【作者】 王建明；

【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】 TLR4信号转导通路活化在胃缺血再灌注损伤中的作用研究研究背景近年来,随着临床医学的进展,缺血再灌注损伤的普遍意义越来越引起人们高度重视。胃粘膜对缺血缺氧高度敏感,是全身最容易受累及表现临床症状的脏器,机体在遭受创伤、疾病和手术等应激原刺激时,胃肠粘膜会发生不同程度的缺血再灌注损伤。本课题组前期的研究表明在大鼠的胃粘膜缺血再灌注损伤中,MAPKs信号通路以及NF-kB信号通路活化参与了损伤过程,抑制p38 MAPK、JNK以及NF-KB信号通路活化能明显减轻胃粘膜糜烂、出血、胃组织中炎症因子表达升高以及凋亡细胞增加。一些证据显示,再灌注损伤在机制方面与炎症损伤有共同之处。Toll样受体4(TLR4)在炎症损伤中起重要作用,它存在于哺乳动物胃粘膜等大多数组织和器官中。细胞膜受体TLR4处于MAPKs以及NF-KB信号通路的上游,其功能缺失对下游信号转导通路的活化有明显的影响,应用TLR4基因敲除或基因突变鼠实验,发现TLR4功能缺失小鼠在脑、心脏、肺脏、肾脏等器官的缺血再灌注中相对于野生型小鼠损伤明显减轻；应用TLR4选择性抑制剂Eritoran也能够明显减轻心脏缺血再灌注损伤以及炎症反应。在胃缺血再灌注损伤过程中,MAPKs信号通路以及NF-kB信号通路活化参与了损伤过程发病过程,但是TLR4基因缺陷影响胃缺血再灌注损伤的机制尚未阐明。因此,本实验第一部分通过夹闭及松开腹腔动脉诱导胃缺血再灌注损伤,应用TLR4基因缺陷鼠与野生型小鼠做对照,观察TLR4基因缺陷对小鼠胃缺血再灌注损伤程度、下游主要信号通路活化、细胞凋亡以及炎症因子表达的影响。现阶段,胃缺血再灌注损伤的防治主要是应用抑酸剂,动物实验表明在缺血再灌注过程中胃酸分泌低于正常状态,应用抑酸剂只是去除胃缺血再灌注损伤的外部因素或加重因素,而未去除致使胃粘膜屏障功能降低的因素。胃缺血再灌注时产生氧化应激等刺激信号,通过细胞内信号通路转导并级联放大,使细胞和组织发生炎症、凋亡,最终导致胃粘膜屏障功能减弱是胃缺血再灌注损伤的致伤因素。本研究的第二部分通过观察抑制538 MAPK活化和胃酸分泌对胃缺血再灌注损伤的影响,进一步阐释TLR4下游相关信号通路活性抑制减轻胃缺血再灌注损伤机制,探讨未来通过在信号转导通路不同层面调控来干预胃缺血再灌注损伤进展和治疗此类疾病的可能性。第一部分TLR4功能缺陷减轻胃缺血再灌注损伤的作用机制目的：对比观察胃缺血再灌注后TLR4基因缺陷小鼠和野生型小鼠胃粘膜损伤差异,并探讨TLR4基因缺陷减轻胃缺血再灌注损伤中的机理。方法：采用夹闭腹腔动脉(celiac artery)30分钟造成小鼠胃缺血,松开动脉夹形成再灌注的胃缺血再灌注损伤模型,8-12周龄的雄性TLR4基因缺陷型小鼠C3H/HeJ和野生型小鼠C3H/HouJ,随机分为TLR4缺陷小鼠(MU)和野生型小鼠(WT)的假手术组、再灌注即刻、0.5小时、1小时、2小时、4小时、以及再灌注12小时组。实验前禁食12小时自由饮水,麻醉用腹腔注射1%戊巴比妥钠(40mgkg),假手术组小鼠经历相同的手术过程,只是不夹闭腹腔动脉,手术后于不同的再灌注时间点深麻醉处死动物。各组取4只小鼠胃标本用10%中性福尔马林液固定5分钟,后沿胃大弯侧剪开平铺于台面拍照观察胃粘膜出血情况,应用Image J图像分析软件分析计算胃粘膜的相对出血面积,后继续用福尔马林固定做病理检查。各组另外6只小鼠取胃标本做分子生物学检测,剖开胃腔用生理盐水漂洗胃内容物,无菌滤纸吸干水滴液氮冻存。采用Masuda E所描述方法进行双盲病理评分,评价各组小鼠胃粘膜损伤程度；Trizol法提取胃组织总RNA后,实时定量PCR分析TNF-α、IL-10、IL-1β以及IFN-y表达水平判断各组胃组织炎症反应的强弱；免疫组织化学的方法检测各组胃组织中TUNEL染色的阳性细胞数,判断各组胃标本的细胞凋亡发生的情况；全细胞裂解法提取各标本总蛋白后,western blot法检测各组肺组织中信号蛋白p38 MAPK、JNK、ERK和IKB-α活化情况,同样检测凋亡通路关键蛋白caspase-8以及Cleaved Caspase-3的含量判断凋亡通路的活化情况；用EMSA方法检测核因子NF-kB的活化情况。结果：(1)相对于各自假手术组TLR4野生型以及缺陷型小鼠,胃缺血30分钟后再灌注1小时,胃粘膜均出现明显的出血及糜烂损伤改变,而TLR4基因缺陷小鼠大体观胃粘膜出血糜烂面积明显小于野生型小鼠；病理评分结果显示,胃缺血再灌注组小鼠胃的病理评分均显著高于各自的假手术组,而TLR4基因缺陷型小鼠胃的病理评分显著低于野生型小鼠评分。(2)胃组织切片TUNEL染色结果显示：两种不同基因型小鼠的假手术组内均有少量的细胞发生凋亡,集中在胃粘膜表层；两种不同基因型小鼠胃缺血再灌注损伤后相对于各自基因型的假手术组,凋亡细胞明显增加；胃缺血再灌注损伤1小时,TLR4基因缺陷型小鼠胃TUNEL染色阳性细胞计数明显低于野生型；凋亡通路起始信号蛋白caspase-8以及最终通路信号蛋白Cleaved Caspase-3表达在灌注1小时组TLR4基因缺陷型明显低于野生型小鼠,这一趋势与TUNEL染色结果一致。TLR4基因缺陷型小鼠再灌注各组胃组织促炎细胞因子TNF-a的表达,以及再灌注1小时IL-1β, IFN-γ表达明显低于野生型小鼠；再灌注1小时组抑炎细胞因子IL-10的表达明显高于野生型小鼠。(3)对MAPKs三条通路的活化情况的检测发现,胃缺血再灌注损伤后TLR4基因缺陷鼠p38 MAPK活化(0,0.5,1,2,4 hours)与JNK早期(0.5 and 1 hours)舌化明显低于野生型小鼠,除再灌注30分钟、2小时、4小时ERK通路活化TLR4基因缺陷鼠明显高于野生性鼠外,其余时间点及假手术组两种基因型小鼠ERK的活化没有明显差别；胃缺血再灌注损伤后TLR4基因缺陷鼠胃组织NF-kB的活化明显低于野生型鼠；胃缺血再灌注损伤后TLR4基因缺陷鼠IKB-α磷酸化活化明显低于野生型手术组。结论：TLR4基因功能缺陷能够明显减轻胃缺血再灌注损伤,可能原因是胃缺血再灌注损伤后,TLR4基因缺陷型小鼠胃组织p38 MAPK以及NF-kB活化明显低于野生型小鼠,凋亡通路活化下降、凋亡发生减少,炎症细胞因子表达减少、组织炎症反应减轻；综上,TLR4可以作为防治胃缺血再灌注损伤的靶点。