Neutrophil Granule Protein Positively Regulates Nitric Oxide Production In Lipopolysaccharide-induced Macrophages By The STAT1/iNOS Pathway

Backgrounds:Sepsis(sepsis)is an uncontrolled and long-lasting inflammatory response produced when the host is infected,resulting in immune disorders and multiple organ dysfunction.The condition is critical and the prognosis is extremely poor.At present,it is generally believed that inflammatory response and disorder of inflammatory cytokine secretion play an important role in the occurrence and development of sepsis.Macrophage(Mj)is an important component of the body’s non-specific immunity,especially in the secretion of inflammatory cytokines.Pro-inflammatory mediators stimulate macrophages to secrete a series of inflammatory mediators such as Nitric Oxide(NO),Interleukin-10(IL-10)and Tumor necrosis factor-α(TNF-α)and so on.NO is not only an important neurotransmitter,it can mediate the transmission of nerve information,regulate vasomotor function and reduce thrombosis,and it is also an important inflammatory mediator that mediates the immune process and inflammatory response.The role of NO in the inflammatory response is like a "double-edged sword".On the one hand,a large amount of NO is released to help resist the invasion of pathogens;on the other hand,excessive increase of NO may produce cytotoxic effects.Therefore,Fine regulation of NO production is essential in the inflammatory response,but at present,the molecular mechanism regulating NO production is unclear.Neutrophil granule protein(NGP)belongs to the family of cysteine protease inhibitors.Studies have confirmed that cysteine protease inhibitors can extensively participate in multiple links in the inflammatory process by regulating the production of cytokines.The previous work of the research group found that the administration of lipopolysaccharide(LPS)to induce the establishment of an inflammation model,NGP can promote the secretion of the anti-inflammatory factor interleukin-10(IL-10)by macrophages,and inhibit the pro-inflammatory factor leukocytes With the expression of Interleukin-1β(IL-1β)and TNF-α,NGP plays an important role in the inflammatory response process involving macrophages.At the same time,we also found that NGP is closely related to NO production in macrophages,but the role and molecular mechanism of NGP regulating NO production are still unclear.This study intends to use NGP high expression and NGP knockout macrophage cell lines and transgenic mouse peritoneal primary macrophages as the research objects,the LPS stimulation model was constructed to explore the effects of NGP on macrophages’ NO production and its regulation Molecular mechanism.Methods:Part Ⅰ: Effects of NGP on nitric oxide production in lipopolysaccharide-induced macrophages.1.NGP highexpression RAW264.7 cells(NGP/RAW)and negative controlempty vector cells(NC/RAW),NGP knockout RAW264.7 cells(NGP KO/RAW)and wild-type cells(WT/RAW)were cultured in vitro.Establish LPS inflammation model.Western blot was used to detect the expression of NGP and i NOS proteins in the cells.Griess method and q RT-PCR were used to detect the expression of NO and i NOS m RNA in the cell supernatant.2.The primary peritoneal macrophages(PMs)of mice with high expression of NGP and C57BL/6 mice were extracted and cultured in vitro,and the LPS in vitro stimulation model was established.Western blot was used to detect the expression of NGP protein in the cells,and the Griess method and q RT-PCR were used to detect Cellular NO and i NOS m RNA expression.Part Ⅱ: NGP positively regulates nitric oxide production in lipopolysaccharide-induced macrophages by activating the STAT1/i NOS pathway.1.NGP/RAW and NC/RAW were cultured in vitro,LPS stimulation model was established,and Western blot was used to detect the expression of phosphorylated STAT1(p-STAT1)protein in cells.2.The primary peritoneal macrophages(PMs)of NGP high-expressing mice and C57BL/6 mice were extracted and cultured in vitro.Establishment of LPS in vitro stimulation model.Western blot was used to detect the expression of p-STAT1 protein in the cells.3.NGP/RAW and NC/RAW were cultured in vitro,the cells were pre-treated with the inhibitor Fludarabine,and then stimulated with LPS.The Griess method and q RT-PCR were used to detect the expression of NO and i NOS m RNA in the supernatant of the cells.Results:Part Ⅰ: NGP up-regulates LPS-induced NO production in macrophages.1.Compared with NC/RAW cells,the expression of NGP protein in NGP/RAW cells was significantly higher.After induction with LPS,the expression of i NOS m RNA and NO in NGP/RAW cells was significantly increased compared with NC/RAW cells.2.Compared with WT/RAW cells,NGP was not expressed in NGP KO/RAW cells.After induction with LPS,the expression of i NOS m RNA and NO in NGP KO/RAW cells was significantly reduced compared with WT/RAWcells.3.In primary peritoneal macrophages,compared with C57BL/6mice,the expression level of NGP protein in peritoneal macrophages of mice with high NGP expression is higher.After induction with LPS,the expression of i NOS m RNA and NO in primary PMs of NGP high-expressing mice was significantly higher than that of C57BL/6 mice.Part Ⅱ: NGP positively regulates nitric oxide production in lipopolysaccharide-induced macrophages by activating the STAT1/i NOS pathway.1.In the RAW264.7 cell line,after LPS induction,the expression of p-STAT1 protein in NGP/RAW cells was significantly increased than that in NC/RAW cells.2.In primary PMs,after induction with LPS,the expression of p-STAT1 protein in primary PMs of NGP high-expressing mice was significantly higher than that of C57BL/6 mice.3.In RAW264.7 cell line,after inhibiting STAT1 phosphorylation,NO production and i NOS expression in NGP/RAW cells decreased significantly,but there was no significant change in NC/RAW cells.Conclusions:1.High expression of NGP increases the NO production of macrophages induced by LPS,and knocking out NGP reduces NO production,suggesting that NGP can positively regulate the level of NO produced in macrophages induced by LPS.2.LPS induces macrophage activation,NGP can up-regulate the phosphorylation level of STAT1 pathway,thereby increasing the expression of i NOS and NO,suggesting that NGP positively regulates the NO production of macrophages induced by LPS through the STAT1/i NOS pathway.