Study Of Novel Protective Methods On Hepatic Injury

Part I Liraglutide protects against ConA-induced lethal and non-lethal acute liver injury in mice[Objective]To investigate whether liraglutide can protects against ConA-induced lethal and non-lethal acute liver injury in mice.[Methods]Murine lethal and non-lethal acute liver injury models were established by intravenous administered with ConA at 15mg/kg or 25mg/kg body weight.Liraglutide(200?g/kg)was administered subcutaneously 3 days before ConA injection.For the lethal acute liver injury model,the survival of the mice was observed within 72 hours after ConA injection.For the non-lethal acute liver injury model,mice were sacrificed at 12 hours after ConA injection and blood plasma was obtained to assess liver function.Liver tissue samples were stained with H&E and TUNEL to detect the degree of liver injury and the numbers of apoptotic cells in liver.The levels of MDA and SOD were measured by total liver protein,and the levels of iNOS and COX-2 were also measured by RT-PCR to reflect the degree of the oxidative stress injury in liver.Finally,RT-PCR was used to detect the changes of inflammatory factors in liver,and the levels of IL-6 and IFN-? in plasma were also detected by ELISA.[Results]The lethal dose of ConA resulted in a large number of mice dead within 72 hours after ConA injection,and the survival rate was significantly improved when mice were pretreated with liraglutide(P<0.05).When mice were intravenously given a non-lethal dose of ConA for 12 hours,its liver function was significantly impaired(P<0.001),however when mice were pretreated with liraglutide,the liver function had a strong protection(P<0.01).H&E staining showed that ConA could induced severely liver damage,and pretreated with liraglutide could alleviate these pathological lesions.TUNEL staining also confirmed that pretreated with liraglutide significantly reduced ConA-induced hepatocellular apoptosis.ConA induced a significant increasing of MDA and decreasing of SOD levels in mice liver tissues,while the contents of MDA and SOD levels were significantly restored when mice were pretreated with liraglutide.RT-PCR analysis of iNOS and COX-2 mRNA changes also confirmed that liraglutide pretreatment could significantly reduced ConA-induced oxidative stress injury.Finally,RT-PCR results showed that ConA significantly up-regulated the expression of IL-2,IL-6,IFN-? and MCP-1,and these inflammatory factors was significantly decreased when mice were pretreated with liraglutide.[Conclusion]Liraglutide significantly attenuates ConA-induced acute liver injury by reducing oxidative stress and inflammatory injury in mice.Part ? Effects of liraglutide on ConA-induced T lymphocyte and macrophage activation[Objective]To investigate the influences of liraglutide on ConA-induced T lymphocyte and macrophage activation in mice.[Methods]Murine non-lethal acute liver injury models were established by intravenous injection with ConA at 15mg/kg body weight.Liraglutide(200?g/kg)was administered subcutaneously 3 days before ConA injection.Mice were sacrificed 12 hours after ConA injection,CD3 and CD68 immunohistochemical staining were performed to detect the infiltration of T lymphocyte and macrophage in liver.The percentage of CD3+CD69+,F4/80+MHC-?+ and F4/80+CD86+ cells were detected by flow cytometry to reflect the activation of T lymphocyte and macrophage in liver.And the percentage of CD3+CD69+,CD4+CD25+,CD4+CD62L+,CD4+CD69+,CD8+CD25+,CD8+CD62L+,CD8+CD69+,F4/80+M HC-?+,F4/80+CD80+ and F4/80+CD86+cells were also detected by flow cytometry to reflect the activation of T lymphocyte and macrophage in spleen.Finally,RT-PCR was used to detect the mRNA changes of TNF-a and IL-1? in liver tissues.[Results]Mice had a severely injury after intravenous injection of ConA,and liraglutide pretreatment significantly protects against mice from ConA-induced liver injury.Immunohistochemical staining showed that the numbers of CD3+T lymphocyte and CD68+macrophage were significantly increased in liver after ConA injection,and the infiltration cells did not decreased in mice pretreated with liraglutide(P>0.05).The percentage of CD3+CD69+,F4/80+MHC-?+ and F4/80+CD86+cells in ConA group did not different from ConA + LRG group,indicating that liraglutide did not inhibit the activation of T lymphocyte and macrophage in liver.The ratio of CD3+CD69+,CD4+CD25+,CD4+CD62L+,CD4+CD69+,CD8+CD25+,CD8+CD62L+ and CD8+CD69+ cells in spleen also showed that ConA could induced T lymphocyte activation and pretreated with liraglutide did not inhibit the activation of these surface markers.The flow cytometry results of F4/80+MHC-?