过度机械拉伸应力通过Piezo1介导肌腱细胞凋亡的作用及机制研究

Excessive mechanical stretch stress induces tendon cell apoptosis via mechanosensitive ion channel protein Piezo1

目的探索机械敏感离子通道蛋白Piezo1在过度机械拉伸应力诱导肌腱细胞凋亡中的作用机制。方法分离并培养8周龄雄性C57小鼠(n=20,体质量22~26 g)的肌腱细胞,采用Flexcell系统建立机械拉伸诱导肌腱细胞凋亡的细胞模型。将肌腱细胞分为对照组、20%拉伸组、20%拉伸+Yoda1组、20%拉伸+GsMTx4组、20%拉伸+Piezo1敲低慢病毒(Lv-Piezo1)组及20%拉伸+对照慢病毒(Lv-Ctrl)组,采用线粒体膜电位、流式细胞仪、Western blot和钙离子荧光探针检测Piezo1在肌腱细胞凋亡中的作用。将肌腱细胞分为20%拉伸组、20%拉伸+siRNA介导Calpain2敲低(si-Calpain2)组、20%拉伸+对照siRNA(si-Ctrl)组,Western blot检测Piezo1下游信号Calpain2/BAX/Caspase3轴的激活情况。结果过度机械拉伸应力作用下,20%拉伸组细胞凋亡水平显著高于对照组(P<0.05),Yoda1进一步促进了机械拉伸应力诱导的肌腱细胞凋亡(P<0.05),而GsMTx4和Lv-Piezo1表现出相反的作用(P<0.05)。过度机械拉伸应力作用下,肌腱细胞Calpain2、BAX和cleaved-Caspase3的表达增强(P<0.05)。20%拉伸+si-Calpain2组BAX和cleaved-Caspase3的表达水平低于20%拉伸组、20%拉伸+对照siRNA组(P<0.05),敲低Calpain2可抑制BAX和cleaved-Caspase3的表达(P<0.05),减轻肌腱细胞凋亡(P<0.05)。结论过度机械牵拉应力通过激活Piezo1和下游Calpain2/BAX/Caspase3通路诱导肌腱细胞凋亡,Piezo1有望成为肌腱病的潜在治疗靶点。

Objective To investigate the role and mechanism of Piezo1,a mechanically-sensitive ion channel protein,in excessive mechanical stretch induced apoptosis of tendon cells.Methods Tendon cells of 8-week-old male C57 mice(n=20,body mass:22~26 g)were isolated and cultured,and a cell model of mechanical stretch induced apoptosis of tendon cells was established using the Flexcell system.Tendon cells were divided into the control group,the 20%elongation group,the 20%elongation+Yoda1 group,the 20%elongation+GsMTx4 group,the 20%elongation+Piezo1 knockdown lentivirus group(Lv-Piezo1)and 20%elongation+control lentivirus group(Lv-Ctrl).Mitochondrial membrane potential assay,flow cytometry assay,Western blot assay and calcium fluorescence probe were used to detect the role of Piezo1 in tendon cell apoptosis.Tendon cells were divided into the 20%elongation group,the 20%elongation+siRNA-mediated Calpain2 knockdown group(si-Calpain2),and the 20%elongation+control siRNA group(si-Ctrl).Western blot assay was used to detect the activation of Piezo1 downstream signal Calpain2/BAX/Caspase3 axis.Results The level of apoptosis with excessive mechanical tensile stress was higher in the 20%elongation group than those in the control group(P<0.05),and Yoda1 promoted mechanical tensile stress induced apoptosis in tendon cells(P<0.05),while GsMTx4 and Lv-Piezo1 showed the opposite effect(P<0.05).The expression of Calpain2,BAX and cleaved-Caspase3 in tendon cells was enhanced by excessive mechanical stretch stress(P<0.05).The 20%elongation+si-Calpain2 group had lower expression levels of BAX and cleaved-Caspase3 than those in the 20%elongation and the 20%elongation+control siRNA group(P<0.05).Knockdown of Calpain2 inhibited the expression of BAX and cleaved-Caspase3(P<0.05)and attenuated apoptosis in tendon cells(P<0.05).Conclusion Excessive mechanical stretch stress can induce apoptosis of tendon cells via the activation of Piezo1 and the downstream Calpain2/BAX/Caspase3 pathways.Piezo1 holds promise as a potential therapeutic target for tendinopathy.