第二部分p38 MAPK活性抑制减轻胃缺血再灌注伤的作用机制目的：对比观察p38 MAPK抑制剂和质子泵抑制剂干预胃缺血再灌注损伤的效果,探讨p38 MAPK抑制剂和质子泵抑制剂干预胃缺血再灌注损伤的分子机制,并为临床防治胃缺血再灌注损伤提供指导。方法：采用第一部分叙述的方法制作动物模型,将8-12周龄的雄C57BL/6J小鼠随机分为假手术组；模型组；质子泵抑制剂组；p38 MAPK抑制剂组。各组动物均禁食自由饮水12小时,手术前1小时分别腹腔注射干预试剂,10ml/kg生理盐水(假手术组和模型组)；20mg/kg Pantoloc(质子泵抑制剂组)；20mg/kgSB239063 (p38 MAPK抑制剂组)。手术方法同前,于再灌注后1小时深麻醉处死动物取材处理标本,假手术组经历同样的手术过程,只是不夹闭腹腔动脉。各组术后取4只小鼠的胃标本10%中性福尔马林液固定5分钟,沿胃大弯侧剪开平铺于台面拍照观察胃粘膜出血情况,应用Image J图像分析软件分析计算胃粘膜的相对出血面积,后继续福尔马林做病理检查。各组另外6只小鼠深麻醉处死后取胃,剖开胃腔用生理盐水漂洗胃内容物吸干水滴后液氮冻存。用Masuda E所描述方法进行双盲病理评分,评价各组小鼠胃粘膜损伤程度；Trizol法提取胃组织总RNA后,逆转录cDNA后将各组模板进行实时定量PCR扩增,分析TNF-αIL-1β、IFN-γ以及ICAM-1表达水平,判断各组胃组织的炎症反应程度；全细胞裂解法提取各标本总蛋白后,western blot法检测各组肺组织中磷酸化p38 MAPK、JNK、ERK以及Cleaved Caspase-3的含量,判断各组胃组织中MAPK信号蛋白的活化情况,以及两种干预措施对胃组织细胞凋亡的影响；比色法测定各组胃组织中脂质过氧化产物丙二醛(MDA)的含量,判断各组胃组织的过氧化损伤程度；测定生理盐水,质子泵抑制剂潘妥洛克以及p38MAPK抑制齐ISB239063腹腔注射一小时后对胃内pH值的影响,观察p38MAPK抑制剂SB239063对小鼠胃酸分泌的影响。结果：(1)C57BL/6J小鼠胃缺血30分钟再灌注1小时大体可见到明显胃粘膜糜烂、出血,应用等剂量的质子泵抑制剂以及p38 MAPK抑制剂干预,发现相对于模型组,两种试剂干预均可明显减小胃粘膜出血面积,相对于p38 MAPK抑制剂组质子泵抑制剂组胃粘膜出血面积减少更明显；病理学评分显示,模型组比假手术组病理评分明显高,两种试剂干预后相对于模型组评分均明显下降。(2)脂质过氧化产物丙二醛(MDA)含量测定,MDA含量模型组明显高于假手术组,两种试剂干预后相对于模型组MDA含量均明显下降,且两种试剂干预组间MDA含量无明显差异。炎症因子检测方面,模型组TNF-a, IL-1β, IFN-γ和ICAM-1的表达比假手术组均明显升高,质子泵抑制剂干预后TNF-a, IL-1β和IFN-y表达比模型组明显降低,p38 MAPK抑制剂干预后TNF-α表达比模型组明显降低。(3)MAPKs信号通路检测,发现缺血再灌注组比假手术组MAPKs三条通路：p38MAKP、JNK以及ERK均明显活化；对比模型组,质子泵抑制剂于预能够明显抑制]p38MAKP、JNK、ERK活化；,对比模型组,应用p38 MAPK抑制剂干预p38 MAPK及JNK活化被明显抑制,对ERK活化没有明显影响。(4)腹腔注射质子泵抑制剂以及p38MAPK抑制剂对胃内pH值的影响,发现质子泵抑制剂能够明显升高胃内pH值,而腹腔注射p38 MAPK抑制剂对胃内pH值没有明显影响。结论：p38 MAPK抑制剂腹腔注射对胃内pH值没有明显影响,却能明显减轻小鼠的胃缺血再灌注损伤,机理可能是其抑制了p38 MAPK以及JNK信号通路活化而对ERK信号通路没有明显影响,减轻了缺血再灌注损伤对胃粘膜屏障的破坏作用；抑制p38 MAPK活化可以作为临床防治胃缺血再灌注损伤的靶点。更多还原

【Abstract】 The Role of Toll Like Receptor-4 Signal Pathways activation in Gastric Ischemia-Reperfusion InjuryBackgroundRecently, along with the progress in clinical medicine, the importance of ischemia-reperfusion injury was generally realized. Gastric mucosal was hypersensitive to redox, and was the most likely be involved in and express some clinical symptoms. Stomach and intestine endured ischemia-reperfusion injury at certain degree, when body suffered from trauma illness and operation stress.Our recent research found that Mitogen-activated protein kinases (MAPKs) and NF-kB signaling pathway were involved in gastric mucosa ischemia-reperfusion injury. Depressing the p38 MAPK, JNK and NF-kB pathways could attenuate stress induced stomach mucosa bleeding, lesion, inflammatory cytokines expression and apoptosis in rats. Some evidences showed the mechanism of ischemia-reperfusion and inflammatory injury were sameToll like receptor 4 (TLR4) played an important role in inflammation, and it had been found in gastric mucosa cells and many other tissues in mammalian. TLR4 was the upstream of MAPKs and NF-kB pathways, and the deficiency of it would