【作者】 姚晓敏；

【导师】 李燕； 程桂芳； 陈晓光； 申竹芳；

【作者基本信息】 中国协和医科大学 ， 药理学， 2009， 博士

【摘要】 肝脏缺血再灌注损伤(Hepatic ischemia reperfusion injury,HIRI)是指不同原因导致肝脏缺血(血流中断或供应不足),当恢复血供再灌注后,不仅不能使肝功能恢复,反而加重肝细胞功能障碍和结构损伤的现象。HIRI是肝脏外科疾病中常见的病理生理过程,如肝切除术、肝移植、肝外伤、失血性和心源性休克等,可引起严重的肝脏和临近器官的衰竭,发病率和死亡率均较高。目前HIRI确切的发病机制仍不十分清楚,也无完全理想的治疗手段,因此寻找有效手段(包括药物)用于HIRI治疗和预防是非常必要的。原发性肝癌是常见肝脏肿瘤之一,首选治疗方案是以外科手术切除为主的综合治疗方案。为减少肝叶切除术后(特别是伴发肝硬变患者)肝衰的发生率,临床多采用局部切除或不规则切除(缩小手术)的措施尽可能保存较多肝组织,但也存在部分患者的肝癌不能得到根治性切除的风险。所以,提高肝癌切除率,同时降低肝叶切除术后肝衰的发生率成为肝外科的难题,如何提高肝脏储备功能、促进残肝组织再生是解决这一问题关键。双环醇是治疗慢性病毒性肝炎的新药。临床治疗表明,双环醇可明显改善慢性乙型肝炎和丙型肝炎患者的临床症状和肝功能损伤。以往药理研究显示,双环醇对多种化学毒物如四氯化碳、乙醇、D—半乳糖胺(D-galactosamine,D-GaIN)等所致肝损伤及脂多糖(Lipopolysaccharide,LPS)、刀豆蛋白A(Concanavalin A,ConA)引起的免疫性肝炎均有明显的保护作用,其保肝作用机制与清除自由基、保护线粒体功能和调节炎症因子表达等密切相关。双环醇对大鼠HIRI和部分肝切除(Partialhepatectomy,PH)后肝脏再生的作用未见报道。本研究根据双环醇的药理作用特点,分别选用大鼠70%HIRI和PH两个模型,研究双环醇对HIRI的保护作用以及对PH大鼠肝脏再生的促进作用,并进一步探讨其相关分子机制。1.双环醇对大鼠HIRI的保护作用及机制研究采用Yoshizumi等方法建立大鼠HIRI模型,即缺血90 min后进行再灌1h、3h、6h和24h。结果表明,缺血再灌(Ischemia/reperfusion,I/R)大鼠出现严重的肝损伤,表现为血清总胆红素(Total bilirubin,TBil)和丙氨酸氨基转移酶(Alanine aminotransaminase,ALT)水平随再灌时间延长而逐渐升高,分别于再灌3h和6h达峰,为SM组的3倍和9倍。同时,肝细胞出现坏死、水样变性、伴大量炎症细胞浸润,动物7天存活率仅为60%。双环醇(100、200、300mg/kg)可显著降低I/R引起大鼠血清ALT和TBil的异常升高,并呈一定的剂量效应关系,上述肝脏病理形态学改变也明显减轻。双环醇(300mg/kg)还可明显减少HIRI引起的动物死亡,使存活率升至90%。已知HIRI导致肝损伤的主要原因是缺血再灌过程中产生大量活性氧(Reactiveoxygen species,ROS)和活性氮(Reactive nitrogen species,RNS)。本研究可见I/R模型组动物肝脏脂质过氧化产物丙二醛(Malondialdehyde,MDA)含量显著升高,约为假手术(Sham operation,SM)组的1.4倍,超氧化物歧化酶(Superoxide dismutase,SOD)活性则下降14%。双环醇(200、300 mg/kg)可显著抑制肝脏和微粒体MDA含量的升高,恢复抗氧化酶SOD的活性。