| Objective:To explore the effects of nicotinamide adenine dinucleotide phosphate oxidase4(NOX4)inhibitor GKT137831 and M2-type macrophages on NOX4,nuclear factor-E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1),oxidative stress markers of HSC T6 in rat liver,to find a new target for the treatment of hepatic fibrosis.Methods:Rat bone marrow macrophages were isolated and induced to differentiate into M2 macrophage phenotypes by IL-4.The expression of CD206 m RNA in M2-type macrophage marker was detected by qRT-PCR.HSC-T6 was activated by 5 μg/L transforming growth factor beta β1(TGF-β1).The proliferation of stimulated rat HSC-T6 cells stimulated was detected by CCK8 method,the concentration gradient of NOX4 inhibitor GKT137831 were 5 μmol/L,10 μmol/L,20 μmol/L,40 μmol/L,80 μmol/L and the optimal drug concentration was selected.Divided into separate culture control HSC group(G1),TGF-β1stimulation group(G2),TGF-β1+GKT137831 stimulation group(G3),co-culture M2 type macrophages +HSC group(G4),M2 type macrophages +TGF-β1 stimulation group(G5),M2 type macrophages +TGF-β1+GKT137831 stimulation group(G6),group G2-G6 were experimental groups.Reactive oxygen species(ROS)production levels were detected by DCFH-DA probe.The levels of niacinamide adenine dinucleotide phosphate oxidase 4(NOX4),α-Smooth muscle actin(α-SMA),nuclear factor E2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)in HSC cells of each group were determined by qRT-PCR and Western Blot.Results:1.CCK8 test results showed that: The survival rate of activated rat HSC cells decreases with the increase of NOX4 inhibitor GKT137831 concentration,and the IC50 of half inhibitory concentration is about 22.86μmol/L after 48 h of culture.In this study,20μmol/L was selected as the concentration dosage of GKT137831 for convenience of subsequent calculation.2.The results of macrophage polarization detection by qRT-PCR showed that :CD206m RNA expression in induced M2-type macrophages was significantly higher than that in M0 stage macrophages,and the difference was significant(P<0.05).3.DCFH-DA probe method was used to detect the production level of reactive oxygen species(ROS)in cells of each group.The results showed that,under fluorescence microscope,the intracellular ROS fluorescence level was significantly increased after TGF-β1 stimulation in the single culture group(P<0.05),ROS level decreased after adding GKT137831(P<0.05),the intracellular ROS production after TGF-β1 stimulation in the co-culture group was lower than that after TGF-β1 stimulation in the single culture group(P<0.05),the intracellular ROS fluorescence level decreased after addition of GKT137831 compared with that after addition of GKT137831(P<0.05).4.NOX4,α-SMA,Nrf2 and HO-1 in HSC cells of each group were detected by qRT-PCR : The results showed that the m RNA expressions of NOX4,α-SMA,Nrf2 and HO-1 in HSC cells were significantly increased after stimulation of TGF-β1(P<0.05);After addition of NOX4 inhibitor GKT137831,NOX4,α-SMA m RNA expression were decreased compared with TGF-β1 stimulation group(P<0.05),m RNA expressions of Nrf2 and HO-1 increased(P<0.05),and the expression of NOX4,α-SMA m RNA after TGF-β1stimulation in co-culture group was significantly decreased compared with that in single culture group(P<0.05),while m RNA expressions of Nrf2 and HO-1 were significantly increased(P<0.05);After addition of NOX4 inhibitor GKT137831,NOX4 m RNA expression of α-SMA in co-culture group were further reduced than that in single culture group(P<0.05),and the m RNA expressions of Nrf2 and HO-1 were further increased(P<0.05).5.NOX4,α-SMA,Nrf2 and HO-1 in HSC cells of each group were detected by Western Blot: The results showed that the protein expressions of NOX4,α-SMA,Nrf2 and HO-1 in HSC cells were significantly increased after stimulation of TGF-β1(P<0.05);After addition of NOX4 inhibitor GKT137831,NOX4,α-SMA protein expression were decreased compared with TGF-β1 stimulation group(P<0.05),protein expressions of Nrf2 and HO-1 increased(P<0.05),and the expression of NOX4,α-SMA protein after TGF-β1stimulation in co-culture group was significantly decreased compared with that in single culture group(P<0.05),while the protein expressions of Nrf2 and HO-1 were significantly increased(P<0.05);After addition of NOX4 inhibitor GKT137831,NOX4 protein expression of α-SMA in co-culture group were further reduced than that in single culture group(P<0.05),and the protein expressions of Nrf2 and HO-1 were further increased(P<0.05).Conclusion:NOX4 inhibitor GKT137831 can reduce ROS and NOX4 and α-SMA levels and increase Nrf2 and HO-1 levels in HSC.After co-culture of M2-type macrophages,GKT137831 assisted to reduce ROS and NOX4 and α-SMA levels,increase Nrf2 and HO-1 levels in HSC,regulate the balance between oxidative stress and anti-oxidative stress systems,and it may be a new therapeutic target for hepatic fibrosis. |
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