| 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-α,IL-1βand IL-10 in LPS-induced NGP/RAW and NC/RAW were measured by qRT-PCR and ELISA;（6）NF-κB,JNK1/AP-1 signaling pathways proteins in LPS-induced NGP/RAW and NC/RAW were compared by Western blot;（7）The binding of NF-κB 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-α,IL-1βlevel,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-κB signaling pathway;（7）LPS induced for 1 h,LSCM showed that NGP overexpression reduced NF-κB translocation,and EMSA showed that NGP overexpression inhibited NF-κB 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-κB signaling pathway and IL-10secretion.Moreover,NGP enhanced the phagocytosis of macrophages to E.coli. |
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