还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶4(NOX4)在肝癌细胞诱导巨噬细胞M2型极化中的作用

Role of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase 4(NOX4)in hepatoma cell-induced M2 polarization of macrophages

目的研究还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶4(NOX4)在肝癌细胞诱导巨噬细胞M2型极化中的作用和机制。方法单核巨噬细胞系THP-1和肝癌细胞系HepG2共培养模拟巨噬细胞的M2型极化,采用靶向NOX4的小干扰RNA(si-NOX4)和NOX4特异性抑制剂GLX351322抑制THP-1中NOX4的表达。将细胞分为对照组、si-NOX4组和GLX351322组。CCK-8法检测细胞活力,流式细胞术检测CD206+F4/80+M2型巨噬细胞比例,试剂盒检测M1型相关分子诱导型一氧化氮合酶(iNOS)、TNF-α的表达,蛋白质免疫印迹法(Western blot)检测M2型标志物精氨酸酶1(Arg1)、CCL22的表达以及TGF-β信号关键蛋白TGF-β1、Smad1的表达,试剂盒检测血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,免疫荧光染色检测CD206的表达。构建肝癌荷瘤小鼠模型,GLX351322干预后检测CD206的表达,Western blot法检测M2型标志物以及TGF-β信号关键蛋白的表达。结果si-NOX4和GLX351322抑制了NOX4的表达,THP-1和HepG2共培养后可显著抑制THP-1细胞活力,si-NOX4组和GLX351322组细胞活力显著低于对照组(P<0.05)。此外,si-NOX4组和GLX351322组CD206+F4/80+M2细胞比例也显著低于对照组(P<0.05)。M1型标志物iNOS和TNF-α的组间比较差异无统计学意义(P>0.05),而对照组中M2型标志物Arg1、CCL22以及TGF-β1、Smad1的表达显著高于si-NOX4组和GLX351322组(P<0.05)。免疫荧光染色结果也显示M2型标志物CD206在si-NOX4组和GLX351322组中表达下调,荧光强度低于对照组(P<0.05)。肝癌荷瘤小鼠模型中,GLX351322干预后肿瘤组织中CD206的表达水平下调,与对照组比较差异有统计学意义(P<0.05);M2型标志物Arg1、CCL22以及TGF-β1、Smad1的表达也显著低于对照组(P<0.05)。结论抑制NOX4表达可显著抑制肝癌细胞诱导的巨噬细胞M2型极化,提示NOX4在肿瘤相关巨噬细胞的转化中具有重要作用。

Objective To study the role of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase 4(NOX4)in M2 polarization of macrophages induced by hepatoma cells and the possible mechanism.Methods Monocyte cell line THP-1 cells and hepatoma HepG2 cells were co-cultured to simulate the M2 polarization of macrophages.Small interfering RNA targeting NOX4(si-NOX4)and GLX351322,a specific inhibitor of NOX4,were used to inhibit the expression of NOX4 in THP-1 cells.Three groups,control group,si-NOX4 group and GLX351322 group,were set up.CCK-8 assay was used to detect the changes in cell viability.Flow cytometry was performed to measure the ratios of CD206+F4/80+M2 macrophages.Expression of M1 macrophage-related molecules,inducible nitric oxide synthase(iNOS)and TNF-α,was analyzed using detection kits.Western blot was used to analyze the expression of arginase-1(Arg1),CCL22,TGF-β1 and Smad1.The expression of vascular endothelial growth factor(VEGF)and CD206 was detected using detection kit and immunofluorescence staining,respectively.A hepatoma-bearing mouse model was established and the expression of CD206,Arg1,CCL22,TGF-β1 and Smad1 were detected after GLX351322 intervention.Results Both si-NOX4 and GLX351322 inhibited the expression of NOX4.The viability of THP-1 cells was significantly inhibited after co-culturing the cells with HepG2 cells.Compared with the control group,the si-NOX4 and GLX351322 groups showed significantly decreased cell viability(P<0.05).Moreover,the percentages of CD206+F4/80+M2 cells in the si-NOX4 and GLX351322 groups were significantly lower than that in the control group(P<0.05).There was no significant difference in the expression of iNOS and TNF-αamong the three groups(P>0.05),while the expression of Arg1,CCL22,TGF-β1 and Smad1 in the control group was significantly higher than that in the si-NOX4 and GLX351322 groups(P<0.05).Immunofluorescence staining results showed that the expression of CD206 was down regulated in the si-NOX4 and GLX351322 groups,and the fluorescence intensity was lower than that in the control group(P<0.05).In the mouse model of hepatoma,CD206 expression was down regulated after GLX351322 intervention,which was significantly different from that in the control group(P<0.05).In addition,the expression of Arg1,CCL22,TGF-β1 and Smad1 was also significantly lower than that in the control group(P<0.05).Conclusions Inhibition of NOX4 expression could significantly inhibit the M2 polarization of macrophages induced by hepatoma cells.NOX4 might play an important role in the transformation of tumor-associated macrophages.