糖尿病早期大鼠视网膜神经细胞凋亡与锰超氧化物歧化酶活性及mRNA表达变化的关系

目的 探讨糖尿病早期大鼠视网膜神经细胞凋亡与锰超氧化物歧化酶(MnSOD)活性及mRNA表达变化的关系.方法 对照实验研究.健康雄性8周龄SD大鼠72只,分为正常对照组及糖尿病组.糖尿病组大鼠给予一次性腹腔注射链脲佐菌素(STZ)60 mg/kg体重,以诱导糖尿病大鼠模型;建模成功后再于建模后4、8、12周3个时间点分为3组,每组各12只大鼠.正常对照组不进行干预,与糖尿病组大鼠同时饲养,也按相同时间点分为4、8、12周3组,每组各12只大鼠.采用TdT介导DNA缺口末端的dUTP标记(TUNEL)法检测视网膜神经细胞凋亡指数,免疫组织化学方法检测半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白表达水平,黄嘌呤氧化酶法检测MnSOD及铜一锌超氧化物歧化酶(Cu-ZnSOD)活性,实时荧光定量聚合酶链反应(PCR)法检测MnSOD mRNA、Cu-ZnSODmRNA及easpase-3 mRNA表达情况.正常对照组与糖尿病组大鼠各时间点视网膜神经细胞凋亡指数、视网膜caspase-3 mRNA表达水平、Cu-ZnSOD和MnSOD活性及mRNA表达水平比较,均采用两因素方差分析,进一步两两比较采用LSD检验法.结果 (1)正常对照组4、8、12周大鼠视网膜均未见神经细胞凋亡.糖尿病组大鼠于建模后4周即出现神经细胞凋亡,凋亡指数为(0.5±1.5)%;8和12周大鼠神经细胞凋亡指数为(5.7±3.9)%和(11.8±5.1)%.糖尿病组与正常对照组大鼠各时间点视网膜神经细胞凋亡指数差异有统计学意义(F=19.5412,P＜0.05).进一步两两比较,8和12周糖尿病大鼠视网膜神经细胞凋亡指数较同期正常对照组大鼠均明显升高(均P=0.000).(2)正常对照组4、8、12周大鼠视网膜均未见caspase-3蛋白阳性表达,糖尿病组大鼠4周即可见caspase-3蛋白阳性表达,8和12周时表达增强.正常对照组4、8、12周大鼠caspase-3 mRNA表达RQ值分别为1.649±0.586、1.526±0.486及1.614±0.296;糖尿病组4、8、12周大鼠caspase-3 mRNA表达RQ值分别为5.672±1.193、12.566±2.272及14.297±2.11;正常对照组与糖尿病组大鼠各时间点视网膜caspase-3 mRNA表达RQ值差异有统计学意义(F=105.175,P＜0.05).进一步两两比较,糖尿病组大鼠各时间点视网膜caspase-3 mRNA表达RQ值均较同期正常对照组明显升高(均P=0.000).(3)正常对照组4、8、12周大鼠MnSOD活性分别为(47.118±5.018)、(46.033±6.835)及(45.813±6.859)U/mg,MnSOD mRNA表达RQ值分别为0.973±0.123、0.974±0.085及0.994±0.074,CuZnSOD活性分别为(113.884±9.07)、(112.301±5.24)及(117.52±7.982)U/mg,Cu-ZnSOD mRNA表达RQ值分别为1.067±0.109、1.055±0.119及1.092±0.180.糖尿病组4、8、12周大鼠MnSOD活性分别为(33.863±6.909)、(22.877±7.875)及(20.034±6.796)U/mg,MnSOD mRNA表达RQ值分别为0.627±0.083、0.333±0.080及0.256±0.057;8和12周大鼠Cu-ZnSOD活性分别为(98.588±9.212)和(78.168±12.180)U/mg,Cu-ZnSOD mRNA表达RQ值为0.829±0.048和0.621±0.033.正常对照组与糖尿病组大鼠各时间点视网膜MnSOD、Cu-ZnSOD活性及mRNA表达RQ值差异有统计学意义(MnSOD:活性F=19.709,P＜0.05;mRNA F=93.352,P＜0.05;Cu-ZnSOD:活性F=16.708,P＜0.05;mRNA F=16.332,P＜0.05).进一步两两比较,糖尿病组大鼠各时间点视网膜MnSOD、Cu-ZnSOD活性及mRNA表达RQ值均较同期对照组明显下降(MnSOD活性:4周P=0.002,8和12周均P=0.000;MnSOD mRNA:4、8、12周均P=0.000;Cu-ZnSOD活性:8周P=0.011,12周P=0.000;Cu-ZnSOD mRNA:8周P=0.001,12周P=0.000).结论 糖尿病早期视网膜神经细胞凋亡可能与MnSOD活性及MnSOD mRNA表达水平下降有关.

