The Effect And Mechanism Of AICAR And Compound C Attenuate Liver Injury In Endotoxemia

Background：Endotoxemia is the clinical challenge with high mortality and poor prognosis,which can be induced during severe trauma, burns, and intestinal infections. As themost potent microbial mediator implicated in endotoxemia, lipopolysaccharide (LPS)can initiate immune cell activation, induce release of large amounts of proinflamma-tory cytokines and chemokines, and trigger multiple organ injury, which is typicallycharacterized with liver injury and dysfunction. Recently, AMP-activated proteinkinase (AMPK) has been reported as one of anti-inflammatory signals, and its ligand5-Aminoimidazole-4-carboxamide (AICAR) has been studied in some animal modelssuch as colitis, asthma. However, it remains to be elucidated whether activation orinhibition of AMPK signal can attenuate endotoxemia-induced immune response andliver injury.Objective：To study the effects of AICAR as AMPK activator and Compound C as AMPKinhibitor on LPS induced liver injury.Methods：(1) RAW264.7cells were treated with AICAR or/and compound C, and theexpression of phospho-AMPKα was determined by western blot after the treatment.(2) RAW264.7cells were divided into five groups as Control, LPS, LPS+AICAR, LPS+Compound C, and LPS+AICAR+Compound C. The cells werepretreated with AICAR or/and compound C1hour before LPS challenge. Theexpression of p-NF-κB p65was determined by western blot1hour after LPSchallenge.(3) BALB/c mice were randomly divided into five groups: Control (i.p. injectionof saline, LPS (i.p. injection of LPS2mg/kg body weight), LPS+AICAR (i.p.injection of AICAR500mg/kg1h before LPS challenge), LPS+Compound C(i.p.injection of Compound C20mg/kg1h before LPS challenge), and LPS+AICAR+ Compound C (i.p. injection of the same doses of both chemicals1h before LPSchallenge). The mice were sacrificed12hours after LPS injection, and tissues andbloods were collected for analysis.(4) The survival experiments were performed in five group mice mentionedabove with injection of LPS (20mg/kg body weight). The injection of AICAR and/orcompound C remained the same dose as above. Survival of mice was monitored for24hours.Result：(1) AICAR induced phospho–AMPKα expression, while compound C inhibitedphospho–AMPKα expression in RAW264.7cells. AICAR and compound C bothinhibited LPS-induced phospho–NFκB p65expression in RAW264.7cells.(2) LPS induced live injury with increased serum ALT, AST, and TNF levels,high histological injury score, apoptosis of hepatocytes, accumulation of macrophageand neutrophil evidenced with increment of CD68expression and MPO activity inliver tissue.(3) AICAR or compound C treatment decreased ALT, AST, and TNF levels inserum, reduced histological injury score, CD68expression, MPO activity, apoptosiscell number in liver of mice with endotoxemia. However, combination of AICAR andcompound C treatment diminished the beneficial effect of each single treatment.(4) In survival experiments, AICAR or compound C treatment improved survivalof endotoxemic mice.Conclusion：AICAR or compound C treatment attenuates LPS-induced liver dysfunction,indicating that activation or inhibition AMPK signal can inhibit endotoxemia-inducedimmune response and liver injury. AMPK signal may provide an alternative to thecurrent clinical treatments for endotoxemia.