| Sepsis is a syndrome of systemic inflammatorome induced by infection, of which the severe complication, sepsis shock, is the major cause of death of the patients suffered from wound, burn, and post operation. The mortality is still up to 35-60% despite the antibiotics are widely used and the intensive care is provided. Statistic indicates that half of the sepses were caused by Gram-negative bacteria, and that lipopolysaccharide (LPS), released from the cytoderm of Gram-negative bacteria, was considered to be the major molecule responsible for the endotoxin sepsis. LPS released after the bacteria is broken then act on multiple organ and tissue, among which endothelium, macrophage and neutrophil, are main targets of its, producing a large number of cytokine, which leads to inflammatory cascade reaction out of control, such as hemorrhage, leukocytes infiltration, vasodilatation, plasma protein extravasation and edema, leading to endotoxin shock, tissue damage and multiple organ dysfunction syndrome at last. Therefore it's an important subject to control the inflammation induced by LPS.Propofol also knows as diprivan,2,6-Diisopropylphenol, is an inert phenolic derivative. Propofol is widely used in clinic for its fast and short effect. Propofol now is used to anesthesia and sedation when patients are in the condition of sepsis, severe trauma and in ICU with monitoring. In recent years, many researches indicate that propofol can inhibit the inflammation induced by LPS in the processes of injury, operation and so on. This effect greatly attracted attention, but its mechanism is not clear. There are severel mechanisms probably involved in this process:a. Propofol can inhibit chemotaxis and adhesion aggregation of neutrophils; b, Propofol can inhibit the release of inflammatory factors and the respiratory burst; c. Propofol can regulate apoptosis of neutrophils.Objective In the process of Sepsis, it can produce a large number of inflammatory mediators which include cytokines, chemokines, complement, etc., these inflammatory mediators can activate endothelial cells to promote the expression of adhesion factors, cause adhesion of platelet and exudation of leukocyte. Though resent researches show that Propofol can inhibit inflammatory response, most observation are merely keen on some clinical indicators, they have not been able to reach into the molecular level. Even some study can reach into the molecular level, they only use some indicators of cytokines, through different interventional ways to detect what changes of cytokines. Therefore, we study the effects of propofol on the serum proteomic profiles in endotoxemic rats with gel-based comparative proteomic approach.Two-dimensional protein electrophoresis is the electrophoresis technolegy of highest resolution and sensitivity. Some new technolegy such as narrow pH gradient gel strip, two-dimensional difference gel electrophoresis were introduced into 2-DE makes it more important in the reasch of proteomics.In order to find those proteins which related with life processes or pathological processes, we use Functional proteomics to find differentially expressed proteins which appear in different life processes or different disease states. Through bio-informatics (bioinformatics) principle, we can divide differentially expressed proteins into a number of feature subsets according to structural domain or motif, and then conduct a deep study to pairs combining the traditional research strategy. If we can get enough information of differential proteins, we can provide an opportunity for finding those proteins which interact in cells. Such research ideas not only increase efficiency of the study, but also provide a framework for our study, so we can through analyze the network of protein function, to better understand the life course or biological phenomena.Method Under the above research mentality instruction, we have established 2-DE MS union Proteomics technology, and use PMF to identify protein, through westernbolt and ELISA to test proteins which we get. To determine the change of serum protein profile in response to LPS and propofol. Experiment equally divided into three groups:In the control group, rats received a continuous infusion of 10% Intralipid(R) (sino-swed pharmaceuticalcorp.ltd, China) (20 mg/kg/h) via the femoral vein to control for EDTA present in the lipid diluents of propofol. In the LPS group, rats were infused with Intralipid(R) immediately following 3 mg/kg LPS i.v., and in the LPS+propofol group rats were injected with LPS followed by propofol at 20 mg/kg/h. After 6 h,3-4 ml of blood from the carotid artery of each rat was collected into Eppendorf tubes and the rats were sacrificed thereafter. only which up-regulated or down-regulated in LPS-stimulated group and show a contrary trend in LPS+ propofol group will be take to identified. The volume of information will be greatly reduced and more accurate to reveal the change of the proteins function. Combining bioinformatics analysis, to determine the change of serum protein profile in response to LPS and propofol.Result Twelve protein spots were found to be significantly altered. All the protein spots of interest were successfully identified by MALDI-TOF-MS and by subsequent comparative sequence search in the Mascot database. Among them, platelet factor 4 (PF4) precursors and major urinary protein precursor were substantially up-regulated while hemoglobin subunit alpha-1/2, hemoglobin subunit beta-1, and peroxiredoxin-2 were modestly altered in the LPS group as compared with the control group. In contrast, propofol significantly down-regulated the expression of platelet factor 4 precursor and major urinary protein precursor induced by LPS. In addition, haptoglobin precursor, urinary protein 1 precursor, apolipoprotein C-â…¢precursor, complement C3 precursor, Ig lambda-2 chain C region were down-regulated whilst parvalbumin alpha was up-regulated in LPS +propofol group as compared with the control and the LPS groups. Neutrophil gelatinase-associated lipocalin precursor had the highest expression in the LPS+ propofol group and the lowest in the control group.Conclusion Activation of coagulation and subsequent fibrin deposition are essential parts of the host defense against infectious agents in an attempt to contain the invading microorganisms and the subsequent inflammatory response. An exaggerated response can lead to a situation in which coagulation itself contributes to disease in its most severe form causing microvascular thrombosis and organ dysfunction, a syndrome known as disseminated intravascular coagulation (DIC). It becomes increasingly clear that components of the coagulation system are able to markedly modulate the inflammatory response. PF4 is an important mediator in coagulation, which is released from alpha-granules of activated platelets during platelet aggregation, and promotes blood coagulation by moderating the effects of heparin-like molecules and plays roles in inflammation and wound repair. It is also a strong chemoattractant for neutrophils, fibroblasts and monocytes. Normally blood contains a very little PF4 and only in pathological states, such as acute tissue injury and sepsis would induce large amounts of PF4 be released from activated platelets. |
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