糖肾煎通过促进SIRT1介导的自噬对糖尿病肾病大鼠的治疗作用

Promotive effect of Tangshenjian on SIRT1-mediated autophagy in diabetic nephropathy rats

目的探究糖肾煎通过促进沉默信息调节因子1(silent information regulator 1,SIRT1)介导的细胞自噬对糖尿病肾病(diabetic nephropathy,DN)大鼠的治疗作用。方法60只大鼠随机均分为对照组、糖尿病肾病(DN)模型组、糖肾煎组及二甲双胍组。对照组用普通饲料喂养,其他三组大鼠使用高糖高脂饲料及腹腔注射1%链脲佐菌素溶液(STZ)建立DN模型。建模成功后,糖肾煎组和二甲双胍组大鼠分别以10 g/(kg·d)糖肾煎和100 mg/(kg·d)二甲双胍灌胃,对照组与模型组大鼠给予同体积生理盐水灌胃,连续8周。末次给药前禁食不禁水12 h,经腹主动脉抽取各组大鼠外周血检测血清空腹血糖(fasting blood glucose,FBG)、尿素氮(BUN)、血尿肌酐(Scr),收集24 h尿液测定24 h尿白蛋白(24 h-UAlb)及胱抑素-c(Cys-c)的含量;给药结束后处死大鼠,取肾脏组织,采用苏木精-伊红(HE)染色观察各组大鼠肾脏组织病理改变,分别采用逆转录-聚合酶链反应(RT-PCR)和免疫印迹法(Western blot)检测各组大鼠肾脏组织中沉默信息调节因子1(SIRT1)、肾病蛋白(Nephrin)、肌间线蛋白(Desmin)、肿瘤坏死因子-α(TNF-α)、锌指蛋白36(TTP)mRNA表达和SIRT1、微管相关蛋白1的轻链3(LC3)、核孔蛋白p62、Desmin、Nephrin蛋白表达。结果模型组大鼠血清FBG、BUG、SCr和24 h-UAlb、Cys-c含量较对照组升高(P<0.05);糖肾煎组和二甲双胍组大鼠血清上述指标含量分别较模型组降低(P<0.05)。与对照组相比,模型组大鼠局部肾小管间质纤维化、部分肾小管上皮细胞肿胀和脱落、肾小球萎缩及部分肾小球系膜细胞增生;糖肾煎组和二甲双胍组大鼠部分肾小管颗粒变性、少量肾小球萎缩及肾小球系膜细胞轻度增生。与对照组比较,模型组大鼠肾脏组织Nephrin、LC3-Ⅱ/LC3-Ⅰ及SIRT1表达量降低(P<0.05),Desmin、p62、TNF-α及TTP表达量增加(P<0.05);与模型组比较,糖肾煎组和二甲双胍组大鼠肾脏组织Nephrin、LC3-Ⅱ/LC3-Ⅰ及SIRT表达量升高(P<0.05),Desmin、TNF-α、TTP和p62表达降低(P<0.05)。结论糖肾煎可通过降低血糖水平保护DN大鼠肾脏,其机制可能与上调SIRT1介导的肾小管自噬有关。

Objective To explore the therapeutic effect of Tangshenjian on diabetic nephropathy(DN)rats through the autophagy mediated by silent information regulator 1(SIRT1).Methods Sixty rats were randomly divided into four groups:control group,diabetic nephropathy(DN)model group,Tangshenjian group and metformin group.Except control group,the rest in the other groups were fed with high-sugar and high-fat diet and intraperitoneally injected with 1%streptozotocin(STZ)to establish DN models.The rats in Tangshenjian group were gavaged with 10 g/(kg·d)Tangshenjian and 10 mg/(kg·d)metformin for 8 weeks in Tangshenjian group and metformin group,respectively.The rats in control group and model group were gavaged with the same volume of saline for 8 weeks.After fasting without water for 12 h before the last administration,the peripheral blood was extracted through abdominal aorta to detect the expression of fasting blood-glucose(FBG),urea nitrogen(BUN),hematuria creatinine(Scr),and 24 h urine was collected to determine the contents of 24 h urinary albumin(24 h-UAlb)and Cystatin-c(Cys-c).HE staining was used to observe the pathological changes of renal tissue.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the mRNA expression of SIRT1,Nephrin,Desmin,tumor necrosis factor-α(TNF-α)and tristetraprolin(TTP)in renal tissue.Western blot was used to detect the protein expression of SIRT1,LC3-Ⅱ,p62,Desmin and Nephrin in renal tissues.Results Compared with control group,the contents of FBG,BUG,Scr in serum and 24 h-UAlb,Cys-c were significantly increased in model group(P<0.05).Compared with model group,the contents of FBG,BUG,Scr in serum and 24 h-UAlb,Cys-c were decreased in Tangshenjian group and metformin group(P<0.05).Compared with control group,renal tubule interstitial fibrosis,swelling and exfoliation of some tubule epithelial cells,glomerular atrophy and proliferation of some mesangial cells were found in model group.In Tangshenjian group and metformin group,some renal tubule granule degeneration,a small amount of glomerular atrophy and mild proliferation of mesangial cells were observed.Compared with control group,the expression levels of Nephrin,LC3-Ⅱ/LC3-Ⅰand SIRT1 were decreased in model group(P<0.05),while the expression levels of Desmin,TNF-α,TTP and P62 were increased(P<0.05).Compared with model group,the expression levels of Nephrin,LC3-Ⅱ/LC3-Ⅰand SIRT were increased in Tangshenjian group and metformin group(P<0.05),while the expression levels of Desmin,TNF-α,TTP and P62 were decreased(P<0.05).Conclusion Tangshenjian can protect the kidney of DN rats by reducing blood-glucose level,which may be related to up-regulation of renal tubule autophagy mediated by SIRT1.