【作者】 田凤军；

【导师】 凌亦凌；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 急性肺损伤（acute lung injury, ALI）可由多种原因引起,主要病理特征为肺泡毛细血管膜和肺泡上皮广泛受损而致的肺水肿和肺不张,临床上可表现为呼吸窘迫和顽固性低氧血症,进一步发展至呼吸衰竭即为急性呼吸窘迫综合征（acute respiratory distress syndrome, ARDS）。多种病因（包括肺内/肺外,感染/非感染）都能直接或间接地引起ALI及ARDS。革兰氏阴性细菌内毒素的主要活性成分脂多糖（lipopolysaccharide, LPS）是导致肺部感染和全身感染的主要因素。LPS由其受体介导启动机体炎症反应,进而导致全身性炎症反应综合征（systemic inflammatory response syndrome, SIRS）,在肺部可表现为ALI。然而对于LPS启动机体炎症反应、导致ALI的具体机制尚未完全阐明。胆囊收缩素（cholecystokinin, CCK）是一种典型的脑肠肽,它不但具有胃肠激素的功能,还以神经递质和神经调质的形式发挥着重要的生理和病理生理作用。八肽胆囊收缩素（cholecystokinin octapeptide, CCK-8）为内源性CCK的重要功能片段。以往对CCK的研究多偏重于其在消化系统、神经系统及内分泌系统等的调节作用。CCK-8在肺脏也有分布。本室一直致力于研究CCK-8的抗内毒素休克（endotoxin shock,ES）及抗炎作用。CCK的作用主要由两种受体介导,即CCK-A受体（CCK-A receptor, CCK-AR）和CCK-B受体（CCK-B receptor, CCK-BR）。但有关CCK-8的抗炎作用及其机制尚未完全阐明,仍存在许多问题亟待解决。内源性气体信号分子是一类具有多种生理和病理生理作用的生物物质,它们的发现为ALI发病机制的研究开辟了一个新的领域。迄今已证实的内源性气体信号分子有三种,即一氧化氮（nitric oxide, NO）、一氧化碳（carbon monoxide, CO）和硫化氢（hydrogen sulfide, H2S）。NO和CO在ALI中的作用已得到了证实。H2S是继NO和CO之后被确认的第三种内源性气体信号分子。它是由含硫氨基酸被磷酸吡多醛-5’-磷酸依赖性酶[包括胱硫醚-β-合酶（cystathionineβ-synthase, CBS）、胱硫醚-γ-裂解酶（cystathionineγ-lyase, CSE）]等催化产生的。关于生理和病理情况下H₂S作用的研究虽刚刚起步,但已证实H₂S参与了神经和血管等的生理功能调节,以及高血压、肺动脉高压、内毒素休克等疾病的发生。那么,探明H₂S在LPS所致ALI中发挥何种作用,是本研究的主要目的之一。研究表明,肺微血管内皮细胞（lung microvascular endothelial cells, LMVEC）的活化和损伤在LPS所致的ALI中发挥重要作用。LPS介导内皮细胞活化的信号通路错综复杂,但目前认为LPS—CD14—TLR4—IRAK—IKK—IκB—NF-κB—proinflammatory cytokines是其中一条关键的信号通路,即LPS与内皮细胞膜表面LPS受体CD14结合后,激活Toll样受体4（Toll like receptor 4, TLR4）并与之结合形成受体复合物,募集适配分子MyD88,MyD88的死亡结构域再募集下游的IL-1受体相关激酶（IL-1 receptor-associated kinase, IRAK）到受体复合物。IRAK随后发生自磷酸化,从受体复合物解离,募集TNF受体相关因子6（TNF receptor-associated factor 6, TRAF6）,顺次激活下游激酶,包括NF-κB诱导激酶（NF-κB-inducing kinase, NIK）和丝裂素活化蛋白激酶/ERK激酶激酶（mitogen-activated protein kinase/ERK kinase kinase 1, MEKK1）。激活的NIK或MEKK1都能独自激活IKK复合物,导致IκB磷酸化降解,NF-κB移位入核,启动目的基因转录。CCK-8对LPS诱导的大鼠肺组织中CSE表达有何作用,它是否通过调控CSE/H₂S来发挥细胞保护作用,以及TLR4/NF-κB通路是否参与CCK-8对LPS诱导的CSE表达的调控均未见报道。本研究在我室以往系列研究的基础上,从整体、细胞及分子水平,观察CCK-8对LPS所致的ALI及LMVEC损伤的改善作用,CCK受体（CCK receptor, CCK-R）在LMVEC中的分布及表达变化,以及CCK-8对LPS诱导的CSE/H₂S的影响及TLR4/NF-κB通路在此过程中的作用,以探讨CSE/H₂S体系在CCK-8抗LPS所致的ALI中的作用,为临床防治ALI提供新的对策。1内、外源性H₂S在CCK-8减轻脂多糖所致急性肺损伤中的作用本部分实验的目的是观察内、外源性H₂S在CCK-8抗LPS所致的ALI中的作用。将84只雄性SD大鼠随机分为7组（每组12只）:①对照组:气管内滴注无热原生理盐水(200μl·只^-1);②LPS组:气管内滴注LPS(200μg·200μl^-1·只^-1);③NaHS+LPS组:气管内滴注LPS前10 min经腹腔注射0.5 mL NaHS(28μmol·kg^-1);④PPG+LPS组:气管内滴注LPS前10 min经腹腔注射0.5 mL PPG(45μmol·kg^-1);⑤CCK-8+LPS组:气管内滴注LPS前10 min经舌静脉注射CCK-8 (40μg·kg^-1);⑥PPG+CCK-8+LPS组:气管内滴注LPS前10 min先腹腔注射PPG（剂量同前）,随即舌静脉注射CCK-8（剂量同前）;⑦CCK-8组:气管内滴注生理盐水前10 min经舌静脉注射CCK-8（剂量同前）。于给药后4 h、8 h观察。留取血浆以检测H₂S含量。制备支气管肺泡灌洗液（bronchoalveolar lavage fluid, BALF）以检测其中中性粒细胞（polymorphonuclear neutrophils, PMN）数目和蛋白含量。未进行支气管肺泡灌洗的动物的动物摘取全肺称重并测定肺系数,留取左肺叶中上部,用于制备肺组织匀浆以测定丙二醛（malondialdehyde, MDA）含量、髓过氧化物酶（Myeloperoxidase, MPO）活性和P-选择素（P-selectin, P-SLT）水平。10%中性甲醛固定左肺叶下部,常规石蜡包埋、切片,经HE染色以观察肺组织形态学改变并进行定量评价指标（index of quantitative assessment, IQA）的测定。结果发现:①气管内滴注LPS后4 h、8 h可见肺组织弥漫性炎细胞浸润,肺泡间隔增宽,部分肺泡结构被破坏,部分肺泡代偿性气肿,且病变随时间延长而有所加重;IQA与对照组相比亦明显增高（P均<0.01）。与对照组比较,LPS可使大鼠肺系数明显增大（P均<0.01）、BALF中PMN数目和蛋白含量显著增加（P均<0.01）、肺组织中脂质过氧化产物MDA含量升高（P<0.05或P<0.01）。②与相应时间点的LPS组相比,CCK-8+LPS和NaHS+LPS组肺组织病变明显减轻,IQA亦下降（P均<0.01）;肺系数明显减小（P均<0.05）,BALF中PMN数目和蛋白含量显著下降（P均<0.01）,肺组织中MDA含量、MPO活性和P-SLT水平明显降低（P<0.05或P<0.01）。PPG可使BALF中PMN数目、肺组织MDA含量、MPO活性和P-SLT水平较相应时间点的LPS组明显增高（P<0.05或P<0.01）,加重LPS导致的肺组织损伤的程度并抑制CCK-8的保护作用。单独应用CCK-8对肺组织无明显影响。③气管内滴注LPS引起ALI时,血浆中H₂S含量减少。CCK-8和NaHS可逆转LPS的上述作用,与LPS组相比,其血浆中H₂S含量增加（P<0.01）。PPG则促进LPS对H₂S的下调作用（P<0.05）。单独应用CCK-8对H₂S无明显影响。以上结果表明,气管内滴注LPS可引起明显的肺组织损伤并下调H₂S的生成量。应用CCK-8和H₂S供体NaHS可上调H₂S,并减轻肺损伤。提示CCK-8细胞保护作用的发挥与H₂S的产生之间可能具有某种内在联系,它可能通过增加H₂S的产生来发挥抗氧化、抑制PMN聚集等作用,从而改善了LPS诱导的肺损伤。2 CSE/H₂S体系在CCK-8减轻脂多糖所致的急性肺损伤中的作用机制2.1 CCK-8减轻脂多糖所致大鼠急性肺损伤过程中肺组织CSE表达的变化本部分实验的目的是观察CCK-8改善LPS所致的肺损伤过程中肺组织CSE活性和CSE mRNA表达的变化,以阐明CCK-8调控LPS诱导H₂S生成的分子机制及其可能的生物学意义,同时在分子水平验证肺的非呼吸功能。将60只雄性SD大鼠随机分为5组（每组12只,给药方法同前）:①对照组:气管内滴注无热原生理盐水(200μl·只^-1);②LPS组:气管内滴注LPS(200μg·200μl^-1·只^-1);③CCK-8+LPS组:气管内滴注LPS前10 min经舌静脉注射CCK-8(40μg·kg^-1);④proglumide（非特异性CCK-R拮抗剂） + CCK-8+LPS组:舌静脉预注射proglumide(1 mg·kg^-1)10 min后注入CCK-8（剂量同前）,再经10 min后气管内滴注LPS（剂量同前）;⑤CCK-8组:气管内滴注生理盐水前10 min经舌静脉注射CCK-8（剂量同前）。