安石榴苷对小鼠巨噬细胞生长因子及M1/M2型极化的影响

Effect of punicalagin on macrophage growth factor and M1/M2 polarization in mice

目的探讨安石榴苷(punicalagin,PUN)对小鼠巨噬细胞中生长因子及M1/M2极化的影响。方法将RAW264.7细胞或小鼠原代腹腔巨噬细胞随机分为正常对照组、LPS(脂多糖,10μg/mL)组、PUN(50μmol/L)组、LPS+PUN组。药物作用4、12h后,采用qRT-PCR检测细胞中生长因子(VEGF、PDGFa、PDGFb等)mRNA的变化,以及M1/M2型相关炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、白细胞介素-10(IL-10)、M1/M2标志物诱导性一氧化氮合酶(iNOS)和精氨酸酶1(Arg-1)mRNA表达的动态变化,采用Western blot法检测蛋白表达变化,采用ELISA检测细胞上清中炎症因子TNF-α、IL-6、IL-1β、IL-10的含量。结果在LPS刺激的RAW264.7细胞中,与对照组比较,PUN(50μmol/L)对上述生长因子并无正性调控作用,差异无统计学意义(P>0.05)。LPS刺激后,与对照组比较,M1巨噬细胞标志物iNOS、IL-6、IL-1β、TNF-α等的表达水平升高,差异有统计学意义(P<0.05),表明模型建立成功。与LPS组比较,PUN干预组在RAW264.7细胞或小鼠原代腹腔巨噬细胞中,M1型巨噬细胞标志物iNOS、IL-6等的表达水平均下降,差异有统计学意义(P<0.05),而M2型巨噬细胞标志物Arg-1和IL-10的表达水平上升,差异有统计学意义(P<0.05),Arg-1的蛋白表达量无统计学差异(P>0.05)。结论 PUN对于炎性巨噬细胞中部分生长因子并无正性调控作用,但可促进巨噬细胞从M1型向M2型转化。

Objective To investigate the effect of punicalagin（PUN）on growth factor and M1/M2 polarization of mouse macrophages.Methods RAW264.7 cells or mouse primary peritoneal macrophages were randomly divided into normal control group,LPS（lipopolysaccharide,10μg/mL）group,PUN（50μmol/L）group and LPS＋PUN group.After 4 hand 12 hof drug treatment,to intervene to detect the mRNA expression of growth factors（VEGF,PDGFa,PDGFb,etc.）in RAW264.7 cells by qRT-PCR.Meanwhile,the biomarkers iNOS,Arg-1,and the M1/M2 type correlated inflammatory cytokines,such as TNF-α,IL-6,IL-1βand IL-10 were tested by dynamic changes of mRNA expression,and iNOS,Arg-1 were tested by Western blot.The cytokines TNF-α,IL-6,IL-1βand IL-10 were tested by ELISA assay.Results In LPS-stimulated macrophages,compared with the control group,PUN（50μmol/L）had no positive regulation on the above growth factors,and the difference was not statistically significant（P 0.05）.After stimulation with LPS,the expression of iNOS,IL-6,IL-1βand TNF-α,which represented the M1 macrophage,increased,and the model was successfully established.However,compared with the LPS group,after being treated with PUN,the expressions of iNOS,TNF-αand IL-6 were obviously decreased,and the difference was statistically significant（P 0.05）.While,the expression of Arg I and IL-10,which represented the M2 macrophage,increased and the difference was statistically significant（P 0.05）.However,there was no significant difference in the expression of Arg-1 protein（P 0.05）.Conclusion PUN has no positive regulation on some growth factors in inflammatory macrophages,but it can promote macrophage transformation from M1 to M2.