曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

Influence of tranilast on the cyclosporine A-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells

目的研究曲尼司特（Tran）对环孢素A（CsA）诱导的人肾小管上皮细胞（HK-2）向间充质转变的影响，并探讨该药抗纤维化的机制。方法所有用于实验的HK-2细胞株均为8～12代细胞，分为4组：（1）空白对照组，收获细胞，不做任何处理；（2）CsA组，加入4．2μmol／LCsA；（3）CsA＋Tran组，预先加入100μmol／LTran，作用2h后再加入4．2μmol／LCsA；（4）Tran组，仅加入100μmol／LTran。72h后于共聚焦显微镜下观察各组细胞形态学变化；用免疫荧光法以及免疫印迹法检测各组钙黏蛋白（E-cadherin）、平滑肌肌动蛋白α（α-SMA）和骨桥蛋白（OPN）的表达。结果HK-2细胞在正常情况下表现为典型的“鹅卵石”样形态，细胞圆钝，且与邻近的细胞连接较为紧密；空白对照组和Tran组细胞表现为典型的HK-2细胞形态；CsA组细胞变狭长，甚至向周边伸出“伪足”样改变，细胞间连接较为稀疏；CsA＋Tran组的细胞形态学改变有明显改善。CsA组细胞E-cadherin荧光表达强度明显弱于对照组，α-SMA、OPN荧光表达强于对照组；CsA＋Tran组细胞Bcadherin荧光表达强于CsA组，α-SMA、OPN荧光表达弱于CsA组。免疫印迹检查中，CsA组细胞E-cadherin的表达明显低于对照组，而α-SMA、OPN的表达明显高于对照组，CsA＋Tran组细胞E-cadherin的表达高于CsA组，而α—SMA、OPN的表达低于CsA组。结论曲尼司特能抑制CsA诱导的HK-2细胞由肾小管上皮向间充质细胞转化的过程，其机制可能与抑制OPN的表达有关。

Objective To study the effect of tranilast on cyclosporine A （CsA）-induced epithelial-to-mesenchymal transition in human renal tubular epithelial ceils, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups： （1） In the control group, cells were treated without any medicine; （2） The cell were treated with CsA （4. 2 μmol/L） for 72 h; （3） The cells were treated with a combination of CsA （4. 2 vmol/L） and tranilast （100μmol/L）; （4） The cells were treated with tranilast （100 μmol/L） alone for 72 h. Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the ,expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The inmaunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA ＋ Tranilast group was higher than the CsA group, while the expression of α-SMA and CPN in the CsA ＋ Tranilast group was lower than the CsA group. Western blotting showed that protein, expression level of E-cadherin in CsA group was dramatically lower than that in the control group （ P〈0. 05）, while that of α-SMA and OPN in CsA group was significantly higher than in the control group （P 〈0. 05 ）. The protein expression level of E-cadherin in HK-2 ceils in the CsA ＋ Tranilast group was markedly higher than in the CsA group （P〈0. 05）, and that of α-SMA and OPN in CsA ＋ Tranilast group was significantly lower than in the CsA group （P〈0. 05）. Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.