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

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, 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. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/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 irmmunofluorescence 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 OPN 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 cells 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.     

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

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, 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. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/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 irmmunofluorescence 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 OPN 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 cells 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.     

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

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/L Tran,作用2 h后再加入4.2 μmol/L CsA;(4)Tran组,仅加入100μmol/L Tran.72 h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E-cadherin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.

Abstract：

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, 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. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/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 irmmunofluorescence 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 OPN 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 cells 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.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.