Study On The Roles And Mechanisms Of Mannan-Binding Lectin And Its Ligand In Inflammatory Disorders

Mannan-binding lectin(MBL)mainly produced by the liver is a crucial component of the complement system,and it is involved in the body infection immunity,tumor immunity,innate immunity,and immunoregulation.In recent years,clinical studies have shown that MBL is closely related to various inflammatory diseases such as hepatitis B virus infection,systemic lupus erythematosus,rheumatoid arthritis,but the specific mechanism of MBL in inflammatory diseases has yet to be identified.This study exploited the effects and mechanisms of MBL and its ligand mannose on inflammatory diseases in acute inflammatory disease ulcerative colitis(UC)and chronic inflammatory disease rheumatoid arthritis(RA).Part Ⅰ Chapter Ⅰ Regulation of MBL on Chronic Inflammatory Diseases and Its MechanismObjective:To investigate the role and mechanism of MBL in the development of adjuvant-induced chronic arthritis.Methods:A mouse model of chronic inflammatory arthritis was established by immunization of wild-type(WT)and MBL-deficient(MBL-/-)mice with Freund’s adjuvant.The mice were sacrificed on the day 30,60,and 90 post immunization,and pathological damages of their knee joints were examined.Human CD 14+monocytes were sorted form the peripheral blood mononuclear cells by magnetic beads,followed by stimulating with recombinant human macrophage colony-stimulating factor(MCSF)and nuclear factor NF-κB receptor activating factor ligand(RANKL),in the presence or absence of human MBL protein,to induce the formation of osteoclasts.Subsequently,tartrate acid phosphatase(TRAP)staining and bone resorption assay were used to determine the osteoclast formation.Gene expression profiles of osteoclast differentiation were examined by real-time quantitative PCR(qPCR).Immunoblotting was used to evaluate the protein level of osteoclast differentiation genes.ELISA was used to detect the levels of MBL in sera from healthy donors and arthritic patients,respectively.The correlations between serum MBL levels with rheumatoid factor(RF),erythrocyte sedimentation rate(ESR),amino-terminal propeptide of type Ⅰ procollagen(P1NP),and C-terminal telopeptide of type Ⅰ collagen(β-CTX)were analyzed respectively.Results:1.Serum MBL levels in patients with rheumatoid arthritis were negatively correlated with disease severity.2.MBL deficiency promoted the development of adjuvant-induced inflammatory arthritis in mice.3.MBL inhibited osteoclast differentiation in vitro.4.MBL affected the expression of osteoclast differentiation related proteins by inhibiting the P38-c-fos-NFATcl signaling pathway.Conclusion:MBL inhibits the differentiation of osteoclasts by affecting the P38-cfos-NFATc1 signaling pathway,thereby alleviating the development of adjuvantinduced arthritis.Part Ⅱ Effect and mechanism of MBL and its ligand mannose on acute inflammatory diseasesObjective:To investigate the effect and mechanism of MBL and its.ligand mannose on dextran sulfate sodium(DSS)-induced acute ulcerative colitis(UC).Methods:The expression of MBL in the colon tissue of mice and the changes in plasma mannose levels in the course of the disease were examined.The correlation between the disease severity and plasma mannose levels in patients with inflammatory bowel disease was analyzed.WT and MBL-deficient mice were received 3%DSS in drinking water for 7 days to establish a mouse model of acute ulcerative colitis.The body weight of the mice was measured every day.On the last day of the experiment,the mice were sacrificed,and the colon length of the mice was measured.Immunoblotting was performed to detect the expression of mouse colon barrier protein.Mice were administrated with 3.0%DSS in the drinking water combined with 5 mg of mannose per day.The body weight of the mice was monitored daily and the mice were sacrificed on day 7 to measure the colon length of the mice.Immunoblotting was performed to detect the expression of mouse colon barrier protein.The human colonic epithelial cells NCM460 were treated with DSS in the presence or absence of mannose and the expression of the barrier protein was detected by immunoblotting.Cellular aerobic respiration of NCM460 cells was measured.Immunofluorescence was used to detect changes in mitochondrial and lysosomal morphology.Immunoblotting was conducted to detect the expression of cathepsin B.Co-immunoprecipitation was performed to detect the binding of AMPK and AXIN on lysosomes in the epithelial cells.Barrier proteins changes were detected by immunoblotting after AXIN knocked out by crispr cas 9 technology.Finally,the therapeutic effects of mannose either alone or combination mesalazine,the first-line treatment for inflammatory bowel disease,on DSS-induced colitis were investigated.Results:1.Mouse plasma mannose levels gradually increased in the course of acute ulcerative colitis.2.Human plasma mannose levels were intimately associated with the pathogenesis of inflammatory bowel disease.3.MBL defeciency protected DSSinduced ulcerative colitis barrier damage caused by DSS.4.Mannose relieved DSSinduced acute ulcerative colitis.5.Mannose protects the colon barrier caused by DSS.6.Mannose restored the decrease in aerobic respiration of mouse colon tissue and colonic epithelial cells induced by DSS.7.Mannose protected colon barrier damage caused by DSS through the MLCK-MLC2 pathway.8.Mannose maintained the stability of colonic epithelial lysosomes via the AMPK-AXIN pathway.9.Mannose up-regulated the expression of aerobic respiration-related enzymes by inhibiting the DSS-induced overproduction of cathepsin B.10.Mannose combined with mesalazine in the treatment of DSS-induced ulcerative colitis.Conclusion:MBL and its ligand mannose are closely related to the development of ulcerative colitis.Mannose maintains lysosomal stability by protecting the AMPKAXIN pathway,afterward it inhibits the excessive release of cathepsin B,increases aerobic respiration-related enzymes,increases aerobic respiration,and protects DSSinduced barrier disruption in ulcerative colitis by the MLCK-MLC2 pathway.