The Function And Mechanism Study Of MDSCs And CXCR4/CXCL12 Axis In The Nash Mice

PartⅠNASH model building in mice with CDAA dietObjective:Building NASH model in mice with CDAA diet（choline-deficient,L-amino acid–defined diet）for further intervention studies.Methods:Choose 6 to 8 weeks of age male SPF C57BL mice,after a 1-week acclimation period on a standard diet,the mice were divided into ordinary normal food group,CDAA diet model group,CSAA diet control group until the end of the 12th at the weekend.At the end of model building,blood samples were collected from each mouse via picking the eyeball blood.Serum levels of alanine amino invertase（ALT）,nmda aminotransferase（AST）,triglycerides（TG）,urea nitrogen（BUN）and Blood Glucose（Blood Glucose）,total cholesterol（T-CHO）,total bilirubin（TBIL）were tested by using an automatic analyser.Liver tissue samples were fixed overnight in10%phosphatebuffered formaldehyde,embedded in paraffin and stained with haematoxylin and eosin.To analyse fibrosis in the sections,Sirius Red stained areas were measured.The pathological changes in mice liver tissue was observated by microscope and graded according to the NAS（NAFLD activity score）.The levels of PGE2,IL-6,INF-γin peripheral blood of the normal group,control group,model group mice were detected by ELISA method.Results:（1）Compared with control group,the mean body weight,liver index of CSAA,CDAA model mice had no significant difference（P>0.05）.In model group,mice liver is dull edge,yellow color,and the flexibility is poor.ALT,AST,Glucose were significantly higher in CDAA model group than normal group（P<0.001）.But,there was no statistically significant difference of BUN,TG,T-CHO,TBIL level in different groups（P>0.05）.（2）ELISA results show that serum inflammatory factors PGE2,IL-6,INF-γlevels in CDAA diet mice were higher than normal diet mice（P<0.001）.（3）Based on observation of H-E stained sections,the liver of mice in CDAA diet group was full of big fat drop,different level lobular inflammation and hepatocyte ballooning.Sirius scarlet dye cases showed different degrees of fibrosis.The score of NAS was above 5 in CDAA diet group.But in CSAA diet group,some liver tissue presents different degree and fatty degeneration and a few inflammatory cells infiltration.The score of NAS was 2-4 in CSAA diet group.Conclusion:A number of biochemical indexes and liver histology change in CDAA diet NASH mouse model accorded with the NAS（NAFLD activity score）criteria.This mouse NASH model was established successfully,which can be used in the further research.Part Ⅱ MDSCs change and function of MDSCs in NASH miceObjective:Objective:Studies confirm that myeloid derived suppressor cells（MDSCs）have a powerful immune regulation function,but the role of MDSCs in nonalcoholic fatty liver（NASH）is unclear.The purpose of this study is to research the distribution,function and variation of MDSCs cells in NASH mice as well as the relationship with T cells subgroup.Methods:The proportion and number of CD11b⁺Gr-1⁺MDSCs in liver,spleen,blood and bone marrow in CDAA NASH model,CSAA control and normal mice were analyzed by flow cytometry.The subgroups of MDSCs in blood,liver,spleen,and bone marrow and the change of peripheral blood T cell subsets in the different groups were detected by flow cytometry.The relationship between MDSCs cells in the body as a whole and serum transaminase ALT,AST level,T cell subsets were analyzed.Gr-1^highLy-6G⁺,Gr-1^dimLy-6G^-MDSCs from bone marrow of mice and CD8+T cells from speed of mice were separated by magnetic bead.NASH mice were divided into the following two groups（n=5 per group）:a control group with PBS injection,a Intervention group with MDSCs injection.In vivo,the highly purified Gr-1^highLy-6G⁺MDSCs was adoptively transfered into NASH mouse model through tail vein injection.Intraperitoneal injection of gemcitabine to delete MDSCs.NASH mice were divided into the following two groups（n=5 per group）:a control group with normal saline（NS）injection,a intervention group with gemcitabine injection.The role of MDSCs in NASH was analyzed by detection of serological indexes of liver function and pathological dyeing.Results:T cell subsets test results show that the number of CD3+CD8+cell is a downward trend..There is cell immune dysfunction in NASH mice.The proportion of CD3+CD8+T cells in peripheral blood and ALT,AST levels were significantly negative correlation（P<0.001）.In NASH mice model induced by CDAA diet,MDSCs proportion in the bone marrow（73.68±4.49）significantly increased compared with normal group（59.71±4.02）,with statistical significance（P<0.05）.Decreased