姜黄素对大鼠肝星状细胞中TGF-β调节的NADPH氧化酶4的激活及Smad信号通路的影响

Curcumin attenuates hepatic fibrogenesis through interrupting TGF-β-mediated NADPH oxidase 4 activation and Smad signaling in vitro

目的研究姜黄素潜在的抗纤维化作用,以及转化生长因子β(TGF-β)诱导大鼠肝星状细胞(HSC-T6)激活的机制。方法不同浓度的姜黄素(0、5、10、20、40μmol/L)处理HSC-T6后,用MTT法测定其对HSC-T6增殖作用的影响;用RT-PCR法检测其mRNA水平的表达;用Westem blot法检测表达α-平滑肌肌动蛋白(α-SMA)、细胞外基质主要成分α1I胶原(Col1α1)、NADPH氧化酶4(NOX 4)、Smad 2、Smad 3及其磷酸化的水平。结果 40μmol/L的姜黄素组刺激HSC-T6 48 h后,其抑制率达到最大,显著低于仅TGF-β1刺激组、5、10及20μmol/L姜黄素组(P<0.05)。姜黄素可显著降低激活型HSC-T6的α-SMA、α1I胶原mRNA与蛋白表达,从而可以降低细胞外基质的沉积;有效降低了NOX 4的蛋白水平,减少了活性氧的水平;还明显降低Smad2、Smad 3的磷酸化程度,呈浓度依赖性。结论姜黄素在体外能够明显地抑制HSC-T6激活,对肝纤维化的发生发展具有一定抑制作用,其机制可能与抑制TGF-β诱导的NOX4的激活及Smad信号通路有关。

Objective To observe the effects of curcumin on activation and proliferation of hepatic stellate cells （HSCs） and, their expression of NADPH oxidase 4 （NOX 4） and smad signaling factors of rat hepatic stellate cells line in vitro using a rat HSC line. Methods Synchronized HSC-T6 cells were treated with different dose of purified curcumin （0, 5, 10, 20, 40μmol/L）. Untreated cells were viewed as controls. MTT technique analysis was used to examine the proliferation of the hepatic stellate cells stimulated by differernt concentration of curcumin;the mR-NA expression ofα-smooth muscle actin （α-SMA） and main constituents in extracellular matrix I procollagen （α1I collagen） were determined by RT-PCR in TGF-β-stimulated HSCs, and the antifibrotic effects on the protein ex-pression of α-SMA, α1I collagen, NADPH oxidase （NOX） protein （NOX 4） and the phosphorylation level of smad 2 and smad 3 was assessd by western blot;beta-actin （β-actin） was detected as the normalization control in the hepatic smllate cells was examined by Western blot analysis. Results The inhibition rate of HSC-T6 cells was the largest with curcumin for 48 h, significantly lower than that of TGF-β-stimulated group, 5 μmol/L, 10 μmol/L and 20 μmol/L group （P 〈0. 05）. Notably, curcumin could obviously reduce the expression ofα-SMA andα1I collagen at both mRNA and protein levels and reduce the deposition of extracellular matrix. Treatment of curcumin markedly blocks up-regulation of NOX4 by TGF-β. It also significantly reduced TGF-β-induced Smad 2/3 phos-phorylation, and this effect observed dose-dependent. Conclusion Curcumin inhibited TGF-β-mediated HSC acti-vation in vitro, which was involved in Smad signaling pathway, and consequently influenced the profibrotic actions of TGF-β on HSC-T6 cells. Our results suggest that curcumin could become potential candidate for the therapeutic applications by inhibiting the TGF-β induced NOX activation and Smad signaling.