姜黄素及其衍生物对TGF-β诱导的LX-2细胞纤维化的抑制作用研究

Inhibitory Effect of Curcumin and Its Derivatives on TGF-βInduced Fibrosis of LX-2 Cells

目的研究姜黄素及其衍生物A和B对TGF-β诱导的LX-2细胞纤维化的抑制作用及其机制。方法采用TGF-β(10 ng·m L^(-1))诱导LX-2细胞肝纤维化模型;CCK-8法检测姜黄素及其衍生物对细胞增殖的影响;流式细胞术检测细胞凋亡率;Western blotting和q-PCR法检测纤维化相关因子CollagenⅠ、CollagenⅣ、Fibronectin、Vimentin、α-SMA、PDGFRβ、TGFβR1、TGFβR2、MMP2、MMP9、TIMP1和TIMP2蛋白表达和基因转录水平。结果姜黄素及其衍生物A和B对正常LX-2细胞的生长具有抑制作用,IC25值分别为15.7、2.6和10.2μmol·L^(-1);与模型组比较,姜黄素(15.7μmol·L^(-1))及其衍生物A(2.6μmol·L^(-1))和B(10.2μmol·L^(-1))对TGF-β诱导LX-2细胞增殖具有抑制作用(P<0.05);姜黄素衍生物B组的细胞凋亡率升高;姜黄素组的Collagen I、Fibronectin、Vimentin、α-SMA、TGFβR1和TIMP-1的蛋白表达水平下降,MMP-9的蛋白表达水平升高(P<0.05);姜黄素衍生物A组的Collagen I、Collagen IV、Fibronectin、Vimentin、α-SMA、TIMP-1、TIMP-2的蛋白表达水平下降,MMP-2的蛋白表达水平升高(P<0.05);姜黄素衍生物B组的Collagen I、Collagen IV、Fibronectin、Vimentin、α-SMA、PDGFRβ、TGFβR1、TGFβR2、TIMP-1、TIMP-2的蛋白表达水平下降,MMP-2的蛋白表达水平升高(P<0.05)。姜黄素组的Collagen I、Fibronectin、α-SMA、TIMP-1的基因转录水平下降(P<0.05)。姜黄素衍生物A组和B组的Collagen I、Fibronectin、α-SMA的基因转录水平下降(P<0.05)。结论姜黄素及其衍生物A和B通过抑制TGF-β诱导的LX-2细胞的异常活化和增殖,抑制其细胞外基质组分的过度分泌和积聚,促进其降解,从而发挥体外抗纤维化作用,尤以姜黄素衍生物B的作用最突出。

OBJECTIVE To study the inhibitory effect and mechanism of curcumin and its derivatives A and B on TGF-βinduced LX-2 cell fibrosis.METHODS Established the liver fibrosis model of LX-2 cells induced by TGF-β(10 ng·mL^(−1)).The effects on cell proliferation were detected by CCK-8.The effects on cell apoptosis was detected by flow cytometry.The effects on fibrosis related factors(Collagen I,CollagenⅣ,Fibronectin,Vimentin,α-SMA,PDGFRβ,TGFβR1,TGFβR2,MMP2,MMP9,TIMP1 and TIMP2)protein expression and gene transcription levels were detected by Western blotting and q-PCR.RESULTS The curcumin and its derivative A and B had the inhibition effects on normal LX-2 cells,and the IC25 values were 15.7,2.6,10.2μmol·L^(−1),respectively.Compared to the model group,the curcumin(15.7μmol·L^(−1))and its derivative A(2.6μmol·L^(−1))and B(10.2μmol·L^(−1))had the significant inhibition effects on cell proliferation of the TGF-βinduced LX-2 cells(P<0.05).The cell apoptosis rate of curcumin derivative B group was higher than the model group(P<0.05).Collagen I,Fibronectin,Vimentin,α-SMA,TGFβR1 and TIMP-1 protein expression levels in curcumin group were lower,while the protein expression level of MMP-9 was higher(P<0.05).The protein expression levels of Collagen I,Collagen IV,Fibronectin,Vimentin,α-SMA,TIMP-1 and TIMP-2 in curcumin derivative A group were lower,while the protein expression level of MMP-2 was higher(P<0.05).The protein expression levels of Collagen I,Collagen IV,Fibronectin,Vimentin,α-SMA,PDGFRβ,TGFβR1,TGFβR2,TIMP-1 and TIMP-2 in curcumin derivative B group were lower,while the protein expression level of MMP-2 was higher(P<0.05).The gene transcription levels of Collagen I,Fibronectin,α-SMA and TIMP-1 in curcumin group were lower(P<0.05).The gene transcription levels of Collagen I,Fibronectin andα-SMA in curcumin derivative A and B groups were lower(P<0.05).CONCLUSION Curcumin and its derivatives A and B inhibit the abnormal activation and proliferation of TGF-β-induced LX-2 cells,inhibit the excessive secretion and accumulation of its extracellular matrix components,and promote its degradation,thus playing an anti-fibrotic effect in vitro,especially the curcumin derivative B.