Study On The Protective Effect And Related Mechanisms Of Bicyclol On Experimental Acute Liver Failure In Mice

Acute liver failure (ALF) is a rapid deterioration of liver function characterizedby hepatic encephalopathy, severe coagulopathy, jaundice, hydroperitoneum, andmultiple organ failure. The emergency liver transplantation is considered as the onlyeffective treatment that radically improves the outcome of ALF, while a highmortality (80-90%) occurred in ALF patients who did not receive livertransplantation. Although the causative agents are frequently known, thepathogenesis of this clinical syndrome remains unclear. Previous studies showed theinvolvement of endotoxemia, over-immunoreactions, proinflammatory cytokines,mitochondrial injury and disorder of energy metabolism in the pathogenesis of ALF.The effective drug therapies are still unavailable at present except antidoteN-acetylcysteine (specific for acetaminophen intoxication).ALF has drawn more and more attention because of its various etiologies,complicated pathogenesis and high mortality in clinic. Therefore, it is of greatimportance to find effective drugs for the treatment of ALF.Bicyclol (4, 4'-dimethoxy-5, 6, 5', 6'-Bis (dimethylene-dioxy)-2-hydroxymethyl-2'-methoxy carbonyl biphenyl; Fig1), a new synthetic anti-hepatitisdrug, has been widely used in patients with chronic viral hepatitis B to improve thedamaged liver function and to inhibit hepatitis B virus replication to a certain extent.Previous pharmacological studies showed that bicyclol significantly protected againstexperimental liver injury induced by certain toxins such as carbon tetracholride,D-galactosamine (GalN) and concanavalin A, etc. The hepatoprotective mechanism ofbicyclol is related to the regulation of cytokine secretion, improvement of impairedmitochondrion function and scavenging free radicals.The most frequent cause of ALF is viral hepatitis, followed by acetaminophen(APAP) overdose. Etiology of ALF is considered as not only one of the best indicators of prognosis, but also the instructions for specific therapy. It was reported thatexperimental liver failure induced by lipopolysaccharide (LPS)/GalN and APAP canbe used to reflect the clinical ALF caused by etiologies mentioned above. Therefore,the purpose of the present study was to investigate the effect and related mechanismsof bicyclol on acute hepatic failure induced by different etiologies in mice.Part one: Hepatoprotective effect of bicyclol on acute hepatic failure inducedby LPS/GalN in miceGalN, a specific hepatotoxic agent, was used to increase the sensitivity to thelethal effects of LPS in mice intoxicated by LPS/GalN. The biochemical andpathological changes in this model mostly resembled ALF caused by virus hepatitisin human.The present study demonstrated that LPS (15ug/kg)/GalN (800mg/kg) causedsevere liver injury including elevation of serum aminotransferase, jaundice,dysfunction of blood coagulation and necrosis of hepatocytes that may ultimatelylead to the lethal shock in mice. As a result, bicyclol showed significant protection asevidenced by the decrease of elevated serum aminotransferases and total bilirubin,and the improvement of liver pathological injury in a dose-dependent manner. Inaddition, a notable change in blood coagulation was observed as indicated by theprolongation of prothrombin time and decrease of prothrombin activity in LPS/GalNintoxicated mice. Administration of bicyclol (300mg/kg) significantly shortened theprolonged prothrombin time by 9-13% and enhanced the prothromibin activity by32-56%, respectively. Pretreatment with bicyclol (300mg/kg) was found to reducethe mortality (bicyclol pretreatment 25% vs LPS/GalN 70%) in ALF mice.Dexamethasone, as a positive control drug, also showed a protective effect on theabove biochemical markers.A proper balance between pro- and anti-inflammatory mediators is necessaryfor modulating an adequate immune response toward LPS. After the injection ofLPS/GalN, there was a massive induction of inflammatory cytokines TNF-αandIFN-γ, which were not counterbalanced by the anti-inflammatory cytokine IL-10. Bicyclol markedly inhibited the elevation of serum TNF-αand IFN-γ, while theIL-10 was enhanced. The above results suggested that the imbalance of pro- andanti-inflammatory mediators induced by LPS/GalN was significantly reversed bybicyclol to a certain extent.Intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associatedantigen 1 (LFA-1) were believed to mediate the cytotoxic injury. During LPS/GalNintoxication, the expressions of ICAM-1 and LFA-1 in hepatocytes andnon-parenchymal cells were enhanced. Bicyclol (300mg/kg) was found to reducethe immunoreactivity of ICAM—1 and LFA-1, except the expression of LFA-1 wasnot affected by three doses of bicyclol.CD14 and TLR4, as the critical components of receptor complex, play animportant role in the signal transduction of LPS. It was found that the expressions ofCD14 and TLR4 mRNA were significantly enhanced after LPS injection in thepresent study. Pretreatment with bicyclol (300mg/kg) can inhibited theover-expression of CD14 and TLR4 mRNA at 6h after LPS/GalN injection.In conclusion, bicyclol had a remarkable hepatoprotective effect on murine liverinjury induced by LPS/GalN, namely improvement of liver function, alleviation ofliver pathological injury and reduction in lethality. The effect of bicyclol was