Protective Effect And Mechanism Of Agmatine On Liver Ischemia-reperfusion Injury In Mice

Objective: Liver ischemia-reperfusion injury(LIRI)is a common problem during liver surgery.It can cause severe inflammatory reactions and cell death,which can eventually lead to severe liver damage,affecting surgical outcome and patient prognosis.Although there are many studies on the mechanism of liver ischemia-reperfusion injury,there is still a lack of effective treatment for it.Agmatine(AGM)is an endogenous polyamine with analgesic,anti-inflammatory and anti-apoptotic effects,and it has been reported to have protective effects on ischemia-reperfusion injury in various organs.However,whether AGM can protect the liver from ischemia-reperfusion injury has not been reported,so this study aims to explore whether AGM can protect liver from ischemia-reperfusion injury in mice and its possible mechanism.Methods: This study was divided into two parts,namely the protective effect of AGM on liver ischemia-reperfusion injury in mice and the mechanism of the protective effect of AGM.The experimental methods of these two parts are described separately below.In the first part,male C57BL/6 mice were randomly divided into three groups: sham group,LIRI group and LIRI+AGM group.The sham group contained 4 mice,and the LIRI group and the LIRI + AGM group each had 6 mice.Mice in LIRI group and LIRI+AGM group were treated with partial hepatic ischemia reperfusion,while mice in LIRI+AGM group were treated with AGM intervention.Serum and liver tissues of mice were obtained 6 hours after reperfusion,and serum ALT and AST levels were measured to detect liver function.Hematoxylin/eosin(HE)staining and terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate notch terminal marker(TUNEL)staining were used to evaluate the extent of liver injury and apoptosis,respectively.Real-time fluorescence quantitative PCR(RT-PCR)technology was used to detect the m RNA expression levels of IL-1? and TNF-?,and myeloperoxidase(MPO)staining was used to detect the inflammation of liver tissue in each group.The expression of apoptotic proteins(Bcl-2,cleaved-caspase3)in each group was analyzed by western blot.The mouse liver cell lines AML12 were cultured in vitro and the cell hypoxia model induced by CoCl₂ was established.The experiment was divided into three groups: normal group,CoCl₂ group and CoCl₂+AGM group.Each group contains at least 3 samples.Cell apoptosis was detected by flow cytometry.Total cell proteins were extracted and the expression levels of apoptotic proteins(Bax,Bcl-2,cleaved caspase3)in each group were analyzed by western blot.In the second part,in order to elaborate the protective mechanism of AGM,the total proteins of liver tissues and cells in each group described in the first part were extracted and the expression of Wnt/?-catenin pathway related molecules(Wnt1,?-catenin,c-Myc,cyclin D1)were analyzed by western blot.To further clarify the correlation between AGM and Wnt/?-catenin pathway during hepatic ischemia-reperfusion injury,two kinds of Wnt/?-catenin pathway inhibitors,XAV-939 and ICG-001,were selected for this study.In vivo experiments were divided into six groups: sham group,LIRI group,LIRI+Vehicle group,LIRI+AGM group,LIRI+AGM+XAV-939 group and LIRI+AGM+ICG-001 group.The sham group contained 4 mice,and the other five groups each had 6 mice.Liver function(ALT,AST),liver damage,inflammatory response,and apoptosis(TUNEL staining)were measured according to the methods described in the first part.In vitro experiments were also divided into six groups: normal group,DMSO group,CoCl₂ group,CoCl₂+AGM group,CoCl₂+AGM+XAV-939 group and CoCl₂+AGM+ICG-001 group.Each group contains at least 3 samples.The apoptosis rate and expression of apoptotic proteins(Bax,Bcl-2,cleaved-caspase3)in each group were analyzed by flow cytometry and western blot.Results: In the first part of the study,it was found that the serum transaminase in the LIRI group increased significantly,but decreased significantly after AGM was used(P<0.05).Compared with the LIRI group,the liver necrosis area in the LIRI + AGM group was significantly reduced,the damage of liver tissue structure was reduced,and the Suzuki's score was significantly reduced(P<0.05).After ischemia-reperfusion,the expression levels of inflammatory cytokines IL-1? and TNF-? in the liver of mice were significantly up-regulated(P<0.05),while the expression levels of inflammatory cytokines were decreased after AGM treatment(P<0.05),and the results of MPO staining were consistent(P<0.05).The apoptosis-related results showed that compared with the LIRI group,the expression of Bcl-2 was increased in the LIRI+AGM group,the expression of cleaved-caspase3 was decreased,and the positive rate of TUNEL staining was also significantly decreased(P<0.05).In addition,the results of in vitro experiments also showed that AGM significantly reduced the increased apoptosis rate and the increased expression of Bax and cleaved-caspase3 induced by the CoCl₂-induced hypoxia model(P<0.05).The second part of the study found that compared with the LIRI group,the protein expression levels of Wnt1,?-catenin,c-Myc and cyclin D1 in the LIRI+AGM group were significantly increased(P<0.05).In vitro,compared with the CoCl₂ group,the expression levels of these protein also significantly increased(P<0.05).After using the inhibitor,compared with LIRI+AGM group,ALT and AST were increased in LIRI+AGM+XAV-939 group and LIRI+AGM+ICG-001 group(P<0.05),the degree of liver tissue damage and Suzuki's score were also increased(P<0.05).The m RNA expression levels of IL-1? and TNF-? and the positive rate of MPO staining were significantly increased(P<0.05),the positive rate of TUNEL staining of liver tissue and the expression of cleaved-caspase3 protein were increased,and the expression of Bcl-2 decreased(P<0.05).In vitro experiments,apoptosis rate and expression of Bax and cleaved-caspase3 were increased in CoCl₂+AGM+XAV-939 group and CoCl₂+AGM+ICG-001 group,while expression of Bcl-2 was decreased(P<0.05).Conclusion: AGM plays significant protective effect on liver injury,inflammatory response and apoptosis induced by ischemia-reperfusion.In addition,AGM can also significantly improve hypoxic damage to liver cells induced by cobalt chloride(CoCl₂).AGM activates the Wnt/?-catenin signaling pathway during liver ischemia-reperfusion injury and hepatocyte hypoxic injury,while the protective effect of AGM is weakened when the Wnt/?-catenin pathway is blocked by inhibitors.This study explored the effect of AGM on liver ischemia-reperfusion injury in mice and its possible mechanism,thus providing a new idea for the treatment of liver ischemia-reperfusion injury in clinic work.