【作者】 崔文瑶；

【导师】 王俊科；

【作者基本信息】 中国医科大学 ， 麻醉学， 2009， 博士

【摘要】 目的内毒素血症常见于严重创伤、烧伤、休克、重症感染等应激状态下,来自于感染部位的LPS大量进入体内,并引起炎症介质的大量合成、释放,使包括肾脏在内的多器官受累,在急性损伤的发生、发展中起着不可忽视的作用。肾脏又是代谢产物及毒素排泄的主要途径,LPS亦可经肾小管重吸收后,使肾小管上皮细胞处在高浓度LPS微环境中,可能对肾小管上皮细胞结构和功能产生重要的影响,在AKI的发生、发展中起着一定的作用。急性肾损伤所致的急性肾功能衰竭的特征性改变就是肾小球滤过率下降及肾小管功能障碍,完善的尿液浓缩与钠离子重吸收的肾小管功能依赖于水通道蛋白的参与及功能性表达。AQPs（水通道蛋白）分布广泛,脑、肺、肾等脏器组织均有不同亚型分布,对水和甘油等小分子物质代谢有重要的调节作用,是保持内环境稳定的重要物质,也受多种因素影响其结构和功能,从而改变对水的通透性,影响器官水和小分子物质的代谢,同时也是导致细胞凋亡的关键因素。1992年Preston等发现肾脏AQP,这一发现合理地解释了肾脏自由水代谢的问题。肾脏是水代谢的主要器官,AQPs在肾脏中的研究对揭示肾脏的病理生理改变具有重要作用。近期有报道药物、缺血等引起的肾功能衰竭中AQP2表达明显下调,AQP2的这一变化与肾集合管上皮细胞损伤程度密切相关,恢复早于形态学改变。已证明AQP2是集合管上皮细胞上最重要,且是惟一受AVP调节的水通道蛋白。分布在细胞顶质膜及细胞内小泡。AVP对AQP2的调节有2种方式:（1）短期调节作用,AQP2氨基酸序列中从253-256位含蛋白激酶A（PKA）磷酸化部位,AVP与V2R结合后激活AC-cAMP-PKA信号传导途径,导致AQP2磷酸化,是含AQP2的细胞内小泡向细胞顶质膜转移并与之熔接,从而使细胞膜对水的渗透性迅速增加,这种调节作用可在数分钟内发生。（2）长期调节作用:V2R稳定激活可导致AQP2合成增加,AVP是通过V2RcAMP-PKA信号传递途经致细胞核内cAMP结合转录因子磷酸化及cjun/cFos基因表达,影响AQP2mRNA转录及AQP2蛋白合成,从而上调AQP2的总丰度,因而改变肾脏集（?）管对水的渗透性,AQP2mRNA的变化可在几个小时内出现。近年研究发现AQPs可以改善一些病理过程中脏器如肺、脑、肾的水及小分子物质的代谢,减少这些相应脏器的水肿和损伤,水通道蛋白介导的细胞移行对急性肾损伤肾小管功能的恢复和重建很重要,与预防和治疗这些重要脏器损伤关系密切,研究其在这些病理过程中功能和结构的变化有助于指导用药。早期发现和治疗肾功能异常不仅对于纠正休克具有作用,对于内脏器官保护具有更加重要的意义。水通道蛋白可以受多种因素影响,但是对其有调节作用的药物并不多,其阻断剂也多为重金属物质,本身就对生物体有损伤,少数药物如糖皮质激素据报道有一定的水通道蛋白调节作用。目前研究表明麻醉药如异氟烷、七氟烷、利多卡因和丙泊酚等对心、脑、肺和肾等脏器的不同损伤具有保护作用,丙泊酚作为一种静脉麻醉药具有镇静催眠作用,广泛应用于临床,有报道在内毒素血症肺损伤时丙泊酚可以通过调节AQP1起到保护作用,但对大鼠内毒素血症肾损伤是否通过调节AQPs起到保护作用尚未见报道。本实验根据文献并模拟病人内毒素血症观察所见建立大鼠内毒素血症模型,12小时后行功能检查并留取肾脏标本,病理检查肾脏损伤情况以及观察肾脏超微结构的改变,应用RT-PCR技术、Western印迹杂交技术、免疫组织化学染色技术等检测AQP2基因mRNA和AQP2蛋白、TNF-α、ICAM-1、Bcl2、Bax、Caspase3蛋白等在大鼠内毒素血症肾损伤过程中表达的变化,细胞凋亡程度;同时观察在大鼠内毒素血症肾损伤过程中应用公认的具有抗炎抗氧化作用的静脉麻醉药丙泊酚,采用预处理,同时给药和后处理的不同时间给药方式,观察其在大鼠内毒素血症肾损伤过程中对肾脏功能和AQP2基因mRNA、AQP2蛋白、ICAM-1、Bcl2、Bax、Caspase3和细胞凋亡程度的影响,从分子生物学角度观察和探讨不同丙泊酚给药时间对大鼠内毒素血症肾损伤的作用,为指导临床用药提供实验依据。本实验包括三部分:1、内毒素血症大鼠肾脏AQP2基因表达及结构功能变化;2、丙泊酚对大鼠内毒素血症肾组织AQP2基因表达及结构功能的影响;3、丙泊酚对内毒素血症大鼠肾脏细胞凋亡的影响。实验材料1实验动物选取48只Wistar大鼠（体重在200g—300g之间）,由沈阳军区总院实验动物中心提供。2实验试剂aquaporin 2一抗:兔抗鼠aquaporin 2,试剂盒（武汉博士德公司）;二抗:羊抗兔IgG,试剂盒（美国西格玛公司）;异丙酚（ASTRA ZENECA）;Bcl-2/Bax兔抗鼠多克隆抗体（即用型）（santa,美国）;caspase-3兔抗鼠多克隆抗体（Neomarker,美国）;Histostain^TM Plus Kits S-P 9000免疫组化染色试剂盒、ZLI-9108浓缩型DAB试剂盒（北京中杉生物公司）;Trizol（Invitrogen,美国）;One step RT-PCR试剂盒（大连宝生物公司）;聚偏二氟乙烯（PVDF）蛋白印迹膜（Bio-Rad,美国）;PBS等其它试剂为国产分析纯。3主要实验仪器（1）振荡水浴箱（GFL THERMOLAB,美国）（2）超低温冰箱（SANYO MDF-U,日本）（3）旋涡振荡器（VORTEX-2 GENE,美国）（4）水平板电泳系统（BIO-RAD Sub-cell GT,美国）（5）通用电泳仪（BIO-RAD PowerPac200,美国）（6）台式低温高速冷冻离心机（Sigma 3K30,美国）（7）显微图像分析系统（Olympus AX70/Coolsnapfx/MetaMorph,日本）（8）电热恒温鼓风干燥箱（余姚TDW,中国）（9）自动电泳凝胶成像分析仪（Alphainnotech ChemiImager 5500,美国）（10）小型垂直电泳仪（Bio-Rad Mini-ProteinⅢ,美国）（11）半干转印仪（Bio-Rad Seimidry transfer system,美国）（12）自动封膜仪（江苏仪器设备公司,中国）（13）HEIDOLPH DIAX900型匀浆机（德国）（14）PTC-100型PCR扩增仪（美国）（15）GLS-700D型数码凝胶扫描分析系统（上海,中国）（16）A-200 Ds电子天平（美国）（17）B-Brown微量泵（德国）（18）超纯水装置（MILLIPORE MILLI-Q,美国）（19）实验室制冰机（ZIGERA 2BE-70-35,德国）（20）小型台式离心机（SIGMA 1-13,美国）（21）PH计（WTW InoLab,德国）（22）电动高压消毒锅（HIRAYAMA HVE-50,美国）（23）HSS-1数字超级恒温浴槽（成都仪器厂,中国）实验方法1.建立模型Wistar大鼠腹腔注射戊巴比妥钠（40mg/kg）进行麻醉诱导及维持,LPS5mg/kg静脉注射建立大鼠内毒素血症模型,按照各组要求给予相应药物静脉注射(异丙酚:持续4小时静脉输注30mg·kg^-1·h^-1)后分别置入各自代谢笼中,以分别收集尿液,正常取食水,12小时后收集尿液和采取血液行功能检查及提取肾脏标本。2.分组和取材实验共分6组,分别为空白对照组（C1组8只）;注射生理盐水和脂肪乳组（C2组8只）;注射内毒素和脂肪乳组（L组8只）;丙泊酚预处理组（P1组8只,注射LPS前1小时开始输注丙泊酚）;丙泊酚同时给药组（P2组8只,注射LPS后立刻继以丙泊酚输注）和丙泊酚后处理组（P3组8只,注射LPS后1小时开始输注丙泊酚）。12小时后收集尿液并记录尿量,然后眼动脉取血3ml置于真空采血管内,静置30 min后在德国Sigma离心机上3000 r/min（r=9cm）离心10min,取上清置于灭菌子弹头内冻于-20℃冰箱内待测,应用7600全自动生化分析仪检测BUN、Crea,折射仪冰点下降法测血、尿渗透压。收集血、尿后断头处死大鼠,严格灭RNA酶操作下取各组肾脏组织,取部分肾脏分离出肾髓质置于灭菌子弹头内用液氮罐转移至-70℃冰箱内待测,一部分放入TRIZOL液中保存;另取部分肾脏经甲醛固定后HE染色以光镜观察肾集合管形态学变化和留待免疫组化分析。为电镜观察肾脏超微结构改变,另横断肾皮质髓质,取肾组织1mm³置25ml/L戊二醛中固定,固定的组织经磷酸盐缓冲液中充分漂洗,锇酸后固定,丙酮酸梯度脱水,环氧数脂618浸透处理,半薄切片定位肾小球,超薄切片,醋酸钠和枸橼酸铅双重染色,透视电镜下观察。3.检测指标（1）RT-PCR:RT-PCR方法检测各组细胞AQP2 mRNA、TNF-α、ICAM-1 mRNA。TRIZOL裂解各组细胞后进行总RNA的提取,一步法RT-PCR,按宝生物公司逆转录反应试剂盒使用说明操作。按试剂盒说明加入反应体系。逆转录合成第一链cDNA,反应条件为42℃30min,95℃5min。直接进入第二链cDNA合成及PCR扩增,反应条件为94℃变性40s,54℃退火40s,72℃延伸60s,38个循环,最后72℃5min结束,4℃保存。取8ulPCR产物于2%的琼脂糖凝胶电泳,在溴酚蓝指示剂到达凝胶底部边缘时停止电泳。以溴化乙啶液（1ug/ul）覆盖凝胶,染色5min自动电泳凝胶成像分析仪下观察拍照。测定电泳条带灰度值,与β-actin灰度值相比以百分数表示。（2）免疫组织化学染色技术:免疫细胞化学染色测定caspase3蛋白、Bcl-2/Bax蛋白和AQP2蛋白表达10%福尔马林溶液中固定肾组织经脱水、切片后,SP法进行免疫细胞化学染色,染色步骤按试剂盒说明书进行操作。DAB显色,苏木素轻度复染,脱水,透明,封片。