摘要
肝脏缺血再灌注损伤(hepatic ischemia reperfusion injury, HIRI)是常见的临床病理生理过程,是肝切除术、肝移植、肝外伤、失血性和心源性休克等疾病共同经历的一个多因素参与的过程。HIRI是术后肝功能异常、原发性肝移植无功能、肝功能衰竭的重要原因。研究表明,HIRI是影响肝移植术后移植物长期存活的危险因素。如何干预HIRI导致的肝功能损害以及保护肝脏功能,愈来愈受到全球重视。探索防治HIRI的药物已成为当前研究热点。
为了减轻肝脏缺血再灌注损伤,许多学者做了大量的研究,如缩短缺血时间、缺血预处理、缺血后处理及药物处理等,从而达到减轻肝细胞损伤的目的。药物处理是针对缺血再灌注的某个或某些机制利用药物的药理作用,减少再灌注损伤过程中产生的氧自由基、钙超载、炎性介质等或利用某些活性物质直接或间接的药理作用来增强肝组织或肝细胞对缺血再灌注损伤的耐受能力,减轻组织或细胞的损伤,从而达到保护的作用。促红细胞生成素(erythropoietin, EPO)由髓质与肾皮质交界处的球旁细胞合成的,能刺激骨髓造血的唾液糖蛋白类激素,促进红细胞的生成是其基本作用。
EPO可通过与中枢神经系统、内皮细胞、实体肿瘤、子宫等多种非红系组织细胞表达的促红细胞生成素受体(erythropoietin recptor, EPOR)结合,形成EPO-EPOR信号传导系统,参与多种非造血生物活动,并对细胞和组织器官有保护作用。
在肝脏疾病的治疗过程中,使用重组人红细胞生成素(recombinant human erythropoietin, rHuEPO)治疗小鼠和大鼠,能够显著降低缺血再灌注所致损伤,包括肝脏组织病理学分数、酶学指标、细胞凋亡、有害细胞内信号转导和活性氧作用。研究表明,rHuEPO体内对缺血再灌注和其他原因引起的肝损伤保护,并不是直接作用于肝细胞,推测rHuEPO可能直接或间接地作用于非肝实质细胞如肝窦内皮细胞和kupffer细胞等减轻肝脏损伤。因此,值得深入探讨rHuEPO对肝脏缺血再灌注损伤的保护作用和机制。
本文采用大鼠自体肝移植动物模型,模拟肝移植过程中肝脏经历缺血再灌注损伤的过程,通过研究血清中肝脏酶学指标的变化,肝脏病理形态学改变,肝细胞凋亡等,观察rHuEPO预处理对肝脏缺血再灌注损伤的保护作用。并结合调控细胞存活的关键信号通路-磷脂酰肌醇3-激酶(Phosphatidylinositol3-kinase, PI3K)信号通路的变化,探讨rHuEPO的作用机制,为研究rHuEPO预处理对肝脏缺血再灌注损伤的保护作用提供科学依据。
第一部分大鼠自体肝移植模型的建立情况
目的
肝移植动物模型是肝移植实验研究的非常重要的基础。Kamada提出的“双套袖法”大鼠肝移植是目前应用较广泛且成熟的肝移植动物模型,但此模型不吻合肝动脉。而本中心建立的大鼠自体肝移植动物模型,能模拟临床肝移植过程,完全排除免疫、血管吻合等因素,全面反映肝脏缺血再灌注损伤的病理生理过程。现总结该动物模型建立情况以及术中、术后管理经验,以期该模型成功率高、重复性好。
方法
1.实验动物SPF级纯系、雄性的SD大鼠55只,其中27只大鼠用于早期动物模型建立训练。另28只大鼠按照实验设计的要求和药物预处理的方案进行实验,其中假手术组(sham)3只,其余各组I/R, I/R+rHuEPO, I/R+DMSO,I/R+rHuEPO+LY294002, I/R+LY294002组,每组5只大鼠。
2.手术操作经术前准备后,充分游离肝周结构,在使肝脏肝素化(含肝素37.5U/m1)后,使用4℃含肝素(12.5U/m1)的复方乳酸林格氏液经门静脉、腹主动脉双重恒压冷灌注。除冷灌注保存以及血管吻合操作外,其余方法同大鼠原位肝移植。
3.观察时间及内容SD大鼠的观察的时间点分别为24h,48h,72h,观察大鼠的生存质量和存活情况。
4.统计方法运用SPSS13.0软件绘制生存曲线。
结果
1.以肝素化大鼠时阻断门静脉到开始冷灌注为热缺血时间,到门静脉开放为无肝期。为标准化研究实验对象,其中热缺血时间为2.5-3.5min、冷灌注时间均统一为15min。将无肝期时间设定为30min。
2.在实验初期,大鼠存活满足实验设计要求达到92.31%。在研究实验药物对大鼠72h生存率影响时,发现使用LY294002(PI3K特异性抑制剂)预处理组的存活率低。
小结
自体移植的动物模型完全符合实验设计要求。手术操作时,应严格按照动物实验的基本原则,注意小细节处理,能显著改善大鼠术后恢复过程,甚至可以直接影响实验动物的存活。
第二部分rHuEPO预处理对大鼠自体肝移植缺血再灌注损伤的保护作用
目的
建立的自体肝移植缺血再灌注损伤模型,从血清中肝脏酶学指标变化、肝脏病理形态改变和肝细胞凋亡三方面,初步探讨rHuEPO对大鼠自体肝移植缺血再灌注损伤的保护作用。
方法
1.建立自体肝移植的动物模型(同第一部分)。
2.实验动物SD大鼠随机60只,分成3组：①Group Ⅰ:假手术组(Sham)20只。本组实验对象仅游离肝脏不给药。②Group Ⅱ:缺血再灌注组(I/R)20只。术前30min经尾静脉给予1ml0.9%生理盐水(normal saline, NS)。③Group Ⅲ: rHuEPO预处理组(I/R+rHuEPO)20只。本组实验对象行自体原位肝移植手术。在术前30min,经尾静脉给予按照体重(Kg)×3000U/kg的rHuEPO并将混合在0.9%NS中,容积为1m1。除Sham组外,所有大鼠行自体肝移植手术,均经历15mmin冷灌注时间,30min的无肝期。
3.标本的采集和处理按照肝脏再灌注后3h,6h,12h,24h各时点每次处死5只大鼠。从1VC穿刺采集血标本4ml后注入血生化管中,室温血液自然凝固后离心,取上层血清标本冻存于-20℃备用。切取大鼠肝中叶部分作为标本检测,分别置液氮冻存和10%中性甲醛固定备用。
4.观察指标血清酶学变化,病理形态学改变以及细胞凋亡指数。
5.统计学分析计量资料用均数±标准差(x±s)表示,用SPSS13.0软件进行统计学分析,组均数间比较采用析因设计的方差分析,组间两两比较采用最小差异t检验(LSD-t),若方差不齐,采用Dunnett's T3检验。P<0.05认为差异有统计学意义。
结果
1.血清酶学指标变化
血清标本在再灌注后3h,6h,12h及24h各时间点取材,比较同一时间点血清ALT、AST及LDH水平差异。在同一时间点,各组动物血清中AST水平,差异无统计学意义(未标明具体数据)。但是血清ALT、LDH的变化差异,有统计学意义(P<0.05)。大鼠经自体肝移植手术后,I/R组和rHuEPO预处理组(I/R+rHuEPO)的血清ALT、LDH水平均较假手术组(Sham)显著升高(P<0.01)。I/R+rHuEPO组同I/R组比较,血清中ALT、LDH水平均显著降低,其差异具有统计学意义(P<0.05)。此外,肝脏经历缺血再灌注后,血清ALT、LDH水平在再灌注后3h内迅速上升,于再灌注后6h达到较高水平后呈下降趋势,但至再灌注后24h为止,血清中ALT、LDH水平均未恢复至正常水平,同Sham组比较差异具有统计学意义(P<0.01)。
2.组织形态学变化
选取再灌注6h后的肝脏标本,作为观察对象。HE染色结果提示：假手术组(Sham)大鼠肝组织结构基本正常,肝窦排列整齐,未见中性粒细胞浸润。大鼠经历缺血再灌注损伤后6h,I/R组肝细胞肿胀,肝细胞边界不清,肝小叶中央静脉和肝血窦淤血明显,肝血窦狭窄,肝细胞索排列紊乱,肝细胞可见不同程度的水肿变性和空泡状变性,偶可见点状坏死,肝组织中有明显中性粒细胞的聚集、浸润,以中央静脉周围为甚。同Sham组比较,I/R组病理形态学评分高,差异具有统计学意义(P<0.05)。但是,经rHuEPO预处理的肝脏(I/R+rHuEPO)组织,肝小叶结构基本正常,小叶中央静脉、肝细胞索、肝血窦结构清晰,细胞变性不明显,中性粒细胞浸润减轻。
3.肝细胞凋亡情况
TUNEL法检测提示：肝脏缺血再灌注时组大鼠肝脏可见凋亡细胞。检测结果表明：缺血再灌注组(I/R)和I/R+rHuPO预处理组TUNEL阳性细胞较Sham组显著升高,差异具有统计学意义(P<0.01); I/R+rHuPO预处理组TUNEL阳性细胞显著低于I/R组(P<0.05); Sham组偶见阳性细胞。
小结
本部分以SD大鼠为实验动物,运用大鼠自体肝移植模型模拟肝脏缺血再灌注损伤。通过检测血清酶学指标、组织病理学及细胞凋亡指数的变化证实了该模型能够反应缺血再灌注损伤的病理生理过程。rHuEPO预处理能够降低血清酶学指标,改善肝脏组织形态,具有减轻缺血再灌注导致的损伤,对大鼠肝脏缺血再灌注损伤有保护作用。
第三部分PI3K/AKT信号通路在rHuEPO预处理对大鼠自体肝移植缺血再灌注损伤保护中的作用
目的