+,F4/80+CD80+ and F4/80+CD86+ cells showed that ConA did not up-regulate the expression of MHC-? but could induce CD80 and CD86 activation,and liraglutide also did not inhibit the activation of these molecules.[Conclusion]Liraglutide had no influences on ConA-induced T lymphocyte and macrophage activation in mice.Part ? Liraglutide protects against ConA-induced acute liver injury by decrease HMGBl nucleocytoplasmic shuttling[Objective]To examine the relationship between the protective effects of liraglutide and translocation of HMGB1 in ConA-induced acute liver injury in mice.[Methods]Murine non-lethal acute liver injury model was established by intravenous administered with ConA at 15mg/kg body weight.Liraglutide(200?g/kg)was administered subcutaneously 3 days before ConA injection.Mice were sacrificed at 6 hours,12 hours and 24 hours after ConA injection and blood plasma was obtained to assess liver function.Immunohistochemical staining of HMGB1 was performed to observe the translocation of HMGB1 at different time points after ConA injection,and the released content of HMGB1 into plasma was also detected by ELISA.Finally,the expression of TLR4 and RAGE in the liver was also detected by RT-PCR.[Results]Mice had severely injury after intravenous injection of ConA,and the degree of liver injury was difference at different ConA injection time points.However,when mice were pretreated with liraglutide,liver injury had a strong protection at all time points.Immunohistochemical staining of liver samples showed that ConA could potently induced HMGB1 nuclear-cytoplasmic translocation in the early stage(6 hours and 12 hours)after ConA injection,at the same time we found that the translocation of HMGB1 at each time points were significantly inhibited when mice pretreated with liraglutide.ELISA results also showed that the serum levels of HMGB1 on liraglutide pretreated mice were much lower than untreated mice.RT-PCR results showed that liraglutide could significantly inhibited TLR4 and RAGE up-regulation which induced by ConA.[Conclusion]The protective effects of liraglutide on ConA induced acute liver injury was achieved by potently inhibiting the nuclear-cytoplasmic translocation of HMGB1.Part ? Down-regulation of nuclear HMGBl protects against liver ischemia-reperfusion injury[Objective]To examine whether down-regulation of nuclear HMGB1 expression by small interfering RNA can protects against liver ischemia-reperfusion injury.[Methods]Murine liver ischemia-reperfusion injury(IRI)model was established by segmental ischemia hepatic for 1 hour and then reperfusion for 6 hours.HMGB1-siRNA was injected into mice 48 hours prior to surgery to achieve the effects of down-regulation of HMGB1 expression in liver.Mice were sacrificed at 6 hours after reperfusion and blood plasma was used to examine the degree of liver injury and the levels of HMGB1 released into the circulation.Liver tissue samples were stained with H&E,MPO and TUNEL to detect the degree of liver injury,the infiltrated numbers of neutrophil and the apoptotic cells in liver.Mice were sacrificed at 0 hour after reperfusion,and liver tissues were used to observe HMGB1 nuclear-cytoplasmic translocation in the ischemic stage.Finally,the changes of HMGB1 related inflammatory factors and receptors in liver tissues were also detected by RT-PCR.[Results]Mice had a severely liver damage after 1 hour of ischemia and 6 hours of reperfusion.HMGB1-siRNA achieved a reduction of?70%HMGB1 expression in the livers at 48 hours post-treatment.Knockdown of nuclear HMGB1 expression dramatically reduced IRI induced liver injury(P<0.01).Liver histopathological examination also confirmed that down-regulation of nuclear HMGB1 expression can reduce IRI-induced hepatocellular necrosis and apoptosis and neutrophil infiltration.Immunohistochemistry and cytoplasmic protein analysis showed that down-regulation of HMGB1 expression can significantly reduce the translocation of HMGB1 in ischemic stage.ELISA results also showed that down-regulation of HMGB1 expression decreased the degree of HMGB1 release into the circulation during reperfusion.RT-PCR analysis also showed that down-regulation of nuclear HMGB1 expression can significantly reduce HMGB1 related inflammatory factors and receptors expression.[Conclusion]Down-regulation of nuclear HMGB1 expression by specific siRNA significantly protects the liver against warm IRI by directly reducing HMGB1 release.