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目的:研究鳖甲提取物相对分子质量<6 k Da肽段(P6)对转化生长因子-β(transforming growth factor-β,TGF-β)诱导的大鼠肝星状细胞HSC-T6的活化增殖与肝纤维化(hepatic fibrosis,HF)相关基因表达的影响,初步探究其抗肝纤维化的作用机制。方法:应用透析法得到P6;细胞增殖与活性检测(CCK8)法检测P6对HSC-T6细胞增殖的影响;实时荧光定量聚合酶反应(Real-time PCR)检测HSC-T6细胞内α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA),I型胶原(collagen type I,Col I),基质金属蛋白酶抑制因子-1(tissue inhibitor of metalloproteinase-1,TIMP-1)以及基质金属蛋白酶(matrix metalloproteinase-2,MMP-2)的mRNA表达水平;蛋白免疫印迹法(Western blot)分析HSC-T6细胞内α-SMA与Col I蛋白的表达。结果:与空白组比较,P6对HSC-T6细胞存活率无明显影响,但却能显著抑制TGF-β诱导的HSC-T6细胞的增殖(P<0.05)。与TGF-β组比较,P6各浓度均能显著降低HSC-T6细胞中α-SMA,Col I,TIMP-1 mRNA的表达,增加MMP-2 mRNA的表达(P<0.05,P<0.01),明显降低α-SMA与Col I蛋白的表达。结论:P6能抑制TGF-β诱导的HSC-T6细胞的活化增殖,减少细胞外基质(extracellular matrix,ECM)生成,促进其降解,发挥抗肝纤维化作用。
OBJECTIVE: To investigate the effect of the relative molecular mass of 6 g Da peptide (P6) on the activation and proliferation of rat hepatic stellate cells (HSC-T6) induced by transforming growth factor-β (TGF-β) And hepatic fibrosis (hepatic fibrosis, HF) related gene expression, to explore its mechanism of anti-hepatic fibrosis. Methods: P6 was obtained by dialysis method; the effect of P6 on the proliferation of HSC-T6 cells was detected by the method of CCK8; the expression of α-smooth muscle in HSC-T6 cells was detected by Real-time quantitative polymerase chain reaction (Real-time PCR) Α-smooth muscle actin (α-SMA), collagen type I (Col I), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase (MMP-2, MMP-2, MMP-2) in HSC-T6 cells. Western-blot was used to analyze the expression of α-SMA and Col I protein in HSC-T6 cells. Results: Compared with the blank group, P6 had no significant effect on the survival rate of HSC-T6 cells, but significantly inhibited the proliferation of HSC-T6 cells induced by TGF-β (P <0.05). Compared with TGF-β group, P6 concentration significantly decreased the expression of α-SMA, Col I, TIMP-1 mRNA and the expression of MMP-2 mRNA (P <0.05, P <0.01) Significantly reduce the expression of α-SMA and Col I protein. Conclusion: P6 can inhibit the activation and proliferation of HSC-T6 cells induced by TGF-β, reduce the formation of extracellular matrix (ECM), promote the degradation of HSC-T6 cells and exert anti-hepatic fibrosis.