青蒿琥酯调节BCL-2家族蛋白平衡诱发肌成纤维细胞凋亡

Myofibroblasts apoptosis induced by artesunate via regulating balance of BCL-2 family

目的研究青蒿琥酯诱导纤维化核心细胞——肌成纤维细胞线粒体凋亡作用并探索其对BCL-2家族表达的影响。方法构建人肺肌成纤维细胞模型,采用CCK-8、流式及JC-1方法检测青蒿琥酯处理后人肺肌成纤维细胞的活性、凋亡启动、凋亡状态的变化,RT-qPCR和western blot检测α-SMA和BCL-2家族基因表达情况。结果人肺成纤维细胞加入TGF-β10 ng/mL刺激48 h成功构建表达α-SMA的人肺肌成纤维细胞模型;青蒿琥酯组细胞活性明显下降(P<0.001),细胞凋亡(Annexin V+)比例明显增加(P<0.001);青蒿琥酯组红色荧光强度明显下降[从(17.58±1.08)降至最低峰(1.28±0.35)],24~36 h降幅最为明显;而对照组红色和绿色荧光强度(JC-1信号)无明显变化。青蒿琥酯组抗凋亡BCL-2 mRNA表达较对照组明显降低(P<0.001),促凋亡BIM(P<0.01)和PUMA、BMF、HRK(P<0.05)mRNA表达升高,而BAX、BAK、BAD、NOXA变化差异无统计学意义。结论青蒿琥酯通过下调BCL-2,上调BIM、PUMA、BMF和HRK基因的表达,调节线粒体BCL-2家族促凋亡/抗凋亡蛋白表达平衡,触发线粒体凋亡启动,从而诱发人肺肌成纤维细胞凋亡。

Objective To study the effect of artesunate on inducing mitochondrial apoptosis of myofibroblasts,the key cells of fibrosis,and to explore the effect on the expression of BCL-2 family.Methods Human pulmonary myofibroblasts model was constructed.CCK-8,flow cytometry and JC-1 were used to detect the changes of cell activity,apoptosis initiation and apoptosis status of the myofibroblasts treated with artesunate.RT-qPCR and western blot were used to detectα-SMA and BCL-2 family gene expression.Results Human pulmonary myofibroblasts expressingα-SMA were successfully constructed by adding 10 ng/mL TGF-βto fibroblasts for 48 h.In artesunate treating group,the cell viability of myofibroblasts was significantly decreased(P<0.001),but thepercentage of apoptotic cells(Annexin V+)was significantly increased(P<0.001).The red fluorescence intensity decreased significantly in the artesunate group[from(17.58±1.08)to(1.28±0.35)],especially at 24~36 h.The red and green fluorescence intensity(JC-1 signal)did not change significantly in the control group.Compared with the control group,the mRNA expression of anti-apoptotic BCL-2 in the artesunate group was significantly decreased(P<0.001),but the mRNA expression of pro-apoptotic BIM(P<0.01)and PUMA,BMF and HRK(P<0.05)were increased.The mRNA expression of BAX,BAK,BAD and NOXA did not change significantly.Conclusion Artesunate regulates the pro-apoptotic/anti-apoptotic balance of mitochondrial BCL-2 family by down-regulating the expression of gene BCL-2,up-regulating the expressions of genes BIM,PUMA,BMF and HRK,and triggering the initiation of mitochondrial apoptosis,thereby inducing the apoptosis of human pulmonary myofibroblasts.