每组各取6只大鼠分别于给药后4 h或8 h处死。取左肺叶中上部,部分用于制备肺组织匀浆CSE活性,部分用于提取组织总RNA以RT-PCR方法检测CSE mRNA的表达。结果如下:与对照组相比,LPS组大鼠肺组织CSE mRNA表达减少,CSE蛋白活性下降,以8h组明显（P<0.05）。与LPS组相比,CCK-8+LPS组肺组织CSE mRNA表达增多,CSE蛋白活性升高;与CCK-8+LPS组相比,proglumide+ CCK-8+LPS组肺组织CSE mRNA表达减少,CSE蛋白活性下降（P均<0.05）。与对照组相比,CCK-8组大鼠肺组织CSE活性及CSE mRNA表达无明显改变（P均>0.05）。结合第一部分的结果提示,H₂S/CSE体系参与了LPS所致ALI的病理生理过程。CCK-8可能通过上调CSE mRNA表达和蛋白活性而发挥肺保护作用。CCK-8增加LPS所致ALI大鼠肺组织中H₂S的产生有可能是通过CSE表达上调而实现的。2.2 NF-κB在CCK-8上调脂多糖所致急性肺损伤大鼠肺组织CSE表达中的作用及对TLR4的影响CCK-8上调LPS所致ALI大鼠肺组织中CSE表达的细胞内信号转导通路有许多问题尚未阐明。NF-κB调控免疫系统许多成份如前炎因子、粘附分子和一些酶类等的表达,它是许多信号通路的枢纽。mCD14是一种通过磷脂酰肌醇基团（phosphotidyl inositol, PI）锚定在细胞膜上的糖蛋白,由于缺乏跨膜区,多数生化和遗传学研究认为还存在第二种分子协助CD14介导LPS的效应。自从1997年首次在人类发现了果蝇Toll的同源物并将其命名为TLR以来,大量研究证实TLR4是参与哺乳动物识别及介导LPS效应的较特异的信号受体。LPS与CD14结合后,激活TLR4,形成LPS-受体复合物,才能介导完整的细胞活化效应。CCK-8上调LPS所致ALI大鼠肺组织中CSE表达是否与TLR4/NF-κB通路有关是一个值得探讨的问题。据此,本实验应用NF-κB特异性抑制剂PDTC进一步探讨了CCK-8上调ALI大鼠肺组织中CSE表达的信号转导机制。将84只大鼠,随机分为7组,每组12只。①对照组:气管内滴注无热原生理盐水(200μl·只^-1);②LPS组:气管内滴注LPS(200μg·200μl^-1·只^-1);③PDTC+LPS组:腹腔注射PDTC(15 mg·kg^-1)2 h后气管内滴注LPS（剂量同前）;④CCK-8+LPS组:舌静脉注射CCK-8(40μg·kg^-1)后10 min气管内滴注LPS（剂量同前）;⑤PDTC+CCK-8+LPS组:腹腔注射PDTC（剂量同前）后2 h注入注入CCK-8,10min后气管内滴注LPS（剂量同前）;⑥CCK-8组:气管内滴注生理盐水前10 min经舌静脉注射CCK-8（剂量同前）;⑦PDTC组:腹腔注射PDTC（剂量同前）,2 h后气管内滴注NS（剂量同前）。于给药后8 h观察。结果发现,NF-κB特异性的抑制剂PDTC进一步上调了CCK-8诱导的CSE mRNA表达和CSE蛋白活性,提示NF-κB通路在CCK-8上调LPS所致ALI大鼠肺组织CSE表达过程中发挥重要作用;PDTC和CCK-8抑制了肺组织TLR4基因的转录和蛋白表达,提示NF-κB对TLR4可能具有重要的调节作用。2.3 H₂S对脂多糖所致急性肺损伤大鼠肺组织CCK-R基因表达的影响我室以往的研究表明肺组织存在CCK受体基因表达,并可被LPS诱导表达增加,提示CCK-8可能通过与其受体相互作用而干预LPS所致的ALI过程,从而调节炎症反应。本部分实验应用RT-PCR方法观察H₂S对脂多糖所致急性肺损伤大鼠肺组织CCK-R基因表达的影响,从另一个角度证实H₂S在CCK-8减轻脂多糖所致急性肺损伤中的作用。将18只大鼠,随机分为6组,每组3只。①对照组:气管内滴注无热原生理盐水(200μl·只^-1);②LPS组:气管内滴注LPS(200μg·200μl^-1·只^-1);③NaHS+LPS组:气管内滴注LPS前10 min经腹腔注射0.5 mL NaHS(28μmol·kg^-1);④PPG+LPS组:气管内滴注LPS前10 min经腹腔注射0.5 mL PPG(45μmol·kg^-1);⑤NaHS组:气管内滴注生理盐水前10 min经腹腔注射NaHS（剂量同前）;⑥PPG组:气管内滴注生理盐水前10 min经腹腔注射PPG（剂量同前）。于给药后4 h观察。结果显示,LPS引起肺损伤的同时诱导CCK-A/BR mRNA的表达;H₂S可上调LPS诱导的CCK-A/BR mRNA表达,提示CCK-8与H₂S可能存在着正反馈的保护机制。3 CSE/H₂S体系在CCK-8减轻脂多糖所致肺微血管内皮细胞损伤中的作用及其机制3.1 CCK-8在减轻脂多糖所致LMVEC损伤中的作用及其对CSE/H₂S体系的调节LMVEC在炎症反应性中起着重要的作用。但CCK-8对ALI动物的保护作用是否与LMVEC有关尚未见报道。CCK-8的作用由CCK-R介导。目前发现,最经典的CCK-R亚型是CCK-AR和CCK-BR,而这两种CCK-R亚型的基因以及CSE基因在LMVEC中是否存在及其在LPS作用下的表达变化尚不清楚。本实验旨在通过培养大鼠LMVEC,观察H₂S在CCK-8减轻LPS所致LMVEC损伤中的作用及其机制以及CCK-R在LMVEC中的分布和表达的变化。分离并培养大鼠LMVEC,实验细胞随机分为5组:①对照组:常规DMEM培养基培养;②LPS组:DMEM培养基中加入LPS (10 mg·L^-1);③LPS+CCK-8组:DMEM培养基中先加入CCK-8 (10-6 mol·L^-1),10 min后再加入LPS;④LPS+ proglumide（丙谷胺,CCK-R阻断剂）组:DMEM培养基中先加入proglumide (10 mg·L^-1),10 min后再加入LPS;⑤CCK-8组:DMEM培养基中加入CCK-8培养。LPS、CCK-8和proglumide预先配成母液,细胞换液后加入到DMEM培养基中使成相应的终浓度。加药后继续培养8 h,应用试剂盒测定细胞MDA含量和乳酸脱氢酶（lactate dehydrogenase, LDH）释放率并检测细胞培养上清中H₂S含量、可溶性E-选择素水平和LMVEC台盼蓝摄取率的变化及LMVEC中CSE活性;采用RT-PCR方法检测细胞CSE mRNA和CCK-R mRNA表达的变化。结果如下:①与对照组相比,LPS孵育可使LMVEC活力下降、LDH释放率和MDA含量增加,抑制CSE mRNA表达和蛋白活性及H₂S生成。与LPS组相比,CCK-8与LPS共同孵育,减轻LPS所致上述细胞损伤指标变化,使细胞活力增强、LDH释放率和MDA含量下降,同时上调CSE mRNA表达,增强CSE活性,促进H₂S产生;而proglumide与LPS共同孵育则加重LPS对LMVEC的损伤作用,进一步下调H₂S/CSE体系;②在本实验条件下各组LMVEC均未见CCK-AR mRNA（1.3 Kb）的表达。正常LMVEC有CCK-BR mRNA （480bp）表达,LPS和CCK-8均可诱导其表达上调。以上结果提示:在培养的LMVEC水平,CCK-8具有改善LPS的损伤作用,此作用可能由CCK-R介导,结合以往的研究结果,提示LMVEC在CCK-8改善ALI中发挥重要作用。首次发现在培养的LMVEC上存在CCK-BR和CSE mRNA,CCK-8上调LPS诱导的LMVEC损伤中CSE/H₂S可能是CCK-8发挥细胞保护作用的另一个环节。3.2 H₂S对脂多糖诱导大鼠LMVEC中NF-κB活性的影响本部分将主要观察H₂S对LPS诱导LMVEC NF-κB活性及IκB蛋白水平的影响,以阐明H₂S的抗炎作用机制。实验细胞随机分为6组:①对照组:常规DMEM培养基培养;②LPS组:培养基中加入LPS(10 mg·L^-1);③NaHS + LPS组:DMEM培养基中加入NaHS（0.5 mM）,10 min后再加入LPS（剂量同前）;④PPG+LPS组:培养基中加入PPG（10 mM）,10 min后再加入LPS（剂量同前）;⑤NaHS组:培养基中加入NaHS（剂量同前）;⑥PPG组:培养基中加入PPG（剂量同前）。LPS、NaHS和PPG预先配成母液,细胞换液后加入到DMEM培养基中使成相应的终浓度。加药后继续培养1 h进行检测。结果显示:①用LPS孵育LMVEC 1 h,细胞内NF-κB活性明显高于溶剂对照组,而用NaHS处理后,抑制了LPS诱导的NF-κB活性增高,NaHS的作用可被PPG所减弱。NaHS和PPG单独孵育对细胞NF-κB活性无明显影响。用同源性寡核苷酸（含NF-κB结合位点）及异源性寡核苷酸（含AP-2结合位点）作为竞争物证实了DNA-蛋白结合的特异性。②LPS孵育LMVEC 1 h,IκB蛋白水平明显降低,NaHS可增加LPS处理的LMVEC内IκB蛋白水平并可被PPG所抑制。NaHSh和PPG单独孵育对细胞IκB蛋白水平无明显影响。上述结果表明,NaHS可抑制LPS诱导的NF-κB活性,其上游信号机制为抑制IκB蛋白降解。3.3 NF-κB在CCK-8上调脂多糖所致LMVEC损伤中CSE表达的作用及对TLR4的影响本部分实验主要通过培养大鼠LMVEC,在细胞水平进一步验证NF-κB通路在CCK-8上调LPS所致LMVEC损伤中CSE表达的作用及对TLR4的影响。