Bicyclol The Protective Effect And Mechanism Of Ethanol Induced Acute Liver Injury And Hepatocyte Apoptosis

ALD is induced by chronic alcohol abuse and represents a major cause of morbidity and mortality in western counties.The incidence of ALD is increasing with the improvement of living conditions in China recently.The clinical manifestation of ALD ranges from minor injury including steatosis to moderate and severe injury characterized by hepatitis and fibrosis/cirrhosis.Moreover,approximately 15%of the patients with established alcoholic cirrhosis develop to hepatocellular carcinoma.The development of ALD is a multifactoral process involving various genetic,nutritional and environmental factors,and its early pathogenesis is closely related with oxidative stress,endotoxin-mediated cytokine release,and apoptosis.Most injuries in early stage of ALD are reversible,so that further aggravation can be prevented by abstinence and symptomatic treatment.Therefore,development of drugs targeting at early injuries of ALD will have great clinical significance.Bicyclol is a novel anti-hepatitis drug for the treatment of chronic viral hepatitis. Clinical data showed that bicyclol can significantly improve the symptoms and damaged liver function in chronic type B and type C viral hepatitis in addition to inhibit the replication of type B hepatitis virus,featured by low rebounding rate,few adverse effects and convenient usage.Previous pharmacological studies have demonstrated that bicyclol offer protective effect on experimental liver injury induced by various chemical toxicants,such as carbon tetrachloride,D-galactosamine, concanavalin A and acetaminophen.The hepatoprotective mechanisms of bicyclol include the clearance of reactive oxygen species,regulation of cytokine secretion,and inhibition of apoptosis induced by immunological injury,etc.An open-labeled,randomized and controlled clinical study has reported that bicyclol had therapeutic effect on ALD,indicating its application perspective in the treatment of ALD.Previous study in our laboratory has also demostrated its role in protecting experimental animals from alcohol-induced steatosis,however,in-depth investigation on related mechanisms are still needed.Therefore,two simple and convenient models of acute alcohol-induced liver injury were established in the present study to observe the protective effect of bicyclol and related mechanisms,so as to provide experimental evidences for its clinical application. Partâ… :Protective effect of bicyclol on acute alcohol-induced liver injury in mice and related mechanismsSingle intragastic administration of alcohol(6g/kg)induced acute liver injury,as evidenced by elevation of serum ALT(2.3 times of control at 6h)and hepatic triglyceride levels(3.6 times of control at 6h),swelling and hydropic degeneration of hepatocytes,which reflected early biochemical and pathological changes in ALD. Bicyclol pretreatment(200,300mg/kg)significantly alleviated ALT elevation(60% and 71%)and hepatic triglyceride accumulation(18%and 35%)in a dose dependent manner.Moreover,bicyclol markedly improve the above liver pathological changes.Oxidative stress is the major pathogenetic factor in acute alcohol-induced liver injury.Hepatic TBARS content was elevated from 6h after alcohol administration and reached 2.7 fold of the control at 12 h.On the contrary,hepatic GSH content was decreased as early as 1.5 h after alcohol treatment and only 40%of control was found at 6 h.Pretreatment with bicyclol(300mg/kg)significantly inhibited the increase of TBARS content(32%)and prevented GSH depletion.In addition,a notable change of liver SOD,CAT,GR and GSH-px was observed as indicated by the decreasing of enzyme activity(35%,18%,49%and 45%)at 1.5 h after alcohol administration. Pretreatment with bicyclol(200,300mg/kg)significantly inhibited the decrease of SOD(returned to 94%and 99%of control,)and GSH-px(returned to 1.3 and 1.5 fold of control)dose-dependently.In addition,bicyclol(300mg/kg)also showed slight protective effect against alcohol-induced decrease of CAT and GR activity,although there was no