depress the downstream pathways activation. TLR4 knockdown or mutant mouse strain showed some protective affection on ischemia-reperfusion stress, in brain, heart, liver and kidney. And TLR4 selected inhibitor Eritoran had been proved effective in protecting heart from ischemia-reperfusion induced injury. MAPKs and NF-kB pathways were involved in gastric ischemia-reperfusion injury (GIRI), but the role of TLR4 in GIRI is still unclear. Thus, the first part of this study was to compare the TLR4 mutant and wild type mice on GIRI, further investigated the activation of critical pathways downstream of TLR4, apoptosis and inflammation at the same time.In clinical cases, antiacid was widely used to prevent and cure gastric ischemia-reperfusion injury, which just get rid of the external part or aggravate factor of gastric ischemia-reperfusion injury. The intrinsic factor of gastric ischemia-reperfusion injury was redox stress, caused pathways activation, then inflammatory cytokines expressing and apoptosis, at last the barrier shield function of gastric mucosa was breakdown. Thus the second parts of the present study were to compare the effect of p38 MAPK inhibitor and proton pump inhibitor on gastric ischemia-reperfusion injury, and to investigate the mechanism of such effect. We expect that awareness of the role that TLR4 downstream signal transduction pathways in gastric ischemia-reperfusion induced gastric injury will add something useful to the pathogenesis of GIRI.Part I:The mechanism of TLR4 deficient reduced gastric ischemia-reperfusion injury in miceObjectives:To observe TLR4 deficient mice on gastric ischemia-reperfusion injury versus wild type mice, and investigate the mechanism of TLR4 deficient attenuated gastric ischemia-reperfusion injury.Methods:Wild type mice C3H/HouJ and TLR4 mutant mice C3H/HeJ,8-12 weeks old, were randomly divided into wild type sham group (WT sham), mutant sham group (MU sham), wild type (WT) and TLR4 mutant (MU) reperfusion Oh, 0.5h, 1h,2h,4h and 12h groups. Mice were starved but allowed free access to water for 12 h before the experiments. I/R injury of the stomach were produced by the occlusion and releasing of the celiac artery as Wada et al. described. Briefly, under pentobarbital sodium (40 mg/kg i.p.) anesthesia, the celiac artery was occluded with a small clamp for 0.5 h, and reperfusion was achieved by removing the clamp. Sham-operated animals underwent the same surgical procedure but without clamping the celiac artery. At different reperfusion time mice were killed, and tissue samples were collected. Four mice stomachs of each semi-group were kept in 10%neutral formalin solution for gastric mucosa bleeding area analysis by Image J 1.4, pathological examination, and the other six stomachs