Research On The Effect Of Mitogen Activated Protein Kinases Signaling Transduction Pathway In A Mouse Model Of Intestinal Ischemia/Reperfusion Injury

IntroductionIschemia/reperfusion(Ⅰ/R) injury is tissue lesions produced during reperfusion were greater than those produced during ischemia,which is a common organ damage that affects both medical and surgical patients.Intestine is one of most vulnerable organs to ischemia injury,and consequently intestinal ischemia/reperfusion(Ⅱ/R) often occurs in severe trauma and burn injury,shock,infection,intestinal transplantation and neonatal necrotizing enterocolitis as well as in patients in intensive care units.Ⅱ/R become the basis of the pathophysiology of numerous diseases and played an initiating role in the development of triggering sepsis and multiple organ failure.Ischemia and reperfusion of the small intestine provoke mucosal barrier dysfunction and augmentation of mucosa permeability,and resulting in bacterial/endotoxic translocation,production of oxygen free radicals,activation of inflammatory responses and uncontrolled release of cytokines and inflammatory mediators.It may lead to injuries in both local and remote organs,particularly in lung, and induces systemic inflammatory response syndrome(SIRS) and multiple organ dysfunction syndromes(MODS),which often progresses to acute respiratory distress syndrome(ARDS) associated with high mortality rate in critically ill patients.The molecular mechanisms ofⅡ/R injury have not been completely defined,and further investigations promote the focuses on the field of intracellular signal transduction pathway in recent years.Mitogen activated protein kinases(MAPKs) are an evolutionarily conserved family of serine/threonine kinases in eukaryotic cells that are primarily composed of three forms of c-Jun NH2 terminal kinase(JNK),extracellular signal regulated kinase (ERK) and p38 MAP kinase(p38 MAPK);MAPKs are activated under the conditions of ischemia/reperfusion injury,oxidative stress,infections and inflammatory cytokines so on.On activation of MAPKs,transcription factors present in the cytoplasm or nucleus are phosphorylated or activated,leading to expression of certain target genes resulting in a biological response,and controlling cell differentiation,cell proliferation,and cell death,and inflammatory response.Although MAP kinases generally function as autonomous signaling modules,the multiple interactions between the different MAP kinase cascades serve to integrate responses and to moderate outputs.Indeed,it has been demonstrated that MAP kinases have overlapping substrate specificities and phosphorylation of regulatory sites is shared among multiple protein kinases.Some studies have proved MAPKs was participated inⅡ/R-induced intestinal injury,however,no systematic evaluation of the expression, activity,or signal transduction of MAPKs has been published so far.It is unclear how MAPKs and its down-stream signal proteins are involved in the development ofⅡ/R. In the first part of this study,we examined the change course of MAPKs activation inⅡ/R-induced intestinal tissues,and explored the potential effects of MAPKs-mediated apoptotic signal pathway in the development ofⅡ/R injury in mice.Lungs are not only the gas exchange place,but also the place some cytokines and hormones are inactived.As the lung is the first station that invasive toxins and endogenous inflammatory agents will visit,as well as its physical and anatomic characters,such as low flow rate and pressure,big area,all those add the risk to be injured.From the most,it is the common sense that the lung is one of the most fragile organs and main target organs following uncontrolled systemic inflammatory response.The mechanism of lung injury induced by intestinalⅠ/R is complicated. Many studies have shown thatⅡ/R induce disruption of the intestinal mucosal barrier, allowing translocation of bacteria and endotoxin,reactive oxygen species(ROS), cytokines and inflammatory mediators originated from the injured intestinal tissue into the circulation,which result in distal organ injury including acute lung injury. MAPKs can also be activated in the lung by a number of pro-inflammatory stimuli, including LPS,cytokines,and oxidative stress;it is therefore plausible to speculate that pulmonary MAPKs activation is involved in the pathogenesis ofⅡ/R-induced lung injury.LPS,ROS and inflammatory cytokines generated during intestinalⅠ/R injury may trigger the activation of p38 signaling cascade in the lung,which may then in turn contribute to the development of lung injury.Acute lung injury is characterized by intensive pulmonary inflammatory response.As we known,many inflammatory cells and inflammatory mediators were involved in the systemic inflammatory response and constitute the inflammatory response network.So it has been emphasized that those factors that were closely related to the inflammatory response, such as TNF-α,IL-1βhave played a centre role.Accordingly,in this study,we examined the time course of p38 activation in lung tissues in mice subjected toⅡ/R. With the use of a potent and selective second-generation inhibitor of p38,SB 239063, we determined the effects of in vivo inhibition of p38 onⅡ/R-induced TNF-α,IL-1βgene expression,and investigated the possible role and mechanisms of p38 MAPK activation in theⅡ/R-induced lung injury.PartⅠRole of activation of MAPKs signaling transduction pathways