实验细胞随机分为5组:①对照组:常规DMEM培养基培养;②LPS组:DMEM培养基中加入LPS(10 mg·L^-1);③PDTC+LPS组:DMEM培养基中先加入PDTC(50μmol·L^-1),2 h后再加入LPS（剂量同前）;④CCK-8+LPS组:DMEM培养基中先加入CCK-8(10-6 mol·L^-1),10 min后再加入LPS（剂量同前）;⑤PDTC+CCK-8+LPS组:DMEM培养基中先加入PDTC（剂量同前）,2 h后加入CCK-8（剂量同前）,其后10 min再加入LPS（剂量同前）;⑥CCK-8组:DMEM培养基中加入CCK-8（剂量同前）培养;⑦PDTC组:DMEM培养基中加入PDTC（剂量同前）培养。LPS、CCK-8和PDTC预先配成母液,细胞换液后加入到DMEM培养基中使成相应的终浓度。应用RT-PCR、Western blot等技术手段,各组于加药后8 h分别测定细胞中CSE基因和TLR4基因和蛋白表达的变化。结果发现,NF-κB特异性的抑制剂PDTC上调了CCK-8诱导的CSE表达,在细胞水平证实了NF-κB通路在CCK-8上调LPS损伤LMVEC中CSE基因表达过程中发挥重要作用;同时PDTC和CCK-8抑制了LMVEC中TLR4基因的转录和蛋白表达,提示在细胞水平NF-κB对TLR4可能存在重要的调节作用。结论本实验从整体、细胞和分子水平较为系统地观察了CSE/H₂S体系在CCK-8减轻LPS所致ALI和LMVEC损伤中的作用,并探讨了在此过程中的信号转导机制和受体机制,为其在临床上应用提供了新的、可靠的动物实验依据。1气管内滴注LPS可引起明显的肺组织损伤,同时血浆H₂S含量下降;抑制内源性H₂S的产生,可加重LPS所致的ALI,提示H₂S具有抗肺损伤作用,其机制可能与其抑制肺内PMN聚集及由此引起的炎症反应和氧化损伤有关。CCK-8可减轻肺损伤的同时上调血浆H₂S含量。CCK-8细胞保护作用的发挥与内源性H₂S生成之间可能具有某种内在联系。2 CCK-8通过上调CSEmRNA表达和CSE蛋白活性增加而发挥肺保护作用。并在整体和细胞水平证实CCK-8上调CSE表达可能是通过NF-κB信号通路进行调控的。在这一过程中,NF-κB对TLR4可能存在重要的的调节作用。H₂S可上调ALI大鼠肺组织CCKA/B R的表达。3在培养的LMVEC水平,CCK-8也可减轻LPS的损伤作用,此作用可能由CCK-R介导,通过上调LPS所致大鼠LMVEC CSE mRNA表达实现的;同时提示LMVEC在CCK-8减轻LPS所致ALI中发挥重要作用。4首次发现在培养的LMVEC上存在CCK-BR。LPS作用下,CCK-BR mRNA表达上调,是内源和外源性CCK-8发挥其改善LPS损伤作用和调控CSE/H₂S体系的重要受体机制。5首次发现在培养的LMVEC上存在CSE mRNA表达,为进一步研究H₂S抗炎作用的分子机制奠定了基础。更多还原

【Abstract】 Acute lung injury （ALI） is mainly characterized by diffusive injuries to lung epithelium and increased permeability of alveolar-capillary membranes caused by various factors, which lead to pulmonary edema and pulmonary closure. It expresses as distress of respiratory and refractory hypoximia clinically. There are many etiological factors that can evoke ALI/acute respiratoty distress syndrome （ARDS） directly or indirectly. Lipopolysaccharide （LPS）, the main component of the cell wall of gram-negative bacteria, is one of the most important factors causing pulmonary infection and systemic infection. LPS plays an important role in initiating inflammatory response through binding to its receptors and causing systemic inflammatory response syndrome （SIRS） which can induce ALI. However, it remains incompletely illuminated the definite mechanism of LPS initiating inflammatory over-reaction and inducing ALI.Cholecyetokinin （CCK）, a typical braingut peptide, is discovered initially in the gut as a gastrointestinal hormone with the function of contracting gallbladder and mediating pancreatic secretion, and subsequently localized in the central and peripheral nervous system as a neurotransmitter or neuromodulator to play a pivotal role in many physiological and pathophysiological processes. Sulfated cholecystokinin-octapeptide （CCK-8） is the minimum sequence for biological activity. Particular emphasis had been laid on its regulatory actions in nervous system, digestive system, and endocrine system in the previous studies of CCK. CCK is also demonstrated to be located in the lung. Our laboratory has been studying the effect of CCK-8 against endotoxin shock （ES） and inflammation. It was well known that the actions of CCK are mainly mediated by two distinct receptors, CCK-A receptor （CCK-AR） and CCK-B receptor （CCK-BR）. However the mechanism of alleviating the inflammatory response by CCK-8 is not clear.Endogenous gaseous transmitters, a unique class of biomaterials in regulating homeostasis, are found to play important roles in a variety of physiological and pathological events. Up to now, three gaseous transmitters have been recognized, namely nitric oxide （NO）, carbon monoxide （CO） and hydrogen sulfide （H₂S）. Many evidence confirmed the important role of NO and CO in ALI. H₂S is the third to be included in the family of endogenous gaseous transmitters, following NO and CO. Two pyridoxal-5’-phosphate-dependent enzymes-cystathionine-synthase （CBS ） and cystathionineγ-lyase （CSE） are responsible for the majority of the endogenous production of H₂S in mammalian tissues that use L-cysteine as the main substrate. Researches about physiological functions and pathological effects of H₂S became frequent only recently. It was proved that H₂S participated in the regulation of neural function and vasomotion, as well as the pathogenesis of hypertension, pulmonary artery hypertension and ES, etc. However, there were few reports about the role of H₂S in LPS-induced ALI, which was the