Regulation of paxillin tyrosine phosphorylation via inhibition of c-Abl kinase to protect ventilator induce lung injury in vivo in rats

Abstract

To determine whether the inhibition of paxillin tyrosine residues 31 and tyrosine residues 118 (Pxn Y31 and Pxn Y118) phosphorylation via inhibition of c-Abl kinase will effectively block its downstream effector molecules vessel endothelium-cadherin (VE-cad), and whether Rho/Rho kinase activation which will induce the vascular barrier dysfunction. Ninety healthy male Sprague-Dawley (SD) rats were randomly divided into nine groups (each n =10). Only tracheotomy was undergone in the sham group. Groups of protective ventilation were set at a volume tidal (VT) of 6 mL/kg, a positive end-expiratory pressure (PEEP) of 5 cmH2O (1 cmH2O = 0.098 kPa) for 1 hour or 2 hours (namely group PVT 1 h and group PVT 2 h), respectively. Groups of high VT were put on mechanical ventilation (MV) at high VT 30 mL/kg, PEEP 0 for 1 hour or 2 hours (namely group HVT 1 h and group HVT 2 h), respectively. Groups UO126 and AG957 pretreatment were set on MV at HVT for 1 hour or 2 hour respectively, but they were given p42/44 mitogen-activated protein kinase (p42/44MAPK) inhibitor UO126 1 mg/kg by intraperitoneal injection or c-Abl kinase inhibitor AG957 10 mL/kg by intragastric injection 1 hour before HVT ventilation. All the animals were sacrificed after experiments and specimens of lung tissues and bronchoalveolar lavage fluid (BALF) were harvested. Pulmonary vascular permeability was measured by Evans blue (EB). The levels of tumor necrosis factor-α (TNF-α) in BALF were measured by enzyme linked immunosorbent assay (ELISA). Then the change of lung tissue pathology was observed with light microscope, diffuse alveolar damage system (DAD) score and lung wet/dry ratio (W/D) were estimated. The myeloperoxidase (MPO) activity was measured by colorimetric analysis, phosphorylations of c-Abl Y245, Pxn Y31, Pxn Y118, VE-cad Y658, p42/44MAPK Y202/Y204, myosin light chain (MLC) and myosin-associated phosphatasetype Y696 (MYPT Y696) were determined by Western Blot. (1)There were no obvious pathological changes in the lung tissue in the sham group and PVT 1 h or 2 h group, and also there were no significant differences in all the parameters between above groups. However, the injury in lung tissue was severe in the HVT groups. In addition, DAD score, lung W/D ratio, EB content, the activity of MPO, and TNF-α in BALF in HVT groups were significantly higher than those in sham group and PVT groups. After pretreatment with AG957 or UO126, all the parameters were significantly decreased as compared with those of groups HVT. (2) The levels of phosphorylation of the proteins in lung tissue in HVT groups were increased as compared with those of group sham and groups PVT, especially at 2 hours of MV. However, compared with groups HVT, the level of p-VE-cad Y658 in lung tissue decreased significantly in group AG957 and group UO126 at 2 hours after HVT. However, the levels of all phosphorylated proteins at 2 hours were significantly lowered in the AG957 group compared with those of the HVT group [p-c-Abl Y245 (gray value): 0.29±0.04 vs. 0.42±0.04, p-Pxn Y31 (gray value): 0.51±0.03 vs. 0.70±0.05, p-Pxn Y118 (gray value): 0.65±0.04 vs. 0.91±0.04, p-VE-cad Y658 (gray value): 0.77±0.07 vs. 1.32±0.07, p-p42/44MAPK Y202/Y204 (gray value): 0.38±0.06 vs. 0.61±0.03, p-MLC (gray value): 0.37±0.04 vs. 0.77±0.05, p-MYPT Y696 (gray value): 0.54±0.05 vs. 0.87±0.06, all P < 0.05]. After pretreatment with UO126, the phosphorylation level of VE-cad in lung tissue at 2 hours was significantly lower than that of HVT group (gray value: 0.74±0.04 vs. 1.32±0.07), and the phosphorylation levels of p42/44MAPK and its downstream effector molecules MLC and MYPT Y696 were also significantly decreased [p-p42/44MAPK Y202/Y204 (gray value): 0.38±0.07 vs. 0.61±0.03, p-MLC (gray value): 0.37±0.04 vs. 0.77±0.05, p-MYPT Y696 (gray value): 0.55±0.05 vs. 0.87±0.06, all P < 0.05]. Pxn Y31 and Pxn Y118 phosphorylation could be blocked by inhibition of c-Abl kinase, which could strengthen VE-cad at attachment junction and might block formation of Pxn-guanine nucleotide-exchange factor H1 (GEF-H1)-p44/42MAPK signalosome which induce activation local Rho signaling, lead to activation of MLC phosphorylation, actomyosin contraction, and increase endothelial permeability.