褪黑素对早期糖尿病大鼠视网膜锰超氧化物歧化酶和铜锌超氧化物歧化酶表达的影响

Effects of melatonin on expression of manganese superoxide dismutase and cuproznic superoxide dismutase in retina in early diabetic rats

目的观察褪黑素对早期糖尿病大鼠视网膜锰超氧化物歧化酶(MnSOD)、铜锌超氧化物歧化酶(Cu/ZnSOD)活性及基因表达变化的影响,以揭示褪黑素对早期糖尿病大鼠视网膜的抗氧化作用机制。方法 54只SD大鼠按随机数字表法分为正常对照组、糖尿病组及褪黑素组,每组18只。糖尿病组及褪黑素组大鼠一次性腹腔注射链脲佐菌素(STZ)60mg/kg,升高血糖至>16.7mmol/L,建立糖尿病大鼠模型,褪黑素组大鼠于STZ注射后第2天给予褪黑素灌胃,每日10mg/kg。于造模后4、8、12周3个时间点处死各组6只大鼠制备视网膜匀浆。应用黄嘌呤氧化酶法检测视网膜组织中总SOD、Cu/ZnSOD及MnSOD的活性;应用实时荧光定量PCR法检测视网膜组织中MnSODmRNA及Cu/ZnSODmRNA的表达量。结果模型建立后4、8、12周时糖尿病组和褪黑素组大鼠血糖水平明显高于正常对照组大鼠,差异均有统计学意义(P<0.01)。造模后4、8、12周时各时间点糖尿病组大鼠视网膜中总SOD、MnSOD活性明显低于正常对照组,8周及12周时褪黑素组大鼠视网膜中总SOD、MnSOD活性明显高于糖尿病组,差异均有统计学意义(P<0.01)。糖尿病组大鼠视网膜中Cu/ZnSOD活性在8周及12周时明显降低,与正常对照组比较差异均有统计学意义(P<0.01),12周时褪黑素组较糖尿病组有所升高,差异有统计学意义(P<0.01)。各时间点糖尿病组大鼠MnSODmRNA在视网膜中的表达量与正常对照组相比明显下降,褪黑素组MnSODmRNA的表达明显高于糖尿病组,差异均有统计学意义(P<0.01);8周及12周时糖尿病组Cu/ZnSODmRNA的表达量较正常对照组明显下降,12周时褪黑素组Cu/ZnSODmRNA的表达明显高于糖尿病组,差异有统计学意义(P<0.01)。结论糖尿病早期,褪黑素主要通过提高视网膜MnSOD的活性及增强其基因表达减轻视网膜的氧化损伤。

Background Oxidative stress is increased in the retina in diabetes mellitus,and antioxidants may inhibit oxidative injury and prevent the development of diabetic retinopathy.Melatonin is an effective scavenger of different reactive oxygen species,and these species cause chronic oxidative stress in diabetic rats.There are few reports about the effects of melatonin on the diabetic retinopathy.ObjectivePresent study was to observe the activity and gene expression of retinal manganese superoxide dismutase（MnSOD）and cuproznic superoxide dismutase（Cu/ZnSOD）in diabetic rats after melatonin treatment and explore the antioxide mechanism of melatonin in retina of diabetic rat.MethodsDiabetes animal models were established in 36 8-week-old clean SD rats by intraperitonial injection of 60mg/kg streptozotocin to elevate the blood glucose to16.7mmol/L.10 mg/（kg·d）melatonin was administered intragastrically at the second day after modeling once per day in 18 models.Eighteen age-matched SD rats were used as normal control group.The rats were sacrificed on the 4,8,12 weeks after establishment of model and eyeballs were obtained for the preparation of retinal homogenate.The content and activity of total SOD,MnSOD and Cu/ZnSOD in retina were detected by Xanthine oxidase method.Expressions of MnSOD mRNA and Cu/ZnSOD mRNA in retina were determined by SYBR Green Real time PCR Master Mix.ResultsThe blood glucose in rats was significantly elevated in various time points in model group and melatonin group compared with control group（P0.01）.The activity and content of the total SOD,MnSOD were considerably lower in model group than the normal control group in various time points（P0.01）,and in 8 and 12 weeks,those of melatonin group were evidently increased in comparison with model group with statistical significance in various time points（P0.01）.The activity and content of Cu/ZnSOD in retina was significant decreased in model group in comparison with the normal control group in 8 and 12 weeks（P0.01）,and melatonin group were evidently increased in comparison with model group with statistical significance in 12 weeks（P0.01）.The expression of MnSOD mRNA were considerably decreased in model group compared with the control group（P0.01）,but that in melatonin group was higher than one of model group from 4 weeks though 12 weeks（P0.01）;the expression of Cu/ZnSOD mRNA were considerably decreased in model group compared with the control group in 8 and 12 weeks,and that in melatonin group was higher than one of model group in 12 weeks（P0.01）.ConclusionIn early stage of diabetes,melatonin protects retina from oxidative damage mainly by enhancing the activity and gene expression of MnSOD in retinal tissue.