Protective Effects And Mechanisms Of Dexmedetomidine-ulinastatin Combination On Lipopolysaccharide-induced Acute Lung Injury In Rats

BackgroundAcute lung injury(ALI) is an early stage of the acute respiratory distress syndrome(ARDS)and pathology of ALI/ARDS is characterised by diffuse alveolar damage, alveolar capillary leakage and protein rich pulmonary oedema leading to the clinical manifestation of poor lung compliance, severe distress of respiration and hypoxaemia. Several aetiological factors associated with the development of ALI are identified with endotoxemia,trauma,shock and so on. The essence is severe inflammatory responses in lung, which readily cause lung injury. ALI remains a significant health burden with substantial morbidity and mortality over the past decade in spite of endeavouring remedy.Pulmonary edema will impair gas exchange function,increase pulmonary shunt, cause hypoxemia,acid poisoning and internal environmental derangement,eventually induce multiple organ injury if pulmonary edema cannot be eliminated quickly. Pulmonary edema is a so much hallmark event in pathology process of ALI that clearance of lung edema is key point in the treatment of ALI as well as necessary measurement of preventing disease development. Although the mechnisms causing pulmonary edema is complicated, the essence is unbalance between alveoli fluid formation and alveoli fluid clearance. The development of pulmonary edema is characterized with an increase in the permeability of the alveoli capillary membrane and impairing alveolar fluid clearance (AFC). For this reason, to decrease the permeability of the alveoli capillary membrane and increase alveolar fluid clearance is beneficial for gas exchange efficiently, and also is an important stratery in the treatment ofALI.-The pulmonary inflammatory response caused by inflammatory cytokines is a important reason,which induce an increase in the permeability of the alveoli capillary membrane and impairing alveolar fluid clearance. Neutrophil plays a key role in progression of pulmonary edema formation.Active neutrophils leads to alveoli capillary membrane damage by release of cytotoxic and immune cell-activating agents such as proteinases, reactive oxygen species (ROS) and proinflammatory cytokines. The aquaporin (AQP), epithelium Na+channelis (ENaC) and Na+-K+-ATPase distributed in alveolar epithelia and capillary endothelia are responsiable for alveolar fluid clearance. In normal conditions, vectorial Na+gating at the apical membrane and its extrusion by Na+-K+-ATPase at the basolateral side create osmotic pressure that drives water from the alveoli to the interstitium and blood capillary through AQP.Inflammatory cytokines such as TNF-α、IL-1β present during lung inflammation and linked with ALI have been found to reduce ENaC、AQP、 Na+K+ATPase expression and activity in lung epithelial cells and endothelial cells, reducing Na+and pulmonary edema clearance. Inhibition of inflammatory cytokines release and pulmonary inflammatory response could be beneficial to AQP, Na+-K+-ATPase expression and increase alveolar fluid clearance.At present no specific treatment is used to cure ALI. It has been confirmed clinically that the protective mechanical ventilation was beneficial to reduce the mortality of ALI. In recent years, treatment of ALI with β2receptor agonist, statins has showed curative effect, but its common use was limited according to clinical test from Ⅲ stage. Therefore it is important to explore new drugs for treating ALI. Mechanical ventilation is widely used as a supportive therapeutic adjunct for treatment of ALI because of dyspnea, especially those patients also need sedation. Dexmedetomidine, a selective agonist of the a2-adrenergic receptors, has sedative action and repress laryngeal reflex, espscially to suit for those who need mechanical ventilation including ALI. For the past few years, increasing evidences indicated that dexmedetomidine possess potent anti-inflammatory capacity through suppressing TNF-α, IL-1β, IL-6and neutrpphile activity. Ulinastatin, a protease inhibitor purified from human urine, has anti-inflammatory activity which may suppressTNF-α, IL-1β, IL-8, and could inhibit neutrophil mediated endothelial cell injury. However, evidence regarding the anti-inflammatory effects of dexmedetomidine-ulinastatin combination is still lacking. NF-κB is known to be one of the crucial transcription factors required for maximal transcription of a wide array of pro-inflammatory molecules, including TNF-α, IL-1β and other mediators. In endotoxemia, NF-κB released from IκB translocates into nucleus, where it enchances the transcription of cytokines, such as TNF-α, IL-1β and IL-6. It has been suggested that Ulinastatin or dexmedetomidine may act at the transcriptional level through inhibition of NF-κB.However, evidence regarding the effects of dexmedetomidine-ulinastatin combination on NF-κB is still lacking.In one word, the