摘要
目的探讨细胞色素P450 1A1(CYP1A1)对脂多糖(LPS)诱导巨噬细胞生成一氧化氮(NO)的影响及作用机制。方法采用10 mg/L的LPS诱导野生型C57BL/6小鼠原代腹腔巨噬细胞(PMs)建立炎症反应模型,检测细胞中CYP1A1的mRNA和蛋白表达。体外培养CYP1A1过表达小鼠巨噬细胞株RAW264.7(CYP1A1/RAW),用10 mg/L的LPS刺激细胞,并设阴性对照细胞株(NC/RAW),检测细胞中激活蛋白-1(AP-1)和诱导型一氧化氮合酶(iNOS)的蛋白与mRNA表达以及细胞上清中NO含量。体外培养RAW264.7细胞,用10 mg/L的LPS和100 nmol/L的12(S)-羟基二十碳四烯酸〔12(S)-HETE〕单独或联合刺激RAW264.7细胞,并设空白对照组,检测细胞中iNOS mRNA表达及细胞上清中NO含量,观察CYP1A1对LPS诱导巨噬细胞生成NO的影响是否与于其代谢产生的12(S)-HETE有关。体外培养CYP1A1过表达同时羟基化酶活性突变的RAW264.7细胞(CYP1A1m/RAW),用10 mg/L的LPS刺激细胞,并设CYP1A1/RAW细胞对照组,检测细胞中iNOS mRNA表达及细胞上清中NO含量,观察CYP1A1羟基化酶活性是否参与CYP1A1对LPS诱导巨噬细胞NO生成的调控。结果与磷酸盐缓冲液(PBS)对照组比较,LPS刺激2 h起小鼠PMs细胞中CYP1A1 mRNA表达即明显升高,并持续至12 h到达峰值〔CYP1A1 mRNA(2-ΔΔCt):6.41±0.98比1.00±0.00,P<0.05〕,6 h CYP1A1蛋白表达也显著增强,并在24 h达高峰,说明LPS诱导巨噬细胞炎症反应时CYP1A1表达水平发生变化。与NC/RAW+LPS组相比,CYP1A1/RAW+LPS组细胞中iNOS mRNA表达及NO的生成均显著增加,分别于12 h和24 h达峰值〔12 h iNOS mRNA(2-ΔΔCt):54.42±8.21比24.22±3.89,24 h NO(μmol/L):66.52±4.09比41.42±2.09,均P<0.05〕,同时iNOS蛋白和AP-1磷酸化亦明显增强,说明CYP1A1可能通过促进AP-1活化和iNOS表达增加,从而使NO生成增多。给予LPS单独或联合12(S)-HETE刺激RAW264.7细胞后,细胞中iNOS mRNA表达和NO的生成并无明显改变,12(S)-HETE+LPS组与LPS组相比差异无统计学意义〔12 h iNOS mRNA(2-
Objective To determine the effects of cytochrome P4501A1(CYP1A1)on nitric oxide(NO)production in lipopolysaccharide(LPS)-induced macrophages and the underlying mechanism.Methods The peritoneal macrophages(PMs)were isolated from healthy C57BL/6 mice and stimulated with 10 mg/L LPS to establish inflammatory response model.The CYP1A1 mRNA and protein expressions in the cells were determined.The mouse macrophages RAW264.7 cell line with CYP1A1 overexpression(CYP1A1/RAW)were cultured in vitro,and they were stimulated by 10 mg/L LPS at logarithmic phase.The negative control-expressed RAW264.7 cells(NC/RAW)were established.The protein and mRNA expressions of activator protein-1(AP-1)and inducible nitric oxide synthase(iNOS)in the cells as well as the content of NO in the cell supernatant were determined.The RAW264.7 cells were cultured in vitro,and they were stimulated by 10 mg/L LPS and 100 nmol/L 12(S)-hydroxyeicosatetraenoic acid[12(S)-HETE]only or in combination at logarithmic phase.The blank control group was set up.The expression of iNOS mRNA in the cells and NO content in the cell supernatant were determined to observe whether the effect of CYP1A1 on LPS induced NO production in macrophages was related to 12(S)-HETE produced by metabolism.The RAW264.7 cells with CYP1A1 overexpression and hydroxylase activity mutation(CYP1A1m/RAW)were cultured in vitro,and they were stimulated by 10 mg/L LPS at logarithmic phase.The CYP1A1/RAW cell control group was set up.The iNOS mRNA expression in the cells and NO content in the cell supernatant were determined to observe the effect of hydroxylase activity of CYP1A1 in regulating NO production in macrophages.Results Compared with the phosphate buffered saline(PBS)control group,the CYP1A1 mRNA expressions were elevated significantly from 2 hours after LPS stimulation and reached a peak at 12 hours[CYP1A1 mRNA(2-ΔΔCt):6.41±0.98 vs.1.00±0.00,P<0.05],while CYP1A1 protein expressions were increased from 6 hours after LPS stimulation and reached a peak at 24 hours,suggesting that
作者
唐欣
陈涛
田李星
梁华平
Tang Xin;Chen Tao;Tian Lixing;Liang Huaping(Department of Intensive Care Unit,the Affiliated Hospital of Zunyi Medical University,Zunyi 563003,Guizhou,China;Department of Wound Infection and Drug,Army Medical Center,Army Medical University,Chongqing 400042,China;Zunyi Medical University,Zunyi 563003,Guizhou,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2020年第5期605-610,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金(81871612)。
