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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。
AIM: To investigate whether asymmetric dimethylarginine (ADMA) up-regulates the expression of inducible nitric oxide synthase (iNOS) in rat peritoneal macrophages under the regulation of NF-κB. Methods: Primary Wistar rat peritoneal macrophages were isolated and cultured. Group: ① normal control group: 10% fetal bovine serum (FBS) DMEM culture medium and macrophage co-incubation; ② oxidized LDL (oxLDL) group: oxLDL50mg / L in the culture medium; ③ A + O group: In the culture medium, ADMA (15μmol / L) and oxLDL (50mg / L) were added; ④P + A + O group: PDTC 25 μmol / L), ADMA (15 μmol / L), and oxLDL (50 mg / L). The expression of iNOS mRNA and protein were detected by real-time fluorescence quantitative PCR and Westen blotting respectively. The activity of NF-κB was detected by electrophoretic mobility shift assay (EMSA) and enhanced chemiluminescence (ECL). Results: ①Compared with normal control group, the expression of iNOS mRNA and protein in oxLDL group and A + O group was enhanced, especially in A + O group, the difference was statistically significant (P <0.05); P + A + O Group iNOS mRNA and protein expression decreased, compared with the A + O group was statistically significant (P <0.05). ② Compared with the normal control group, the activity of NF-κB in oxLDL group and A + O group was enhanced, especially in A + O group, the difference was statistically significant (P <0.05); P + A + O group NF-κB activity decreased, compared with A + O group, the difference was statistically significant (P <0.05). ③ Correlation analysis: NF-κB activity and iNOS mRNA and protein expression were positively correlated. CONCLUSION: ADMA may enhance iNOS expression through NF-κB pathway and promote the conversion of macrophages into foam cells and promote the development of atherosclerosis.