阿魏酸钠对乙醛诱导的肝星状细胞增殖、活化和胶原合成的影响

Effect of Sodium Ferulate on Proliferation,Activation and Collagen Synthesis of Hepatic Stellate Cells Induced by Acetaldehyde

目的：研究阿魏酸钠（SF）对体外乙醛诱导的大鼠肝星状细胞（HSC-T6）增殖、活化、胶原合成的影响及其作用机制。方法：体外培养HSC-T6细胞,建立乙醛诱导的HSC-T6增殖模型,设空白组、乙醛组、秋水仙碱组（2.5μmol·L^-1）及SF组（1,10,25,50,100,200,400μmol·L^-1）,通过细胞增殖和毒性检测（MTS）比色法检测细胞增殖,筛选合适的SF浓度。蛋白免疫印迹法（Western blot）检测SF（400,200,100μmol·L^-1）对乙醛诱导的HSC-T6细胞α-平滑肌肌动蛋白（α-SMA）表达;酶联免疫吸附测定法（ELISA）检测Ⅰ型,Ⅲ型胶原浓度,酸水解法检测羟脯氨酸（Hyp）含量,实时荧光定量聚合酶链式反应（Realtime PCR）检测转化生长因子-β1（TGF-β1）,信号转导蛋白Smad2,Smad3,Smad4,Smad7,基质金属蛋白酶-1（MMP-1）和金属蛋白酶抑制剂-1（TIMP-1）基因mRNA表达变化。结果：与空白组比较,200μmol·L^-1的乙醛能显著诱导HSC-T6体外增殖（P〈0.01）;与空白组比较,模型组HSC-T6增殖显著增加（P〈0.01）,α-SMA和I型,Ⅲ型胶原和Hyp含量显著增加（P〈0.01）,TGF-β1,Smad2,Smad3,Smad4,Smad7 mRNA的表达显著上调（P〈0.01）,MMP-1和TIMP-1 mRNA表达显著上调（P〈0.01）。与模型组比较,400,200,100μmol·L^-1浓度的SF能明显抑制乙醛诱导的HSC-T6增殖（P〈0.05,P〈0.01）,显著降低α-SMA和Ⅰ型,Ⅲ型胶原和Hyp含量（P〈0.01）,下调TGF-β1,Smad2,Smad3,Smad4基因mRNA的表达（P〈0.05,P〈0.01）,上调Smad7基因mRNA表达（P〈0.01）,并明显上调MMP-1 mRNA表达（P〈0.05,P〈0.01）和下调TIMP-1 mRNA表达（P〈0.01）。结论：SF能通过调控TGF-β1/Smad和MMP-1/TIMP-1信号通路,抑制乙醛诱导的体外HSC-T6细胞的增殖,活化和胶原分泌。

Objective： To investigate the effect of sodium ferulate（ SF） on the proliferation,activation and collagen synthesis of rat hepatic stellate cells（ HSC-T6） induced by acetaldehyde in vitro and explore its mechanism. Method： HSC-T6 cells were cultured in vitro to establish acetaldehyde-induced HSC-T6 proliferation models. The rats were divided into blank group,acetaldehyde group,colchicine group（ 2. 5 μmol·L^-1）,and SF groups（ 1,10,25,50,100,200,400 μmol·L^-1）. HSC-T6 proliferation was measured by MTS method and the appropriate SF concentration was screened. The effect of SF（ 400,200,100 μmol·L^-1） on the expression of α-smooth muscle actin（ α-SMA） in HSC-T6 cells induced by acetaldehyde was detected by Western blot; type I and type Ⅲ collagen concentrations were detected by Enzyme-linked immunosorbent assay（ ELISA）; the Hydroxyproline（ Hyp） content was detected by hydrolysis method; mRNA expression levels of transforming growth factor-β1（ TGF-β1）,signal transducers Smad2,Smad3,Smad4,Smad7,Matrix metallo proteinase 1（ MMP-1）and tissue inhibitors of metallo proteinase-1（ TIMP-1） were detected by real-time fluorescence quantitative polymerase chain reaction（ Real-time PCR）. Result： As compared with the blank group,200 μmol·L^-1 acetaldehyde significantly induced HSC-T6 proliferation in vitro（ P〈0. 01）. As compared with the blank group,the proliferation of HSC-T6 in model group was significantly increased（ P〈0. 01）,and the contents of α-SMA,type I,type Ⅲ collagen and Hyp were significantly increased（ P〈0. 01）,the mRNA expression levels of TGF-β1,Smad2,Smad3,Smad4 and Smad7 as well as mRNA expression levels of MMP-1 and TIMP-1 were significantly upregulated（ P〈0. 01）. As compared with the model group,SF at concentrations of 400,200,100 μmol·L^-1 significantly inhibited acetaldehyde-induced HSC-T6 proliferation（ P〈0. 05,P〈0. 01）,significantly decreasedα-SMA,type I and type Ⅲ collagen and Hyp contents（ P〈0. 05,P〈0. 01）,down-regulated the mRNA expression of TGF-β1,Smad2,Smad3 and Smad4（ P〈0. 05,P〈0. 01）,up-regulated the mRNA expression of Smad7 mRNA（ P〈0. 05, P〈0. 01） and down-regulated the mRNA expression of TIMP-1（ P〈0. 01）.Conclusion： SF can inhibit proliferation,activation and collagen secretion of HSC-T6 induced by acetaldehyde in vitro by regulating TGF-β1/Smads and MMP-1/TIMP-1 signaling pathways.