核因子-κB介导非对称性二甲基精氨酸上调大鼠腹腔巨噬细胞诱导型一氧化氮合酶的表达

Asymmetric dimethylarginine induces inducible nitric oxide synthase expression via NF-κB pathway in rat peritoneal macrophages

目的:探讨非对称性二甲基精氨酸(ADMA)上调大鼠腹腔巨噬细胞诱导型一氧化氮合酶(iNOS)的表达是否受核因子-κB(NF-κB)调控。方法:分离培养原代Wistar大鼠腹腔巨噬细胞。分组:①正常对照组:以含10%胎牛血清(FBS)DMEM培养液与巨噬细胞共孵育;②氧化型LDL(oxLDL)组:在培养液中加oxLDL50mg/L;③A+O组:在培养液中加ADMA(15μmol/L)和oxLDL(50mg/L);④P+A+O组:在培养液中加NF-κB特异性抑制剂吡咯烷二硫代氨基甲酸盐(PDTC,25μmol/L)、ADMA(15μmol/L)和oxLDL(50mg/L)。以实时荧光定量PCR和Westen blotting分别检测iNOSmRNA和蛋白表达,以电泳迁移率变动分析(EMSA)和增强化学发光法(ECL)检测NF-κB活性。结果:①与正常对照组相比,oxLDL组与A+O组iNOSmRNA和蛋白表达增强,以A+O组尤为明显,2组比较差异有统计学意义(P<0.05);P+A+O组iNOSmRNA和蛋白表达降低,与A+O组比较差异有统计学意义(P<0.05)。②与正常对照组相比,oxLDL组与A+O组NF-κB活性增强,以A+O组尤为明显,2组相比差异有统计学意义(P<0.05);P+A+O组NF-κB活性降低,与A+O组相比差异有统计学意义(P<0.05)。③相关分析:NF-κB活性与iNOSmRNA和蛋白表达量均呈正相关。结论:ADMA可能通过NF-κB途径增强iNOS表达而促进巨噬细胞转化为泡沫细胞、促进动脉粥样硬化的发生发展。

Objective：To investigate whether asymmetric dimethylarginine（ADMA）upregulated the expression of inducible nitric oxide synthase（iNOS）in rat peritoneal macrophages is modulated by nuclear factor kappa B （NF-κB）pathway.Method：Primary cultured Wistar rat peritoneal macrophages were divided into four groups：① Control group：macrophages incubated in DMEM media with 10%fetal bovine serum（FBS）without any intervention;②oxLDL group：macrophages incubated in DMEM media with 10% FBS and oxidized low density lipoprotein（oxLDL,50mg/L）;③A＋O group：macrophages incubated in DMEM media with 10% FBS,ADMA （15 μmol/L）and oxLDL（50mg/L）;④P＋A＋O group：macrophages incubated in DMEM media with 10% FBS, PDTC（25μmol/L）,ADMA（15μmol/L）and oxLDL（50mg/L）.The mRNA and protein expression of iNOS were detected by real time fluorescent quantitative PCR（real-time PCR）and Westen Blotting respectively.The activity of NF-κB was detected by electrophoretic mobility shift assay（EMSA）and enhanced chemiluminescent （ECL）technique.Result：①Compared with control group,the mRNA and protein expression of iNOS were significantly increased in oxLDL group and A＋O group,especially in A＋O group（P0.05）;the mRNA and protein expression of iNOS in P＋A＋O group were significantly decreased than those in A＋O group（P0.05）.②The NF-κB activity in both A＋O group and oxLDL group was extremely higher than that in control group,especially in A＋O group（P0.05）;and the NF-κB activity in P＋A＋O group was significantly lower than that in A＋O group（P0.05）.③Correlative analysis：The activity of NF-κB was positively correlated with the mRNA and protein expression of iNOS（P0.05）.Conclusion：ADMA upregulates the iNOS expression through the NF-κB activity pathway in rat peritoneal macrophages,which might be one of mechanisms of foam cell formation and atherosclerosis.