Relationship between retinal neurons apoptosis and changes of manganese superoxide dismutase activity and mRNA expression in early diabetic rats

Objective To study the relationship between the apoptosis of retinal neurons and changes of manganese superoxide dismutase (MnSOD) activity and mRNA expression in early diabetic rats.Methods Controlled experimental study.Seventy two male 8-week-aged SD rats were divided into control group and diabetic mellitus group.Each group were subdivided into 4,8 and 12 weeks groups (each group,n = 12).The diabetic group rats were injected with a single dose of streptozotocin (60 mg/kg) to induce the diabetic model,the control group rats were raised without any intervention.Apoptosis of retinal neurons was detected by TdT-mediated dUTP nick end label (TUNEL) assay.The protein expression of caspase-3 was detected by immunohistochemistry.Xanthine oxidase method was used to measure the activity of MnSOD and copper-znic superoxide dismutase (Cu-ZnSOD).MnSOD mRNA,Cu-ZnSODmRNA and caspase-3 mRNA levels were determined by SYBR Green Realtime PCR Master Mix.ANOVA was used to test the comparisons of the apoptosis ratio of retinal ganglion cells,the levels of caspase-3 mRNA,and the activity and mRNA levels of Cu-ZnSOD and MnSOD in the control groups and the diabetic groups,and LSD was used to test multiple comparisons.Results (1) The difference of the retinal ganglion cells apoptosis ratio in the control groups and the diabetic groups had statistical significance at 4,8,12 weeks (F = 19.5412,P ＜ 0.05).There were no apoptosis neurons in control groups' retina at 4,8,12 weeks.Apoptosis of the retinal neurons occurred 4 weeks after the onset of diabetes.The apoptosis ratio of retinal ganglion cells in rats that had diabetes for 8 and 12 weeks was(5.7 ±3.9)% and (11.8±5.1)%,respectively,and was significantly higher than that of age-matched control groups (8 and 12 weeks;P = 0.000).(2) Caspase-3 protein expression was not observed in the control rats' retina at 4,8,12 weeks.Positive staining of caspase-3 occurred 4 weeks after the onset of diabetes,and enhanced at 8 and 12 weeks.The difference of caspase-3 mRNA levels in the control groups and the diabetic groups had statistical significance at 4,8,12 weeks (F = 105.175,P＜0.05).In control rats at 4,8 and 12 weeks,caspase-3 mRNA levels were 1.649 ±0.586,1.526 ±0.486,1.614 ±0.296,respectively.Caspase-3 mRNA levels in diabetic rate that had diabetes for 4,8 and 12 weeks were 5.672 ± 1.193,12.566 ±2.272,14.297 ±2.11,respectively,which were greater than that in the control groups (4,8 and 12 weeks;P =0.000).(3) The difference of the activity and mRNA levels of MnSOD and Cu-ZnSOD in the control groups and the diabetic groups had statistical significance at 4,8,12 weeks (MnSOD:activity:F = 19.709,P＜0.05,mRNA:F = 93.352,P＜0.05;Cu-ZnSOD:activity:F=16.708,P＜0.05,mRNA:F = 16.332,P ＜0.05).In the control groups at 4,8 and 12 weeks,the activity of MnSOD was (47.118 ± 5.018) ,(46.033 ± 6.835) and (45.813 ±6.859)U/mg,respectively;and MnSOD mRNA levels were 0.973 ±0.123,0.974 ±0.085 and 0.994 ±0.074,respectively.The activity of Cu-ZnSOD was (113.884 ± 9.07),(112.301 ± 5.24) and (117.52 ±7.982) U/mg,respectively;and Cu-ZnSOD mRNA levels of were 1.067 ±0.109,1.055 ±0.119,1.092 ±0.180,respectively.In the rats had diabetes for4,8 and 12 weeks,the activity of MnSOD was (33.863 ±6.909) ,(22.877 ±7.875) and (20.034 ±6.796) U/mg,respectively;and MnSOD mRNA levels were 0.627 ± 0.083,0.333 ± 0.080,0.256 ± 0.057,respectively;these data were less than those in the age-matched control groups (activity:4 weeks:P=0.002,8 and 12 weeks;P= 0.000;mRNA:4,8 and 12 weeks:P = 0.000).The activity (109.793 ± 7.468) U/mg and mRNA level (0.976 ± 0.108) of Cu-ZnSOD in rats had diabetes for 4 weeks showed no significant difference as compared to age-matched control group (activity;P=0.426;mRNA:P = 0.172).In diabetic rats at 8 and 12 weeks,the activity of Cu-ZnSOD was (98.588 ±9.212) and (78.168 ± 12.180) U/mg,respectively;Cu-ZnSOD mRNA levels were 0.829 ±0.048 and 0.621 ± 0.033,respectively;these data were less than those in the age-matched control group (activity;8 weeks;P =0.011,12 weeks;P =0.000;mRNA:8 weeks;P =0.001,12 weeks;P= 0.000).The changes of MnSOD occurred as early as 4 weeks after the onset of diabetes,while the changes of the Cu-ZnSOD occurred later,mainly at 8 and 12 weeks after the onset of diabetes.Conclusions Apoptosis of the retinal neurons in early diabetic rats may correlate with the decline of the activity and mRNA expression of MnSOD.