M2型巨噬细胞与GKT137831对肝星状细胞氧化应激的影响

Effects of M2-type macrophages and GKT137831 on oxidative stress in hepatic stellate cells

目的探讨还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)抑制剂GKT137831与M2型巨噬细胞对大鼠肝星状细胞(HSC)系(HSC-T6)氧化应激指标NOX4、核因子E2相关因子2(Nrf2)和血红素加氧酶1(HO-1)的影响。方法分离大鼠骨髓巨噬细胞,用白细胞介素(IL)-4诱导其分化为M2巨噬细胞表型。选5μg/L转化生长因子β1(TGF-β1)激活HSC-T6,采用细胞计数(CCK-8)法检测5~80μmol/L浓度梯度下NOX4抑制剂GKT137831刺激活化的大鼠HSC-T6细胞48 h后细胞增殖情况,选定最适药物浓度。分单独培养HSC组(对照组)、TGF-β1刺激组、TGF-β1+GKT137831刺激组、共同培养M2型巨噬细胞+HSC组、M2型巨噬细胞+TGF-β1刺激组、M2型巨噬细胞+TGF-β1+GKT137831刺激组,后5组为实验组。采用DCFH-DA探针法检测各组细胞活性氧(ROS)产生水平,采用qRT-PCR法和蛋白质印迹法检测各组HSC细胞NOX4、α-平滑肌肌动蛋白(α-SMA)、Nrf2和HO-1水平。两组数据间的比较采用t检验,多组间比较行One-way ANOVA法分析。结果TGF-β1刺激后细胞内ROS显著升高,各组细胞ROS相对水平分别为1.03±0.11、3.88±0.07、2.90±0.08、0.99±0.06、3.30±0.05、2.21±0.11,F=686.1,P=0.001;NOX4、α-SMA、Nrf2、HO-1 mRNA和蛋白表达显著升高(P<0.05),加入GKT137831后,ROS及NOX4、α-SMA mRNA和蛋白表达较TGF-β1刺激组降低(P<0.05),Nrf2和HO-1 mRNA及蛋白表达升高(P<0.05)。共培养组中TGF-β1刺激后HSC产生的ROS与NOX4、α-SMA mRNA及蛋白表达较单独培养组中TGF-β1刺激后显著降低(P<0.05),而Nrf2和HO-1 mRNA及蛋白表达显著升高(P<0.05),加入GKT137831后,共同培养组中ROS与NOX4、α-SMA mRNA及蛋白表达较单独培养组进一步降低(P<0.05),而Nrf2和HO-1 mRNA及蛋白表达进一步升高(P<0.05)。结论NOX4抑制剂GKT137831能降低HSC中ROS及NOX4、α-SMA水平和升高Nrf2、HO-1水平;M2型巨噬细胞共培养后辅助GKT137831降低HSC中ROS及NOX4、α-SMA水平,升高Nrf2、HO-1水平,调节了氧化应激与抗氧化应激系统间的平衡,从而拮抗纤维化进程。

Objective To investigate the effects of reduced nicotinamide adenine dinucleotide phosphooxidase 4(NOX4)inhibitors GKT137831 and M2-type macrophages on oxidative stress markers NOX4,nuclear factor E2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)in the rat hepatic stellate cell line(HSC-T6).Methods Rat bone marrow macrophages were extracted and induced using interleukin(IL)-4 to differentiate them into M2 phenotype macrophages.HSC-T6 activation was performed with 5μg/L transforming growth factorβ1(TGF-β1).The proliferation condition of HSC-T6 cells stimulated by the NOX4 inhibitor GKT137831 at a concentration gradient of 5 to 80μmol/L after 48 hours was detected using the Cell Counting Kit-8(CCK-8)assay.The optimal drug concentration was chosen and divided into an HSC co-culture group(the control group)and five experimental groups:the TGF-β1 stimulation group,the TGF-β1+GKT137831 stimulation group,the M2-type macrophage+HSC co-culture group,the M2-type macrophage+TGF-β1 stimulation group,and the M2-type+TGF-β1+GKT137831 stimulation group.Reactive oxygen species(ROS)production level was detected in each cell using the DCFH-DA probe method.NOX4,α-smooth muscle actin(α-SMA),Nrf2,and HO-1 levels in each group of HSC cells were detected using the qRT-PCR method and the Western blot method.The t-test was used to compare the two groups.The one-way ANOVA method was used to compare multiple groups.Results Intracellular ROS increased significantly following TGF-β1 stimulation.ROS relative levels in each cell group were 1.03±0.11,3.88±0.07,2.90±0.08,0.99±0.06,3.30±0.05,2.21±0.11,F=686.1,P=0.001,respectively.The mRNA and protein expressions of NOX4,α-SMA,Nrf2,and HO-1 were significantly increased(P<0.05).After the addition of GKT137831,ROS,and NOX4,α-SMA mRNA and protein expression were comparatively decreased in the TGF-β1 stimulation group(P<0.05),while mRNA and protein expressions of Nrf2 and HO-1 were increased(P<0.05).The expression of ROS and NOX4,as well asα-SMA mRNA and protein,produced by HSC were significantly decreased in the co-culture group compared to the single culture group after TGF-β1 stimulation(P<0.05).After the addition of GKT137831,ROS,NOX4,α-SMA mRNA,and protein expression were further reduced in the co-culture group compared with the single culture group(P<0.05),while the mRNA and protein expression of Nrf2 and HO-1 were further increased(P<0.05).Conclusion NOX4 inhibitor GKT137831 can reduce RO,NOX4,andα-SMA levels while increasing Nrf2 and HO-1 levels in hepatic stellate cells.After M2-type macrophage co-culture,GKT137831 assists in lowering ROS,NOX4,andα-SMA levels while accelerating Nrf2 and HO-1 levels in hepatic stellate cells,which regulates the balance between oxidative stress and anti-oxidative stress systems,thereby antagonizing the fibrosis process.