在第二部分实验的基础上,实验拟利用大鼠自体肝移植的模型及PI3K特异性抑制剂LY294002,通过检测血清酶学指标的变化,肝脏病理形态改变和AKT蛋白的激活情况,探讨PI3K/AKT信号通路是否参与rHuEPO预处理对肝脏缺血再灌注损伤的保护作用。
方法
1.自体肝移植动物模型的建立(同第一部分)。
2.实验动物①Group Ⅰ:Sham组；②Group Ⅱ:缺血再灌注组(I/R)；③Group Ⅲ:rHuEPO预处理组(I/R+rHuEPO),上述三组第二部分实验已完成,本部分一并纳入分析。本部分实验采用60只SD大鼠,随机分入下列组别：④Group Ⅳ:I/R+DMSO组(20只),术前30min,经尾静脉给予1ml2%(v/v) DMSO;⑤Group V:I/R+LY294002组(20只),术前60min,按照1mg/kg的LY294002将其溶解于1ml2%(v/v) DMSO中经尾静脉给药：⑥Group Ⅵ: I/R+rHuEPO+LY294002组(20只),术前60min,按照1mg/kg的LY294002将其溶解于0.5ml2%(v/v) DMSO中经尾静脉给药,术前30min再按照体重(Kg)×3000U/kg rHuEPO并将混合在0.5ml的0.9%NS中,经尾静脉再次给药。上述各组动物,均行自体肝移植手术。
3.标本的采集和保存(同第二部分实验)。
4.观察血清酶学的指标检查,肝脏组织形态学评分(同第二部分实验)。
5. Western Blot检测rHuEPO和LY294002单独及联合预处理对总AKT和磷酸化AKTSer473蛋白表达的影响。
6.统计学分析分析方法(同第二部分实验)。
结果
1.P13K抑制剂LY294002预处理组对肝脏酶学指标的影响
在肝脏再灌注(无肝期结束开始计时)3h,6h,12h及24h各时间点,I/R组,I/R+LY2940002组,Sham组,I/R+DMSO血清ALT、LDH水平均有差异,且有统计学意义(P<0.01)。自体肝移植大鼠在经历缺血再灌注损伤后,其血清中ALT、LDH均较Sham组显著升高,差异有统计学意义(P<0.01)。其中(?)[/R+DMSO组同I/R组比较,结果显示两组SD大鼠在经历肝脏缺血再灌注损伤后,其血清中ALT、LDH的变化均无显著的统计学差异(P>0.05)。但是I/R+LY294002组,血清ALT、LDH水平在肝脏再灌注后上述时间点较I/R组和I/R+DMSO组均显著升高,其差异有统计学意义(P<0.05)。此外,I/R+LY294002组SD大鼠经历缺血再灌注后,血清ALT、LDH水平在再灌注后3h内迅速上升,其后上升趋势变缓,血清ALT、LDH水平于再灌注后6h达到较高水平后逐渐下降,但至再灌注24h为止,血清ALT、LDH水平均未恢复至正常水平,同Sham组比较差异具有统计学意义(P<0.01)。
2.药物预处理对肝脏组织形态学的影响
为了进一步观察PI3K抑制剂LY294002预处理对肝脏缺血再灌注的作用,我们对复灌后6h各组大鼠肝脏的组织形态学变化进行量化比较。再灌注后6h,在I/R+DMSO组,肝细胞肿胀,肝窦内淤血及少量炎性细胞浸润。LY294002预处理组(I/R+LY294002)大鼠病理学检查可见肝细胞索结构明显破坏、大量的肝细胞变性、溶解,其病理损伤评分较高。在灌注后6h,同I/R+DMSO组,I/R+LY294002+rHuEPO组比较,I/R+LY294002组组织病理学评分高,差异具有统计学意义。但LY294002+rHuEPO联合预处理的SD大鼠,灌注结束后6h,少量坏死肝细胞形态不可辨别,细胞崩解、坏死,细胞核分解。综合分析实验各组(Sham、I/R、I/R+DMSO、I/R+LY29400、I/R+rHuEPO、 I/R+LY294002+rHuEPO)的组织病理学染色结果：在肝脏灌注结束后6h,使用LY294002预处理,反而加重了肝脏缺血再灌注后肝脏组织结构的破坏,而联合使用rHuEPO预处理后,肝脏病理损伤有所减轻。rHuEPO预处理组病理损伤评分低,提示其对肝脏的组织形态有保护作用。
3. rHuEPO联合LY294002预处理对肝缺血再灌注损伤的影响
在实验设计中,将rHuEPO和LY294002联合使用对自体肝移植大鼠进行预处理。研究发现在再灌注后同一时点,I/R+rHuEPO+LY294002组同I/R+rHuEPO组比较,血清ALTvLDH水平显著升高,其差异具有统计学意义(P<0.05)。但是IR+rHuEPO+LY294002组与I/R+LY294002组比较时,发现血清ALT、LDH水平在HuEPO联合LY294002预处理组有所降低,其差异具有统计学意义(P<0.05)。
4.药物预处理对总AKT和磷酸化AKTser473蛋白表达的影响
以相应蛋白条带平均光强度值来表示AKT和p-AKT活化水平的相对强度。通过p-AKT ser473/β-actin和AKT/β-actin平均光强度值的比值,反映PI3K/AKT信号通路的活化情况。肝脏灌注结束后6h, Sham、I/R、I/R+DMSO、I/R+LY29400. I/R+rHuEPO、I/R+LY294002+rHuEPO,各组总AKT表达的差异无统计学意义(P>0.05)。但是各组磷酸化AKTser473和总AKT (p-AKT ser473/AKT)进行比较,发现肝脏经历缺血再灌注损伤后,p-AKTser473磷酸化水平增高,同sham组比较,其差异具有统计学意义(P<0.05)。与I/R组比较,rHuEPO预处理组磷酸化AKTser473的增高有统计学意义(P<0.05)。LY294002预处理组同I/R组比较,其磷酸化的AKTser473水平,显著降低,提示LY294002预处理能够抑制缺血再灌注以及rHuEPO预处理诱导肝组织中的磷酸化AKTser473蛋白增高。
小结
1.通过蛋白质印迹技术发现,rHuEPO预处理诱导磷酸化AKT ser473表达增加与rHuEPO预处理引起的组织病理学损伤,肝脏酶学的释放显著减少相一致。
2.P13K阻断剂LY294002显著抑制了rHuEPO预处理诱导磷酸化AKTser473表达的增加和对肝脏的保护。rHuEPO联合LY294002预处理,能减轻LY294002预处理对肝脏缺血再灌注的损伤。
3.进一步支持P13K/AKT信号通路的激活介导了rHuEPO预处理的肝脏保护作用。肝脏组织中磷酸化AKTser473、总AKT的蛋白表达的变化证实了rHuEPO预处理对肝脏缺血再灌注损伤的保护作用有PI3K/AKT细胞信号通路参与。
第四部分eNOS在rHuEPO预处理对大鼠自体肝移植缺血再灌注损伤保护中的作用
目的
本部分实验通过检测肝脏组织标本中总eNOS、磷酸化eNOSSer1177的蛋白表达,eNOSmRNA、EPOR mRNA的变化,以及血清中NO和ET-1含量变化,探讨PI3K/AKT/eNOS细胞信号通路参与rHuEPO预处理对肝脏缺血再灌注损伤的保护的作用机制。
方法
1.收集肝脏组织标本和血清样本(第二、三部分实验保存)。
2.实时荧光定量PCR检测eNOS mRNA, EPOR mRNA表达。
3.Western Blot检测肝脏组织中总eNOS和p-eNOSSer1177的蛋白表达。
4.硝酸盐还原酶法测定大鼠血清样本中NO含量。
5.酶联免疫吸附法测定大鼠血清样本中ET-1含量。
6.统计学分析分析方法(同第二部分实验)。
结果
1.rHuEPO预处理对肝脏组织中eNOS mRNA含量的影响
肝脏再灌注后6h,与假手术组(Sham)比较,经历缺血再灌注损伤各组肝脏组织内eNOS mRNA含量显著增高,其差异具有统计学意义(P<0.05)。同其I/R, I/R+DMSO和I/R+LY294002组比较, I/R+rHuEPO预处理组肝脏组织内eNOS mRNA含量显著增高,其差异具有统计学意义(P<0.05)。但是肝脏组织内eNOS mRNA含量在I/R+rHuEPO和I/R+LY294002+rHuEPO组,差异无统计学意义(P>0.05)。
2. rHuEPO预处理对肝脏组织中EROP mRNA含量的影响
实时荧光定量PCR结果提示：与假手术组(Sham)比较,缺血再灌注6h后的肝脏组织内EPOR mRNA均显著升高,其差异有统计学意义(P<0.05),提示缺血再灌注能够增加肝脏组织中EPOR mRNA含量。同各组缺血再灌注比较,rHuEPO预处理组(I/R+rHuEPO)并不能增加肝脏组织内EPOR mRNA含量。使用PI3K抑制剂LY294002预处理亦不能增加或减少肝脏组织内EPOR mRNA含量。
3.药物预处理对总eNOS,磷酸化eNOS Ser1177蛋白表达的影响
