Li Xiao-yan, Zhang Mao-nian, Pi Yu-li
Department of Ophthalmology, the First Hospital Affiliated to PLA General Hospital, Beijing 100048, China.
Zhonghua Yan Ke Za Zhi. 2010 Apr;46(4):347-54.
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.
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.
(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 rats 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 that had diabetes for 4, 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.
Apoptosis of the retinal neurons in early diabetic rats may correlate with the decline of the activity and mRNA expression of MnSOD.
研究早期糖尿病大鼠视网膜神经元凋亡与锰超氧化物歧化酶（MnSOD）活性及mRNA表达变化的关系。
对照实验研究。将72只8周龄雄性SD大鼠分为对照组和糖尿病组。每组再分为4周、8周和12周组（每组n = 12）。糖尿病组大鼠单次注射链脲佐菌素（60 mg/kg）诱导糖尿病模型，对照组大鼠无任何干预正常饲养。采用TdT介导的dUTP缺口末端标记（TUNEL）法检测视网膜神经元凋亡。免疫组织化学法检测caspase-3蛋白表达。采用黄嘌呤氧化酶法测定MnSOD和铜锌超氧化物歧化酶（Cu-ZnSOD）活性。用SYBR Green Realtime PCR Master Mix检测MnSOD mRNA、Cu-ZnSODmRNA和caspase-3 mRNA水平。采用方差分析检验对照组和糖尿病组视网膜神经节细胞凋亡率、caspase-3 mRNA水平以及Cu-ZnSOD和MnSOD活性及mRNA水平的差异，并用LSD检验进行多重比较。
（1）对照组和糖尿病组在4周、8周、12周时视网膜神经节细胞凋亡率差异有统计学意义（F = 19.5412，P < 0.05）。对照组4周、8周、12周时视网膜均无凋亡神经元。糖尿病发病4周后视网膜神经元开始凋亡。糖尿病8周和12周大鼠视网膜神经节细胞凋亡率分别为（5.7±3.9）%和（11.8±5.1）%，显著高于同年龄对照组（8周和12周：P = 0.000）。（2）对照组大鼠4周、8周、12周时视网膜均未观察到caspase-3蛋白表达。糖尿病发病4周后出现caspase-3阳性染色，8周和12周时增强。对照组和糖尿病组在4周、8周、12周时caspase-3 mRNA水平差异有统计学意义（F = 105.175，P < 0.05）。对照组大鼠4周、8周、12周时caspase-3 mRNA水平分别为1.649±0.586、1.526±0.486、1.614±0.296。糖尿病4周、8周和12周大鼠caspase-3 mRNA水平分别为5.672±1.193、12.566±2.272、14.297±2.11，均高于对照组（4周、8周和12周：P = 0.000）。（3）对照组和糖尿病组在4周、8周、12周时MnSOD和Cu-ZnSOD活性及mRNA水平差异有统计学意义（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）。对照组4周、8周、12周时MnSOD活性分别为（47.118±5.018）、（46.033±6.835）和（45.813±6.859）U/mg；MnSOD mRNA水平分别为0.973±0.123、0.974±0.085和0.994±0.074。Cu-ZnSOD活性分别为（113.884±9.07）、（112.301±5.24）和（117.52±7.982）U/mg；Cu-ZnSOD mRNA水平分别为1.067±0.109、1.055±0.119、1.092±0.180。糖尿病4周、8周和12周大鼠MnSOD活性分别为（33.863±6.909）、（22.877±7.875）和（20.034±6.796）U/mg；MnSOD mRNA水平分别为0.627±0.083、0.333±0.080、0.256±0.057，均低于同年龄对照组（活性：4周：P = 0.002，8周和12周：P = 0.000；mRNA：4周、8周和12周：P = 0.000）。糖尿病4周大鼠Cu-ZnSOD活性（109.793±7.468）U/mg和mRNA水平（0.976±0.108）与同年龄对照组相比无显著差异（活性：P = 0.426；mRNA：P = 0.172）。糖尿病8周和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水平分别为0.829±0.048和0.621±0.033，均低于同年龄对照组（活性：8周：P = 0.011，12周：P = 0.000；mRNA：8周：P = 0.001，12周：P = 0.000）。MnSOD的变化在糖尿病发病4周时即出现，而Cu-ZnSOD的变化出现较晚，主要在糖尿病发病8周和12周时。
早期糖尿病大鼠视网膜神经元凋亡可能与MnSOD活性及mRNA表达下降有关。
