【作者】 王海萍；

【导师】 关广聚； 杨天新；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 硝基油酸对内毒素血症性休克及多器官功能不全综合征的保护作用及机制的研究前言内毒素血症(Endotoxemia)是由于血中细菌或病灶内细菌释放出大量内毒素至血液,或输入大量内毒素污染的液体而引起的一种病理生理表现。内毒素血症通常导致致死性感染性休克、多器官功能衰竭、弥漫性血管内凝血等,病死率极高。其中多器官功能不全综合征(Multiple organ dysfunction syndrome, MODS)是内毒素血症患者后期死亡的主要原因,内毒素血症患者一旦发生感染性休克和MODS,其死亡率将增高至60%-70%。内毒素血症常损伤多器官功能,其中心脏、肝脏及肾脏为最常受累器官。而内毒素性急性肾衰竭是感染性休克患者死亡的重要原因之一,约有50%的感染性休克患者并发急性肾功能衰竭(acute renal failure, ARF)。虽然近几十年来,关于ARF病理生理及发病机制的研究取得了长足的进步,但ARF的防治形势依然十分严峻。ARF的死亡率仍居高不下,高达50%左右。防治内毒素性急性肾功能衰竭对降低感染性休克患者的死亡率具有十分重要的意义。因此,寻找有效的药物防治内毒血症性ARF及多器官功能衰竭对内毒性血症的治疗具有十分重要的临床意义。脂肪酸经过氧化亚氮衍生物的硝化作用,产生亲水性的硝基脂肪酸。现已证明,硝基脂肪酸,如硝基油酸,硝基亚油酸存在于正常人的血液、尿液、细胞膜及组织中。利用荧光素酶报告基因技术,观察到硝基脂肪酸是过氧化物酶体增殖物激活受体γ(PPARγ)的强效激动剂,而对PPARα、PPARβ的激动活性稍弱。自硝基油酸被证明体内天然存在的PPAR受体激动剂以来,其具有的无明显毒副作用、在体内天然存在的特点,和广阔的应用前景,引起科学家的浓厚兴趣。硝基脂肪酸可通过激活PPARγ,介导细胞增殖及炎症信号传导通路,与其他已报道的内源性PPARγ激动剂相比较(15 PGJ2),硝基脂肪酸在极低的生物学浓度内(100nM)即可激活PPARγ。另外,越来越多的研究证明,硝基油酸可通过其它非PPARγ受体介导的通路而发挥抗炎作用。硝基脂肪酸通过其亲电子活性,调节转录因子如：核受体-κB的亚单位P65的表达,从而调节其他炎症因子的表达,进而抑制炎症级联反应。另外,硝基脂肪酸是细胞信号传导通路的中间介质,可以参与多种生物学活性,并可通过其亲电子特性进行蛋白修饰,诱导蛋白功能失活,从而发挥抗炎作用。硝基油酸因其在体内天然存在,且无明显毒副作用,具有广泛的生物学活性,故其有广阔的临床开发利用前景。但是,目前针对硝基油酸的实验研究多致力于体外细胞研究,而动物实验较少。那么,硝基油酸在体内的治疗效果究竟如何?其临床开发利用价值到底有多大?其对内毒素血症性休克是否有抗炎保护作用?目前均无研究加以详细阐明。针对硝基油酸临床开发利用的问题,我们设计了以下实验。目的1.构建LPS内毒素休克模型；2.观察硝基油酸是否对LPS诱导的内毒素休克模型具有保护作用；3.观察硝基油酸是否对常受累器官(如：肾脏、心脏及肝脏)具有保护作用。方法1.LPS小鼠模型构建8周龄C57BL／6小鼠适应性喂养7天后,21只小鼠随机分为以下各组：对照组(n=4),LPS组(n=8), LPS+OA-NO2组(n=9)。实验处理如下：对照组、LPS组埋置内含100% DMSO的微量泵,LPS+OA-NO2组小鼠埋植内含100%DMSO+OA-NO2 (0.2μg/kg/d)(硝基油酸溶于100% DMSO溶液,浓度1mg/ml)的微量泵,预处理48h。48h后,LPS组及LPS+OA-NO2组小鼠予以腹腔注射LPS (10mg/Kg/d)(以0.9%的生理盐水溶解,配制成lmg/ml LPS溶液),对照组小鼠注射等量生理盐水。注射LPS 18h后,处死小鼠,采集血液及心肝肾脏组织。另取20只小鼠随机分为以下各组：对照组(n=5),LPS组(n=5),LPS+OA-NO2组(n=5),LPS+OA组(n=5)。对照组、LPS组埋置内含100%DMSO的微量泵,LPS+OA-N02组和LPS+OA组小鼠分别埋植内含100%DMSO+OA-NO2(0.2μg/kg/d).100%DMSO+OA(0.2μg/kg/d)(硝基油酸或油酸溶于100% DMSO溶液,浓度1mg/ml)的微量泵,预处理48h,其余实验处理同前描述。2.超声心动图检查另取28只雄性C57BL／6小鼠随机分以下各组：对照组(n=5),LPS组(n=11),LPS+OA-NO2组(n=12),各组小鼠微量泵的埋置、药物预处理时间及LPS注射同前描述。小鼠注射LPS 18h后,进行超声心动图检查。3.生物遥测记录血压及心率另取23只雄性C57BL／6小鼠随机分以下各组：对照组(n=5),LPS组(n=8),LPS+OA-NO2组(n=10)。通过颈动脉插管埋置遥测元件(telemetry device),步骤简要如下：使用乙醚麻醉小鼠后,行颈正中切口,分离颈动脉,植入导管,其余传输导管的埋置按厂家说明书操作。导管埋植术后恢复一周后,开启遥测装置连续记录小鼠血压及心率。之后,小鼠微量泵的埋置、药物预处理时间及LPS注射同前描述。4.温度及红细胞压积的测量小鼠注射LPS 18h后,用JM222便携式数字温度计测量大鼠肛温。用玻璃毛细管自尾静脉采血2～2.5cm,后置于Thermo IEC微量离心机内离心。离心结束后,取出玻璃毛细管,用标尺测量红细胞长度及样本总长度,两者比值即红细胞长度／样本总长度即为红细胞压积。5.血液生化指标检测检测血浆尿素氮(BUN)、肌酐(Cr).AST.ALT和LDH。6.实时定量RT-PCR检测取新鲜肝肾组织,提取RNA,实时荧光定量RT-PCR检测肝肾组织β-actin, TNF-α,iNOS,MCP-1,ICAM-1,VCAM-1,MCP-1 mRNA的表达水平。7.Western Blot检测取新鲜肾组织,提取蛋白质,Western Blot检测肾组织COX-2,iNOS蛋白的表达水平。8. ELISA检测用相应的ELISA试剂盒检测血浆、心肝肾组织中TNF-a的蛋白表达水平,检测肾组织内PGE2和cGMP的生成。所有操作均严格按照ELISA试剂说明书进行。结果1.硝基油酸预处理48h对体温及红细胞压积的影响腹腔注射LPS18h后,LPS组小鼠体温较对照组显著下降约7℃(P<0.01)；预处理硝基油酸(OA-NO2) 48h能明显改善LPS诱导的低体温(P<0.05)。LPS组小鼠红细胞压积较对照组显著下降(P<0.01),硝基油酸的预处理能明显改善LPS诱导的红细胞压积降低(P<0.01)。2.硝基油酸预处理48h对肾脏功能的保护作用与正常对照组小鼠相比较,LPS组小鼠BUN约升高4倍,Cr值约升高1.5倍(P<0.05)。硝基油酸预处理可显著降低BUN (P< 0.05),且几乎使Cr降至正常水平(P<0.05)。而与肾功能的恶化程度相比,LPS组小鼠的肾组织病理损伤只是轻度损伤,因此本研究未将病理改变作为治疗评估指标,故病理结果未在本论文中显示。3.硝基油酸预处理48h对炎症因子及粘附因子的影响LPS可诱导肾脏TNF-a mRNA及蛋白分别升高14倍及4倍,预处理硝基油酸明显降低其高表达(P<0.01)。硝基油酸同样显著降低LPS诱导的循环血中TNF-a的升高(P<0.05)。LPS可诱导肾脏MCP-1 mRNA升高27.1倍(P<0.01),预处理硝基油酸后使此升高降至9.7倍(P<0.05)。LPS可诱导肾脏ICAM-1 mRNA较对照组升高12.0倍(P<0.01),预处理硝基油酸后使此升高降至4.8倍(P<0.01)。同时,LPS诱导肾脏VCAM-1 mRNA较对照组升高4.2倍(P<0.01),预处理硝基油酸后使此升高降至1.8倍(P<0.01)。4.硝基油酸预处理48h对肾脏诱导型一氧化氮合酶(iNOS)表达的影响LPS组小鼠肾脏iNOS mRNA表达急剧升高,高达1000倍,而硝基油酸预处理能80%降低此高表达。iNOS蛋白水平的表达变化与mRNA的表达变化基本一致,LPS可诱导iNOS蛋白表达上调70倍,而硝基油酸可将LPS诱导的iNOS蛋白的高表达降至7倍。LPS可诱导cGMP生成增加(P<0.05),硝基油酸的预处理可降低LPS诱导的cGMP生成增加,接近统计学差异(P=0.05)。5.硝基油酸预处理48h对肾脏环氧合酶-2(COX-2)及前列腺素E-2(PGE2)表达的影响与对照组比较,LPS分别诱导COX-2 mRNA及蛋白表达升高8.3倍及17.1倍,而硝基油酸可分别使此高表达降至2.1倍及8.9倍。PGE2的ELISA结果显示,LPS组肾组织PGE2生成显著增多(P<0.05),硝基油酸的预处理可降低其生成(P<0.05)。6.硝基油酸预处理48h对肝脏损伤的保护作用与对照组比较,LPS诱导AST、ALT升高(P<0.05),硝基油酸对肝脏有一定程度保护作用。