dexmedetomidine-ulinastatin may reduce the permeability of the alveoli capillary membrane and increasing alveolar fluid clearance through suppressing inflammatory cytokines and relieving pulmonary inflammatory response, thus relieve lung edema and ALI.So we hope to duplicate acute lung injury modle by administration of lipopolysaccharide to investigate effects of dexmede-tomidine-ulinastatin combination on acute lung injury in rats. According to the pulmonary inflammatory response caused by inflammatory cytokines is a important factor in lung edema formation, we intisvetigate effects of dexmedetomidine-ulinastatin combination on pulmonary inflammatory cytokines and NF-κB expression in rats with acute lung injury.Part IProtective effects of dexmedetomidine-ulinastatin combination on lipopolysaccharide-induced acute lung injury in ratsObjectiveTo intisvetigate effects of dexmedetomidine-ulinastatin combination on acute lung injury induced by Lipopolysaccharide in rats.MethodsMale Wistar rats were randomly divided into five groups(n=8,per group):Saline control group (NSgroup) received saline (infusion at0.5ml-kg-1·h-1)and saline (2ml-kg-1,ip) immediately after saline(5ml·kg-1,iv). Lipopolysaccharide(LPS) group (L group) received saline (infusion at0.5ml·kg-1·h-1)and saline (2ml·kg-1,ip) immediately after LPS (10mg·kg-1,iv). LPS-dexmedetomidine group(L+D group) received dexmedetomidine (infusion at lμg-kg-1·h-1) and saline (2ml·kg-1,ip) immediately after LPS (10mg·kg-1,,iv). LPS-ulinastatin group(L+U group) received saline (infusion at0.5ml·kg-1·h-1)and ulinastatin(2000·kg-1,ip) immediately after LPS (10mg·kg-1,iv). LPS-dexmedetomidine+ulinastatin group(L+D+U group) received dexmedetomidine (infusion at1μg-kg-1·h-1)and ulinastatin(2000U·kg-1,ip) immediately after LPS (10mg·kg-1,iv). The animals were sacrificed at6h after LPS or NS administration. PaO2, PH and BE were measured, the lungs were removed for evaluation of histological characteristics and determination the lung wet/dry weight ratio(W/D),the concentrations of macrophage inflammatory protein-2(MIP-2)、 malondialdehyde (MDA), the activity of myeloperoxidase (MPO)and superoxide dismutase(SOD), the expression of intercellular adhesion molecule-1(ICAM-1).ResultsCompared with NS group, PaO2, PH, BE was lower in L group,which was increased by dexmedetomidine-ulinastatin combination but not by dexmede-tomidine or ulinastatin alone. Compared with NS group, LPS induced marked lung histological injury, which were less pronounced in dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone.The W/D and the concentrations of MDA, MIP-2of pulmonary tissue increased in the L group compared with NS group, which was reduced by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone.The MPO activity increased and the SOD activity decreased of pulmonary tissue in the L group compared with NS group,which was reversed by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone.The ICAM-1expression of pulmonary tissue increased in the L group compared with NS group, which was reduced by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone.ConclusionsDexmedetomidine-ulinastatin combination has protective effects against lipopo-lysaccharide-induced acute lung injury in rats, which is partially mediated by suppressing ICAM-1, MIP-2expression and an influx of neutrophils into the interstitium and broncheoalveolar space. Part ⅡEffects of dexmedetomidine-ulinastatin combination on membrane permeability of alveolar capillaries in rats with acute lung injury induced by lipopolysaccharideObjectiveTo intisvetigate effects of dexmedetomidine-ulinastatin combination on membrane permeability of alveolar capillaries with acute lung injury induced by Lipopolysaccharide(LPS) in rats.MethodsMale Wistar rats were randomly divided into five groups(n=16, per group): Saline control group (NSgroup) received saline (infusion at0.5ml·kg-1·h-1)and saline (2ml·kg-1,ip) immediately after saline(5ml·kg-1,iv). Lipopolysaccharide(LPS) group (L group) received saline (infusion at0.5ml·kg-1·h-1)and saline (2ml·kg-1,ip) immediately after LPS (10mg·kg-1,iv). LPS-dexmedetomidine group(L+D group) received dexmedetomidine (infusion at1μg·kg·h-1)and saline(2ml·kg-1,ip) immediately after LPS (10mg·kg-1,iv). LPS-ulinastatin group(L+U group) received saline (infusion at0.5ml·kg-1·h-1)-and ulinastatin(2000U·kg-1,ip) immediately after LPS (10mg·kg-1,ⅳ). LPS-dexmedetomidine+ulinastatin group(L+D+U group) received dexmedetomidine (infusion at1μg·kg-1·h-1)and ulinastatin(2000U·kg-1,ip) immediately after LPS (10mg·kg-1,iv). The animals were sacrificed at6h after LPS or NS administration. Eight animals in each group received Evans blue20mg/k g30minutes before sacrifies, lung tissues were obtained for determination of Evans blue concentrations. Lungs were removed from the rest of eight animals in each group before sacrifies, protein concentrations, total cell counts and neutrophil counts in