The Regulation Effects Of Co-Releasing Molecules (CORM) 2 On Coagulation System In Sepsis And Molecular Mechanisms

Sepsis is a severe inflammatory response that results from a variety of microorganisms or its toxin which reside in blood or tissue. It will be to become complications of lots of severe clinical insults, such as bacterial infections, trauma, shock, pancreatitis, or major surgical interventions. If they cause a systemic hyper-inflammatory immune response, this exaggerated host inflammatory response may not lead to an efficient elimination of the infectious agent; rather, it may contribute to a state of immune deactivation. A severe inflammatory response has been implicated in the pathogenesis of systemic inflammatory response syndrome (SIRS), sepsis or multiple organ dysfunction syndrome (MODS). Sepsis will trigger the development of coagulation system, meanwhile, fibronolysis system will be inhibited in some degree. So, blood is in state of hyper-coagulable, and capillary vessels emerge well-bound microthrombus. After capillary vessels failure, that is septic shock. In sepsis, endothelial cells will be activated, then, the level of expression of tissue factor (TF) also will be increased. It's well known that TF is the primary initiator of the exogenous coagulation cascade. Otherwise, platelet will be activated then adhesion and aggregation are to happen to form pathological thrombosis, and this course plays a significant role in the interaction between inflammation and coagulation.It is well known that endogenous carbon monoxide (CO), a bi-product of inducible heme oxygenase (HO-1) can modulates inflammation. In addition, some experiments have determined that the administration of exogenous CO can well inhibit Lipopolysaccharide (LPS)-induced production of cytokines both in vivo and in vitro, and consequently results of our experiments also showed that CO exhibits important cytoprotective function and anti-inflammatory properties that are beneficial for the resolution of acute inflammation. However, the mechanisms of CO-anti-inflammatory activity are not fully understood yet.At present, achievements about anti-coagulation properties in sepsis from CO are not so many. And recently, transitional metal carbonyls have been identified as potential CO-releasing molecules (CORMs) with the potential to facilitate the pharmaceutical use of CO by delivering it to tissues and organs, for example, CORMs have been shown to act pharmacologically in rat aortic and cardiac tissue where liberation of CO produced vasorelaxant effects and decreased myocardial ischemia-reperfusion damage, respectively. Based on these preliminary observations, in this study, we employed tricarbonyldichlororuthenium (Ⅱ) dimer (CORM-2), one of the novel group of CORMs, to determine whether exogenous CO can exert the potential characteristics to inhibit the early hyper-coagulable state on LPS-induced activation of endothelial cells(HUVEC) and assess the effects and potential mechanisms of it on anti-coagulation in the blood of CLP-induced mice.Part I Carbon monoxide releasing molecule (CORM)2-liberated CO attenuates TF expression of LPS-induced activation of HUVECObjective:1. Establish and vertify the HUVEC model by drawing the materials from mature infant umbilical cord; 2. Establish sepsis cells model and make definition the best concentration of LPS; 3. Make definition the best concentration of CORM-2; 4. Detect whether CORM-2 can attenuate LPS-induced activation of HUVEC (TF and NF-κB).Methods:Human umbilical vein endothelial cells(HUVEC) were harvested from the fresh human umbilical vein of newborns by collagenase in 6 hours. HUVEC cells were grown in Medium 199 which was changed 1 days later when the cells were attached and every 2 days, until the cells reached confluence. The cell cultures were expanded by trypsin and passaged by 1:3. HUVEC were identified by morphologic character and membrane antigenⅧfactor by immunofluorescent assay. And after HUVEC were plated, media within the wells was removed.HUVEC were treated with 1μg/ml,10μg/ml,100μg/ml LPS for 4h, and the expression some inflammation relevant factors were assessed, then choosing the optimization concentration. Whereafter, HUVEC was treated with CORM-2 at the concentration of 10μM,50μM,100μM,200μM for 4h. The cell cultures were incubated in room air with 5% C02at 37℃and 95% humidity. Observe the cytotoxicity then select the optimization concentration. Before and after intervention by CORM-2, detect the activity and expression of TF and NF-κB by ELISA and EMSA, respectively.Results:HUVEC model were established successfully in our laboratory. At confluence (6-8 days), the cultured cells had a paving stone appearance with a strict monolayer growth