Study On Protection Against Mammalian Acute Liver Injury By Adenosine5’-monophosphate

Liver disease is one of the most common diseases which threaten our health. Liver injury is widespread due to its universality and diversity of incentives. Liver injury causes many complicating diseases including infection complications, liver laceration and bile leakage, secondary hemorrhage, acute liver/kidney/lung failure. And some severe patients will result in fulminant liver failure, which had high mortality. There are various types of liver injury which divided according to their causes, e.g. immunological liver injury, drug-induced liver injury, chemical liver injury, ischemia-reperfusion liver injury, viral liver injury, radiation-induced liver injury, alcohol liver injury, nonalcoholic fatty liver disease. Moreover the safe and effective drugs which can be used in therapy of acute liver injury are very limited in clinic setting. It’s necessary to find some safe and effective method for prevention and protection against liver injury. Here we used C57BL/6wide-type mice to get different acute liver injury model to investigate the protection against different acute liver injury models by5’-AMP through different mechanisms.C57BL/6J mice were given an intraperitoneal dose of D-galactosamine/Lipopolysaccha-ride (GalN (500mg/kg body weight)/LPS (5μg/kg body weight)) to induce acute liver injury.5’-AMP (5mg/20g) was administered to mice at30min before GalN/LPS treatment. The GalN/LPS-treated mice began to die at6h after GalN/LPS and survival rate was33.3%(24h), whereas survival rate in5’-AMP+GalN/LPS treated mice was93.3%(24h). Liver injury was assessed biochemically and histologically at4h after GalN/LPS challenge. Injection with5’-AMP significantly reduced activities of serum aspartate transaminase (AST) and alanine transaminase (ALT), mitigated hepatic necrosis, attenuated inflammatory cell infiltration. Quantitative RT-PCR analysis revealed that5’-AMP pretreatment significantly reduced hepatic mRNA expressions of tumor necrosis factor a (TNF-a), interleukin-6(IL-6), interleukin-1β (1L-1β), and monocyte chemotactic protein-1(MCP-1). Moreover, administration of GalN/LPS had similar lethality rate in both WT mice and adenosine receptor-deficient mice (A1AR-/-、 A2aAR-/-、A2bAR-/-and A3AR-/-mice). Pretreatment with5’-AMP also increased survival rate in GalN/LPS-induced acute liver injury in adenosine receptor-deficient mice. In addition, western blotting analysis revealed that5’-AMP failed to attenuate LPS-induced NF-κB p65nuclear translocation; chromatin immunoprecipitation (ChIP) assay demonstrated that5’-AMP reduced LPS-induced recruitment of NF-κB p65to inflammatory gene (TNF-a, IL-6, and IL-1/3) promoters. 5’-AMP markedly elevated intracellular level of adenosine, decreased the ratio of AdoMet to AdoHcy, which presents the intracellular potential of methylation, and inhibited the LPS-induced binding of H3K4dimethylation to the TNF-a promoter. An in vitro biochemical analysis revealed that adenosine directly attenuated LPS-induced recruitment of NF-κB to the TNF-a and IL-6promoters. Our findings demonstrate that elevated cellular adenosine effectively inhibited GalN/LPS-associated intrahepatic inflammatory responses, representing a potential protective agent for prevention of GalN/LPS-induced liver injury.Overnight fasting C57BL/6J mice were treated with acetaminophen (APAP300mg/kg body weight) and together with5’-AMP or30min pretreated with5’-AMP. Liver injury was assessed biochemically and histologically at24h after APAP challenge. Mice treated with APAP showed evidence of severe liver injury indicated by the significant increase of serum AST and ALT levels and large areas of severe extent of necrosis. Treatment with5’-AMP significantly reduced serum AST and ALT values and attenuated the area and extent of necrosis. In addition, in HepG2cells, MTT analysis showed that Cell viability was increased in5’-AMP-treated cells after APAP exposure. Propidium iodide (PI) staining revealed that5’-AMP markedly suppressed cell death after APAP treatment. At the early stage of APAP treatment (0-4h), it is well established that APAP hepatotoxicity causes cell death with depletion of hepatic GSH and ATP. The GSH and ATP levels in the livers of both mice treated with APAP and APAP plus5’-AMP were robustly reduced. Treatment with5’-AMP did not result in an early GSH and ATP recovery but improved at the late time point (24h). The hepatic GSH and ATP levels were significantly higher in5’-AMP-treated mice24h after APAP challenge. Moreover, quantitative RT-PCR analysis revealed that expressions of c-Jun and c-Fos mRNA were significantly elevated in APAP mice and suppressed by5’-AMP treatment. Furthermore, western blotting analysis showed that treatment of mice with5’-AMP significantly decreased the levels of phospho-JNK, phospho-MKK4, phospho-ASK1, and total ASK1while total JNK and MKK4levels were unaffected. In addition, co-immunoprecipitation analysis indicated that the basal ubiquitination of ASK1was in a low level after APAP challenge, and plus5’-AMP treatment significantly increased ASK1ubiquitination, which stimulated the degradation of ASK1, then the loss of total ASK1protein could decrease the phosphorylation of ASK1. Formalin test revealed that pretreatment with APAP or APAP plus5’-AMP markedly reduced the cumulative response time of formalin responses. Therefore, these data revealed that5’-AMP protects against APAP-induced acute liver injury, suggesting5’-AMP can be used with APAP to form a pharmaceutical composition as a potential protective agent for amelioration of APAP-induced liver injury.Furthermore, C57BL/6J mice were given an intraperitoneal dose of carbon tetrachloride (CCl4,300μL/kg body weight) to induce acute liver injury.5’-AMP (5mg/20g) was administered to mice at30min before CCl4challenge. Liver injury was assessed biochemically and histologically at0h,12h, and24h after CCI4treatment. Mice treated with CCl4showed evidence of liver injury indicated by the significant increase of serum AST, ALT and LDH activities and large areas of necrosis. Pretreatment with5’-AMP significantly reduced serum AST and ALT values and attenuated the extent of necrosis. In addition, CCl4induced lipid peroxidation, MDA as one of the final product of lipid peroxidation was markedly increased and GSH as clearance of free radical was markedly reduced, whereas5’-AMP treatment could significantly reduced the hepatic MDA levels and increased GSH levels. Moreover, quantitative RT-PCR analysis revealed that expressions of TNF-α, IL-6, and IL-1β mRNA were significantly elevated in CCl4mice and suppressed by5’-AMP treatment at24h after CCl4treatment. Furthermore, HPLC data showed that the hepatic ATP levels was markedly reduced after CCl4challenge and significantly improved by5’-AMP treatment. In addition, quantitative RT-PCR analysis indicated that5’-AMP significantly decreased UCP2mRNA expression and transcription factor peroxisome proliferator-activated receptor alpha (PPARa) mRNA expression. Our results indicated that5’-AMP protects against CCl4-induced acute liver injury, suggesting that5’-AMP as a potential protective agent for protection against CCl4-induced chemical acute liver injury.In conclusion, our study demonstrated that protection against mammalian acute liver injury by5’-AMP, suggesting5’-AMP may represent a potential therapeutic agent for amelioration of liver injury.