氯沙坦联合表没食子儿茶素没食子酸酯对大鼠糖尿病肾病治疗的研究

Treatment of the combination of EGCG and Losartan on rat diabetic nephropathy

目的观察氯沙坦联合表没食子儿茶素没食子酸脂（EGCG）对糖尿病肾病大鼠的肾脏保护作用。方法采用链脲佐菌素（60mg·kg^-1）腹腔注射建立糖尿病大鼠模型，模型成功后分为正常组、糖尿病组、氯沙坦组、EGCG组、氯沙坦＋EGCG组。EGCG组和氯沙坦＋EGCG组每周给予ECd2G10mg·kg^-1腹腔注射，氯沙坦组和氯沙坦＋EGCG组每天给予氯沙坦40rag·kg^-1灌胃。分别测定各组4、8、12周时血肌酐（Cr）、尿素氮（BUN）、葡萄糖（GLU）、甘油三酯（TG）、总胆固醇（CHOL）、高低密度脂蛋白（HDL）、低密度脂蛋白（LDL）、白蛋白（Alb）、24h尿蛋白定量（24hPro）。应用RealtimePCR法检测肾皮质活性氧（ReactiveOxygenSpecies，ROS）产生的主要通路NADPH氧化酶Nox4mRNA表达、Westernblot法检测肾皮质蛋白NADPH氧化酶Nox4的表达。结果糖尿病组相对于正常对照组Cr，BUN，TG，CHOL，HDL，LDL，24hPro水平均显著升高（P〈0．05）。与糖尿病组比较，氯沙坦组、EGCG组、氯沙坦＋ECa2G组大鼠Cr、24hPro有显著减少（P〈0．05），但GLU，BUN，TG，CHOL，HDL，LDL均无明显减少（P〉0．05）。与氯沙坦组和EGCG组比较，氯沙坦＋ECA2G组Cr，GLU，BUN，TG，CHOL，HDL，LDL，ALB，24hPro均有差异但差异不明显（P〉0．05）。在第4和12周与正常组比较，糖尿病组大鼠肾小球硬化明显；与糖尿病组比较，氯沙坦组、EGCG组、氯沙坦＋EGCG组大鼠肾小球硬化均显著改善；与氯沙坦组比较，EGCG组、氯沙坦＋EGCG组大鼠。肾小球硬化缓解更加明显；在电子显微镜观察下与正常组比较，糖尿病组大鼠肾小球足细胞明显融合，氯沙坦组少量融合，而EGCG组和氯沙坦＋EGCG组足细胞基本完好。糖尿病组Nox4蛋白表达最高；与EGCG组和氯沙坦组比较，氯沙坦＋EGCG组Nox4蛋白表达均降低，并且随着时间的推移降低更加显著（P〈0．05）。第4、8、12周NADPH氧化酶Nox4mRNA表达均降低，但不同时间点同组之间差异不是很明显（P〉0．05）。结论EGCG协同氯沙坦通过下调NAD—PH氧化酶Nox4的表达，抑制氧化应激对糖尿病大鼠肾脏的损伤，对糖尿病肾病具有良好的保护作用。

Objective To observe the protection of Losartan combined epigallocatechin gallate （EGCG） in rat diabetic nephropathy. Methods STZ （60 mg. kg^-1 ） were injected intraperitoneal to get typeldiabetic nephropathy rats. All subjects were divided into 5 groups： normal group, diabetic group, Losartan group, EGCG group, Losartan ＋ EGCG group. EGCG group and Losartan ＋ EGCG group were given EGCG10 mg-kg^-1 . wk^-1 intraperitoneal injection once a week, Losartan group and Losartan ＋ EGCG group were given losartan 40rag orally daily. We measured blood Glucose（GLU）, Creatinine（Cr）, Blood urea nitrogen（ BUN）, Triglycerides（ TG）, Total cholesterol（ CHOL）, High density lipoprotein cholesterol（ HDL）, Low density lipoprotein cholesterol（LDL）, Albumin（ ALB）,24 h proteinuria at 4,8,12wk. Real time PCR and Western blot were used to detect renal cortical NADPH oxidase Nox4 mRNA and protein expression. Results Compared to the control group, the Cr, BUN, GLU,TG, CHOL, HDL, LDL, 24-hour proteinuria of the diabetic group were signifi- cantly increased （P〈0.05）. Compared to the diabetic group, the Losartan group, EGCG group and losartan ＋ EGCG group had lower Cr and 24-hour proteinuria （P 〈0.05）. The Cr, GLU, BUN, TG, CHOL, HDL, LDL, Alb, 24-hour proteinuria in Losartan ＋ EGCG group were not significant- ly different from that in Losartan group and EGCG group （P〉0.05）. Compared to the control group, the glomerular sclerosis in diabetic group was obvious; and compared to the diabetic group, the glomerular sclerosis in Losartan group, EGCG group and Losartan ＋ EGCG group were signifi- cantly improved, especially in the EGCG group and Losartan ＋ EGCG group. Under the electron mi- croscope, the diabetic group had obvious podocyte fusion, while the Losartan group had a little of fu- sion, and the EGCG group and Losartan ＋ EGCG group had almost intact podocytes. Compared to the diabetic group, all the other groups had lower Nox4 protein expression levels. And compared to the Losartan group and EGCG group, the Losartan ＋ EGCG group had a lower Nox4 protein expres- sion. Compared to the diabetic group, all the other groups had lower Nox4 mRNA expression levels （P〈0.05）. And compared to the Losartan group and EGCG group, the Losartan ＋ EGCG group had a lower Nox4 mRNA expression. But no difference was found in the same group at different times （P〉0.05）. Conclusions EGCG cooperated with Losartan could reduce the expression of NADPH oxidase Nox4, thereby inhibit the oxidative stress damages to kidneys in diabetic rats, so it had pro- tective effects to diabetic nephropathy.