没食子酸对M1型巨噬细胞极化的影响

Effect of gallic acid on polarization of M1 macrophages

目的:体外建立M1型巨噬细胞极化模型,探讨没食子酸(GA)对M1型巨噬细胞极化的影响。方法:选用8周龄C57BL/6雌性小鼠,取腹腔巨噬细胞进行体外原代培养,通过对细胞培养基进行不同的处理,将实验分为对照组(不做处理)、M1极化模型组[加入脂多糖(LPS,100μg·L^(-1))和干扰素γ(IFN-γ,20μg·L^(-1))诱导腹腔巨噬细胞M1型极化]和GA组[在M1极化模型组的基础上,加入GA(37.5 mg·L^(-1))与LPS和IFN-γ共处理腹腔巨噬细胞]。采用倒置显微镜观察细胞形态表现,Annexin Ⅴ-PE/7-AAD双染法检测各组细胞凋亡率,流式细胞术检测F4/80+iNOS+M1型巨噬细胞阳性表达率,实时荧光定量PCR(RT-qPCR)法检测各组细胞中诱导型一氧化氮合酶(iNOS)和白细胞介素1β(IL-1β)mRNA表达水平。结果:倒置显微镜观察,对照组细胞主要呈圆形或椭圆形,M1极化模型组细胞主要表现为梭形;与M1极化模型组比较,GA组可见梭形细胞减少,圆形细胞增多。3组巨噬细胞凋亡率比较差异无统计学意义(P>0.05)。与对照组比较,M1极化模型组中F4/80+iNOS+M1型巨噬细胞阳性表达率升高(P<0.01),GA组中F4/80+iNOS+M1型巨噬细胞阳性表达率升高,但差异无统计学意义(P>0.05);与M1极化模型组比较,GA组中F4/80+iNOS+M1型巨噬细胞阳性表达率明显降低(P<0.01)。与对照组比较,M1极化模型组细胞中iNOS和IL-1βmRNA表达水平升高(P<0.01);与M1极化模型组比较,GA组iNOS和IL-1βmRNA表达水平明显降低(P<0.01),与对照组比较,GA组iNOS和IL-1βmRNA表达水平差异无统计学意义(P>0.05)。结论:GA对巨噬细胞凋亡无明显影响,但可抑制巨噬细胞向M1型的极化。

Objective:To establish the polarization models of the M1 macrophages in vitro,and to investigate the effect of gallic acid(GA)on the polarization of the M1 macrophages.Methods:The eight-week-old C57BL/6 female mice were selected for primary culture of peritoneal macrophages,and the experiment was divided into control group(untreated),M1 polarization model group[adding lipopolysaccharide(LPS,100μg·L-1)and interferon-γ(IFN-γ,20μg·L-1)to induce the M1 polarization of peritoneal macrophages]and GA group[adding GA(37.5 mg·L-1)to co-treat the peritoneal macrophages with lipopolysaccharide and IFN-γon the basis of M1 polarization model group].The morphology of cells was observed by inverted microscope,AnnexinⅤ-PE/7-AAD double dyeing method was used to detect the apoptotic rates in various groups,the positive expression rates of F4/80+iNOS+M1 macrophages were detected by flow cytometry,and the expression levels of inducible nitric oxide synthase(iNOS)and interleukin-1β(IL-1β)in the cells in various groups were detected by Real-time fluorescence quantitative PCR(RT-qPCR)method.Results:The results of inverted microscope showed that the cells in control group were mainly round or oval;in M1 polarization model group,the cells were mainly spindle-shaped;compared with M1 polarization model group,the spindle cells were decreased and the round cells were increased in GA group.There were no significant differences in the apoptotic rate of macrophages between three groups(P>0.05).Compared with control group,the positive expression rate of F4/80+iNOS+M1 macrophages in M1 polarization model group was increased significantly(P<0.01);the positive expression rate of F4/80+iNOS+M1 macrophages in GA group increased,but the difference was not statistically significant(P>0.05).Compared with M1 polarization model group,the positive expression rate of F4/80+iNOS+M1 macrophages in GA group was decreased significantly(P<0.01).Compared with control group,the expression levels of iNOS and IL-1βmRNA in the cells in M1 polarization model group were significantly increased(P<0.01);compared with M1 polarization model group,the expression levels of iNOS and IL-1βmRNA in the cells in GA group were decreased significantly(P<0.01);but there was no significant difference in the expression levels of iNOS and IL-1βmRNA between GA group and control group(P>0.05).Conclusion:GA has no obvious effect on the macrophage apoptosis,but it can inhibit the polarization of macrophages to type M1.