褪黑素对早期糖尿病大鼠视网膜氧化应激反应的影响

摘要

背景 氧化应激是糖尿病并发症的早期发生机制,而褪黑素是迄今已知最高效的抗氧化物质之一,目前国内就褪黑素对糖尿病并发症的防治机制研究尚不多见.目的 研究褪黑素对早期糖尿病大鼠视网膜丙二醛(MDA)、还原型谷胱甘肽(GsH)含量及神经胶质纤维酸性蛋白(GFAP)表达的影响,探讨褪黑素对早期高血糖诱导的视网膜氧化损伤的作用.方法 清洁级6周龄SD大鼠48只尾静脉注射质量分数2%链脲佐菌素(STZ)制作糖尿病模型,按随机数字表法分为模型对照组和褪黑素治疗组;尾静脉注射等量柠檬酸缓冲液的大鼠24只作为正常对照组.褪黑素治疗组每日腹腔注射褪黑素10 mg/kg,正常对照组和模型对照组每日给予等量的生理盐水腹腔注射.于4、6、8周时采用MMT比色法检测视网膜组织匀浆中MDA、还原型GSH的含量,并应用Western blot和免疫组织化学法分析视网膜GFAP的表达量.结果 造模后4、6、8周时褪黑素治疗组视网膜MDA含量均明显低于相应时间点模型对照组(P<0.05);4周、6周时与正常对照组比较差异无统计学意义(P>0.05),8周时高于正常对照组(P<0.05).模型对照组视网膜还原型GSH的质量分数均明显低于同时间点正常对照组(P<0.05);4、6、8周时褪黑素治疗组视网膜还原型GSH质量分数均明显高于同时间点模型对照组(P<0.05),4周、6周时与正常对照组比较差异无统计学意义(P>0.05),8周时低于正常对照组(P<0.05).4、6、8周时褪黑素治疗组视网膜GFAP的表达量均低于同时间点模型对照组(P<0.01);4周、6周时与正常对照组比较差异均无统计学意义(P>0.05),8周时高于同时间点正常对照组(P<0.05).结论褪黑素能降低糖尿病大鼠视网膜MDA的含量,增加GSH水平,抑制糖尿病大鼠视网膜GFAP的过度表达,进而减轻氧化应激引起的视网膜损伤.%Background Oxidative stress is one of the pathogenesis mechanisms of diabetic complications.Melatonin is the most powerful antioxidant in living organism.Seldom study on the effects of melatonin on diabetic complications is found in China now.Objective This study aimed to detect the expression and alteration of malonadehyde(MDA),glutathione(GSH)and glial fibrillary acidic protein(GFAP)in retina with experimental diabetes and explore the effects of melatonin on retinal oxidative stress in diabetic rats. Methods The animal models of diabetes were established by injection of 2％ streptozocin via caudal vein(STZ,45 mS/kg)in 48 6-week-old clean SD rats.Isometric citric acid buffer solution wag injected in 24 normal.rats as control group.Seven days after injection of STZ,melatonin(10 mg/kg)was injected into the abdominal cavity of 24 diabetic rats in melatonin group dailv,and the equal volume of normal saline was injected in other 24 diabetic rats(model rats)and 24 rats in normal control group.The animals were sacrificed and retinas were obtained in three groups in 4,6 and 8 weeks respectively.Immunohistochemistry and Western blot were used to detect the expression of GFAP in rat retina.The levels of MDA and GSH in retina homogenate were assayed by spectrophotometer.This experiment complied the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results The expression of GSH in model group was lower than that in melatonin group or control group at 4,6 and 8 weeks (all P<0.05).However,no statistically significant difference was seen in the expression level of GSH between melatonin group and normal control group at 4 and 6 weeks(P>0.05),and in the eighth week,expression of GSH in melatonin group was lower than that in control group(P<0.05).The expressions of MDA and GFAP were more prominent in the model group than in melatonin group and control group in 4,6 and 8 weeks(P<0.05),but no significant difference was found between melatonin group and control group at 4 and 6 weeks(P>0.05).The expressions of MDA and GFAP in melatonin group were higher than those in control group at 8 weeks(P<0.05).Conclusion Melatonin plays a powerful effect in protecting the retina from oxidative stress in diabetes retina by downregulating the MDA level,upregulating GSH level and inhibiting GFAP expression.