bronchoalveolar lavage fluid(BALF)were obtained, and the expression of iNOS and NO concentration in lung tissues was determined.ResultsThe protein concentrations, total cell counts and neutrophil counts in BALF in L group were higher than those in NS group, while these parameters in dexmede-tomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone group were significantly lower than those in L group.The concentrations of evans blue, NO and the expression of iNOS in L group were higher than those in NS group, while these parameters in dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone group were significantly lower than those in L group.ConclusionsDexmedetomidine-ulinastatin combination attenuated alveoli-capillary permea-bility in rats with acute lung injury induced by lipopolysaccharide, which is partially mediated by suppressing iNOS/NO expression. PartⅢEffects of dexmedetomidine-ulinastatin combination on alveolar fluid clearance in rats with acute lung injury induced by lipopolysaccharideObjectiveTo intisvetigate effects of dexmedetomidine-ulinastatin combination on alveolar fluid clearance in rats with acute lung injury induced by Lipopolysaccharide(LPS). MethodsMale Wistar rats were randomly divided into five groups(n=8, per group): Saline control group (NSgroup) received saline (infusion at0.5ml·kg-1·h-1)and saline^ml-kg’^ip) immediately after saline(5ml·kg-1,iv). Lipopolysaccharide(LPS) group (L group) received saline (infusion at0.5ml·kg-1·h-1)and saline (2ml·kg-1,ip) immediately after LPS (10ml·kg-1iv). LPS-dexmedetomidine group(L+D group) received dexmedetomidine (infusion at1μg·kg-1·-h-1)and saline (2ml·kg-1,ip) immediately after LPS (10mg·kg-1,iv). LPS-ulinastatin group(L+U group) received saline (infusion at0.5ml-kg-1-h-1)and ulinastatin (1000U·kg-1,ip) immediately after LPS (10mg·kg-1,iv). LPS-dexmedetomidine+ulinastatin group(L+D+U group) received dexmedetomidine (infusion at lμg·kg-1·h-1)and ulinastatin(2000U·kg-1,ip) immediately after LPS (10mg·kg-1,iv). The animals were sacrificed at6h after LPS or NS administration and the lungs were removed for evaluation the expression of aquaporin-1and aquaporin-5and the activity of Na+-K+-ATPase in lung tissue.ResultsThe expression of aquaporin-1and aquaporin-5and the activity of Na+-K+-ATPase in ALI group were lower than those in NS group,the expression of aquaporin-1and aquaporin-5and the activity of Na+-K+-ATPase in dexmed-etomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone group were higher than those in ALI group.ConclusionDexmedetomidine-ulinastatin combination increase alveolar fluid clearance in rats with acute lung injury induced by lipopolysaccharide,which findings was caused in part by up-regulation the expression of AQP-land AQP-5and the activity of Na+-K+-ATPase. Part ⅣEffects of dexmedetomidine-ulinastatin combination on inflammatory cytokines in lung tissues of lipopolysaccharide-induced acute lung injury in ratsObjectiveTo intisvetigate effects of dexmedetomidine-ulinastatin combination on pul-monary inflammatory cytokines in rats with acute lung injury induced by Lipopolysaccharide(LPS).MethodsMale Wistar rats were randomly divided into five groups(n=8, per group):Saline control group (NSgroup) received saline (infusion at0.5ml·kg-1·h-1)and saline (2ml·kg-1,ip) immediately after saline(5ml·kg-1,iv). Lipopolysaccharide(LPS) group (L group) received saline (infusion at0.5ml·kg-1·h-1)and saline (2ml·kg-1,ip) immediately after LPS (10ml·kg-1,iv). LPS-dexmedetomidine group(L+D group) received dexmedetomidine (infusion at1μg·kg-1·h-1)and saline (2ml·kg-1,ip) immediately after LPS (10mg·kg-1,iv). LPS-ulinastatin group(L+U group) received saline (infusion at0.5ml·kg-1·h-1) and ulinastatin(2000U·kg-1,ip) immediately after LPS (10mg·kg,iv). LPS-dexmedetomidine+ulinastatin group(L+D+U group) received dexmedetomidine (infusion at1μl·kg-1·h-1) and ulinastatin(2000U·kg-1,ip) immediately after LPS (10ml·kg-1,iv). The animals were sacrificed at6h after LPS or NS administration and the lungs were removed for evaluation the concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6(IL-6), interleukin-10(IL-10) and the expression of nuclear factor-kappa B (NF-κB)in lung tissues. ResultsCompared with NS group, concentrations of TNF-α、IL-1β、IL-6、IL-10of lung tissue was increased in L group. The concentrations of TNF-α、IL-1β、IL-6of lung tissue was decreased and the concentrations of IL-10was increased further in dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone group. The nuclear localization of p65increased markedly in the L group and this enhancement of nuclear p65expression was much less in the dexmede-tomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone group.ConclusionDexmedetomidine-ulinastatin combination suppresses TNF-α, IL-1β, IL-6overproduction and increase IL-10production in rats with acute lung injury induced by lipopolysaccharide, which is partially mediated by inhibition of NF-κB.