Acute Liver Injury In Mice And The Protective Effects Of Arctigenin

The liver is not only the most important metabolic organ of the human body,but also biological transformation organ of many non nutritive substances such as drugs,poisons and toxic metabolites,so the liver is also known as detoxification organ.In recent years,more and more studies have shown that the liver is also an important immune organ of the human body.The liver includes a variety of immune cells,such as Kupffer cells,dendritic cells and lymphocytes,and hepatic sinusoid endothelial cells and hepatic stellate cells also have immune function.Acute liver injury refers to a variety of pathogenic factors that infringe the liver to cause liver cell damage,causing the liver to not play normal function,and then cause a series of symptoms,and even cause death.Common pathogenic factors include viruses,bacteria,drugs,chemical poisons and alcohol.Immune hepatitis and drug hepatitis are common in the clinic.Acute exacerbation can easily lead to acute liver failure;it is easy to cause death when the liver compensatory function is insufficient.Liver transplantation is the best treatment.However,due to lack of donor,it often delays the time of treatment and eventually loses life.Therefore,prevention and treatment of liver failure is an urgent problem.It is of great clinical importance to find effective drugs to treat acute liver injury.Acetaminophen(APAP)is an effective antipyretic-analgesic that is commonly used,but it can cause severe hepatic necrosis when taken overdose.The mechanism underlying APAP-induced acute liver failure(ALF)particularly the factors involved in necrosis has remained to be investigated.In this study,we investigated the effect and mechanism of ARC on APAP-induced ALF mice model.300 mg·kg-1 of APAP was injected intraperitoneally in male ICR mice to induce AIF.While 200 mg·kg-1 of APAP was only induce mild liver injury.Quantitative proteome analysis of liver tissues was performed by 2-Nitrobenzenesulfenyl(NBS)tagging,2D-nano HPLC separation and MALDI-TOF/TOF MS analysis.The IHC method was performed to validate some highlighted proteins.36 differentially expressed proteins were identified at 24h after APAP(200 mg·kg-1)administration,in which 24 ones were up-regulated and 12 ones were down-regulated.44 differentially expressed proteins were identified at 24h after APAP(300 mg·kg-1)administration,in which 32 ones were up-regulated and 12 ones were down-regulated.The IHC analysis confirmed that Stat3 was overexpressed in necrotic regions,whose expression was positively correlated with necrosis;serially cut sections showed that the distribution of Stat3 was totally different with PCNA and Hsp70.While the p-Stat3 was negative in necrotic regions but positive in mild damage or recovered hepatocytes,the alternation from Stat3 to P-Stat3 in necrotic regions represented hepatocytes from death to renewal.The pathological changes of acute liver injury caused by APAP are mainly manifested by hepatocyte necrosis,and the main role of the inflammatory cells in the recovery period is to clear up the necrotic cell fragments.The Stat3 might be a DAMP molecular which attacted inflammatory cells to necrosis region.The high expression of P4hα1,Ncam,a-SMA and Cygb were involved in liver repair,they were not only the marker of the activation of HSC but also represented the intermediate stage of hepatocytes from damage or necrosis to renewal.The positive staining of Hsp70,p-Stat3,Ncam,α-SMA and c-kit in perivenous cells indicated a progenitor response of these cells to defense against toxicant.The expression of P4hα1,Ncam,α-SMA and Cygb allowed us to re-evaluate the role of hepatocytes in liver self-repair.This study confirmed the beneficial role of inflammatory cells,especially macrophages in AILI,not only through phagocytosis to remove necrotic cell debris,but also through the secretion and release of IL-6 to promote the activation of Stat3 and activation of Hsp70 to promote liver regeneration,and promote the expression of P4h to promote liver cell repair,so anti-inflammatory therapy for AILI is not necessary.ConA(Concanavalin A)induced autoimmune hepatitis is a well-established experimental model for immune-mediated liver injury.It has been widely used in