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-β induced fibrosis of LX-2 cells***1, ***1, ***1, ***1, ***1, ***1, ***1, ***1, ***1*(1.*****, *** ******)

关键词:  姜黄素  衍生物  肝纤维化  LX-2  TGF-β

Key words:  Curcumin  Derivative  Liver Fibrosis  LX-2  TGF-β

摘要:

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 CCK-8法检测姜黄素及其衍生物对正常LX-2细胞生长抑制率的影响,并计算各化合物的IC25值；利用TGF-β(10ng/ml)诱导LX-2细胞建立肝纤维化模型,CCK-8法检测姜黄素及其衍生物对细胞增殖的影响；流式细胞术检测姜黄素及其衍生物对TGF-β诱导的LX-2细胞凋亡率的影响；WB法和QPCR法检测姜黄素及其衍生物对纤维化相关因子collagen Ⅰ、collagen Ⅳ、Fibronectin、vimentin、α-SMA、PDGFRβ、TGFβR1、TGFβR2 、MMP2、MMP9、TIMP1和TIMP2的蛋白表达和基因转录水平的影响。结果 姜黄素及其衍生物A和B对正常LX-2细胞的生长抑制率随着浓度的增加而升高,其IC25值分别为15.7μM、2.6μM和10.2μM；利用TGF-β诱导LX-2细胞建立体外肝纤维化模型,并测得姜黄素及其衍生物A和B组的细胞增值率分别94.15%、89.28%和91.53%,相较模型组的128.53%更低,差异具有统计学意义(p＜0.05)；姜黄素及其衍生物A和B组的细胞凋亡率分别为8.34%、11.00%和10.99%,相较正常组的6.55%和模型组的6.01%有所增加,但差异暂无统计学意义；姜黄素组的collagen I、Fibronectin、vimentin、α-SMA、TGFβR1和TIMP-1的蛋白表达水平较模型组均有所下降,MMP-9的蛋白表达水平较模型组略有升高,差异具有统计学意义(P＜0.05)。姜黄素衍生物A组的collagen I、collagen IV、Fibronectin、vimentin、α-SMA、TIMP-1、TIMP-2的蛋白表达水平较模型组均有所下降,MMP-2的蛋白表达水平较模型组升高,差异均具有统计学意义(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的蛋白表达水平较模型组均有所下降,MMP-2的蛋白表达水平较模型组升高,差异均具有统计学意义(P＜0.05)。姜黄素组的collagen I、Fibronectin、α-SMA、TIMP-1的基因转录水平较模型组均有所下降,差异具有统计学意义(P＜0.05)。姜黄素衍生物A组和B组的collagen I、Fibronectin、α-SMA的基因转录水平较模型组均有所下降,差异具有统计学意义(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ECM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。

Abstract:

ABSTRACT: OBJECTIVE To study the inhibitory effect and molecular mechanism of curcumin and its derivatives A and B on TGF-β induced LX-2 cell fibrosis. METHODS The effects of curcumin and its derivatives on the growth inhibition rate of normal LX-2 cells were detected by CCK-8 method, and the IC25 values of each compound were calculated. Liver fibrosis model was established in LX-2 cells induced by TGF-β (10ng/ml), and the effects of curcumin and its derivatives on cell proliferation were detected by CCK-8 method. The effect of curcumin and its derivatives on TGF-β-induced LX-2 cells apoptosis was detected by flow cytometry. The effects of curcumin and its derivatives on fibrosis related factors (collagen I, collagen Ⅳ, Fibronectin, vimentin, α-SMA, PDGFRβ, TGFβR1, TGFβR2, MMP2, MMP9, TIMP1 and TIMP2) protein expression and gene transcription levels were detected by WB and QPCR. RESULTS The growth inhibition rates of curcumin and its derivatives A and B on normal LX-2 cells increased with increasing concentration, and their IC25 values were 15.7μM, 2.6μM and 10.2μM, respectively. LX-2 cells were induced by TGF-β to establish in vitro liver fibrosis model, and the proliferation rates of curcumin and its derivatives in groups A and B were 94.15%, 89.28% and 91.53%, respectively, which were lower than 128.53% in the model group, the difference was statistically significant (p<0.05). The apoptosis rates of curcumin and its derivatives A and B groups were 8.34%, 11.00% and 10.99%, respectively, which were increased compared with 6.55% in normal group and 6.01% in model group, but the difference was not statistically significant. collagen I, Fibronectin, vimentin, α-SMA, TGFβR1 and TIMP-1 protein expression levels in curcumin group were decreased compared with those in the model group, while the protein expression level of MMP-9 was slightly increased, with statistical significance (P<0.05). The protein expression levels of collagen I, collagen IV, Fibronectin, vimentin, α-SMA, TIMP-1 and TIMP-2 in curcumin derivative group A were decreased compared with the model group, while the protein expression levels of MMP-2 was increased compared with the model group, with statistical significance (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 group B were decreased compared with those in model group. The protein expression level of MMP-2 was higher than that of model group, and the differences were statistically significant (P<0.05). The gene transcription levels of collagen I, Fibronectin, α-SMA and TIMP-1 in curcumin group were lower than those in the model group, with statistical significance (P < 0.05). The gene transcription levels of collagen I, Fibronectin and α-SMA in curcumin derivative groups A and B were lower than those in model group, with statistical significance (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 ECM components, and promote its degradation, thus playing an anti-fibrotic effect in vitro, especially the curcumin derivative B.