were kept in liquid nitrogen for further analysis. Thereafter, the gastric injury was evaluated by Masuda E’s double-blind pathological score. The total RNA was extracted from the stomach tissues using Trizol and the TNF-a, IL-10 IL-1βand IFN-γmRNA expression was tested by the meaning of real time PCR. The location and counts of apoptosis cell in stomach was detected by TUNEL staining. Western blot was accessed to exam the protein content of caspase-8, Cleaved Caspase-3 and phosphorylation of p38 MAPK, JNK, ERK, IKB-a in stomach tissue. Activation of NF-kB was dectected by EMSA.Results:(1)The stomach showed significant bleeding area after 1 hour of reperfusion injury versus sham group both in wild type and mutant mice, and mutant mice showed obvious less bleeding than wild type mice after 1 hour of reperfusion injury. Results of Masuda E’s double-blind pathological scores evaluation showed that, the sores of ischemia-reperfusion group were significant higher than the sham group’s, at the same time mutant mice showed obvious lower scores than wild type mice after 1 hour of reperfusion injury. (2)According to the TUNEL examination results, TUNEL stained positive cells had been found expressed in the nucleus of stomach mucosa epithelial cells, and the positive cells count result was corresponding with caspase-8 and Cleaved caspase-3 expression. The apoptosis of ischemia-reperfusion group were significant higher than the sham group’s, at the same time mutant mice showed obvious lower apoptosis than wild type mice after 1 hour of reperfusion injury. (3)TNF-a mRNA from the wild type 0.5 hours and 1 hour reperfusion groups expressed significantly higher than the wild type sham group, TNF-a mRNA from the mutant mice 0.5 hours and 1 hour reperfusion groups expressed significantly lower than the wild type mice to each correspond time point did by real time PCR test. IL-10 mRNA from the wild type 0.5 hours reperfusion groups expressed significantly higher than the wild type sham group, IL-10 mRNA from the mutant mice 0.5 hours reperfusion groups expressed significantly lower than the wild type mice, and 1 hour reperfusion groups expressed significantly higher than the wild type mice did by real time PCR test. IL-1(3 and IFN-γmRNA expression in TLR4 mutant mice were lower than wild type mice after 1 hour of rperfusion. (4)The expression of phosphorylated p38 MAPK (0,0.5,1,2,4 hours) and JNK (0.5 and 1 hours) were significant higher in wild type mice than in mutant mice after reperfusion. The expression of phosphorylated ERK was significant lower in wild type mice than in mutant mice after 0.5,2 and 4 hours of reperfusion, and there is no difference in other reperfusion group. EMSA analysis showed the activation of NF-kB was significant