statistical significance.TNF-αand IL-1βare two key inflammatory cytokines in ALD.Hepatic TNF-αand IL-1βlevels were elevated at 1.5 h and reached peak level at 12 h after alcohol administration.Bicyclol(200,300mg/kg)significantly alleviated hepatic TNF-αproduction by 33%and 47%,and IL-1βby 26%and 46%,respectively.The mRNA expression of TNF-αand IL-1βwas increased to 2 and 2.4 times of control by alcohol administration and bicyclol significantly inhibited such changes of two cytokines. Furthermore,bicyclol(300mg/kg)pretreatment can decrease the positive TNF-αstaining on Kupffer cells. Endotoxin is the main factor responsible for the activation of Kupffer cell.It was found that plasma endotoxin level was markedly increased at 1.5 h(9.6 fold of control) and declined to the normal level at 6 h after alcohol treatment in the present study. Bicyclol(200,300 mg/kg)significantly inhibited the elevation of plasma endotoxin level by 79%and 60%,respectively.CD-14 is the critical receptor in activation of Kupffer cell by endotoxin.Expression of CD-14 can be greatly induced at12h after alcohol intoxication and bicyclol(300mg/kg)reduced the over-expression of CD-14 significantly.In conclusion,bicyclol showed significant protective effect on acute alcohol-induced liver injury.The hepatoprotective action of bicyclol is mostly mediated by its ability to attenuate oxidative stress,suppress cytokine expression at both protein and gene level,and inhibit the activation of Kupffer cell by decreasing plasma endotoxin level and CD 14 expression.Partâ…¡:Effect of bicyclol on hepatocyte apoptosis in alcohol-intoxicated mice and related mechanismsThree consecutive intragastric administration of alcohol(6g/kg)in 12h interval resulted in an obvious liver injury and inflammation in mice,as indicated by the elevation of serum alanine transaminase(2.2 fold of control),increased hepatic COX-2 protein expression(2.2 fold of control),as well as liver pathological changes characterized by myelonic degeneration,vacuolar degeneration and inflammatory infiltration.Alcohol-intoxicated mice also showed obvious hepatocyte apoptosis,as positive cells in TUNEL staining were significantly increased.Bicyclol pretreatment can markedly decrease elevated serum transaminase,inhibit over-expression of COX-2 protein,and alleviate pathological changes.Furthermore,bicyclol diminished the number of TUNEL positive cells and staining intensity,suggesting its role in protection against alcohol-induced hepatocyte apoptosis.The metabolism of alcohol produces toxic metabolite-acetaldehyde and generates large amount of ROS at the same time,which is the priming factor for successive oxidative and nitrosative stress.Compared with control group,liver cytosolic ADH activity was increased by 46%after alcohol administration,while liver microsomal CYP2E1 activity and protein expression was increased to 2.2 fold and 2.6 fold of control,respectively.Pretreatment with bicyclol(300mg/kg)can decrease ADH and CYP2E1 activity by 20%and 25%respectively,and return CYP2E1 protein expression to approximate normal level.Moreover,liver mitochondrial and cytosolic ALDH activity in model group was elevated by 27%and 66%,respectively.Bicyclol (300mg/kg)can significantly regulate the increased cytosolic ALDH activity.Oxidative stress is an important pathogenetic factor in early stage of ALD.Liver oxidized protein content was increased to 1.7 fold,while cytosolic NAD⁺/NADH and mitochondrial GSH content was decreased by 30%and 10%by alcohol,when comparing with control group.Bicyclol pretreatment(200,300mg/kg)notably inhibited the elevation of protein carbonyl content,antagonized the change in NAD⁺/NADH,and maintained GSH at normal level.In addition to oxidative stress,alcohol can also induce nitrosative stress.After three consecutive alcohol administration,hepatic NO content,iNOS activity and protein expression,and NT protein expression was increased to 1.5,2.6,3.2 and 2.5 fold of control,respectively.Bicyclol pretreatment(200,300mg/kg)can effectively reversed the elevated NO content to normal level,and inhibited the over-expression of iNOS