达格列净通过Rffl抑制STAT1/TGF-β1信号通路改善糖尿病肾病肾小管上皮细胞EMT和纤维化

Dapagliflozin improves renal tubular epithelial EMT and fibrosis in diabetic nephropathy by inhibiting STAT1/TGF⁃β1 signaling pathway via Rffl

目的:探讨达格列净对糖尿病肾病肾小管上皮细胞的上皮细胞-间充质转化和纤维化的影响及分子机制。方法:体外培养人肾小管上皮细胞HK-2,分为对照组、高糖组、低剂量达格列净+高糖组和高剂量达格列净+高糖组。用Western blot与RT-PCR分别检测E3泛素连接酶Rffl的表达水平。Western blot检测各组上皮细胞钙黏蛋白(epithelial cadherin,E-cadherin)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤连蛋白(Fibronectin)、转化生长因子-β1(transforming growth factorβ1,TGF-β1)、信号转导与转录激活因子1(signal transducer and activator of transcription 1,STAT1)的表达水平。在HK-2细胞中过表达Rffl后,Western blot检测E-cadherin、α-SMA、Fibronectin、TGF-β1、STAT1的表达水平。结果:高糖组中Rffl的表达水平显著低于对照组,加入达格列净后Rffl表达升高;与对照组相比,高糖组细胞中E-cadherin表达水平降低,Fibronectin、α-SMA表达水平升高,过表达Rffl后E-cadherin表达水平升高,Fibronectin、α-SMA表达水平降低;与高糖组相比,低剂量达格列净+高糖组和高剂量达格列净+高糖组中E-cadherin表达水平均升高,Fibronectin、α-SMA表达水平均降低,且呈一定的剂量依赖性;与对照组相比,高糖组细胞中STAT1、TGF-β1的表达水平升高,而在过表达Rffl或达格列净作用后则明显降低。结论:达格列净通过上调Rffl的表达抑制STAT1/TGF-β1信号通路,改善糖尿病肾病肾小管上皮细胞的上皮细胞-间充质转化和纤维化。

Objective:The current study aims to investigate the effect and molecular mechanism of dapagliflozin on epithelial⁃mesenchymal transformation and fibrosis of renal tubular epithelial cells in diabetic kidney disease.Methods:Human renal tubular epithelial cells HK⁃2 were cultured in vitro and divided into control group,high glucose group,low dose dapagliflozin+high glucose group and high dose dapagliflozin+high glucose group.The expression levels of Rffl were detected by Western blot and RT⁃PCR,respectively.The expression levels of epithelial cadherin(E⁃cadherin),α⁃smooth muscle actin(α⁃SMA),Fibronectin,transforming growth factorβ1(TGF⁃β1)and signal transducer and activator of transcription 1(STAT1)were detected by Western blot.After Rffl was overexpressed in HK⁃2 cells,the expression levels of E⁃cadherin,α⁃SMA,Fibronectin,TGF⁃β1 and STAT1 were detected by Western blot.Results:The expression level of Rffl was significantly lower in the high glucose group than in the control group,and the expression of Rffl was increased after adding dapagliflozin.Compared with the control group,the expression level of E⁃Cadherin in the high glucose group was decreased,while the expression level of Fibronectin andα⁃SMA was increased.After Rffl was overexpressed in cells,the expression level of E⁃Cadherin was increased,while the expression levels of Fibronectin andα⁃SMA were decreased.Compared with high glucose group,the expression levels of E⁃Cadherin in low⁃dose dapagliflozin+high glucose group and high⁃dose dapagliflozin+high glucose group were increased,but the expression levels of Fibronectin andα⁃SMA were decreased in a dose⁃dependent manner.Compared with the control group,the expression levels of STAT1 and TGF⁃β1 were increased in the high glucose group,but significantly decreased after overexpression of Rffl or addition of dapagliflozin.Conclusion:Dapagliflozin inhibits STAT1/TGF⁃β1 signaling pathway by up⁃regulating Rffl expression,and improves epithelial⁃mesenchymal transformation and fibrosis of renal tubular epithelial cells in diabetic kidney disease.