瞬时受体电位M2抑制剂A10对缺糖缺氧后复糖复氧细胞的保护作用

Protective effect of transient receptor potential melastatin 2 inhibitor A10 on oxygen glucose deprivation/reperfusion model

目的:探讨瞬时受体电位M2(TRPM2)抑制剂A10对缺糖缺氧后复糖复氧(OGD/R)细胞模型的保护作用。方法:采用SH-SY5Y细胞系制备OGD/R损伤模型。将细胞随机分为空白对照组、模型对照组和A10组。细胞计数试剂盒8检测细胞存活率;活性氧检测试剂盒检测细胞活性氧水平;四甲基罗丹明甲酯法检测线粒体膜电位;一步法TUNEL细胞凋亡检测试剂盒检测凋亡细胞数量;蛋白质印迹法测定cleaved caspase 3蛋白表达。结果:相对于3、20、30、50和100μmol/L,10μmol/L浓度的A10具有较低的细胞毒性及较好的通道活性抑制作用。与模型对照组比较,A10组活性氧水平降低(P<0.05),线粒体膜电位降低程度改善(P<0.05),凋亡细胞数减少(P<0.05),凋亡相关蛋白cleaved caspase 3表达减少(P<0.05)。结论:A10可以通过抑制TRPM2通道功能、减少细胞外钙离子内流、降低细胞活性氧水平、稳定线粒体膜电位水平和减少细胞凋亡缓解OGD/R后细胞的损伤。

Objective:To investigate the effect of transient receptor potential melastatin 2(TRPM2)inhibitor A10 on oxygen glucose deprivation/reperfusion(OGD/R)injury in SH-SY5Y cells.Methods:Human neuroblastoma SH-SY5Y cells were subject to OGD/R injury,and then were divided into blank control group,model control group and A10 group randomly.The cell survival rate was detected by cell counting kit 8(CCK-8);the level of cellular reactive oxygen species(ROS)was detected by reactive oxygen detection kit;the mitochondrial membrane potential was detected by tetramethylrhodamine(TMRM)method;the number of apoptotic cells was detected by TUNEL apoptosis assay kit;the protein expression level of cleaved caspase 3 was detected by Western blot.Results:Compared with 3,20,30,50,100μmol/L,10μmol/L A10 has lower cytotoxicity and better inhibition effect on channel activity.Compared with the model control group,ROS level was reduced,the mitochondrial membrane potential was improved,the number of apoptosis cells was reduced,and the expression of cleaved caspase 3 was significantly reduced in the A10 group(all P<0.05).Conclusion:A10 can alleviate cell damage after OGD/R by inhibiting TRPM2 channel function,reducing extracellular calcium influx,reducing cell ROS levels,stabilizing mitochondrial membrane potential levels,and reducing apoptosis.