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目的研究丹皮酚对肝星状细胞活化及胶原合成的影响,进一步阐明其抑制肝纤维化的机制,为抗肝纤维化提供新的成分明确的中药单体。方法采用TGF-β刺激培养的人肝星状细胞,观察丹皮酚在上述条件下对肝星状细胞表型转化、胶原合成的影响,以探讨丹皮酚在体外抗纤维化作用并初步研究其机制。实时荧光定量PCR、Western Blotting检测肝星状细胞α-SMA mRNA及蛋白水平表达,明确丹皮酚对TGF-β刺激下肝星状细胞表型转化的影响。羟脯氨酸法检测细胞培养上清总胶原的表达。结果在TGF-β刺激作用下,α-SMA基因表达明显上调,加入不同浓度的丹皮酚后,α-SMA基因表达下调,并且具有丹皮酚剂量依赖性。在TGF-β刺激作用下,肝星状细胞培养上清中的羟脯氨酸含量明显上升,加入不同浓度的丹皮酚后明显下降。结论 TGF-β刺激使得α-SMA基因表达上调,加入丹皮酚表达下调,丹皮酚对肝星状细胞表型转化有促进其纤维化作用。TGF-β刺激作用下,肝星状细胞培养上清中的羟脯氨酸含量明显上升,提示肝星状细胞胶原合成增加。
Objective To study the effect of paeonol on the activation of hepatic stellate cells and collagen synthesis, to further clarify its mechanism of inhibiting hepatic fibrosis and to provide a new composition of traditional Chinese medicine monomer for anti-hepatic fibrosis. Methods The cultured human hepatic stellate cells were stimulated with TGF-β, and the effect of paeonol on the phenotype and collagen synthesis of hepatic stellate cells under the above conditions was observed to investigate the anti-fibrotic effect of paeonol in vitro Its mechanism. The expression of α-SMA mRNA and protein in hepatic stellate cells was detected by real-time quantitative PCR and Western Blotting, and the effect of paeonol on the phenotype of hepatic stellate cells stimulated by TGF-β was clarified. Hydroxyproline method was used to detect the expression of total collagen in cell culture supernatant. Results The expression of α-SMA gene was significantly up-regulated under the stimulation of TGF-β. The expression of α-SMA gene was down-regulated after the addition of paeonol at different concentrations, and the dose of paeonol was dose-dependent. Under the stimulation of TGF-β, the content of hydroxyproline in the culture supernatant of hepatic stellate cells increased obviously, but the content of paeonol decreased significantly after the addition of different concentrations of paeonol. Conclusion TGF-β stimulates the up-regulation of α-SMA gene expression and down-regulation of paeonol expression. Paeonol can promote hepatic stellate cell phenotype to promote fibrosis. TGF-β stimulation, hepatic stellate cell culture supernatant of hydroxyproline content increased significantly, suggesting that hepatic stellate cell collagen synthesis increased.