and contact inhibition. HUVEC were positive staining with theⅧfactor for 95%.10μg/ml LPS for sepsis cells model was the optimization concentration. Treatment of HUVEC with different concentration of CORM-2 combined with CLP had demonstrated less oxidation as a concentration-dependent manner. PMN accumulation and activation of NF-κB (EMSA) were prevented markedly compared to the LPS. The expression of ICAM-1,iNOS and HO-1 are decreased compared with CLP-challenged. Then 10μM,50μM,100μM are the effective concentrations for our research.Conclusions:The cultured cells and its sepsis model are successful, and the method of identification is practical and convenient. The results of our experiments show that CORM-2 can not only inhibit the inflammation response, but also decrease the expression and activity of TF and NF-κB. Thus we could conclude exogenous CO is useful for inhibiting the development of coagulation cascade.PartⅡCarbon monoxide releasing molecule(CORM)2-derived CO attenuates TF expression of CLP-induced of miceObjective:To determine the circumstance of coagulation system in sepsis, we established mice model with CLP-challenged. And by detecting the severe inflammation response of liver to make sure that our CLP model was successful. Then investigated the relationship of TF expression between in vivo and in vitro. In addition, assessed the effects and potential mechanisms of exogenous CO on exogenous coagulation system in sepsis. Methods:Mice were underwent CLP-challenged. CORM-2 (8mg/kg; i.v.) was administrated immediately after induction of CLP-challenged injury. Blood sample was obtained by cardiac puncture from the left ventricle. Evaluation of hepatocellular injury was performed by determinations of the enzymatic activity of the alanine aminotransferase (ALT) in serum samples using a commercial kit. To determinate the TNF-αand IL-1 levels, serum TNF-αand IL-1βexpressions were assayed by enzyme-linked immunosorbent assay kits. PMN accumulation (MPO assay) was assessed in mice liver, lung and heart. Expression levels of TF and TFPI in sample were assessed.Results:Expression levels of serum hepatic transaminase,TNF-α,IL-1βand activity of AP-1,NF-κB in liver were markedly increased. This was accompanied by a increase of the expression of TF in the plasma, but the level of TFPI was in contrary. Treatment of CLP-induced mice with CORM-2 can decrease the expression of TF and up-regulate the level of TFPI.Conclusions:The sepsis model of mice was successful. The results of our study showed that exogenous CO could obviously down-regulate the expression of TF and effectually upgrade the level of TFPI. In addition, CORM-2 can also inhibit the activity of NF-κB which can mediate genetic transcription of TF and other many inflammatory factors, thus demonstrating exogenous CO can inhibit the activation of exogenous coagulation system through down-regulating the activity of NF-κB.Part 3 Preconditioning of hyperxia combined with carbon monoxide releasing molecule(CORM)2-derived CO regulate platelets of CLP-induced of miceObjective:1. Detect the effects of intervention about hyperxia combined with CORM-2 towards blood coagulation factors of CLP-induced of mice; 3. Investigate the effects about hyperxia combined with CORM-2 towards platelet membrane glucoproteins and its functions of CLP-induced of mice.Methods:Settling mice be in condition of hyperxia,administrating with CORM-2 and handling with CLP as preparations. Then, we detected the percentage of carbon monoxide hemoglobin (HBCO) in different groups by spectrophotometer and counted the survival rate. And detecting the level of coagulation factors (FIB,D-D) in whole blood; To investigated activation of platelet in sepsis, essaying the expression of platelet glucoproteins (CD41,CD61,CD62p) in platelet-rich plasma by flow cytometry; To determined whether there were obviously influences about hyperxia combined with CORM-2 towards CLP-induced of mice, we detected the rates of platelet adhesion and platelet aggregation by glass-bulb method and platelet-aggregation meter, respectively. Finally, we made the curve of packing fraction.Results:After intervention by hyperxia combined with CORM-2, the expressions of HBCO,FIB and D-D were highly decreased, in contrary, the survival rate of mice were increased. This was accompanied by a downregulation of the functions of platelet adhesion and platelet aggregation in the plasma. Conclusions:In this study, the results of intervention with CORM-2 and hyperxia togather were better than using CORM-2 only. Meanwhile, we could conclude that hyperxia combined with CORM-2 can effectively inhibit the activation of blood coagulation system in sepsis in some degree. And expand new methods for treatment for sepsis.