基于JAK/STAT的栀子苷体外抑制巨噬细胞M2极化作用

Geniposide Inhibiting M2 Polarization of Macrophages in vitro by JAK/STAT

目的:研究栀子苷对巨噬细胞M2极化的影响,并探讨其可能机制。方法:以巨噬细胞系RAW 264.7为研究对象,以不同浓度的栀子苷(0~25μmol·L^-1)预孵后加白细胞介素4(IL-4)/细胞介素13(IL-13)(20 ng·ml^-1)刺激并培养至相应时间点,MTT法检测栀子苷对巨噬细胞的活力,蛋白质印迹法(Western Blot)测定巨噬细胞M2极化标记精氨酸酶1(ARG1)、类几丁质酶3样分子(YM1)以及Janus激酶1(JAK1)/信号转导和转录激活因子6(STAT6)信号通路(p-STAT6、STAT6和p-JAK1)的蛋白表达。实时荧光定量PCR(RT-qPCR)测定巨噬细胞M2极化标记ARG1、炎症区域1(FIZZ1)、YM1、甘露糖受体C1(MRC1)、干扰素调节因子4(IRF4)和巨噬细胞半乳糖-C型凝集素-2(MGL-2)的基因表达。结果:与未用IL-4或IL-13处理相比,L-4或IL-13处理组典型M2标记物ARG1、FIZZ1、YM1、MRC1、IRF-4和MGL-2基因表达升高,ARG1和YM1的蛋白水平升高;相反,与IL-4或IL-13处理组相比,栀子苷预处理显著抑制上述标记物的基因表达,抑制ARG1和YM1的蛋白水平,且呈剂量依赖性(P<0.05或P<0.01)。此外,IL-4处理诱导巨噬细胞JAK-STAT活化,与仅IL-4处理组相比,栀子苷预处理显著抑制巨噬细胞JAK-STAT活化(P<0.05或P<0.01)。结论:栀子苷可能通过抑制JAK-STAT抑制小巨噬细胞M2极化。

Objective:To study the effect of geniposide on M2 polarization of macrophages and explore its possible mechanism.Methods:Macrophage cell line(RAW264.7)was used as the research object.After being pre-incubated with geniposide(0-25μmol·L^-1)at different concentrations,the cells were stimulated with interleukin 4(IL-4)/interleukin 13(IL-13)(20 ng·ml^-1 l)and cultured to the corresponding time points.MTT assay was used to detect the viability of geniposide on macrophages,and the protein expressions of M2 polarized markers arginase 1(ARG1),chitinase 3-like(YM1)and janus kinase 1(JAK1)/signaltrans ducerand activator of transcription 6(STAT6)signaling pathway(p-STAT6,STAT6 and p-JAK1)were determined by Western blot.RT-qPCR was used to determine the expressions of ARG1,found in inflammatory zone 1(FIZZ1),YM1,mannose receptor C type 1(MRC1),interferon regulator factor 4(IRF4)and macrophage galactose type C-type Lectin 2(MGL-2).Results:Compared with those without IL-4 or IL-13,the gene expressions of typical M2 markers ARG1,FIZZ1,YM1,MRC1,IRF4 and MGL-2 in IL-4 or IL-13 treatment group were increased,and ARG1 and YM1 protein levels were increased.In contrast,compared with IL-4 or IL-13 treatment group,geniposide pretreatment significantly inhibited the gene expressions of the above markers,inhibited the protein levels of ARG1 and YM1,and the effects were in a dose-dependent manner(P<0.05 or P<0.01).In addition,IL-4 treatment induced the activation of JAK-STAT in macrophages.Compared with IL-4 treatment group,geniposide pretreatment significantly inhibited the activation of JAKSTAT in macrophages(P<0.05 or P<0.01).Conclusion:Geniposide may inhibit M2 polarization of macrophages by inhibiting JAKSTAT in vitro.