芪玉三龙汤调节小鼠腹腔巨噬细胞M2/M1型极化及其对CD4^(+)、CD8^(+)T细胞的影响

Role of Qiyu Sanlong Decoction in Regulating Mouse Peritoneal Macrophage M2/M1 Polarization and Its Effect on CD4^(+)and CD8^(+)T Cells

目的探讨芪玉三龙汤在体外条件下对小鼠腹腔巨噬细胞M2/M1型极化的调控作用及其对CD4^(+)T、CD8^(+)T细胞的影响。方法分离培养小鼠腹腔巨噬细胞,并通过流式细胞术(PE-CD11b/和FITC-F4/80染色)鉴定其纯度。采用白细胞介素(interleukin,IL)-4刺激小鼠腹腔来源的巨噬细胞构建M2型巨噬细胞模型,芪玉三龙汤处理M2型巨噬细胞,分别于不同时间点应用流式细胞术检测细胞表面膜分子CD16/32、CD206、CD155、PD-L1、Galectin-9的表达水平;qRT-PCR检测诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、IL-12p40、IL-1β、精氨酸酶1(arginase1,Arg1)、FIZZ1、IL-10 mRNA的表达水平。采用芪玉三龙汤处理后的M2型巨噬细胞分别与CD4^(+)、CD8^(+)T细胞体外共培养,流式细胞术检测TIGIT的表达水平。结果成功分离出小鼠腹腔巨噬细胞,并鉴定其纯度高达89.8%。芪玉三龙汤处理M2型巨噬细胞后,细胞表面膜分子CD16/32的表达水平升高,而CD206表达水平降低;处理24 h后细胞iNOS、IL-12p40及IL-1βmRNA表达水平上升,Arg-1、FIZZ-1、IL-10 mRNA表达水平降低;处理24 h后细胞表面CD155、PD-L1、Galectin-9表达水平降低。经芪玉三龙汤处理的M2型巨噬细胞分别与CD4^(+)、CD8^(+)T淋巴细胞共培养后,CD4^(+)、CD8^(+)T细胞表面TIGIT的表达受到明显抑制。结论芪玉三龙汤能够诱导小鼠腹腔M2型巨噬细胞向M1型极化,并且极化后的细胞能够抑制T细胞负性调节分子的表达。

Objective To investigate the regulatory role of Qiyu Sanlong Decoction in mouse peritoneal macrophage M2/M1 polarization and its effect on CD4^(+)T cells and CD8^(+)T cells.Methods Mouse peritoneal macrophages were isolated and cultured,and flow cytometry was used to identify purity.Mouse peritoneal-derived macrophages were stimulated by IL-4 to establish an M2 macrophage model,and then M2 macrophages were treated by Qiyu Sanlong Decoction;flow cytometry was used to measure the expression levels of the cell surface membrane molecules CD16/32,CD206,CD155,PD-L1,and Galectin-9 at different time points,and qRT-PCR was used to measure the expression levels of inducible nitric oxide synthase(iNOS),IL-12p40,IL-1β,arginase-1(Arg 1),FIZZ1,and IL-10.M2 macrophages treated by Qiyu Sanlong Decoction were co-cultured with CD4^(+)and CD8^(+)T lymphocytes,respectively,in vitro,and then flow cytometry was used to measure the expression level of TIGIT,an immunosuppressive molecule on the surface of T cells.Results Mouse peritoneal macrophages were successfully isolated with a purity as high as 89.8%.After M2 macrophages were treated by Qiyu Sanlong decoction,there was a continuous increase in the expression level of the cell surface membrane molecule CD16/32 and a reduction in the expression level of CD206,and after 24 hours of treatment,there were increases in the mRNA expression levels of iNOS,IL-12 p40,and IL-1βand reductions in the mRNA expression levels of Arg1,FIZZ1,and IL-10,as well as reductions in the expression levels of CD155,PD-L1,and Galectin-9 molecules on cell surface.After M2 macrophages treated by Qiyu Sanlong Decoction were co-cultured with CD4^(+)and CD8^(+)T lymphocytes,respectively,the expression of TIGIT on the surface of CD4^(+)and CD8^(+)T cells were significantly inhibited.Conclusion Qiyu Sanlong Decoction can induce M1 polarization of mouse peritoneal M2 macrophages,and the polarized cells can inhibit the expression of negative regulatory molecules in T cells.