醛糖还原酶和晚期糖化终产物受体对糖尿病视网膜病变神经细胞凋亡、自噬和高尔基应激水平的影响

Effects of aldose reductase and receptors for advanced glycation end products on neuronal apoptosis,autophagy and Golgi stress levels in diabetic retinopathy

目的探讨醛糖还原酶和晚期糖化终产物受体对糖尿病视网膜病变神经细胞凋亡、自噬和高尔基应激水平的影响。方法实验中使用C57BL/6J小鼠,动物研究符合ARVO关于在眼科和视觉研究中使用动物的声明,该研究方案得到了当地动物研究伦理委员会的批准。将实验小鼠分为对照组,模型组,观察组。通过蛋白印迹分析大鼠视网膜组织中AR和RAGE的蛋白表达。通过PCR分析大鼠视网膜裂解组织中自噬相关蛋白Beclin-1、LC3Ⅱ/Ⅰ和p62/SQTSM1的mRNA表达。通过ERG检测各组小鼠的视网膜受损情况。通过免疫荧光分析大鼠视网膜组织中Caspase-8和Caspase-3的蛋白表达。TUNEL分析了小鼠视网膜神经细胞的凋亡。通过ELISA分析血清炎性因子TNF-α、IL-6、IL-1β及VEGF的分泌水平。通过PCR分析高尔基应激反应相关因子DR4、TRAPPC13、丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)和ETS的转录水平。结果模型组较对照组AR和RAGE的蛋白表达升高(P<0.05),观察组较模型组AR和RAGE的蛋白表达降低(P<0.05)。模型组较对照组Beclin-1、LC3 II/I和p62/SQTSM1的mRNA表达升高(P<0.05),观察组较模型组表达降低(P<0.05)。模型组较对照组暗视b波和暗视a波的振幅降低(P<0.05),观察组较模型组升高(P<0.05)。模型组较对照组Caspase-8和Caspase-3的蛋白表达升高(P<0.05),观察组较模型组的蛋白表达降低(P<0.05)。与对照组相比,链脲佐菌素诱导的DR神经细胞凋亡量增加(P<0.05),当AR和RAGE收到抑制后,阳性细胞数量降低(P<0.05)。模型组较对照组TNF-α、IL-6、IL-1β及VEGF的水平升高(P<0.05),观察组较模型组降低(P<0.05)。模型组较对照组DR4、TRAPPC13、MAPK和ETS的mRNA表达升高(P<0.05),观察组较模型组表达降低(P<0.05)。结论降低RAGE和AR体内的表达可显著改善小鼠的视力情况,抑制视网膜病变神经细胞凋亡、自噬和高尔基应激水平的发生。

Objective To investigate the effects of aldose reductase(AR)and receptors for advanced glycation end products(RAGE)on neuronal apoptosis,autophagy and Golgi stress levels in diabetic retinopathy(DR).Methods C57BL/6J mice were used in the experiment.The animal research complied with Association for Research in Vision and Ophthalmology(ARVO)statement on the use of animals.The research protocol was approved by the local animal research ethics committee.The experimental mice were allocated to control group,model group and observation group.The protein expressions of AR and RAGE in mouse retina were analyzed by Western blot.The mRNA expressions of autophagy-related proteins(Beclin-1,LC3Ⅱ/Ⅰand p62/SQTSM1)in mouse retina were analyzed by polymerase chain reaction(PCR).The damage to the retina of each group of mice was detected by electroretinogram(ERG).Positive expressions of Caspase-8 and Caspase-3 in rat retina were analyzed by immunofluorescence.Terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)assay was performed to analyze the apoptosis of mouse retinal nerve cells.The secretion levels of serum inflammatory factors(Tumor necrosis factor-alpha[TNF-alpha],Interleukin 6[IL-6],interleukin-1beta[IL-1β],and vascular endothelial growth factor[VEGF])were analyzed by enzyme-linked immunosorbent assay(ELISA).The transcription levels of Golgi stress response factors(DR4,TRAPPC13,MAPK and ETS)were analyzed by PCR.Results The protein expressions of AR and RAGE,and the mRNA expressions of Beclin-1,LC3Ⅱ/Ⅰand p62/SQTSM1 in the model group were significantly higher than those in the control group(all P<0.05),but which in the observation group were significantly lower than those in the model group(P<0.05).Compared with those in the control group,the amplitudes of the b-wave and a-wave of scotopic vision in the model group were significantly lower than those in the control group(P<0.05),which in the observation group were significantly higher than those in the model group(P<0.05).The protein expressions of Caspase-8 and Caspase-3 in the model group were significantly higher than those in the control group(P<0.05),which in the observation group were significantly lower than those in the model group(P<0.05).Compared with that in the control group,the amount of neuronal apoptosis in diabetic retinopathy induced by streptozotocin(STZ)significantly increased(P<0.05),which decreased significantly after silence of AR and RAGE(P<0.05).The positive levels of TNF-α,IL-6,IL-1βand VEGF in the model group were significantly higher than those in the control group(P<0.05),which were significantly lower in the observation group than those in the model group(P<0.05).Compared with those in the control group,the mRNA expressions of DR4,TRAPPC13,MAPK and ETS in the model group were significantly higher(P<0.05),and which in the observation group were significantly lower than those in the model group(P<0.05).Conclusion Silence of RAGE and AR in vivo can significantly improve the visual acuity of DR mice,and inhibit the occurrence of neuronal apoptosis,autophagy and Golgi stress levels.