白藜芦醇通过上调自噬发挥对糖尿病小鼠足细胞保护作用的机制研究

Mechanism of resveratrol′s protective effect on diabetic mice podocytes by upregulating autophagy

目的探讨白藜芦醇通过上调1型糖尿病小鼠肾脏自噬水平减轻足细胞损伤的机制。方法选取健康雄性无特定病原体级昆明小鼠28只,以8只健康昆明小鼠作为正常对照组(NC组),剩余20只小鼠给予链脲佐菌素建立1型糖尿病小鼠模型,将造模成功的18只小鼠随机分为糖尿病组(DM组,n=9)和白藜芦醇干预组(Res组,n=9)。12周末检测24 h尿蛋白、血清肌酐、尿素氮水平,采用实时荧光定量聚合酶链反应、免疫印迹法检测沉默信号调节因子1(SIRT1)、叉头状转录因子O1(FoxO1)、微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、自噬接头蛋白(P62)、裂孔膜蛋白肾病蛋白(nephrin)及足突蛋白(podocin)的表达水平及磷酸化FoxO1(p-FoxO1)/FoxO1。免疫组织化学法观察足细胞nephrin、podocin的表达,应用HE染色及电镜的方法观察肾小球形态及超微结构的变化。多组间比较采用方差分析,组内比较采用LSD-t法。结果与NC组相比,DM组肌酐、尿素氮、24 h尿蛋白升高(t=-39.57、-28.17、-57.31,均P<0.05),SIRT1、LC3Ⅱ、nephrin及podocin表达减少,P62表达增多(均P<0.05),p-FoxO1/FoxO1升高(0.71±0.03比0.32±0.01,t=-38.81,P<0.05),光镜及电镜显示肾小球、基底膜及足细胞结构损伤。Res组与DM组相比,肌酐、尿素氮、24 h尿蛋白降低(t=30.89、16.39、49.64,均P<0.05),SIRT1、LC3Ⅱ、nephrin及podocin表达增高,P62表达减少(均P<0.05),p-FoxO1/FoxO1降低(0.42±0.01比0.71±0.03,t=27.47,P<0.05),光、电镜显示以上损伤减轻。结论白藜芦醇通过激活SIRT1/FoxO1通路提高肾脏自噬水平,对糖尿病小鼠肾脏足细胞起保护作用。

Objective The role of resveratrol in reducing podocyte damage by regulating autophagy in the kidney of mice with type 1 diabetes mellitus.Methods A total of 28 healthy male Kunming mice without specific pathogen were selected,of which eight healthy Kunming mice were included in normal control group(NC group).The remaining twenty Kunming mice were given streptozotocin to establish the model of type 1 diabetes mellitus.Eighteen mice successfully induced with diabetes were randomized into diabetes mellitus group(DM group,n=9)and resveratrol intervention group(Res group,n=9).At the end of the 12th week,the urine was collected to detect 24-hour urine protein,and the serum was used to detect creatinine and urea nitrogen.Real-time fluorescent quantitative polymerase chain reaction and Western blotting method were used to detect the expression levels of silent information regulator 1(SIRT1),forkhead transcription factor O1(FoxO1),microtubule-associated protein 1 light chain 3(LC3)Ⅱ,P62,nephrin,podocin and phosphorylation of FoxO1(p-FoxO1)/FoxO1.Immunohistochemistry was used to observe the expression levels of nephrin and podocin in podocytes.The morphology and ultrastructure of glomerular were observed by Hematoxylin-eosin staining and the method of electron microscopy.Analysis of variance was used for comparison in multiple groups.LSD-t method was used for comparison between two groups.Results Compared with NC group,the level of plasma creatinine,plasma urea nitrogen and 24-hour urine protein in DM group were increased(t=-39.57,-28.17,-57.31,all P<0.05).The expression levels of SIRT1,LC3Ⅱ,nephrin and podocin were decreased,where as the expression level of P62 was increased in DM group compared with NC group(all P<0.05)The ratio of p-FoxO1/FoxO1 was also increased in DM group(0.71±0.03 vs 0.32±0.01,t=-38.81,P<0.05).Results from light microscope and electron microscope showed that the glomerulus,basement membrane and podocyte structures were damaged.Compared with DM group,plasma creatinine,plasma urea nitrogen and 24-hour urine protein in Res group were decreased(t=30.89,16.39,49.64,all P<0.05).In Res group,the expression levels of SIRT1,LC3Ⅱ,nephrin and podocin were increased,but the expression level of P62 were decreased compared with DM group(all P<0.05).Beside,resveratrol treatment also decreased the ratio of p-FoxO1/FoxO1(0.42±0.01 vs 0.71±0.03,t=27.47,P<0.05).Results from light and electron microscopy showed that above damages were reduced.Conclusion Resveratrol protects podocytes by activating SIRT1/FoxO1 pathway and increasing the level of autophagy in the kidney in mice with diabetes mellitus.