major objective of this study.Recent years, a series of studies showed that the activation and damage of lung microvascular endothelial cells （LMVEC） has the effect in ALI induced by LPS. The signal transduction mechanisms through which LPS activate endothelial cells are anfractuous. However it is undoubtful that the LPS—CD14—TLR4—IRAK—IKK—IκB—NF-κB—proinflammatory cytokines pathway plays a pivotal role in the endothelial cells activation induced by LPS. NF-κB is a center of various signal pathways. LPS binds to its receptor CD14 on the membrane of endothelial cells and activates TLR4 to form LPS-receptor complex. Upon activation, TLR4 most likely form homodimers itself, resulting in the recruitment of an adapter named MyD88. The death domain of MyD88 then recruits downstream IL^-1 receptor–associated kinase （IRAK） to the receptor complex. IRAK is then autophosphorylated and dissociated from the receptor complex, and recruits TNF receptor-associated factor 6 （TRAF6） that in turn activates downstream kinases. Several such kinases have been found to be involved in TLR/NF-κB signaling pathways including NF-κB-inducing kinase （NIK） and mitogen-activated protein kinase/ERK kinase kinase 1 （MEKK1） . Activated MEKK1 or NIK are individually capable of activating the IKK complex. Subsequently, IκB is phosphorylated and degraded, leading NF-κB to translocate to the nucleus, and initiating the related gene transcription.However, it hadn’t been reported that whether CCK-8 could influence CSE expression induced by LPS in the lung of rats and exert its action by regulating CSE/H₂S, and whether TLR4/NF-κB signal pathway was involved in the regulatory effect of CCK-8 on CSE expression induced by LPS. The present study was designed to observe the ameliorative effect of CCK-8 on ALI and injury of LMVEC induced by LPS, the location and expression of CCK-R in LMVEC, the regulatory effect of CCK-8 on the expression of CSE/H₂S and the role of TLR4/NF-κB signal pathway at different levels including in vivo and in vitro, in order to explore the role of CSE/H₂S system in the ameliorative effect of CCK-8 ALI induced by LPS and provide new method for clinical doctor to prevent and treatment ALI.1 The roles of endogenous and exogenous H₂S in the ameliorative effect of CCK-8 on ALI induced by LPSThe objective of this study was to investigate the role of endogenous and exogenous H₂S in the ameliorative effect of CCK-8 on ALI induced by LPS. Eighty-four SD rats were divided into seven groups randomly （n=12 in each group）:①control group: pyrogen-free normal saline （NS, 200μl per rat） was instilled intratracheally;②LPS group: LPS (200μg·200μl^-1 each) was instilled intratracheally;③NaHS （sodium hydrosulfide, donor of H₂S）+LPS group: 0.5 mL of NaHS (28μmol·kg^-1) was injected intraperitoneally 10 min before LPS instillation;④PPG （L-propargylglycine, CSE inhibitor）+LPS group: 0.5 ml of PPG (45μmol·kg^-1) was injected intraperitoneally 10 min before LPS instillation;⑤CCK-8+LPS group: a bolus dose (40μg·kg^-1) of CCK-8 was injected through lingual vein 10 min before LPS instillation;⑥PPG+CCK-8+LPS group: PPG was injected 20 min before LPS administration, and CCK-8 was injected 10 min after PPG administration;⑦CCK-8 group: a bolus dose (40μg·kg^-1) of CCK-8 was injected through lingual vein 10 min before NS instillation. Each parameter was observed respectively 4 h, 8 h after LPS administration. Blood sample was collected to test the H₂S concentration in plasma. Bronchoalveolar lavage （BAL） was done on half of the rats in each group to detect polymorphonuclear neutrophils （PMN） number and protein content in bronchoalveolar lavage fluid （BALF）. As for the other rats in each group, which did not receive BAL, the ratio of lung weight to body weight （LW/BW） was calculated, and lung MDA content, MPO activity and P-selectin （P-SLT） level were determined. The morphological changes and the index of quantitative assessment （IQA） of lung injuries were also observed through light microscopy.The results as follows:①diffusive inflammatory cellular infiltration was observed 4 h and 8 h after LPS instillation, accompanied with widened alveolar septum, damaged alveolar structure and local emphysema, and the process was worsened as time lengthened; compared to control group, the IQA, LW/BW, PMN and protein content in BALF and MDA content, MPO activity and P-SLT level in lung tissue were all increased in LPS group （P<0.05 or P<0.01）.