Effect Of IL-22 On Expression Of Inflammatory Factors In Bone-marrow-derived Macrophages

Background & Aims: To investigate the effect of IL-22 on expression of iNOS,IL-1? and TNF-? in bone-marrow-derived macrophages associated inflammatory factors.Method: Bone marrow cells were isolated from the femur of mice,and bone marrow cells were stimulated with macrophage colony-stimulating factor(M-CSF).Bone marrow-derived macrophages(M0 macrophages)were obtained after 7 days of differentiation culture,and Lipopolysaccharide(LPS)was used.Stimulating M0 macrophages to induce M1 type macrophages.Iatex beads were added to bone marrow-derived macrophages culture medium,and the phagocytosis of bone marrow-derived macrophages was observed with a microscope to calculate the phagocytosis rate of bone marrow-derived macrophages.Bone marrow-derived macrophages were treated with different concentrations of 2.5 ng/ml,5 ng/ml,10 ng/ml,20 ng/ml,40 ng/ml,80 ng/ml,160 ng/ml IL-22 for 36 h,and CCK8 assay was used to detect Effect of 22 on the activity of bone marrow-derived macrophages.Flow cytometry was used to detect the cell markers of M0 macrophages and M1 macrophages.Afterinterfering with M1 macrophages with IL-22(20ng/ml)for 36 h,the LPS group was detected by flow cytometry.Markers of M1 macrophages in LPS+IL-22group;IL-1? and TNF-? secretion levels in M1 macrophage culture supernates of LPS group and LPS+IL-22 group were detected by ELISA;IL-M1 macrophages were intervened at 22 h,24h and 36 h at different time points.RT-PCR was used to detect the mRNA expression levels of M1macrophage-associated inflammatory factors iNOS,IL-1? and TNF-? in LPS group and LPS+IL-22 group,respectively.Result: The morphology of M0 macrophages and M1 macrophages was clearly observed by microscopy.After 7 days of differentiation culture,the purity rate of bone marrow-derived macrophages was 99.3±0.4%,and high-purity mature M0-type macrophages were successfully obtained.The phagocytosis rate of bone marrow-derived macrophages was observed by fluorescence microscopy to be 99%,indicating that the induced bone marrow macrophages have normal phagocytic function.After CCK8 was used to detect the activity of bone marrow-derived macrophages,the concentration of IL-22 was 20 ng/ml,which did not affect the viability of bone marrow-derived macrophages.After intervention with IL-22(20ng/ml)for 36 h,the ratio of CD11b+F4/80+iNOS+ cells in LPS+IL-22 group was 68.8.9±2.6%,which was lower than that in LPS group(P<0.01).),the difference is statistically significant.Compared with the LPS group,the LPS+IL-22 group pro-inflammatory cytokine IL-1?(431±47 vs 296±55 pg/ml,t=3.214,P=0.032)and TNF-?(629±74 vs 274±24 pg/ml,t = 7.896,P = 0.001)The secretion level decreased,and the difference was statistically significant.IL-22(20ng/ml)interfered with M1 macrophages at 12 h,24h and 36 h,respectively,and compared with LPS group,proinflammatory cytokines iNOS,IL-1?,TNF-? inLPS+IL-22 group.The mRNA expression levels were decreased at different levels.The expression level of TNF-? mRNA decreased with the prolongation of IL-22(P <0.01).The mRNA expression levels of IL-1? in 24 h and 36 h groups decreased(P <0.05).The mRNA expression levels of iNOS in 24 h and36h groups were also decreased(P <0.05),the difference was statistically significant,while the mRNA expression level of iNOS 12 h group was increased(P > 0.05),the difference was not statistically significant.Conclusions: LPS induced M0 macrophages to M1 macrophages,and the expression of inflammatory factors iNOS,IL-1? and TNF-? increased.IL-22 can inhibit the expression of M1 macrophage inflammatory factors iNOS,IL-1?and TNF-? induced by LPS,reduce its inflammatory response and inhibit the progression of liver fibrosis.The results of this study demonstrate the anti-inflammatory effects of IL-22 on macrophages and provide a solid experimental basis for the development of anti-fibrosis-related drugs in the future.