proportion of MDSCs was detected in the peripheral blood of CDAA model group,but no significant statistical difference compared with normal group（P>0.05）.Compared with normal group,MDSCs proportion in the liver tissue of NASH model group c decreased significantly（P<0.001）.There has certain relations between NASH lesion degree and the proportion of MDSCs.The proportion of MDSCs in the peripheral blood,NAS<7 case is greater than that the NAS>7 points.MDSCs subgroup analysis shows,according to the analysis of the subgroup of MDSCs,the proportion of CD11b⁺Gr-1^highLy-6G⁺（G-MDSC）in CDAA model group（76.67±12.64）,CSAA control group（69.42±13.69）significantly increased compared with normal group（2.57±0.93）in bone marrow.There is the significant difference significance（F=42.152,P<0.001）.MDSCs and CD8+T cells in peripheral blood has a negative correlation（r=-0.701,P=0.024）.Through adoptive transfer of normal mice bone marrow sources of Gr-1^highLy-6G⁺MDSCs,serum AST,ALT levels in NASH model were significantly lowered（P<0.001）,and hepatic lesions was improved.In vivo,application of gemcitabine to delete MDSCs does not affect the proportion of T cells in the body.But Compared with normal group,NASH model serum AST,ALT levels influence has no obvious statistical significance（P>0.05）.Conclusion:In vivo,the numbe,subtype of MDSCs in CDAA diet induced NASH model were showing a different degree of change.MDSC can affect cellular immune function,especially the function of CD8+T cells.Gr-1^highLy-6G⁺MDSCs from normal mice bone marrow has alleviate effect in NASH lesions induced by CDAA diet.The immunosuppressive function of MDSCs is expected as a kind of immune intervention methods applied in the treatment of NASH.Part Ⅲ The function of CXCR4/CXCL12 axis in NASH mice and influence on migration of MDSCsObjective:Objective:The second part of the research results show that MDSCs in mice NASH has played an important role in the process,can inhibit the immune and reduce the liver pathological damage.However,the migration mechanism of MDSCs in NASH is unclear.The purpose of this part of the study is to investigate the change of CXCL12,CXCR4 in NASH.The role of CXCR4/CXCL12 axis in MDSCs migration and settled in the liver is researched.Methods:The CXCR4 receptor expression on MDSCs of blood,bone marrow,liver and spleen in CDAA,CSAA and normal diet mice were detected by flow cytometry technology.Immunohistochemical techniques to detect the expression of CXCL12,CXCR4,STAT3 and p-STAT3 in liver tissue.In vitro,immune magnetic beads technology was used to purificate bone marrow-derived Gr-1^highLy-6G⁺MDSCs.The chemotaxis role of CXCR4/CXCL12 axis for MDSCs was tested by transwell experiment.In order to characterize the relevance of the CXCR4/CXCL12 chemokine axis during NASH,AMD3100,a CXCR4 small molecule inhibitor,was used in models of CDAA,CSAA,normal diet mice.And then,flow cytometry technology was used to detect MDSCs in blood,bone marrow.Serum ALT,AST and the liver pathological changes were detected after AMD3100 used.Results:Flow cytometry results show that compared with normal mice,the express CXCR4average fluorescence intensity（MFI）increased significantly on blood,bone marrow,liver,spleen MDSCs of CSAA contrast and NASH model group.Statistical results show that compared with normal group the CXCR4 expression level on bone marrow,liver,spleen MDSCs has significant differences in CSAA contrast and NASH model group（P<0.05）.The expression of CXCL12,CXCR4 were higher in NASH liver tissue than in normal liver tissue.Statistical analysis showed statistically significant difference（P<0.05）.The protein expression of STAT3,p-STAT3 increased significantly in NASH liver tissue.Compared with normal group,the differences were significant（P<0.01）.In vitro transwell experimental results show that the Gr-1^highigh Ly-6G⁺MDSCs migration ability has increased with the increase of concentration of CXCL12（P<0.05）,and the ability to migrate was inhibited by AMD3100.Compared with control group,the proportion of MDSCs of the blood increased significantly in 3 consecutive days to give AMD3100 group（P<0.01）.While,the proportion of MDSCs of the bone marrow decreased significantly in AMD3100 group（P<0.01）.However,NASH lesions and ALT,AST level analysis did not see obvious change after application AMD3100.Conclusion:BonemarrowGr-1^highLy-6G⁺MDSCsmigrationabilityregulationby CXCR4/CXCL12 pathway.CXCL12,CXCR4 expression in NASH liver tissue plays an important role in MDSCs directional migration.But the application AMD3100 for3 consecutive days is no obvious therapeutic effect for mice NASH lesions.