细胞色素酶P4501A1对巨噬细胞向M2型极化的调控作用

Regulation of cytochrome P4501A1 on M2 macrophage polarization

目的探讨细胞色素酶P4501A1(CYP1A1)对巨噬细胞向M2型极化的调控作用及其分子机制。方法所有实验采用完全随机设计。①实验1:选择6~8周龄健康雄性C57BL/6J小鼠,提取原代腹腔细胞,将细胞分为磷酸盐缓冲液(PBS)对照组和白细胞介素-4(IL-4)组。IL-4组用10 mg/L的M2型巨噬细胞诱导剂IL-4刺激细胞;PBS对照组给予等量PBS。采用定量反转录-聚合酶链反应(RT-qPCR)检测IL-4刺激2、4、6 h后细胞内M2型极化标志分子精氨酸酶-1(Arg-1)、几丁质酶3样蛋白1(YM1)及CYP1A1的mRNA表达;采用蛋白质免疫印迹试验(Western Blot)检测IL-4刺激6、12、24 h后细胞内酪氨酸蛋白激酶1/信号转导和转录激活因子6(JAK1/STAT6)通路磷酸化及CYP1A1、Arg-1蛋白表达。②实验2:体外培养CYP1A1高表达的RAW264.7细胞(CYP1A1/RAW)及其阴性对照细胞(NC/RAW),取对数生长期细胞分为PBS对照组和IL-4组,处理方法同实验1。采用Western Blot法检测IL-4刺激1 h、2 h后不同细胞内JAK1/STAT6及磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)通路磷酸化;同时采用RT-qPCR及Western Blot法检测IL-4刺激2 h、4 h后细胞内通路下游产物Arg-1的mRNA和蛋白表达。结果①实验1:经IL-4刺激2 h后,小鼠原代腹腔巨噬细胞内Arg-1、YM1的mRNA表达即较PBS对照组明显增加〔Arg-1 mRNA(2-ΔΔCt):1.75±0.82比1.00±0.21,YM1 mRNA(2-ΔΔCt):2.58±0.53比1.00±0.20,均P<0.05〕,说明IL-4诱导巨噬细胞向M2型极化成功;同时,极化的小鼠原代腹腔巨噬细胞内CYP1A1的mRNA表达也较PBS对照组明显增加,4 h达峰值〔CYP1A1 mRNA(2-ΔΔCt):2.25±0.69比1.00±0.17,P<0.01〕,说明巨噬细胞向M2型极化时CYP1A1表达增强。IL-4刺激6 h后,小鼠原代腹腔巨噬细胞内磷酸化JAK1(p-JAK1)、磷酸化STAT6(p-STAT6)、Arg-1及CYP1A1的蛋白表达即较PBS对照组明显增强,以12 h更为显著,之后逐渐减弱,说明CYP1A1高表达时M2型巨噬细胞内JAK1/STAT6通路磷酸化增强,通路被激活。②实验2:经IL-4刺激2 h后,CYP1A1/RAW细胞内Arg-1 mRNA表达即较NC/RAW细胞明显增加(2-ΔΔCt:3.02±0.60比1.47±0.43,P<0.05),蛋白表达也较NC/RAW细胞明显增强,以4 h最为显著,说明CYP1A1可促进巨噬细胞向M2型极化。IL-4刺激2 h后,两种细胞内p-JAK1、p-JAK3蛋白表达均较PBS对照组明显增强,但CYP1A1/RAW细胞内p-STAT6的增强幅度较NC/RAW细胞更加明显,说明CYP1A1通过促进JAK1/STAT6通路磷酸化来促进巨噬细胞极化;同时,CYP1A1/RAW细胞内PI3K/Akt通路下游分子Akt蛋白表达较NC/RAW细胞明显减弱,说明CYP1A1不通过该通路促进巨噬细胞极化。结论CYP1A1可促进巨噬细胞向M2型极化,其机制与促进JAK1/STAT6通路磷酸化有关。