higher in wild type mice than in mutant mice after 1 hour of reperfusion. The expression of phosphorylated IKB-a were significant higher in wild type mice than in mutant mice after 1 hour of reperfusion, which were corresponding with the activation of NF-kB.Conclusions:TLR4 deficient reduced gastric ischemia-reperfusion injury versus wild type mice. The possible mechanisms are that the activation of p38 MAPK and NF-kB pathway lower in TLR4 mutant mice than in wild type mice, and TLR4 mutant mice has less apoptosis than wild type mice after gastric ischemia-reperfusion injury. TLR4 might be a potential strategy for prevention the pathological changes in gastric ischemia-reperfusion injury.Part II:The mechanism of p38 MAPK inhibitor reduced gastric ischemia-reperfusion injury in miceObjectives:To observe the effect of p38 MAPK inhibitor and proton pump inhibitor on gastric ischemia-reperfusion injury, and investigate the mechanism of attenuate effect on gastric ischemia-reperfusion injury of two reagents in mice. Then give some guideline on prevention and cure of gastric ischemia-reperfusion injury in clinic casesMethods:C57BL/6J mice,8-12 weeks old, were randomly divided into sham group (sham), ischemia-reperfusion+vehicle group (I/R+vehicle), ischemia-reperfusion+proton pump inhibitor group (I/R+proton pump inhibitor) and ischemia-reperfusion+p38 MAPK inhibitor group (I/R+p38 MAPK inhibitor). Mice were starved but allowed free access to water for 12 h before the experiments. Physiological saline (as vehicle) (0.9%NaCl lOml/kg), Pantoloc solution (20ml/kg) or SB239063 solution (20ml/kg,) were injected intraperitoneally 1 hour before the operation. I/R injury of the stomach were produced by the occlusion of the celiac artery as Wada et al. described. Briefly, under pentobarbital sodium (40 mg/kg i.p.) anesthesia, the celiac artery was occluded with a small clamp for 0.5 h, and reperfusion was achieved by removing the clamp. Sham-operated animals underwent the same surgical procedure but without clamping. After 1 hour of reperfusion mice were killed, and tissue samples were collected, four mice stomachs of each semi-group were kept in 10%neutral formalin solution for gastric mucosa bleeding area analysis by Image J 1.4, pathological examination, and the other six stomachs were kept in liquid nitrogen for further analysis. Thereafter, the gastric injury was evaluated by Masuda E’s double-blind pathological score. The total RNA was extracted from the stomach tissues using Trizol and the TNF-a, IL-1β, INF-y and ICAM-1 expression was tested by the meaning of real time PCR. Western blot was accessed to exam the protein content of Cleaved Caspase-3 and phosphorylation of p38 MAPK, JNK and ERK in stomach tissue. For investigating the effect