the therapeutic studies of immune hepatitis.The in-depth analysis of dysregulated proteins from comparative proteomic results indicated that the activation of immune system resulted in the deregulation of autophagy.The upregulation of Adrml increased the release of ubiquitin by activating Uch37,a deubiqitinating enzyme.The Isg15 conjugation pathway overlapped ubiquitin conjugation pathway,leading to ubiquitin stress.The P62 would undergo ubiquitylation under ubiquitin stress and activate selective autophagy.In addition,the Isg15 and ISGylation led to upregulation of P62 and LC3 Ⅱ,the Isg15-congugated proteins were also degraded by lysosome through conjugation with P62.The upregulations of Pkr and Atg7 might lead to the increase of LC3.The over expression of P62 represented the accumulated autophagosome and a blockage of autophagic flux,eventually leading to autophagic cell death.It was Statl mediated the actication of immune system.Follow-up study validated that some immune related proteins,including Statl,Pkr,Atg7 and Adrml,were indeed upregulated.The accumulation of LC3B-Ⅱ and P62 was confirmed by IHC and WB.The pathological changes of ConA mediated immune liver injury were mainly manifested by the apoptosis of liver cells and the infiltration of inflammatory cells.ConA-induced liver injury is mainly mediated by the activation of the immune system mediated by IFN-y/Statl signaling pathway.IL-6 plays an inflammatory role by activating Statl in the presence of IFN-y.It is suggested that IFNy/IL-6/Statl signaling mediated the upregulation of those immuny related proteins.The enhanced Statl signaling pathway should be mainly regulated in the treatment of immunological liver disease.Arctigenin(ARC)is a biologically active lignan extracted from the seeds of arctium lappa L.(Compositae).Arctigenin was shown to have distinct anti-inflammatory properties.It could inhibite T lymphocyte proliferation,macrophage activation,and IFN-y/Statl and IL-6/Stat3 signalings.The therapeutic effects of arctigenin against concanavalin A(ConA)-induced hepatitis were studied in mice.Arctigenin pretreatment significantly elevated the survival rate of mice after ConA adminstration.Arctigenin pretreatment significantly alleviated the liver injury,as evidenced by biochemical and histopathological investigations,whose protective effects were comparable with PS and CsA.Arctigenin pretreatment decreased the synthesis and release of IL-6 and IFN-γ,but increased the ones of IL-10.Next,the quantitative proteomic analysis demonstrated that arctigenin pretreatment suppressed the activation of immune system through the inhibition of IFN-1 signaling,when it downregulated the protein expressions of Stat1,P-Stat1,Pkr,P-Pkr,Bnip3,Beclinl,Atg7,LC3B,Adrm1 and P62.Meanwhile,ARC pretreatment also reduced the gene expressions of Statl,Pkr and Atg7.These results suggested that arctigenin alleviated autophagy as well as apoptosis through inhibiting IFN-γ/IL-6/Statl and IL-6/Bnip3 pathways.In summary,the comparative proteomic analysis revealed that the activation of immune system led to ConA-induced hepatitis.The autophagy as well as apoptosis both has important clinical implications for the treatment of immune hepatitis.ARC might exert great therapeutic potential in immune-mediated liver injury.In conclusion,the significance of this study is to analyze the factors of injury,necrosis and repair and regeneration in drug-induced hepatitis with proteomics analysis and immunohistochemical technique,to study the pathogenesis of immune hepatitis and to draw the conclusion of inflammation in two kinds of hepatitis through the study and comparison of models.It was speculated that arctigenin with anti-inflammatory effect had protective effect on ConA-induced acute liver injury in mice.The therapeutic effect of arctigenin on immune hepatitis was verified by experiments.Recent evidence suggested that regulation of autophagy might be a potential strategy for viral hepatitis.It may be a new strategy for the treatment of immune hepatitis especially when the AIH and virus hepatitis occur simultaneously.Here,the protective mechanism of ARC on ConA-induced hepatitis indicated it might be a candidate for both viral hepatitis as well as autoimmune hepatitis.It deserves further study.