LPS诱导肝TNF-α蛋白表达升高(P<0.05),硝基油酸可显著降低肝脏TNF-α的高表达(P<0.05)。实时定量RT-PCR结果显示,LPS诱导TNF-α、IL-1β、ICAM-1、MCP-1、iNOS和COX-2高表达,硝基油酸可下调这些炎症因子的高表达(与LPS组比较,均P<0.05)7.硝基油酸预处理48h对心血管系统的保护作用超声心动图结果显示LPS可显著降低EF(P<0.01),硝基油酸明显改善左室收缩功能(P<0.05)。同时,LPS诱导心肌酶谱LDH显著升高(P<0.05),硝基油酸几乎使LDH降至正常(P<0.05)。LPS注射一小时后,血压急剧下降,6小时后从约103 mmHg降至约72 mmHg(P<0.01),其后血压一直未能恢复。硝基油酸可轻度改善LPS诱导的低血压：LPS硝基油酸治疗组比LPS组血压持续高10 mmHg左右。8.硝基油酸与油酸预处理的作用效果比较本研究另设一组实验,比较硝基油酸预处理与油酸预处理治疗对内毒素血症的治疗效果,以确定硝基油酸的治疗效果是否依赖于其硝基化。实验结果显示,油酸预处理治疗后,无论对系统性炎症反应,或急性肾衰竭均无保护治疗作用,故硝基油酸对内毒素血症的系统及局部炎症的治疗作用是依赖于油酸的硝基化。结论1.首次通过动物实验证实硝基油酸在LPS诱导的内毒素血症模型中具有显著抗炎作用。预处理硝基油酸48h,可减轻小鼠系统性及局部性炎症反应,并改善内毒素血症诱导的多器官功能不全综合征。2.初步探讨硝基油酸的抗炎作用机制。硝基油酸通过参与调节多个炎症反应信号通路,抑制炎症介质及炎症因子的产生及释放,如TNF-α、MCP1、ICAM-1、VCAM-1、iNOS和COX-2,从而发挥抗炎作用。硝基油酸对顺铂所致肾损伤的保护作用及机制的研究前言顺铂(cis-diamminedichloroplatinum, CDDP)因其抗瘤作用强、抗瘤谱广,是临床应用较广泛的一种肿瘤化疗药,但其毒副作用限制了其临床的广泛应用。CDDP对肾脏的累积性毒性损伤,可致患者出现急性肾功能衰竭的严重后果,有报道CDDP造成的肾毒性约25-35%发展为急性肾功能衰竭,大大限制其在临床大剂量和长期的应用。迄今,临床仍无有效的防治CDDP肾毒性的治疗方法。因此,如何减轻或者预防治疗CDDP所致肾损伤成为基础和临床研究的热点。我们通过研究证明,硝基油酸通过抑制多种炎症介质和炎症因子的产生及释放,对脂多糖诱导的内毒素血症性肾衰竭有保护作用。因硝基油酸在体内天然存在,在极低的血药浓度范围内即可发挥生物学作用,另外其具有无明显毒副作用的优点,有极大的开发利用前景。那么,硝基油酸对LPS诱导的肾毒性的保护作用是否具有特异性?是否在其他急性肾损伤中也发挥有效的保护作用?为解答以上问题,同时为进一步探索硝基油酸的治疗作用,更为其在临床的开发应用提供更有力、更全面的证据,我们通过复制顺铂致肾毒性模型,观察硝基油酸是否在此模型中同样具有肾脏保护作用。另外,为进一步探讨硝基油酸的作用机制,我们利用诱导型PPARγ全身基因敲除模型,来初步明确硝基油酸的肾保护作用是否依赖PPARγ受体途径介导。目的1.构建顺铂性肾损伤模型；2.观察硝基油酸是否对顺铂诱导的肾损伤具有保护作用；3.观察硝基油酸是否对诱导型PPARγ全身基因敲除小鼠的顺铂性肾损伤仍具有保护作用。方法1.小鼠顺铂肾毒性模型构建8周龄C57BL／6小鼠适应性喂养7天后,18只小鼠随机分为以下各组：对照组(Cont组,n=6),CDDP组(n=6), CDDP+OA-NO2(n=6)。实验处理如下：对照组、CDDP组埋置内含100% DMSO的微量泵,CDDP+OA-NO2组小鼠埋植内含100% DMSO+OA-NO2 (20mg/kg/d)(硝基油酸溶于100%DMSO溶液,浓度100mg/ml),预处理48h。48h后,CDDP组和CDDP+OA-NO2组小鼠予一次性腹腔注射CDPP(20mg/Kg)(以0.9%的生理盐水溶解,配制成2mg/ml CDDP溶液),对照组小鼠注射等量生理盐水。注射CDDP 3天后,处死小鼠,采集血液及心肝肾脏组织。另取23只小鼠随机分为以下各组：对照组(n=5), CDDP组(n=6),CDDP +OA-NO2 (n=6), CDDP+OA组(n=6)。对照组、CDDP组、CDDP+OA-NO2及CDDP+OA组小鼠分别埋植内含100% DMSO、100% DMSO.100% DMSO+OA-NO2 (20mg/kg/d)及100% DMSO+OA (20mg/kg/d)的微量泵,预处理48h。其余实验步骤同前描述。另取21只野生型LOXP小鼠,随机分为：对照组,CDDP组,CDDP+OA-NO2组；21只PPARy全身基因敲除小鼠,分组同上。硝基油酸预处理及顺铂注射步骤及时间均同前描述。2.一般情况观察及体重测量小鼠注射CDDP 3天后,观察小鼠一般状况,并将小鼠置于天平上测量体重。3.血液生化指标检测检测血浆尿素氮(BUN)、肌酐(Cr)。4.实时定量RT-PCR检测取新鲜肝肾组织,提取RNA,实时荧光定量RT-PCR检测肝肾组织GAPDH、TNF-α、IL-β、MCP-1、ICAM-1、VCAM-1、MCP-1、COX-2、mPGES-1、P47phox及gp91phox mRNA的表达水平。5. Western Blot检测取新鲜肾组织,提取蛋白质,Western Blot检测肾组织TNF-α、COX-2、P47phox及gp91phox蛋白的表达水平。6.病理学检测肾组织经固定、石蜡包埋后行石蜡切片,切片经苏木素-伊红(HE)染色后,观察肾组织损伤情况。7.ELISA检测用相应的ELISA试剂盒检测血浆中TNF-a的蛋白表达水平,检测肾组织内PGE2和TBARS的生成。所有操作均严格按照ELISA试剂说明书进行。8.统计学处理采用SPSS11.0软件包进行统计分析,组间均数的比较采用方差齐性检验与单因素方差分析,定量数据用均数±标准差(x±s)表示,检验水平a=0.05。结果1.小鼠的一般情况与对照组相比,CDDP组小鼠状态较差,进食、活动等明显减少,精神萎靡。各组小鼠自顺铂注射后体重均有下降,硝基油酸治疗对体重的下降无明显影响。2.硝基油酸对肾脏系数的影响与对照组相比,CDDP组小鼠的肾脏色苍白、略肿胀,肾脏系数增大(P<0.05),硝基油酸治疗组小鼠的肾脏系数减少(P<0.05),颜色跟对照组无明显差异。3.硝基油酸对肾功能及肾组织变化的影响CDDP组小鼠的血尿素氮及肌酐较对照组分别升高约4.5倍及4倍(P<0.05),硝基油酸的预处理治疗,可明显改善CDDP诱导的肾衰竭(P<0.05)。CDDP小鼠肾内可见较多渗出的中性粒细胞,肾小管上皮细胞坏死、脱落,可见较多的蛋白管型。硝基油酸预治疗组小鼠肾内中性粒细胞渗出较少,上皮细胞浊肿,偶见散在空泡,未见明显蛋白管型。4.硝基油酸对肾脏氧化应激影响与对照组相比,CDDP组小鼠的肾脏TBARS显著升高(P<0.01),硝基油酸预处理治疗可明显改善CDDP诱导的氧化应激损伤(P<0.05)。p47phox和gp91phox mRNA及蛋白的表达变化及硝基油酸的治疗效果与TBARS结果一致。5.硝基油酸对肾脏炎症反应影响CDDP可显著升高肾脏TNF-αmRNA、蛋白的表达水平(P<0.01),硝基油酸可明显降低其高表达(P<0.05)。另外,CDDP上调IL-1β至2.8倍,硝基油酸可使其降至正常水平。ICAM-1、VCAM-1及MCP-1分别上调至3.8倍、5倍和5.1倍,硝基油酸预处理治疗可将MCP-1降至正常水平,将ICAM-1和VCAM-1分别降至1.8倍和1.7倍。6.硝基油酸对肾脏COX-2和PGE2生成的影响与对照组相比,CDDP可诱导COX-2 mRNA、蛋白分别高表达6倍、4倍,而硝基油酸可分别下调其表达至1.6倍及2倍。另外,CDDP可上调mPGES-1的表达,增加前列腺素E-2 (PGE2)生成(P<0.05),硝基油酸治疗组小鼠肾脏PGE2生成水平明显下降,几乎降至对照组小鼠水平。7.油酸与硝基油酸治疗效果比较本研究另设一组实验,比较硝基油酸预处理与油酸预处理对CDDP诱导的急性肾损伤的治疗效果,以确定硝基油酸的治疗效果是否依赖于其硝基化。实验结果显示,油酸预处理治疗后,无论对肾功能指标或肾脏病理组织变化均无保护治疗作用,故硝基油酸对CDDP诱导的急性肾损伤的治疗保护作用依赖于油酸的硝基化。8.硝基油酸对全身PPARgama基因敲除小鼠的治疗效果与C57BL／6小鼠相似,硝基油酸同样可改善CDDP诱导的野生型及全身PPARY基因敲除小鼠的急肾衰,两组治疗效果无明显差别。故说明硝基油酸的肾脏保护作用,并非依赖PPARy受体途径介导。结论1.硝基油酸可明显改善顺铂诱导的肾功能不全及肾脏病理改变。2.硝基油酸通过其抗炎、抗氧化作用对顺铂肾损伤发挥保护作用。即硝基油酸通过抑制炎症介质、炎症因子及NADPH同工酶的产生及释放,如TNF-α、MCP1、ICAM-1、VCAM-1及p47phox、gp91phox从而发挥抗炎、抗氧化作用。