基于HIF-1α/SLC7A11轴介导肝星状细胞铁死亡探讨香芹酚抗肝纤维化的作用机制

Mechanism of carvacrol against liver fibrosis based on HIF-1α/SLC7A11 axis mediated ferroptosis in hepatic stellate cell

目的探究香芹酚对肝星状细胞(hepatic stellate cell,HSC)铁死亡的影响及其抗肝纤维化的作用机制。方法体内实验以四氯化碳诱导的小鼠肝纤维化模型为研究对象,设置对照组、模型组和香芹酚低、中、高剂量(25、50、100 mg/kg)组。采用苏木素-伊红(hematoxylin-eosin,HE)和Masson染色观察各组小鼠肝组织病理变化;采用试剂盒检测小鼠血清中丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)活性和羟脯氨酸(hydroxyproline,HYP)水平;采用Western blotting检测小鼠肝组织谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、I型胶原α1(collagen type 1α1,Col1α1)蛋白表达。体外实验以HSC-T6细胞为研究对象,用不同浓度(100、200、300、400、500、600μmol/L)的香芹酚或铁死亡抑制剂Ferrostatin-1(Fer-1)或缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)稳定剂DMOG(1 mmol/L)进行干预,CCK-8检测细胞活力;试剂盒检测HSC-T6细胞中Fe^(2+)、谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)和活性氧(reactive oxygen species,ROS)含量;Western blotting检测HSC-T6细胞中GPX4、α-SMA、Col1α1、HIF-1α、溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)蛋白表达。结果与模型组比较,香芹酚组小鼠血清中ALT、AST活性和HYP水平显著降低(P<0.05、0.01),肝脏肿胀、炎性细胞浸润和胶原沉积减少,肝组织α-SMA、COL1α1和GPX4蛋白表达水平显著降低(P<0.05、0.01)。香芹酚可降低HSC-T6细胞中GSH水平(P<0.05、0.01),并提高Fe^(2+)、MDA、ROS的含量(P<0.05、0.01),其对HSC-T6细胞活力的抑制作用可被Fer-1逆转(P<0.05、0.01);香芹酚对HSC-T6细胞中HIF-1α总蛋白水平无显著影响,但可降低细胞核中的HIF-1α水平以及细胞SLC7A11总蛋白水平(P<0.01);HIF-1α稳定剂DMOG可阻断香芹酚对HSC-T6细胞铁死亡的诱导作用(P<0.05、0.01)。结论香芹酚能够通过HIF-1α/SLC7A11轴诱导HSC铁死亡,进而发挥抗肝纤维化的作用。

Objective To explore the effect of carvacrol on ferroptosis in hepatic stellate cell(HSC)and its mechanism against liver fibrosis.Methods In vivo experiment was conducted on a mouse liver fibrosis model induced by carbon tetrachloride.The control group,model group,and carvacrol low-,medium-,high-dose(25,50,100 mg/kg)groups were set up,hematoxylin-eosin(HE)and Masson staining were used to observe the pathological changes of liver tissue in each group of mice;Kits were used to detect the activities of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and hydroxyproline(HYP)level in serum of mice;Western blotting was used to detect the protein expressions of glutathione peroxidase 4(GPX4),α-smooth muscle actin(α-SMA)and collagen type 1α1(Col1α1)in liver tissue of mice.In vitro experiments were conducted on HSC-T6 cells,and different concentrations(100,200,300,400,500,600μmol/L)of carvacrol or ferroptosis inhibitor ferrostatin-1(Fer-1)or hypoxia inducible factor-1α(HIF-1α)stabilizer DMOG(1 mmol/L)were used for intervention.Cell viability was detected by CCK-8;The reagent kits were used to detect the levels of Fe^(2+),glutathione(GSH),malondialdehyde(MDA)and reactive oxygen species(ROS)in HSC-T6 cells;Western blotting was used to detect the protein expressions of GPX4,α-SMA,Col1α1,HIF-1αand solute carrier family 7 member 11(SLC7A11)in HSC-T6 cells.Results Compared with model group,the activities of ALT,AST and level of HYP in serum of mice in carvacrol group were significantly reduced(P<0.05,0.01),while liver swelling,inflammatory cell infiltration and collagen deposition were reduced;The protein expression levels ofα-SMA,COL1α1 and GPX4 in liver tissue were significantly reduced(P<0.05,0.01).Carvacrol could reduce GSH level in HSC-T6 cells(P<0.05,0.01)and increase the contents of Fe^(2+),MDA and ROS(P<0.05,0.01);Its inhibitory effect on HSC-T6 cell viability could be reversed by Fer-1(P<0.05,0.01);Carvacrol had no significant effect on the total protein level of HIF-1αin HSC-T6 cells,but could reduce the HIF-1αlevel in nucleus and the total protein level of SLC7A11 in HSC-T6 cells(P<0.01);The HIF-1αstabilizer DMOG could block the induction effect of carvacrol on ferroptosis in HSC-T6 cells(P<0.05,0.01).Conclusion Carvacrol can induce ferroptosis in HSC through HIF-1α/SLC7A11 axis,thereby exerting an anti-liver fibrosis effect.