Objective To investigate the potential effects of cytochrome P4501A1(CYP1A1)in regulating macrophages polarize to M2 type and explore the molecular mechanism.Methods All trials were completely randomized.①Experiment 1:6-8 weeks old healthy male C57BL/6J mice were collected,and primary peritoneal cells were extracted,then the cells were divided into phosphate buffered saline(PBS)group and interleukin-4(IL-4)group.The cells in the IL-4 group were stimulated with 10 mg/L IL-4(M2 macrophage inducer);and those in the PBS group were given with an equal amount of PBS.The mRNA expressions of intracellular M2 type polarized marker molecules including arginase-1(Arg-1)and chitinase 3 like protein 1(YM1)at 2,4,6 hours after IL-4 challenge were determined by quantitative reverse transcription-polymerase chain reaction(RT-qPCR).The phosphorylation of tyrosine protein kinase 1/signaling transcriptional and transduced activator 6(JAK1/STAT6)signaling pathway and protein expressions of CYP1A1 and Arg-1 at 6,12,24 hours after IL-4 challenge were determined by Western Blot.②Experiment 2:RAW264.7 cells with high expression CYP1A1(CYP1A1/RAW)and their negative control cells(NC/RAW)were cultured in vitro.The cells in logarithmic growth phase were collected,and then they were divided into PBS control group and IL-4 group.The treatment method was the same as experiment 1.The phosphorylations of intracellular JAK1/STAT6 and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)pathways in different cells at 1 hour and 2 hours after IL-4 challenge were determined by Western Blot.The mRNA and protein expressions of Arg-1 in different cells at 2 hours and 4 hours after IL-4 challenge were determined by RT-qPCR and Western Blot,respectively.Results①Experiment 1:after IL-4 challenge for 2 hours,the mRNA expressions of Arg-1 and YM1 in the primary peritoneal macrophages of mice were significantly increased as compared with the PBS control group[Arg-1 mRNA(2-ΔΔCt):1.75±0.82 vs.1.00±0.21;YM1 mRNA(2-ΔΔCt):2.58±0.53 vs.1.00±0.20,both P<0.05]which indicated that IL-4 induced macrophage to M2 type polarization successfully.Meanwhile,the mRNA expression of CYP1A1 in polarized mouse peritoneal primary macrophages was also elevated as compared with the PBS control group,and peaked at 4 hours[CYP1A1 mRNA(2-ΔΔCt):2.25±0.69 vs.1.00±0.17,P<0.01].The results indicated that CYP1A1 expression was enhanced when macrophages polarized to M2 type.Compared with the PBS control group,the bands of phosphorylated JAK1(p-JAK1),phosphorylated STAT6(p-STAT6),Arg-1 and CYP1A1 were enhanced in primary peritoneal macrophages of mice in the IL-4 group,and reached the peak value at 12 hours then gradually decreased.This result indicated that the phosphorylation of JAK1/STAT6 pathway was enhanced in M2 macrophages with high expression of CYP1A1,and the pathway was activated.②Experiment 2:after IL-4 challenge for 2 hours,the expression of Arg-1 mRNA in CYP1A1/RAW cells was significantly higher than that in NC/RAW cells(2-ΔΔCt:3.02±0.60 vs.1.47±0.43,P<0.05),and the protein band signal was also stronger,and both peaked at 4 hours.This indicated that CYP1A1 could promote the polarization of macrophages to M2.After IL-4 challenge for 2 hours,the expression of p-JAK1 and p-JAK3 protein bands in both cells were significantly enhanced as compared with the PBS control group,but the enhancement of p-STAT6 band in CYP1A1/RAW cells was stronger than that of NC/RAW cells,indicating that CYP1A1 promoted macrophage polarization by promoting phosphorylation of the JAK1/STAT6 pathway.In the meantime,the protein band of Akt,a downstream protein of the PI3K/Akt pathway,in CYP1A1/RAW cells was significantly lower than that of NC/RAW cells,indicating that CYP1A1 did not promote macrophage polarization through this pathway.Conclusions CYP1A1 promotes the polarization of macrophage to M2 type.The mechanism is related to promoting the phosphorylation of JAK1/STAT6 pathway.