含氢培养基调节巨噬细胞RAW264.7极化的机制研究

The regulation of hydrogen-rich medium on the polarization of macrophage RAW264. 7

目的观察含氢培养基对巨噬细胞RAW264.7极化的影响,并初步探讨巨噬细胞的功能。方法分别采用常规培养和含氢培养基培养巨噬细胞RAw264.7(M0),予以脂多糖(lipopolysac-charide,LPs)20ng/mL和干扰素-γ(interferon gamma,IFN-γ)50ng/mL诱导M1型极化,白细胞介素4(interleukin,IL-4)20ng/mL诱导M2型极化,采用相差显微镜观察细胞形态学变化,CCK-8法检测细胞活力,实时荧光定量PCR(quantitative real-time,qPCR)检测相关炎症基因表达情况,流式细胞仪检测M1型巨噬细胞膜蛋白CD16/32和M2型巨噬细胞膜蛋白CD206比例变化,westem blot检测核转录因子(nuclear factor-kappa B,NF-κB)表达变化,划痕实验检测细胞迁移能力。结果巨噬细胞予以LPS和IFN-γ诱导后显示M1型极化,表现为轴突细而长;予以IL-4诱导后显示M2型极化,表现为轴突短而小。炎症因子(iNOS、TNF-α、和IL-1β)在M1型巨噬细胞中以及IL-10和YM-1在M2型巨噬细胞中表达显著增加(P<0.05)。氢气对RAw264.7的活力无显著影响。含氢培养基可降低M1型巨噬细胞促炎症因子表达和膜蛋白CD16/32表达下降,核转录因子NF-κB(P65)入核减少,并降低M1型巨噬细胞的迁移能力。但对M2型巨噬细胞的极化及功能无显著影响(P>0.05)。结论含氢培养基减少M1型巨噬细胞炎症因子表达和膜蛋白CD16/32比例,并降低M1型巨噬细胞迁移能力,可能与核转录因子NF-κB(P65)入核减少有关。

Objective To observe the effect of hydrogen-containing medium on the polarization of macrophage RAW264. 7 and to explore the function of macrophages. Methods RAW264. 7 macrophage induced by lipopolysaccharide LPS(20 ng/mL) and IFN-γ(50 ng/mL) or by IL4(20 ng/mL) were cultured in medium with or without hydrogen. The cell viability was measured through cell counting kit-8 ( CCK-8 ) assay. The markers of macrophages were assayed with flow cytometry. The levels of inflammatory cytokines were determined by quantitative PCR( qPCR). The proteins of nuclear transcription factor-κB( NF-κB ) was detected by western blotting. The migration capacity of macrophages was measured with wound scratch assay. Results Macrophages were polarized to Ml induced by LPS and IFN-γ. The axons were small and elongated. Macrophages were polarized to M2 induced by IL-4,which contained large axons. The mRNA levels of iNOS,TNF-a,and IL-1β in M1 were significantly up-regulated. The mRNA levels of Arg-1 ,IL-10,and YM-1 in M2 were also significantly up-regulated( P<0.05). After treatment with hydrogen, the cell viability was not affected, while the levels of inflammatory factors and the markers of macrophsge CD16/32 were significantly lower than that of the control cells. NF-κB (p65) was de-creased significantly as compared to the control cells. The migration ability of Ml macrophages was inhibited. However, the polarization and function of M2 macrophages was not affected by hydrogen (P>0.05). Conclusion The present study showed that hydrogen could inhibit Ml polarization. The levels of inflammatory factors secreted by Ml macrophages and the percentage of CD16/32 were decrease after treatment with hydrogen. The migration ability of Ml may be associated with the levels of NF-κB(p65) in the nucleus.