摘要目的: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。

abstractsObjective: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-α among 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.