通过免疫印迹分析比较缺血再灌注6h后的肝脏组织中eNOS蛋白和p-eNOSSer1177的变化,以各组磷酸化eNOSSer1177/β-actin与总eNOS/β-actin的比值进行比较。研究发现各组间总eNOS蛋白表达的差异无统计学意义(P>0.05)。磷酸化eNOS Ser1177在假手术组(Sham)中有较少的表达。但同I/R组比较,在rHuEPO预处理组使肝脏磷酸化eNOSSer1177增加,其差异具有统计学意义(P<0.05)。但是在使用PI3K特异性抑制剂LY294002预处理,磷酸化eNOSSer1177的表达并没有增加。联合使用rHuEPO和LY294002预处理发现,rHuEPO预处理能改善LY294002对p-eNOS Ser1177的抑制。p-eNOSSer1177在缺血再灌注各组中的变化趋势同AKT及磷酸化AKTser473的趋势一致。rHuEPO预处理能够激活肝脏组织中磷酸化的AKTser473和eNOSSer1177蛋白表达增加。
4.rHuEPO预处理对大鼠血清中NO含量的影响
用硝酸酶还原法进行检查肝脏灌注后3h,6h,12h,24h血清样本中NO含量的变化。与假手术组(Sham)比较,经历缺血再灌注各组血清内NO含量减少,其差异具有统计学意义(P<0.05)。在同一时间点,rHuEPO预处理组血清中NO含量显著增加。I/R+LY294002组的血清NO含量显著降低,同其他各组(Sham, I/R, I/R+DMSO, I/R+rHuEPO)比较差异具有统计学意义。在缺血再灌注6h后,经历缺血再灌注的各组大鼠的血清NO含量较低。这一变化趋势与肝脏酶学指标的释放,组织病理形态学的改变相一致。随着时间的推移,大鼠血清NO水平逐渐升高, rHuEPO预处理组的血清NO水平,在灌注24h后,与假手术组的大鼠血清NO水平基本接近。
5.rHuEPO预处理对大鼠血清中ET-1含量的影响
用ELASA法进行检查肝脏灌注后3h,6h,12h,24h血清样本中ET-1含量的变化。与假手术组比较,缺血再灌注损伤组血清内ET-1含量显著增加(P<0.05)。在同一时间点,rHuEPO预处理组降低了血清中ET-1含量。I/R+LY294002组的血清ET-1含量显著升高,同其他各组(Sham, I/R, I/R+DMSO, I/R+rHuEPO)比较差异具有统计学意义。在缺血再灌注3h,经历缺血再灌注的各组大鼠的血清ET-1含量较高,随着时间的推移,大鼠血清ET-1水平逐渐降低,rHuEPO预处理组的血清ET-1水平,再灌注24h后,与假手术组血清ET-1水平基本接近。
小结
1.rHuEPO预处理并不能增加肝脏组织内EPOR mRNA含量,但能增加肝脏组织内eNOS mRNA含量。LY294002预处理组对EPOR mRNA含量无影响。
2. rHuEPO预处理增加肝脏组织p-eNOSser1177蛋白的表达,LY294002预处理组抑制了p-eNOSser1177蛋白的表达,与AKT及p-AKTser473的趋势一致,提示PI3K/AKT/eNOS细胞信号通路参与了rHuEPO对肝脏缺血再灌注损伤的保护。
3.当肝脏经历缺血再灌注损伤后,其血清中NO和ET-1之间的平衡关系被破坏。rHuEPO预处理组能够显著增加血清NO的水平,调节血清中NO和ET-1间的平衡,证实了rHuEPO预处理通过激活p-eNOSser1177表达和增加NO的释放,减轻肝脏缺血再灌注导致的损伤。
结论
1.自体肝移植的动物模型,能够模拟肝缺血再灌注损伤的病理生理过程。肝脏经历缺血再灌注后,血清酶学指标(ALT、LDH)显著升高,病理形态发生改变,肝细胞有坏死和凋亡发生。
2. rHuEPO预处理可显著抑制肝脏缺血再灌注损伤导致的血清中ALT、 LDH水平的升高,减轻肝脏的病理损伤,改善肝脏组织形态结构,抑制肝细胞凋亡。
3.LY294002预处理显著抑制了rHuEPO对肝脏缺血再灌注损伤的保护。提示rHuEPO预处理可能通过激活PI3K/AKT细胞信号通路发挥对肝脏缺血再灌注损伤的保护。
4.LY294002预处理显著抑制了rHuEPO诱导的p-eNOSser1177表达的增加。提示rHuEPO预处理可能通过激活PI3K/AKT/eNOS细胞信号通路发挥对肝脏缺血再灌注损伤的保护作用。
5. rHuEPO预处理通过激活肝脏组织p-eNOSser1177,增加血清中NO含量,调节血清中NO与ET-1之间的平衡,发挥对肝脏缺血再灌注损伤的保护作用。
Hepatic ischemia reperfusion injury(HIRI) is a frequently encountered complication in a variety of clinical scenarios including hepatectomy, liver transplantation, liver trauma, hemorrhagic shock and cardiogenic shock. It is also the most important reason for post-operation liver dysfunction, primary graft failure and a risk factor influencing the long-term graft survival. How to intervene liver failure caused by HIRI and protect the liver attracts more and more global attention. To explore the drug to prevention and control of HIRI has become a research hotspot.
In order to reduce HIRI, many scholars have done a lot of researches. They shorten ischemia time, perform ischemia preconditioning and ischemia posttreatment so as to reduce the injury. Exploring the drug to prevent HIRI has become a research hotspot. Drug treatment aims at some mechanism during HIRI, utilizing its pharmacological effects to reduce oxygen free radicals, calcium overloading, inflammatory mediators. Or it takes advantage of certain active substance to enhance the tolerance of liver cell to HIRI directly and indirectly. Thus, injury can be relieved so as to achieve the protection. Erythropoietin (EPO) is a salivary glycoprotein hormone synthesized mainly by the juxtaglomerular cells located in the junction of renal cortex and medulla which can stimulate hematopoiesis in bone marrow. Its primary duty is to promote erythropoiesis.
EPO can form EPO-EPOR signaling system through combining with erythropoietin recptor(EPOR) expressed by some kinds of non-erythroid organs, tissues, and cells, such as central nervous system, endothelial cells, uterus. Then it can involve in varieties of non-hematopoietic biological activity. More and more evidences show that EPO has the protective function of organs, tissues and cells. However, this function is independently of its hematopoietic effect.
