【作者】 刘文军；

【导师】 夏照帆； 付晋凤；

【作者基本信息】 昆明医学院 ， 外科学， 2008， 博士

【摘要】 一、研究背景一些严重创伤及大面积深度烧伤病人由于早期得不到及时有效的复苏治疗,伤员只能得到延迟复苏,往往得不到预想的疗效,休克的发生率高,病人在后续病程中多脏器功能衰竭发生率高,死亡率也高。夏照帆等早在1991年就提出了延迟复苏可以引起脏器的再灌注损伤。其中肾为高灌注器官,对缺血以及再灌注造成的损伤均敏感。造成缺血再灌注损伤(ischemia reperfusion injury,IRI)的相关因素很多,其中IRI引起的炎症级联反应和活性氧氧化导致的细胞凋亡是人们所关注的两个主要因素。研究证明抑制与炎症反应明确相关的MARK等信号通路,对炎性介质和氧化损伤引起的细胞损伤具有明确的保护作用。三七皂甙R1(notoginsenoside R1,NR1)是五加科人参属植物三七根部提取物三七总皂甙的主要成分之一,现代医学研究表明,NR1具有抗炎、抗氧化、保护心肌、抗心率失常、抗休克、降低动脉血压、抑制血小板聚集、增加心脑血流量等多方面的作用。而且最近有文献报道NRl可以通过ROS/ERK信号转导通路抑制TNF-α诱导的炎症反应,明显改善心脑IRI损伤。因此本实验一采用大鼠右侧肾摘除左肾动静脉夹闭45 min制备缺血再灌注模型,系统观察MAPK信号通路的变化,并采用NR1干预,探讨其在肾IRI损伤中的作用及机制。严重创伤、休克和重度烧伤等重症患者,内毒素血症引起的肝功能衰竭也是很常见的。严重创烧伤引起的肠源性感染已经日益受到临床的重视,由于这些患者往往合并有肠源性内毒素血症,更易导致临床肝功能衰竭。韩德五等在1995年就已经提出了肝功能衰竭的内毒素机制,认为内毒素血症特别是肠源性内毒素血症是肝功能衰竭发生的物质基础。内毒素引起的肝损伤的机制包括直接损伤和间接损伤,直接损伤指内毒素通过细胞膜特异性受体介导及非特异性胞膜结合两种方式进人细胞内,造成肝细胞的损伤,间接损伤指由内毒素介导的炎症介质的释放对肝组织的毒性作用以及所引发的细胞凋亡和坏死。圆盘状受体1(Discoidin Domain receptors,DDR1)属于酪氨酸激酶受体家族,在介导细胞粘附、迁移、增殖、凋亡、浸润以及胶原的合成和代谢中担当重要角色,最近研究发现体内DDR1可通过核转录因子NF-κB/Fas信号通路在细胞凋亡中起重要作用,并且在动物实验证实抑制DDR1的激活可减轻依赖p38MAPK信号通路的肺部炎症。为此本实验二采用脂多糖/D-半乳糖胺(LPS/D-GaIN)诱导的急性小鼠肝损伤模型,观察急性肝损伤后DDRl是否参与内毒素诱导的肝损伤,并采用体内DDR1 siRNA干扰技术,观察急性肝损伤后DDRl对LPS诱导急性肝损伤细胞凋亡及炎症相关丝裂原活化蛋白激酶(MAPK)细胞信号通路的影响,以了解DDR1和相关信号转导通路在急性肝损伤中的可能发病机制。二、研究目的1.观察三七皂甙R1在肾IRI中对MAPK信号转导通路及细胞凋亡的影响。探讨中药防治肾缺血再灌注损伤的机制及未来通过在信号转导通路不同层面调控来干预肾IRI进展和治疗此类疾病的可能性。2.采用体内DDR1 siRNA干扰技术,观察急性肝损伤后DDR1对LPS诱导急性肝损伤细胞凋亡及炎症相关MAPK细胞信号通路的影响,以了解DDR1和相关信号转导通路在急性肝损伤中的可能作用机制及体内DDR1 siRNA干扰技术对其的影响和作用。三、研究内容实验一三七皂甙R1在大鼠肾缺血再灌注损伤中的作用机制第一部分大鼠肾IRI后MAPK细胞信号转导通路的活化目的:系统观察肾IRI后p38MAPK、JNK、ERK蛋白激酶的动态活化,为进一步研究肾脏IRI机制提供理论基础。方法:雄性SD大鼠70只,随机将实验动物分为手术对照组;肾缺血再灌注(IRI)组,再分为再灌注后5、10、15、30、45、60、120 min七个时相点(n=5每个时相点)。采用右侧肾摘除左肾蒂夹闭左肾动脉、静脉45 min后松开制备缺血再灌注模型;对照组除未给予左肾动静脉夹闭外,余操作同IRI模型组,分别于再灌注各时相点处死动物取材。病理切片检查及Western blot法检测肾组织中p38MAPK、JNK、ERK激酶的活化情况。结果:IRI可造成早期肾脏病理损害,并且早期2h内随着再灌注时间的延长肾脏病理损害呈渐进性加重;IRI后5min大鼠肾组织p38很快被诱导活化,45min达到峰值,随后p38活化减弱,但仍明显高于正常水平(P＜0.05);JNK在再灌注10min开始轻度被诱导活化,45min活化明显增强,而且持续增高至再灌注后2h,明显高于正常水平(P＜0.05);再灌注后各个时相点5min,10 min,15 min,30 min,45 min,1 h,2 h大鼠肾组织ERK活化相比均未有明显差异(P＞0.05)。结论:采用大鼠右侧肾摘除左肾蒂夹闭左肾动脉、静脉45min后松开行再灌注可以造成肾脏病理损害并且早期2h内随着再灌注时间延长病理损害呈渐进性加重。MAPK信号通路中p38MAPK、JNK信号通路都可能参与了肾IRI损伤,而ERK可能未参与此损伤。第二部分三七皂甙R1在大鼠肾缺血再灌注损伤中作用机制及对p38MAPK、JNK信号转导通路的影响目的:观察MAPK信号通路在肾IRI后的变化及NR1干预对其的影响,探讨其在肾IRI损伤中的作用及机制。方法:雄性SD大鼠30只,随机分为手术组对照组(Control组,n=10)、缺血再灌注损伤组(IRI组,n=10)和NR1处理组(NR1组,n=10)。采用右侧肾摘除左肾蒂夹闭左肾动脉、静脉45 min后松开制备缺血再灌注模型,术前给予0.2ml等渗生理盐水尾静脉注射;NR1处理组术前给予NR1(40mg/kg)溶于0.2ml等渗生理盐水尾静脉注射,余操作同IRI模型组;Control组除未给予左肾动静脉夹闭外,余操作同IRI模型组。再灌注45min后处死动物取材检测肾功、MDA含量、凋亡细胞TUNEL分析、p38MAPK和JNK活化及炎症细胞因子TNF-α,IL-1β蛋白表达。结果:NR1可以明显改善IRI早期病理损害,但对早期肾功能恢复没有作用;IRI组MDA与对照组相比明显增高(P＜0.05)。