楮实子对肝星状细胞增殖、凋亡及TLR4/NF-κB/TNF-α信号通路的影响

Effects of fructus broussonetiae on the proliferation,apoptosis and TLR4/NF-κB/TNF-αsignal pathway of hepatic stellate cells

目的 探究楮实子对肝星状细胞(HSC)增殖、凋亡及Toll样受体4(TLR4)/核因子-κB(NF-κB)/肿瘤坏死因子-α(TNF-α)信号通路的影响。方法 体外培养大鼠肝星状细胞系(HSC-T6),正常培养细胞作为空白组;使用转化生长因子-β1(TGF-β1)刺激细胞24 h后,分别添加不同质量浓度(0、1、5、10 g/L)的楮实子培养细胞,依次作为对照组、低剂量组、中剂量组、高剂量组。MTT法、Annexin V-FITC/PI双染法检测楮实子对HSC-T6细胞增殖、凋亡的影响;ELISA法检测HSC-T6细胞上清液中TNF-α、白细胞介素-6(IL-6)含量;Western blotting法检测HSC-T6细胞中TLR4、NF-κB、TNF-α蛋白表达。结果 楮实子可抑制活化的HSC-T6细胞增殖,促进其凋亡,且呈质量浓度依赖性(P<0.05);楮实子可降低活化的HSC-T6细胞上清液中TNF-α、IL-6水平,下调细胞中TLR4、NF-κB、TNF-α蛋白水平,并均呈质量浓度依赖性(P<0.05)。结论 楮实子可抑制HSC-T6细胞增殖,促进HSC-T6细胞凋亡,其机制可能与抑制TLR4/NF-κB/TNF-α信号通路激活有关。

Aim To investigate the effects of fructus broussonetiae on proliferation and apoptosis of hepatic stellate cells(HSC)and toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)/tumor necrosis factor-α(TNF-α)signal pathway.Methods Rat hepatic stellate cell line HSC-T6 was cultured in vitro,and the normal cultured cells were used as the blank group;after stimulating the cells with transforming growth factor-β1(TGF-β1)for 24 h,different concentrations(0,1,5,and 10 g/L)of fructus broussonetiae were added to cultured cells to serve as the control group,low concentration group,middle concentration group,and high concentration group.MTT assay and Annexin V-FITC/PI double staining method were used to detect the effects of fructus broussonetiae on the proliferation and apoptosis of HSC-T6 cells;ELISA method was used to detect the contents of TNF-αand Interleukin-6(IL-6)in the supernatant of HSC-T6 cells;Western blotting method was used to detect the protein expression of TLR4,NF-κB and TNF-αin HSC-T6 cells.Results Fructus broussonetiae could inhibit the proliferation of activated HSC-T6 cells and promote their apoptosis in a concentration-dependent decline or increase manner(P<0.05);Fructus broussonetiae could reduce the levels of TNF-αand IL-6 in the supernatant of activated HSC-T6 cells,and down-regulate the levels of TLR4,NF-κB,and TNF-αproteins in the cells,all in a concentration-dependent decline manner(P<0.05).Conclusion Fructus broussonetiae could inhibit the proliferation of HSC-T6 cells and promote the apoptosis of HSC-T6 cells.The mechanism may be related to inhibiting the activation of the TLR4/NF-κB/TNF-αsignaling pathway.