Mechanisms Of C-Src Tyrosine Kinase In Ventilator-induced Lung Injury

ObjiectiveVentilation induced-lung injury was one of acute lung injuries which caused by mechanical ventilation and primary pulmonary disease used with respirator. Mechanical factors could destroy the complete destruction of alveolar and the permeability of membrane, cause alveolar edema, and cause lung injury. Occludin which is one of tight junction protein is an important protein to maintain alveolar epithelial permeability and the barrier function. Therefore, this study simulated ventilation induced-lung injury by the vitro experiments (cyclic mechanical stretch induced pulmonary alveolar epithelial cells) and vivo experiments (different volume mechanical ventilation) to investigate the expression of tight junction protein Occludin and tyrosine kinase c-Src, the changes of c-Src kinase phosphorylation state and the expression of Occludin protein which MLE-12 was pretreated with c-Src inhibitor, and the role of c-Src kinase in VILI.MethodIn vitro, MEL-12 cells were randomly divided into 5 groups:control group (group C), mechanical stretch group (group S), dimethyl sulfoxide group (group D), tyrosine kinase (c-Src) inhibitor (PP2) group (group P), small interference RNA (siRNA-Occludin) group (group Si). MLE-12 cells were pretreated with the c-Src kinase inhibitor PP2 (100umol/L) and DMSO (30ul/ml) before cyclic stretch 30 min in group P and group D. Group C was not treated with cyclic stretch. MLE-12 cells were treated with frequency 0.5HZ, amplitude 8%,20% of cyclic stretch in group S. MLE-12 cells were treated with cyclic stretch of frequency 0.5HZ, stretch amplitude of 20% in group D and group P. The stretch time in group S was 1 h,2 h,4 h. The stretch time in group D and group P was 4 h. Group Si was treated with siRNA-Occludin. After the pretreatment and cyclic stretch, parts of MLE-12 cells were lysed rapidly on the ice to pick up the total protein to measure the expression of Occludin protein and c-Src kinase by Western Blotting; parts of MLE-12 cells were fixed by 4% paraformaldehyde for 10 min, and observed the distribution of occluding by immunofluorescence.In vivo, thirty Wistar rats, weighing 250-300g, were randomly divided into 5 groups (n=6):the control group (group C), low tidal volume group (group M), high tidal volume group (group H), low tidal volume+c-Src kinase inhibitor group (group M+P), high tidal volume+c-Src kinase inhibitor group (group H+P). The rats in group C did not have mechanical ventilation; breathing parameters in group M, group H, group M+P, and group H+P were that respiratory frequency for 40/min, respiratory ratio (I:E) of 1:2, and the inspired oxygen concentration for 21%, the ventilation time for 4h. The tidal volume was 7mL/kg in group M and group M+P, and 42mL/kg in group H and group H+P. The rats in group M+P and group H+P were pretreated with the c-Src inhibitor PP2 (lug/kg) for 1 h. Lung injury score was recorded after mechanical ventilation. The upper lobe of the right lung was rapidly put into liquid nitrogen freezing to measure the expression of occcludin and c-Src kinase by western blotting; the other part of the right lung was fixed in 4% paraformaldehyde to stain by HE; the left lung was evaluated the pulmonary edema by measuring wet/dry weight ratio (W/D).ResultIn vitro1. Immunofluorescence showed that the expression of occludin was reduced and time-dependent after 20% cyclic stretch. After pretreated with PP2, the expression of occludin was increased.2. Western blotting showed that, compared with Oh, the expression of occludin was not significant changed at 1 h,2 h, and 4 h on 8% cyclic stretch (P>0.05); compared with Oh, the expression of occludin was reduced (P<0.05) and the expression of c-Src was increased (P,0.05) at 1 h,2 h, and 4 h on 20% cyclic stretch. Compared with 4 h in group S, the expression of occludin was not significant changed in group D (P>0.05); Compared with 4 h in group S, the expression of occludin was increased in group P (P<0.05).In vivo1. HE staining showed that high tidal volume mechanical ventilation could cause alveolar congestion seriously, hemorrhage, alveolar wall thickening, and hyaline membrane formation. After pretreated with PP2, alveolar congestion and bleeding could reduce, and alveolar wall could reduce.2. Western blotting showed that, compared with group C and group M, the expression of occludin was reduced (P<0.05), and total and phosphorylated c-Src kinase expression were increased (P<0.05) in group H; compared with group H, the expression of occludin was increased (P<0.05) and total and phosphorylated c-Src kinase expression were reduced (P<0.05).ConclusionHigh tidal volume mechanical ventilation could lead to VILI, and the c-Src kinase could take part in VILI. The phosphorylation of c-Src could cause the activation of c-Src kinase, and the activation of c-Src kinase could lead to the decreasion of occludin, and cause lung injury.