采用显微图像分析系统（Olympus AX70/Coolsnapfx/MetaMorph）图像处理分析仪检测,每组在高倍镜下（×400）随机选择6个视野,测出caspase3、Bcl-2、Bax和AQP2蛋白等的平均光密度。肾组织AQP一2蛋白表达检测常规固定包埋后切片,以二甲苯脱蜡和梯度酒精脱水,0.3过氧化氢处理后,热抗原修复,反复加热3次,待切片冷却后按SABC免疫组织化学试剂盒和DAB显色试剂盒（北京中杉金桥生物技术有限公司）提供的方法,即予以一抗（羊抗小鼠AQP2多克隆抗体,美国SANTA公司）1:100稀释后孵育（4℃,过夜）,二抗（辣根过氧化物酶标记的兔抗山羊IgG,杭州联科生物技术有限公司）以1:500稀释后孵育（37℃,60min）,DAB显色,苏木素轻度复染,常规树脂封片后观察。AQP-2阳性部位呈棕黄色染色。（3）Western印迹杂交技术:肾AQP2、TNF-α、ICAM-1蛋白表达的检测采用Western blot技术,A.蛋白质样品的制备①每管肾组织中加入蛋白裂解缓冲液1.0ml,用研磨器反复混匀,冰浴30分钟后,12000rpm,4℃离心15分钟,收集上清,分装后保存于-70℃。②蛋白质浓度测定:在紫外分光光度计于570nm测定吸光度值,并根据标准曲线得出实际蛋白含量。B.SDS-PAGE电泳①将玻璃板用清洗剂洗净,晾干。配制12%的分离胶,将分离胶注入玻璃板夹层中,上面流出灌注积层胶所需空间（梳子的齿长再加上1cm）。用吸管在胶面上覆盖一层超纯水,保持胶面平整,将凝胶垂直放置在室温。②待分离胶完全聚合后（30分钟）,倾出覆盖层的液体,用去离子水洗涤凝胶顶部数次以除去未聚合的丙烯酰胺。配置好一定浓度的浓缩胶（4%）,将其直接灌注在已聚合的分离胶面上,立即在浓缩胶中插入干净的梳子,小心避免混入气泡,在加入浓缩胶液以充满梳子间的空隙,将凝胶垂直放置在室温下。③待浓缩胶完全聚合时,将待分析的蛋白质样品置于1×SDS凝胶加样缓冲液中,100℃加热3～5分钟使蛋白质变性,插入冰浴冷却后上样。④在电泳槽中加入电泳缓冲液,小心拔出上样梳,然后用注射器洗干净加样孔,用微量加样器分别吸取待分析样品,根据蛋白质的浓度和加样孔体积决定加样量。⑤加样完毕后,接通电源,起始时用低电压或低电流,当样品在浓缩胶部分浓缩成一条直线,进入分离胶后,将电源电压或电流提高,继续电泳直至溴酚蓝指示剂到达分离胶底部边缘时即可停止电泳。⑥从电泳装置上卸下玻璃板,放在纸巾上,撬开两层玻璃,取出凝胶。C.转膜①将电泳后的胶取出浸入转移缓冲液中,水平摇床缓慢摇30分钟,裁取相同大小的聚偏二氟乙烯（PVDF）蛋白印迹膜,甲醇浸透,弃去甲醇后浸入蒸馏水或1×TBS中,再将PVDF膜浸入转移缓冲液中至少5分钟。②用转移缓冲液浸湿一张厚滤纸,铺于半干转印仪的底层电极板上,将PVDF膜铺于滤纸上,勿留气泡,将凝胶贴于PVDF膜上,不留气泡,润湿另一张滤纸,盖在凝胶上,用玻璃棒赶去气泡。盖上顶层电极板,接通正负极电源,15V（不超过25V）转印10～30分钟。D,杂交①取出PVDF膜,1×TBS洗一次,和胶相邻面向上浸入封闭液中置于水平摇床缓慢摇1小时至过夜。②倒掉封闭液,用洗膜液略洗一下,加入用杂交液按1:500稀释的一抗（iNOS、eNOS和ET-1兔抗鼠多克隆抗体）,在摇床摇床上室温杂交1小时。取出PVDF膜,用洗膜液洗3次,每次5～15分钟。③倒掉洗膜液,加入用杂交液按比例（1:30000）稀释的二抗（碱性磷酸酶标记羊抗兔IgG,稀释度为1:5000）,摇床上杂交45分钟。取出膜,用洗膜液洗3次,每次5～15分钟。E.显色取出PVDF膜放入碱性磷酸酶显色液中,显色5-30min,显色明显后取出PVDF膜,晾干,备扫图,图像分析软件分析各电泳条带灰度值,半定量测定蛋白含量。（4）TUNEL法检测鼠肾脏细胞凋亡程度:10%福尔马林溶液中固定肾组织经脱水、切片后,SP法进行免疫细胞化学染色,染色步骤按试剂盒说明书进行操作。DAB显色,苏木素轻度复染,脱水,透明,封片。每组在高倍镜下（×400）随机选择6个不重复视野,计数鼠肾小管细胞凋亡数量,以细胞核呈棕黄色为阳性即凋亡细胞。（5）HE染色观察肾脏病理学改变:取肾组织10%中性甲醛固定,脱水后常规石蜡包埋,切片厚度4μm,行HE染色,用普通光学显微镜在400倍光镜下,随机选取5个视野,每个视野10个肾小管计分,光镜下肾小管病变程度采用Paller氏标准评分,以上检查交由病理专业人士进行,采用单盲法;电镜观察肾脏超微结构改变,标本处理:低温下横断肾皮质髓质,取肾组织1mm³置25ml/L戊二醛中固定,固定的组织经磷酸盐缓冲液中充分漂洗,锇酸后固定,丙酮酸梯度脱水,环氧数脂618浸透处理,半薄切片定位肾小球,超薄切片,醋酸钠和枸橼酸铅双重染色,透视电镜下观察。（6）肾脏功能学检测:收集尿液计量后冻存,眼动脉抽血3ml离心后取上清-20℃保存,留待血肌酐、尿素氮含量测定,血、尿渗透压测定采用折射仪冰点下降法测量。4统计学处理实验数据采用均数±标准差（（?）±s）表示,采用SPSS 12.0统计软件进行单因素方差分析,方差齐性采用LSD检验,方差不齐采用Dunnett T3检验,P＜0.05差异具有显著性。实验结果1.与两个对照组相比内毒素组尿量减少,血肌酐、尿素氮、血渗透压均明显提高,而尿渗透压明显下降,不同时间应用丙泊酚后出现不同程度的改善,尿量增加,血肌酐、尿素氮、血渗透压下降,尿渗透压提高。2.与两个对照组相比内毒素组大鼠肾脏AQP2 mRNA和蛋白的表达减少（P＜0.01）,、TNF、ICAM-1的mRNA和蛋白表达增多（P＜0.01）,而应用丙泊酚预处理、同时处理和后处理可以不同程度的纠正上述变化。3.与两个对照组比较内毒素血症大鼠肾脏caspase3和bax蛋白等促凋亡物质表达增加,bcl2作为一种抗凋亡物质表达减少,bcl2/bax比值下降,细胞凋亡明显增加,而经过丙泊酚处理后减轻上述表现,bcl2/bax比值得到恢复。4.肾组织超微结构的改变对照组肾组织超微结构基本正常,肾小管上皮细胞及细胞器结构正常。肾小管细胞核形态完整,无肿胀,大量线粒体、核糖体存在,无空泡变性,无断裂,粗面内质网无扩张,核糖体存在无脱落;内毒素组肾小管细胞受损,胞膜破裂,细胞核圆形其中异染色质边集,细胞膜表面微绒毛破坏,线粒体肿胀,破碎,嵴部分消失,粗面内质网扩张。P₁和P₂组肾小管细胞绝大部分结构正常或基本正常,肾小管细胞核形态、结构存在,核仁清楚,线粒体嵴稍扩张,粗面内质网和微绒毛稍扩张,较内毒素组超微结构明显改善。P₃组肾小管损伤略重,细胞器减少空泡变性明显,但与内毒素组比较也有改善。5.肾组织病理改变对照组形态学观察未见明显异常;内毒素组肾小管上皮细胞变性、坏死,细胞脱落,肾小管管腔变窄,肾间质水肿、充血,P₁和P₂组肾小管排列基本正常,以肿胀为主,肾间质水肿、充血,炎性细胞浸润不明显。P₃组肾小管的改变也有改善。光镜下肾小管损伤Paller氏评分,P₁和P₂组显著低于L组（P＜0.01）,P₃组也有差异（P＜0.05）。讨论本实验根据文献报道模拟内毒素血症病人观察所见建立内毒素血症大鼠模型,研究丙泊酚不同给药时间对肾脏功能学的影响并提取肾脏标本,行病理学检查及超微结构观察并采用免疫组化和western blot等技术检测水通道蛋白、肿瘤坏死因子、细胞间黏附分子、bcl2/bax以及caspase3水平和凋亡细胞数量,诣在探讨丙泊酚的肾脏保护作用及可能机制,为指导临床麻醉用药提供依据。结果显示与对照组相比,内毒素血症大鼠12小时后血Cr、BUN等水平显著升高,血渗透压升高、尿渗透压下降,出现AKI的负水平衡及高渗改变;透射电镜观察肾集合管超微结构发现,内毒素组肾小管细胞核、线粒体、粗面内质网、微绒毛等均遭破坏,但紧密连接尚在;光镜结果显示:内毒素组肾小管上皮细胞变性、坏死,细胞脱落,肾小管管腔变窄,肾间质水肿、充血;免疫组化等检查发现,内毒素组水通道蛋白含量明显下降,肿瘤坏死因子、细胞间黏附蛋白等表达增多,Bcl2/Bax比值下降,caspase3表达增加,细胞凋亡增加,不同时间给予丙泊酚后,各种功能学检查以及肾集合管的超微结构和病理改变均得到改善,水通道蛋白的表达增加,上调了抗凋亡基因Bcl-2mRNA的表达,肿瘤坏死因子、细胞间黏附分子、caspase3等表达下降,肾小管凋亡细胞减少。上述结果表明,不同时间应用丙泊酚给药能对抗内毒素血症肾损伤,有利于肾结构和功能的恢复。内毒素血症常见于严重创伤、烧伤、休克、重症感染等应激状态下,来自于感染部位的LPS大量进入体内,并引起炎症介质的大量合成、释放,导致细胞变性和坏死,表现为组织结构的损害和器官功能的衰退;LPS引起肾小球尤其是入球卸鍪账?使肾内血流再分布,造成肾血流及肾小球滤过率下降,从而诱发肾缺血再灌注损伤,同样的在急性肾损伤的发生、发展起着不可忽视的作用;肾脏又是代谢产物及毒素排泄的主要途径,LPS亦可经肾小管重吸收后,使肾小管上皮细胞处在高浓度LPS微环境中,可能对肾小管上皮细胞结构和功能产生重要的影响,在AKI的发生、发展中也起着一定的作用。丙泊酚是一种临床常用静脉麻醉药,其特有的功能和结构使其具有抑制炎性反应、减轻炎性因子释放、抑制炎症细胞的聚集活化从而减轻炎症反应强度、增强抗炎功能并减少坏死及凋亡,具有保护脏器作用,有研究表明丙泊酚可以在内毒素血症肺损伤时通过调节AQP1并减少局部TNF等物质产生减轻肺泡上皮细胞损伤的作用,本实验观察到在内毒素血症急性肾损伤过程中应用丙泊酚处理的几组均表现出不同程度的损伤减轻表现,而脂质溶剂组则没有这种效果,说明是丙泊酚本身可能通过调节局部的水通道蛋白表达及相关因子水平和肾的灌注而起到了肾脏的保护作用。内毒素血症肾损伤所致的急性肾功能衰竭的特征性改变就是肾小球滤过率下降及肾小管功能障碍,完善的尿液浓缩与钠离子重吸收的肾小管功能依赖于水通道蛋白及其它离子转运蛋白的参与及功能性表达。