The Inhibition Effects Of Preconditioning Of CO-releasing Molecules (CORM)2 On Early Inflammatory Responses In Sepsis And Molecular Mechanisms

The severe inflammatory response constitutes the primary host response to a variety of severe clinical insults, such as bacterial infections, trauma, burns, pancreatitis, or major surgical interventions. If they cause a systemic hyperinflammatory 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. It is characteristic of hypernomic inflammatory trauma because immunity system is out of control. Polymorphonuclear neutrophils(PMN) is the main effector cell in the development of inflammation. It's sequestration and the subsequent generation of oxidants and inflammatory mediators play the key roles in the pathogenesis of severe inflammatory response, for which influence occurrence, development and turnover.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 inhibits Lipopolysaccharide (LPS)-induced production of cytokines both in vivo and in vitro, and consequently 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.Recently, transitional metal carbonyls have been identified as potential CO-releasing molecules (CO-RMs) with the potential to facilitate the pharmaceutical use of CO by delivering it to tissues and organs. CO-RMs 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 the preconditioning of endothelial cells (HUVEC) with it can exert the potential characteristics to modulate the inflammatory response on LPS-induced activation of endothelial cells(HUVEC) and assesse the effects and potential mechanisms of it on inflammatory responses in the liver of CLP-induced mice.Part 1 The establishment and verification of HUVEC modelObjective: Vascular endothelial cells(VEC) are unique multifunctional cells with critical basal and inducible metabolic and synthetic and immune functions. They from the lining of the circulatory system, constitute the interface between the blood and the tissue.Mature infant umbilical cord is homogeneous cell ,easy and facility to be obtained. We use HUVEC as a unique in vitro cell mode to investigate the mechanism of inflammatory response,. We must determine the special conditions of the cell culture in our laboratory because some distinctions are obvious in different laboratory. In this study, we must determine the ideal model of HUVEC.Methods: Human umbilical vein endothelial cells(HUVEC) were harvested from the fresh human umbilical vein of newboms by collagenase in 6 hours. HUVEC cells were grown in Medium 199 supplemented with 20% heat-inactivated fetal calf serum(FCS), 10 IU/ml heparin sodium, anbitiotics(100 IU/ml penicillin and 100μg/ml streptomycin), 1.5μg/ml fungizone and 80μg/ml endothelial mitogen.(Complete M199). The medium was changed 1 days later when the cells were attached and every 2 days, until the cells reached confluence.The cell cultures were incubated in room air with 5% CO₂ at 37℃and 95% humidity and expanded by brief typsinization with 0.25% trypsin in PBS containing 0.025% EDTA.HUVEC were identified by morphologic character and membrane antigen VIII factor by immunofluorescent assay.Results: HUVEC model were established successfully in our laboratory. At confuluence(6-8 days),the cultured cells had a cobbestone appearance with a strict monolayer growth and contact inhibition. HUVEC were positive staining with the Vm factor.Conclusions: The cultured cells are endothelial cells, and the method of identification is practical and convenient.Part 2 Preconditioning of endothelial cells(HUVEC) with carbon monoxide(CO) liberated by CO-releasing molecules(CORM)2 attenuates LPS-induced activation of HUVECObjective: To determine the mechanism of inflammatory response, we established HUVEC model with LPS stimulation. and the inhibitory effects of exogenous CO on LPS-induced activation of HUVEC. carbon monoxide (CO), a bi-product of inducible heme oxygenase (HO-1) can exhibits important cytoprotective function and anti-inflammatory properties. However, the mechanisms of CO-anti-inflammatory activity are not fully understood yet. To access the effects and potential mechanisms of CORM-liberated CO on LPS-induced activation of endothelial cells.In this study,we investigated whether exogenous CO released acts protectively against inflammatory response in vitro, and studied the mechanisms involved in it.Methods: Establishment of sepisis cells model: after HUVEC were plated, media within the wells was removed.HUVEC were treated with 10μg/ ml (?) LPS for 4h. The cell cultures were incubated in room air with 5% CO₂ at 37℃and 95% humidity.HUVEC was pretreated with CORM-2 at the concentration of 50,100μg/ ml for 2h, washed and stimulated with 10μg/ml LPS for 4h. PMN accumulation (MPO assay) was assessed in HUVEC. Activation of HUVEC was assessed by measuring intracellular oxidation of DHR123 and nitration of DAF-FM, specific ROS and NO fluorochromes,respectively. In addition,the expression of ICAM-1,HO-1 and iNOS protein (Western blotting) and activation of inflammation-relevant transcription factor, NF-κB (EMSA) were also assessed.Results: Preconditioning of HUVEC with different concerntration of CORM-2 induced less DHR oxidation compared to those LPS-stimulated HUVEC, thus demonstrating less oxidation as a concentration-dependent manner, PMN accumulation and activation of NF-κB (EMSA) were prevented markedly compared to the LPS. This was accompanied by a decrease of the expression of ICAM-1 and iNOS while an increase of the expression of HO-1. In parallel, PMN adhesion to HUVEC stimulated by LPS in the presense of preconditioning of CORM-2 was markedly decreased compared to those LPS- stimulated HUVECs .Conclusions: CORM-released CO modulates inflammation in LPS-stimulated HUVEC by interfering with NF-κB activation, protein expression of ICAM-1, iNOS and HO-1 and therefore suppressing endothelial cells pro-adhesive phenotype.Part 3 Carbon monoxide releasing molecule(CORM)2-liberated CO attenuates inflammatory response of CLP-induced of miceObjective: To determine the mechanism of inflammatory response in severe trauma, we established mice model with CLP-challenged. In this study, we investigated whether exogenous CO released acts protectively against inflammatory response in vitro, and assessed the effects and potential mechanisms of it on inflammatory responses in the liver of CLP-induced mice.Methods: Mice were underwent CLP- challenged. CORM-2 (8mg/kg; i.v.) was administrated immediately after induction of CLP- challenged injury. Blood samples obtained by cardiac puncture of 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 For determination of TNF-αand IL-1 levels, serum TNF-αand IL-1βconcentrations were assayed by enzyme-linked immunosorbent assay kits. PMN accumulation (MPO assay) was assessed in mice liver, lung and heart. Activation of NF-κB, an inflammation-relevant transcription factor, expression levels of ICAM-1 in liver were assessed. Results: Treatment of CLP-induced mice with CORM-2 decreased serum hepatic transaminases , attenuated PMN accumulation and prevented activation of NF-κB (EMSA) in the liver compared to the CLP-induced. This was accompanied by a decrease of the expression of ICAM-1 in the liver tissue.Conclusions: These findings indicate that CORM-released CO modulates inflammation by interfering with NF-κB activation, protein expression of ICAM-1 and therefore suppressing endothelial cell pro-adhesive phenotype. Of course, further studies are now required to understand the detail mechanisms of anti-inflammatory effects mediated by CORMs, and to contribute to the development of a therapeutic approach to protect the tissue or organs during severe inflammation.