基于SIRT1/TGF-β1/Smad通路研究健脾固肾化瘀方治疗糖尿病肾病机制

The mechanism of Jianpi Gushen Huayu Decoction in the treatment of diabetic kidney disease based on SIRT1/TGF-β1/Smad pathway

关键词:  健脾固肾化瘀方  糖尿病肾病  上皮间质转化  去乙酰化酶1/转化生长因子-β1/Smad通路

Key words:  Jianpi Gushen Huayu Decoction  diabetic kidney disease  epithelial interstitium transition  SIRT1/TGF-β1/Smad pathway

摘要:

[目的] 研究健脾固肾化瘀方（JPGS）治疗糖尿病肾病（DKD）效果，并从去乙酰化酶1（SIRT1）/转化生长因子-β1（TGF-β1）/Smad通路介导的上皮间质转化（EMT）研究JPGS治疗DKD的作用机制。[方法] 60只C57BL/6J小鼠适应性喂养1周，高脂饮食喂养联合链脲佐菌素腹腔注射造模，并随机平均分为正常组、模型组、SIRT1激动剂组（50 mg/kg，灌胃）、低剂量JPGS组（2.4 g/kg，灌胃）、中剂量JPGS组（4.8 g/kg，灌胃）、高剂量JPGS组（9.6 g/kg，灌胃）。治疗8周后，收集小鼠24 h尿液样本、血液样本、肾脏进行分析测定。使用试剂盒测定小鼠24 h尿蛋白定量（24 h-UPQ）、血清肌酐（Cr）、尿素氮（BUN）水平以评估肾功能。对肾脏同一部位进行苏木精-伊红（HE）、Masson染色，观察其病理学变化。采用实时荧光定量聚合酶链反应（RT-qPCR）以及蛋白免疫印迹（Western Blot）法检测EMT相关因子[E-钙黏蛋白（E-cad）、紧密连接蛋白-1（ZO-1）、波形蛋白（VIM）、α-平滑肌肌动蛋白（α-SMA）]表达情况，以评估JPGS对EMT的影响。运用Western Blot法检测SIRT1和TGF-β1蛋白表达水平，以及Smad2、Smad3磷酸化水平，评估JPGS对SIRT1/TGF-β1/Smad通路的调控作用。[结果] 与模型组比较，经JPGS治疗后，DKD小鼠Cr、BUN、24 h-UPQ降低，肾组织病理损伤好转，E-cad、ZO-1表达升高，VIM、α-SMA表达降低，SIRT1表达上调，TGF-β1、磷酸化Smad2/Smad2、磷酸化Smad3/Smad3表达下调。[结论] JPGS可以抑制SIRT1/TGF-β1/Smad通路介导的EMT，以缓解DKD肾损伤。

Abstract:

[Objective] To investigate the effect of Jianpi Gushen Huayu Decoction（JPGS） in the treatment of diabetic kidney disease（DKD） and the mechanism of JPGS in the treatment of DKD from the direction of epithelial interstitium transition（EMT） and SIRT1/TGF-β1/Smad pathway. [Methods] The 60 C57BL/6J mice were fed adaptive diet for 1 week and HFD for 8 weeks. After STZ modeling，they were randomly divided into normal group（C），model group（M），SIRT1 agonist group（S，50 mg/kg，gavage），low-dose JPGS group（JL，2.4 g/kg，gavage），medium-dose JPGS group（JM，4.8 g/kg，gavage），and high-dose JPGS group（JH，9.6 g/kg，gavage）. After 8 weeks of treatment，24 h urine samples，blood samples and kidney tissues of mice were collected for analysis and determination. Levels of 24 h-UPQ，serum Cr and BUN were measured by the kit to evaluate the renal function of mice. The pathological changes of the kidney were observed by HE and Masson staining. RT-qPCR and Western Blot were used to detect the expression of EMT-related factors（E-cad，ZO-1，VIM and α-SMA） to evaluate the effect of JPGS on EMT. Western Blot was used to detect the expressions of SIRT1，TGF-β1，P-Smad2/Smad2 and P-Smad3/Smad3 to evaluate the effect of JPGS on SIRT1/TGF-β1/Smad pathway. [Results] Compared with the model group，the levels of Cr，BUN and 24 h-UPQ in DKD mice treated with JPGS were decreased in varying degrees，the pathological damage of renal tissue was improved in varying degrees，the expression of E-cad and ZO-1 was increased，the expression of VIM and α-SMA was decreased，and the expression of SIRT1 was increased，the expressions of TGF-β1，P-Smad2/Smad2 and P-Smad3/Smad3 were down-regulated. [Conclusion] JPGS inhibited EMT by activating SIRT1/TGF-β1/Smad pathway to alleviate kidney injury in DKD.