3.硝基油酸的肾保护作用是通过非PPARγ受体途径介导的。硝基油酸对DOCA-盐型高血压小鼠肾损伤的保护作用及机制的研究前言近年来,醛固酮在高血压发病机制中的作用引起人们的广泛关注。长期、大样本的追踪调查显示,体内高水平的醛固酮可预示高血压的发生及发展。人工合成醛固酮激素-DOCA(脱氧皮质酮)-盐型高血压模型,是盐型继发型高血压动物模型,因此模型跟人原发性醛固酮增多症相似,目前已被广泛应用于研究。由于原发性醛固酮增多症发病率逐年升高,且研究证实其发病机制仅与醛固酮升高有关,无遗传学因素参与。另外,特异性受体阻滞剂的开发利用,为治疗原发性醛固酮增多症提供了可能,越来越多的研究着重于醛固酮与肾功能损伤关系。醛固酮直接作用或可通过升高血压,而诱导肾脏炎症反应,肾脏氧化应激状态及肾纤维化。研究证实,醛固酮受体阻滞剂可在多种高血压动物模型减轻肾脏损伤。我们通过研究证明,硝基油酸可在LPS和顺铂诱导的急性肾损伤模型中,通过抗炎、抗氧化作用机制发挥肾脏保护作用。故本实验研究目的：拟通过建立DOCA-盐型高血压小鼠模型,观察硝基油酸是否对此模型的肾损伤同样具有保护作用。目的1.构建DOCA-盐型高血压小鼠模型；2.观察硝基油酸是否对DOCA-盐型高血压小鼠肾损伤具有保护作用；3.初步探讨硝基油酸对DOCA-盐型高血压小鼠肾损伤的保护作用机制。方法1.DOCA-盐型高血压小鼠模型构建8周龄C57BL／6小鼠适应性喂养7天后,27只小鼠随机分为以下各组：对照组(Cont组,n=7), DOCA组(n=10), DOCA+OA-NO2组(n=10)。各组小鼠处理如下：对照组、DOCA组和DOCA+OA-NO2组小鼠分别埋植内含100%DMSO、100% DMSO和100%DMSO+OA-NO2 (20mg/kg/d)(硝基油酸溶于100%DMSO溶液,浓度10mg/ml)的微量泵,预处理48h。48h后,DOCA组和DOCA +OA-NO2组小鼠予皮下埋置DOCA缓释片,对照组小鼠仅行同样手术操作。DOCA组和DOCA+OA-NO2组小鼠饲以1%NaCl、含1.5%盐的胶体饲料。对照组小鼠予以标准饮水、标准胶体饲料喂养。DOCA缓释片埋置术后,于2周后采集标本,处死小鼠。2. Tail-Cuff法测量小鼠血压于随机分组后,皮下埋置微量泵前一周,将所有小鼠分别置于Tail-Cuff测量台上适应性测量一周。后DOCA埋植术后第10天行Tail-Cuff测量小鼠血压,连续测量4天,每天两次,记录血压及心率,并取其平均值。3.收集小鼠24小时新鲜尿液并测量进食量及饮水量DOCA处理两周后,将小鼠置于代谢笼中收集24小时尿液,并测量小鼠的进食量及饮水量。4. ELISA测量24h尿蛋白、尿8-isoprostane、肾组织TBARS及血TNFα均用相应的ELISA试剂盒测量。5.实时定量RT-PCR检测取新鲜肾组织,提取RNA,实时荧光定量RT-PCR检测肾组织GAPDH、TNF-α、IL-β、MCP-1、ICAM-1、VCAM-1、MCP-1、COX-2、mPGES-1、P47phox gp91phox及nephrin mRNA的表达水平。6.Western Blot检测取新鲜肾组织,提取蛋白质,W Western Blot检测肾组织TNF-α、COX-2、nephrin、P47phox及gp91phox蛋白的表达水平。7病理学检测肾组织经固定、石蜡包埋后行石蜡切片,切片经苏木素-伊红(HE)、PAS染色后,观察肾组织损伤情况。8 ELISA检测用相应的ELISA试剂盒检测血浆中TNF-α的蛋白表达水平,检测肾组织内PGE2和TBARS的生成。所有操作均严格按照ELISA试剂说明书进行。9电镜检测电镜检测足细胞损伤情况。10统计学处理采用SPSS11.0软件包进行统计分析,组间均数的比较采用方差齐性检验与单因素方差分析,定量数据用均数±标准差(x±s)表示,检验水平α=0.05。结果1.小鼠的血压情况埋置DOCA缓释片2周后,DOCA组小鼠血压显著升高约15mmHg(P<0.05),而硝基油酸预处理治疗可显著降低小鼠血压(P<0.05)。2.小鼠的进食、饮水量及体重所有动物自由饮食无明显差异,DOCA组小鼠饮水量较对照组明显增多,硝基油酸组小鼠饮水较DOCA组少,但无统计学差异。DOCA组小鼠体重较埋置DOCA前明显减轻(P<0.05),硝基油酸组小鼠与实验前基础体重无显著性差异。3.小鼠尿量及尿蛋白情况DOCA组小鼠尿量及尿蛋白显著增多(P<0.01),与DOCA组比较,硝基油酸预处理可使尿量减少,但无统计学差异,但可显著降低蛋白尿(P<0.05)。4.肾脏系数及其组织病理改变DOCA组小鼠肾脏肥大、色苍白,硝基油酸组小鼠肾脏色较DOCA组红润,肾脏系数减少(P<0.01)。PAS染色显示,与对照组比较,DOCA组小鼠肾皮质及肾外髓质小管扩张、肥厚,肾小管基底膜萎缩,刷状缘不连续,而硝基油酸预处理治疗可改善DOCA诱导的小管损伤。5.肾足细胞损伤DOCA组小鼠足细胞损伤明显,足突广泛融合、消失,硝基油酸组小鼠足细胞损伤明显改善。肾脏实时定量PCR结果显示,足突特异蛋白nephrin表达减少。硝基油酸干预治疗组,足细胞损伤减轻。6.肾脏氧化应激DOCA可明显诱导肾脏氧化应激,肾组织TBARS及尿8-isoprostane明显升高,P<0.05；同时P47phox和gp91phox的mRNA及蛋白均明显高表达。而硝基油酸组小鼠的肾脏氧化应激减轻,其相应氧化应激指标明显下降。7.肾脏炎症改变DOCA诱导小鼠肾TNF-α在mRNA及蛋白水平高表达(P<0.01),IL-1β、MCP-1、ICAM-1、VCAM-1、MCP-1、COX-2 mRNA高表达(P<0.01),硝基油酸可减轻DOCA诱导的炎症反应。结论1.硝基油酸可降低DOCA-盐诱导的小鼠高血压。2.硝基油酸可显著减少DOCA-盐高血压小鼠的尿蛋白排泄,显著改善足细胞损伤。3.此保护作用机制仍然通过抗炎、抗氧化作用介导。更多还原

【Abstract】 Endotoxemia is a systemic inflammatory response to a blood-borne infection that is associated with an extremely high rate of morbidity and mortality. Endotoxemia often leads to sepsis, DIC and the failure of multiple organs, which can cause the mortality rate as high as 60%-70%.Endotoxemia often cause multiple organ damage, especially the heart, liver and kidney. ARF is considered a critical prognostic factor in endotoxic shock, with the 50% coincidence, the mortality rate for septic patients with acute renal failure (ARF) is approximately doubled compared with patients with sepsis alone. Although there is great progress in the pathology and physiology of the ARF, but the management of sepsis and sepsis-induced ARF is largely supportive. Therefore, novel therapies to prevent or treat this devastating disease are urgently required.Recently, nitrated free fatty acid (NO2-FA), notably nitroalkene derivatives of linoleic acid (nitrolinoleic acid; L NO2) and nitro-oleic acid (OA-NO2), are identified as endogenous molecules with several attractive signaling properties. These derivatives are formed via NO-dependent oxidative reactions. They were detected in healthy human blood, indicating their capability to act in physiological concentration ranges. To