of the assay of RA-FLSs proliferation ability,the optimal concentration of triptolide was selected to treat RA-FLSs. After 24, 48, 72 h treatment, the autophagy fluorescence intensity of RA-FLSs was detected. The protein expression levels of LC3Ⅱ/LC3Ⅰ, Beclin1, Bax and Caspase-3 were detected by Western blotting.Results The proliferation of RA-FLSs cells was inhibited by triptolide in a dose-dependent manner.When the concentration of triptolide was 40 ng/mL, the cell proliferation activity was significantly decreased(P<0.001), and the IC_(50) values for 24, 48, 72 h were 42.31、40.71、42.71 ng/mL, respectively. Therefore, the concentration of triptolide was40 ng/mL in subsequent experiments. Compared with blank group, 40 ng/mL triptolide could decrease the autophagy fluorescence of LC3Ⅱ, significantly decrease the expression levels of autophagy protein LC3Ⅱ/LC3Ⅰ and Beclin1(P<0.05),and significantly increase the expression levels of apoptotic protein Bax and Caspase-3(P<0.05) after 24 h, 48 h and 72 h treatment of RA-FLSs cells. At 48 h, 40 ng/mL triptolide showed stronger inhibition of autophagy and promotion of apoptosis than that at 24 h and 72 h.Conclusions The mechanism of triptolide in the treatment of rheumatoid arthritis may be related to the inhibition of RA-FLSs proliferation and promotion of RA-FLSs apoptosis through inhibition of RA-FLSs autophagy.This finding indicates that triptolide is a potential therapeutic agent for RA.