The Regulatory Effects And Preliminary Mechanism Of RHP-NAP On The Polarization And Metabolism Of Macrophages

Background: Helicobacter pylori is one of the common infectious pathogens,it can induce chronic inflammation,and is the causative factor of duodenal ulcer,gastric ulcer and stomach cancer.In China,the infection rate of the general population is relatively high,reaching 50 % ~ 80 %,and still increasing at a rate of 1 % ~ 2 %.Helicobacter pylori infection can induce a strong immune response,but generally does not cause bacterial clearance.The invasion of Helicobacter pylori into gastric epithelial cells can induce a variety of host signal transduction events,and its virulence factors can also cause a variety of biological responses.The pathogenesis and disease outcome of Helicobacter pylori is mediated by complex interactions between bacterial virulence factors,host and environmental factors.The relationship between bacterial virulence factors and Helicobacter pylori infection has been extensively studied.For example,virulence factors Cag A,cytotoxin A(Vac A)and Helicobacter pylori neutrophil activating protein(HP-NAP),which regulate cell proliferation,movement,polarity,regulate the phenotype of host cells and induce inflammation reaction etc.The virulence factor Helicobacter pylori neutrophil activating protein(HP-NAP)can stimulate neutrophils to produce hyperoxia free radicals,leading to local tissue damage.In addition to stimulating the production of reactive oxygen species,HP-NAP also induces the expression and release of MIP-1α(also known as CCL3)and MIP-1β(also known as CCL4).It has been reported that HP-NAP is also involved in the adhesion of Helicobacter pylori to host cells and is involved in the induction of the production of various inflammatory cytokines,which also may be involved in the induction of chronic inflammation.It is seen that HP-NAP has important role in the process of Helicobacter pylori infection.In response to various signals from the extracellular environment,macrophages can be polarized into different subtypes,exhibiting different phenotypic,receptor and cytokine secretion patterns.Macrophages activated by IFN-γ or the like,were called M1 type macrophages,play a pro-inflammatory function,and macrophages activated by Th2 type cytokines etc,were called M2 type macrophages,and have antiinflammatory functions.During H.pylori infection,macrophages play an important role in the host’s defense against bacterial infections and regulation of inflammation.During HP infection,macrophages are central regulators of the immune response,promoting a high load of HP rather than promoting bacterial clearance.Related studies have shown that the expression of M1 and M2 markers are elevated in the antrum of patients with HP-infected uncomplicated gastritis,and in patients with atrophic gastritis,M1 polarization is enhanced,such as highly expressed i NOS.This reveals that macrophage polarization is a complex process in HP infection,and how HP infection affect macrophages polarization remains to be studied.The polarization of macrophages is closely related to cellular metabolism,and cell fate and function are regulated by metabolism.Metabolic processes such as glycolysis,tricarboxylic acid cycle and fatty acid metabolism have a highly specific effect on macrophage function,and metabolic reprogramming drives specific effector functions in macrophages.The interaction between metabolic pathways,metabolites and their intracellular signaling cascades in turn affects cellular transcription events,leading to different polarization states of macrophages.There is evidence that metabolic enzymes and regulatory factors play a direct role in regulating macrophage function.It can be seen that the metabolic changes of macrophages play an important role in inducing the polarization of macrophages.Based on the unique immunological and biological properties of HP-NAP,and the complex polarization state of macrophages in HP infection,the virulence factorHP-NAP is highly likely to participate in the progression of HP infection by affecting the polarization of macrophages.This study will investigate the effects of HP-NAP on the polarization of RAW264.7 macrophages,and explore the metabolic reprogramming of inducing macrophage polarization,to investigate whether and how HP-NAP regulates macrophage polarization will provide an important theoretical basis for the role of HP-NAP in the pathogenesis of diseases caused by H.pylori.Methods: 1.The prokaryotic expression system was used to express and purify the r HPNAP protein and remove endotoxin.2.CCK-8 assay was used to detect the cell viability of RAW264.7 macrophages.3.Using the Griess method to detect the change of NO content in RAW264.7 macrophage culture medium.4.ELISA was used to detect the expression of TNF-α in RAW264.7 macrophage culture medium.5.Flow cytometry was used to detect the expression of CD86 and membrane transporter(GLUT1),and to detect the phagocytic ability of RAW264.7 macrophages.6.The expression of macrophage M1 cytokines(i NOS,TNF-α,IL-1β)and M2 cytokines(Arg-1,IL-10),the relative metabolic enzymes and transcription factors of glycolytic pathway,pentose phosphate pathway and fatty acid decomposition pathway(HK,LDH,u-PFK-2,PKM,PDK,G6 PD,CPT1,ATGL,HSL,CD36)were detected by real-time quantitative PCR(q RT-PCR).7.Using micro-determination to detect the content of lactic acid(LA),coenzyme II(NADPH),free fatty acid(FFA)and the activity of hexokinase(HK).8.Western blot was used to detect the phosphorylation of ERK,JNK,p38 MAPK signaling pathway and the expression of transcription factors HIF1α,c-Myc and PPARγ.9.Immunofluorescence staining was used to detect the translocation of NF-κB p65 into nucleus in RAW264.7 macrophages.Results: 1.We successfully expressed and purified the protein of rHP-NAP.2.rHP-NAP significantly increased the phagocytic capacity of RAW264.7 macrophages,promoted the secretion of NO and TNF-α protein,and increased the expression of M1 cytokines(i NOS,TNF-α and IL-1β)and molecular Marker CD86,had no significant effect on the expression of M2 type cytokines(Arg-1 and IL-10),so that rHP-NAP induced M1 macrophage polarization.3.rHP-NAP activated the MAPK signaling pathway of macrophages,promoted the translocation of NF-κB p65 into the nucleus and increased the transcriptional activity of NF-κB.4.rHP-NAP increased the expression of glucose transporter(GLUT1)protein,increased the relative expression of glycolysis and pentose phosphate pathway-related metabolic enzymes and membrane receptors,and increased the activity of the glycolytic pathway rate-limiting enzyme hexokinase(HK),promoted the production of lactic acid(LA)and NADPH.5.rHP-NAP significantly reduced the content of free fatty acids(FFA)and decreased the relative expression of lipid catabolic hydrolase ATGL and transporter CPT1.6.rHP-NAP up-regulated the expression of transcription factors HIF1α and cMyc,and inhibited the expression of PPARγ.7.Blocking the glycolytic pathway reduces the expression of M1 marker i NOS,TNF-α,IL-1β and CD86,decreases the phosphorylation of ERK in RAW264.7 macrophages.Conclusion: This study found that rHP-NAP induces M1 polarization of macrophages by activating MAPK and NF-κB signaling pathways.We also found that rHP-NAP enhances the glycolytic pathway and the pentose phosphate pathway by up-regulating the expression of HIF1α and c-Myc,reduces the lipid catabolic pathway by inhibiting the expression of PPARγ,triggering macrophages metabolic reprogramming and thus regulating the polarization of macrophages.This reveals that the virulence factor HPNAP can induce inflammatory response though inducing macrophage polarization in HP infection.