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

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 5 μg/L transforming growth factor β1 (TGF-β1). The proliferation condition of HSC-T6 cells stimulated by the NOX4 inhibitor GKT137831 at a concentration gradient of 5 to 80 μmol/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 as α-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, and α-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, and α-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.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5 μg/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5～80 μmol/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。.