线粒体钙单向转运体在氧糖剥夺-复氧复糖SH-SY5 Y细胞线粒体自噬中的作用

摘要

Objective To evaluate the role of mitochondrial calcium uniporter ( MCU) in mitoph-agy in SH-SY5Y cells subjected to oxygen-glucose deprivation and restoration (OGD∕R). Methods SH-SY5Y cells were cultured in vitro, seeded in 96-well plates at a density of 2×105cells∕ml, and randomly di-vided into 4 groups (n=6 each) using a random number table method: control group (group C), group OGD∕R, OGD∕R plus MCU inhibitor group ( group OGD∕R + Ru360) and MCU inhibitor group ( group Ru360) . Cells were cultured in normal culture medium in group C. Cells were subjected to O2-glucose dep-rivation for 6 h followed by restoration of O2-glucose supply for 24 h in group OGD∕R. In group OGD∕R+Ru360, Ru360 at a final concentration of 10 μmol∕L was added at 30 min before O2-glucose deprivation, and the other treatments were similar to those previously described in group OGD∕R. Ru360 was added at a final concentration of 10 μmol∕L, and 30 min later cells were cultured under normoxic conditions in group Ru360. At 24 h of restoration of O2-glucose supply, cell counting kit-8 assay was used to detect the cell survival rate, JC-1 assay was used to detect mitochondrial membrane potential ( MMP ) , the ultrastructure of cells was observed with a transmission electron microscope, and the expression of p62, Tom20 and Bec-lin-1 was detected by Western blot. Results Compared with group C, no significant change was found in each parameter in group Ru360 ( P>0. 05) , the cell survival rate and MMP were significantly decreased, the expression of Tom20 and p62 was down-regulated, Beclin-1 expression was up-regulated (P<0. 01), the mitochondria swelled, and mitochondrial autophagosomes were increased in group OGD∕R. Compared with group OGD∕R, the cell survival rate and MMP were significantly increased, the expression of Tom20 and p62 was up-regulated, Beclin-1 expression was down-regulated (P<0. 01), the mitochondrial mor-phology kept well, and mitochondrial autophagosomes were decreased. Conclusion MCU is involved in the process of mitophagy in SH-SY5Y cells subjected to OGD∕R.%目的 评价线粒体钙单向转运体(MCU)在氧糖剥夺-复氧复糖SH-SY5Y细胞线粒体自噬中的作用.方法 离体培养SH-SY5Y细胞,以2×105个∕ml的密度接种于96孔培养板,采用随机数字表法分为4组(n=6):对照组(C组)、氧糖剥夺-复氧复糖组(OGD∕R组)、氧糖剥夺-复氧复糖+MCU抑制剂组(OGD∕R+Ru360组)和MCU抑制剂组(Ru360组).C组正常培养;OGD∕R组行氧糖剥夺6 h后,复氧复糖24 h;OGD∕R+Ru360组氧糖剥夺前30 min加入Ru360,终浓度为10μmol∕L,其余处理同OGD∕R组;Ru360组加入Ru360,终浓度为10μmol∕L,30 min后恢复正常培养.于复氧复糖24 h时采用CCK-8法检测细胞存活率;JC-1法检测细胞线粒体膜电位(MMP);透射电镜下观察细胞超微结构;Western blot法检测线粒体自噬相关蛋白p62、Tom20和Beclin-1的表达.结果 与C组比较,Ru360组各项指标差异无统计学意义(P>0.05),OGD∕R组细胞存活率和MMP下降,Tom20和p62表达下调,Beclin-1表达上调(P<0.01),线粒体肿胀,线粒体自噬体增多;与OGD∕R组比较,OGD∕R+Ru360组细胞存活率和MMP升高,Tom20和p62表达上调,Beclin-1表达下调(P<0.01),线粒体形态较完整,线粒体自噬体数量减少.结论 MCU参与了氧糖剥夺-复氧复糖SH-SY5Y细胞线粒体自噬的过程.