Study On The Protection Of Renal Ischemia Reperfusion Injury And Acute Liver Injury After Severe Trauma

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...