氧化/硝化应激是缺血再灌引起肝损伤的重要机制之一。I/R大鼠肝脏髓过氧化物酶(Myeloperoxidase,MPO)和诱导型一氧化氮合酶(Inducible nitric oxide synthase,iNOS)活性明显升高,分别为SM组的1.3倍和1.6倍,同时肝脏亚硝酸盐/硝酸盐含量也显著升高,为SM组的1.7倍。双环醇(100、200、300 mg/kg)对肝脏MPO、iNOS活性和亚硝酸盐/硝酸盐水平升高具有明显的抑制作用,并呈一定剂量效应关系。缺血再灌诱导肝损伤的机制中,炎症因子和抗炎因子水平失衡是产生炎性损伤的主要因素。I/R大鼠血清肿瘤坏死因子α(Tumor necrosis factor-α,TNF-α)随再灌时间延长而逐渐升高,抗炎因子白细胞介素10(Interleukin-10,IL-10)的表达也明显增加。同时,模型组动物肝脏TNF-α和IL-10基因表达明显高于SM组。双环醇(300 mg/kg)可抑制I/R大鼠血清和肝脏TNF-α过表达,并进一步上调血清和肝脏IL-10表达。此外,双环醇(300 mg/kg)还可减少I/R大鼠肝脏细胞间粘附分子(Intercellular adhesion molecule,ICAM-1)和P-selectin的表达。肝脏缺血再灌时肠道革兰氏阴性杆菌释放的内毒素(脂多糖)可进一步加重肝损伤。I/R模型组血浆内毒素水平于再灌6h达峰,为SM组的16.5倍,24h时降至最低。双环醇(300 mg/kg)可明显抑制I/R大鼠血浆内毒素水平的异常升高。Toll样受体4(Toll-like receptor 4,TLR4)是LPS受体的关键组成部分,在LPS信号转导途径中起着重要作用。I/R大鼠肝组织TLR4蛋白表达显著升高,双环醇(300mg/kg)可明显下调I/R大鼠肝组织TLR4蛋白表达。核因子-κB(Nuclear factor-kappaB,NF-κB)是细胞内重要的转录因子之一,可引发一系列炎症因子的表达,从而加重肝损伤。I/R模型组肝脏核转录因子NF-κB的表达明显增加,同时胞浆中核因子-κB抑制因子(Inhibitor of NF-kappaB,IκB)表达则显著降低。双环醇(300 mg/kg)可明显抑制肝细胞核中NF-κB的表达,并增加胞浆中IκB的表达。由此可见,双环醇对大鼠HIRI具有显著的保护作用,其肝保护作用机理与抑制氧化应激,降低内毒素水平,调控TLR4和NF-κB表达,调节炎症因子/抗炎因子表达失衡密切相关。2.双环醇对HIRI大鼠肝脏CYP450活性和表达的影响细胞色素P450(Cytochrome P450,CYP450)是一种多功能药物代谢酶系,参与内外源性化合物的体内生物转化。缺血再灌产生的ROS和炎症因子等可损伤肝微粒体CYP450蛋白,从而影响肝脏的代谢功能。因此,本研究拟从CYP450同工酶活性、mRNA和蛋白表达的角度,反映双环醇对HIRI导致肝功能损伤的改善作用。肝微粒体CYP450含量可初步反映肝脏依赖CYP450催化的氧化活性。I/R大鼠肝微粒体CYP450含量于再灌3h和24h均显著下降(32%和45%)。双环醇(300mg/kg)对I/R大鼠肝微粒体CYP450含量的下降具有明显的保护作用。四种常见CYP450同工酶(CYP2C6、2C11、2E1和3A1/2)的活性和表达在HIRI大鼠出现差异性改变。CYP2C家族中2C6和2C11参与多种药物的体内代谢。I/R大鼠肝微粒体CYP2C6活性和mRNA表达均下降,而2C11虽活性下降,但mRNA表达无显著改变。