Protective Effects And Molecular Mechanisms Of Berberine On Concanavalin A-induced Liver Injury In Mice

Background and Objective Autoimmune hepatitis(AIH)is a chronic inflammation of the liver that occurs when the body’s immune system attacks hepatocytes.Rapid progression to cirrhosis and liver failure is observed in patients with severe AIH.The diagnostic detection rate of AIH has increased;however,it is still difficult to be cured.The pathological features of concanavalin A(Con A)-induced hepatic injury in mice are similar to those of human AIH.Therefore,Con A-injected mice serve as a well-defined animal model of AIH.Elevated levels of proinflammatory cytokines have been documented in Con A-injected mice and patients with AIH,contributing greatly to hepatic injury.Berberine(BBR)has several pharmacological actions against bacterial infection,inflammation,hyperglycemia,hyperlipidemia,oxidative stress,and tumor.It has been used extensively in clinical practice,with few severe adverse effects.BBR was found to suppress immune reactions by activating adenosine 5′-monophosphate-activated protein kinase(AMPK.However,no reports are available about the protective effects of BBR on immune liver disease such as AIH.This study aimed to explore the protective effects of BBR on Con A-induced hepatic injury and the mechanisms in mice.Methods Male BALB/c(aged 6–8 weeks)were injected intravenously with Con A(20 mg/kg)and then exposed to BBR [50,100,and 200 mg/(kg × day)] by oral gavage for 3 days.The plasma levels of aminotransferases were measured after 8 h using a biochemical analyzer.The liver tissue injury was observed by macroscopic and histological analyses using hematoxylin and eosin staining.The release and expression of cytokines were detected using enzyme-linked immunosorbent assay and real-time polymerase chain reaction.Using Western blot analysis,AMPK activation was analyzed by detect ing the phosphorylation of acetyl coenzyme A carboxylase(ACC),a downstream target protein of AMPK.Con A-injected mice were pretreated with BBR combined with Compound C(20 mg/kg),a special inhibitor of AMPK,to investigate whether the protective effects of BBR on hepatic injury involved AMPK pathway.The mice were then injected intraperitoneally with Acadesine(AICAR)(50,100,and 200 mg/kg).The plasma levels of aminotransferases,lesions in liver tissue,and release and expression of cytokines were detected.Results Con A injection induced the swelling and disorganization of hepatocytes with multiple local necrosis.Also,plasma aminotransferases and proinflammatory cytokines were augmented significantly in Con A-challenged mice.Compared with Con A-challenged group,BBR at moderate and high doses(100 and 200 mg/kg)reduced histological changes and plasma levels of aminotransferases and proinflammatory cytokines significantly,increased the release of anti-inflammatory cytokines,and in turn attenuated Con A-induced hepatic injury.Nevertheless,no significant difference was observed between the two groups,namely,moderate-dose and high-dose BBR.Compared with the Con A-challenged group,the levels of aminotransferases and pro-inflammatory cytokines in the low-dose berberine-treated group(50 mg/kg)showed a decreasing trend,which was not statistically significant.Compared with the control group,the aminotransferase levels did not change significantly when the mice were pretreated with high-dose berberine alone(200 mg/kg).Compared with the con-trol and Con A-challenged groups,BBR pretreatment elevated the ratio of phosphorylated ACC and total ACC(p ACC/t ACC),suggesting the activation of AMPK by BRR.Compound C reduced p ACC/t ACC,confirming that it effectively inhibited AMPK activation by BBR.Compared with BBR plus Con A group,hepatic injury was exacer bated,levels of aminotransferases and proinflammatory cytokines increased,and release of anti-inflammatory cytokines decreased in Con A-challenged mice pretreated with BBR combined with Compound C.Compared with the control group,AICAR elevated p ACC/t ACC in liver tissue,suggesting that it effectively activated AMPK pathway.AICAR pretreatment attenuated Con A-induced hepatic injury and reduced the levels of aminotransferases and proinflammatory cytokines elevated by Con A challenge.Also,AICAR increased the release of anti-inflammatory cytokines.ConclusionsBBR pretreatment led to the reduction of proinflammatory cytokines,in turn,attenuating hepatic injury in Con A-challenged mice.Compound C reversed the protective effects of BBR on Con-A induced injury.AICAR had protective effects similar to those of BBR.Therefore,AMPK signaling was involved in BBR protection against Con-A induced injury.This study demonstrated the protective effects of BBR on hepatic injury induced by immune reactions.The findings might suggest a novel approach to treating AIH.