类胰蛋白酶通过PAR2激活JAK-STAT通路促进小鼠骨髓来源的巨噬细胞向M1表型转换

Tryptase transforms murine bone marrow derived macrophages to M1 macrophages subtype by activating JAK-STAT pathway through PAR2 in mice

目的研究类胰蛋白酶促进小鼠巨噬细胞表型转换的作用及其机制。方法体外分离培养小鼠巨噬细胞(对照组),并诱导为M1和M2亚型,经类胰蛋白酶和PAR2激动剂分别作用后,通过real-time PCR和Western blot检测巨噬细胞亚型标志物以及JAK-STAT信号通路的变化。结果成功分离培养小鼠骨髓来源的巨噬细胞,并诱导其分化为M1和M2亚型。PAR2激动剂(2-Furoyl-LIGRLO-amide)和类胰蛋白酶分别与M1型巨噬细胞分别作用后,M1型标记物iNOS和IL-12高于对照组,而M2型标记物arg1和mrc1与对照相比无明显变化,提示类胰蛋白酶和PAR2激动剂可以促进巨噬细胞向M1型转化,而不会促进M1型向M2型转化。PAR2激动剂和类胰蛋白酶分别与M2型巨噬细胞作用后,M2型标记物arg1和mrc1明显下降,而M1型标记物iNOS和iL-12有所增加,提示类胰蛋白酶和PAR-2激动剂均可促进M2型向M1型转化。类胰蛋白酶作用于M1型巨噬细胞后,JAK2磷酸化水平、STAT和p-STAT的表达水平均无明显变化。类胰蛋白酶作用于M2型巨噬细胞后,JAK2的磷酸化随着时间延长明显升高,STAT的磷酸化水平同时升高,提示类胰蛋白酶可以通过和PAR-2结合而激活JAK2-STAT信号通路,进而引起M2型向M1型转化。结论类胰蛋白酶可能通过与其受体PAR2相互作用后激活JAK-STAT通路,促进小鼠骨髓来源的巨噬细胞向M1亚型转化,起到促进炎症发展的作用,从而进一步加重动脉粥样硬化的发展。

Objective To investigate the effects and mechanism of tryptase in macrophages phenotype transformation in mice. Methods The molecular markers of macrophages subtype were detected by real - time PCR , and the activation of JAK - STAT pathway was detected by Western blot. Results The murine bone marrow derived macrophages were isolated and induced to M1 or M2 subtype successfully. After PAR2 agonist （2-Furoyl-LIGRLO-amide） or tryptase were added into M1 macrophages, the expression levels of the M1 markers iNOS and IL-12 were increased obviously, while the M2 markers arg 1 or mrcl had no obvious change. It suggested that tryptase and PAR2 agonist promoted macrophages differentiating to M1, but did not promote the transformation from M1 to M2. On the other hand, after adding PAR2 agonist or tryptase into M2 macrophages, the expression levels of the M2 markers argl and mrcl were decreased obviously, meanwhile the expression level of M1 markers iNOS and IL-12 increased . It indicated that tryptase or PAR-2 agonists promoted M2 transformating to M1. The expression levels of p-STAT , STAT or JAK2 were not change after tryptase incubated with M1 macrophages. However, incubated with tryptase, the phosphorylation levels of JAK2 and STAT were significantly increased in M2 macrophages, which indicated that tryptase may activated the JAK2-STAT signaling pathway by binding to PAR-2, and then resulted in the conversion M2 to M1 macrophages. Conclusions Tryptase may activate JAK-STAT pathway through the interaction with its receptor PAR2, which results in the murine bone marrow derived macrophages differentiating to M1. This effect plays a role in promoting the development of inflammation, further aggravates the development of atherosclerosis.