AQPs（水通道蛋白）分布广泛,脑肺肾等脏器组织均有不同亚型分布,对水和甘油等小分子物质代谢有重要的调节作用,是保持内环境稳定的重要物质,也受多种因素影响其结构和功能,由于AQPs对多种病理过程和很多细胞因子炎症因子的敏感性,从而改变对水的通透性,影响器官水和小分子物质的代谢,同时也是导致细胞凋亡的关键因素。肾脏AQP的发现合理地解释了肾脏自由水代谢的问题。正是因为肾脏是水代谢的主要器官,AQPs在肾脏中的研究对揭示肾脏的病理生理改变具有重要作用。近期有报道药物、缺血等引起的肾功能衰竭中AQP2表达明显下调,AQP2的这一变化与肾集合管上皮细胞损伤程度密切相关,恢复早于形态学改变。已证明AQP2是集合管上皮细胞上最重要的水通道蛋白。分布在细胞顶质膜及细胞内小泡,其丰度影响集合管对水的通透性,几分钟内即可发生变化。近年研究发现AQPs可以改善一些病理过程中脏器如肺、脑、肾的水及小分子物质的代谢,减少这些相应脏器的水肿和损伤,水通道蛋白介导的细胞移行对急性肾损伤肾小管功能的恢复和重建很重要,与预防和治疗这些重要脏器损伤关系密切,研究其在这些病理过程中功能和结构的变化有助于指导用药。本实验中我们观察到与对照组比较,注射脂质溶剂+内毒素组的水通道蛋白含量减少,提示水通道蛋白的减少可能是急性肾损伤的一个因素,而应用丙泊酚的几组中水通道蛋白2的含量不同程度的均有提高,提示丙泊酚本身具有直接的或间接的作用调整水通道蛋白2从而起到肾脏保护的作用,当然,局部的炎性反应、其它的水通道蛋白和离子通道也参与其中,确切机制有待于进一步研究。研究表明大量细胞因子参与了内毒素血症的过程,其中包括IL-1β,IL-6,ICAM-1,VCAM-1和TNF-a,这些炎性因子诱导炎性瀑布,放大炎性反应。TNF-α是一种17kd的多肽,被认为是全身炎性反应的启动和关键因子。有研究表明,TNF-α对肾组织有较强毒性,具有直接的细胞毒性,直接引起细胞坏死,还可以通过微循环障碍导致细胞坏死,在内毒素刺激下与其它炎症介质相互激发,可诱导内皮细胞和巨噬细胞释放IL-1,IL-1随后又刺激产生其它细胞因子的生物合成,如加强ICAM-1mRNA的表达,使炎症信号进一步扩大,反过来增强组织对TNF-α的敏感性。给动物注射适量的TNF-α能引起急性肾损伤,如给以TNF-α抗体或是采取措施减少其产生能阻断内毒素的损伤作用。本研究证实,内毒素血症后,肾皮质TNF-αmRNA表达明显增加,表明其参与了AKI的病理过程,而给予异丙酚能显著减少TNF-αmRNA和TNF-α蛋白表达,从而明显减少肾组织中TNF-α的含量,减轻肾损伤。ICAM-1是细胞表面的糖蛋白,是内皮细胞结构的一部分。内毒素血症后,LPS刺激及机体的应激反应产生大量的炎性介质和细胞因子,这些物质在激活白细胞的同时,也激活了血管内皮细胞,前者在细胞表面表达CD11/CD18糖蛋白黏附复合物,后者表达ICAM-1,二者相互结合,引起白细胞与内皮细胞的黏附,促进白细胞聚集活化,释放炎性介质。正常肾组织缺乏ICAM-1表达,LPS及细胞因子均能使ICAM-1mRNA表达上调,导致循环白细胞在局部黏附,于外髓质形成梗阻,同时使内皮通透性增加,且浸润、激活的白细胞还可导致直接的组织损伤,红细胞凝聚导致再灌注后的外髓失灌注,加重内毒素血症病理过程中的缺血再灌注损伤,而减少局部和循环系统中的ICAM-1含量能减少血管内皮细胞的活性和损伤,其含量能较好地反应损伤状况。Kelly等研究发现,ICAM-1缺陷大鼠能明显减轻肾缺血再灌注损伤,给予ICAM-1抗体同样可减轻缺血再灌注引起的肾损伤。本实验RT-PCR结果表明,内毒素血症12小时后肾皮质ICAM-1mRNA表达增强,说明ICAM-1也是急性肾损伤的一个因素。丙泊酚不同时间给药可不同程度减少肾组织ICAM-1mRNA和ICAM-1蛋白的表达,减轻肾损伤。肾脏作为内毒素血症的重要靶器官,其损伤机制尚不完全明了,许多因素参与其中,作为区别于坏死的一种细胞死亡形式,凋亡是否参与其中并导致损伤有待进一步研究。细胞凋亡是机体内衰老的、无用的或某些损伤细胞死亡的一种不同于细胞坏死的特殊的细胞死亡形式,细胞发生特异性的降解,形态上表现为核固缩、胞膜发泡和凋亡小体形成,是由特定基因调控的,没有明显细胞溶解的过程。Caspase3是半胱氨酸蛋白酶家族的重要成员,是凋亡过程中的最主要的终末执行酶,抑制其活性可能减少凋亡。Bcl-2mRNA是该基因家族中重要的抗细胞凋亡基因,与促凋亡基因间的平衡影响着凋亡的发生,对Caspase依赖和非依赖的凋亡均有抵抗作用。研究结果显示,与对照组比较,LPS组凋亡细胞的明显增加也提示凋亡参与了急性肾损伤的过程,而丙泊酚不同时间给药后可程度不同的增加bcl-2、减少Caspase3的表达,减少促凋亡物质,增加抗凋亡物质的表达,从而减少细胞的凋亡。由此可见,丙泊酚对肾细胞的保护作用部分是通过调节bcl-2/bax及Caspase3的表达,减少细胞凋亡,来发挥其抗损伤作用。综上所述,本研究发现,内毒素血症时给予丙泊酚对急性肾损伤有明显的保护作用,其可能的机制是通过调节水通道蛋白表达、下调黏附分子和肿瘤坏死因子等炎性因子,以及影响促/抗凋亡因素等环节发挥作用;与预先和同时给与丙泊酚组相比,延迟给药组亦有此作用但作用略弱,说明早期后处理（一小时内）也具有一定的保护作用,为临床麻醉用药提供了理论依据。结论1,内毒素血症大鼠肾功能、形态及一系列分子生物学发生改变,表明内毒素血症时存在急性肾损伤。2,在此过程中采用不同时间应用丙泊酚处理可以不同程度地减轻肾功能和形态的改变,对肾组织具有一定程度的保护作用,后处理尽管改善效果不如预处理和同时处理组明显,但也具有一定的保护作用。3,内毒素血症大鼠肾脏凋亡因子增加,细胞凋亡增加,不同时间应用丙泊酚处理包括后处理后减少凋亡从而起到此过程中肾脏保护的作用。更多还原

【Abstract】 PurposeEndotoxemia is often present in stress conditions such as severe trauma,burn, shock,severe infection,etc.In these conditions,plentiful LPS from the infective areas enter the body and cause the mediators of inflammationsynthesizing and releasing abundantly.Consequently,multiple organs including kidney are involved.Accordingly, endotoxemia plays an critical role in the onset and development in acute injury.Kidney is the major route for the excretion of metabolic product and toxin.The LPS reabsorbed by renal tubule can place the renal tubular epithelium cells in a high lipopolysaccharide（LPS） concentration microenvironment,which might have a great effect on the function and structure of renal tubular epithelium cells.It also acts on the onset and development of acute kidney injury（AKI）.The characteristic change of acute kidney failure caused by AKI is the decline of glomerular filtration rate（GFR） and renal tubule dysfunction.The consummate renal tubular function including urine concentration and reabsorption of K⁺ depends on the involvement and functional expression of Aquaporin（AQP）.AQPs distribute widely and possess various subtypes in many organs and tissues such as brain,lung,and kidney.AQPs play an important part in regulation of water and glycerol metabolism.They are vital substances for maintaining a stable internal environment,whose structure and function is influenced by many ingredients as well.AQPs can change water permeability and affect the metabolism of water and micromolecules.AQPs are also the key fact influencing cell apoptosis simultaneously. In 1992,Preston found out kidney AQP.This finding