委陵菜酸通过阻断核因子κB(NF-κB)信号通路抑制LX-2人肝星状细胞增殖并促进其凋亡

Tormentic acid inhibits proliferation and promotes apoptosis of LX-2 human hepatic stellate cells via blocking NF-κB signaling pathway

目的研究委陵菜酸(TA)对核因子κB(NF-κB)信号通路的调控作用,分析其对LX-2人肝星状细胞增殖和凋亡的影响及机制。方法将培养的LX-2细胞分为正常组、血小板源性生长因子BB(PDGF-BB)刺激组(20 ng/mL)、1-吡咯烷二硫代羧酸铵盐(PDTC)组(PDGF-BB联合10 mmol/L PDTC)、TA处理组[PDGF-BB分别联合(35、25、15)μmol/LTA]。噻唑蓝(MTT)法检测TA对细胞增殖的影响,试剂盒检测天冬氨酸氨基转移酶(AST)、丙氨酸转氨酶(ALT)和总胆红素(TBIL)的活性;流式细胞术检测细胞凋亡情况。实时定量PCR检测核因子κBp65(NF-κB p65)mRNA水平,Western blot法检测NF-κB p65、磷酸化的NF-κB p65(p-NF-κB p65)、NF-κB p65抑制蛋白α(IκBα)、α平滑肌肌动蛋白(α-SMA)和转化生长因子β(TGF-β)、B细胞淋巴瘤因子2(Bcl2)、Bcl2相关X蛋白(BAX)的蛋白表达。结果与模型组相比,TA明显抑制LX-2细胞的活化增殖,降低α-SMA和TGF-β的蛋白表达;同时,TA处理可降低细胞中ASL、ALT和TBIL的水平,减少细胞损伤;TA明显诱导LX-2细胞凋亡,下调Bcl2表达同时上调BAX的表达。TA能明显抑制NF-κB p65 mRNA和蛋白表达,抑制NF-κB p65和IκBα蛋白的磷酸化。结论TA通过阻断NF-κB信号通路抑制肝星状细胞增殖,促进细胞凋亡。

Objective To investigate the regulating function of tormentic acid(TA)on the NF-κB signaling pathway and analyze its effect on proliferation and apoptosis of LX-2 human hepatic stellate cells and its mechanism.Methods The cultured LX-2 cells were randomized into normal control group,platelet-derived growth factor BB(PDGF-BB)stimulated group(20 ng/mL),PDTC group(20 ng/mL of PDGF-BB combined with 10 mmol/L of PDTC),and TA treated groups(20 ng/mL of PDGF-BB combined with 35,25,15μmol/L of TA).The effects of TA on cell proliferation were detected by MTT assay.The activities of ASL,ALT,and TBIL were detected by using commercially available kits.Cell apoptosis was determined by flow cytometry.The mRNA of NF-κB p65 was detected by real-time quantitative PCR;and the protein expressions of NF-κB p65,phosphorylated NF-κB p65(p-NF-κB p65),IκBα,α-SMA,TGF-β,Bcl2,and Bax were detected by Western blot.Results Compared with PDGF-BB,TA significantly inhibited the activation and proliferation of LX-2 cells,and decreased the protein expressions ofα-SMA and TGF-β.TA treatment reduced the levels of ASL,ALT,and TBIL in the cells and the cellular damage.TA significantly induced LX-2 cells apoptosis by down-regulating the expression of the anti-apoptotic protein Bcl2 and up-regulating the expression of the pro-apoptotic protein Bax.In addition,TA markedly inhibited the expressions of NF-κB p65 mRNA and protein,and the phosphorylation of NF-κB p65 and IκBαproteins.Conclusion TA inhibits proliferation and promotes apoptosis of LX-2 cells by blocking the NF-κB signaling pathway.