【作者】 郑德义；

【导师】 夏照帆； 付晋凤； 路卫；

【作者基本信息】 昆明医学院 ， 外科学， 2010， 博士

【摘要】 前言缺血再灌注（ischemia/reperfusion;Ⅰ/R）损伤是组织器官在缺血的基础上恢复血流后损伤反而加重的现象,是内、外科常见的一种损伤。肠道对缺血最敏感的组织器官之一,肠缺血再灌注常见于严重创（烧）伤、休克、感染、肠移植、新生儿缺血坏死性肠炎及危重病患者,肠缺血再灌注（intestinalischemia/reperfusion;Ⅱ/R）常常原发或继发性肠道疾病,是临床众多疾病的病理生理基础,是引发脓毒症及多器官功能衰竭的始动因素之一。缺血再灌注肠损伤后肠黏膜屏障功能受损,通透性增加,促使肠道细菌及内毒素移位、大量氧自由基生成、炎性细胞浸润增加,细胞因子和炎症介质的失控性释放,不仅引起肠道局部损伤,而且造成远隔器官损害,尤其是肺损伤,诱发全身炎症反应综合症及多器官功能衰竭,病情凶险、严重,在临床上具有极高的发病率和病死率。由于肠缺血再灌注肠损伤在各类临床疾病中均可能原发或继发性发生,对于其发病机制的探讨一直是学者们的研究热点。丝裂原活化蛋白激酶（Mitogen activated protein kinases;MAPKs）是真核细胞内一组高度保守的丝/苏氨酸蛋白激酶,目前已知MAPKs有c-Jun氨基末端激酶（JNK）、细胞外信号蛋白激酶（ERK）和p38丝裂原活化蛋白激酶（p38 MAPK）三种主要形式。在损伤应激、氧自由基、感染和炎症等刺激条件下,细胞内MAPKs蛋白的苏氨酸/酪氨酸双位点磷酸化反应从而被顺序激活,通过细胞内信号传递级联效应使基因表达发生变化,在调节组织炎症反应、细胞增殖与凋亡和免疫反应等病理生理过程发挥重要作用。由于各种MAPK本身和其作用底物各有特点,MAPKs三个主要成员成具有相对独立而又相互联系的信号传递网络,三种形式的MAPKs执行不同的生理功能。有研究表明,MAPKs参与了缺血再灌注肠损伤的发病过程,但是,在肠缺血再灌注早期,有关MAPKs蛋白表达及活化过程尚不清楚,MAPKs蛋白及其下游信号通路参与肠缺血再灌注损伤的机制尚未阐明。采用夹闭肠系膜上动脉方法造成小鼠肠缺血再灌注损伤,本实验第一部分观察损伤后肠组织MAPKs及其下游信号通路的变化,探讨小肠组织MAPKs活化及其介导凋亡信号通路在小鼠小肠缺血再灌注损伤中的可能作用。肺脏不仅是气体交换的器官,也是一些细胞因子和激素生与灭活的场所,经过各种途径入血的毒素、内源性炎性介质等物质经静脉回流后都要进入肺循环,加上肺脏本身在解剖结构上相对较脆弱,肺循环具有面积大、流速慢、压力低的特点,使肺脏实质细胞暴露在毒性物质的机会和时间增加,因此,在诸多脏器中,肺脏最易受到各种致病因素的打击,也是机体失控炎性损害的主要靶器官之一。尽管目前对肠缺血再灌注肺损伤进行大量的研究,但是其分子机理尚未完全阐明。肠缺血再灌注导致肠粘膜损,肠屏障功能破坏,引起细菌/内毒素移位,氧自由基、细胞因子及炎性介质等有害物质释放并进入全身循环,导致远隔器官损害;同时,它们又都是刺激MAPKs信号通路活化的有力因素。我们由此推测,肠缺血再灌注可能通过激活肺组织MAPKs信号通路活化,参与肠缺血再灌注肺损伤的发生。肺损伤特征性主要表现为肺部剧烈炎症反应,炎症细胞浸润和炎性细胞因子合成、释放增加,其中细胞因子TNF-α、IL-1β在肺组织炎症反应中起着关键作用。第二部分通过观察p38 MAPK信号通路活化在肠缺血再灌注肺损伤的活化过程,使用特异性p38 MAPK抑制剂SB239063抑制p38 MAPK蛋白活性,探讨其在小肠缺血再灌注肺损伤中的作用及可能机制。第一部分MAPKs蛋白活化在小肠缺血再灌注损伤的作用目的:观察小肠缺血再灌注后小肠组织MAPKs蛋白活化过程及凋亡相关蛋白Bcl-2、Bax和活化型Caspase-3的变化,探讨MAPKs活化及其介导凋亡信号通路在小肠缺血再灌注损伤中的可能作用。方法:采用夹闭肠系膜上动脉（superior mesenteric artery;SMA）后再灌注造成小肠缺血再灌注损伤小鼠动物模型,经腹腔注射1%戊巴比妥钠50mg.kg^-1麻醉。（1）8-10周龄的雄性C57 BL/6小鼠随机分为肠缺血30min、40min、50min和60min,每组8只,观察14d内肠缺血再灌注后小鼠生存率。（2）小鼠随机分为假手术组（sham）,肠缺血再灌注处理组（Ⅱ/R）:分再灌注即刻（0）、0.5h、1h、4h、6h和12h不同再灌注时间组。夹闭SMA 40min再灌注造成小鼠Ⅱ/R模型,假手术组同样进行开腹手术但不夹闭动脉。假手术后及肠缺血再灌注后不同时间相处死小鼠取小肠标本,回肠组织10%中性福尔马林液固定待病理检查,用Chiu双盲病理评分,评价肠损伤情况程度;原位末端标记（TUNEL）法对肠组织凋亡细胞进行定位检测;全细胞裂解法提取小肠总蛋白,Western blot蛋白免疫印迹法检测小肠组织JNK、ERK和p38 MAPK蛋白,磷酸化JNK、ERK和p38 MAPK蛋白,以及与凋亡相关蛋白Cleaved-caspase-3、Bcl-2和Bax表达水平,以磷酸化MAPKs蛋白代表其活化水平。结果:（1）肠缺血30 min小鼠生存率100%,随着肠缺血时间的增加,动物生存率逐渐降低,肠缺血60 min动物生存率仅约30%。（2）肠组织大体表现及病理评分结果均显示,肠缺血40 min后再灌注早期肠损伤严重,尤以再灌注1h肠组织病理损伤最重,至12h肠组织结构基本恢复正常;TUNEL法定位凋亡细胞结果表明,凋亡细胞分布广泛,包括黏膜、黏膜固有层,黏膜下层,甚至肌层均存在凋亡细胞,涉及细胞成分多样,以肠上皮细胞凋亡为主。肠缺血再灌注损伤细胞凋亡与坏死并存。（3）肠缺血再灌注早期促凋亡的活化型caspase-3蛋白表达增加,与假手术组比较,再灌注0.5h、1h小肠组织活化型caspase-3明显增加（P＜0.01）,与小肠组织病理损害时相过程基本一致,同时抗凋亡Bcl-2蛋白明显下调（P＜0.01）;各组间促凋亡蛋白Bax表达无统计学差异。（4）肠缺血再灌注不影响肠组织JNK、ERK、p38 MAPK蛋白表达,而磷酸化JNK、ERK、p38 MAPK蛋白表达明显增加。与假手术对照组比较,肠缺血及再灌注早期,小肠组织JNK持续活化,差异有统计学意义（P＜0.01）;肠缺血再灌注导致磷酸化ERK显著增加,至再灌注4h下降至接近正常,然后ERK活化再次明显增加（P＜0.01）,ERK活化呈现明显上升-下降-再上升动态活化过程;肠缺血p38 MAPK活化明显减少（P＜0.05）,再灌注0.5 h肠组织磷酸化p38 MAPK急剧上升（P＜0.01）,其后p38 MAPK持续活化,尽管与假手术对照组差异无显著性。结论:肠缺血再灌注损伤细胞凋亡与坏死并存;肠缺血再灌注早期小肠组织JNK、p38 MAPK活化、可能通过下调抗凋亡蛋白Bcl-2表达,参与了Caspase-3依赖的细胞凋亡和组织损害,ERK的活化与受损肠组织及时修复有关。