5,6-二氢环戊烯并1,2-二硫杂环戊烯-3-硫酮对2型糖尿病大鼠视网膜核因子NF-E2相关因子及血红素氧合酶-1表达的影响

摘要

目的 观察Ⅱ相酶诱导剂5,6-二氢环戊烯并1,2-二硫杂环戊烯-3-硫酮(CPDT)对2型糖尿病大鼠视网膜核因子NF-E2相关因子/抗氧化反应元件(Nrf2/ARE)信号通路及氧化应激的影响,探讨CPDT保护糖尿病大鼠视网膜的机制.方法 35只Wistar雄性大鼠随机分为正常组、造模组2个组,造模组中造模成功者再随机分为糖尿病组、CPDT干预组2个组.正常组、造模组分别有8、27只大鼠.糖尿病组和CPDT干预组给予高脂高糖饲料饲养2个月,大鼠空腹12 h后按体重35 mg/kg腹腔注射链脲佐菌素诱导2型糖尿病大鼠模型.CPDT干预组成模后1周开始在高脂高糖饲料中添加CPDT.8周后检测3组大鼠血糖、血清丙二醛(MDA)含量,检测正常组和糖尿病组大鼠血脂含量.逆转录聚合酶链反应检测3组大鼠视网膜Nrf2、血红素氧合酶-1(HO-1)mRNA表达,蛋白免疫印迹法检测Nrf2、HO-1蛋白表达,免疫组织化学法检测Nrf2、HO-1在视网膜的表达.结果 25只大鼠成功建立2型糖尿病大鼠模型,成功率为92.6％.糖尿病组大鼠血脂水平较正常组显著升高(F总胆固醇=65.866,F甘油三酯=25.441,F低密度脂蛋白=38.889,P=0.000).各组间大鼠血糖值比较,差异有统计学意义(x2=25.812,P=0.000),且糖尿病组大鼠血糖显著高于正常组和CPDT干预组.各组间大鼠血清MDA含量比较,差异有统计学意义(F=59.545,P=0.000),糖尿病组大鼠血清MDA高于正常组(t=10.523,P=0.000)和CPDT干预组(t=7.766,P=0.000).各组大鼠视网膜Nrf2、HO-1 mRNA比较,差异有统计学意义(FNrf2=19.503,PNrf2=0.000;FHO-1 =9.737,PHO-1 =0.001).与糖尿病组相比,CPDT干预组大鼠视网膜Nrf2、HO-1 mRNA表达明显增高(tNrf2 =3.399,PNrf2=0.002;tHo-1=2.167,PHO-1=0.039).各组大鼠视网膜Nrf2、HO-1蛋白比较,差异有统计学意义(FNrf2=112.823,FHO-1 =119.361,P=0.000).与糖尿病组相比,CPDT干预组大鼠视网膜Nrf2、HO-1蛋白表达明显增高(tNrf2=6.203,tHO-1=6.388,P=0.000).免疫组织化学检测结果显示,Nrf2、HO-1蛋白主要表达于视网膜神经节细胞层、内丛状层、内核层,各组大鼠视网膜Nrf2、HO-1蛋白表达量比较,差异有统计学意义(FNrf2=16.206,FHO-1=46.790,P=0.000).CPDT干预组大鼠视网膜Nrf2、HO-1蛋白的表达量高于糖尿病组(tNrf2 =3.172,PNrf2 =0.003;tHO-1 =6.321,PHO-1=0.000),糖尿病组高于正常组(tNrf2=2.679,PNrf2=0.011; tHO-1=3.482,PHO1=0.001).结论 CPDT可能通过激活Nrf2/ARE信号通路,诱导Nrf2和HO-1的表达,降低血糖、血清MDA含量,从而减轻2型糖尿病大鼠视网膜的氧化应激损伤.%Objective To observe the effect of phase Ⅱ enzyme inducer 5,6-dihydrocyclopenta 1,2-dithiole-3-thione (CPDT) on nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signal pathway and oxidative stress in the retina of type 2 diabetic rats.Methods Thirty-five male Wistar rats were randomly divided into two group,normal group and model group.Model group were further randomly divided into two group,diabetic group and CPDT intervention group.There were 8 rats in the normal group and 27 rats in the model group.Diabetic group and CPDT intervention group were given high fat and high sugar diet for 2 months.After 12 hours of fasting,type 2 diabetic rat model was induced by intraperitoneal injection of low dose of streptozotocin.CPDT was added into the high fat and high sugar diets at 1 week after the diabetic model was established in the CPDT intervention group.Eight weeks after CPDT treatment,blood glucose,serum malondialdehyde (MDA),blood lipid [total cholesterol (TC),triglyceride (TG),low density lipoprotein-cholesterol (LDL-C)],Nrf2 and hemeoxygenase-1 (HO-1) expression were evaluated.Results Type 2 diabetic model was successfully established in 25 rats,the success rate was 92.6％.The level of blood lipid of diabetic group was higher than those of the normal group (FTC =65.866,FTG =25.441,FLDL-C =38.889 ; P =0.000).Blood glucose was significant different between all groups (x2 =25.812,P=0.000),and was significantly higher in diabetic group than that in normal group and CPDT intervention group.The serum MDA content was significant different between all groups (F=59.545,P=0.000),and was significantly higher in diabetic group than that in normal group (t=10.523,P=0.000) and CPDT intervention group (t=7.766,P=0.000).The mRNA level of retinal Nrf2 and HO-1 was significant different between all groups (FNrf2 =19.503,PNrf2 =0.000; FHO-1 =9.737,PHO-1 =0.001),and was higher in CPDT intervention group than the diabetic group (tNrf2 =3.399,PNrf2 =O.002;tHO-1 =2.167,PHO-1 =0.039).The protein level of retinal Nrf2 and HO-1 was significant different between all groups (FNrf2 =112.823,FHO-1 =119.361; P=0.000),and was higher in CPDT intervention group than the diabetic group (tNrf2 =6.203,tHO-1 =6.388; P=0.000).Immuno-staining showed that Nrf2 and HO-1 were mainly expressed in retinal ganglion cell layer,inner plexiform layer and inner nuclear layer,and were significant different between all groups (FNrf2 =16.206,FHO-1 =46.790 ; P=0.000).They also were higher in CPDT intervention group than the diabetic group (tNrf2 =3.172,PNrf2 =0.003 ; tHO-1 =6.321,PHO-1 =0.000),was higher in diabetic group than that in normal group (tNrf2 =2.679,PNrf2 =0.011;tHO-1 =3.482,PHO-1 =0.001).Conclusion CPDT may activate Nrf2/ARE pathway,induce Nrf2 and HO-1 expression,decrease serum MDA and blood glucose,and thus reduce oxidative stress injury in the retina of type 2 diabetic rats.