丹皮酚对人肝星状细胞α-SMA表达的影响

Research of Paeonol affect α-SMA expression in hepatic stellate cells

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 This paper intends to study the effect of paeonol on hepatic stellate cells activation and collagen synthesis,and further indicate the mechanism of inhibiting liver fibrosis,so as to provide new ingredients of the clear Chinese herbal monomer for anti-hepatic fibrosis.Methods In this study,TGF-β was used to stimulate and culture human hepatic stellate cells.Observing the effects of paeonol on hepatic stellate cell phenotype transformation and collagen synthesis to investigate the influencing mechanism of paeonol on anti-fibrosis in vitro.Real time fluorescence quantitative PCR and Western Blotting were utilized to detect the hepatic stellate cellular α-SMA mRNA expression,which can further clarify the effect of paeonol on hepatic stellate cell phenotype conversion in the case of TGF-β stimulation.Hydroxyproline assay method was used to detect the total collagen expression of cell culture supernatant.Results TGF-β stimulation inceased the α-SMA gene expression,and subsequently,adding different concentrations of paeonol decreased the α-SMA gene expression.The α-SMA gene expression had a certain dependency uponpaeonol dose.Hydroxyproline content in the hepatic stellate cell culture supernatant increased significantly in the case of TGF-β stimulation,and significantly decreased after adding different concentrations of paeonol.Conclusion TGF-β stimulation makes a positive effect on α-SMA gene expression,while the adding of paeonol has a negative effect.Paeonol may promote the fibrosis of hepatic stellate cell.Hydroxyproline content in the hepatic stellate cell culture supernatant increased significantly in the case of TGF-β stimulation,indicating an increase of hepatic stellate cells collagen synthesis.