explained the problem of renal free water metabolism rationally.Kidney is the major organ of water metabolism.The research of AQPs in kidney operates a crucial fact on revealing renal physiological and pathological change.It is reported the expression of AQP is decreased apparently in renal failure caused by drugs,ischemia etc.This change of AQPs is close related to the injury degree of renal tubular epithelium cell,whose convalescence occurs earlier than morphological change.It has been proved thatAQP2 is the most important aquaporin which is unique regulated by AVP.It is distributed in cellular cell apicoplasmatic membrane and intracellular vesicle.There are two regulation ways of AVP to AQP2:（1） Short regulation action:253-256 sites in amino acid sequence of AQP2 contain protein kinase（PKA） phosphorylation position.The combination of AVP and V2R activates the AC-cAMP-PKA signal conduction pathway and causes the phosphorylation of AQP2.Then it makes intracellular vesicles containing AQP2 moving and fritting to cell apicoplasmatic membrane.Consequently,the permeability of cell membrane to water increases rapidly,which happens in several minutes.（2） Long regulation action:the stable activation of V2R can cause increased synthesis of AQP2.AVP phosphrylates intranuclear cAMP and stimulates the expression of cjun / cFos gene by V2RcAMP-PKA signal conduction pathway and then affects the RNA transcription and protein synthesis of AQF2.Accordingly,the total abundance of AQP2 is up-regulated and the permeability of renal collecting tube to water is changed.The alteration of AQP2 RNA can appear in several hours.Recent years’ researches show that AQPs can improve micromolecule and water metabolism of lung,brain,kidney in some pathological process and relieve the edema and injury of these corresponding organs.The cell migration inedited by AQP is important for the recovery and reconstruction of renal tubular function in acute renal injury.It is close relative to the prophylaxis and treatment of these vital organs injury. Researching the changes of function and structure in these pathological processes can assist guiding drugs usage.Discovering and treating renal function disorder early can not only retrieve shock,but also protect viscera better. The researches shows that anesthetics such as isoflurane,sevoflurane,lidocaine, propofol,etc can protect heart,brain,kidney,lung from varied injuries.As a widely used clinical intravenous anesthetic,Propofol’s function is sedative and hypnosis.It has been reported that propofol has its protective action through regulating AQP1 when confronting lung injury in endotoxemia,but still unsure whether it will go well for the rat’s kidney injury in endotoxemia.Based on literature,this experiment imitates the observation of human endotoxemia in order to establish rats endotoxemia model.Examine function and acquire kidney specimen after 12 hours.Find out renal injury condition by pathological examination and observe the alteration of ultramicrostructure in kidney.Detect the changes in expression of AQP2 mRNA,AQP2 protein,tumor necrosis factor-α（TNF-a）, intercellar adhesion molecule-1（ICAM-1）,B-cell lymphoma/leukemia-2（Bel2）,Bax and Caspase3 and cell Apotosis Indexin in rats endotoxemia’s kidney injury process by RT-PCR,Western-blotting hybridization skill,immuno-histology chemical staining skill,etc.Meanwhile,observe the effect of the well known anti-inflammation and anti-oxydation intravenous anesthetic--propofol in the rats endotoxemia’s kidney injury. Take three different ways:pretreatment,use drugs simultaneously,aftertreatment.And then observe propofol’s effect on rensl function,AQP2mRNA,AQP2protein,ICAM-1, Bcl2,Bax,Caspase3 and cell apoptosis Index in rats eodotoxemia’s kidney.Observe and discuss the effects of different dosage time of propofol on rats endotoxemia’s kidney injury from molecular biology aspect and provide experimental basis for guiding clinical dosage.The experiment includes three parts:1.the alteration of gene expression of AQP2,structure and function in endotoxemia rats’ kidney.2.the effect of propofol on the gene