| Hepatic ischemia-reperfusion injury(HIRI) is a phenomenon whereby cellular damage in ischemic liver can be accentuated after the reestablishment of oxygen flow.It has been implicated in the pathogenesis of a variety of clinical conditions,such as liver resection,liver transplantation,hypovolemic shock and trauma,and so on.In more severe cases,the issue of ischemia-reperfusion injury includes liver failure in association with remote organ failure,both of which result in high morbidity and mortality.At present, the exact pathogenesy remains unclear,and therapeutic method is also not ideal completely.Accordingly,development of new pharmaceutical strategies to ameliorate HIRI is always very important for achieving a better clinical outcome.Primary hepatic carcinoma is one of the common tumors,the preferred treatment of which is a comprehensive therapy with surgical excision.To reduce the incidence rate of liver failure after hepatolobectomy,especially in patients with liver fibrosis,local or irregular excision is adopted to keep more hepatic tissue.In this way,some patients can't gain the radical excision.Therefore,elevating resect rate and simultaneously cutting down liver failure after hepatolobectomy is a difficult problem in liver surgery.The key point to solve the problem is how to elevate liver functional reserve and promote liver regeneration.Bicyclol,4,4'-dimethoxy-5,6,5',6'-bis(dimethylene-dioxy)-2-hydroxymethyl -2'-methoxy carbonyl biphenyl,is an anti-hepatitis drug for the treatment of chronic hepatitis B patients in China.Previous studies have shown that bicyclol markedly alleviated experimental liver injury induced by certain toxins such as CCl4, D-galactosamine,acetaminophen,alcohol and concanavalin A,and its mechanism of hepatoprotection was related with its antioxidation,regulation of cytokine secretion and inhibition of apoptosis,etc.However,there was no report about the effect of bicyclol on HIRI-or PH-induced liver injury and on regenerative capacity.Therefore,70%HIRI and PH models in rats were selected to investigate the potential of bicyclol to alleviate liver injury induced by HIRI or PH,enhance the regenerative capacity,and possible mechanism in the present study. Part one:Protective effect of bicyclol on liver injury induced by hepatic warm ischemia/reperfusion in rats and related mechanismsThe model of HIRI in rats was established by the method of Yoshizumi et al.Rats were subjected to 90 minutes of hepatic ischemia followed by reperfusion for 1,3,6 and 24 h.The results of present study demonstrated that serum total bilirubin(TBil) and ALT levels were raised gradually with the increasing of reperfusion time in I/R rats,the peak levels of which were located at 3 and 6 h,and increased by 3 and 9 fold compared with sham-operated rats,respectively.The pathological changes induced by HIRI included the inflammation and necrosis of hepatocytes.Additionally,the 7-day survival rate was 60% in I/R rats.Bicyclol pretreatment(100,200 and 300 mg/kg) significantly inhibited the elevation of serum ALT and TBil levels in a dose-dependent manner,as well as improved liver histopathological changes and 7-day survival(90%).The productions of ROS and RNS were the major causes for liver injury during ischemia/reperfusion.Our data indicated that lipid peroxidation occured in hepatic I/R rats as evidenced by a significant increase(38%) of hepatic MDA content and a notable decrease of liver SOD activity(14%) in I/R rats.Bicyclol pretreatment(100,200 and 300 mg/kg) significantly inhibited the elevation of MDA in both liver tissues and microsomes, and restored liver SOD activity dose-dependently.It was reported that oxygen/nitrogen stress might play a decisive role in HIRI.A remarkable increase(28%and 61%) of liver MPO and iNOS activities was found at 3 h after reperfusion.Meanwhile,liver nitrate and nitrite levels were increased by 1.7 times in I/R rats compared with sham-operated rats.Pretreatment with bicyclol(100,200 and 300mg/kg) markedly suppressed the elevated activities of MPO and iNOS,and inhibited nitrate and nitrite levels dose-dependently.Disequilibrium between pro-and anti-inflammatory mediators is an important factor in modulating inflammatory injury during I/R.Our results showed that serum level of TNF-αwas gradually increased at 1,3,6 and 24 h after reperfusion in I/R rats,and IL-10 level was also significantly raised.Meanwhile,liver injury induced by I/R resulted in an increase of TNF-αmRNA expression.IL-10 mRNA expression was also raised in the early phase after reperfusion.Bicyclol pretreatment significantly down-regulated TNF-αexpression and further up-regulated IL-10 expression in I/R rats.In addition,high expressions of ICAM-1 and P-selectin,which were predominantly expressed in the vascular endothelium,were observed in I/R rats.Bicyclol pretreatment(300mg/kg) markedly inhibited the over-expressions of ICAM-1 and P-selectin.Endotoxin is a critical mediator to aggravate liver injury induced by hepatic I/R.A significant increase of plasma endotoxin was found in I/R rats and peak level(16.5 fold) was located at 6 h after reperfusion.At 24 h after reperfusion,endotoxin level in I/R rats was decreased to minimum.Pretreatment with bicyclol(300mg/kg) significantly inhibited the elevation of plasma