IL-22 Inhibits The Differentiation Of M1 Macrophage And Reduces Liver Fibrosis By CCL2-CCR2 Axis

Objective : To study the effect and mechanism of IL-22 regulating liver macrophages to reduce liver fibrosis through CCL2-CCR2 pathway,and to further clarify the role of IL-22 in inhibiting liver fibrosis.Methods:M-CSF(50ng/ml)was used to induce mice bone marrow cells to differentiate into macrophages.By using lipopolysaccharide(1ug/ml)to stimulate bone marrow-derived macrophages,the bone marrow macrophages are polarized into M1-type macrophages.The flow cytometry was used to detect the polarization of macrophages and the induction rate of macrophages.Interfere M1 macrophage with IL-22,IL-22 concentration group 0ng / ml,20 ng / ml,40 ng / ml;use CCR2 antagonist to intervene M1 macrophage,CCR2 antagonist concentration group 0ng / ml,10 ng / ml,20 ng / ml,40 ng / ml,80 ng / ml,160 ng /ml,320 ng / ml.CCK8 method was used to detect the cell proliferation induced by stimulation.Flow cytometry was used to detect the induction rate of macrophages and the content of CCR2.Transwell was used to detect the effect of CCL2-CCR2 on the migration ability of macrophages.Immunofluorescence technique was used to detect the effect of IL-22 and CCR2 antagonists on the number of CCR2 receptors in macrophages.The model of mouse fibrosis was established by intraperitoneal injection of CCl4.C57 BL / 6 mice were randomly divided into five groups:(1)blank control group: no treatment was given;(2)liver fibrosis group: intraperitoneal injection of CCl4;(3)liver fibrosis + IL-22 treatment group: intraperitoneal injection of CCl4,IL-22 injection after 30minutes;(4)liver fibrosis + CCR2 antagonist treatment group: intraperitoneal injection of CCl4,CCR2 antagonist injection after 30 minutes;(5)liver fibrosis +IL-22 + CCR2 antagonist treatment group: CCl4 was intraperitoneally injected,IL-22 was injected 30 minutes later,and CCR2 antagonist was injected 30 minutes later.The concentration of CCl4 was 20%,the injection concentration of IL-22 was 100 ng/ml;the injection concentration of CCR2 antagonist was 1 m M;the injection dose of IL-22 and CCR2 antagonist was 0.06 ml per mouse.The effects of IL-22 and CCR2 antagonists on the migration ability of liver macrophages were detected by immunofluorescence technology.HE and Masson pathological staining to identify the degree of liver fibrosis in animal models.Results:The results of HE showed that the liver of the model mice in the liver fibrosis group had obvious absence of central veins,unclear structures of liver lobules and manifolds,and formed pseudo-lobular structures with different shapes,that is,the fibrosis model was successfully established;IL-22 Intraperitoneal injection of fibrotic mouse liver HE staining results showed that less areas formed pseudolobular structure;CCR2 antagonist intraperitoneal injection of fibrotic mouse liver HE staining results also showed less areas of pseudolobular structure formation.Tip IL-22 and CCR2 antagonists can reduce liver fibrosis.The results of Masson trichrome staining experiment showed that a large amount of blue-stained collagen fibers were deposited in the liver of model liver fibrosis model mice,extending outward from the area around the manifold,and the collagen fibers were wrapped to form pseudolobules,that is,the fibrosis model was successfully established;IL-22 Masson staining results of intraperitoneally injected fibrotic mouse liver showed less blue-stained collagen fiber deposition,and the structure of pseudolobules was smaller;Masson staining results of CCR2 antagonized intraperitoneally injected fibrotic mouse liver also saw less blue-stained collagen fiber Due to the deposition,the area of the pseudolobular structure is small,suggesting that IL-22 and CCR2 antagonists can reduce the degree of liver fibrosis.Immunofluorescence results of mouse liver tissue showed that the i NOS antibody-positive area could be seen in the fibrotic mouse liver manifold;IL-22 intraperitoneal injection of fibrotic mouse liver immunofluorescence results showed that the i NOS positive area was reduced and the CCR2 receptor was antagonized The number of protein markers of the agent is also decreasing;the liver immunofluorescence results of CCR2 antagonistic intraperitoneal injection of fibrosis mice show that the area of i NOS positive is reduced,and the number of protein markers of CCR2 receptor antagonist is also decreasing,suggesting IL-22 and CCR2 antagonist Can reduce the degree of liver fibrosis.According to the CCK8 method,the optimal concentrations of LPS,IL-22 and CCR2 antagonists for cell proliferation were 1000ng/ml,20ng/ml and20 ng / ml,respectively.The results of flow cytometry showed that the result of adding LPS to the cultured bone marrow-derived macrophages,the percentage of M1 macrophages polarized was 89.1%,and the percentage of M1 macrophages decreased to 76.8% after the addition of CCR2 antagonists,and M1 after IL-22 was added The percentage of macrophages decreased by 67.3%,and after treatment with IL-22 and CCR2 antagonists,the percentage of M1 macrophages decreased by 64.2%.It was concluded that IL-22 and CCR2 antagonists inhibited the polarization of macrophages.Function.In addition,the results of immunofluorescence measurement of macrophages showed that the number of i NOS specific antibodies on the surface of M1 macrophages in the LPS and IL-22 intervention group showed a downward trend,and the content of CCR2 receptors also showed a downward trend;LPS and CCR2 antagonized The number of i NOS on the surface of M1 macrophage-specific antibody in the coadministration group showed a downward trend,and the content of CCR2 receptor also showed a downward trend;the surface of M1 type macrophages in the LPS and IL-22,CCR2 antagonist co-intervention group was specific The number of sexual antibodies i NOS showed a downward trend,and the CCR2 receptor content also showed a downward trend.IL-22 can reduce the expression of CCR2 on the outer surface of M1 macrophages and the polarizing ability of M1 macrophages.Our data also show that high concentrations of IL-22 and CCR2 antagonists can inhibit the proliferation of M1 macrophages.Transwell image and data analysis showed that the migration ability of M1 type macrophages is stronger than that of M0 type macrophages.When IL-22 intervenes in M1 type macrophages,its migration ability decreases,and it drops to-%;when CCR2 antagonists intervene in M1 type macrophages,its migration ability also decreases,which drops to-%;when used After IL-22 and CCR2 antagonists intervened in M1 type macrophages,their migration ability decreased,and dropped to-%.The combined ability of the two shows that the blockage of IL-22 and CCR2-CCL2 on M1 type Macrophage migration inhibition has similarities.Combined with the previous results of macrophage immunofluorescence,the inhibitory effect of IL-22 on M1 macrophages may be related to the CCL2-CCR2 ligand receptor.The expression of i NOS and CCR2 in the liver of mice was detected by immunofluorescence technology,and it was found that the liver fibrosis group injected with CCl4 was co-injected with CCl4 and IL-22,the group co-injected with CCl4 and CCR2 antagonists,CCl4 with IL-22 and CCR2 Compared with the mice in the antagonist co-injection group,the expression of i NOS and CCR2 was significantly down-regulated,which was consistent with the previous immunofluorescence results of M1-type macrophages.Conclusion : IL-22 inhibits the binding of macrophage CCL2-CCR2 ligand receptor,inhibits the macrophage polarization to M1 type and reduces the migration and aggregation of macrophages to the liver,thereby reducing liver inflammation to liver fibrosis development of.