expression of AQP2,structure and function in endotoxemia rats’ kidney.3.the effect of propofol on cell apoptosis in endotoxemia rats’ kidney. Materials1.Animals48 Wistar rats（body weight:200-300g）,provided by Experimental Animal Center,Shenyang General Military Hospital2.Main reagents and apparatusaquaporin 2 antibody,propofol,Bcl-2/Bax multiclonal antibody, caspase-3 multiclonal antibody,PCR machine,stable temperature water bath, electrophoretic machine,eletroalance,liquid nitrogen tank,section machine,inverted phase contrast microscope,MDF-Ultra deep-freeze equipment,micro-image analysis system,etc.Methods1.Establishment of animal modelAnesthesia of the rats was induced and maintained by intraperitoneal injection of pentobarbital,LPS 5mg/kg intravenous injection was given to establish endotoxin model.2.Experiment design48 wistar rats were divided into 6 groups.Group C（sham group,n=8）,Group Saline and Fat Milk（n=8）,Group Endotoxin and Fat Milk（n=8）,Group Propofol Predisposal （n=8,propofol infusion was given one hour before LPS injection）,Group Propofol（n=8, propofol infusion began immediately after LPS injection）,Group Propofol Postprocessing（n=8,ropofol infusion was given one hour after LPS injection）.12 hours later,urine and 5ml of blood from ophthalmic artery were collected.After the collection of urine and blood,the rats were killed and their kidney medulla was sampled.3.Detection parameter（1） Morphological changes of renal collecting tube by light microscope （HE stain）（2） Ultrastructural morphological changes of kidney by electron microscope（3） Measurement of BUN,Cr and osmotic pressure of urine and blood（4） AQP2 mRNA,TNF-α,ICAM-1,caspase3,Bcl-2/Bax mRNA by RT-PCR and Western blot（5） Expression level of caspase3 protein,Bcl-2/Bax protein and AQP2, TNF-α,ICAM-1 by Immunocytoehemical stain（6） Renal apoptosis detection by TUNEL method4.Statistical analysisAll values were presented as mean SE and subjected to one-factor analysis of variance using SPSS 12.0 software.Homoscedasticity was checked by LSD method and Dunnett T3 method was adopted to check the heterogeneity of variance. Differences were considered significant at P＜0.05.Results1.compared with control group,in endotoxin group,the expression of rats’ kidney AQP2,bcl-2 mRNA and protein was downregulated（P＜0.05）,the expression of TNF, ICAM-1,caspase3,bax mRNA and protein was upregulated（P＜0.01）,and propofol predisposal,coprocessing and postprocessing can retrieve those changes.2.Compared with control group,in endotoxin group,the urine volume decreas ed,the Cr,BUN and blood osmotic pressure increased significantly while the urine osmotic pressure decreased,propofol infusion can relieve those reactions.3.Compared with control group,the amount of apoptotic cell in endotoxin group increased significantly,propofol infusion can relieve those reactions.4.Ultrastructual morphological changes:The ultrastructure of nephridial tissue in control group was fundamentally normal, renal tubular epithelial cell and its organelle structures were normal.In endotoxin group, renal tubular cell demonstrated injury,nucleus degeneration and irregularity,proximal convoluted tubule microvillus damaged,mitochondfia swelled,broken,some of the cristae disappeared,PER dilated.In Group P1 and P2,most of renal tubular cell structure showed normal or essentially normal,tubular cell nucleus showed normal morph and structure,nucleoli were clear,mitochondria cristae swelled slightly,RER and microvillus dilated slightly,ultrastructural morphologh improved significantly.In Group P3,renal tubule damage was slightly severer than that of endotoxin group.1.Kidney histological morphologic changes:There was no manifest morphologic abnormality in control group.In endotoxin group,the renal tubular cells showed degeneration,necrosis and falling off. The lumens of renal tubule were narrowed,renal interstitial showedobvious edema,congestion and infiltration of inflammatory cell.The alignment of renal tubule in Group P1 and P2 were fundamentally normal,morphologic changes consisted of swell and small amounts of congestion and infiltration of inflammatory