Effect Of RAGE Deficiency On The Proteome Of Lung Tissue Of Mice With Septic Shock

Sepsis is a systemic inflammatory response syndrome caused by microbial infection,which is one of the serious complications of serious infection,severe trauma and burn,shock,major surgery of clinical critical patients.It will go further leading to septic shock,ARDS,MODS and so on.It is an overreaction of the organism to microbial products and also an uncontrolled amplifying immune response.Sepsis and its septic shock are a hard task for critical medicine to face.It has been one of the main reason for death in clinical critical patients.Although we have achieved some process in the pathogenesis of sepsis,we did not get any satisfying answer from the clinic treatment.The researchers have done lots of animal experiments and clinical trials through the antibody to LPS and the antagonists of tumor necrosis factor and foundation support auxiliary therapy,while there was no satisfying answer.The mortality rate caused by sepsis remain high.All of these show us that we need further research on the awareness of sepsis especially the septic shock.A growing of evidence shows that the vital of organisms such as lungs plays an vital role in the development of sepsis,while so far the molecular mechanism of pathophysiology is still unclear.Animal models of sepsis is a necessary experimental platform to research the pathogenesis and clinical prevention and treatment of sepsis,as well as a difficult point in the study of sepsis.Now there are a variety of methods of constructing animal models of sepsis,including cecal ligation and puncture(CLP)abdominal cavity infection sepsis model,ascending colon stenting abdominal sepsis infection model of peritonitis,bacteria or bacterial endotoxin attacking model.CLP model is a model of sepsis caused by a variety of microbial,which is induced by surgery acute suppurative peritonitis and forming a persistent infection,a large number of bacteria and toxins are quickly absorbed into the bloodstream and spread to the whole body,each viscera appeared a severe inflammation reaction and the levels of inflammatory cytokines and endotoxin in lung,intestinal,liver tissue increased significantly,the tissue damaged seriously and finally formed MODS.It largely simulates lipopolvsaccharide(LPS)relentlessly striking when human have sepsis and thus can fits body well.Therefore,it becomes more and more popular among sepsis research.In the early research,scientists found that the signal transduction pathway of Toll-like receptor is an important way to make the activation of early-stage sepsis and inflammatory reaction.In recent years,after the deep research in pathogenesis of sepsis,more and more evidence show that high mobility group box 1(HMGB1),as the potential medium of late inflammation,involves in the pathogenesis of sepsis.Although nowadays the scientific field has the new understanding of mechanism of action on how HMGB1 involves in the late inflammation,for example there is a report that HMGB1 can involve in the late inflammation through action by combining Toll-like receptor 4/2,receptor for advanced glycation end products(RAGE)as well as other cell surface receptors to activate downstream signaling pathways.There are still many unknowns and doubts caused by the cell signaling pathways which mediates late inflammation.And there remains to in-depth research in the downstream near-end signal mediated by HMGB1 and its receptors.RAGE as one of the HMGB1 conventional signal transduction pathway,belongs to the immunoglobulin super family.It widely distributes in many kinds of cell surface,highly expressed in the lung tissues,and can combine with many ligands.Yet the affinity with HMGB1 is much higher than other ligands.In the pathogenesis of sepsis,the earlier animal experiments proved that HMGB1 gene expression in liver,lung and intestinal tissue was significantly increased after severe burns and abdominal infection 6～24h,which continued until 72 hours after injury.HMGB 1 induction at local tissue is closely related to LPS-mediated organ damage.At the same time,It reported that the activation of RAGE plays an important role in the CLP-induced abdominal infection and further tissue injury.By knockouting RAGE gene in mice and preventing interaction between HMGB1 and RAGE receptor,it can reduce tissue damage to a large extent,and then improve the survival rate of animals to protect the body.Therefore,the research on the process of development of late sepsis by HMGB1 with RAGE-mediated downstream signaling pathway has a very important clinical significance.The lung tissue is one of the earliest and most vulnerable organs to be damaged from inflammation.After establishment of CLP sepsis model,myeloperoxidase,total protein in the bronchioalveolar lavage and inflammatory mediators in lung raise rapidly,changes of pathology are significant,the damaged lung tissue is involved in the pathophysiological process of sepsis and septic shock.At the septic shock,afterRAGE removed,we are awarded of little about what happen to the function of lungtissue and the molecular mechanism,which will help us to know well about pathogenesis of sepsis at a molecular level.As we all know,protein is the final implementor of a life,what we have to study on the functional changes of the lung tissue after septic shock,in fact,is the protein changes of lung tissue.While the differential proteome research provides a goodmethod to analyze the change of protein during septic shock through comparing