高迁移率族蛋白B-1和NADPH氧化酶衍生活性氧对糖尿病视网膜病变大鼠视网膜电图波幅和玻璃体通透性的影响

Effects and mechanism of high mobility group box-1 and nicotinamide adenine dinucleotide phosphate oxidase on electroretinogram amplitude and vitreous permeability of diabetic retinopathy rats

目的:探究高迁移率族蛋白B-1(HMGB1)和还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶衍生活性氧(ROS)对糖尿病视网膜病变(DR)大鼠视网膜电图波幅和玻璃体通透性的影响及机制。方法:选取40只雄性SD大鼠,根据实验目的将大鼠分为对照组、DR模型组、HMGB1注射组和ROS抑制剂组。使用链脲佐菌素诱导大鼠糖尿病,进而发展DR模型。通过2’-7’-二氯荧光素-二乙酸酯(DCFH-DA)检测大鼠视网膜组织匀浆中ROS的生成。通过蛋白印迹分析视网膜组织中Nox2、HMGB1、Occludin和ZO-1的蛋白表达。通过PCR分析大鼠视网膜组织匀浆中PARP-1和Caspase 3的mRNA表达。通过视网膜电图分析大鼠a波和b波振幅。通过测量FITC-BSA荧光评估大鼠玻璃体通透性。结果:DR模型组和HMGB1注射组较对照组大鼠视网膜ROS水平升高(均P<0.05),ROS抑制剂组较DR模型组和HMGB1注射组ROS水平降低(均P<0.05)。DR模型组和HMGB1注射组较对照组Nox2和HMGB1的蛋白表达升高(均P<0.05),ROS抑制剂组较HMGB1注射组和DR模型组Nox2和HMGB1的蛋白表达降低(均P<0.05)。HMGB1注射组和DR模型组较对照组大鼠视网膜PARP-1和Caspase 3的mRNA表达升高(均P<0.05)。HMGB1注射组和DR模型组较对照组a波和b波振幅减少(均P<0.05),ROS抑制剂组较HMGB1注射组和DR模型组振幅升高(均P<0.05)。HMGB1注射组和DR模型组较对照组Occludin和ZO-1的蛋白表达降低(均P<0.05),ROS抑制剂组较HMGB1注射组和DR模型组Occludin和ZO-1的蛋白表达升高(均P<0.05)。DR模型组和HMGB1注射组较对照组荧光强度升高(P<0.05),ROS抑制剂组较DR模型组和HMGB1注射组荧光强度降低(P<0.05)。结论:糖尿病视网膜组织中HMGB1和Nox衍生的ROS之间存在正反馈,这可能是促进糖尿病诱导的视网膜电图波幅降低和玻璃体通透性增加的机制。

Objective:To explore the effects and mechanism of high mobility group box-1(HMGB1)and nicotinamide adenine dinucleotide phosphate(NADPH)oxidase on the amplitude of electroretinogram and the permeability of vitreous of diabetic retinopathy(DR)rats.Methods:Forty male SD rats were divided into control group,DR model group,HMGB1 injection group and ROS inhibitor group.Streptozotocin was used to induce diabetes in rats,and DR model was developed.ROS in rat retinal homogenate was detected by DCFH-DA.The protein expressions of Nox2,HMGB1,Occludin and ZO-1 in retinal tissue were analyzed by Western blot.The mRNA expressions of PARP-1 and Caspase-3 in rat retinal homogenate were analyzed by PCR.The amplitude of a wave and b wave in rats was ananlyzed by electroretinogram.Rat vitreous permeability was evaluated by measuring FITC-BSA fluorescence.Results:The level of ROS in the retina of DR model group and HMGB1 injection group was higher than that of the control group,and the level of ROS in ROS inhibitor group was lower than that of DR model group and HMGB1 injection group(all P<0.05).The expression levels of Nox2 and HMGB1 proteins in DR model group and HMGB1 injection group were higher than those in control group,and the expression levels of Nox2 and HMGB1 proteins in ROS inhibitor group were lower than those in HMGB1 injection group and DR model group(all P<0.05).The expression levels of PARP-1 and Caspase-3 mRNAs in retina of rats in HMGB1 injection group and DR model group were higher than those in control group(all P<0.05).Compared with the control group,the amplitude of a wave and b wave in HMGB1 injection group and DR model group decreased,and the amplitude of ROS inhibitor group increased(all P<0.05).The expression levels of Occludin and ZO-1 proteins in HMGB1 injection group and DR model group were lower than those in control group,and the expression levels of Occludin and ZO-1 proteins in ROS inhibitor group were higher than those in HMGB1 injection group and DR model group(all P<0.05).The fluorescence intensity of DR model group and HMGB1 injection group was higher than that of the control group,and the fluorescence intensity of ROS inhibitor group was lower than that of DR model group and HMGB1 injection group(all P<0.05).Conclusion:There is a positive feedback between HMGB1 and Nox-derived ROS in the diabetic retina,which may be the mechanism that promotes the decrease in the amplitude of the diabetic-induced electroretinogram and the increase in the permeability of the vitreous.