In the process of liver disease treatment, using recombinant human erythropoietin (rHuEPO) treatment in mice and rats, and can significantly reduce the ischemia/reperfusion(I/R) injury, including hepatic histopathological scores, enzymology indexes, apoptosis, harmful cells signal transduction and the function of reactive oxygen species. Decisive pathophysiological mechanisms contributing to the in vivo I/R injury to the liver are mediated or enhanced by endothelial and Kupffer cells, i.e. by liver cells other than hepatocytes rHuEPO may directly or indirectly act on these cells and thus influence the injurious response. Thus, elucidating the protective mechanism of rHuEPO on liver I/R injury is necessary.
In this study, we employed liver auto-transplantation rat model to imitate the HIRI process. We discussed the protective effect of rHuEPO against HIRI through studying the changes in liver enzyme indicators, pathological change and cell apoptosisin liver cell and cell apoptosis. We also studied the PI3K signal pathway at gene transcription and protein expression level, maneged to find a possible rHuEPO protection mechanism and also hope to provide some scientific evidence for further understanding of the rHuEPO preconditioned protection.
Part1liver auto-transplantation model of rats
Purpose
Ideal animal model is the basis of liver transplantation experimental study."double sleeve" rat model raised by Kamada is a mature and wide-used animal model for liver transplantation. However, this model dose not anastomosis liver artery, can not be completely ruled out the immune factors, thus can't fully represent HIRI pathophysiology. So we choose the HIRI model built in our center to imitate clinical liver transplantation, ruled out the immune and anastomosis liver artery factors. Now we summarize the experience obtained during building the model, management before and after the operation to expect the model has a high success rate and repeatability.
Method
1. Animals55pure SPF male SD rats were divided into two groups.27of them were trained for early model building. The other28were experimented following the experimental design and drug preconditioning method. Among the28rats group, three were in sham group, the other were equally divided into I/R, I/R+rHuEPO, I/R+DMSO, I/R+rHuEPO+LY294002and I/R+LY294002group, five each.
2. Preoperative preparation was done. After the liver and related structureswere fully freed, the rats were heparinized with37.5U/ml heparin. Then4℃lactated Ringer's solution which contains12.5U/ml heparin were perfused through portal vein and abdominal aorta using double constant pressure cold perfusion. Methods were the same as rat autologous transplantation model except preservation in cold reperfusion and vascular anastomosis operation.
3. Observation time and content life quality and survival rate of rats were observed at24h,48h and72h.
4. Statistical analysis SPSS13.0was employed to draw the survival curve.
Result
1.Warm anhepatice phase was from blocking the PV of heparinized rats to the beginning of cold reperfusion while anhepatic phase lasted until reopening of PV. To standardized research subjects, we set the anhepatic phase to same30min. Wherein warm ischemia time was2.5-3.5min and cold perfusion time was unified to15min.
2. Early in the experiment, survival rate of the rats achieves92.31%that met the design requirement. Modeling process was also successful and no modeling-caused death appeared. Studying the impact of experimental drug on the rat72h survival rate, there was no death after42h except the LY294002proconditioned group. In I/R+LY2940002group,72-hour survival rate is low and it could be promoted when combined with rHuEPO.
Summary
liver auto-transplantation model simulated the whole clinical liver transplantation process. Thus we can observe HIRI more directly and objectively. When animal model fully met this experimental design, operations followed the primary principle of animal experiment strictly and operators payedattention to proper handling of the details, we can improve the post-operation recovery process significantly and even influence the survival of the rats directly.
Part2the protective effect of rHuEPO preconditioning of liver auto-transplantation in rats
Purpose
We managed to observe protective effect of rHuEPO preconditioning through liver auto-transplantation HIRI rat model and preliminarily discussed the protective effect at liver enzyme indicator level, pathomorphology and liver cell apotosis in rats.
Method
1. Established liver auto-transplantation rat model.
2. Animals60rats were randomly assigned into three groups.Group Ⅰ:Sham group (n=20).Laparotomy and dissociation of the liver only. Group Ⅱ:I/R group (n=20). The rats received1ml saline via the tail vein30min before the operation. Group Ⅲ:I/R+rHuEPO group (n=20). The rats were treated the same as group Ⅱ but received a single intravenous dose of weight(kg)×3000U/kg rHuEPO intravenously via the tail vein30min before the operation. The rHuEPO was diluted in1ml saline.Except sham group, during orthotopic autologous transplantation, all liver all liver went through15min cold reperfusion and30min anhepatic phase.
3.Sample collection and management
In each group,5rats were humanely killed after3,6,12and24h from the beginning of reperfusion.4ml blood samples were collected via the inferior vena cava and then injected into biochemical pipe. Let the blood coagulated under room temperature, centrifuged, then took the upper serum sample to store at-20℃for further analysis.The left lobe of the liver was removed, snap frozen in liquid nitrogen and stored in10%buffered formalin.
4. Measurement of Serum ALT, AST, LDH, histopathologic analysis and TUNEL assay to detect the liver cell apoptosis
5. Statistical analysis measurement data were presented as mean±standard deviations. The groups were compared using factorial design analysis of variance. A p value<0.5is considered statistically significant. All the analyses are performed using SPSS13.0.
RESULT
1. Alterations in the serum AST, ALT and LDH levels
The serum levels of AST were not significantly different among the groups at the same time point (data not shown), but the serum levels of ALT and LDH were significantly different among the groups (P<0.05). The changes in the serum levels of ALT and LDH after the operation, compared with the shamgroup, were significantly increased in I/R+rHuEPO group and were statistically different (P<0.05). However, at the same point after hepatic ischemia reperfusion, serum ALT and LDH levels significantly decreased in I/R+rHuEPO group compared with I/R group and were statistically different(P<0.05).The serum levels of ALT and LDH increased rapidly3hafter reperfusion and reached a peak at6h in all groups and then decreased. It did not come back to common level yet24h after reperfusion and were significantly different compared with sham group(P<0.05).
2. Histopathological alteration
Sectionsof the liver obtained at6h after reperfusion were evaluated for histopathological analysis. The liver tissue in sham group showed basically normal structure. The liver sinusoidals were arranged in order with no neutrophil infiltration. after6h reperfusion, I/R group showed severe swelling of liver cells, ill-liver cells, obvious hepatic lobule central vein and hepatic sinusoid congestion, narrow hepatic sinusoid and disorders liver cells, varying degrees of edema and vocuolar degeneration, possible punctate necrotic. Significant neutrophil aggregation and infiltration can be seen, especially around the central vein.The histological scores for I/R injury were significantly higher than sham groupand were statistically different(P<0.05). However, in I/R+rHuEPO group, the liver congestion got some relief. Hepatic lobule was basically normal.Lobule central vein, hepatic cell cord, liver sinusoids got clear structure. Cell degeneration neutrophil infiltration is not obvious.
3. liver cell apoptosis
Choose the liver tissue sample which has bear6h HIRI to check. TUNEL positive cells in I/R and I/R+rHuEPO was much larger than sham group(P<0.01). Apoptotic index of I/R+rHuEPO group is significantly lower than I/R group(P<0.01). Few TUNEL positive cell was seen in sham group.
Summary
We established liver auto-transplantation experimental model rats to simulatethe process of HIRI. Changes in enzyme indicator level, pathomorphology level and liver cell apoptosis level indicated that this model represented pathophysiological process of HIRI well. rHuEPO preconditioning can decrease serum ALT and LDH, reduce cell apoptosis and structure damage and express protective function of HIRI caused by transplantation.
Part3PI3K/AKT signal pathway in the protective effect of rHuEPO preconditioning against HIRI
Purpose
On the basis of Part2, we employed the model the same as Part1and PI3K inhibitor LY294002. We measured the serum enzymology, tissue morphology and the protein expression of AKT and p-AKTser473to discuss whether rHuEPO involved in the protection against HIRI.
Method
1. Model (similar to Part1)
2. Animals Group Ⅰ:Sham group (n=20); Group Ⅱ:I/R group (n=20); Group Ⅲ: I/R+rHuEPO group (n=20); Group Ⅳ:I/R+DMSO group (n=20);GroupⅤ:I/R+LY294002group (n-20);Group Ⅵ:I/R+rHuEPO+LY294002group (n=20). All the disposal is similar to Part1. As Group Ⅰ, Ⅱ and Ⅲ have been done in Part2, we just need to collect the data and analysis.
