| The permeability of pulmonary microvascular endothelial cells increases markedly after acute lung injury via paracellular gap. Connexin40 is a primary component of pulmonary microvascular endothelial cells gap junction channel and mediates intercellular communication. However, the relationship between Connexin40 and the permeability of pulmonary microvascular endothelial cells is still unknown. Therefore, we determined whether Connexin40 affected rabbits'pulmonary microvascular endothelial cells permeability after acute lung injury induced by gunshot trauma.Part I: rabbit lung tissue permeability and the expression of Cx40 in ALIObjective Establish acute lung injury animal model following gunshot chest trauma and investigate the relationship of Connexin40 inmunohistochemistry and lung tissue permeability.Methods: We used an acute lung injury model in New Zealand rabbits following gunshot chest trauma and correlated Connexin40 inmunohistochemistry in gunshot lung tissue with Evans blue leak rate. Lung tissue water content and serum concentration of TNF alfa and IL 8 were measured.Results: Lung tissue water content and serum concentration of TNF alfa and IL 8 increased significantly in post time compared with pre gunshot (p<0.05). Connexin40 expression time dependently decreased, whereas Evans blue leak rate increased. Connexin40 expression and Evans blue leak rate exhibited a strong inverse correlation (γ=-0.934, p <0.05).PartⅡ: The relationship of Cx40 and rabbit pulmonary microvascular endothelial cells permeability in acute lung injuryObjective: Investigate the relationship of gap junction channel Connexin40 and rabbit pulmonary microvascular endothelial cells permeability in acute lung injuryMethods: Cultured pulmonary microvessel endothelial cells were divided into three groups, control (Gcontrol), injured serum (Gserum), and blocker agent (Gblocker). Gap junction channel function was assessed by scrape-loading and dye transfer techniques. Pulmonary microvessel endothelial cells permeability was measured by Evans blue-labeled albumin transfer, and expression of Cx40 was measured by western blot.Results: Injured serum decreased gap junction channel function and the level of Cx40 and the gap junction channel blocker aggravated this effect. Similarly, pulmonary microvascular endothelial cells permeability increased significantly in Gserum and Gblocker compared with Gcontrol (p <0.05). PartⅢ: The relationship of PMVECs Cx40 and change of intracullar free calcium concentration in acute lung injuryObjective: Investigate the relationship of PMVECs Cx40 and intracullar free calcium concentration in cellsMethods: Cultured pulmonary microvessel endothelial cells were cultured and divided into three groups as described in partⅡ. Intracullar free calcium concentration was measured by fluo-3am, and the degree of cell injury was assessed by LDH concentration in medium.Results: When intervented by injured serum and gap junction blocker in vitro, intracellular free calcium concentration increased (p<0.05), medium LDH concentration in medium increased and the degree of cell injury deteriorated (p<0.05).Conclusions:1. Down-regulation of pulmonary microvascular endothelial cells Connexin40 expression contributes to the increase of lung tissue microvascular permeability after acute lung injury.2. Down-regulation of PMVECs gap junction Cx40 expression and gap junction channel opening contribute to the increase of PMVECs permeability after ALI.3. Down-regulation of PMVECs Cx40 expression and gap junction channel opening contribute to the increase of intracellular calcium concentration, and overload calcium increased PMVECs permeability and made cells injured.
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