of proton pump inhibitor or p38 MAPK inhibitor on gastric fluid pH, mice were randomly divided into physiological saline group; Proton pump inhibitor group and p38 MAPK inhibitor group. Physiological saline (as vehicle), Pantoloc solution or SB239063 solution was administered immediately after pylorus was ligated. Mice were sacrificed 1 hour after solution injection and the stomachs were washed with 3 ml deionized water for measurement of gastric fluid pH.Results:(1)The stomach showed significant bleeding area after 1 hour of reperfusion injury versus sham group, both proton pump inhibitor and p38 MAPK inhibitor pretreatment group showed obvious less bleeding area than I/R+vehicle group. Results of Masuda E’s double-blind pathological scores evaluation showed that, the sores of I/R+vehicle group were significant higher than that of sham group, at the same time pretreatment with proton pump inhibitor and p38 MAPK inhibitor showed obvious lower scores than I/R+vehicle. (2)MDA content from I/R+vehicle group expressed significantly higher than the sham group. At the same time pretreatment with proton pump inhibitor and p38 MAPK inhibitor showed obvious lower MDA content than I/R+vehicle. TNF-a, IL-1β, INF-y and ICAM-1 expression mRNA from I/R+vehicle group expressed significantly higher than the sham group, pretreatment with proton pump inhibitor significantly reduced TNF-α, IL-1βand INF-y mRNA expression versus I/R+vehicle group, pretreatment with p38 MAPK inhibitor significantly reduced TNF-a, mRNA expression versus I/R+vehicle group did by real time PCR test. (3)The expression of phosphorylated p38 MAPK, JNK and ERK from I/R+vehicle group expressed significantly higher than those of sham group, pretreatment with proton pump inhibitor significantly reduced activation of p38 MAPK, JNK and ERK versus I/R+vehicle group, pretreatment with p38 MAPK inhibitor significantly reduced activation of p38 MAPK and JNK versus I/R+vehicle group. Cleaved caspase-3 from I/R+vehicle group expressed significantly higher than the sham group, pretreatment with proton pump inhibitor had no obvious effect on Cleaved caspase-3 expression versus I/R+vehicle group, pretreatment with p38 MAPK inhibitor significantly reduced Cleaved caspase-3 expression versus I/R+ vehicle group.Conclusions:The p38 MAPK inhibitor attenuated gastric ischemia-reperfusion injury and had no obvious affect on gastric fluid pH in mice, the possible reasons were that p38 MAPK inhibitor depressing the p38 MAPK and JNK pathways, but had on obvious affect on activation of ERK pathway, finally breakdown of mucosa barrier was attenuated. Inhibition of p38 MAPK activation might be a potential strategy for prevention the pathological changes in gastric ischemia-reperfusion injury.更多还原