激活Sonic hedgehog通路改善缺血缺氧心肌细胞DNA损伤

Activation of Sonic hedgehog signaling in ventricular cardiomyocytes exerts DNA protection against hypoxia

目的探究Sonic hedgehog通路在糖氧剥夺(oxygen and glucose deprivation,OGD)诱导的乳鼠心肌细胞DNA损伤和细胞凋亡中的作用及机制。方法原代培养乳鼠心肌细胞,建立OGD的缺血缺氧模型,分别给予Sonic hedgehog通路激动剂SAG1.3(0.002μmol·L^(-1))、抑制剂GANT61(0.005μmol·L^(-1))处理6、12 h,MTT法检测细胞生存率;Western blot检测DNA损伤标志物γH2AX、DNA损伤反应蛋白ATM、ATR,以及细胞凋亡相关蛋白p53、凋亡标志物cleaved-caspase-3、Bcl-2、Bax表达;免疫荧光检测γH2AX表达。结果与正常组相比,OGD组心肌细胞γH2AX、p-ATM表达呈时间依赖性增加,OGD 6 h达到高峰,OGD 12 h后表达逐渐下降;p-p53、cleaved-caspase-3表达增加,Bcl-2/Bax比例下降;与OGD 6 h、OGD 12 h组相比,给予SAG1.3组OGD心肌细胞γH2AX、p-ATM表达明显下降,心肌细胞存活率上升,p-p53、cleaved-caspase-3表达下降,Bcl-2/Bax比例上升;GANT61组OGD心肌细胞较OGD组γH2AX、p-ATM表达明显上升,细胞存活率下降,p-p53、cleaved-caspase-3表达上升,Bcl-2/Bax比例下降。结论激活Sonic hedgehog通路可以通过抑制ATM磷酸化,减少γH2AX表达,改善缺血缺氧诱导的心肌细胞DNA损伤,抑制细胞凋亡。

To investigate the protective roles of sonic hedgehog(Shh)signaling pathway in hypoxia-induced DNA damage with the neonatal rat cardiomyocytes.Methods The hypoxia model on neonatal cardiomyocytes was established with one to two days old Sprague Dawley rats by deprivation of oxygen and glucose(OGD).After pretreated with Shh pathway agonist SAG1.3 or antagonist GANT61,the survival rates of cardiomyocytes were assayed by MTT after OGD 6 hours or 12 hours.The protein levels of Shh pathway,phosphorylated histone H2AX at serine 139(γH2AX),phosphorylated ATM(p-ATM),phosphorylated p53(p-p53),cleaved-caspase-3,Bcl-2 and Bax were detected by Western blot.TheγH2AX foci was detected by immunofluorescence.Results Compared to control group,the protein expression ofγH2AX,p-ATM,cleaved-caspase-3,p-p53 in OGD cardiomyocytes significantly increased,and Bcl-2/Bax ratio proportionally decreased.Particularly,the expression ofγH2AX,p-ATM was highest at OGD 6 h,and then gradually declined after OGD 12 h.After SAG1.3 pretreatment,the expression ofγH2AX,p-ATM,cleaved-caspase-3 and p-p53 dramatically decreased and the Bcl2/Bax ratio increased in OGD 6 h or OGD 12 h cardiomyocytes.On the contrary,in GANT61 pretreatment group,the expression ofγH2AX,p-ATM,cleaved-caspase-3 and p-p53 significantly increased and the Bcl-2/Bax ratio decreased compared to the OGD 6 h or OGD 12 h cardiomyocytes.Conclusion The activation of Shh pathway protects cardiomyocytes against hypoxia-induced apoptosis through inhibition of DNA damage.