3. Sample collection and reservation (similar to Part2)
4. Alterations in serum enzyme and histological score (similar to Part2)
5. We employed western blot to analysis the expression of AKT and p-AKTser473in six groups Relative active level of AKT and p-AKTser473is expressed by corresponding protein bar average light intensity value. Activation of AKT/p-AKTser47signal pathway is reflected by the ratio of p-AKTser473/β-actin and AKT/β-actin average value.
6. Statistical analysis The statistical method is similar to Part2. All the analyses are performed using SPSS13.0.
RESULT
1. Effect of LY294002(a PI3K inhibitor) preconditioning on liver enzymes
The serum levels of AST, LDHwere significantly different among group I/R, I/R+LY2940002and Sham at3h,6h,12h and24h after reperfusion(timing begins at the end of anhepatic phase)(P<0.01). After being through HIRI, the serum ALT and LDH of the orthotopic autologous transplantation ratswere significantly different when compared with sham group(P<0.01). Wherein changes in the serum levels of ALT and LDH between I/R+DMSO and Ⅰ/Rgroup is not statistically different(P>0.05). Increment of the serum ALT and LDH level in I/R+LY294002group is much higher than I/Rand I/R+DMSO group at the time point referred before(P<0.05). However, serum levels of ALT and LDH increase rapidly in3hafter reperfusion and reached a peak at6h in I/R+LY294002group and then decreased. It still did not come back to common level until24h after reperfusion and were significantly different compared to sham group (P<0.01)
2. Effect of pharmaceuticals pretreatment on the liver tissue morphology
HE staining results suggest:after6h reperfusion, in I/R+DMSO group, liver structure is near-normal. However, hepatic sinusoid congestion and light inflammatory infiltration can be seen. LY294002preconditioning group showed hepatocellularcode damage, vast cell degeneration, dissolved and got high histological score. The score was statistically deferent when compared to I/R+DMSO and I/R+LY294002+rHuEPO group. After6h reperfusion, the SD rats pretreated with I/R+LY294002+rHuEPO showed light cell degeneration, near-normalhepatic lobule, and light neutrophil aggregation and infiltration. But necrosis was also can be seen locally. The structure of necrotic liver cell can not be extinguished. All the results reveal that rHuEPO preconditioning can significantly improve the histology of liver after6h reperfusion. Damage was aggravated in I/R+LY294002group, but can be relieved when combined with rHuEPO.
3. Effect of LY294002combined with rHuEPO preconditioning against liver I/R injury
The serum levels of AST and LDH increased significantly in group I/R+rHuEPO+LY294002when compared with I/R+rHuEPO. It was statistically different(P<0.05). But the serum levels of AST and LDH decreased when compared with I/R+LY294002group at the same time point. It was also statistically different (P<0.05).
4. Effect of pharmaceuticals pretreatment on AKT and p-AKTser473rHuEPO preconditioning
After6h reperfusion, difference of AKT expression among Sham, I/R, I/R+DMSO, I/R+LY29400, I/R+rHuEPO and I/R+LY294002+rHuEPO groups was not statistically significant(P>0.05). Butthe ratio of p-AKTser473and total AKT, p-AKTser473phosphorylation increased significantly after HIRI compared with sham group. And the difference was statistically significant(P<0.05). p-AKTser473increased after rHuEPO preconditioning and when compared with I/R the increment of rHuEPOgroupwasstatistically significant(P<0.05). In I/R+LY294002, we observed LY294002inhibit the activation of p-AKTser473and when compared with I/R group, p-AKTser473decreased obviously. LY294002inhibit the p-AKTser473expression induced by rHuEPO preconditioning after HIRI at the same time.
Summary
1.Western bloting found that rHuEPO preconditioning induced the p-AKTser473expression increased significantly.Increment of p-AKTser473expression, histological damage and decrement of liver enzyme induced by rHuEPO preconditioning were corresponding to each other.
2. LY294002(a specific PI3K inhibitor) inhibit p-AKTser473expression induced by rHuEPO and protective effect of rHuEPO.This experiment through the joint use of rHuEPO and LY294002for rats pretreatment, the research results show that can eliminate rHuEPO preconditioning on liver I/R injury of protection.
3. To further support PI3K/AKT signaling pathway activation mediates the rHuEPO pretreatment of liver protection. The protein of p-AKTser473, total AKT protein expression changes confirmed the PI3K/AKT signal pathway involvement in protective effect of rHuEPO preconditioning against HIRI.
Part4The role of eNOS in protective effect of rHuEPO preconditioning against HIRI
Purpose
To study the PI3K/AKT signal pathway involvement in protective effect of rHuEPO preconditioning, this part we discussed the protective mechanism of rHuEPO againt HIRI though detecting the tissue sample, p-eNOSser1177and eNOS protein expression, transcription level, content of NO and ET-1in serum sample.
Method
1. Liver tissue and serum sample (collected in part2and part3)
2. Transcription level of EPOR and eNOS in the liver sample was detected by qPCR.
3. Western blot to detect-protein expression of total eNOS and p-eNOSser1177.
4. The serum NO level was detected by Nitrate reduction method.
5. ET-1in the serum sample was detected by biotin double antibody sandwich ELISA method.
6. Statistical analysis The statistical method is similar to Part2. All the analyses are performed using SPSS13.0.
RESULT
1. eNOS mRNA in liver tissue alternation by rHuEPO preconditioning
After6h reperfusion, eNOS mRNA in I/R group increased significantly compared with Sham group. The difference was statistically significant(P<0.05). eNOS mRNA in I/R+rHuEPO group increased significantly compared with I/R, I/R+DMSO and I/R+LY294002group. The differences were statistically significant (P<0.05). But the difference of eNOS mRNA content between I/R+rHuEPO and I/R+LY294002+rHuEPO group was not significant (P>0.05).
2. EROP mRNA in liver tissue alternation by rHuEPO preconditioning
After6h reperfusion, EROP mRNA increased significantly compared with Sham group. The difference was statistically significant (P<0.05). We found this through real-time PCR. When compared with the HIRI group, we did not find content of eNOS mRNAcan be increased by rHuEPO preconditioning or can be decreased by LY294002.
3. Effect of pharmaceuticals pretreatment on eNOS protein expression and p-eNOSSer1177
After6h reperfusion were detected by western blot. We compare the ratio of p-Enos Ser1177and total Enos. It turned out to be no significant difference of Enos protein expression among each group (P>0.05).p-eNOSSer1177was seldom expressed in sham group. However p-eNOSSerll77expressed in rHuEPO preconditioning group increased significantly compared with I/R group.The difference was statistically significant (P<0.05). But p-eNOSSer1177did not increase in I/R+LY294002 group.rHuEPO can improve LY294002inhibition of p-eNOSSer1177, we found this when combine use of rHuEPO and LY29400. The alteration trend of p-EnosSer1177in HIRI groups is in accord with alteration trend of AKT及p-AKTser473. These indicated that P13K signal pathway involved in the adjustment of p-eNOSSer1177.rHuEPO can activate phosphorylation of p-AKTser473and p-eNOSSer1177.
4. The influence of rHuEPO pretreatment on the serum NO in rats
Content of serum NO in liver tissue sample at3h,6h,12h,24h after reperfusion was detected by Nitric acid enzyme reduction method. NO in HIRI groups decreased significantly when compared with Sham group (P<0.05). rHuEPO preconditioning can increase NO significantly at the same time point.NO in I/R+LY294002decreased significantly and it were statistically different compared with other groups (P<0.05). After6h ischemic reperfusion, NO of the rats in I/R groups decreased to nadir. Then NO increased as time went by. NO of the rHuEPO preconditioning groups was very close to that of Sham group after24h reperfusion.
5. Effect of rHuEPO pretreatment on the serum ET-1in rats
Content of serum ET-1in liver serum sample at3h,6h,12h,24h after reperfusion was detected by ELISA method. ET-1in I/R groups increased significantly when compared with Sham group (P<0.05). rHuEPO preconditioning can decreaseET significantly at the same time point.ET in I/R+LY294002increased significantly and it were statistically different compared with other groups (P<0.05). After3h reperfusion, in I/R groups, the serum ET-1increased to a high level. Then the serum ET-1decreased as time went by. The serum ET-1in the rHuEPO preconditioning groups was very close to that in the sham group after24h reperfusion.