对进一步研究MAPKs信号通路活化在Ⅱ/R肠损伤中的作用具有指导意义。第二部分p38 MAPK活化在肠缺血再灌注肺损伤的作用目的:观察小肠缺血再灌注后肺组织p38 MAPK活化规律、以及肠和肺组织炎性细胞因子TNF-α、IL-1β基因转录水平的变化,探讨p38 MAPK蛋白活化在小鼠小肠缺血再灌注肠道本身及肺损伤中的作用及可能机制。方法:为探讨肠缺血再灌注后肺组织p38 MAPK活化动态过程,10周龄健康雄性C57 BL/6小鼠随机分为假手术组（sham）,肠缺血再灌注组（Ⅱ/R）:再分为肠缺血45min再灌注即刻（0）、0.5h、4h和6h。为了进一步观察p38 MAPK活化在场缺血再灌注肺损伤的可能作用,动物随机分为假手术组（sham）、缺血再灌注对照组（Ⅰ/R）和缺血再灌注+SB239063处理组（Ⅰ/R+SB239063组）,Ⅰ/R+SB239063组于术前1h腹腔注入p38 MAPK抑制剂SB239063(3 mg.kg^-1),另两组注入等量生理盐水。采用夹闭C57BL/6小鼠肠系膜上动脉45 min后再灌注6h的造成肠缺血再灌注损伤模型。于再灌注6h麻醉后处死小鼠,取小肠、肺标本,Western blot蛋白质印迹法检测肺组织磷酸化p38 MAPK蛋白水平;RT-PCR法检测小肠、肺组织TNF-α、IL-1βmRNA表达;HE染色,光学显微镜观察回肠及肺组织病理学改变,分别用Chiu方法肠损伤、Gloor方法肺损伤病理评分,评价回肠及肺损伤严重程度。结果:（1）肠缺血后再灌注导致肺组织p38 MAPK蛋白快速、持续活化,与假手术组比较,差异有显著性（P＜0.01）。（2）肠缺血再灌注引起肠道局部及肺损伤,小肠、肺组织TNF-α、IL-1β基因表达水平显著升高。（3）在肠缺血后再灌注6h,SB239063抑制肺组织p38MAPK活化,下调肺组织TNF-α、IL-1βmRNA表达水平,减轻肠缺血再灌注肺损伤（P＜0.05）;SB239063显著下调小肠组织IL-1β基因表达,尽管不影响TNF-αmRNA表达水平,减轻肠缺血再灌注肠损伤。结论:p38 MAPK活化在小肠缺血再灌注肺损伤中起重要作用,抑制p38MAPK降低小肠及肺部分促炎性细胞因子基因转录水平,减轻肠缺血再灌注引起的肠道本身及肺损伤,抑制p38 MAPK活化可以作为预防肠缺血再灌注肺损伤的治疗靶点。更多还原

【Abstract】 IntroductionIschemia/reperfusion（Ⅰ/R） injury is tissue lesions produced during reperfusion were greater than those produced during ischemia,which is a common organ damage that affects both medical and surgical patients.Intestine is one of most vulnerable organs to ischemia injury,and consequently intestinal ischemia/reperfusion（Ⅱ/R） often occurs in severe trauma and burn injury,shock,infection,intestinal transplantation and neonatal necrotizing enterocolitis as well as in patients in intensive care units.Ⅱ/R become the basis of the pathophysiology of numerous diseases and played an initiating role in the development of triggering sepsis and multiple organ failure.Ischemia and reperfusion of the small intestine provoke mucosal barrier dysfunction and augmentation of mucosa permeability,and resulting in bacterial/endotoxic translocation,production of oxygen free radicals,activation of inflammatory responses and uncontrolled release of cytokines and inflammatory mediators.It may lead to injuries in both local and remote organs,particularly in lung, and induces systemic inflammatory response syndrome（SIRS） and multiple organ dysfunction syndromes（MODS）,which often progresses to acute respiratory distress syndrome（ARDS） associated with high mortality rate in critically ill patients.The molecular mechanisms ofⅡ/R injury have not been completely defined,and further investigations promote the focuses on the field of intracellular signal transduction pathway in recent years.Mitogen activated protein kinases（MAPKs） are an evolutionarily conserved family of serine/threonine kinases in eukaryotic cells that are primarily composed of three forms of c-Jun NH2 terminal kinase（JNK）,extracellular signal regulated kinase （ERK） and p38 MAP kinase（p38 MAPK）;MAPKs are activated under the conditions of ischemia/reperfusion injury,oxidative stress,infections and inflammatory cytokines so on.On