date, there are three major mechanisms by which nitroalkenes appear to mediate cell signaling. LNO2 and OA-NO2, at physiological concentrations, serve as potent ligands for proxisome proliferator-activated receptor subtype gamma (PPARy). Moreover, nitroalkenes are electrophiles and thus can nitroalkylate proteins and small peptides such as glutathione through reaction with cysteine thiols and histidine., which is independent on proxisome proliferator-activated receptor pathway. Increasing in vitro evidence demonstrates that nitroalkenes exert potent anti-inflammatory actions.Therefore, the nitroalkenes has multiple advantage, for example, endogenous molecules without side effects, and notably several attractive signaling properties. Nitrooleate arouses the widespread interest because of its content rich, the structure simple, and it will benefit the patients if it is applied in clinic. The present study seeks to examine the potential therapeutic effects of OA-NO2 in LPS-induced inflammation and renal injury.Objectives1. TO produce LPS-induced endotoxemia model in mice.2. To illustrate the potential therapeutic effects of OA-NO2 in LPS-induced inflammation and renal injury.3. To illustrate the potential therapeutic effects of OA-NO2 in LPS-induced heart and liver injury. Methods1. Animal protocolMale C57BL/6 mice (8-week-old) were maintained on a standard rodent chow and had free access to water. OA-NO2 was dissolved in 100% DMSO at 1 mg/ml. Mice were pretreated for 48 h with DMSO (LPS vehicle) or OA-NO2 (LPS OA-NO2) at 0.2 mg/kg/d via a micro-osmotic pump, and then both groups were treated with a single intraperitoneal injection of LPS at 10 mg/kg. The third group received an i.p. injection of saline only and served as controls. Functional studies were done at 18 h after LPS injection.A separate experiment was performed to compare the anti-inflammatory effects of OA-NO2 versus OA; OA was delivered at the same dose (0.2 mg/kg/d) via the same route as OA-NO2. 2. Echocardiography.In vivo cardiac function was assessed using echocardiography.28 male C57BL/6 mice (8-week-old) divided into cont, LPS and LPS+OA-NO2 groups, and the OA-NO2 pretreatment, LPS injection were the same as the previous.3. Blood pressure measurements.MAP was determined by telemetry. The radiotelemetric device was implanted into mice through catheterization of the carotid artery.Animals were allowed to recover from surgery for 1 week. MAP was recorded 2 days prior to and 18 h after LPS injection.4. Measurement of body temperature and hematocritRectal temperature was measured before and 18 h after LPS injection using a digital thermometer. At 18 h after LPS injection, hematocrit was determined.Briefly, blood was collected from tail cut using a capillary glass. The tube was centrifuged in a microcentrifuge machine. The total height of sample and height of the red blood cell column were measured.5. Measurement of biochemical parameters.Plasma BUN、Cr、AST、ALT and LDH were measured.6. Real-time RT-PCR.Total RNA was isolated from fresh tissue, and the mRNA expression ofβ-actin, TNF-α, iNOS, MCP-1, ICAM-1, VCAM-1, MCP-1 were determined.7. Westen BlotThe protein was isolated from fresh tissue, and protein expression of COX-2, iNOS in kidney were determined.8. ELISA measurementThe plasma, heart, liver and kidney TNF-acontent, also the kidney PGE2 content were measured using EIA kits, respectively.9. Statistical analysisValues shown represent means±SE. Data were analyzed using unpaired t test or ANOVA followed by a Bonferroni posttest. A P value<0.05 was considered significant. Results1. Body temperature and hematocritThe body temperature decreased significantly at 18 h after LPS injection (P< 0.01). Pretreatment of OA-NO2 for two 48h significantly improved LPS-induced hypothermia (P< 