而NR1处理组与IRI组相比明显降低,但仍高于手术对照组(P＜0.05);IRI组肾组织细胞凋亡明显,AI值明显高于对照组,差异显著(P＜0.05),NRI预处理组亦出现凋亡细胞,但凋亡程度即AI值明显轻于IRI组,差异显著(P＜0.05);IRI组phospho-p38、phospho-JNK在再灌注45min时相点与对照组相比表达明显增强(P＜0.05),NRI处理组phospho-p38、phospho-JNK与IRI组相比表达明显减弱(P＜0.05);TNF-α、IL-1β在缺血再灌注损伤后45min时相点IRI组与对照组相比明显升高(P＜0.05),NR1处理组与IRI组相比表达减弱(P＜0.05),但仍高于对照组(P＜0.05)。结论:NR1在IRI引起的肾损伤中可能通过抑制p38MAPK和JNK信号通路的活化,减少炎症细胞因子TNF-α、IL-1β的表达,减轻组织受自由基攻击损伤的程度,从而减少肾脏损伤后细胞的凋亡,改善IRI引起的病理损害。实验二体内RNA干扰抑制盘状受体1对LPS/D-GaIN诱导小鼠急性肝损伤的保护作用及机制研究第一部分盘状受体1蛋白在急性肝损伤后的表达变化目的:观察LPS/D-GaIN诱导的急性小鼠肝损伤后DDR1蛋白的表达变化,以为更深一步研究DDR1是否参与急性肝损伤的发病机制提供理论依据方法:雄性BALB/c小鼠80只,随机将实验动物分为急性肝损伤组;生理盐水对照组。每组进一步分为0、0.5、1、3、6、12、24、48h八个时相点(n=5每个时相点)。肝损伤组小鼠均以LPS(10μg/kg)+D-GaIN(800 mg/kg),用1ml无菌生理盐水溶解后腹腔内注射,诱发小鼠肝损伤模型;对照组小鼠仅腹腔内注射1ml生理盐水。分别于注射后0、0.5、1、3、6、12、24、48h时相点分批麻醉后处死小鼠,每时相点5只,下腔静脉抽血后,迅速取出肝脏置于冰上,冰冷PBS冲洗后,部分肝组织10%中性福尔马林液固定待病理检查,剩余组织置液氮冻存备用。检测各组ALT、AST变化及病理分析,同时Western Blot检测DDR1蛋白表达变化。采用单因素方差分析判断组间差异,所有统计均经SPSS11.0统计软件处理完成。结果:肝损伤后小鼠血ALT在3h开始轻度升高,6h即呈现显著的升高,24h达到峰值,48h仍维持于高水平,小鼠血AST在1h即开始轻度升高,同ALT在24h达到峰值,48h仍维持于高水平;在HE染色下显示LPS/D-GaIN诱导肝脏损害后24、48h时相点病理损害明显;小鼠DDR1蛋白在0h、0.5h、1h与对照组无差异(P＞0.05),3h开始轻度升高,在肝损伤早期6h才开始明显增高,24h达到高峰,48h后又明显减少,但仍明显高于正常水平(P＜0.05)。结论:LPS/D-GaIN诱导的急性小鼠肝损伤模型可造成内毒素相关肝脏急性病理损害。DRR1表达在肝损伤后明显增强,并在24h达到高峰,因此推测DDR1在内毒素诱导的急性肝损伤中起重要作用。第二部分体内盘状受体1 siRNA干扰在急性肝损伤中的保护作用及机制目的:观察体内DDRI siRNA干扰对LPS/D-GaIN诱导急性肝损伤后细胞凋亡及炎症相关细胞信号通路的影响。方法:设计合成DDRI siRNA。雄性BALB/c小鼠35只,随机将实验动物分为生理盐水对照组;急性肝损伤组;DDR1siRNA1处理组;DDR1siRNA2处理组;非抑制性双链RNA(Non-silencing siRNA)组;暴发性肝衰组;DDR1siRNA1+暴发性肝衰组。n=5每组。其中前5组用来研究DDR1siRNA对急性肝损伤的影响。暴发性肝衰两组用来观察DDR1siRNA对暴发性肝衰后小鼠生存期的影响。急性肝损伤组以1.5ml生理盐水经小鼠尾静脉快速注射,5秒内注射完毕,8h及24h分别再重复注射一次,最后一次注射后24h,采用LPS(10μg/kg)+D-GaIN(800 mg/kg),用1ml无菌生理盐水溶解后腹腔内注射,诱发小鼠肝损伤模型;生理盐水对照组造模前同肝损伤组,生理盐水最后一次注射后24h,只给予1ml等量生理盐水腹腔注射;其余siRNA处理组分别将DDR1siRNA1、DDR1siRNA2、Non-silencing siRNA各50μg溶于1.5ml生理盐水中,按分组情况经小鼠尾静脉快速注射,5秒内注射完毕,8h及24h分别再重复注射一次,最后一次注射后24h操作同肝损伤组;暴发性肝衰组,操作同肝损伤组,但采用LPS(100μg/kg)+D-GaIN(800 mg/kg)剂量;DDR1siRNA1+暴发性肝衰组操作同DDR1siRNA1处理组,但采用LPS(100μg/kg)+D-GaIN(800 mg/kg)剂量诱发小鼠暴发性肝衰模型。前5组小鼠处理后24h,每只小鼠尾静脉注射印度墨汁0.05ml/10g,分别在注射后1min和5min用吸管(预先用肝素溶液湿润)经眶静脉丛取血20μl,用于炭粒廓清实验。然后处死取材,下腔静脉抽血后迅速取出肝脏置于冰上,冰冷等渗盐水冲洗后,部分肝组织10%中性福尔马林液固定待病理检查,剩余组织置液氮冻存备用。Western Blot检测DDRI蛋白、p38、pho-p38、JNK、pho-JNK表达变化,逆转录PCR法检测DDR1mRNA表达变化,TUNEL法检测肝细胞凋亡情况,EMSA法检测NF-κB的DNA结合活性变化,ELISA法检测血清内TNF-α、IL-1β蛋白表达,炭粒廓清实验测定库普弗细胞吞噬功能。结果:DRR1-siRNA序列1和2都能够明显抑制DDR1蛋白和mRNA的表达;DDR1-siRNA体内干扰可以明显抑制肝损伤后ALT、AST的升高,减少细胞坏死及炎性浸润;TUNEL法检测细胞凋亡明显减轻;免疫组化可见肝损伤后Fas蛋白在肝组织且呈弥漫性表达增加,DDR1-siRNA体内干扰抑制Fas蛋白的表达;DDR1-siRNA体内干扰不仅抑制了肝损伤后p38MAPK、JNK激酶和NF-κB的活化,而且体内TNF-α、IL-1β的蛋白表达也被DDR1siRNA的干预所抑制,同时抑制了库普弗细胞吞噬指数升高,提高了急性肝损伤小鼠的中位生存时间。结论:尾静脉高压注射DRR1-siRNA能够明显抑制小鼠肝脏DDR1基因和蛋白的表达,采用小鼠尾静脉高压注射DDR1siRNA体内干扰是可行的。DDR1siRNA体内干扰可能通过降低肝细胞、血管内皮细胞及粒细胞等多种细胞Fas蛋白的表达,间接抑制库普弗细胞的吞噬功能,抑制p38、JNK、NF-κB等信号通路的激活从而减少TNF-α、IL-1β炎症介质的释放,进一步减轻了炎症介质释放的级联反应及细胞凋亡引起的损伤。更多还原

