吡非尼酮对人肝星状细胞LX2增殖、活化以及糖酵解途径的影响

Effects of pirfenidone on proliferation,activation,and glycolytic pathway of human hepatic stellate cell line LX2

目的 探讨吡非尼酮(PFD)对人肝星状细胞LX2增殖、活化以及糖酵解途径的影响,分析其抗肝纤维化的作用途径。方法 用10μg/L转化生长因子-β1(TGF-β1)激活LX2细胞,将LX2细胞分为正常组[0.1%二甲基亚砜(DMSO)]、对照组(10μg/L TGF-β1+0.1%DMSO)及实验组(10μg/L TGF-β1+2、4、6及8 mmol/L PFD);用CCK-8法和平板克隆实验评价LX2细胞增殖能力,试剂盒检测细胞培养上清中的葡萄糖及乳酸水平,蛋白免疫印迹法(Western blot)检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(COL1A1)、葡萄糖转运蛋白1(Glut1)、己糖激酶2(HK2)、血小板型磷酸果糖激酶(PFKP)、M2型-丙酮酸激酶(PKM2)、乳酸脱氢酶A(LDHA)以及单羧酸转运蛋白1(MCT1)蛋白表达,实时荧光定量PCR(RT-qPCR)检测α-SMA、COL1A1、Glut1、HK2、PKM2、LDHA mRNA表达。结果 与正常组比较,对照组LX2细胞的增殖能力增强,α-SMA、COL1A1蛋白及mRNA表达增加(P<0.05),葡萄糖消耗及胞外乳酸积累增多,Glut1、HK2、PFKP、PKM2、LDHA、MCT1蛋白及Glut1、HK2、PKM2、LDHA mRNA水平升高;与对照组比较,实验组LX2细胞增殖受到抑制,α-SMA、COL1A1蛋白表达降低,细胞葡萄糖消耗及胞外乳酸积累减少,LX2细胞Glut1、HK2、PFKP、PKM2、LDHA、MCT1的蛋白及Glut1、HK2、PKM2、LDHA mRNA水平下降(P<0.05)。结论 PFD能抑制LX2细胞的增殖及活化,减少细胞外基质分泌,其抗纤维化能力可能与糖酵解水平下调、细胞能量代谢受扰有关。

Objective To explore the effects of pirfenidone(PFD)on the proliferation,activation and glycolytic pathway of human hepatic stellate cell line LX2.Methods LX2 cells were treated with 10μg/L transforming growth factor-β1(TGF-β1)for activation.LX2 cells were divided into normal group(0.1%DMSO),control group(10μg/L TGF-β1+0.1%DMSO)and experimental groups(10μg/L TGF-β1+2,4,6,8 mmol/L PFD,respectively).LX2 cell proliferation ability was evaluated by CCK-8 assay and colony formation assay.Glucose and lactic acid levels were detected in cell culture supernatants by the corresponding kits.Western blot was used to detect protein expression ofα-smooth muscle actin(α-SMA),collagen typeⅠAlpha 1(COL1A1),glucose transporter 1(Glut1),hexokinase 2(HK2),phosphofructokinase platelet-type(PFKP),pyruvate kinase M2(PKM2),lactate dehydrogenase A(LDHA),and monocarboxylate transporter 1(MCT1).Quantitative Real-time PCR was applied to measure mRNA expression levels ofα-SMA,COL1A1,Glut1,HK2,PFKP,PKM2,LDHA,and MCT1.Results When compared with normal group,LX2 cells in control group exhibited enhanced proliferation ability,increased protein and mRNA expression levels ofα-SMA and COL1A1(P<0.05),increased glucose consumption and accumulation of extracellular lactate,elevated protein expression levels of Glut1,HK2,PFKP,PKM2,LDHA,and MCT1 as well as elevated mRNA expression levels of Glut1,HK2,PKM2,and LDHA.When compared with control group,LX2 cells in experimental groups displayed inhibited proliferation,reduced protein expression levels ofα-SMA and COL1A1,decreased glucose consumption and accumulation of extracellular lactate,reduced protein expression levels of Glut1,HK2,PFKP,PKM2,LDHA,and MCT1 as well as reduced mRNA expression levels of Glut1,HK2,PKM2,and LDHA(P<0.05).Conclusion PFD can inhibit the LX2 cell proliferation and activation,reduce the secretion of extracellular matrix.Its anti-fibrotic ability may be related to the downregulation of glycolysis levels and disruption of cellular energy metabolism.