| The antioxidant activity of bronchial epithelial cells (EEC) plays a essential role in maintenance of integrity of the airway epithelium in structure and function, and preventing the epithelial cells from damage. Integrins expressed on EEC are considered as receptors of extracellular matrix such as fibronectin (Fn). The interaction of integrins with their ligands maybe involve in many aspect of cell biology including proliferation, differentiation and metabolism, etc. In order to test the hypothesis that activation of integrin molecules may exert protection effects on bronchial epithelial cells from oxidant injury, the present study, based on a cell-injury model made with ozone (1.5ppm) exposure, investigated the protective effect of fibronectin, a kind of ligand of integrin, and it's specific sequence Arg-Gly-Asp (ROD peptide ) which is a recognizable domain for integrin on EEC. 3H release, LDH and MDA were measured as indexes of cellular damage. The antioxidant ability has been shown as the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and the level of glutathione (GSH) in cells. Detect the NO released from primary cultured rabbit bronchial epithelial cells and the activity of NO synthesase (NOS) in cells .Use RT-PCR to investigate the gene expression of catalase. ResultsI . The protective effect of fibronectin on bronchial epithelial cellsOzone exposure induced a significant increase of H release (PO.01), pretreatment with Fn(10 U g/ml) or RGD(15 u g/ml) markedly attenuated the injury (respectively P<0.05and PO.01), and the effects were reversed by W7 ,a calmodulin inhibitor; Ozone exposure increasedLDH activity (P<0.01), treatment with Fn and RGD decreased the LDHIVactivity and the effect was abolished by W7 (P<0.01); The content of MDA was altered as similar as 3H release and LDH parallel. Treatment with Fn and ROD both decreased the elevation of MDA induced by ozone (PO.05).II. Integrin-fibronetin binding reaction upregulates the antioxidantactivity of rabbit bronchial epithelial cellsDose-dependent increase of GSH level were observed both in Fn (5-20 u g/ml) (i=0.82) and in RGD( 15-60 u g/ml)treatment (r=0.84); Fn binding increased significantly the activity unit of GSH-Px (P<0.05) while was inhibited by W7 (P<0.05).Both Fn and ROD show a dose-dependent upregulatory effect on GSH-Px activity (respectively r=0.93 and r=0.73); Catalase activity could be also stimulated by Fn (P<0.05) and reversed by W7; Treatment with Fn increased SOD activity (P <0.01) and the effect was abolished by W7(P<0.01).III. Fibronectin or RGD peptide promotes nitric oxide synthesis of rabbitbronchial epithelial cellsOzone stress enhanced NO release from BEC(P<0.01), and treatment with Fn also promoted the NO production(P<0.01). The effect of Fn could be blocked by calmodulin inhibitor W-(P<0.01); Either Fn or RGD showed a dose-dependent promotion on NO release (respectively r=0.85 and r=0.89); The NOS activity was significantly elevated in Fn treatment group(P<0.01) and the effect of Fn was abolished by W. 7(P<0.01). A dose-response relation in NOS activity could be observed either in Fn treatment or in RGD treatment(respectively r=0.93 and r=0.90).IV.Upregulation of catalse gene expression in bronchial epithelial cells by fibronetin-integrin reactionFn could promote transcription of the catalase encoding mRNA (P<0.01). However, this effect was abolished either by inhibitor of protein-tyrosine kinase, Genistein or by the inhibitor of calmodulin, W7. Conclusion:The data suggests that the binding of integrin with Fn improve the antioxidant ability of EEC and lighten the damage induced by oxidant attack. The protective mechanism of Fn-integrin binding may be relatedvwith calmodulin. The promotion of catalase activity was due to the upregulation of catalase gene expression via Fn-integrin reaction, in which tyrosine phosphorylation and calmodulin are requisite. |
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