| Popis: |
Pulmonary edema (PE) is attributed to ruptured alveolar-epithelial barrier integrity together with impaired alveolar fluid clearance (AFC). Disruption of tight junction (TJ) integrity can occur from loss of TJ protein expression and/or disorganization. TJ determine the barrier properties of cell-cell contact existing between two neighbouring cells and regulate paracellular permeability. The TJ disruption has been related to multiple signal transduction pathways; understanding such mechanisms in hypoxia induced disruption of epithelial and endothelial barrier functions is likely to provide insight into the pathogenesis of various inflammatory diseases, and may form a basis for the design of treatment strategies for different diseases involving barrier dysfunction. The present study was undertaken to determine whether differential expression of tight junction protein(s) is a mechanism for regulation of hypoxia-induced pulmonary epithelial permeability both in-vitro and in-vivo. For in-vitro studies, Human lung adenocarcinoma cell line (A549) was exposed to 3% O2 for different time periods viz. 1 h, 3h, 6h, 12h, 24h and 48 h. For in-vivo studies male Sprague Dawley rats were used as an animal model and exposed them to 25000 ft at 25°C for 6 hrs. Simultaneously, to rule out the fact that inflammation causes impaired tight junction proteins integrity leading to pulmonary edema, A549 cells were pre-treated with 10 µM curcumin and the rats were pre-treated with curcumin (50 mg/kg body weight) 1 h prior to 6-h hypoxia. Hypoxia exposure resulted into increased RBC, WBC, Hb, monocytes, lymphocytes and HCT. Blood gas analysis revealed that PaO2 mmHg, PaCO2 mmHg and SaO2 (%) were significantly (P≤0.001) altered compared to control. Hypoxia induced a progressive time dependent decrease in TJ proteins ZO-1, Claudin-4, and JAM-C proteins expression over control. Further, hypoxic exposure also increased the NFkB, TNF-α, Claudin -5 and Occludin protein expression levels with time as compared to control. However prior treatment with curcumin both in-vitro and in-vivo showed significant changes in tight junction proteins integrity and attenuated NFkB activity with reduced expression of its regulatory genes in lung tissue, serum and BALF. Further we confirmed by pre-treatment with NFkB inhibitor MG132 or siRNA mediated knock down of p65 significantly abrogated (p≤0.001) hypoxia induced NFkB expression followed by significant reduction in (p ≤0.05 ) in dextran FITC efflux in to the lungs. Similarly, HIF-1α reporter gene studies further, confirmed that curcumin stabilized the lung HIF-1α levels under hypoxia. Immunohistochemistry and immunofluorescence observations supports the notion that curcumin restored the brain ZO-1, JAMC, claudin 4 and claudin 5 levels (p ≤ 0.001) under hypoxia. These results indicate that curcumin is a potent drug in amelioration of pulmonary edema as it effectively attenuated inflammation with enhanced alveolar fluid clearance via maintaining the tight junction proteins integrity under hypoxia. |
Full Text from ScienceDirect