cell.Morphologic improvement of renal tubule was also observed in Group P3.Under light microscope,the Paller’s Score of renal tubule damage in Group P1 and P2 were significantly lower than that of Group L（P＜0.01） and Group P3（P＜0.05）.DiscussionBased on literature,this experiment imitates the observation of human endotoxemia in order to establish rats endotoxemia model.Research the effect of different dosage time of propofol on renal function and acquire kidney specimen.Operate pathological examination and ultramicrostrcture observation as well as immuno-histology chemical skill and western-blotting skill to detect the change of aquaporin,TNF-a,ICAM-1, bcl2/bax,caspase3,etc,aiming to discuss propofors kidney protection and mechanism and provide experiment basis for guiding clinical anesthetic dosage.Compared to control group,the result shows that endotoxemia rats’ blood Cr,BUN increase significantly.Serum osmolarity(S_osm) also increases while Urine osmolarity(U_osm) decreases.AKI’s negative water equilibrium and high osmotic pressure are present. Observe ultramicrostructure of renal tubular Through transmission electron microscope we can find that endotoxemia group encounter severe injury of renal tubular nucleus, mitochondria,RER,microvillus,etc.Through light microscope we can find that endotoxemia group encounter renal tubular epithelium cell’s degeneration,necrosis, defluxion,the stenosis of renal tubular lumen,the edema of renal interstitium and congestion with inflammatory cell infiltration apparently.The immuno-histology chemical examination finds that the content of aquaporin in endotoxemia group declines significantly while TNF-a,ICAM-,caspase3 express increasely.The ratio of Bcl2/Bax decreases and the apoptosis of cell increase.After utilizing propofol,all kinds of functional examination and ultramicrostructure as well as pathological change get great improvement,the expression of aquaporin increases.The expression of anti-apoptosis gene Bcl-2mRNA is up-regulated while that of caspase3 is down-regulated.The apoptosis of renal tubular cells decrease.From the results mentioned above,we can see that different dosage time of utilizing propofol can antagonize kidney injury in endotoxemia and assist recovering renal structure and function.Endotoxemia is often present in stress conditions such as severe trauma,burn, shock,severe infection,etc.In these conditions,plentiful LPS from the infective areas enter the body and cause the mediators of inflammation synthesizing and releasing abundantly,which causes cell degeneration and necrosis.These manifestations are expressed by the following symptoms:the impairment of tissue and structure the deterioration of organ function;the blood redistribution of kidney resulting from renal glomerulus especially renal arteriole contraction caused by LPS,which causes the decline of renal blood flow and GFR,hence,inducing renal ischemia-reperfusion injury. It also plays a vital role in the onset and development of AKI.Kidney is the major route for the excretion of metabolic product and toxin.The LPS reabsorbed by renal tubule can place the renal tubular epithelium cells in a high lipopolysaecharide（LPS） concentration microenvironment,which might have a great effect on the function and structure of renal tubular epithelium cells.It also acts on the onset and development of acute kidney injury（AKI）.Propofol is a common clinical intravenous anesthetic,whose unique function and structure enable it possess the ability that inhibits inflammatory reaction,relieves inflammatory ingredients releasing,inhibits inflammatory cell congregation and activation to relieve inflammatory intensity,potentiates anti-inflammatory function and reduces necrosis as well as apoptosis,protects organs,etc.There are researches showing that propofol can relieve alveolar epithelium injury in endotoxemia’lung injury by regulating AQP1 and reducing local TNE In this experiment,we can see that the groups utilizing proprofol all manifest relieving injury symptoms to different extent in endotoxemia’s AKI.However,the groups using lipid solvent don’t have the same