mainlythrough suppressing the transcription of LPS receptors, reversing the imbalance ofpro-and anti-inflammatory cytokines which may lead to the inhibition of adhesionmolecules expression.Part two: Hepatoprotective effect of bicyclol on acute hepatic failure inducedby APAP in miceAPAP has been used as an analgesic and over-the-counter drug for more than 40years; however, overdose with APAP is the most common cause of ALF in Denmark,the United Kingdom and the United States. The late poisoning of APAP is believed tobe similar to ALF induced by virus hepatitis and the accumulation of reactivemetabolite N-acetyl-p-benzoquinone (NAPQI) is considered as the main cause in theearly stage of APAP intoxication. Since the protective effect of bicyclol against LPS/GalN-induced ALF had been proved; the study aimed to investigate further theeffect and related mechanisms of bicyclol on ALF caused by APAP in mice.The liver injury induced by APAP was judged by the elevation of serumaminotransferase, jaundice and necrosis of hepatocytes that may ultimately lead to thelethal shock. Bicyclol significantly alleviated the hepatotoxicity by the decrease ofelevated aminotransferases and total bilirubin, and the improvement of liverpathological injury in a dose-dependent manner. Bicyclol (300mg/kg) also reducedthe mortality (bicyclol pretreatment 20%, bicyclol treatment 40% vs APAP 90%) inAPAP-treated mice.It was reported that oxygen/nitrogen stress might play a decisive role in initiationof APAP hepatotoxicity, because the increased free radicals may oxidize the lipids,nucleic acids and proteins. In the present study, nitric oxide and myeloperoxidaselevels in APAP-treated mice were significantly increased by 0.5 and 1.3 fold overthose in normal mice, while the activity of iNOS and the expressions of liver iNOSand nitrotyrosine were also enhanced after injection of APAP. Bicyclol (300mg/kg)markedly inhibited the elevation of nitric oxide and myeloperoxidase. Meanwhile, theactivity and the over-expression of iNOS and nitrotyrosine were also suppressed bybicyclol. In addition, the cytotoxicity, the elevation of ALT and the depletion of GSHinduced by APAP were also inhibited by bicyclol (5, 10, 50 and 100uM) in isolated rathepatocytes in vitro.Mitochondrial injury and disorder of energy metabolism are found to be involvedin the APAP hepatotoxicity and appear to be the key events. During APAPintoxication, liver glycogen and proteins contents in mice were decreased by 34%and 68%, respectively, while activity of ATPase was also reduced. Administration ofbicyclol significantly inhibited the reduction of glycogen and proteins and increasedthe activity of liver ATPase in APAP-intoxicated mice.Mitochondria permeability transition (MPT) is the principal mechanism inAPAP-induced liver injury. NAPQI is considered as a candidate to open the transitionpore and lead to a lethal event for the cells. MPT is usually represented by theabnormal changes of membrane depolarization and mitochondrial swelling after intoxication. The present study showed that isolated mitochondria had a rapid onset ofMPT as evidenced by the decreased uptake of the cationic dye Rodamine 123 and thelowered sensitivity to Ca²⁺ and pH after APAP injection. Bicyclol protectedmitochondria function by maintaining the normal transmembrane potential and theintegrity of membrane in APAP-intoxicated mice. The decreased uptake of thecationic dye and the lowered sensitivity to Ca²⁺ and pH were markedly improved bybicyclol.As the pro-inflammatory cytokines, serum IL-1βand macrophage inflammatoryprotein-2 (MIP-2) were markedly increased in APAP-treated mice (20000 and 1500fold of the control). Bicyclol significantly suppressed the increase of serum IL-1βandMIP-2 (53% and 84% by three doses, 40% and 78% by single dose of bicyclol).Therefore, bicyclol had a remarkable protective effect on murine liver injuryinduced by APAP, namely improvement of liver function, alleviation of liverpathological injury and reduction in lethality. The effect of bicyclol was mainlythrough suppressing the oxidative stress and the production of nitrotyrsine, theregulation of pro-inflammatory cytokines secretion and the improvement of impairedmitochondrion function.In conclusion, bicyclol had a remarkable protective effect against ALF in miceinduced by two frequent etiologies, namely LPS/GalN and APAP. The relatedmechanisms were summarized as follows:1) Regulation on the expressions of LPS receptors: down-regulating of CD14and TLR4 mRNA expressions.2) Maintenance of pro- and anti-inflammatory cytokines balance:down-regulating of TNF-α, IFN-γ, IL-1βand MIP-2 and up-regulating ofIL-10.3) Inhibition of adhesion molecules expression: down-regulating of theexpressions of ICAM-1 and LFA-1.4) Inhibition of oxygen/nitrogen stress: suppressing the activity and theexpression of iNOS to reduce the nitric oxide level; decreasing mycloperoxidase level and inhibition of liver GSH depletion; and preventingthe production of nitrotyrsine.5) Improvement of mitochondrion function: inhibition of glycogen and proteinsreduction; increasing the activity of ATPase and maintaining normaltransmembrane potential and the integrity of membrane of mitochondrion.All above results will provide valuable experimental evidences for furtherinvestigation of the hepatoprotective effect of bicyclol and the possibility of clinicalapplication for ALF.