activation of MAPKs,transcription factors present in the cytoplasm or nucleus are phosphorylated or activated,leading to expression of certain target genes resulting in a biological response,and controlling cell differentiation,cell proliferation,and cell death,and inflammatory response.Although MAP kinases generally function as autonomous signaling modules,the multiple interactions between the different MAP kinase cascades serve to integrate responses and to moderate outputs.Indeed,it has been demonstrated that MAP kinases have overlapping substrate specificities and phosphorylation of regulatory sites is shared among multiple protein kinases.Some studies have proved MAPKs was participated inⅡ/R-induced intestinal injury,however,no systematic evaluation of the expression, activity,or signal transduction of MAPKs has been published so far.It is unclear how MAPKs and its down-stream signal proteins are involved in the development ofⅡ/R. In the first part of this study,we examined the change course of MAPKs activation inⅡ/R-induced intestinal tissues,and explored the potential effects of MAPKs-mediated apoptotic signal pathway in the development ofⅡ/R injury in mice.Lungs are not only the gas exchange place,but also the place some cytokines and hormones are inactived.As the lung is the first station that invasive toxins and endogenous inflammatory agents will visit,as well as its physical and anatomic characters,such as low flow rate and pressure,big area,all those add the risk to be injured.From the most,it is the common sense that the lung is one of the most fragile organs and main target organs following uncontrolled systemic inflammatory response.The mechanism of lung injury induced by intestinalⅠ/R is complicated. Many studies have shown thatⅡ/R induce disruption of the intestinal mucosal barrier, allowing translocation of bacteria and endotoxin,reactive oxygen species（ROS）, cytokines and inflammatory mediators originated from the injured intestinal tissue into the circulation,which result in distal organ injury including acute lung injury. MAPKs can also be activated in the lung by a number of pro-inflammatory stimuli, including LPS,cytokines,and oxidative stress;it is therefore plausible to speculate that pulmonary MAPKs activation is involved in the pathogenesis ofⅡ/R-induced lung injury.LPS,ROS and inflammatory cytokines generated during intestinalⅠ/R injury may trigger the activation of p38 signaling cascade in the lung,which may then in turn contribute to the development of lung injury.Acute lung injury is characterized by intensive pulmonary inflammatory response.As we known,many inflammatory cells and inflammatory mediators were involved in the systemic inflammatory response and constitute the inflammatory response network.So it has been emphasized that those factors that were closely related to the inflammatory response, such as TNF-α,IL-1βhave played a centre role.Accordingly,in this