Effect of Pimozide on the Regulation of Inducible Nitric Oxide Synthetase and Its Mechanism in RAW264.7 Cells Induced by Lipopolysaccharide
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 10 μmol·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+2.5 μmol·L-1),middle(LPS+5 μmol·L-1)and high(LPS+10 μmol·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+2.5 μmol·L-1),medium(LPS+5 μmol·L-1)and high levels(LPS+10 μmol·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 related to the inhibition of signal transduction and the phosphorylation of activator of transcription-5 pathway.
PimozidelipopolysaccharideRAW264.7 cellsignal transducer and activator of transcription pathwayinducible nitric oxide synthase
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