②Compared with LPS group at the same time points, the severity of lung injuries and the IQA were decreased in NaHS+LPS group and CCK-8+LPS group. NaHS and CCK-8 also lead to a decrease in LW/BW, PMN number and protein content in BALF, as well as lung MDA content, MPO activity and P-SLT level compared with those of LPS group （P<0.05 or P<0.01）. PPG could enhance the rise of PMN numbers, MDA content, MPO activity and P-SLT level in the lung （P<0.05 or P<0.01）, worsen the injury induced by LPS and inhibit the protective action of CCK-8. CCK-8 alone had no effect on the lung of rats.③Compared with control group, LPS instillation resulted in a decrease in plasma H₂S concentration. Pretreatment with CCK-8 and NaHS reversed the changes caused by LPS administration. It could elevate the H₂S content in plasma （P<0.01）. However, PPG could enhance the effect of downregulating H₂S content in plasma （P<0.05）. CCK-8 alone had no effect on H₂S level.The results above indicated that, LPS instillation could cause obvious lung injuries and downregulate H₂S concentration in plasma. NaHS, the H₂S donor and CCK-8 could upregulate the H₂S content and aggravated the injuries. There maybe exist internal relationship between the protective effect of CCK-8 and H₂S. CCK-8 could attenuate LPS-induced ALI by means of anti-oxidation and inhibition of PMN aggregation, which were both mediated by H₂S.2 The mechanisms of CSE/H₂S system in CCK-8 attenuating ALI induced by LPS2.1 Changes of CSE mRNA expression in rats with CCK-8 attenuating ALI induced by LPSIn the present study, we investigated CSE activity and the expression of CSE mRNA by RT-PCR in the lung during the process of CCK-8 attenuating lung injury induced by LPS, to explore the molecular mechanism and biology significance of CCK-8 regulating LPS-induced H₂S production and to verify the non-respiratory function of the lung at molecular level. Sixty SD rats were divided into five groups randomly （n=12 in each group）:①control group: pyrogen-free normal saline （NS, 200μl per rat） was instilled intratracheally;②LPS group: LPS (200μg·200μl^-1each) was instilled intratracheally;③CCK-8+LPS group: a bolus dose (40μg·kg^-1) of CCK-8 was injected through lingual vein 10 min before LPS instillation;④proglumide（CCK-R-non-specific inhibitor） +CCK-8 +LPS group: proglumide(1mg·kg^-1) was injected 20 min before LPS administration, and CCK-8 was injected 10 min after proglumide administration;⑤CCK-8 group: a bolus dose (40μg·kg^-1) of CCK-8 was injected through lingual vein 10 min before NS instillation. 4 h or 8 h after administration, each rat was executed. Part of mid-upper of left lung lobe was used to prepare homogenate of lung tissue for detecting CSE activity, part to extract total RNA and to test CSE mRNA expression with RT-PCR method.Results were as follows: compared with control group, CSE mRNA expression, CSE protein activity in the lung of LPS group were reduced, especially significantly in groups of 8 h （p<0.05）; compared to LPS group, CSE mRNA expression, CSE protein activity of CCK-8+LPS were all increased （all p<0.01）; compared with CCK-8+LPS group, CSE mRNA expression, CSE protein activity of proglumide+CCK-8+LPS group were reduced （p<0.05）; compared with control group, there were no significant changes of CCK-8 group about CSE activity and CSE mRNA expression （all p>0.05）.It suggested that CSE/H₂S system participated in the pathophysiological process of ALI caused by LPS. CCK-8 could enhance the increased expression of CSE mRNA and CSE activity in rats with ALI induced by LPS. CCK-8 enhanced the H₂S content through up-regulating CSE expression2.2 The role of NF-κB in CCK-8 enhancing CSE mRNA expression in the lung of rats with ALI induced by LPS and its influence on TLR4It was not elucidated about the