Cu-ATSM保护内皮细胞糖氧剥夺再灌注损伤的机制

The mechanism of Cu-ATSM in protecting human umbilical vein endothelial cells from oxygen-glucose deprivation and reperfusion injury

目的:探讨铜-二乙酰-2(N4-甲基氨基硫脲)(Cu-ATSM)对糖氧剥夺再灌注(OGD/R)后人脐静脉内皮细胞(HUVECs)凋亡的作用及其潜在机制。方法:将体外培养的HUVECs分为Control组、OGD/R组、OGD/R+Cu-ATSM组;采用流式细胞术检测HUVECs的凋亡情况;采用Mito-tracker染色检测线粒体形态;JC-1染色分析各组线粒体膜电位的变化;采用蛋白质印迹法(Western blot)检测各组自噬相关蛋白如ATG5、Beclin1、LC3Ⅰ/Ⅱ的激活情况;采用免疫荧光检测各组细胞LC3Ⅱ的表达情况。结果:流式细胞术结果显示,与Control组比,OGD/R损伤后HUVECs凋亡数目显著上升,而当Cu-ATSM治疗后,凋亡的HUVECs显著减少(均P<0.05)。Mito-tracker及JC-1染色结果显示,与Control组比,OGD/R组损伤后线粒体的形态明显受到损伤,线粒体功能受损,膜电位下降,而OGD/R+Cu-ATSM组治疗后能够改善损伤的线粒体功能,维持线粒体形态和线粒体膜电位(均P<0.05)。Western blot结果显示,与Control组比,OGD/R组损伤后自噬相关信号通路如ATG5、Beclin1、LC3Ⅱ蛋白的表达量增多,而OGD/R+Cu-ATSM组治疗后的自噬相关蛋白表达量较OGD/R组下降(均P<0.05)。免疫荧光结果显示,与Control组比,OGD/R组损伤后LC3Ⅱ的表达显著增多,而OGD/R+Cu-ATSM治疗后的LC3Ⅱ显著减少。结论:Cu-ATSM能够改善HUVECs OGD/R损伤后的线粒体功能,减少HUVECs凋亡,其机制可能是通过抑制自噬实现的。

Objective:To investigate the effect of copper-diacetyl-2(N~4-methylthiosemicarourea)(CuATSM)on human umbilical vein endothelial cells(HUVECs)apoptosis and its underlying mechanisms after oxygen-glucose deprivation/reperfusion(OGD/R).Methods:HUVECs were divided as the control,OGD/R and OGD/R+Cu-ATSM groups.Flow cytometry was used to detect the effect of Cu-ATSM on OGD/R induced cell apoptosis.Mito-tracker staining was used to detect the effect of Cu-ATSM on mitochondrial morphology.JC-1 staining was used to detect the change of mitochondrial membrane potential.Western blot was used to detect the expression of autophagy related proteins such as ATG5,Beclin1 and LC3Ⅰ/Ⅱ.Immunofluorescenc was used to detect the expression of LC3Ⅱ.Results:Flow cytometry results showed that the number of HUVECs apoptosis was significantly increased in OGD/R group,compared with the control group.However,after Cu-ATSM treatment the apoptosis cells were significantly decreased(P<0.05),indicating that Cu-ATSM treatment inhibited the HUVECs cell apoptosis after OGD/R.Mito-tracker and JC-1 staining results suggested that mitochondrial morphology and function were significantly damaged and membrane potential decreased in the OGD/R group,compared with the control group.However,the Cu-ATSM treatment ameliorated mitochondrial function and maintained mitochondrial morphology and mitochondrial membrane potential.Western blot results suggested that compared with the control group,the expression of autophagy related protein such as ATG5,Beclin1 and LC3Ⅱincreased in OGD/R group,while the expression of autophagy related proteins decreased after Cu-ATSM treatment(P<0.05).Immunofluorescence results showed that compared with the control group,the number of LC3Ⅱwas significantly increased after OGD/R injury,while the number of LC3Ⅱwas significantly decreased after CuATSM treatment.Conclusion:Cu-ATSM improves mitochondrial function and inhibits apotosis on HUVECs after OGD/R,which may be achieved by inhibiting autophagy.