脂多糖通过上调CYP1A1环氧化酶活性抑制巨噬细胞精氨酸酶-1的表达

LPS inhibits arginase-1 expression in macrophages by up-regulating activity of cytochrome P4501A1 epoxidase

目的探索细胞色素P4501A1(cytochrome P4501A1,CYP1A1)对脂多糖(lipopolysaccharide,LPS)诱导时巨噬细胞中精氨酸酶-1(Arginase-1,Arg-1)的调控作用及机制。方法使用LPS刺激小鼠单核巨噬细胞细胞系RAW264.7(RAW),分为0 h组、12 h组、24 h组(每组设3复孔,n=3),采用实时荧光定量PCR(RT-qPCR)及蛋白免疫印迹实验(Western blot)检测CYP1A1、Arg-1 mRNA及蛋白表达;构建阴性对照和高表达、敲除CYP1A1的RAW(NC/RAW、CYP1A1/RAW、CYP1A1 KO/RAW)细胞系以及突变CYP1A1羟化酶活性的RAW(CYP1A1m/RAW)细胞系,分为NC/RAW+LPS组、CYP1A1 KO/RAW+LPS组、CYP1A1/RAW+LPS组、CYP1A1m/RAW+LPS组,使用LPS刺激后检测Arg-1 mRNA、蛋白表达及信号转导和转录活化因子6(signal transducer and activators of transcription-6,STAT6)活化水平;使用STAT6抑制剂AS1517499作用CYP1A1敲除RAW细胞,设立NC/RAW+PBS+LPS组、NC/RAW+AS1517499+LPS组、CYP1A1 KO/RAW+PBS+LPS组、CYP1A1 KO/RAW+AS1517499+LPS组,检测Arg-1 mRNA、蛋白表达;使用CYP1A1环氧化酶活性抑制剂甲二氢愈创木酸(Nordihydroguaiaretic Acid,NDGA)及12(S)-羟基二十碳四烯酸[12(S)-HETE]作用RAW细胞,设立RAW+PBS+LPS组、RAW+12(S)-HETE+LPS组、RAW+NDGA+LPS组,检测Arg-1 mRNA、蛋白表达及STAT6活化水平。结果LPS可诱导RAW中的CYP1A1及Arg-1呈错峰高表达(P<0.05);CYP1A1高表达可抑制LPS诱导时RAW中Arg-1表达(P<0.05)及STAT6活化,敲除则可增强上述效应(P<0.05),而STAT6抑制剂AS1517499可消除CYP1A1敲除带来的增强效应(P<0.05);突变CYP1A1的羟化酶活性或使用其代谢产物12(S)-HETE刺激均不影响Arg-1表达(P>0.05),而使用CYP1A1环氧化酶抑制剂NDGA可增强Arg-1表达(P<0.05)及STAT6活化。结论CYP1A1可通过抑制LPS诱导时巨噬细胞中STAT6活化水平以降低Arg-1表达,但此效应与CYP1A1羟化酶活性及其致炎产物12(S)-HETE无关,而可能与CYP1A1环氧化酶活性有关。

Objective To explore the regulative effect and mechanism of cytochrome P4501A1(CYP1A1)on regulating lipopolysaccharide(LPS)-induced arginase-1(Arg-1)expression in macrophages.Methods After mouse macrophage-like cell line RAW264.7(RAW)was treated with 10 mg/L LPS for 0,12 and 24 h,respectively(n=3 for each time point),the expression of CYP1A1 and Arg-1 at mRNA and protein levels were measured by RT-PCR and Western blotting.Negative control RAW(NC/RAW)cells,CYP1A1-overexpression RAW(CYP1A1/RAW)cells,CYP1A1-knockout RAW(CYP1A1 KO/RAW)cells and CYP1A1 hydroxylase-activity mutant RAW(CYP1A1m/RAW)cells were constructed,respectively,and then the cells were treated with LPS and further detected with RT-PCR and Western blotting for the expression and activation levels of Arg-1 and signal transducer and activators of transcription-6(STAT6).Then STAT 6 inhibitor AS1517499 was added to treat the CYP1A1 KO/RAW cells and NC/RAW cells,and the mRNA and protein expression of Arg-1 was measured again after LPS or PBS treatment.CYP1A1 epoxidase inhibitor,nordihydroguaiaretic acid(NDGA)and 12(S)-HETE were introduced to treat normal RAW cells,the mRNA and protein levels of Arg-1 and activity of STAT6 were detected after LPS or PBS treatment.Results LPS-induced CYP1A1 and Arg-1 were highly expressed in staggering peaks in RAW cells(P0.05).Overexpression of CYP1A1 inhibited LPS-induced Arg-1 expression and STAT6 activation in RAW cells,while CYP1A1 knockout enhanced above effects(P0.05).STAT6 inhibitor AS1517499 abolished the enhancement of CYP1A1 knockout(P0.05).CYP1A1 hydroxylase-activity mutation or 12(S)-HETE simulation had no effect on the expression of Arg-1(P0.05),while CYP1A1 epoxidase-activity inhibitor NDGA enhanced Arg-1 expression and STAT6 activation(P0.05).Conclusion CYP1A1 can inhibit Arg-1 expression by reducing STAT6 activation in LPS-induced macrophages,and CYP1A1 hydroxylase-activity or its proinflammatory metabolism 12(S)-HETE has no effect on this regulation,while CYP1A1 epoxidase-activity may be involved in this effect.