The Effects And Mechanisms Of Cholinergic Alpha7Receptor Agonist On Vascular Endothelial Cells In The Shock Stage Of Severe Burn-blast Combined Injury

Objective The severe shock of burn-blast combined injury was a major cause of deathin the early stage and an important predisposing factor of complication in the later stage.Vascular endothelial cells (VECs) plays a critical role in the shock. In this study, the nicotinicalpha7acetylcholine receptors (α7NAchR) agonist was used in the resuscitation of shock.The therapeutic effects of resuscitation with the agonist was observed. Then in vitro, theeffects and mechanisms of α7NAchR regulating on the junction proteins of umbilical veinendothelial cells (UVECs) were investigated.MethodsⅠ：1. A severe burn-blast combined injury animal canine model injuried byhexogen explosion and napalm burning, which was proved by gross anatomy andpathological examination of lung tissue.2. Thirty six canines were randomized to six groups:①Sham group.②Burn group.③Blast group.④Burn-blast combined injury group.⑤Burn-blast combined injury without resuscitation group.⑥Burn-blast combined injury withPNU282987treatment group. Hemodynamics parameters, vascular permeability and PVwere measured. The urine output of24hours was collected. The levels of inflammatorymediators in serum were detected. Canines were sacrificed by phlebotomy at24hours post-injury. Then the visceral water content was measured. Microvascular endothelial cellsconnexin in the lung was examined by pathology and immunohistologyMethodsⅡ：1. The VECs were isolated from these umbilical cords digested bycollagenase A with different time. After cultured, the vascular endothelial cells wereidentified by morphology, immunofluorescence and flow cytometry.2. The UVECs werestimulated bythe serum of burn-blast combined injury canines, and intervened by nicotine,blockers and agonists of protein kinase C or Rho kinase. The permeability of the monolayerof UVECs was measured and the expression of connexin of UVECs and upstream regulatoryfactor were detected by western blotting and immunofluorescence. ResultⅠ:1. The canine with50cm away from the explosion of hexogen was diagnosedas moderated blast injury by gross anatomy and pathological examination of lung tissue. Asevere burn-blast combined injury animal model was established by the blast injury andsevere burn caused by napalm.2. After burn-blast combined injury, VECs were damaged,which induced the vascular permeability rising, tissue edema, blood volume lowering,circulatory system disequilibrium and organ ischemia and severe hypoxia. The α7NAchRagonist alleviated the vascular permeability and tissue edema, increased the blood volume,enhanced hemodynamics and pulmonary ventilation ventilation function, and improvedorgan ischemia and hypoxia.ResultⅡ:1. Maximal VECs can be achieved with7-minute digestion.2. After culturedby the serum of canines with burn-blast combined injury, the permeability of the monolayerof UVECs was increased and the expression of connexin was decreased significantly.However, the permeability was reduced and the expression of connexin was increased bynicotines or PNU282987or inhibitor of PKC or Rho kinase, which can be broken down byα-BGT or agonist of PKC or Rho kinase.Conclusion The VECs were damaged after burn-blast combined injury, and thenexpression of connexin was reduced, barrier functions of VECs were unbalanced, whichled to the increase of vascular permeability, tissue edema, blood volume plummeted, andanoxaemia. By inhibiting protein kinase C, the α7NAchR agonist can increase theexpressions of VECs’connexins, protect its barrier functions, reduce vascular permeabilityand tissue edema significantly, increase blood volume, repair hemodynamics andpulmonary ventilation ventilatory function, and improve the ischemia and hypoxia.