【Abstract】 Study on the Protection of Renal Ischemia Reperfusion Injury and Acute Liver Injury After Severe Trauma1.IntroductionMany severe trauma and burned patients if not to obtain promptly effective resuscitation therapy,only gained delayed fluid resuscitation, seldom got anticipatively therapeutic effect and obtained high incidence of shock,and got high incidence of MODS and mortality in post-course of disease.Xia ZF etal proposed delayed fluid resuscitation induced reperfusion injury of organs on 1991.Kidney is a hyperperfusion organ and it was rather sensitive to ischemia and reperfusion.There are many reasons for ischemia reperfusion injury(IRI)and apoptosis induced by inflammation cascade reaction and oxidative damage are the two main factors.It have been verificated inhibit MAPK signal transduction have the protection effects on cell injury induced by inflammation cascade reaction and oxidative damage.Notoginsenoside R1(NR1),a major active component of the traditional Chinese medicine herb panax notoginseng. Modern medicine research indicated that NR1 have many effects on anti-inflammatory,anti-inflammatory,protective myocardium,anti- heart rate upset,antishock,depress arterial blood pressure,inhibit platelet aggregation,increase cerebral blood flow etal.Furthermore,it reported NR1 can inhibit inflammation induced by TNF-αdependent on ROS/ERK signal transduction and ameliorate heart and brain IRI.So we apply IRI model was induced by renal pedicle ligation followed by reperfusion 45min along with a contralateral nephrectomy,and to observe the changes of MAPK signal transduction,then investigate the effect of NR1 on renal IRI.Liver function failure induced by endotoxemia of severe trauma,shock and severe burned patients are common.Now it pay more attention to enterogenic infection induced by severe trauma and burned.Complicating with enterogenic infection endotoxemia is more easily to be to lead to liver function failure.Nan DW etal proposed endotoxin mechanism of liver function failure on 1995,considered endotoxemia and especially enterogenic infection endotoxemia are the substance fundament of liver function failure.Hepatic injury induced by endotoxin including direct injury and indirect injury,direct injury to count on to cause hepatocellular injury induced by endotoxin through cell membrane specificity and non-specificity acceptor,and indirect injury indicate to cause hepatocellular injury induced by mediators of inflammation. Discoidin domain receptor 1(DDR1)is a receptor tyrosine kinase that is activated on binding to its ligand-collagen,DDR1 