in intestinal tissue after intestinal ischemia/reperfusion in miceObjective:To observe the dynamic changes of activation of MAPKs and apoptotic proteins of Bcl-2,Bax and Caspase-3 in intestinal tissue after intestinal ischemia/reperfusion(Ⅱ/R) in mice.To explore the potential role of MAPKs-dependent apoptotic signal pathway inⅡ/R-induced mice.Methods:AnimalⅡ/R model was established with clamping the superior mesenteric artery(SMA).Mice were intraperitoneally anesthetized with 1%solution of pentobarbital sodium(50 mg.kg^-1).First,forty male C57BL/6 mice,8-10 weeks, were randomly divided into five group:30 min,40 min,50 min and 60 min of intestinal ischemia,and survival rate was observed within fourteen days after intestinal reperfusion.Second,mice were randomly divided into sham-operated group (n=6) andⅡ/R groups(n=36);the latter was further divided according to time after reperfusion(0,0.5,1,4,6 and 12 h) after reperfusion.AnimalⅡ/R model was established with clamping SMA for 40 minutes.Animals in the sham-operated mice received no clamping.The expression of JNK and phosphorylation(phospho-) JNK, ERK and phospho-ERK and p38 MAPK and phospho-p38 MAPK,cleaved caspase-3, Bcl-2 and Bax proteins in the intestinal tissue was examined by Western blotting analysis,and the pathological change of ileum tissue was observed by optical microscope.Phospho-MAPKs by Western blot analysis was applied to reflect the activation status of MAPKsResults:(1) All animals were survived in the group of 30 minute intestinal ischemia,and animal survival rates decreased gradually followed by increase of ischemia time.In contrast,approximately 70%animals died in the group of 60 minute of intestinal ischemia.(2) Most sereve intestinal injury was induced by intestinal ischemia/reperfusion at the early stage of reperfusion,which peaked at 1 h,and intestinal tissue almost recovered 12 h later.Localization of apoptotic cells,as detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL), showed apoptotic cells were scattered in the intestinal tissues including mucosa, propria lamina,mucosa underlayer even and muscular layer.(3) Cleaved-caspase-3 was significantly increased inⅡ/R group at 0.5h,lh after reperfusion compared to the sham-operated group(P＜0.01);at the same time,the expression of Bcl-2 protein inⅡ/R group was significantly decreased compared with sham group(P＜0.01),and there was no significant difference in Bax expression between different group.(4)Ⅱ/R caused an early activation in all members of the MAPK family,whereas there was no significant change in JNK,ERK and p38 MAPK level at various time points afterⅡ/R. the phospho-JNK in the intestine was significantly elevated within 1 h after reperfusion compared with sham group(P＜0.01).when compared with sham group, the expression of phospho-ERK protein in the intestine was significantly increased within 1 h after reperfusion(P＜0.01),sharp decreased to the normal level at 4 h of reperfusion,and again elevated significantly at 6 h,12 h after reperfusion(P＜0.01); the phospho-p38 MAPK protein was stringly decreased in the ischemia intestinal tissue(P＜0.05),in contrast,significantly elevated at 0.5 h after reperfusion(P＜0.01), and kept at this high level up to 12 h of reperfusion(P＞0.05).Conclusions:phosphorylation of JNK and p38 MAPK plays an essential role in the intestinal damages induced byⅡ/R,possibly through down-regulation of Bcl-2 protein and caspase-3 dependent proapoptotic pathway;and ERK phosphorylation may contribute to restitution of damaged intestine. PartⅡInhibition of p38 mitogen activated protein kinase attenuates local and pulmonary injury following intestinal ischemia/reperfusion in miceObjectives:to investigate the potential roles of p38 mitogen-activated protein kinase(MAPK) in lung injury following intestinalⅠ/R.Methods:First,to determine the dynamic changes of pulmonary p38 MAPK activation.Twenty Male C57BL/6 mice were randomly divided into sham-operated group,and 0,0.5,4 and 6 h groups of reperfusion after 45 minute intestinal ischemia (n=4 in each group).To further examine the role of p38 MAPK activation in intestinalⅠ/R-induced lung injury.Mice were randomly divided into sham-operated, intestinalⅠ/R plus vehicle treatment and intestinalⅠ/R plus p38 MAPK inhibitor SB239063 treatment(3 mg/kg,i.p.) groups;intestinalⅠ/R was performed with 45 minutes intestinal ischemia followed by 6 hours reperfusion.The pulmonary p38 MAPK activation was observed by western blot after intestinalⅠ/R;and histopathology and production of TNF-αand IL-1βmRNA in the intestinal and pulmonary tissues were also analyzed at 6 hour of reperfusion after 45 min intestinal ischemia.Results:IntestinalⅠ/R caused a rapid activation of p38 MAPK in lung tissues, and which lasted to 6 hour of reperfusion.Selective inhibition of p38 MAPK with SB 239063 could down-regulate an elevated IL- 1βmRNA level induced by intestinalⅠ/R, and significantly attenuated the intestinal injury in the mouse model ofⅡ/R.Further, blockade of p38 MAPK activation not only inhibited the up-regulation of pulmonary TNF-αand IL-1βmRNA levels induced by intestinalⅠ/R,but also significantly attenuated the pulmonary pathological changes.Conclusions:Activation of p38 MAPK plays an important role in intestinalⅠ/R-induced lung injury in mice.Inhibiting p38 MAPK activation might be a potential strategy for prevention the pathological changes in lung after intestinalⅠ/R.