Summary
1. rHuEPO preconditioning does not increase the liver tissues EPOR mRNA levels, but can increase the eNOS mRNA levels. No influence about the EPOR mRNA levels in liver, when combined use of PI3K inhibitor LY294002.
2. rHuEPO pretreatment increase the expression of p-eNOSser1177protein and LY294002pretreatment inhibits the expression of p-eNOSser1177protein in liver tissue. It indicates that the PI3K/AKT/eNOS cell signaling pathway is involved in the protective effect in the liver I/R injury.
3. Balance between the serum NO and ET-1was broken after HIRI. rHuEPO preconditioning can increase the serum NO significantly and adjust the balance between the serum NO and ET-1. It confirmed the rHuEPO pretreatment by activating p-eNOSser1177increases the release of NO, play on the protective in liver ischemia-reperfusion injury.
Conclusion
1. liver auto-transplantation model was employed in this experiment.it can simulate pathophysiological process of HIRI, increment of liver enzyme indicator, alteration of liver morphology and occurrence of apoptosis and necrosis also occurred in this model.
2. rHuEPO pretreatment can obviously inhibit the liver I/R injury caused by the increase of serum ALT, LDH level, and improve the morphological structure of liver tissue, inhibiting hepatocellular apoptosis and relieve the liver pathological damage.
3. Specific PI3K blocker LY294002significantly inhibited the increase of the p-AKTser473expression induced by rHuEPO and its protective effects. These indicated that the rHuEPO preconditioning may protect liver against HIRI through activating PI3K/AKT signal pathway.
4. LY294002pretreatment significantly inhibited the increase of rHuEPO preconditioning induced p-eNOSser1177expression in liver tissue. It indicates that the PI3K/AKT/eNOS cell signaling pathway is involved in the protective effect in the liver I/R injury.
5. rHuEPO preconditioning increased NO through activating p-eNOSser1177to adjust the ratio of NO/ET-1,protect liver against HIRI.
引文
1.赵宏峰,周杰.大鼠自体原位肝移植胆道缺血再灌注损伤模型的建立[J].第二军医大学报,2006,27(4)：429-30.
2. Peng Y, Gong JP, Yan LN, et al. Improved two-cuff technique for orthotopic liver transplantation in rat [J]. Hepatobiliary Pancreat Dis Int,2004,3(1):33-7.
3. Madrahimov N, Dirsch O, Broelsch C, et al. Marginal hepatectomy in the rat: from anatomy to surgery [J]. Ann Surg,2006,244(1):89-98.
4. Chimalakonda AP, Montgomery DL, Weidanz JA, et al. Attenuation of acute rejection in a rat liver transplantation model by a liver-targeted dextran prodrug of methylprednisolonel [J]. Transplantation,2006,81(5):678-85.
5. 朱佳庚,钱立新,贺厚光,等.改良“二袖套法”制备大鼠原位肝移植动物模型[J].医学研究生学报,2004,17(8)：698-700.
6. Engemann R, Ulrichs K, Thiede A, et al. Value of a physiological liver transplant model in rats. Induction of specific graft tolerance in a fully allogeneic strain combination. [J]. Transplantation,1982,33(8):566-8.
7. Pichlmayr L, Bretschneider HJ, Kirchner E, et al. Ex situ operation on the liver: A new possibility in liver surgery[J].Langenbecks Arch Chit,1988,73(2):122-6.
8. Gruttadauria S, Marsh JW, Bartlett DL, et al. Ex situ resection techniques and liver autotransplantation:last resource for otherwise unresectable malignancy [J]. Dig Dis Sci,2005,50(10):1829-35.
9. Pichlmayr R, Grosse H, Hauss J, et al.Technique. and preliminary results of extracorporeal liver surgery(bench procedure)and of surgery on the in situ perfused liver [J]. Br J Surg,1990,77(1):21-6.
10.叶启发,陈晚平。自体肝移植的技术要点及现状[J].中华外科杂志,2008,46(2)：1-3.
11. Peng Y, Gong J P, Yan L N, et al. Improved two-cuff technique for orthotopic liver transplantation in rat[J]. Hepatobiliary Pancreat Dis Int,2004,3(1):33-7.
12. Tokunaga Y, Ozaki N, Wakashiro S, et al. Effects of perfusion pressure during flushing on the viability of the procured liver using noninvasive fluorometry [J].Transplantation,1988,45(6):1031-5.
13. Fudaba Y, Tashiro H, Ohdan H, et al. Prevention of warm ischemic injury in rat liver transplantation by geranylgeranylacetone [J]. Transplant Proc,2000,32(7): 1615-6.
14. Schemmer P, Schoonhoven R, Swenberg JA, et al. Gentle in situ liver manipulation during organ harvest decreases survival after rat liver transplantation:role of Kupffer cells [J].Transplantation,1998,65(8):1015-20.
15. Kobayashi E, Kamada N, Goto S, et al. Protocol for the technique of orthotopie liver transplantation in the rat[J].Microsurgery,1993,14(8):541-6.
16.孙君泓,吴孟超,曾琪华.300次大鼠原位肝移植[J].中华器官移植杂志,1990,11(1)：19-21.
1. Serracino-Inglott F, Habib NA, Mathie RT. Hepatic ischemia-reperfusion injury [J]. Am J Surg,2001,181 (2):160-6.
2. Totsuka E, Fung U, Hakamada K, et al. Analysis of clinical variables of donors and recipients with respect to short-term graft outcome in human liver transplantation [J].Transplant Proc,2004,36(8):2215-8.
3. Ghaboura N, Tamareille S, Ducluzeau PH, et al. Diabetes mellitus abrogates erythropoietin-induced cardioprotection against ischemic-reperfusion injury by alteration of the RISK/GSK-3β signaling [J]. Basic Res Cardiol,2011,106(1): 147-62.
4. Oba S, Suzuki E, Nishimatsu H, et al. Renoprotective effect of erythropoietin in ischemia/reperfusion injury:possible roles of the Akt/endothelial nitric oxide synthase-dependent pathway [J]. Int J Urol,2012,19(3):248-55.
5. Moore EM., Bellomo R, Nichol AD, et al. Erythropoietin as a novel brain and kidney protective agent [J]. Anaesthesia and Intensive Care,2011,39(3):356-72.
6. Sorg H, Harder Y, Krueger C, et al. The Nonhematopoietic Effects of Erythropoietin in Skin Regeneration and Repair:From Basic Research to Clinical Use [J]. Med Res Rev.2012,doi:10.1002/med.21259. [Epub ahead of print].
7. Parsa CJ, Matsumoto A, Kim J, et al. A novel protective effect of erythropoietin in the infarcted heart [J]. J Clin Invest.2003,112(7):999-1007.
8. Takeda K, Jin MB, Fujita M, et al. A novel inhibitor of Rho-associated protein kinase, Y-27632, ameliorates hepatic ischemia and reperfusion injury in rats [J]. Surgery,2003,133(2):197-206.
9. Suzuki S, Toledo-Pereyra LH, Rodriguez FJ, et al.Neutrophil infiltration as an important factor in liver ischemia and reperfusion [J].Transplantation,1993, 55 (6):1265-72.
10. Walsh KB, Toledo AH, Rivera-Chavez FA, et al. Inflammatory mediators of liver ischemia-reperfusion injury [J]. Exp Clin Transplant,2009,7(2):78-93
11. Fernandez L, Heredia N, Grande L, et al.Preconditioning protects liver and lung damage in rat liver transplantation:role of xanthine/anthine oxidase [J].Hepatology, 2002,36(3):562-72.
12. Jaeschke H. Molecular mechanisms of the hepatic ischemia reperfusion injury and preconditioning [J]. Am J Physiol Gastrointest Liver Physiol,2003,284(1):G15-26.
13. Glanemann M, Volllnar B, NussIer AK, et al. Ischemic preconditioning protects from hepatic ischemia-repermsion injury by preselVation of microcirculation and mitochondrial redox-state [J]. J Hepatol,2003,38(1):59-66.
14. Banga NR, Homer-Vanniasinkam S, Graham A, et al.Ischaemic preconditioning in transplantation and major resection of the liver [J]. Br J Surg.2005,92(5):528-38.
15. EI-Badry AM, Moritz W, Contaldo C, et al.Prevention of reperfusion injuryand microcirculatory failure in macrosteatotic mouse liver by omega-3 fatty acids [J]. Hepatology,2007,45(4):855-63.
16.赵宏峰,周杰.大鼠自体原位肝移植胆道缺血再灌注损伤模型的建立[J].第二军医大学报,2006,27(4)：429-30.