effect. Consequently,propofol might protect kidney by regulating local aquaporin expression and relative facts as well as renal perfusion per se.The characteristic change of acute renal failure caused by kidney injury of endotoxemia is the decline of GFR and renal tubular dysfunction.The consummate renal tubular function including urine concentration and reabsorption of K⁺ depends on the involvement and functional expression of Aquaporin（AQP） and other ion transportation protein.AQPs distribute widely and possess various subtypes in many organs and tissues such as brain,lung,and kidney.AQPs play an important part in regulation of water and glycerol metabolism.They are vital substances for maintaining a stable internal environment,whose structure and function is influenced by many ingredients as well.Because AQPs are sensitive to multiple pathological process and celluar inflammatory ingredients,AQPs can change water permeability and affect the metabolism of water and micromolecules.AQPs are also the key fact influencing cell apoptosis simultaneously.The discovery of kidney AQP explained the problem of renal free water metabolism rationally.Kidney is the major organ of water metabolism.The research of AQPs in kidney operates a crucial fact on revealing renal physiological and pathological change.It is recently reported the expression of AQP is decreased apparently in renal failure caused by drugs,ischemia etc.This change of AQPs is close related to the injury degree of renal tubular epithelium cell,whose convalescence occurs earlier than morphological change.It has been proved thatAQP2 is the most important aquaporin which is distributed in cellular cell apicoplasmatic membrane and intracellular vesicle.Its abundance influences the permeability of collecting tube to water,which can happen in several minutes.Recent years’ researches show that AQPs can improve micromolecule and water metabolism of lung,brain,kidney in some pathological process and relieve the edema and injury of these corresponding organs. The cell migration medited by AQP is important for the recovery and reconstruction of renal tubular function in acute renal injury.It is close relative to the prophylaxis and treatment of these vital organs injury.Researching the changes of function and structure in these pathological processes can assist guiding drugs usage.In this experiment,compared to control groups,the groups injecting lipid and endotoxin encounter the decline of aquaporin,which indicates that the decline of aquaporin might be a fact of AKI.However,the content of AQP2 in the groups utilizing propofol all increase to different extent,which indicates that propofol possess direct or indirect action on regulating AQP2 itself to protect kidney.Of course,local inflammatory reaction other aquaporin and ion channels are also involved.In all,the definite mechanism is still to be researched.There are researches showing that plentiful cytokines participate the endotoxemian process,which includes IL-1β,IL-6,ICAM-1,VCAM-1 and TNF-a.These inflammatory induce and amplify inflammatory cascade reaction.TNF-a is a 17kd polypeptide which is considered as the priming and key fact for systematic inflammatiry reaction.It is reported that TNF-a has strong toxicity to kidney.This cytokine possesses direct cytotoxicity and induces cell necrosis directly.TNF-a also can cause cell necrosis by microcirculation disorder.Stimulated by endotoxin,TNF-a and other inflammatory medium activate each other,inducing epithelium cell and macrophage cell releasing IL-1.IL-1 stimulates the biological synthesis of other cytokines.For example,intensify the expression of ICAM-1mRNA to amplify inflammatory signal,which enhance s the sensitiveness of tissue to TNF-a in turn. Injecting appropriate TNF-αto animals can cause AKI.If given TNF-αantibody or take measures to reduce TNF-αproduction,the injury caused by eodotoxin can be blocked.It is demonstrated in this experiment that the expression of TNF-αmRNA increased sharply in renal cortex after endotoxemia.This phenomenon shows that TNF-αparticipate the pathological process of AKI.However,giving propofol