双环醇(300 mg/kg)对CYP2C6和2C11活性和表达的下降均具有明显的抑制作用。CYP2E1可氧化乙醇、四氯化碳和其他引起肝毒性的小分子化合物。I/R大鼠肝微粒体CYP2E1活性于再灌3h和24h均显著降低(16%和40%),同时伴随蛋白表达的下降,但mRNA表达无明显改变。双环醇(300 mg/kg)可进一步下调肝脏CYP2E1的蛋白表达,但对其活性的改变无显著影响。CYP3A亚家族在临床口服药物的代谢中发挥重要作用。I/R大鼠肝微粒体CYP3A1/2活性明显下降(20%),同时CYP3A1 mRNA表达也减少,而CYP3A2mRNA表达无显著改变。CYP3A蛋白表达的改变与mRNA和活性改变的趋势一致。双环醇(300 mg/kg)对CYP3A1/2活性、3A1 mRNA和3A蛋白表达的下降具有显著的抑制作用。由此可见,双环醇预防给药可明显减轻HIRI时CYP450同工酶活性和表达的改变。作用机制可能与其抗氧化和抗炎作用密切相关。3.双环醇对PH大鼠肝脏再生的作用及机制研究PH模型是研究肝细胞再生的理想动物模型,本研究采用Higgins和Anderson方法建立大鼠70%PH模型,研究双环醇对PH大鼠肝脏再生的作用及其相关机制。PH术后大鼠肝重、肝重/体重和肝再生率随时间延长而逐渐增加,双环醇(300mg/kg)则可进一步提高PH大鼠肝重、肝重/体重和肝再生率,分别提高25%、26%和23%,并呈一定的剂量效应关系。此外,给药组PH大鼠残肝组织的分裂指数(Mitotic index,MI)(1.87±0.43)也显著高于对照组(1.16±0.27)。PH可导致大鼠出现严重的肝损伤,表现为术后6h时血清ALT和TBil水平显著升高,分别为假手术(Sham hepatectomy,SH)组的1.7和3.5倍,同时肝细胞出现水样和空泡样变性等形态学改变。双环醇(200、300 mg/kg)可剂量依赖性抑制PH大鼠血清ALT和TBil水平的异常升高,并减轻肝脏病理学改变。肝细胞增殖指数代表处于S期和G2-M期肝细胞的比例,可反映细胞分裂程度。PH模型组S和G2-M期的比例显著增加,为SH组的2.1倍,G0/G1期比例相应下降至SH组的64%。双环醇(300 mg/kg)给药组肝细胞G0/G1期比例下降至PH组的53%,S和G2-M期的比例则增加为PH组的1.4倍,提示双环醇可明显促进肝细胞增殖。增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)是与细胞增殖密切相关的核内酸性蛋白质,其表达于细胞周期的G1晚期和S期。PH大鼠肝组织PCNA表达显著增加,即有丝分裂期(G1和S期)的肝细胞数目明显增多,而双环醇(300mg/kg)可进一步上调PH大鼠肝组织PCNA的表达。肝糖原可通过糖酵解途径,三羧酸循环和柠檬酸途径等氧化分解,产生ATP来维持肝细胞的功能和完整性。术后6h、24h和48h时肝糖原水平显著降低至SH组的36%、31%和40%。双环醇(200、300 mg/kg)可剂量依赖性抑制PH大鼠肝糖原水平的下降。肝脏是蛋白质代谢非常旺盛的器官,是合成自身蛋白和大部分血浆蛋白的主要场所。PH术后各组大鼠血清总蛋白(Total protein,TP)和白蛋白(Albumin,ALB)水平均无显著改变,且肝组织总蛋白含量均在410±45～505±57 mg prot/g liver tissue之间,组间无显著差异。ROS可介导细胞生长停滞,还可激活细胞分裂周期的抑制性蛋白,从而阻碍肝细胞增殖。PH大鼠肝匀浆MDA含量于术后6h、24h和48h时分别升高为SH组的1.4倍、1.2倍和1.3倍,肝微粒体MDA含量在前两个时间点比SH组分别增加63%和48%,而48h时其含量与SH组基本相同。