endotoxin in certain extent.TLR4 is a critical component of receptor complex which show a key role in signal transduction of LPS.It was found that the expression of TLR4 was up-regulated in I/R rats compared with sham-operated rats.The over-expression of TLR4 in I/R rats was markedly down-regulated by bicyclol(300mg/kg).NF-κB,as a capital transcription factor in cell,may induce the expression of inflammatory cytokines,thereby mediate liver injury.The expression of NF-κB and the degradation of IκBαin I/R rats was remarkably increased in the present study.Bicyclol pretreatment(300mg/kg) significantly suppressed the activation of NF-κB and the degradation of IκBαinduced by HIRI in rats.In conclusion,bicyclol has showed an overall protective effect against HIRI in rats. The hepatoprotective effect of bicyclol is mostly related to its ability to attenuate oxidative stress and endotoxin elevation,to partially regulate the expression of TLR4 and NF-κB,and thereby to modulate the expression of inflammatory/anti-inflammatory cytokines.Part two:Effects of bicyclol on the activity and expression of liver microsomal cytochrome P450 in hepatic warm ischemia/reperfusionCytochrome P450(CYP450),as a major multifunctional drug-metabolizing enzyme, is involved in the biotransformation of endogenous and exogenous compounds.Hepatic ischemia reperfusion produced certain amount of ROS that may damage liver microsomal CYP450 protein,thereby influenced liver biotransformation function. Therefore,the present study was designed to investigate the effect of bicyclol on hepatic CYP450 activity,gene and protein expressions during hepatic ischemia and reperfusion (I/R) in rats.In general,total hepatic CYP450 content was believed to reflect the overall activity of CYP450-dependent oxidation.Liver microsomal CYP450 content was found to be significantly decreased by 32%and 45%at 3 and 24 h after reperfusion compared with sham-operated rats,respectively.Bicyclol administration(300mg/kg) markedly prevented such decrease.The activities and expressions of four major CYP450 isozymes(CYP2C6,2C11, 2E1 and 3A1/2) were changed in different extents by HIRI,while the activities of CYP2C6 and 2C11 were strikingly decreased in I/R rats.The decrease of CYP2C6 mRNA expression was in the same tendency with the change of activity,however, CYP2C11 mRNA expression was not alreated in I/R rats compared with sham-operated rats.The decrease of activity and gene expression of CYP2C6 and 2C11 can be inhibited by bicyclol pretreatment(300mg/kg).CYP2E1,an ethanol-inducible isoform of CYP450 isozymes,can oxidize ethanol, carbon tetrachloride and other chemical compounds to contribute to their hepatotoxicity. The activity of CYP2E1 was significantly suppressed at 3 and 24 h after reperfusion, with a coincidental reduction in its protein expression,while the mRNA expression of CYP2E1 was unchanged in I/R rats.Bicyclol pretreatment(300mg/kg) further reduced the protein expression of CYP2E1,whereas it had no influence on its activity.CYP3A subfamily was a major enzyme to be responsible for the metabolism of most oral drugs.In the present study,the activity of CYP3A1/2 and mRNA expression of CYP3A1 were found to be diminished after reperfusion,while that of CYP3A2 was unchanged in I/R rats.As for the change of CYP3A protein expression,there was a similar tendency to the change of the mRNA expression and activity.The above decreases of CYP3A1/2 were prevented by bicyclol pretreatment(300mg/kg).Our results suggested that bicyclol pretreatment may ameliorate the reduction of CYP450 isoforms in both activity and expression levels during HIRI,and this protection is likely due to its antioxidive property and inhibiting the expression of inflammatory cytokines.Part three:Effects of bieyclol on liver regeneration after partial hepatectomy in ratsPH is generally regarded as the strongest reproducible stimulus to hepatocyte proliferation,so 70%PH of Higgins and Anderson was selected in the present study to investigate the effect of bicyclol on liver regeneration.After hepatectomy,liver weight, liver weight/body weight and liver regeneration rate were gradually raised time-dependently in rats,which can be further augmented by bicyclol pretreatment(200 and 300 mg/kg) in a dose-dependent manner.Additionally,mitotic index in PH and bicyclol-pretreated rats was 1.16±0.27 and 1.87±0.43 at 48 h after PH,respectively.Liver injury induced by PH was indicated by the increasing of serum ALT and TBil levels,while the peak levels were 1.7 and 3.5 fold of that in SH rats at 6 h after reperfusion.Meawhile,liver pathological changes characterized by hydropic and vacuolar degeneration were also observed in PH rats.Bicyclol pretreatment(200 and 300 mg/kg) not only remarkably inhibited the elevation of serum ALT and TBil levels,but also attenuated the liver pathological changes,respectively.Proliferation index(PI) was equal to the sum of S phase%and G2-M phase%.The proportion of S and G2-M phase(PI) in PH rats was significantly increased by 2.1 fold at 48 h,and G0/G1 phase decreased by 36%compared with SH rats.Bicyclol(300 mg/kg) resulted in a further increase(44%) of PI,suggesting that bicyclol can promote hepatocyte proliferation.PCNA,as an intranuclear acidic protein associated with cell proliferation,is