17. Man K, Ng KT, Lee TK, et al. FTY720 attenuates hepatic ischemia-reperfusion injury in normal and cirrhotic livers [J]. Am J Transplant,2005,5(1):40-9.
18. WollmanY, Westphal G, blumM, et al. The effectof human recombinant erythropoietion the growth of a human neuroblastom a cell line [J]. LifeScience, 1996,59 (4):315-22.
19. Anagnostou A, LeeE S, KessimianN, et al. Erythropoietin has a mitogenic and positive chemotacticeffect on endothelialcells [J].Froc Natl Acad Sci USA,1990,87 (15):5978-82.
20. Arcasoy MO, Amin K, VollmerRT, et al. Erythropoietin and erythropoietin receptor in human prostate cancer [J]. Mod Patho1,2005,18(3):421-30..
21. McBroom JW, AcsG, Rose GS, et al. Erythropoeitin receptor function and expression in epithelialovarian carcinoma [J]. GynecolOncol,2005,99(3):571-7.
22. Dagnon K, Pacary E, Commo F, et al. Expression of erythropoietinand erythropoietin receptor in non-small cell lung carcinomas [J]. Clin Cancer Res, 2005, 11(3):993-9.
23. Brines M, Cerami A. Discovering erythropoietin's extra-hematopoietic functions: biology and clinical promise [J]. Kidney Int,2006,70(2):246-50.
24. Joyeux-Faure M. Cellular protection by erythropoietin:new therapeutic implications? [J]. J Pharmacol Exp Ther,2007,323(3):759-762.
25. Mocini D, Leone T, Tubaro M, Santini M, et al. Structure, production and function of erythropoietin:implications for therapeutical use in cardiovascular disease [J]. Curr Med Chem,2007,14(21):2278-87.
26. Hiroshi F, Koichi S, Shin-lchi H, et al, Essential role for nuclear factorκB in ischemic preconditioning for ischcmia rcperfusion mjury of the mouse liver [J]. Transplantation,2002,74(4):551-6.
27. Yakamoto K, Wilson D, Brumal R. Outer medullary circulatory defect in ischemic acute renal failure [J]. Am J Pathol,1984,116(2):253-61.
28. Schmeding M, Hunold G, Ariyakhagorn V, et al. Erythropoietin reduces ischemia-reperfusion injury after liver transplantation in rats [J]. Transpl Int,2009,22(7):738-46.
29. Yilmaz S, Ates E, Tokyol C, et al. The protective effect of erythropoietin on ischemia/reperfusion injury of liver [J]. HPB,2004,6(3):169-73.
30. Brines M, Ghezzi P, Keenan S, et al. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury [J].Proc Natl Acad Sci USA,2000, 97(19):105-26.
31. Siren A, Fratelli M, Brines M, et al. Erythropoietin prevents neuronal apoptosis after cerebral ischemia andmetabolic stress [J]. Proc Natl Acad Sci USA.2001, 98(7):4044-9.
32. Cariou A, Claessens YE, Pene F, et al. Early high-dose erythropoietin therapy and hypothermia after out-of-hospital cardiac arrest:a matched control study [J]. Resuscitation,2008,76(3):397-404.
33. Hochhauser E, Pappo O, Ribakovsky E, et al. Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model [J]. Apoptosis,2008,13(1):77-86.
34. Shawky HM, Younan SM, Rashed LA, et al. Effect of recombinant erythropoietin on ischemia-reperfusion-induced apoptosis in rat liver [J]. J Physiol Biochem,2012, 68(1):19-28.
35. Kato M, Sawada T, Kita J, et al. Erythropoietin ameliorates early ischemia-reperfusion injury following the Pringle maneuver [J]. World J Gastroenterol, 2010,16(38):4838-45
36. Besarab A, Bolton WK, Browne JK, et al. The effects of normal as compared with low hematocrit values in patients with cardiac disease who are receiving hemodialysis and epoetin [J]. N Engl J Med.1998;339(9):584-90.
37. Bohlius J, Langensiepen S, Schwarzer G, et al. Recombinant human erythropoietin and overall survival in cancer patients:Results of a comprehensive meta-analysis [J]. J Natl Cancer Inst,2005,97(7):489-98.
38. Khuri FR. Weighing the hazards of erythropoiesis stimulation in patients with cancer [J]. N Engl J Med.2007,356(24):2445-8.
1. Colletti LM, Kunkel SL, Walz A, et al. The role of cytokine networks in the local liver injury following hepatic ischemia/reperfusion in the rat[J]. Hepatology,1996, 23(3):506-14.
2. Bramey T, Freitag P, Fandrey J, et al. No evidence for protective erythropoietin alpha signalling in rat hepatocytes [J]. BMC Gastroenterol,2009,9:26. doi:10. 1186/1471-230X-9-26.
3. Scarabelli TM, Stephanou A, Pasini E, et al. Different signaling pathways induce apoptosis in endothelial cells and cardiac myocytes during ischemia/ reperfusion injury[J]. Circ Res,2002,90(6):745-8.
4. Westenfelder C, Biddle DL, Baranowski RL. Human, rat, and mouse kidney cells express functional erythropoietin receptors [J]. Kidney Int,1999,55(3):808-20.
5. Sharples EJ, Thiemermann C, Yaqoob MM. Mechanisms of disease:Cell death in acute renal failure and emerging evidence for a protective role of erythropoietin [J]. Nat Clin Pract Nephrol,2005,1(2):87-97.
6. Reed JC. Apoptosis-regulating proteins as targets for drug discovery [J]. Trends Mol Med,2001,7(7):314-9.
7. Chen C, EdelsteinLC, Gelinas C. The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor Bcl-x(L)[J]. Mol Cell Biol,2000,20(8): 2687-95.
8. Yatsiv I, Grigoriadis N, Simeonidou C, et al. Erythropoietin is neuro-protective, improves functional recovery, and reduces neuronal apopto-sis and inflammation in a rodent model of experimental closed head injury[J]. Faseb J,2005 19(12): 1701-3.
9. Haroon ZA, Amin K, Jiang X, et al. A novel role for erythropoietin during fibrin-induced wound-healing response [J]. Am J Pathol,2003,163(3):993-1000.
10. Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signaling cascades[J]. Nature,2001, 412(6847):641-7.
11. Joneja B, Wojchowski DM. Mitogenic signaling and inhibition of apoptosis via the erythropoietin receptor Box-1 domain [J]. J Biol Chem,1997,272(17):11176-84.
12. Zhuang H, Niu Z, He TC, et al.Erythropoietin-dependent inhibition of apoptosis is supported by carboxyl-truncated receptor forms and blocked by dominant-negative forms of Jak2 [J]. J Biol Chem,1995,270(24):14500-4.
13. Xu Q, Simpson SE, Scialla TJ, et al. Survival of acute myeloid leukemia cells requires PI3 kinase activation [J]. Blood,2003,102(3):972-80.
14. Kubota Y, Ohnishi H, Kitanaka A, et al. Constitutive activation of P13K is involved in the spontaneous proliferation of primary acute myeloid leukemia ceils:direct evidence of P13K activation [J]. Leukemia,2004,18(8):1438-40.
15. Goswami R, Kilkus J, Dawson SA, et al. Overexpression of Akt(protein kinase B)confers protection against apoptosis and prevents formation of ceramide in response to pro-apoptotic stimuli[J]. J Neurosei Res,1999,57(6):884-93.
16. Yamaguchi A,Tamatani M, Matsuzaki H, et al. Akt activation protects hippocampal neurons from apoptosis by inhibiting transcriptional activity of p53[J]. J Biol Chem,2001,276(7):5256-64.
17. Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in theprevention ofapoptosis by nerve growth factor [J]. Science,1995,267(5206): 2003-6.
18. Uchiyama T, Engelman RM, Maulik N, et al. Role of Akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning [J]. Circulation,2004,109(24):3042-9.
19. Carini R, Grazia De Cesaris M, Splendore R, et al. Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning [J]. Gastroenterology, 2004,127 (3):914-23.
20. Ke B, Shen XD, Kamo N, et al. beta-Catenin regulates innate and adaptive immunity in mouse liver ischemia-reperfusion injury [J]. Hepatology, 2013,57(3):1203-14.
21.朱宇麟,李静,吕毅,等.缺血后处理对肝缺血再灌注损伤后磷脂酰肌醇-3激酶和细胞外信号调节激酶的影响和意义[J].南方医科大学学报,2012,32(5)：677-80.