0.05). Hematocrit was significantly decreased that was attenuated by pretreatment with OA-NO2 (P< 0.01).2. Renal functionLPS injection elevated plasma BUN and Cr 4-,1.5-fold (P< 0.05).Pretreatment with OA-NO2 attenuated the rise of plasma BUN (P< 0.05) and almost completely normalized plasma creatinine levels (P< 0.05). While, renal histological changes in response to LPS were not evident and thus were not used as a parameter for evaluating the effect of OA-NO2 (data not shown).3. Proinflammatory cytokines, chemokines, and adhesion moleculesLPS induced renal TNF-a mRNA and protein 14-fold,4-fold increase. Pretreatment with OA-NO2 attenuated the rise of TNF-a(P< 0.01). Similar results were obtained concerning circulating TNF-a (P< 0.05).The fold inductions between LPS vehicle and LPS OA-NO2 groups were 27.1-vs.9.7-fold for MCP-1,12.0-vs. 4.8-fold for ICAM-1, and 4.2-vs.1.8-fold for VCAM-1.4. Renal iNOS expression.At 18 h post LPS, renal iNOS mRNA exhibited a more than 1000-fold increase that was reduced by 80% in LPS OA-NO2 mice. Similarly, LPS injection induced a 70-fold increase in renal iNOS protein expression that was reduced by 90% with OA-NO2. Renal cGMP content exhibited a similar pattern of changes as renal iNOS expression.5. Renal COX-2 expressionRenal COX-2 mRNA and protein increase 8.3-and 17.1-fold, respectively, by LPS injection; they were attenuated to 2.1-and 8.9-fold by OA-NO2.while renal content of PGE2 exhibited a 1.5-fold increase that was almost normalized by pretreatment with OA-NO2 (P< 0.05). 6. Hepatic injuryLPS injection elevated plasma AST, ALT strikingy (P< 0.05). Pretreatment with OA-NO2 lowered plasma AST and ALT. By ELISA, the increase in hepatic TNF-a content was less in LPS OA-NO2 vs. LPS vehicle mice (Fig.7C). LPS injection induced parallel increases in mRNA expression of hepatic TNF-a, IL-1, ICAM-1, MCP1, iNOS and COX-2; these increases were all attenuated by OA-NO2 (P<0.05).7. Cardiac injury and hypotentionEchocardiography revealed that LPS injection significantly reduced the ejection fraction (EF) (P< 0.01). Plasma LDH was elevated by LPS injection (P< 0.05) that was attenuated by OA-NO2 (P< 0.05). LPS-induced hypotension was evident in both groups. MAP in LPS OA-NO2 mice tended to be consistently-10 mmHg higher than in LPS vehicle animals.8. Effectiveness of OASeparate experiments determined whether the protective effect of OA-NO2 in endotoxin-induced endotoxemia was specific to the nitrated form. The indices of systemic inflammation (body temperature and hematocrit) and renal dysfunction (plasma BUN and creatinine) in the endotoxemic mice were significantly attenuated by OA-NO2 but was unaffected by OA, documenting the lack of anti-inflammatory effect of OA. This finding suggests that the protective effect of OA-NO2 in endotoxic shock is attributable to the nitration of the fatty acid.Conclusion1. Pretreatment with OA-NO2 for 48 h attenuated the systemic inflammation and improved the multiple organ dysfunction in the LPS induce endotoxemia in mice.2. The data showed the mechanism about the anti-inflammation of the OA-NO2, which was via suppress the production and release of the inflammation markers, such as TNF-α、MCP1、ICAM-1、VCAM-1、iNOS and COX-2. Protective Effects of Nitro-Oleic Acid Against Cisplatin-induced Nephrotoxicity in MiceBackgroundCisplatin(cis-diamminedichloroplatinum, CDDP) is widely used in the treatment of a variety of malignancies. Howerer, the full clinical utility of the drug is limited by its adverse effects. One of the most common side effects is cisplatin nephrotoxicity. Cisplatin is a potent toxin to renal tubules and is associated with a cumulative decline in renal function. Approximately 25-35% of patients administered with cisplatin develop acute renal failure (ARF). But the management