signal pathway underlying CCK-8 enhancing CSE expression with ALI induced by LPS. NF-κB regulates expression of numerous components of the immune system. These include proinflammatory cytokines, chemokines, adhesion molecules and inducible enzymes. NF-κB acts at the crossroads of many signalling pathways. The continuing efforts to increase our molecular appreciation of the regulation of NF-κB will be of great value in learning to fully exploit this transcription factor as a therapeutic target. mCD14 is a surface glycoprotein anchored to the plasma membrane by a phosphotidy inositol without transmembrane domain. Several independent lines of evidence from biochemical and genetic studies support the hypothesis that LPS functions by interacting with a second CD14-associated signaling receptor. The homologue of Drosophila Toll （dToll） in human was first identified in 1997 and was named as Toll like receptor （TLR）. Then several lines of evidence suggest that TLR4 functions as a specific co-receptor of CD14 in recognizing and mediating the effect of LPS in mammals. These data support an LPS signaling complex that minimally includes CD14 and TLR4 to respond to LPS to elicit the full spectrum of gene expression. It was worthwhile to study whether TLR4/NF-κB was involved in the up-regulation of CSE expression by CCK-8 in ALI rats induced by LPS. In the present study, we therefore have studied the signal pathway involved in CCK-8 up-regulating CSE expression using NF-κB inhibitor PDTC. Eighty-four SD rats were divided into seven groups randomly （n=12 in each group）:①control group: pyrogen-free normal saline （NS, 200μl per rat） was instilled intratracheally;②LPS group: LPS (200μg·200μl^-1 each) was;③PDTC + LPS group: a bolus dose (15 mg·kg^-1, ip) of PDTC was injected 2 h before LPS administration;④CCK-8+LPS group: a bolus dose (40μg·kg^-1) of CCK-8 was injected through lingual vein 10 min before LPS instillation;⑤PDTC+CCK-8+LPS group: PDTC was injected 2 h and 10 min before LPS administration, and CCK-8 was injected 2 h after PDTC administration;⑥CCK-8 group: a bolus dose (40μg·kg^-1)) of CCK-8 was injected through lingual vein 10 min before NS instillation;⑦PDTC group: a bolus dose (15 mg·kg^-1, ip) of PDTC was injected 2h before NS administration. Animals were sacrificed 8 h after agent instillation.The results showed that PDTC upgraduated the CSE mRNA expression and CSE activity induced by CCK-8, which suggested that NF-κB signal pathway played an important role in CCK-8 up-regulating CSE expression in the lung of rats with ALI induced by LPS; PDTC and CCK-8 inhibited the TLR4 expression induced by LPS, so maybe there is modulate mechanism between NF-κB and TLR4 in LPS-induced ALI.2.3 The effect of H₂S on CCK R mRNA expression with ALI induced by LPSOur laboratory previous reported that lung tissue expressed CCK receptor gene, and the expression could be upregulated by LPS, indicating that CCK-8 might bind to CCK receptors and interfere with inflammatory response induced by LPS. The aim of this study was to investigate the effect of H₂S on CCK R mRNA expression with ALI induced by LPS, using RT-PCR, in order to verify that the roles of H₂S in the ameliorative effect of CCK-8 on ALI induced by LPS from another point of view. Eighteen SD rats were divided into six groups randomly （n=3 in each group）:①control group: pyrogen-free normal saline （NS, 200μl per rat） was instilled intratracheally;②LPS group: LPS (200μg·200μl^-1 each) was instilled intratracheally;③NaHS + LPS group: 0.5 mL of NaHS (28μmol·kg^-1) was injected intraperitoneally 10 min before LPS instillation;④PPG+LPS group: 0.5 mL of PPG (45μmol·kg^-1) was injected intraperitoneally 10 min before LPS instillation;⑤NaHS group: 0.5 mL of NaHS (28μmol·kg^-1) was injected intraperitoneally 10 min before NS instillation;⑥PPG group: 0.5 mL of PPG (45μmol·kg^-1) was injected intraperitoneally 10 min before NS instillation. Animals were sacrificed 4 h after agent instillation.The results showed that LPS upregulated CCK-A/BR mRNA expression, and H₂S could enhance the increased expression of CCK-A/BR mRNA induced by LPS. It suggested that there is positive feedback protective mechanism between H₂S and CCK-8. 