Study On The Role And Mechanism Of MBL In Liver Fibrosis

Liver fibrosis is a common pathological process of chronic liver injury and it is also an inevitable process in the development of cirrhosis from various chronic liver diseases.The network composed of various cells-molecules-extracellular matrix in the liver microenvironment determines the development of liver fibrosis.Therefore,timely intervention of the liver micro-environment is expected to alleviate or reverse liver fibrosis,which is essential for controlling the progression of liver-related diseases.Mannose-binding lectin(MBL)is a key molecule in innate immunity mainly produced from the liver.It plays an important role in host immune system,defense against exogenous infection and the organism homeostasis,and is also an important molecule in the liver microenvironment.The MBL gene polymorphism and its serum level are related to the development of liver fibrosis,but its role and mechanism in liver fibrosis have not been elucidated.Therefore,in this study,we applied the MBL knockout mice(MBL-/-)and recombinant MBL protein to explore the role and mechanism of MBL in liver fibrosis.Part Ⅰ Mannan-binding lectin influenced the progression of liver fibrosis by direct regulation of hepatic stellate cellsOBJECTIVE:To study the role and mechanism of MBL in regulating liver fibrosis through hepatic stellate cells.METHODS:In vivo experiment:1.WT and MBL-/-mice were applied to construct a liver fibrosis model by intraperitoneal injection of 25%CCl4 at a dose of 1(μl/g for 6 weeks.2.Mice were sacrificed 2 days after the last injection of CCl4 then assessed the fibrosis indicators:masson staining,sirius red staining,levels of ALT,LDH in serum,mRNA levels of collA1 and Fn-1.3.Immunohistochemistry,Q-PCR,WB meansured the activated hepatic stellate cell(HSC)marker α-SMA.To measured the senescence of hepatic stellate cell,β-galactosidase staining,ki-67 staining,mRNA levels of p21,p53,p16,CCL2,analysis the co-localization of α-SMA with p21 via immunofluorescence were examined.Purified mouse primary HSC then meansured the indicators related to senescence.4.Analysis of MBL expression in LX-2 cells by immunoblotting.5.Three weeks after injected pAAV-TBG-MBL-EGFP to MBL-/-mice,the liver fibrosis model was established.The liver fibrosis and HSC senescence indicators were measured after the mice sacrificed.In vitro experiment:1.LX-2 cells were cultured with hydrogen peroxide and MBL or not for 48-72h,subsequently,cell cycle,β-galactosidase staining,mRNA levels of p53,p21,p16,MMPs,SASP were meansured.2.After stimulated with p53 inhibitor PFT,LX-2 cultured with hydrogen peroxide and MBL protein for different time then examined LX-2 cell cycle,β-galactosidase staining,p53,p21 protein and mRNA levels.3.After treated with mTOR inhibitor rapamycin,LX-2 was cultured with hydrogen peroxide and MBL protein,then examined the levels of p53 and p21.RESULTS:In vivo experiments:1.MBL-/-mice aggravated CCl4-induced liver fibrosis compared with WT mice,as evidenced by increase collagen deposition,inflammatory response,and activated HSC.2.Further studies on HSC revealed that MBL-/-mice decreased senescent HSC in liver fibrosis model.3.It was found that MBL may contact with activated HSC by immunofluorescence.In vitro experiments:1.βgalactosidase staining positive cells,cell cycle arrest,expression of senescenceassociaed secretory factors were increased while extracellular matrix decreased after incubated with hydrogen peroxide and MBL protein.2.MBL promotion of LX-2 cells senescence were inhibited by p53 inhibitor.3.The role of MBL promoting LX-2 cells expressing p53,p21 were inhibited by mTOR inhibitor Rapamycin.CONCLUSION:MBL may promote hepatic stellate cell senescence via influenced the mTOR/p53/p21 axis,thereby eliminated CCl4-induced liver fibrosis.These results suggested that the role and mechanism of MBL in liver fibrosis,which may lead to new ideas of immunotherapy for liver fibrosis patients with MBL deficiency or low expression.Part Ⅱ Preliminary study on the function of mannan-binding lectin in anti-liver fibrosis through the other immune cellsOBJECTIVE:Preliminary study on the function of mannan-binding lectin in anti-liver fibrosis through the other immune cells.METHODS:The liver fibrosis model was established and pAAV-TBG-MBL-EGFP were used to restored MBL expression in MBL-/-mice,analyse macrophages and NK cells via flow cytometry.RESULTS:1.In liver fibrosis,NK cells and total macrophages increased in MBL-/mice,while IFN-y secreted by NK cells and F4/80intCD11bhiLy6Clo macrophage which associated with fibrosis remission were reduced in MBL-/-mice.It was found that NK cell and its function was restored after restored MBL expression in MBL-/-mice.CONCLUSION:MBL may play a role in fibrosis alleviation via NK cells and F4/80intCD1 lbhiLy6Clo macrophages which beneficial to fibrosis remission.