Inhibition Effects Of Carbon Monoxide Releasing Molecules-2 Combined Oxygen Preconditioning On Inflammatory Responses Of Lung In Sepsis And Molecular Mechanisms

Sepsis is a kind of systemic infection,it shows uncontrollable inflammation. Inflammation is a clinical pathologic process resulted from infection or uninfection factors, such as bacterial infections, trauma, burns, major surgery, etc. It stimulates the host immune system, releases inflammation cytokines, then leads to illness status.As a representative factors in cells, cytokines were released freely and lead to unduly inflammation, they failed to remove the pathogenic factors, continued to suppress the immune system.Finally,they caused systemic inflammatory response syndrome (SIRS), sepsis and multiple organ dysfunction, joint pathophysiological characteristics is a runaway of immune system, PMN activation, overproduction of systemic inflammation cytokines,then caused multiple organ damage.It is well known that endogenous carbon monoxide (CO), a bi-product of inducible heme oxygenase (HO-1), was regarded as a harmful substance damaging cellular respiration. In addition, some experiments have determined that administration of exogenous CO inhibits Lipopolysaccharide (LPS)-induced production of cytokines both in vivo and in vitro.Consequently, exogenous CO exhibits important cytoprotective function and anti-inflammatory properties that are beneficial for the resolution of acute inflammation. CO 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. However, the mechanisms of CO-anti-inflammatory activity are not fully understood yet.Based on these preliminary observations, in recent study, we employed tricarbonyldichlororuthenium (Ⅱ) dimer (CORM-2), one of the novel group of CORMs, to determine whether the preconditioning of oxygen combined CORM-2 can exert the potential characteristics to modulate the inflammatory response and molecular mechanisms in lungs of CLP-induced mice in vivo. Objective:To explicit the protection effects of high oxygen preconditioning in sepsis animal models and the usage of CORM-2, we establish the cecal ligation and puncture model, which is similar to sepsis or septic shock caused by human peritonitis.Methods:1) Select Kunming mice, clean level,6-8weeks, male, weight 20+2g, all mice were offered by animal experiment center of Jiangsu University. To adaptive breed in this lab for a week, no diet 12 hours before operation. Put the mice of oxygen group in the oxygen airtight container to precondition for 30 minutes.2) Anesthesia mice by isoflurane inhaling, make a 1cm incision on medioventral line, go into the abdominal step by step, identify the cecum end and separate it softly, ligate the cecum proxima with thin lines, puncture the cecum distal away from blood vessels by 20 needles, crowd some contents, then return the bowel to the abdominal cavity,suture with 3-0 surgical suture to cover the wound, inject saline 1.0 ml to each mouse intraperitoneally so as to resist shock,breed in single cage, free food, adequate water, incision is coated with sufficient iodine to fight for infection.3) According to specification,10.25mg CORM-2 reagent is dissolved in 500ul DMSO, mixed into 500ul CORM-DMSO solution,8ul CORM-DMSO solution was dissolved in 152ul saline water for tail intravenous injection immediately. CORM-2 was dissolved when used.Results:The establishment of cecal ligation and puncture model was thought to be scientific adaptable. Mice's survival rate was increased after preconditioned by oxygen. The preparation of CORM-2 meets the experimental requirements, and CORM-2 was safe for experiment.Conclusions:Sepsis is the common cause of death in gravely illness, the serious infection or damage leads to systematic inflammation,.Finally,it causes the multiple organ dysfunction syndrome. This sepsis model is thought to be scientific,by way of surgical operation process, some inactive animal tissues exists in abdomine, a little mixed bacterial content overflow into the intestines continuously which causes celiac infection. In this model, each index detected is clear.The model meets the requirements under the damaging circumstance, and can be used in animal experiments for the research of inflammation mechanism.In recent years, the newly synthesis carbon monoxide releasing molecule-2,when dissolved,it releases CO to show some physiological functions.Within certain physiological does,it do not raise the HbCO concentration of animals. Different model in this lab was identified that CORM-2 may be safe and effective for the inflammatory response intervention. Objective:To explore the effects of oxygen preconditioning combined CORM-2 intervention to the percentage of HbCO and the survival rate of sepsis mice, we make sure the safe range of CORM-2, let it provide suitable theoretical guidance for clinical work.Methods:12 mice were randomly divided into three groups:CLP model group, CORM-2 group and oxygen group. We made a plastic box suited emergency oxygen bag as oxygen tank (oxygen was from center oxygen in ward). Mice of oxygen group were put into the tank so as to inhale oxygen 30 minutes. Then we made CLP model, prepared CORM-2 and record the injection time. Respectively, at 1.5h,3h,6 