鹅不食草心菊内酯对肝星状细胞活化的抑制作用机制研究

目的研究鹅不食草心菊内酯(helenalin)对LX-2人肝星状细胞(HSCs)活化的抑制作用及其机制。方法用白细胞介素-1β(IL-1β)20 ng·mL^-1刺激LX-2细胞建立体外细胞模型,另取未刺激细胞作为正常组。将损伤细胞分为5组:模型组、阳性对照组LY294002(磷脂酰肌醇3激酶/丝苏氨酸蛋白激酶通路抑制剂)20μmol·L^-1]和高、中、低3个剂量实验组(helenalin:2.0,1.0,0.5μmol·L^-1),均干预24 h。用四甲基偶氮唑盐法检测细胞的增殖抑制率,流式细胞术检测细胞凋亡率,实时荧光定量-PCR法检测微小RNA-21(miR-21)基因的表达,蛋白质印迹法检测α-平滑肌肌动蛋白(α-SMA)、I型胶原和Ⅲ型胶原蛋白的表达(光密度值)。结果Helenalin作用24 h时的IC₅₀为6.98μmol·L^-1,根据细胞的增殖抑制率,选择低于IC₅₀的3个浓度(2.0,1.0,0.5μmol·L^-1)作为后续实验的给药浓度。正常组、模型组和高、中、低3个剂量实验组的miR-21基因相对表达量分别为0.99±0.15,1.71±0.07,1.03±0.04,1.01±0.02和1.19±0.12;正常组、模型组、阳性对照组和高、中、低3个剂量实验组的总凋亡率分别为(1.45±0.33)%,(1.93±0.55)%,(23.33±0.49)%,(19.77±0.65)%,(10.70±0.75)%和(3.01±0.38)%;上述6组的α-SMA蛋白相对表达量分别为0.19±0.02,0.26±0.04,0.15±0.02,0.15±0.02,0.18±0.04和0.20±0.04;上述6组的I型胶原蛋白相对表达量分别为0.15±0.02,0.39±0.05,0.19±0.01,0.24±0.04,0.37±0.04和0.38±0.06;上述6组的Ⅲ型胶原蛋白相对表达量分别为0.07±0.01,0.31±0.04,0.09±0.01,0.05±0.01,0.05±0.01和0.18±0.02。上述指标:模型组与正常组相比,差异均有统计学意义(均P<0.01);3个剂量实验组与模型组相比,差异均有统计学意义(P<0.05,P<0.01)。结论Helenalin抗肝纤维化的作用机制可能与抑制肝星状细胞的增殖活化、诱导细胞凋亡和减少活化细胞内胶原的合成并下调miR-21基因的表达有关。

Inhibition effect and mechanism of helenalin on the activation of hepatic stellate cell

Objective To investigate the inhibitory effect and its mechanism of helenalin on the activation of LX-2 human hepatic stellate cells(HSCs).Methods Interleukin-1 beta(IL-1β)20 ng·mL^-1 was used to stimulate LX-2 cells to construct in vitro cell model,and unstimulated cells were regarded as the normal group.The damage cells were divided into five groups,including model group,positive control groupLY294002(phosphatidylinositol 3 kinase/serine protein kinase pathway inhibitor)20μmol·L^-1],and experimental-H,experimental-M and experimental-L groups(helenalin:2.0,1.0,0.5μmol·L^-1),all groups were intervened for 24 h.Methyl thiazolyl tetrazolium(MTT)assay was used to determine the inhibitory rate of cell proliferation to investigate the effect of helenalin on LX-2 cytotoxicity.Flow cytometric analysis was used to estimate apoptotic rate.Real-time quantitative-PCR was used to detect the expression levels of microRNA-21 mRNA(miR-21 mRNA).Western blot was used to evaluate the expression levels(OD value)ofα-smooth muscle kinesin(α-SMA),Collagen type I(Collagen-Ⅰ)and Collagen typeⅢ(Collagen-Ⅲ)proteins.Results The IC50 of helenalin for 24 h was 6.98μmol·L^-1,based on the rate of inhibition of cell proliferation,the three concentrations(2.0,1.0 and 0.5μmol·L^-1)lower than IC50 were chosen as the drug concentration for the following experiments.The relative expression levels of miR-21 mRNA in normal group,model group,experimental-H,experimental-M and experimental-L groups were 0.99±0.15,1.71±0.07,1.03±0.04,1.01±0.02,and 1.19±0.12,respectively.The percentage of total apoptotic cells in normal group,model group,positive control group,experimental-H,experimental-M and experimental-L groups were(1.45±0.33)%,(1.93±0.55)%,(23.33±0.49)%,(19.77±0.65)%,(10.70±0.75)%,(3.01±0.38)%,respectively;the relative expression ofα-SMA protein in the above six groups were 0.19±0.02,0.26±0.04,0.15±0.02,0.15±0.02,0.18±0.04,0.20±0.04,respectively;the relative expression of Collagen-Ⅰprotein in the six groups were 0.15±0.02,0.39±0.05,0.19±0.01,0.24±0.04,0.37±0.04,0.38±0.06,respectively;the relative expression of Collagen-Ⅲprotein in the above six groups were 0.07±0.01,0.31±0.04,0.09±0.01,0.05±0.01,0.05±0.01,0.18±0.02,respectively.Comparison between model group and normal control group,the difference of the above indicators was significant(all P<0.01);comparison between experimental-H,experimental-M and experimental-L groups and model group,the difference of the above indicators was significant(P<0.05,P<0.01).Conclusion The mechanism of helenalin against hepatic fibrosis may be related to inhibition of proliferation and activation of hepatic stellate cells,induction of cell apoptosis,reduction of collagen synthesis in activated cells,and down-regulation of miR-21 mRNA expression.