[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 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.


CONCLUSIONS
Apoptosis of the retinal neurons in early diabetic rats may correlate with the decline of the activity and mRNA expression of MnSOD.
Li
Xiao-yan
XY
Department of Ophthalmology, the First Hospital Affiliated to PLA General Hospital, Beijing 100048, China.
Zhang
Mao-nian
MN
Pi
Yu-li
YL
D000818:Animals; D017209:Apoptosis; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism* / Q000473:pathology; D008297:Male; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000378:metabolism / Q000473:pathology*; D013482:Superoxide Dismutase / Q000378:metabolism*
D012333:RNA, Messenger; D013482:Superoxide Dismutase