3 The roles of CSE/H₂S system in CCK-8 attenuating LMVEC injury induced by LPS and its mechanisms3.1 CCK-8 attenuating LPS-induced LMVEC injury and modulating CSE/H₂S systemLMVEC played an important role in anti-inflammation during ALI. However, it was not reported whether LMVEC was involved in the protective effect of CCK-8. The effect of CCK-8 was mediated by its receptor. There are two main isoforms of CCK receptor （CCK-R）, CCK-AR and CCK-BR. But whether the two kinds of CCK-R and CSE exist in LMVEC and the change of its expression induced by LPS were not elucidated. The purpose of the present study was to observe the roles of H₂S in CCK-8 attenuating LMVEC injury induced by LPS and its mechanism, as well as location and expression of CCK-AR and CCK-BR in LMVEC. The cultured LMVEC were divided randomly into five groups treated with different agents: Control group, LPS group, CCK-8+LPS group, CCK-8 group and LPS+proglumid （CCK-R inhibitor） group. For group receiving LPS or CCK-8, LPS (10 mg·L^-1) or CCK-8 (10-6 mol·L^-1) was added into the culture medium （DMEM） respectively. For group receiving CCK-8 plus LPS or LPS plus proglumide, CCK-8 or proglumide (10 mg·L^-1) was added into the culture medium （DMEM） respectively 10 min before LPS administration. Negative control group received saline. Then cells of each group were incubated for eight hours. MDA content and release rate of lactate dehydrogenase （LDH） of LMVEC were investigated using test kit. H₂S content and soluble E-selectin （sE-SLT） in the superment and the rate of trypan blue uptake and CSE activity of LMVEC were also detected. Expression of CSE mRNA and CCK-R of LMVEC was examined by RT-PCR.The results were as follows:①compared with control group, the cell vitality was reduced, while the LDH release rate and MDA content of LMVEC were increased in LPS group. Meanwhile, LPS lead to a decrease in CSE mRNA expression, CSE activity and H₂S concentration. Compared with LPS group, the cell vitality was reduced, while the LDH release rate and MDA content of LMVEC were increased in proglumide +LPS group. The changes of the cell vitality, LDH release rate and MDA content caused by LPS were attenuated and the CSE/H₂S system was upregulated in CCK-8+LPS group. CCK-8 alone had no effect.②There was no CCK-AR mRNA （1.3 Kb） expression in each group. CCK-BR mRNA （480bp） expression products were observed in control group and increased significantly in CCK-8+ LPS group as much as that of control group. LPS upregulated the expression of CCK-BR mRNA also.These data confirmed that CCK-8 functioned the cytoprotective actions for mitigating lipoperoxide damage of LMVEC induced by LPS, which was mediated by CCK-R. It suggested that LMVEC played an important role in CCK-8 attenuating ALI referring to the results of the former part. It was firstly found that there existed CCK-BR in LMVEC and the cytoprotection of CCK-8 may be mediated by up-regulating CCK-BR mRNA expression. It was also firstly found that there existed CSE mRNA in LMVEC. The underlying mechanism of CCK-8 for cytoprotection was also associated with enhancing the generation of CSE/H₂S induced by LPS.3.2 Effect of H₂S on NF-κB binding activity in the rat LMVEC stimulated by LPSTo elucidate the anti-inflammatory mechanism of H₂S in the present study, NF-κB binding activity was analyzed by electrophoretic mobility shift assay （EMSA） and the IκBαprotein level in the cytoplasma was detected by Western blot. The cultured LMVEC were divided randomly into six groups:①control group: incubated with regular culture medium;②LPS group: LPS (10 mg·L^-1) was added to the culture medium;③NaHS+LPS group: NaHS （0.5 mM） was added to the culture medium 10 min before LPS (10 mg·L^-1) addition;④PPG+LPS group: PPG （10 mM） was added to the culture medium 10 min before LPS (10 mg·L^-1) addition;⑤NaHS group: NaHS （0.5 mM） was added to the culture medium;⑥PPG group: PPG （10 mM） was added to the culture medium. Then cells of each group were incubated for one hour.Results:①The NF-κB binding activity was significantly higher in LMVEC stimulated with LPS in comparison with unstimulated cells, and additional treatment with NaHS markedly reduced the binding activity. The effect of NaHS was abrogated by PPG. NaHS or PPG alone had no effect on the NF-κB binding activity. The binding specificity was confirmed by using homologous （NF-κB） and nonhomologous （AP-2） oligonucleotides as competitors.