study,we examined the time course of p38 activation in lung tissues in mice subjected toⅡ/R. With the use of a potent and selective second-generation inhibitor of p38,SB 239063, we determined the effects of in vivo inhibition of p38 onⅡ/R-induced TNF-α,IL-1βgene expression,and investigated the possible role and mechanisms of p38 MAPK activation in theⅡ/R-induced lung injury.PartⅠRole of activation of MAPKs signaling transduction pathways in intestinal tissue after intestinal ischemia/reperfusion in miceObjective:To observe the dynamic changes of activation of MAPKs and apoptotic proteins of Bcl-2,Bax and Caspase-3 in intestinal tissue after intestinal ischemia/reperfusion（Ⅱ/R） in mice.To explore the potential role of MAPKs-dependent apoptotic signal pathway inⅡ/R-induced mice.Methods:AnimalⅡ/R model was established with clamping the superior mesenteric artery（SMA）.Mice were intraperitoneally anesthetized with 1%solution of pentobarbital sodium(50 mg.kg^-1).First,forty male C57BL/6 mice,8-10 weeks, were randomly divided into five group:30 min,40 min,50 min and 60 min of intestinal ischemia,and survival rate was observed within fourteen days after intestinal reperfusion.Second,mice were randomly divided into sham-operated group （n=6） andⅡ/R groups（n=36）;the latter was further divided according to time after reperfusion（0,0.5,1,4,6 and 12 h） after reperfusion.AnimalⅡ/R model was established with clamping SMA for 40 minutes.Animals in the sham-operated mice received no clamping.The expression of JNK and phosphorylation（phospho-） JNK, ERK and phospho-ERK and p38 MAPK and phospho-p38 MAPK,cleaved caspase-3, Bcl-2 and Bax proteins in the intestinal tissue was examined by Western blotting analysis,and the pathological change of ileum tissue was observed by optical microscope.Phospho-MAPKs by Western blot analysis was applied to reflect the activation status of MAPKsResults:（1） All animals were survived in the group of 30 minute intestinal ischemia,and animal survival rates decreased gradually followed by increase of ischemia time.In contrast,approximately 70%animals died in the group of 60 minute of intestinal ischemia.（2） Most sereve intestinal injury was induced by intestinal ischemia/reperfusion at the early stage of reperfusion,which peaked at 1 h,and intestinal tissue almost recovered 12 h later.Localization of apoptotic cells,as detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling（TUNEL）, showed apoptotic cells were scattered in the intestinal tissues including mucosa, propria lamina,mucosa underlayer even and muscular layer.（3） Cleaved-caspase-3 was significantly increased inⅡ/R group at 0.5h,lh after reperfusion compared to the sham-operated group（P＜0.01）;at the same time,the expression of Bcl-2 protein inⅡ/R group was significantly decreased compared with sham group（P＜0.01）,and there was no significant difference in Bax expression between different group.