of cisplatin nephrotoxicity is largely supportive. Therefore, novel therapies to prevent or treat this devastating disease are urgently required.Our previous study indicated that nitro-oleic acid (OA-NO2) protected against LPS induced endotoxic ARF via suppress the production and release of inflammation marker and mediators. As the OA-NO2 is characterized with the endogenous molecules, high capability to act in low concentration ranges, and without any side effects so far. So it may provide the novel therapeutic drug for the clinic. But we ask ourselves several questions:Are the protective effects of the OA-NO2 specific to the LPS induced ARF? Does the drug protect against other model of renal injury? To answer these questions, we design the present study to explore more details of the drug. We investigated whether OA-NO2 ameliorates cisplatin-induced renal dysfunction in mice. To explore whether the protective effect was PPAR gamma dependent, we repeated the model in inducible PPAR gamma knockout mice. Objectives1. To produce cisplatin-induced renal dysfunction in mice.2. To illustrate the potential therapeutic effects of OA-NO2 in cisplatin-induced renal dysfunction.3. To illustrate the whether the protective effect was on PPAR gamma dependent.Methods1. Animal protocolMale C57BL/6 mice (8-week-old) were maintained on a standard rodent chow and had free access to water. OA-NO2 was dissolved in 100% DMSO at 100 mg/ml. Mice were pretreated for 48 h with DMSO (DDP vehicle) or OA-NO2 (DDP+OA-NO2) at20 mg/kg/d via a micro-osmotic pump, and then both groups were treated with a single intraperitoneal injection of DDP at 20 mg/kg. The third group received an i.p. injection of saline only and served as controls. Functional studies were done at 18 h after DDP injection.A separate experiment was performed to compare the anti-inflammatory effects of OA-NO2 versus OA; OA was delivered at the same dose (20mg/kg/d) via the same route as OA-NO2.A separate experiment was performed in wild type and inducible PPAR gamma knockout mice. The pretreatment of the OA-NO2 and the DDP was the same as the described above.2. General condition and body weightAt 3days after DDP injection, evaluate the general condition and weighted the mice.3. Measurement of biochemical parameters.Plasma BUN and Cr were measured.4. Real-time RT-PCRTotal RNA was isolated from fresh tissue, and the mRNA expression of GAPDH, TNF-α,IL-β、MCP-1、ICAM-1、VCAM-1、MCP-1、COX-2、mPGES-1、P47phox and gp91phox mRNA were determined. 5. Westen BlotThe protein was isolated from fresh tissue, and protein expression of TNF-α、COX-2、P47phox and gp91phox in kidney were determined.6. Histological examinationKidney tissue was fixed, embedded in paraffin and cut into sections. The kidney damage was examined in haematoxylin-eosin(HE) stained sections.7. ELISA measurementThe plasma TNF-a content, and the kidney PGE2, TBARS content were measured using EIA kits, respectively.8. Statistical analysisValues shown represent means±SE. Data were analyzed using unpaired t test or ANOVA followed by a Bonferroni posttest. A P value<0.05 was considered significant.Results1. General condition and body weightAfter DDP injection, the mice were sick with less food and water intake less and their activity decrease obviously. The body weight decrease were no statistical difference between the DDP and OA-NO2 treatment groups.2. Kidney indexDDP induced the kidney hypertrophy and swelling, with increased kidney index (P<0.05). Pretreatment with OA-NO2 attenuated the rise of kidney index. 