②The IκBαprotein level in LMVEC was markedly decreased 1 h after incubation with LPS , and NaHS obviously increased IκBαprotein level in LMVEC stimulated by LPS. The effect of NaHS was attenuated by PPG. NaHS or PPG alone had no effect on the IκBαprotein level.These results showed that NaHS inhibited NF-κB activity and IκBαdegradation in rat LMVEC.3.3 The role of NF-κB in CCK-8 enhancing CSE mRNA expression in the rat LMVEC stimulated by LPS and its effect on TLR4The purpose of the study was to verify that NF-κB signal pathway was involved in CCK-8 enhancing CSE expression in LMVEC induced by LPS and its effect on TLR4. The cultured LMVEC were divided randomly into seven groups treated with different agents: control group, LPS group, LPS+PDTC group, CCK-8+LPS group, CCK-8+LPS+PDTC group, CCK-8 group and CCK-8+PDTC group. For group receiving LPS or CCK-8, LPS (10 mg·L^-1) or CCK-8 (10-6 mol·L^-1) was added into the culture medium （DMEM） respectively. For group receiving CCK-8 plus LPS, CCK-8 was added into the culture medium （DMEM） 10 min before LPS administration. PDTC (50μmol·L^-1) was added 2 h before LPS or CCK-8 administration. Negative control group received saline. Then cells of each group were incubated for eight hours. CSE mRNA, TLR4 mRNA and protein expression was detected using RT-PCR, Western blot methods. The results showed that PDTC upregulated CSE expression induced by CCK-8, which demonstrated that NF-κB signal pathway played an important role in CCK-8 up-regulating CSE expression in LMVEC; meanwhile, CCK-8 and PDTC inhibited the expression of TLR4 mRNA and TLR4 protein, so maybe there is a modulate mechanism between NF-κB and TLR4 in LPS-induced LMVEC.CONCLUSIONThe roles of CSE/H₂S system in CCK-8 attenuating ALI and injury of LMVEC induced by LPS was examined systemically in vivo and in vitro, its signal pathway and receptor mechanisms were explored as well. The results provided novel and reliable experimental evidence for the clinical application.1 Intratracheal instillation of LPS could cause severe lung injuries and downregulate the H₂S content in plasma. Inhibition of endogenous H₂S by PPG exacerbated the lung injuries induced by LPS, while exogenous H₂S ameliorated the lung injuries. It was suggested that abnormally low levels of H₂S might contribute to the ALI induced by LPS, exogenous H₂S could protect the lung against the injuries, the possible mechanism of which may be associated with the role of H₂S in inhibiting PMN recruitment and the following inflammation and oxidation. CCK-8 could alleviate the lung injury while upregulated the H₂S content in plasma.There maybe exist internal relationship between the protective actions of CCK-8 and H₂S.2 CCK could enhance the expression of CSE mRNA in the lung of ALI induced by LPS. CCK-8 enhanced LPS-induced CSE expression in the lung of rat and in LMVEC through NF-κB signal pathway. Maybe there is a modulate mechanism between NF-κB and TLR4 in this process. H₂S could upregualte the expression of CCKA/BR mRNA in the rat lung of ALI. 3 CCK-8 functioned the cytoprotective actions for mitigating lipoperoxide damage of LMVEC induced by LPS, which was mediated by CCK-R and upregualte the expression of CSE mRNA. It suggested that LMVEC played an important role in CCK-8 attenuating ALI.4 There existed CCK-BR in LMVEC and LPS could directly increase CCK-BR mRNA expression, which is one of the important receptor mechanism underlying endogenous and exogenous CCK-8 affording cytoprotection for mitigating lipoperoxide damage of LMVEC induced by LPS and regulating CSE/ H₂S system.5 There existed CSE mRNA in LMVEC, thus providing a basical work for developing the molecular mechanisms of anti-inflammation in H₂S.更多还原