地塞米松对马兜铃酸诱导的人肾小管上皮-间充质转化的作用及可能机制

The effect and mechanism of dexamethasone on AA induced epithelial-mesenchymal transition in HKC cells

目的探讨地塞米松(Dex)对马兜铃酸诱导的肾小管上皮-间充质转化(EMT)的影响及可能机制。方法体外培养的人肾小管上皮细胞(HKC)分为阴性对照组、马兜铃酸(AA,10μg/ml)作用组、Dex(100μmol/L)作用组、AA(10μg/ml)+无水乙醇(100μmol/L)组、AA(10μg/ml)+Dex(100、10、1、0.1、0.01μmol/L)共同作用组,培养细胞48h,应用激光共聚焦显微镜(CFMS)观察α-平滑肌肌动蛋白(α-SMA)、E-钙黏素(E-cadherin)的表达,采用Real-timePCR和WesternBlot方法检测α-SMA、E-cadherin、p-Smad3、转化生长因子-β1(TGF-β1)、Smad7mRNA和蛋白的表达。结果 AA作用48h后,CFMS观察发现HKC细胞E-cadherin表达减弱而α-SMA表达增强;地塞米松干预后,E-cadherin表达逐渐增强而α-SMA表达减弱,且呈剂量依赖性。Real-timePCR、WesternBlot结果均显示,与阴性对照组相比,AA组α-SMA、p-Smad3、TGF-β1mRNA和蛋白表达水平均显著增强,E-cadherin、Smad7mRNA和蛋白的表达均减弱(P<0.05)。同AA作用组比较,AA+Dex共同作用组随Dex剂量增加,α-SMA、p-Smad3、TGF-β1mRNA和蛋白的表达逐渐减弱,E-cadherin、Smad7mRNA和蛋白的表达逐渐增强(P<0.05),而无水乙醇却无此作用。结论 Dex可抑制AA诱导HKC细胞发生EMT,并同时下调EMT过程中TGF-β1和p-Smad3的表达、上调Smad7分子的表达,提示TGF-β1/Smad下调可能是Dex抑制EMT发生的机制之一。

Objective To explore the effect and mechanism of dexamethasone （Dex）on AA induced epithelial-mesenchymal transition in HKC ceils. Methods Cultured HKC cells were divided into five groups： negative control group, incubated with AA（10 μg/ml） alone group, incubated with Dex （100 μmol/L） alone group, incubated with AA （10 μg/ml） group and absolute ethanol （100 μmol/L）group, or incubated with AA （10 μg/ml） and Dex group at different concentrations （100, 10, 1, 0.1, 0.01 μmol/L） for 48 h. Confocal microscope was used to detect expressions of α-SMA and E-cadherin. The mRNA and protein expressions of α-SMA, E-cadherin, TGF-β1, p-Smad 3 and Smad 7 were assessed with real-time PCR and western Blot, respectively. In the other experiment, HKC cells were incubated with AA （10 μg/ml） in the absence or presence of Dex （100 μmol/L）, respectively. The mRNA and protein expressions of α-SMA, E-cadherin, p-Smad 3, TGF-β1 and Smad 7 were assessed with real-time PCR and western Blot at different time points respectively. Results As revealed by confocal microscope, AA inhibited E-cadherin expression and enhanced α-SMA expression in HKC cells. Dex reversed AA-induced inhibition of E-cadherin and promotion of α-SMA expressions. Real-time PCR and western Blot showed that mRNA and protein expressions of α-SMA, p-Smad 3 and TGF-β1 significantly decreased, E-cadherin and Smad 7 significantly increased in ceils incubated with AA and Dex, compared with that treated with AA alone （P 〈 0.05）. There was no significant difference between the group of AA alone and the group treated with AA and absolute ethanol for mRNA and protein expression of α-SMA, p-Smad 3 and TGF-β1 in HKC cells. Conclusions The results of the present study suggest that Dex may partially inhibit AA-induced EMT of HKC cells in vitro, which is probably contributed to the repression of TGF-β1 and p-Smad 3, up-regulation of Smad 7 expressions. Further study is needed.