同时,肝脏SOD活性分别下降35%、17%和16%,GSH含量仅于6h时显著下降(49%),24h和48h时逐渐恢复。双环醇(300 mg/kg)不仅可抑制PH大鼠肝匀浆和微粒体中MDA的生成,还可恢复肝脏SOD活性,抑制肝脏GSH耗竭。由此可见,双环醇可显著提高PH大鼠残肝组织的再生能力,并可明显改善PH引起的肝损伤,其作用机制可能通过抑制氧化损伤以减轻ROS对肝细胞增殖的抑制作用,从而促进肝细胞增殖。4.双环醇对PH大鼠肝脏CYP450活性和表达的影响肝微粒体中CYP450主要参与各种内外源物在肝脏的生物转化作用。PH引起的氧化应激和炎症因子不仅阻碍肝细胞增殖,还可损伤肝微粒体CYP450酶。本研究可见PH大鼠肝微粒体CYP450含量于术后48h时下降至SH组的64%。双环醇(300mg/kg)对PH大鼠肝微粒体CYP450含量下降具有明显的抑制作用。由于CYP450同工酶的组成与其代谢功能密切相关,因此,进一步从CYP450同工酶(2C6、2C11、2E1和3A1/2)活性、mRNA和蛋白表达的角度,研究双环醇对PH引起肝功能损伤的改善作用。CYP2C6和2C11参与多种药物的体内代谢。PH大鼠肝微粒体CYP2C6活性于术后6h和48h时分别降至SH组的66%和27%,mRNA表达分别减少了24%和41%,双环醇(300mg/kg)可明显抑制CYP2C6活性和mRNA表达的下降。此外,PH大鼠肝微粒体CYP2C11活性于48h时也明显降低(58%),mRNA表达无显著改变。双环醇(300 mg/kg)对CYP2C11活性的下降无明显作用。CYP2E主要通过催化前致癌物去烷基、去硝基等反应而使之转化为致癌物。PH大鼠肝微粒体CYP2E1活性和mRNA表达均无显著改变,但其蛋白表达却于PH早期显著下降,双环醇(300 mg/kg)可明显下调CYP2E1蛋白表达。CYP3A是肝脏中含量最丰富的CYP450形式,参与大部分口服药物的首过效应。PH大鼠肝微粒体CYP3A1/2活性于48h时显著下降(36%),其mRNA和蛋白表达无明显改变。双环醇(300 mg/kg)可明显上调肝脏CYP3A1 mRNA和蛋白表达,并对CYP3A1/2活性的下降也有明显抑制作用。由此可见,双环醇对PH大鼠肝脏CYP450酶及部分同工酶活性和表达的改变有明显改善作用,其作用机制可能与其抗氧化、抗炎作用和酶诱导作用密切相关。综上所述,双环醇对大鼠HIRI具有显著的保护作用,并可明显提高PH大鼠肝脏的再生能力,还可改善I/R和PH大鼠肝脏CYP450活性及表达的改变。其作用机制可归纳为:(1)抗氧化作用:抑制MDA、NO生成和GSH耗竭,恢复SOD活性。(2)调控中性粒细胞聚集和粘附分子表达:抑制MPO和iNOS活性,下调ICAM-1和P-selectin表达。(3)抑制细菌内毒素转位:降低血浆内毒素水平。(4)调控炎症因子/抗炎因子平衡:下调TNF-α,上调IL-10表达。(5)调节TLR4信号通路:下调TLR4和NF-κB蛋白表达。(6)增加肝细胞分裂:上调PCNA表达,增加分裂指数MI和增殖指数。(7)改善CYP450同工酶改变:抑制肝微粒体CYP450含量和部分同工酶活性、mRNA及蛋白表达的改变。上述研究为进一步了解双环醇的肝保护作用特点以及临床上治疗HIRI和肝脏再生的可能性提供了有参考价值的实验依据。更多还原

【Abstract】 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.更多还原