益气通络方通过调控miR-21a-3p/TGF-β1/Smad3轴改善单侧输尿管梗阻大鼠肾间质纤维化的机制研究

Yiqi Tongluo Formula alleviates renal fibrosis in UUO rats via the miR-21a-3p/TGF-β1/Smad3 axis

关键词:  肾间质纤维化  单侧输尿管梗阻  益气通络方  miR-21a-3p/TGF-β1/Smad3

Key words:  renal interstitial fibrosis  unilateral ureteral obstruction  Yiqi Tongluo Formula  miR-21a-3p/TGF-β1/Smad3 axis

摘要:

[目的] 探讨miR-21a-3p对转化生长因子-β1（TGF-β1）诱导的大鼠肾小管上皮细胞（NRK-52E）纤维化模型及单侧输尿管梗阻（UUO）大鼠模型的影响及益气通络方通过调控miR-21a-3p /TGF-β1/Smad3轴抗肾纤维化的作用机制。[方法] 首先在Gene Expression Omnibus（GEO）数据库筛选出miR-21a-3p为在肾纤维化大鼠模型中显著上调的基因，随后构建TGF-β1刺激的NRK-52E纤维化模型进行验证，细胞实验分组如下：正常对照组、疾病模型组、抑制剂阴性对照组、miR-21a-3p抑制剂组，采用逆转录-定量聚合酶链式反应（RT-qPCR）检测miR-21a-3p表达水平，蛋白免疫印迹法（Western blot）检测细胞纤维化标志物1型胶原蛋白（COL1）、纤连蛋白（FN）及上皮-间充质转化（EMT）标志物α-平滑肌（α-SMA）、E-钙黏着蛋白（E-cadherin）蛋白表达水平，明确miR-21a-3p在NRK-52E纤维化模型中的明显上调趋势；动物实验共设6个组别（假手术组、模型组、益气通络方低/中/高剂量组、氯沙坦钾组），每组10只大鼠，模型为UUO大鼠，对应组别给药2周后取材，通过苏木精-伊红（HE）与Masson染色，对肾组织的病理学改变进行观察，RT-qPCR检测大鼠肾脏组织miR-21a-3p、COL1、FN、TGF-β1 mRNA 表达；Western blot法检测大鼠肾脏组织TGF-β1、Smad3、P-Smad3、α-SMA、E-cadherin表达。[结果] 在细胞实验中，对比正常对照组，模型组及抑制剂阴性对照组的miR-21a-3p、COL1、FN、α-SMA表达均显著上调，而E-cadherin的表达则显著下调；与模型组及抑制剂阴性对照组相比，miR-21a-3p抑制剂组miR-21a-3p、 COL1、FN、α-SMA表达则均明显降低，E-cadherin明显升高。在动物实验中，模型组相比假手术组，模型组肾组织病理结果纤维化明显，胶原容积升高（P<0.001），miR-21a-3p、COL1、FN、TGF-β1mRNA 表达水平升高（P<0.05），TGF-β1、P-Smad3/Smad3、α-SMA的蛋白表达显著上调，而E-cadherin的蛋白表达则显著下调（P<0.001）；益气通络方各剂量组及氯沙坦钾组干预后肾间质纤维化程度均呈现不同程度减轻，胶原容积下降明显（P<0.001），miR-21a-3p、COL1、FN、TGF-β1mRNA 表达水平均有所下降（P<0.05），TGF-β1、P-Smad3/Smad3、α-SMA的蛋白表达明显降低（P<0.001），而E-cadherin的蛋白表达则被上调（P<0.05）。[结论] 益气通络方可能通过抑制miR-21a-3p的表达，干扰TGF-β1/Smad3通路的信号转导，抑制细胞外基质生成及肾小管上皮细胞-间充质转分化发挥抗肾纤维化作用。

Abstract:

[Objective] This study investigates the effect of miR-21a-3p on transforming growth factor-β1（TGF-β1）-induced renal tubular epithelial cell（NRK-52E） fibrosis and unilateral ureteral obstruction（UUO） rat models，and explores the mechanism of Yiqi Tongluo Formula in anti-renal fibrosis via the miR-21a-3p/TGF-β1/Smad3 axis. [Methods] The miR-21a-3p was first identified as significantly upregulated in renal fibrosis rat models through the Gene Expression Omnibus（GEO） database. A TGF-β1-induced NRK-52E fibrosis model was established for validation. Cell experiments included normal control，disease model，inhibitor negative control，and miR-21a-3p inhibitor groups. RT-qPCR measured miR-21a-3p expression，while Western blot detected fibrosis markers（collagen I，fibronectin） and epithelial-mesenchymal transition（EMT） markers（α-SMA，E-cadherin）. Animal experiments divided rats into six groups：sham-operated，model，low/medium/high-dose Yiqi Tongluo Formula，and losartan potassium groups（n=10 per group）. UUO model rats received interventions for two weeks. HE and Masson staining evaluated renal pathology，RT-qPCR measured miR-21a-3p，collagen I，fibronectin，and TGF-β1 mRNA levels，and Western blot assessed TGF-β1，Smad3，p-Smad3，α-SMA，and E-cadherin protein expression. [Results] In cells，the model and inhibitor negative control groups showed significantly upregulated miR-21a-3p，collagen I，fibronectin，and α-SMA with downregulated E-cadherin compared to normal controls. The miR-21a-3p inhibitor group reversed these changes. In UUO rats，the model group exhibited severe fibrosis，increased collagen volume，and elevated miR-21a-3p，collagen I，fibronectin，and TGF-β1 expression versus sham group. Yiqi Tongluo Formula and losartan potassium alleviated fibrosis，reduced collagen volume，and downregulated miR-21a-3p，TGF-β1，p-Smad3/Smad3，and α-SMA while upregulating E-cadherin. [Conclusion] Yiqi Tongluo Formula may inhibit renal fibrosis by suppressing miR-21a-3p expression，blocking TGF-β1/Smad3 signaling，and reducing extracellular matrix production and EMT.