| ObjectiveTo explore the effects of colquhounia root adjusting expression of Claudin and ZO-1 in lung tight junction protein on oleic acid induced acute lung injury(ALI)of rats and its possible mechanisms,especially the mechanisms by means of observing the expression and changes of Claudin-2、Claudin-5 and ZO-1 in lung tissues of the models of ALI and it’s a significant impact brought by colquhounia root.It will provide a theoretical evidence to treat ALI with colquhounia root tablets in clinic practice.Methods1.A total of 70 male Sprague-Dawley(SD)rats were randomly divided into five groups: control group(group C,n=10),ALI group(group A,n=15)and treatment groups(colquhounia root with different dosages,groups H)which included colquhounia root 500mg·kg-1·d-1(group H1,n=15),colquhounia root 600mg·kg-1·d-1(group H2,n=15),colquhounia root 700mg·kg-1·d-1(group H3,n=15).Drug intervention for 10 days.2.In group A,sodium chloride 2mL were instilled into the stomach of each rat for 10 consecutive days until oleic acid(0.04 mL·kg-1)was injected into caudal vein to establish ALI models;in groups H,colquhounia root with 500mg·kg-1·d-1(group H1),600mg·kg-1·d-1(group H2)and 700mg·kg-1·d-1(group H3)were instilled respectively into the stomach of each rat for 10 consecutive days before intravenous injection of same dose oleic acid with ALI group;in group C,sodium chloride 2mL were instilled into the stomach of each rat for 10 consecutive days before intravenous injection of sodium chloride(0.04 mL·kg-1).3.After 6h of establishing model,all animals were anesthetized.Collect bronchoalveolar lavage fluid(BALF)and blood specimen from abdominal aorta to test lung permeability index(LPI)by BCA;collect lung tissues of rats to test lung wet-to-dry weight ratio(W/D).4.Lung tissues was fixed-embedded to do hematoxylin-eosin(HE)staining to test lung injury score(LIS),and morphologic lung changes were also observed.The pathological changes in ultrastructure of lung tissues in the five groups were observed with electron microscopes.5.The expression and localization of Claudin-2、Claudin-5 and ZO-1 in lung tissues were observed by immunohistochemistry methods.6.The expressions of Claudin-2、Claudin-5 and ZO-1 in lung tissues were tested by Western Blot..Results1.The change of pathomorphism features in lung tissue of rats1.1 General observation of samples: In group C,the lung tissue did not change significantly.In group A,the lung tissue was swelled,and there were widespread bleeding points on the surface.In groups H,lung tissue was swelled slightly,especially in group H2.1.2 Light microscope(HE): The structure of alveolar epithelium was integrity,and there was no exudation in alveolar space in group C.In group A,the alveolar wall was not complete,there was alveolar collapse,interalveolar septum thickening,interstitial lung edema,hemorrhage diffuse,and infiltration of a lot of inflammatory cells and leakage of erythrocyte into the alveolar lumen while plasma protein exudation increased.The histopathology changes of the lung tissue was significantly reduced in each group H,especially in group H2.Compared with group C,the LIS increased significantly in group A(P<0.01);compared with group A,the LIS of group H was significantly decreased(P<0.05),especially in group H2(P<0.01).1.3 Electron microscopy: In group C,the pulmonary microvascular basement membrane,endothelial cells and alveolar the type II epithelial cells were normal.In group A,there are bleeding in alveolar cavity,capillary endothelial cell damage,basement membrane destruction,partial emptying of lamellar bodies in alveolar the type II epithelial cells,the structural integrity of intercellular tight junction was destruction.In group H,the lung injury was alleviated signally,and the pulmonary microvascular basement membrane was relatively complete and intercellular tight junction was relative integrity,especially in group H2.2.The change of wet-to-dry weight ratio(W/D)and lung index(LPI)Compared with group C,the W/D ratio and LPI of group A were significantly increased(P<0.01).Compared with group A,the ratio of W/D and LPI were decreased significantly in each groups H(P<0.05),especially in group H2(P<0.01).3.The expression and changes of Claudin-2、Claudin-5 and ZO-1 in lung tissue of rats3.1 In the rats of acute lung injury was induced by oleic acid,the expressions of Claudin-2 and ZO-1 were decreased in rats’ bronchial mucosa epithelial cells(P<0.01),but increased in three groups with colquhounia root intervened before oleic acid injected(P<0.01).And Claudin-2 and ZO-1 expression levels were higher in group H2 than in groups H1 and H3(P<0.05).3.2 In the rats of acute lung injury was induced by oleic acid,the expression of Claudin-5 was increased,while ZO-1 was decreased in alveolar epithelial cells(P<0.01).However,in three groups with colquhounia root intervened before oleic acid injected,the expression of Claudin-5 was decreased,while ZO-1 was increased(P<0.01).And the changes of Claudin-5 and ZO-1 in group H2 were better than in groups H1 and H3(P<0.05).3.3 In the rats of acute lung injury induced by oleic acid,the expressions of Claudin-5 and ZO-1 were decreased in pulmonary vascular endothelial cells(P<0.01),but increased in three groups with colquhounia root intervened before oleic acid injected(P<0.01).And for Claudin-5 and ZO-1 expression levels were higher in group H2 than in groups H1 and H3(P<0.05).Conclusions1.The experiment confirmed that in ALI,the protein expression of Claudin-2 and ZO-1 in rats’ bronchial mucosa epithelial cells was down-regulated,the expression of Claudin-5 and ZO-1 in pulmonary vascular endothelial cells was down-regulated,and the expression of Claudin-5 in alveolar epithelial cells was up-regulated.2.Colquhounia Root could up-regulate the expression of the tight junction protein Claudin-2 in rats’ bronchial mucosa epithelial cells,Claudin-5 and ZO-1 in pulmonary vascular endothelial cells,and down-regulate Claudin-5 in alveolar epithelial cells in order to decrease alveolar epithelial and pulmonary vascular permeability and recover alveolar epithelial and vascular endothelial barrier function.3.Colquhounia Root could play a role in pulmonary protection of acute lung injury rats and the optimum bolus dose is 600 mg(kg·d)-1. |
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