3. Renal function and histologyLPS injection elevated plasma BUN and Cr 4.5-,4-fold (P< 0.05).Pretreatment with OA-NO2 improved the renal dysfunction (P< 0.05). Histological examination revealed necrosis, protein cast, vacuolation and desquamation of epithelial cells in renal tubules after cisplatin injection. However, pretreatment with OA-NO2 dramatically improved the histological damage.4. kidney oxidative stress conditionCisplatin elevated the kidney TBARS and induced the mRNA and protein expression of the p47 phox and gp91 phox subunits. Pretreatment with OA-NO2 attenuated the rise of kidney TBARS, p47phox and gp91phox subunits expression. 5. Proinflammatory cytokines, chemokines, and adhesion moleculesCisplatin induced renal TNF-a mRNA and protein expression increase, also the circulating TNF-a. Pretreatment with OA-NO2 attenuated the rise of TNF-a(P< 0.01). The fold inductions between DDP vehicle and DDP+ OA-NO2 groups were 3.8-vs. 1.8-fold for ICAM-1, and 5.1-vs.1.7-fold for VCAM-1. Pretreatment with OA-NO2 almost normalized DDP induced the increase of MCP-1 and IL-1β.6. Renal COX-2 expression.Renal COX-2 mRNA and protein increase 6- and 4- fold, respectively, by cisplatin injection; they were attenuated to 1.6- and 2-fold by OA-NO2.while renal content of PGE2 exhibited a strikingly increase that was almost normalized by pretreatment with OA-NO2 (P< 0.05).7. Effectiveness of OASeparate experiments determined whether the protective effect of OA-NO2 in cisplatin nephrotoxicity was specific to the nitrated form. The indices of renal dysfunction (plasma BUN and creatinine, histological changes) in the mice were significantly attenuated by OA-NO2 but was unaffected by OA, documenting the lack of anti-inflammatory effect of OA. This finding suggests that the protective effect of OA-NO2 in cisplatin nephrotoxicity is attributable to the nitration of the fatty acid.8. Effectiveness of OA-NO2 in whole body inducible PPAR y knockout mice Similarly, pretreatment with OA-NO2 for 48 h attenuates the renal dysfunction in both the wild type and knockout mice, indicating the protective effects of the OA-NO2 was independent on the PPARy pathway.Conclusion1. Pretreatment with OA-NO2 for 48h attenuated the cisplatin nephrotoxicity in mice.2. The mechanism of the protection of the OA-NO2 was via anti-inflammation and anti-oxidative stress. 3. The protective effects of the OA-NO2 was independent on the PPARy pathway. Renoprotective effect of Nitro-Oleic Acid in DOCA-salt hypertensive MiceBackgroundThe role of aldosterone in hypertension has received increasing attention during recent years. High serum levels of the hormone preceded the development of hypertension in a population-based, long-term study. The use of a synthetic mineralocorticoid, DOCA, which is an aldosterone analog, together with a high-salt diet (DOCA-salt), is a well-established means of inducing hypertension. This hypertension model is relevant to human primary aldosteronism The increasingly frequent diagnosis of primary hyperaldosteronism, the recognition of non-genomic effects of the hormone and the availability of the more specific receptor blocker eplerenone have also drawn attention to the role of aldosteron mineralcorticoid hypertension with renal dysfunction. Aldosterone induces inflammation, oxidative stress and fibrosis in the kidney dependent or independent on blood pressure, and blockade of the hormone reduces nephrosclerosis in several models of hypertensive renal damage.Our previous study demonstrated that nitro-oleic acid protect against LPS-induced and cisplatin induced kidney injury via anti-inflammation and anti-oxidative stress. So this current study was undertaken to investigate whether the nitro-oleic acid ameliorates DOCA-salt induced kidney injury in mice.Objectives1. TO produce DOCA-salt hypertensive model in mice.2. To illustrate the potential therapeutic effects of OA-NO2 in DOCA-salt induced renal injury. 