is constitutively expressed in the normal tissues of organs such as the lungs,kidneys,colon, liver and brain,and DDR1 contribute to important role on cell adhesion, migration,generation,apoptosis,infiltrating as well as collagenous synthesis.Recently research shows that DDR1 play an important role in cell apoptosis dependent on NF-κB/Fas signal transduction passageway,and confirmed that suppress DDR1 can relieve lung inflammation dependent on p38MAPK signal transduction passageway.So we applied acute liver injury induced by LPS/D-GaIN to investigate whether DDR1 participate the injury,as well as applied DDR1 siRNA interference to observe the effects of DDR1 on cell apoptosis and MAPK signal transduction passageway,then to understand the possible pathogenesy related with DDR1 and signal transduction passageway.2.Objectives1)To observe the effects of NR1 on MAPK signal transduction passageway and cell apoptosis after renal IRI.Investigate the mechanism of traditional Chinese medicine prevention and cure of renal IRI and the possibility of apply traditional Chinese medicine to regulate signal transduction passageway to interfere progress of renal IRI and cure this kind of disease.2)To cbserve the effects of DDR1 siRNA invivo interfere on cell apoptosis and MAPK signal transduction passageway in acute liver injury induced by LPS,so to understand the pathogenesy of acute liver injury related with DDR1 and signal transduction passageway.3.ContentsTest 1 The Mechanism of NR1 on Renal Ischemia Reperfusion Injury in RatsPartⅠThe Activation of MAPK Signal Transduction passageway After IRIObjectives:To investigate the activation of p38MAPK,JNK and ERK protein kinase after renal IRI,so to provide rationale for further study on renal IRI.Materials and methods:Seventy male Sprague-Dawley rats randomly separated into two groups:sham operated group control group;IRI group,then subdivided into 5、10、15、30、45、60、120 min time point groups (n=5).IRI model was induced by renal pedicle ligation followed by reperfusion 45min along with a contralateral nephrectomy,and then scarrifice animals for biopsy,the activation of p38MAPK,JNK and ERK protein kinase after renal IRI injury detected by Western blot.Results:IRI can induce renal early pathologicallesion;p38 be activited early at 5min after IRI,to achieve peak at 45min,then begain to weaken(p＜0.05).JNK be activated at 10min after IRI,then persistet increase until 2h after IRI compared with control group(P＜0.05).Conclusions:IRI model induced by renal pedicle ligation followed by reperfusion along with a contralateral nephrectomy can induce renal early pathologicallesion.p38MAPK and JNK participate renal IRI