and NT by 40%and 53%respectively.Both oxidative and nitrosative stress in ALD can induce liver mitochondrial injury,leading to the activation of endogenous apoptosis signaling pathway.The uptake of R123 by liver mitochondria in model group was less than that in control group,and the sensitivity to high level of calcium-induced swelling was also decreased(about 50%of control group),suggesting the occurrence of membrane permeability transition.Bicyclol(300mg/kg)markedly alleviated injured mitochondrial function,as manifested by recovery of R123 uptake and increased changing amplitude in response to high calcium concentration.In addition,the function of mitochondrial respiratory chain was affected.MRCâ… andâ…£activity was decreased to 58%and 30%of control by alcohol administration,which was recovered to 65%and 85%of control by bicyclol(300mg/kg).Mitochondrial injury will ultimately lead to the release of cytochrome C into cytoplasm,as reflected by the two-fold increase of cytochrome C protein content in alcohol group.Bicyclol showed inhibitive effect on the release of cytochrome C.The expression of Bax and Bcl-X_S/L, as the pro-apoptotic and anti-apoptotic protein,were both up-regulated in alcohol-intoxicated mice(1.6 and 3.1 fold of control).Bicyclol(300mg/kg) pretreatment suppressed the over-expression of both Bax and Bcl-X_s/L.Fas and FasL are believed to be involved in death-receptor mediated exogenous apoptosis pathway.Protein expression of Fas,mFasL,and sFasL were all elevated in alcohol-intoxicated mice(1.5,4 and 2 fold of control).Bicyclol(300mg/kg)decreased the expression of Fas by 55%and recovered the expression of mFasL and sFasL to normal level.Furthermore,protein level of NF-κB in nucleus was decreased to 16% of control,while cytosolic ptotein level of I-κB was increased by 15%compared with control.Bicyclol(300mg/kg)can maintain the protein expression of NF-κB at normal level,and inhibit the over-expression of I-κB simultaneously(50%).In summary,bicyclol had significant protective effect on alcohol-induced hepatocyte apoptosis.Its mechanisms were related to its influence on alcohol metabolizing enzymes,inhibition on oxidative/nitrosative stress,improvement of mitochondrial injury,and alleviation of Fas/FasL over-expression,thereby suppressing both endogenous and exogenous apoptosis signaling pathways.In conclusion,bicyclol had a notable protective effect on alcohol-induced liver injury and hepatocyte apoptosis in mice.It can not only significantly inhibit elevation of serum transaminase and accumulation of hepatic triglyceride,but also improve histopathological changes,including hepatocyte swelling,hydropic degeneration, myelonic degeneration,vacuolar degeneration and inflammatory infiltration.In addition,bicyclol markedly decreased the number of TUNEL positive cells and attenuated staining intensity.The possible mechanisms included:1)Regulation on alcohol metabolizing enzyme:inhibit ADH and ALDH activity, inhibit CYP 2E1 activity as well as its protein expression2)Inhibition of oxidative stress:attenuate lipid peroxidation,restore GSH content,improve the activity of antioxidant enzymes,including SOD,GSH-px,GR, and CAT,decrease the content of oxidized protein,and restore NAD⁺/NADH3)Inhibition of nitrosative stress:reduce NO content,and inhibit over-expression of iNOS and nitrotyrosine4)Regulation on inflammatory cytokines:down-regulate both protein and mRNA expression of TNF-αand IL-1β5)Suppression of Kupffer cell activation:decrease endotoxin level,and inhibit CD-14 over-expression6)Attenuation of mitochondrial injury:maintain mitochondrial membrane integrity and normal membrane potential,restore mitochondrial GSH content, increase MRCâ… and MRCâ…£activity,and reduce cytochrome C release7)Regulation on expression of apoptosis-related proteins:down-regulate Fas, mFasL,sFasL,Bax,Bcl-X_S/L,IκB,while up-regulate NF-κBWith the results mentioned above,the present study will provide valuable experimental evidences for further investigation on the hepatoprotective effect of bicyclol and the possibility of clinical application in the treatment of ALD.