摘要
目的运用脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7细胞株,探究匹莫齐特(Pimozide)对一氧化氮和诱导型一氧化氮合成酶(iNOS)合成的影响和作用机制。方法用含10%胎牛血清、100 U·mL^(-1)的青链霉素DMEM培养液将RAW264.7细胞稀释为每孔2×105个接种于24孔板中进行培养,分为空白对照组(仅含DMEM培养液+RAW264.7细胞)、Pimozide组(仅含RAW264.7细胞和10μmol·L^(-1)Pimozide培养液)、LPS诱导(LPS 1 mg·L^(-1))组(LPS+RAW264.7细胞)、药物处理组[含细胞和不同浓度药物,包括Pimozide低(LPS+2.5μmol·L^(-1))、中(LPS+5μmol·L^(-1))、高(LPS+10μmol·L^(-1))组]。各组培养上清液中一氧化氮水平测定采用Griess法进行检测。采用实时定量聚合酶链反应(RT-PCR)法和免疫蛋白印迹法分别检测iNOS mRNA表达水平和iNOS和磷酸化的信号传导及转录激活因子通路-5的蛋白表达相对水平。结果用含10%胎牛血清、100 U·mL^(-1)的青链霉素DMEM培养液培养RAW264.7细胞24 h后,各组培养上清液一氧化氮表达水平差异有统计学意义(F=25.69,P<0.05);Pimozide低(LPS+2.5μmol·L^(-1))、中(LPS+5μmol·L^(-1))、高(LPS+10μmol·L^(-1))组一氧化氮释放的抑制率差异有统计学意义(F=132.49,P<0.05)。各组iNOS mRNA和蛋白水平表达差异有统计学意义(F=118.59和23.37,P<0.05),同时发现各组磷酸化的信号传导及转录激活因子通路-5/信号传导及转录激活因子通路-5比值差异有统计学意义(F=12.07,P<0.05)。结论Pimozide可抑制RAW264.7细胞中iNOS表达和一氧化氮的生成,其作用机制可能与抑制磷酸化的信号传导及转录激活因子通路-5的生成相关。
Objective To investigate the effects of Pimozide on the synthesis of nitric oxide and inducible nitric oxide synthetase(iNOS)in mouse macrophage RAW264.7 cells induced by lipopolysaccharide(LPS).Methods RAW264.7 cells were diluted to 2×105 cells per well in penicillin DMEM medium containing 10%fetal bovine serum and 100 U·mL^(-1)and inoculated into 24-well plates,they were divided into blank control group(DMEM+RAW264.7 cells only),Pimozide group(RAW264.7 cells only and 10μmol·L^(-1)Pimozide medium),LPS-induced(LPS 1 mg·L^(-1))group(LPS+RAW264.7 cells),drug treatment group[containing cells and different concentrations of drugs,including low(LPS+2.5μmol·L^(-1)),middle(LPS+5μmol·L^(-1))and high(LPS+10μmol·L^(-1))Pimozide groups].The levels of nitric oxide in the culture supernatants were determined by Griess method.Real-time quantitative polymerase chain reaction and western blot were used to detect the expression of iNOS mRNA and the relative levels of inducible nitric oxide synthetase and phosphorylated signal transducer and activator of transcription-5 protein,respectively.Results After RAW264.7 cells were cultured in penicillin DMEM medium containing 10%fetal bovine serum and 100 U·mL^(-1)for 24 hours,the nitric oxide expression levels in the supernatants of each group were different(F=25.69,P<0.05).There was significant difference in the inhibition rate of nitric oxide release among the groups with low(LPS+2.5μmol·L^(-1)),medium(LPS+5μmol·L^(-1))and high levels(LPS+10μmol·L^(-1))of Pimozide(F=132.49,P<0.05).The mRNA and protein levels of inducible nitric oxide synthase in each group were significantly different(F=118.59 and 23.37,P<0.05).It was also found that the ratio of phosphorylated signal transducer and activator of transcription-5/signal transducer and activator of transcription-5 was significantly different among the groups(F=12.07,P<0.05).Conclusion Pimozide can inhibit the expression of inducible nitric oxide synthase and the production of nitric oxide in RAW264.7 cells,which may be
作者
刘佳
LIU Jia(Department of Blood Transfusion,the Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《河南医学研究》
CAS
2024年第7期1175-1180,共6页
Henan Medical Research