expressed in the late G1 phase and S phase.In the present study,PCNA positive hepatocytes were notably increased in PH rats.Bicyclol can further stimulate the increasing of PCNA in PH rats.It has been established that hepatocellular glycogen level is a potentially important and manageable factor in maintaining hepatocellular integrity and function by generating ATP.It was found that hepatic glycogen was significantly decreased by 64%,69%and 60%at 6,24 and 48 h after PH.Bicyclol(300 mg/kg) remarkably inhibited the decrease of hepatic glycogen in a dose-dependent manner.Liver is considered as a very vigorous organ in protein synthesis and metabolism.Our results showed that serum TP and ALB levels were not changed in PH rats compared with SH control.Meanwhile,total protein contents in liver tissues were 410±45～505±57 mg prot/g liver tissue in different experimental groups.It has been reported that ROS mediate cell growth arrest and activate proteins inhibiting cell cycle,thereby may influence liver regeneration.The results showed that lipid peroxidation occurred in PH rats,as indicated by the elevation of MDA content in liver homogenate and microsomes.Additionally,a notable decrease of liver SOD activity was observed(35%,17%and 16%) at different time points after PH.Liver GSH content was also markedly decreased by 49%at 6 h after reperfusion.Bicyclol(300 mg/kg) significantly inhibited the elevation of MDA,restored SOD activity and protected against GSH depletion.Therefore,bicyclol has showed a beneficial effect on regenerative capacity of the remnant liver tissue and ameliorated the injury induced by PH,probably due to its antioxidative property.Part four:Effects of bicyclol on the activity and expression of liver microsomal cytochrome P450 after partial hepatectomy in ratsLiver microsomal CYP450 plays an important role in biotransformation of both endogenous and exogenous compounds.Oxidative stress after PH can not only impede hepatocellular proliferation,but also damage liver microsomal CYP450.As we found in the present study,hepatic CYP450 content was significantly decreased by 46%in PH rats, while the decrease of liver microsomal CYP450 content can be prevented by bicyclol (300 mg/kg).The effect of bicyclol on CYP450 isozymes were further investigated in rats after PH,especially the profile of isozymes activity,as well as gene and protein expressions.CYP2C6 and CYP2C11 belonging to CYP2C subfamily were involved in the metabolism of several oral drugs.A 34%decrease of CYP2C6 activity was found at 6 h and subsequently decreased to 73%at 48 h after PH in rats.At the same time,the mRNA expression of CYP2C6 was remarkably reduced by 24%and 41%at 6 and 48 h. Moreover,a decrease of CYP2C11 activity(58%) was also observed at 48 h,and the mRNA expression was not changed in PH rats.Bicyclol(300 mg/kg) showed the protection on the inhibition of CYP2C6 and had no influence on CYP2C 11 activity.CYP2E can catalyze and transform precarcinogen to cancerogen by removing aldyl and nitro.The activity and gene expression of CYP2E1 were not changed in PH rats. Bicyclol pretreatment(300 mg/kg),however,can suppress CYP2E1 protein expression.CYP3A is the most redundant subfamily in liver tissue,which participates in the first pass effect of most oral drugs in clinical application.A 36%decrease of CYP3A1/2 activity was observed at 48 h after PH in rats.No significant changes on gene and protein expressions of CYP3A were found in all experimental groups.Bicyclol(300 mg/kg) can up-regulate the expression of CYP3A1,and inhibit the decrease of CYP3A1/2 activity in PH rats.Therefore,our results suggested that bicyclol pretreatment can improve the abnormalities of activity and expression of CYP isoforms after PH,and the protection is likely due to its antioxidantive,anti-inflammatory properties and enzyme induction.In conclusion,bicyclol had a remarkable hepatoprotective effect against HIRI and can enhance liver regenerative capacity after PH in rats.Furthermore,bicyclol improve the abnormalities of activity and expression of CYP450 isoforms in above models.The related mechanisms were summarized as follows:(1) Inhibition of oxidative stress:suppressing the formation of MDA and NO; inhibition of liver GSH depletion;and restoring the activity of SOD.(2) Modulation on accumulation of neutrophil and expression of adhesion molecule: inhibiting the activities of MPO and iNOS;down-regulating the expressions of ICAM-1 and P-selectin. (3) Prevention the translocation of bacterial endotoxin:inhibiting plasm endotoxin.(4) Maintenance of pro-and anti-inflammatory cytokines balance:down-regulating of TNF-αand up-regulating of IL-10.(5) Regulation on TLR4 signal pathway:down-regulating of TLR4 and NF-κB protein expressions.(6) Increasing cell division in liver:up-regulating of PCNA expression;increasing mitotic index(MI) and proliferation index(PI).(7) Improvement on the changes of CYP450 isozymes:restoring the microsomal total CYP450 content,regulating the mRNA and protein expressions of certain CYP450 isozymes.All above results provided the valuable experimental evidences for further investigation of the hepatoprotective effect of bicyclol and the possibility of clinical application for HIRI and PH.
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