but not for ERK.PartⅡThe Mechanism of NR1 on Renal Ischemia Reperfusion Injury and Effects on p38MAPK and JNK Signal Transduction Passageway in Rats Objectives:To observe the changes of MAPK signal transduction passageway and the effects of NR1 interferein it.To investigate the role of NR1 in IRI.Materials and methods:Thirty male Sprague-Dawley rats randomly separated into three groups:sham operated group control group(n=10);IRI group(n=10);NR1+IRI group(n=10).IRI model was induced by renal pedicle ligation followed by reperfusion 45min along with a contralateral nephrectomy,NR1 group received tail vein injection of NR1(40mg/kg)in 0.2ml saline,sham group treated as IRI group but not with a contralateral nephrectomy.Then scarrifice animals for biopsy at 45min after IRI,the activation of p38MAPK and JNK protein kinase after renal IRI injury detected by Western blot,cell apoptosis analysised by TUNE1,and analysised expressing of MDA,TNF-α,IL-1β.Results:NR1 can improve the renal pathologicallesion but not for functional recovery.The administration of NR1 decreased MDA and cell apoptosis compared with IRI group(P＜0.05).Activation of p38MAPK and JNK was markedly inhibited by NR1(P＜0.05),so as expression of TNF-α,IL-1βcompared with IRI group(P＜0.05).Conclusions:NR1 can Inhibit activation of p38MAPK and JNK so as to decrease TNF-α,IL-1βrelease,thus relieve the injury of free radical attack,thereby decrease cell apoptosis and amelioration pathologicallesion.Test 2 The Protection of Invivo Delivery DDR1siRNA on Acute Liver Injury Induced by LPS/D-GaIN in MicePartⅠThe Changes of Expression of DDR1 After Acute Liver InjuryObjectives:To observe the changes of DDR1 after acute liver injury induced by LPS/D-GaIN,thus to provide theory for furthermore study on the role of DDR1 in acutr liver injury.Materials and methods:eighty male BALB/c randomly separated into two groups:saline control group;acute liver injury group,then subdivided into 0、5、10、15、30、45、60、120 min time point groups(n=5).Acute liver injury group received intraperitoneal injection of LPS(10μg /kg)+D-GaIN(800 mg/kg)in 1ml saline induce acute liver injury,control group animals only received 1ml saline.Sacrifice animals at different time points.ALT and AST are detected,the exrepressin of DDR1 detected by Western Blot.Results:Plasma ALT begain to step up at 3h and reach peak at 24h,then to keep high level at 48h.Plasma AST begain to step up at 1h and reach peak at 24h too.Pathologicallesion was obviously after LPS/D-GaIN induced liver injury.The expression of DD1 step up at 3h and reach peak at 24h too,then begain to decrese at 48h compared with control group.Conclusion:The model of