The Interaction Between ROS And HIF-1? Regulates Helicobacter Pylori-Induced Macrophage Polarization

Background and objective:Helicobacter pylori(H.pylori)is a gram-negative bacterium,which can cause chronic gastritis,peptic ulcer,gastric adenocarcinoma and so on.H.pylori infection can induce the host's innate and adaptive immune response,and macrophages are recruited to the gastric mucosa to produce pro-inflammatory cytokines and chemokines.Macrophages have heterogeneity and plasticity,and can be polarized into different phenotypes.They are not only the essential cells for host defense against pathogens and the primers of adaptive immune response,but also can promote the proliferation and proliferation of cancer cells.At present,many studies have found that H.pylori can affect the polarization of macrophages,and is associated with gastrointestinal diseases and various immune metabolic diseases.Innate immune cells promote reactive oxygen species(ROS)production,which then causes oxidative stress and plays a role in inflammation and tumors.In our previous study,it was found that H.pylori infection can increase the ROS level of gastric epithelial cells and cause DNA damage,and the ROS produced by H.pylori infection can enhance hypoxia inducible factor-1?(HIF-1?)of gastric mucosa.We hypothesized that H.pylori may participate in macrophage polarization through the interaction between ROS and HIF-1?,but the specific mechanism still needs further study.Therefore,this study intends to explore the role and mechanism of ROS/HIF-1? regulating macrophage polarization in the pathogenesis of H.pylori at the cellular level,providing a new theory and basis for the prevention,diagnosis and treatment of H.pylori infection related diseases.Materials and methods:1.Effect of H.pylori infection on macrophage polarization(1)Macrophage RAW264.7 was co-cultured with different multiplicity of infection(MOI)H.pylori for 9h.(2)PCR and ELASA were used to detect the expression of iNOS,CD86 and arg-1,CD206,markers of M1 and M2 macrophages,respectively.2.Effects of H.pylori infection on ROS and HIF-1? in macrophages(1)Different MOI H.pylori was co-cultured with macrophage RAW264.7 for 9 h,and the expression of ROS in macrophages was detected by loading fluorescent probe DCFH-DA.(2)Different MOI H.pylori was co-cultured with macrophage RAW264.7 for 9 h,and the expression of HIF-1? in macrophages was detected by PCR and Western-blot.3.Interaction between ROS and HIF-1? during H.pylori infection(1)Immunohistochemistry was used to detect the expression of HIF-1? in gastric mucosa of H.pylori-infected Balb/c mice.(2)After the macrophages RAW264.7 were treated by NAC,the expression of ROS and HIF-1? was detected.(3)After treatment of macrophage RAW264.7 by YC-1,the expression of ROS and HIF-1? was detected.4.Role of ROS and HIF-1? in H.pylori-induced macrophage polarization(1)After ROS inhibitor NAC treatment of macrophages RAW264.7,the expression levels of M1 macrophage markers iNOS,CD86 and M2 macrophage markers Arg-1 and CD206 were detected by PCR and ELASA,respectively.(2)After HIF-1? inhibitor YC-1 treatment of macrophages RAW264.7,the expression levels of M1 macrophage markers iNOS,CD86 and M2 macrophage markers Arg-1 and CD206 were detected by PCR and ELASA,respectively.Results:1.Effect of H.pylori infection on macrophage polarization(1)With the increase of H.pylori MOI,the expression levels of CD86 and iNOS in M1 macrophages increased gradually.The expression levels of CD86 and iNOS in H.pylori infection group were higher than those in control group(P< 0.05).(2)Compared with the control group,the expression levels of CD206 and Arg-1 in M2 macrophages were increased.In the low MOI(MOI 25,50,100)H.pylori group,CD206 gradually increased,and after reaching a certain level,the expression levels of CD206 and Arg-1 gradually decreased with the increase of MOI(MOI 200 groups compared with MOI 100 group,P<0.01).2.Effects of H.pylori infection on ROS and HIF-1? in macrophages(1)The expression level of ROS in macrophages increased with the increase of H.pylori MOI.(2)Compared with the control group,the expression of HIF-1? in H.pylori infection group was increased(P<0.05),and in MOI 25,50,100 group,HIF-1? expression was gradually increased,while the expression of HIF-1? was decreased in the MOI 200 group(P<0.01).3.Interaction between ROS and HIF-1? during H.pylori infection(1)The expression of HIF-1? in gastric mucosa of H.pylori-infected Balb/c mice increased,and the expression of HIF-1? in NAC-treated group was lower than that in H.pylori-infected group(P<0.001).(2)After treatment of macrophages with ROS inhibitor NAC,the expression of ROS in the NAC-treated group was decreased compared with the control group,and the expression of HIF-1? in the NAC-treated group was inhibited(P<0.05).(3)After treatment of macrophages with HIF-1? inhibitor YC-1,the expression of HIF-1? was inhibited in the YC-1 treated group compared with the control group(P<0.01),and the expression level of ROS was decreased.4.Role of ROS and HIF-1? in H.pylori-induced macrophage polarization(1)After adding NAC treatment,the expression of iNOS and CD86 was inhibited compared with H.pylori infection group(P<0.01),while the expression of Arg-1 and CD206 was increased(P<0.01).This suggests that inhibition of ROS inhibits macrophage polarization toward M1,but promotes macrophage polarization toward M2.(2)Compared with H.pylori infection group,the expression of iNOS,CD86,Arg-1 and CD206 was inhibited after YC-1 treatment(P<0.01).Conclusions:1.H.pylori infection can induce macrophage polarization,and the polarization phenotype of macrophage may be related to the MOI of H.pylori.2.H.pylori infection affects the expression of macrophage ROS and HIF-1?,and the interaction between ROS and HIF-1? regulates H.pylori-induced macrophage polarization.