不同运动干预2型糖尿病模型小鼠的肾间质纤维化

Effects of different exercises on renal interstitial fibrosis in type 2 diabetic mice

背景:2型糖尿病并发症肾间质纤维化日益高发,然而有关2型糖尿病并发症肾间质纤维化及运动对其作用的影响机制尚待揭示。目的:探究不同方式运动对2型糖尿病小鼠肾间质纤维化的影响,及Klotho介导转化生长因子β1/Smad3途径在此过程中的调控机制。方法:44只4周龄C57BL/6雄性小鼠,1周适应性喂养后,随机分为正常对照组和2型糖尿病造模组。利用高脂膳食和一次注射链脲佐菌素法制备2型糖尿病小鼠模型,并随机分为模型对照组、高强度间歇训练组和下坡跑组,进行8周运动干预。结束后,检测肾功能生化指标;利用苏木精-伊红染色检测肾组织微细结构变化;Masson染色检测肾间质纤维化水平;实时定量PCR检测相关因子mRNA表达;Westernblotting检测相关因子蛋白表达;免疫组织化学染色检测肾中Ⅰ型胶原蛋白的蛋白表达。结果与结论:①与正常对照组相比,模型对照组血肌酐、尿素氮和24 h尿蛋白水平均显著升高;肾盂扩张、肾实质变窄、肾小管数量减少;胶原面积百分比升高;肾组织中Klotho mRNA和蛋白表达水平下调,转化生长因子β1、Ⅰ、Ⅲ型胶原蛋白mRNA和蛋白表达水平上调,Smad3 mRNA和p-Smad3蛋白表达水平均显著上调;肾组织中Ⅰ型胶原蛋白的蛋白表达阳性区域显著增加;②与模型对照组相比,高强度间歇训练组Klotho、Ⅰ型胶原蛋白和Ⅲ型胶原蛋白mRNA表达出现显著变化,转化生长因子β1和Ⅲ型胶原蛋白的蛋白表达上调;③与高强度间歇训练组相比,下坡跑组血肌酐、尿素氮和24 h尿蛋白水平下降;肾盂、肾小管、肾小球等结构病变被显著改善;胶原面积百分比显著降低;Klotho mRNA和蛋白表达上调,转化生长因子β1、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白mRNA和蛋白表达水平均下调,p-Smad3蛋白表达下调;Ⅰ型胶原蛋白的蛋白表达阳性区域显著减少;④提示2型糖尿病小鼠肾间质发生了纤维化;下坡跑通过上调Klotho并抑制转化生长因子β1/Smad3途径进而改善2型糖尿病小鼠肾间质纤维化,但高强度间歇性运动的作用效果不显著。

BACKGROUND:The incidence of renal interstitial fibrosis as a complication of type 2 diabetes is increasing.However,the mechanism by which exercises intervene with renal interstitial fibrosis as a complication of type 2 diabetes remains to be revealed.OBJECTIVE:To explore the effects of different exercises on renal interstitial fibrosis in type 2 diabetic mice and the regulatory mechanism of Klotho-mediated transforming growth factor-β1/Smad3 pathway in this process.METHODS:Forty-four 4-week-old C57BL/6 male mice were randomly divided into a normal control group and a type 2 diabetic model group after 1 week of adaptive feeding.The mouse model of type 2 diabetes mellitus was prepared by high-fat diet and one injection of streptozotocin,and the mouse models were randomly divided into a model control group,a high-intensity intermittent training group,and a downhill running group,followed by 8 weeks of exercise intervention.After the end,ELISA was used to detect the biochemical indicators of renal function;hematoxylin-eosin staining was used to detect renal tissue structure;Masson staining was used to detect the degree of renal interstitial fibrosis;RT-PCR was used to detect the mRNA expression of relevant factors;western blot assay was used to detect the protein expression of relevant factors;and immunohistochemical staining was used to detect the expression of type I collagen protein in the kidney.RESULTS AND CONCLUSION:Compared with the normal control group,the levels of serum creatinine,urea nitrogen and 24-hour urine protein were significantly increased in the model control group;the renal pelvis was expanded,the renal parenchyma narrowed,and the number of renal tubules decreased;the percentage of collagen area increased;the expression of Klotho mRNA and protein in the kidney was down-regulated and the expressions of transformed growth factorβ1,type I collagen,type III collagen at mRNA and protein levels were up-regulated,and the expressions of Smad3 mRNA and p-Smad3 protein were significantly up-regulated;the positive area of type I collagen protein expression in the kidney was significantly increased.Compared with the model control group,the mRNA expression of Klotho,type I collagen,and type III collagen in the high-intensity interval training group showed remarkable changes,and the expression of transforming growth factorβ1 and type III collagen was up-regulated.Compared with the high-intensity intermittent training group,the levels of serum creatinine,urea nitrogen and 24-hour urine protein were significantly down-regulated in the downhill running group;renal structural lesions involving the renal pelvis,renal tubules,and glomeruli were remarkably improved;the percentage of collagen area was significantly reduced;Klotho mRNA and protein expression was up-regulated,the expressions of transforming growth factorβ1,type I collagen and type III collagen at mRNA and protein levels were down-regulated,the expressions of Smad3 mRNA and p-Smad3 protein were significantly down-regulated;the positive area of type I collagen protein expression was significantly decreased.To conclude,type 2 diabetic mice develop fibrosis in the renal interstitium;downhill running improves renal interstitial fibrosis in type 2 diabetic mice by up-regulating Klotho and inhibiting the transforming growth factor-β1/Smad3 pathway;however,high-intensity intermittent exercises have no remarkable effects.