The Role Of Mannan-binding Lectin In Hepatocellular Carcinoma And Carbon Tetrachloride-induced Acute Liver Injury

MBL is a kind of soluble PRR,which belongs to the collection group of the C-type lectin family.MBL is synthesized mainly by hepatocytes thought to be an important component of the innate immune system.MBL and MBL2 gene polyrmorphism are associated with liver diseases,such as acute liver failure(ALF),HBV-related liver diseases,HCV-related liver diseases,hepatocellular carcinoma(HCC).However,its precise role in these diseases has not been determined.In this paper,we choose MBL-/-mice as an animal model to study the role of MBL in hepatocellular carcinoma(HCC)and carbon tetrachloride-induced acute liver injury(ALI).We aim at providing a new perspective for the auxiliary treatment of HCC and ALI.Part ? The Role of Mannan-binding Lectin in Hepatocellular CarcinomaObjective:In this study,we investigated whether and how MBL influenced HCC development in mice.Methods:To explore the role of MBL in an experimental model of HCC progression,we established murine orthotopic HCC model by implanting Hepal-6 cells intravenously into WT and MBL-/-.mice.The mice were sacrificed 3 weeks later.Their livers were weighed and removed to analyze tumor size,number of tumor nodules.CCK8,flow cytometry,scratch assay were used to determine whether MBL could directly effect on the proliferation,apoptosis,migration of hepatoma carcinoma cells.Leukocytes from livers of tumor-bearing mice were collected and analyzed by FACS.Then using immunohistochemistry analysis MDSCs.Expression of a-SMA in liver tumors,a marker of aHSCs,was detected by WB,RT-PCR and immunohistochemistry.Then we used confocal analysis to detect COX2-PGE2 pathway in aHSCs.Lastly,pAAV-con or pAAV-MBL2(1 × 1010 genome copies/mouse)was delivered by tail vein injection 3 weeks before Hepal-6 cells injected.LX2 cells were untreated or treated with MBL(20 ?g/ml)in the absence or presence of HepG2-CM(20%)for 24 h.Expression of a-SMA and COX2 was detected by WB,RT-PCR.PGE2 levels in LX2 cells culture supernatants was detected by ELISA.Results:Our results demonstrated that MBL deficiency promoted tumor growth in murine HCC model,which was attributed to elevated frequency of MDSCs that colud suppress anti-tumoral immune responses by inhibiting of CD8+T cell responses and inducing of Tregs.Indeed,MBL-/-mice increased activation of HSCs in the liver,accompanied by more PGE2 produced in aHSCs,which involved in the induction of MDSC,via inducing the expression of COX2.The administration of pAAV-MBL2 reduced tumor growth in MBL-/-mice.MBL inhibited the activation of LX2 cells.Conclusion:MBL deficiency is associated with HCC susceptibility.Together,our results reveals a novel mechanism involving cross-talk among HSCs,PGE2 and MDSCs.These findings also offer new insights into how MBL influences tumor immunity,with potential implications for the development of tumor immunotherapy.Part ? The Role of Mannan-binding Lectin in carbon tetrachloride-induced Acute Liver InjuryObjective:In this study,we investigated whether and how MBL influenced carbon tetrachloride-induced acute liver injury in mice.Methods:We choose MBL-/-mice as an animal model to study the role of MBL in ALI.Mice received a single dose at 1?l/g body weight of CCl4 administered by intraperitoneal injection.Blood was drawn from the orbital venous and livers were removed at 24 hours after CCl4 challenge.Serum was used to detect the ALT,LDH activity and the inflammatory cytokines levels.Livers were fixed in 4%paraformaldehyde,then used for HE staining or TUNEL staining to observe the pathological changes of liver tissue and the necrosis of liver,respectively.Part of liver tissue was used to measure the inflammatory cytokines expression by RT-PCR method.Expression of CYP2E1,required to bioactivate CCl4,was analysis by WB and RT-PCR.Leukocytes from livers were collected and analyzed by FACS.Then using immunohistochemistry analysis macrophages and neutrophils.We used anti-Ly6G Ab to neutralizate neutrophils,and the analysised the liver injury between WT mice and MBL-/-mice.Using RT-PCR analysis the expression of CXCL1,CXCL2.Then we used WB,RT-PCR and confocal analysis to detect the express of CXCL2 in macrophages.We used clodronate liposomes to deplete macrophages,and then analysised the liver injury between WT mice and MBL-/-mice.pAAV-con or pAAV-MBL2(1 × 1010 genome copies/mouse)was delivered by tail vein injection 3 weeks before CCl4 injected.Results:Our results demonstrated that MBL deficiency exacerbated liver Injury in CCl4-induced ALI model.The increased injury in MBL-/-mice was not due to an altered ability to bioactivate CCl4,but due to more neutrophilic infiltrates.Indeed,MBL-/-mice increased infiltration of neutrophils in the liver via inducing the expression of CXCL2 in macrophages,which involved in the induction of neutrophils.MBL deficiency exacerbates CCl4-induced acute liver injury in mice,the most possible mechanism of which may be the functional change of macrophages.The administration of pAAV-MBL2 attenuated liver injury in MBL-/-mice.Conclusion:MBL deficiency exacerbates CCl4-induced acute liver injury in mice through induction of CXCL2 in macrophages.That is MBL may play a protection role in acute liver injury,which provides a new perspective for prevention and treatment of acute liver injury in clinical.