（4）Ⅱ/R caused an early activation in all members of the MAPK family,whereas there was no significant change in JNK,ERK and p38 MAPK level at various time points afterⅡ/R. the phospho-JNK in the intestine was significantly elevated within 1 h after reperfusion compared with sham group（P＜0.01）.when compared with sham group, the expression of phospho-ERK protein in the intestine was significantly increased within 1 h after reperfusion（P＜0.01）,sharp decreased to the normal level at 4 h of reperfusion,and again elevated significantly at 6 h,12 h after reperfusion（P＜0.01）; the phospho-p38 MAPK protein was stringly decreased in the ischemia intestinal tissue（P＜0.05）,in contrast,significantly elevated at 0.5 h after reperfusion（P＜0.01）, and kept at this high level up to 12 h of reperfusion（P＞0.05）.Conclusions:phosphorylation of JNK and p38 MAPK plays an essential role in the intestinal damages induced byⅡ/R,possibly through down-regulation of Bcl-2 protein and caspase-3 dependent proapoptotic pathway;and ERK phosphorylation may contribute to restitution of damaged intestine. PartⅡInhibition of p38 mitogen activated protein kinase attenuates local and pulmonary injury following intestinal ischemia/reperfusion in miceObjectives:to investigate the potential roles of p38 mitogen-activated protein kinase（MAPK） in lung injury following intestinalⅠ/R.Methods:First,to determine the dynamic changes of pulmonary p38 MAPK activation.Twenty Male C57BL/6 mice were randomly divided into sham-operated group,and 0,0.5,4 and 6 h groups of reperfusion after 45 minute intestinal ischemia （n=4 in each group）.To further examine the role of p38 MAPK activation in intestinalⅠ/R-induced lung injury.Mice were randomly divided into sham-operated, intestinalⅠ/R plus vehicle treatment and intestinalⅠ/R plus p38 MAPK inhibitor SB239063 treatment（3 mg/kg,i.p.） groups;intestinalⅠ/R was performed with 45 minutes intestinal ischemia followed by 6 hours reperfusion.The pulmonary p38 MAPK activation was observed by western blot after intestinalⅠ/R;and histopathology and production of TNF-αand IL-1βmRNA in the intestinal and pulmonary tissues were also analyzed at 6 hour of reperfusion after 45 min intestinal ischemia.Results:IntestinalⅠ/R caused a rapid activation of p38 MAPK in lung tissues, and which lasted to 6 hour of reperfusion.Selective inhibition of p38 MAPK with SB 239063 could down-regulate an elevated IL- 1βmRNA level induced by intestinalⅠ/R, and significantly attenuated the intestinal injury in the mouse model ofⅡ/R.Further, blockade of p38 MAPK activation not only inhibited the up-regulation of pulmonary TNF-αand IL-1βmRNA levels induced by intestinalⅠ/R,but also significantly attenuated the pulmonary pathological changes.Conclusions:Activation of p38 MAPK plays an important role in intestinalⅠ/R-induced lung injury in mice.Inhibiting p38 MAPK activation might be a potential strategy for prevention the pathological changes in lung after intestinalⅠ/R.更多还原