3. To illustrate the potential mechanism of the protective effects.Methods1. Animal protocolMale C57BL/6 mice (8-week-old) were maintained on a standard rodent chow and had free access to water. OA-NO2 was dissolved in 100% DMSO at 10mg/ml. Mice were pretreated for 48 h with DMSO (DOCA vehicle) or OA-NO2 (DOCA+ OA-NO2) at 2 mg/kg/d via a micro-osmotic pump, and then both groups were implanted with a DOCA pellet together with a high-salt diet for two weeks. The third group received an surgery only and served as controls. Functional studies were done at 2 weeks after DOCA-salt treatment.2. Blood pressure measurementSystolic blood pressure was measured by a tail-cuff method. All animals were habituated to the blood pressure measurement device for 7 days. At 11 day post DOCA treatment, they all underwent 2 cycles of 20 measurements recorded per day for 4 days.3. Food and water intake, urine collectionThe mice were put in the metabolic cages to collect urine and measure food and water intake at 14 days post DOCA-salt treatment.4. ELISA measurementThe plasma and kidney TNF-αcontent, also the urine albumin and 8-isoprostane were measured using EIA kits, respectively.5. Real-time RT-PCR.Total RNA was isolated from fresh tissue, and the mRNA expression of GAPDH、TNF-α、IL-β、MCP-1、ICAM-1、VCAM-1、MCP-1、COX-2、mPGES-1、P47phox、gp91phox、podocin and nephrin were determined.6. Westen BlotThe protein was isolated from fresh tissue, and protein expression of TNF-α、COX-2、nephrin、P47phox and gp91phox in kidney were determined. 7. EM examinationEM was performed to examined the injury of the podocyte.8. Statistical analysisValues shown represent means±SE. Data were analyzed using unpaired t test or ANOVA followed by a Bonferroni posttest. A P value<0.05 was considered significant.Results1. Measurements of blood pressureAfter DOCA-salt treatment for 14 days, the systolic blood pressure increased strikingly. Pretreatment with OA-NO2 decrease a small degree but significant systolic blood pressure.2. Food and water intake, body weightThere were no differences of the food intake in all mice. DOCA-salt increased the water intake strikingly. Pretreatment with OA-NO2 decreased the water intake but not significant. DOCA-salt decreased the body weight significantly, while pretreatment with OA-NO2 attenuated the decrease of body weight.3. Urine volume and urine albuminDOCA-salt increased the urine volume and urine albumin strikingly. Pretreatment with OA-NO2 attenuated the rise of the urine albumin.4. Kidney histologyDOCA-salt induced the kidney hypertrophy with pale outlook. Pretreatment with OA-NO2 improved the kidney outlook and hypertrophy. According to PAS staining, DOCA-salt induced kidney cortical and outer medulla tubules dilation and hypertrophy, the basement membrane atrophy and brush border discontinuity. Pretreatment with OA-NO2 attenuated the kidney injury.5. Podocyte injuryDOCA-salt induced the podocytic process widely fuge and disappear, while the podocyte markers nephrin and podocin decrease. Pretreatment with OA-NO2 improved podocyte injury. 6. Kidney oxidative stress conditionDOCA-salt elevated the kidney TBARS and urine 8-isoprostane, together with high expression of the p47 phox and gp91 phox subunits. Pretreatment with OA-NO2 attenuated the rise of kidney TBARS, urine 8-isoprostane, p47phox and gp91phox subunits expression.7. Kidney inflammationDOCA-salt induced renal TNF-a mRNA and protein expression increase. DOCA-salt also increased the mRNA expression of IL-1β、MCP-1、ICAM-1、VCAM-1、MCP-1、COX-2. Pretreatment with OA-NO2 improved the inflammation induced by DOCA-salt.Conclusion1. Pretreatment with OA-NO2 for 48 h decreased the systolic blood pressure induced by DOCA-salt.2. Pretreatment with OA-NO2 attenuated the urine albumin and podocyte damage induced by DOCA-salt.3. The data showed the effects of the OA-NO2 via anti-inflammatory and anti-oxidative stress更多还原