NADPH氧化酶与线粒体的相互作用调控糖尿病大鼠视网膜细胞突触连接的机制

The interaction between NADPH oxidase and mitochondria regulates the mechanism of synaptic connections in retinal cells of diabetic rats

目的:探讨NADPH氧化酶与线粒体的相互作用调控糖尿病大鼠视网膜细胞突触连接的机制。方法:大鼠随机分为3组:对照组(正常饲养同龄的大鼠,n=10),观察组(使用链脲佐菌素诱导糖尿病大鼠模型,n=10),治疗组(胃部进行牛磺酸注射治疗,n=10)。通过组织学定量分析视网膜切片上视锥细胞和神经节细胞。通过蛋白印迹分析大鼠视网膜中突触前标记-代谢型谷氨酸受体6(mGluR6)、突触后密度蛋白95(PSD95)、Bcl-2、Bax和caspase-3表达。通过乳酸脱氢酶(LDH)释放测定法测定细胞毒性水平。通过RT-PCR分析相对线粒体DNA(mtDNA)拷贝数以及细胞中SIRT1、SIRT3和PARP的mRNA表达。测定NADP + / NADPH比率。结果:观察组较对照组视锥细胞和神经节细胞数量降低,治疗组较观察组视锥细胞和神经节细胞升高。观察组较对照组MGluR6、PSD95、Bcl-2 / Bax的蛋白降低,caspase-3蛋白升高。治疗组较观察组MGluR6、PSD95、Bcl-2 / Bax的蛋白升高,caspase-3蛋白降低。观察组较对照组LDH水平升高,治疗组较观察组LDH水平降低,观察组较对照组mtDNA拷贝值降低,治疗组较观察组mtDNA拷贝值升高。观察组较对照组SIRT1和SIRT3的mRNA表达降低,治疗组较观察组SIRT1和SIRT3的mRNA表达升高,观察组较对照组PARP mRNA表达升高,治疗组较观察组PARP mRNA表达降低。观察组较对照组NADPH和GSH降低,治疗组较观察组NADPH和GSH升高,观察组较对照组NADP+和GSSG升高,治疗组较观察组NADP +和GSSG降低。结论:NADPH氧化酶与线粒体的相互作用,通过调节细胞凋亡和视网膜突触蛋白以及SIRT1、SIRT3和PARP表达,调控糖尿病大鼠视网膜细胞突触连接。

Objective To investigate the mechanism by which the interaction of NADPH oxidase and mitochondria regulates the synaptic connection of retinal cells in diabetic rats.Methods Rats were randomly divided into three groups:control group (normal feeding rats of the same age,n=10),observation group (streptozotocin induced diabetic rat model,n=10),treatment group (taurine injection in stomach,n=10).The cone cells and ganglion cells in retinal slices were quantitatively analyzed by histology.Western blotting was used to analyze the expression of mGluR6,PSD95,Bcl-2,Bax and Caspase-3 in rat retina.The cytotoxicity level was determined by lactate dehydrogenase (LDH) release assay.The relative copy number of mitochondrial DNA (mtDNA) and the mRNA expression of SIRT1,SIRT3 and PARP were analyzed by RT-PCR.NADP + / NADPH ratio was determined.Results The number of cones and ganglion cells in the observation group was lower than that in the control group,and the cones and ganglion cells in the treatment group were higher than that in the observation group.Compared with the control group,the protein expression of MGluR6,PSD95,and Bcl-2/Bax in the observation group decreased,and the expression of caspase-3 protein increased.Compared with the observation group,the protein expression of MGluR6,PSD95,and Bcl-2/Bax increased in the treatment group,and the protein expression of caspase-3 decreased.The LDH level of the observation group was higher than that of the control group,and the LDH level of the treatment group was lower than that of the observation group.The mtDNA copy value of the observation group was lower than that of the control group.The copy value increased.The mRNA expression of SIRT1 and SIRT3 in the observation group was lower than that in the control group,the mRNA expression of SIRT1 and SIRT3 in the treatment group was higher than that in the observation group,and the expression of PARP mRNA in the observation group was higher than that of the control group,the expression of PARP mRNA in the treatment group was lower than that in the observation group.Compared with the control group,the observation group has lower NADPH and GSH levels,the treatment group has higher NADPH and GSH levels than the observation group,and the observation group has higher NADP+ and GSSG levels than the control group.The NADP + and GSSG levels of the treatment group were lower than those of the observation group.Conclusion The interaction between NADPH oxidase and mitochondria regulates the synaptic connections of retinal cells in diabetic rats by regulating cell apoptosis and the expression of retinal synaptic proteins,as well as SIRT1,SIRT3 and PARP.