acute liver injury induced by LPS/DaIN can duplicate gut origin endotoxin liver injury.DDR1 paticipate pathogenesy of acute liver injury,so we suppose DDR1 play key role in acute liver injury.PartⅡThe Role of Invivo Delivery DDR1siRNA in Acute Liver InjuryObjectives:To observe the effects of DDR1 on cell apoptosis and signal transduction passageway related with inflammation.Materials and methods:To design and synthetic of DDR1siRNA.Thirty five male BALB/c randomly separated into seven groups(n=5):Acute liver injury group treated with 1.5ml saline rapidly injected into the tail vein.The injection was repeated 8 and 24h later.24h after the last of three injections LPS(10μg/kg)+D-GaIN(800 mg/kg)dissolved in 1 ml saline intraperitoneal injection induce acute liver injury;Control mice were injected with an equal volume of normal saline.DDR1siRNA1、DDR1siRNA2、 Non-silencing siRNA groups underwent identical procedures to acute liver inury mice and pre-teated with different sequence of siRNA respectively; underwent identical procedures to acute liver inury mice but administated dose of LPS(100μg/kg)+D-GaIN(800 mg/kg)differently;DDR1siRNA1+ fluminating liver failer group underwent identical procedures to fluminating liver failer group and pre-teated with siRNA1.The first 5 groups received tail vein injection of India ink 0.05ml/10g after 24h respectively and collected 20μl blood via orbital for carbon clearance test,then collected kidney sample for test.Activation of DDR1,p38MAPK and JNK were assessed by Western Blot.Cell apoptsis analyzed by TUNEL and NF-κB activation detected by EMSA.Expression of TNF-α、IL-1βassessed by ELISA and phagocytosis function of Kupffer’s cell detection by carbon clearance test.Results:The expression of DDR1 protein and mRNA were inhibited by DDR1siRNA sequence 1 and 2.Invivo delivery DDR1siRNA can inhibit the increase of ALT and AST,so as to decrease cell apoptosis in Acute Liver Injury.Not only activation of DDR1,p38MAPK,JNK and NF-κB were inhibited,but also expression of TNF-α,IL-1βdecreased too and inhibit phagocytosis function of Kupffer’s cell at the same time.The median Survival time of mice were obviously improved.Conclusion:The expression of DDR1 protein and mRNA were inhibited by invivo delivery DDR1siRNA,so it is feasible to interfere DDR1 by invivo delivery DDR1siRNA.Invivo delivery DDR1siRNA can ihibit expression of Fas on hepatocyte,vascular endothelial cell and granulocyte etal,indirect inhibit phagocytosis function of Kupffer’s cell,then inhibit activation of p38,JNK and NF-κB,thereby decrease releasing of TNF-α,IL-1β,so relieve cell apoptosis by cascade reaction of mediators of inflammation. 属性不符合更多还原