基于 KLF4/SMAD 通路研究金蝉益肾通络方抑制糖尿病肾病小鼠上皮细胞间质化的机制

Mechanism of action of Jinchan Yishen Tongluo Formula in inhibiting epithelial mesenchymal transition of epithelial cells in mice with diabetic kidney disease through KLF4/SMAD pathway

关键词:  糖尿病肾病  上皮细胞间质化  金蝉益肾通络方  KLF4/SMAD通路

Key words:  diabetic kidney disease  epithelial mesenchymal transition  Jinchan Yishen Tongluo Formula  KLF4/SMAD pathway

摘要:

[目的]糖尿病肾病（DKD）是糖尿病的重要微血管并发症。金蝉益肾通络方（JCYSTL）临床治疗糖尿病肾病效果显著，但具体的作用机制还有待进一步探讨。[方法]以高脂饮食联合链脲佐菌素诱导C57BL/6J小鼠的DKD模型为研究对象，并使用JCYSTL进行干预。通过检测24 h-尿蛋白（24 h-UPQ）、血肌酐（Scr）、尿素氮（BUN）和苏木精-伊红染色（HE）、Masson染色，评估JCYSTL对DKD小鼠的治疗作用。通过蛋白免疫印迹法（Western blot）检测上皮细胞间质化标志物[闭锁小带蛋白1（ZO-1）、上皮性钙黏附蛋白（E-cad）、波形蛋白（VIM）、α-平滑肌肌动蛋白（α-SMA）]的表达水平，明确JCYSTL对DKD小鼠肾组织上皮细胞间质化的影响。通过RT-qPCR及Western blot检测Kruppel样因子4（KLF4）、Sma和Mad相关蛋白7（SMAD7）、Sma和Mad相关蛋白3（SMAD3）的表达，观察JCYSTL对KLF4/SMAD通路主要蛋白表达水平的影响。[结果]结果发现JCYSTL显著降低DKD小鼠血糖及24 h-UPQ、Scr、BUN水平，改善肾小球基底膜增厚及胶原沉积。Western blot显示，JCYSTL上调ZO-1、E-cad表达，抑制VIM、α-SMA，同时促进KLF4、SMAD7表达并抑制SMAD3表达。[结论] JCYSTL可能通过激活KLF4/SMAD通路抑制EMT，从而缓解DKD。

Abstract:

[Objective] Diabetic kidney disease（DKD）is an important microvascular complication of diabetes mellitus. The clinical effect of Jinchan Yishen Tongluo Formula（JCYSTL） in the treatment of diabetic kidney disease is significant，but its specific mechanism of action remains to be further explored. [Methods] In this experiment，we took the DKD model of C57BL/6J mice induced by high-fat diet combined with streptozotocin as the research object and used JCYSTL for intervention. The therapeutic effect of JCYSTL on DKD mice was evaluated by detecting 24-hour urinary protein（24 h-UPQ），serum creatinine（Scr），blood urea nitrogen （BUN），hematoxylineosin staining（HE）and Masson staining. The expression levels of epithelial-mesenchymal transition markers [zonula occludens-1（ZO-1），E-cadherin（E-cad），vimentin（VIM），α-smooth muscle actin（α-SMA）] were detected by Western blot to clarify the effect of JCYSTL on epithelial-mesenchymal transition in renal tissue of DKD mice. The expressions of Kruppel-like factor 4 （KLF4），Sma and Mad-related protein 7（SMAD7）and Sma and Mad-related protein 3（SMAD3）were detected by RT-qPCR and Western blot to observe the effect of JCYSTL on the expression levels of main proteins in the KLF4/SMAD pathway. [Results] Our results showed that JCYSTL significantly reduced the levels of blood glucose，24 h-UPQ，Scr and BUN in DKD mice，and improved glomerular basement membrane thickening and collagen deposition. Western blot showed that JCYSTL up-regulated the expressions of ZO-1 and E-cad，inhibited VIM and α-SMA，promoted the expressions of KLF4 and SMAD7，and inhibited the expression of SMAD3. [Conclusion] Therefore，we believe that JCYSTL may alleviate DKD by activating the KLF4/SMAD pathway to inhibit EMT.