利拉鲁肽和艾塞那肽对2型糖尿病肾病大鼠肾脏结构和功能的影响

Effect of liraglutide and exenatide on renal structure and function of diabetic nephropathy rats

目的探讨利拉鲁肽和艾塞那肽在糖尿病肾病(DN)发病过程中对肾脏结构和功能的保护作用及两者是否存在差异。方法将雄性Sprague-Dawley大鼠40只随机分为正常对照组、模型组、利拉鲁肽治疗组和艾塞那肽治疗组,每组10只。模型组、利拉鲁肽治疗组、艾塞那肽治疗组大鼠给予高糖高脂饲养6周后空腹腹腔注射链脲佐菌素(STZ)35 mg·kg^(-1)诱导DN模型,之后3、7 d各测1次清晨空腹尾静脉血糖,2次血糖均≥16.7 mmol·L-1为DN造模成功。正常对照组、模型组大鼠给予皮下注射生理盐水10μL·kg^(-1)·12 h^(-1);艾塞那肽治疗组大鼠给予皮下注射艾塞那肽10μL·kg^(-1)·12 h^(-1);利拉鲁肽治疗组大鼠给予皮下注射利拉鲁肽0.3 mg·kg^(-1)·12 h^(-1),共用药8周。第8周末采集大鼠血液标本、肾脏标本。血液标本离心后收集血清,使用全自动生物化学分析仪检测大鼠血尿素氮(BUN)和血肌酐(Scr)水平;肾脏标本制作成蜡块,采用过碘酸雪夫染色(PAS)观察肾脏组织结构变化;免疫组织化学法检测大鼠肾脏组织中转化生长因子β_1(TGF-β_1)和还原性辅酶Ⅱ氧化酶4(NOX4)蛋白表达水平,使用Image-pro Plus软件对TGF-β_1和NOX4在肾脏组织中蛋白表达水平进行半定量分析。结果与正常对照组比较,模型组和艾塞那肽治疗组大鼠空腹血糖、BUN、Scr及肾组织中TGF-β_1、NOX4蛋白表达水平均显著升高(P<0.05);利拉鲁肽治疗组大鼠空腹血糖和肾组织中TGF-β_1蛋白水平显著升高(P<0.05);模型组、利拉鲁肽治疗组和艾塞那肽治疗组大鼠体质量显著降低(P<0.05)。模型组、利拉鲁肽治疗组和艾塞那肽治疗组大鼠肾小球系膜明显增厚,其中模型组大鼠增厚最明显,利拉鲁肽治疗组大鼠最轻。与模型组比较,利拉鲁肽治疗组和艾塞那肽治疗组大鼠BUN、Scr和肾组织中TGF-β_1、NOX4蛋白水平显著降低(P<0.05);利拉鲁肽治疗组大鼠Scr和肾组织中TGF-β_1、NOX4蛋白水平显著低于艾塞那肽治疗组(P<0.05)。结论利拉鲁肽和艾塞那肽均能通过减轻氧化应激和下调炎性因子表达改善大鼠肾脏功能;利拉鲁肽较艾塞那肽可更好地下调炎性因子TGF-β_1和减轻NOX4介导的氧化应激。

Objective To investigate the protective effect of liraglutide and exenatide on renal structure and function of diabetic nephropathy（ DN） rats and whether there was a difference between the two treatments. Methods Forty male Sprague-Dawley rats were randomly divided into normal control group,model group,liraglutide treatment group and exenatide treatment group,with 10 rats in each group. Rats in model group,liraglutide treatment group and exenatidet treatment group were feeded with high glucose and high fat diet for 6 weeks,then fasting intraperitoneal injection of streptozotocin（ STZ） 35 mg·kg^-1to induced DN model. On the third day and the seventh day,the fasting caudal veins blood glucose was measured,and both the blood glucose value ≥16. 7 mmol·L-1confirmed that the DN model was established successfully. The rats in normal control group and model group were given subcutaneous injection of saline（ 10 μL·kg^-1·12 h^-1） for 8 weeks; the rats in the exenatide treatment group was given subcutaneous injection of exenatide（ 10 μL·kg^-1·12 h^-1） for 8 weeks; the rats in the liraglutide treatment group was given subcutaneous injection of liraglutide（ 0. 3 mg·kg^-1·12 h^-1） for 8 weeks. At the end of the eighth week,the blood samples and kidney specimens were collected. Serum were collected after centrifugation blood samples,then the automatic biochemical analyzer was used to detect the blood urea nitrogen（ BUN） and serum creatinine（ Scr）.Kidney specimens were made into wax pieces,and the changes of renal tissue structure were observed by Periodic Acid-Schiff stain（ PAS）; the expression of transforming growth factor-β1（ TGF-β1） and nicotinamide adenine dinucleotide phosphate oxidase 4（ NOX4） protein in the kidney tissues of rats was detected by immunohistochemistry method; the semi-quantitative analysis of TGF-β1and NOX4 protein levels in renal tissue was performed by using Image-pro Plus software. Results Compared with the normal control group,the levels of fasting blood glucose（ FBG）,BUN,Scr and TGF-β1,NOX4 protein in renal tissue of rats were significantly increased in model group and exenatide treatment group（ P〈0. 05）; the levels of FBG and TGF-β1protein in renal tissue of rats were significantly increased in liraglutide treatment group（ P〈0. 05）. Compared with the normal control group,the body mass of rats in the model group,liraglutide treatment group and exenatide treatment group was significantly decreased（ P〈0. 05）. The mesangium of rats in model group,liraglutide treatment group and exenatide treatment group was thickened,the thickening was most obvious in the model group and was the lightest in the liraglutide treatment group. Compare with model group,the levels of BUN,Scr and TGF-β1,NOX4 protein in renal tissue of rats were significantly decreased in liraglutide treatment group and exenatide treatment group（ P〈0. 05）. The levels of Scr and TGF-β1,NOX4 protein in renal tissue of rats in liraglutide treatment group were significantly lower than those in the exenatide treatment group（ P〈0. 05）.Conclusion Both liraglutide and exenatide can improve renal function through down-regulating the inflammatory factor expression and reduce oxidative stress; liraglutide can better down-regulate the expression of TGF-β1 and lessen the oxidative stress mediated by NOX4.