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目的探讨淫羊藿苷(ICA)对脂多糖/TLR4途径诱导的肝星状细胞的活化和自噬的作用,并进一步研究是否与FXR蛋白相关。方法通过脂多糖刺激肝星状细胞-T6细胞株并通过TLR4 siRNA和淫羊藿苷干预。吖啶橙(AO)染色和MDC染色观察自噬体改变;激光共聚焦观察Beclin-1和LC3B的表达;流式细胞仪检测细胞线粒体膜电位;Western检测NF-κB(p65)、LC3B-Ⅱ、胶原蛋白Ⅰ、TGF-β1和α-SMA蛋白变化。另对淫羊藿苷干预组用Z-Guggulsterone(Z-Gug)进行干预,Western检测LC3B、Beclin-1和FXR的改变。结果脂多糖刺激肝星状细胞-T6自噬体数量增加、上调Beclin-1和LC3B表达、降低细胞线粒体膜电位和增加NF-κB(p65)、LC3B-Ⅱ、胶原蛋白Ⅰ、TGF-β1和α-SMA表达。该作用可被TLR4 siRNA和淫羊藿苷剂量依赖性阻断;淫羊藿苷可以增加FXR表达且被Z-Gug阻断。结论淫羊藿苷通过减少自噬降低脂多糖/TLR4途径诱导的肝星状细胞的活化和FXR有关。
Objective To investigate the effect of icariin (ICA) on activation and autophagy of hepatic stellate cells induced by lipopolysaccharide / TLR4 pathway and to further investigate whether it is related to FXR protein. Methods Lipopolysaccharide was used to stimulate hepatic stellate cell-T6 cells and intervened by TLR4 siRNA and icariin. The changes of autophagosomes were observed by acridine orange (AO) staining and MDC staining. The expression of Beclin-1 and LC3B was observed by laser scanning confocal microscope. The mitochondrial membrane potential was detected by flow cytometry. The expressions of NF-κB (p65), LC3B- , Collagen Ⅰ, TGF-β1 and α-SMA protein changes. In addition, the intervention group with Icariin intervention was treated with Z-Guggulsterone (Z-Gug), Western analysis LC3B, Beclin-1 and FXR changes. Results Lipopolysaccharide stimulated the increase of the number of autophagosomes of T6 in hepatic stellate cells, up-regulated the expression of Beclin-1 and LC3B, decreased the mitochondrial membrane potential and increased the expressions of NF-κB, LC3B-Ⅱ, collagenⅠ, TGF- α-SMA expression. This effect could be dose-dependently blocked by TLR4 siRNA and icariin; icariin increased FXR expression and was blocked by Z-Gug. Conclusion Icariin is related to FXR by decreasing autophagy and decreasing the activation of hepatic stellate cells induced by lipopolysaccharide / TLR4 pathway.