22. Fujio Y, Nguyen T, Wencker D, et al. Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-repefusion injury in mouse heart [J]. Circulation,2000,101(6):660-7.
23. Manickavasagam S, Ye Y, Lin K, et al. The cardioproteetive effect of a statin and cilostazol combination:relationship to Akt and endothelial nitric oxide synthase activation[J]. Cardiovasc Drugs Ther,2007,21(5):321-30.
24. Takahama H, Minamino T, Hiram A, et al. Granulocyte colony-stimulating factor mediates cardioprotection against ischemia/reperfusion injury via phosphatidylinositol-3-kinase/Akt pathway in canine hearts [J]. Cardiovasc Drugs Ther,2006,20(3):159-65.
25. Bouchard V, Harnois C, Demers MJ, et al. B1 integrin/Fak/Src signaling in intestinal epithelial crypt cell survival:integration of complex regulatory mechanisms[J]. Apoptosis,2008,13(4):531-42.
26. Chang F, Lee JT, Navolanic PM, et al. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation:a target for cancer chemotherapy[J]. Leukemia,2003,17(3):590-603.
27. Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia-reperfusion injury:targeting the Reperfusion Injury Salvage Kinase(RISK)-pathway[J].Cardiovase Res,2004,61(3):448-60.
28. Bell RM, Yellon DM. Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart:the role of P13K, Akt and eNOS [J]. J Mol Cell Cardiol,2003,35(2):185-93.
29. Argaud L, Gateau-Roesch O, Muntean D, et al. Specific inhibition of the mitochondrial permeability transition prevents lethal reperfusion injury [J]. J Mol Cell Cardiol,2005,38(2):367-74.
1. de Groot H, Rauen U. Ischemia-reperfusion injury:processes in pathogenetic networks:a review [J]. Transplant Proc,2007,39(2):481-84.
2. Colletti LM, Kunkel SL, Walz A, et al.The role of cytokine networks in the local liver injury following hepatic ischemia reperfusion in the rat [J]. Hepatology 1996, 23(3):506-14.
3. Clavien PA. Sinusoidal endothelial cell injury during hepatic preservation and reperfusion [J]. Hepatology,1998,28(2):281-85.
4. Huet PM, Nagaoka MR, Desbiens G, et al. Sinusoidal endothelial cell and hepatocyte death following cold ischemia-warm reperfusion of the rat liver [J]. Hepatology,2004,39(4):1110-19.
5. Gao W, Bentley RC, Madden JF, et al. Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation [J]. Hepatology, 1998,27(6):1652-60.
6. Enomoto K, Nishikawa Y, Omori Y, et al. Cell biology and pathology of liver sinusoidal endothelial cells [J]. Med Electron Microsc 2004,37(4):208-15.
7. Filip Braet, Eddie Wisse. Structural and functional aspects of liver sinusoidal endothelial cell fenestrae:a review [J]. Comparative Hepatology,2002,1(1):1.
8. Morisue A,Wakabayashi G, Shimazu M, et al.The role ofnitric oxide after a short period ofliver ischemia-reperfusion [J]. J Surg Res,2003,109(2):101-9.
9. Haynes MP,Sinha D, Russell KS, et al. Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the P13-kinase-Akt pathway in human endothelial cells [J]. Circ Res,2000,87(8):677-82.
10. Zeng G, Nystrom FH, Ravichandran LV, et al.Roles for insulin rceptor, P13-kinase,and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells [J].Circulation,2000,101(13):1539-45.
11. WollmanY, Westphal G, Blum M, et al.The effectof human recombinant erythropoietion the growth of a human neuroblastom a cell line [J]. LifeScience, 1996,59(4):315-22.
12. Anagnostou A, LeeE S, KessimianN, et al. Erythropoietin has a mitogenic and positive chemotacticeffect on endothelialcells [J]. FrocNatlAcadSciUSA,1990, 87(15):5978-82.
13. Kupiec-Weglinski JW. Busuttil RW. Ischemia and reperfusion injury in liver transplantation [J]. Transplant Proc,2005,37(4):1653-6.
14. Terajima H, Enders G, Thiaener A, et al. Impact of hyperthermic preconditioning on postischemic hepatic microcirculatory disturbances in an isolated perfusion model of the rat liver [J]. Hepatology,2000,31(2):407-15.
15. Shah V, Haddad FG, Garcia-Cardena G, et al. Liver sinusoidal endothelial cells are responsible for nitric oxide modulation of resistance in the hepatic sinusoids [J]. J Clin Invest.1997; 100(11):2923-30.
16. Vajdova K, Heinrich S, Tian Y, et al. Ischemic preconditioning and intermittent clamping improve murine hepatic microcirculation and Kupffer cell function after ischemic injury [J]. Liver Transpl,2004,10(4):520-8.
17. Lu P, Liu F, Wang CY, et al. Gender differences in hepatic ischemic reperfusion injury in rats are associated with endothelial cell nitric oxide synthase-derived nitric oxide [J]. World J Gastroenterol,2005, 11(22):3441-5.
18. Burwell LS, Brookes PS. Mitochondria as a target for the cardioprotective effects of nitric oxide in ischemia-reperfusion injury [J]. Antioxid Redox Signal, 2008,10(3):579-99.
19. Rachmilewitz D, Kameli F, Eliakim R, et al. Enhanced gastric nitric oxide synthase activity in duodenal ulcer patients[J].Gut,1994,35(10):1394-7.
20.冯志博,陈子琏.脑缺血及再灌流早期大鼠海马CA1区一氧化氮合酶表达的变化[J].新乡医学院学报,2002,19(5)：366-68.
21. Sorg, H., Y. Harder, Krueger C, et al. The Nonhematopoietic Effects of Erythropoietin in Skin Regeneration and Repair:From Basic Research to Clinical Use [J]. Med Res Rev.2012. [Epub ahead of print]
22. Patel NS, Kerr-Peterson HL, Brines M,, et al. Delayed administration of pyroglutamate helix B surface peptide (pHBSP), a novel nonerythropoietic analog of erythropoietin, attenuates acute kidney injury [J]. Mol Med,2012,18(1):719-27.
23. Oba S, Suzuki E, Nishimatsu H, et al. Renoprotective effect of erythropoietin in ischemia/reperfusion injury:possible roles of the Akt/endothelial nitric oxide synthase-dependent pathway [J]. Int J Urol,2012,19(3):248-55.
24. Lu MJ, Chen YS, Huang HS, et al. Erythropoietin alleviates post-ischemic injury of rat hearts by attenuating nitrosative stress [J]. Life Sci,2012,90(19-20):776-84.
25. Teng R, Calvert JW, Sibmooh N et al. Acute erythropoietin cardioprotection is mediated by endothelial response [J]. Basic Res Cardiol,2011,106(3):343-54.
26. Mihov D, Bogdanov N, Grenacher B, et al. Erythropoietin protects from reperfusion-induced myocardial injury by enhancing coronary endothelial nitric oxide production [J]. Eur J Cardiothorac Surg,2009,35(5):839-46.
27. Corson MA, James NL, Latta SE, et al. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress [J].Circ Res,1996,79(5):984-91.
28. Fleming I, Bauersachs J, Fisslthaler B, et al. Ca2+-Independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress [J]. Circ Res,1998,82(6):686-95.
29. Fulton D, Gratton JP, McCabe TJ, et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt [J]. Nature,1999,399(6736):597-601.
30. Dimmeler S, Fleming I, Fisslthaler B, et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation [J]. Nature,1999,399 (6736): 601-5.
31. Acquaviva R, Lanteri R, Li Destri G, et al. Beneficial effects of rutin and L-arginine coadministration in a rat model of liver ischemia-reperfusion injury [J]. Am J Physiol Gastrointest Liver Physiol.2009,296(3):G664-70.
32. Su KH, Shyue SK, Kou YR, et al. β Common Receptor Integrates the Erythropoietin Signaling in Activation of Endothelial Nitric Oxide Synthase [J]. J Cell Physiol,2011,226(12):3330-9.
33. Kaplowitz, N. Mechanisms of liver cell injury [J]. J Hepatol,2000,32(1 Suppl): 39-47.
34. Crompton, M. The mitochondrial permeability transition [J]. Biochem J,1999, 341(Pt 2):233-49.
35. Kobayashi H, Miura T, Ishida H, et al. Limitation of infarct size by erythropoietin is associated with translocation of Akt to the mitochondria after reperfusion [J]. Clin Exp Pharmacol Physiol,2008,35(7):812-9.