h and 24 h, we recorded the number of living mice, collected blood for 0.5 ml in each time point. Same materials were put into four clean tubes:10mg low sodium sulfite deoxidization,10ml ammonia,50ul anticlotting, after blending,538nm and 578nm spectrophotometer were chosen. We determined the OD within 10 minutes, made records and charts in the end.Results:In this experiment, we found that, compared with the model group, after the intervention of CORM-2, the percentage of HbCO was increased; Compared with the CORM-2 intervention group, the percentage of HbCO of mice in oxygen group declined obviously in 1.5h and 3h(P＜0.05). After 10h to 48h of CLP, the survival rate of CORM-2 group is higher than CLP model group; the survival rate of oxygen pretreatment group is higher than CLP model group and CORM-2 intervention group obviously, it still maintain a higher level later.Conclusions:Under the severe traumatic circumstances, by way of oxygen inhalation,COHb dissociation was accelerated, CO eliminating was promoted, Hb carried oxygen normally, thereby improved blood pressure,increased blood-oxygen content, the tissues get sufficient oxygen, reduced the dependence of Hb transported oxygen greatly, which corrected the possibility of hypoxemia quickly, reduced tissue damage effectively,enhanced the survival rate in the end. The research certified that, under the normal circumstances, the percentage of HbCO of mice stained a low level, after the intervention of CORM-2, the percentage of HbCO increased, but is far less than critical poisoning level. After the pretreatment of oxygen, the percentage of HbCO decreased significantly, it shows that the pretreatment of oxygen could reduce the percentage of HbCO levels,then reduced the tissue damage in the early stage of inflammation. In this research, within the safe range of CORM-2, oxygen pretreatment decreased the percentage of HbCO, quicken the CO eliminating, reduced the negative effects of CO which may exist, enhanced its anti-inflammatory effects and the survival rate in the end. Objective:We adopted CLP models of mice to explicit the effects of oxygen preconditioning combined CORM-2 intervention on lung inflammation after severe traumatic, to explicit the target of CORM-2 in IKK-activation, IκBαdegradation and NF-κB activation signaling pathways so as to clarify the (part) molecular mechanism in inflammatory response.Methods:30 Kunming mice were divided into 5 groups at random:sham group, CLP model group, CORM-2 intervention group, iCORM-2 group and oxygen preconditioning group. Respectively,at 6 h,12h and 24 h after CLP operation,we anesthesia and collect plasma and lung tissue samples of those mice, to detect the tumor necrosis factor-a,the Interleukin-1βand macrophage inflammatory protein-2 with Enzyme-linked immunosorbent assay method, to detect the Myeloperoxidase activity of lung tissues and the neutrophils infiltration, to determine the wet-to-dry weight ratio of lung tissues;The pathology changes of lung tissues were observed by hematoxylin-eosin dyeing, the expression of intercellular adhesion molecule-1 and surfactant protein-A were observed by immunohistochemistry,the level of IKK activation,IκBαdegradation were detected by Western blot and NF-κB activation was detected by EMSA.Results:This research shows that, compared with sham group,the permeability of lung tissues of CLP mice was increasing, alveolar wall thickening, intraalveolar edema and neutrophil infiltration,etc. MPO activity, the expression of ICAM-1 and inflammatory cytokines enhanced obviously;SP-A was decreased,IKK-IκBαdegradation-NF-κB activation signal pathway were activated(P＜0.05).Contrarily, compared with the CLP group, the permeability of lung tissues in CORM-2 group decreased, alveolar wall thickening bettered, intraalveolar edema and neutrophil infiltration alleviated; MPO activity, the expression of ICAM-1 and inflammatory cytokines decreased obviously;SP-A was inhanced,IKK activation-IκBa degradation-NF-κB activation signal pathway were inhibited apparently(P＜0.05).After preconditioned by oxygen,those indexes were inhibited further on the basis of CORM-2 group.However, compared with the CLP group,the iCORM-2 group showed not too much changes.Conclusions:The CLP model of this lab is scientific, simple as well as repeatable. Each index is accurate which meet the demands under the damage conditions. Thus, the model can be used in the studies of inflammatory reaction about animals. There is growing evidence that acute lung injury is the early sign of systemic disease. When faced severe traumatic, it is significant to make correct diagnosis and treatment early on lung injury. This research shows that, oxygen preconditioning combined CORM-2 intervention leads to the corresponding changes of CLP mice in functions, expression of various inflammatory cytokines and adhesion molecules,etc.The IKK activation-iKBoc degradation-NF-KB activation signaling pathway was also inhibited in different degrees, which showed that oxygen preconditioning combined CORM-2 intervention could inhibit the inflammation more apparently.The result provides important theoretical guidance for the treatment to severe patients.Thus,it has important clinical values.