protein expression level between different samples and finally found the difference to solve various of biological problems.The innovation of differential proteomics technology makes it possible to study generally proteome from the lung in physiology and pathology.At present there are many novel differential proteomics analysis methods,among which the difference gel electrophoresis(DIGE)technology becomes one of the most popular in quantitative proteomics areas,because it’s not only have the high-resolution feature,inherited from two-dimensional gel electrophoresis(2-DE),but also have high throughput,high reproducibility and high dynamic range.DIGE is a method which can labele protein samples with different fluorescent dyes before 2-D electrophoresis,and can separate up to three different protein samples at the same time in one two-dimensional gel.The application of the internal standard could prevent systematic error of the result and ensure the test result is true and reliable.In this research,a septic shock model was established after exploration at first,and then used a technology platform based on difference gel electrophoresis,MALDI TOF/TOF mass spectrometry and bioinformatics analysis to find the Proteomic map associated with RAGE from lung of mice with septic shock.Firstly we selected normal(RAGE+/+)mice and RAGE gene knockout(RAGE-/-)C57/BL6 mice and which four mice in a group and they were randomly divided into control group and experimental group,the control group was received sham operation(Sham group)and the latter received cecal ligation and puncture(CLP group),totally constituting N-Sham,N-CLP,KO-Sham and KO-CLP four groups.Each group mice were put to death after operation at 8h and the lung tissue and total protein was extracted.After separating by DIGE,we found 72 differentially expressed proteins spots through DeCyder 2D Differential Analysis Software,of which there are 5 proteins spots presenting a significant up-regulated expression change and 22 proteins spots which are down-regulated in N+CLP/N+Sham,11 proteins spots are up-regulated and 4 proteins spots are down-regulated in KO+CLP/KO+Sham,there are 25 spots associated with RAGE.We finally have identified 61 proteins by use of mass spectrometry,of which there are 25 proteins associated with RAGE.Then we carried out the subcellular localization analysis to the 25 differential proteins using with software WOLF PSORT,The results indicated that 15 proteins predicted localized in the cytoplasm;13 proteins predicted localized in nucleus;8 proteins existed in the cytoplasm and nucleus shuttle;9 proteins predicted localized in mitochondria;6 proteins predicted localized in extracellular;3 protein cytoplasm and mitochondrial shuttle;1 protein predicted localized in cytoplasm or membrane;4 localized in skelemin and 4 localized in endoplasmic reticulum.It can be found that many differentially expressed proteins localized in the cytoplasm and nucleus,which may be associated with the regulation of function event from lung tissue with septic shock.Mass spectrometry indicated that three different protein spots were the same protein Krt7,with the same trend and obvious differential expression,which indicated that the protein may have post-translational modification.After reading literature,we finally selected it to be further verification.Therefore,we used the Western blot to verify expression of Krt7 in changes and then verify DIGE results.The results showed that the changes of Krt7 protein is consistent with DIGE result.Real-time quantitative PCR(Q-PCR)was used to verify the changes of protein expression on the mRNA.The results showed that there was significantly down-regulated in N-CLP compared with N-Sham group,while almost no difference was observed between KO-CLP and KO-Sham group.The results are the same as DIGE.Then we detected the location and expression of exogenous Krt7 by immunofluorescence in NIH 3T3 cells,After constructing the eukaryotic expression vector of pcDNA3-Krt7-HA and then transfected NIH 3T3 cells,we found that it was mainly localized at the cytoplasm.The vector was constructed correctly.Through the research,four conclusions are drawn as follows:1.Successfully established cecal ligation and puncture model of sepsis.2.Established differential proteome map from lung tissue with septic shock between normal and RAGE gene knockout C57/BL6 mice.Differences software analysis showed that there were 72 differential protein spots.61 differential proteins were identified by mass spectrometry,of which 25 proteins were controlled by RAGE.3.The expression of the target protein Krt7 at the protein and gene level were consistent with DIGE results with using Western blot and Q-PCR.4.The Krt7-HA fusion protein has been successfully constructed and effectively expressed in NIH 3T3 cells with a correct localization,and it can serve as a useful tool for the further study of these biological functions of Krt7 in septic shock.In this thesis,from the "omics" and living tissue level,based on the 2D-DIGE protein separation and biological mass spectrometry identification technology,we have obtained differential proteome map associated with RAGE from lung tissue with septic shock and verified the target differential protein which are associated with and mediated by RAGE.It can not only provide new experimental data for in-depth study of RAGE and its ligand-mediated signaling pathways in sepsis and septic shock,but also supply for support in the pathogenesis of sepsis and its prevention strategies.