can reduce the expression of TNF-αmRNA and TNF-αprotein.Conaequently,the content of TNF-αin kidney decreased significantly and relieve renal injury.ICAM-1 is a glycoprotein on cell surface as a part of epithelium cellular structure. The stimulation off LPS and organism stress reaction engender abundant inflammatory mediums and cytokines after endotoxemia.These substances activate WBC as well as blood vessel endothelium（BVE） cell.WBC express CD11/CD18 glycoprotein adhering complex on the cell surface and BVE cell express ICAM-1.These expression products combine with each other,which induce WBC and epithelium cell adhering and promote WBC congregating,activating and releasing inflammatory medium.Normal renal tissue lack the expression of ICAM-1.LPS and cytokines both can up-regulate the expression of ICAM-1mRNA,causing WBC in circulation adhering at local sites.This adhesion forms obstruction in outer medulla and increase the permeability of epithelium.Moreover,these infiltrated and activated WBC can also induce direct tissue injury.The congregation of RBC leads to absent perfusion of outer medulla after reperfusion and aggravates the ischemia-reperfusion injury in the pathological process of endotoxemia.However,reducing local and circulated ICAM-1 can reduce the activity and injury of epithelium cell,whose content can reflect injury condition well. Kelly,etc researched and found that ICAM-I lacking rats can relieve renal ischemia-repeffusion injury obviously.Giving rats ICAM-1 antibody will also go well for that injury.The result of RT-PCR in this experiment shows that the expression of ICAM-1 in renal cortex increased 12 hours after endotoxemia onsets,which illustrates that ICAM-1 is a fact influencing AKI as well.Different dosage of propofol can reduce the expression of ICAM-1mRNA and ICAM-1 protein in kidney by different degrees to relieve the kidney injury.Kidney is an important target organof endotoxemia.Its injury mechanism is still not definite for many ingredients participating the process.As a kind of cell death form distinct from necrosis,whether apoptosis participates and induce sinjury needs researched.Cell apoptosis is a special cell deathform distinct from necrosis,referring to aging,useless,injured cell death in the organism.Cell apoptosis has special degradation,whose morphology appears that karyopycnosis,cell membrane effervesce, form apoptosis body.This process is regulated by special gene without definite cell dissolving process.Caspase3 is an important member of cycteine proteinase group.It is the most important terminal executing enzyme in the cell apoptosis.Inhibiting Caspase3’s activity may impede apoptosis.Bcl-2mRNA is an important anti-apoptpsis gene in this gene group.The equilibrium between promoting apoptosis influences the onset of apoptosis and antagonizes the apoptosis dependent or independent on Caspase. Researches shows that the obvious Increase of apoptosis cell in LPS group indicates that apoptosis participates AKI process compared t o control group.The different dosage of propofol can increase the expression of bcl-2 and decrease that of Caspase3 to reduce cell apoptosis.Consequently,the protection of propofol to renal cell is partly through regulating the expression of bcl-2 and Caspase3 to impede cell apoptosis and antagonize injury.From all mentioned above,this research find that propofol can protect kidney from AKI in endotoxemia.The possible mechanism might be through regulating aquaporin, down-regulating adhering molecule and TNF,influencing apoptosis,etc.Compared to preadministration and simultaneous administration,delay-administration can also work but weak.This result shows that early aftertreatment（in an hour） possesses some protect action,providing theory basis for clinical anesthetic administration.ConclusionThe change of renal function,morphology and molecular biology of rats in endotoxemia indicates that AKI exits in endotoxemia.In this process,different dosage of propofol can relieve inflammatory reaction by different degrees and impede renal function and morphology change,which protects the kidney to some extent.更多还原