Changes Of NGP Expression In Inflammatory Macrophages And Its Effect On Proinflammatory Mediators And Phagocytic Function

Objective:To study the changes of Neutrophilic granule protein?NGP?expression in mice peritoneal macrophages?PMs?under E.coli infection and lipopolysaccharide?LPS?-stimulated inflammation model,and to explore the effects of NGP overexpression on inflammatory mediators generation and phagocytosis in macrophages.Methods:?1?In vivo,WT mice were intraperitoneally injected with E.coli or LPS?in vitro:isolated PMs were stimulated with heat-killed E.coli or LPS?,and the dynamic changes of NGP mRNA in PMs were observed by qRT-PCR;?2?In vivo,WT mice and TLR4^-/-mice were intraperitoneally injected with E.coli or LPS(in vitro:isolated WT mice PMs and TLR4^-/-mice PMs were stimulated with heat-killed E.coli or LPS),and qRT-PCR was used to detect NGP mRNA expression in PMs;?3?The expression of the NGP mRNA in the overexpressed RAW264.7?NGP/RAW?and the negative control RAW264.7?NC/RAW?was detected by qRT-PCR,and the cell viability of LPS-induced NGP/RAW and NC/RAW was measured by MTT;?4?TLR4 mRNA and protein in LPS-induced NGP/RAW and NC/RAW were compared by qRT-PCR and Western blot;?5?TNF-?,IL-1?and IL-10 in LPS-induced NGP/RAW and NC/RAW were measured by qRT-PCR and ELISA;?6?NF-?B,JNK1/AP-1 signaling pathways proteins in LPS-induced NGP/RAW and NC/RAW were compared by Western blot;?7?The binding of NF-?B and DNA in LPS-induced NGP/RAW and NC/RAW was analyzed by EMSA,and images of phosphorylated p65 and p50 expression patterns were observed by laser scanning confocal microscope?LSCM?;?8?The phagocytosis of NGP/RAW and NC/RAW was compared by detection of bacterial burden.Results:?1?In vivo,PMs NGP mRNA in mice with E.coli or LPS challenge groups was significantly higher than that of negative control group,and the difference was statistically significant?P<0.05?;In virto,heat-killed or LPS-induced isolated PMs NGP mRNA was significantly increased?P<0.05?;?2?In vivo injected with E.coli or LPS,compared with WT mice,NGP mRNA in PMs of TLR4-/-mice was significantly reduced,P<0.05;In virto heat-killed E.coli or LPS induced PMs,TLR4-/-PMs had lower NGP mRNA when compared with WT PMs,P<0.05;?3?The expression of NGP mRNA in NGP/RAW was significantly higher than that in NC/RAW,P<0.05;MTT showed that compared with NC/RAW,NGP overexpression had no obvious effect on cell viability,and the LPS had no effect on both group cell viability,P>0.05;?4?qRT-PCR and Western blot showed that NGP overexpression promoted TLR4 mRNA and protein expression,P<0.05;?5?Compared with LPS-induced NC/RAW,LPS-induced NGP/RAW had lower TNF-?,IL-1?level,P<0.05,and higher IL-10 expression P<0.05;?6?For JNK/AP1 signaling pathway,there is no significant difference in the expression of fos,p-fos,jun,p-jun,JNK1,and p-JUK1between LPS-induced NC/RAW and NGP/RAW,P>0.05,but p-p65 and p-p50 were lower in NGP/RAW for NF-?B signaling pathway;?7?LPS induced for 1 h,LSCM showed that NGP overexpression reduced NF-?B translocation,and EMSA showed that NGP overexpression inhibited NF-?B binding to DNA,P<0.05;?8?The bacterial burden experiment confirmed that NGP/RAW had stronger ability to phagocytose E.coli than NC/RAW,P<0.05?Conclusion:The high expression of NGP depended on TLR4,NGP overexpression promoted the expression of TLR4,a positive feedback loop existed between NGP and TLR4 in response to E.coli or LPS stimulation.NGP played a critical role in the anti-inflammatory response by targeting the NF-?B signaling pathway and IL-10secretion.Moreover,NGP enhanced the phagocytosis of macrophages to E.coli.