The Role Of Rac1 Protein In Ventilator-induced Lung Injury Of Mechanism

OBJECTIVE:To investigate the role of Rac1 protein in the pathogenesis of ventilator-induced lung injury.METHOD: 30 healthy and clean grade Sprague Dawley rats(male and female)were randomly divided into spontaneous breathing group(group A)and normal tidal volume(VT)group(group B,n=6 ml·kg-1)and the high VT group(group C,n=40 ml·kg-1),10 in each group.Groups of rats by intraperitoneal injection of anesthesia after tracheotomy nasal intubation.Which keep rats spontaneous breathing in group A,group B according to 6 ml·kg-1 tidal volume mechanical ventilation,group C is 40 ml·kg-1 given mechanical ventilation tidal volume,240 min after the heart bleed to death in the rats.Respectively collect groups of rats serum,bronchoalveolar lavage fluid(BALF)and lung tissue samples,using the determination of trace electronic scale lung wet dry weight(W/D).To observe the ultrastructure of alveolar epithelial cells by transmission electron microscope(TEM).To observe the rat lung tissue pathology by the hematoxylin-eosin stain(HE)and optical microscope.Use the method of enzyme-linked immunosorbent(ELISA)to detect interleukin 1 beta(IL-1 beta),interleukin 6(IL-6),tumor necrosis factor alpha(TNF alpha),myeloperoxidase(MPO)and macrophage inflammatory protein-2(MIP-2)content changes in the BALF and serum.To detect Rac1,phosphorylated extracellular signal regulating kinase(p-ERK)and fiber actin(F-actin)expression in lung tissue By immune histochemical.To detect the expression differences of protein Rac1,p-ERK and F-actin of lung tissue in each group of rats by western blot.Use the confocal microscope to observe the expression of Rac1 and F-actin for all lung tissues by immunofluorescence technique(IFT).Application of real-time fluorescent quantitative reverse transcription polymerase chain reaction(qRT-PCR)to detect Rac1,ERK and F-actin gene mRNA expression level.RESULT: Compared with group A and group B,group C W/D ratio in the lung tissue of rats with significantly higher(6.64±0.88 vs.1.79±0.36,4.76±0.73,P < 0.05).Optical microscope observation HE staining showed that A group has no obvious lung tissue pathology change;Group B with mild lung tissue edema and a small amount of inflammatory cell infiltration;Group C lung tissue edema,pulmonary interval broadening,alveolar congestion,accompanied by a large number of inflammatory cells infiltration,alveolar structure disorder。Lung tissue pathology grade C group was obviously higher than that in group A and group B(score: 12.00±2.00 vs.6.00±1.51,1.51±0.53,both P < 0.05).TEM detection results suggest,alveolar epithelial cell ultrastructure of group A has no obvious change;Group B alveolar epithelial cell organelles such as corpuscle layer decreases,the plate membrane fur owe uniformly distributed;Alveolar epithelial cells form in group C,nucleus pycnosis,cytoplasmic corpuscle of medium plate layer and organelles such as mitochondria significantly reduce the number and distribution.ELISA results showed that,compared with group A and group B,group C rats serum and BALF of beta,IL-6,IL-1,TNF-alpha,MPO and MIP-2 were significantly increased[In BALF IL-1beta(ng/L): 121.51±25.62 vs.24.03±7.46,7.46±18.10;IL-6(ng/L): 136.65±32.65 vs.31.35±10.51,10.51 ±6.95,TNF-alpha(ng/L): 97.96±14.75 vs.10.12±2.58,2.58±16.89,MPO(ng/L): 0.8±0.31 vs.0.08±0.04,0.04±0.09,MIP-2(ng/L): 144.41±28.86 vs.41.22±20.65,79.23±8.85.in serum IL-1 beta(ng/L): 104.18±15.10 vs.20.31±8.25,8.25±15.97;IL-6(ng/L): 46.57±11.52 vs.22.65±7.49,7.49±13.27;TNF-alpha(ng/L): 39.41±6.53 vs.5.37±1.87,1.87±4.55;MPO(ng/L): 0.66±0.24 vs.0.06± 0.03,0.03±0.07;MIP-2(ng/L): 109.20±25.78 vs.22.77±8.37,8.37±24.51].Compare the difference between groups was statistically significant(P< 0.05).Immune histochemical method detection,according to A set of rat lung tissue only very small amounts of p-ERK,Rac1 and F-actin positive expression;Group B increased positive expression;In group C p-ERK,Rac1 and F-actin protein positive expression were significantly increased.Also of Western Blot results suggest: the lung tissue of rats in group C p-ERK,Rac1 and F-actin protein expression was obviously higher than that of group A and group B(p-ERK protein ratio of grey value: 1.15±0.36 vs.0.61±0.23,0.88±0.22;Rac1 protein ratio of grey value: 0.91 ±0.16 vs.0.48±0.11,0.11±0.10;F-actin protein ratio of grey value: 0.70±0.09 vs.0.49±0.08,0.55±0.04,Comparison between groups P < 0.05).Confocal microscope IFT show the group C in the lung tissue of Rac1 and F-actin widely distributed in the cell membrane skeleton and cytoplasm,more significant than group A and group B.The qRT-PCR detection of ERK and Rac1 mRNA level,according to the group C than in group A and group B ratio was also higher[ERK mRNA(2-ΔΔCt):8.23±2.83 vs.13.02±1.38,Rac1 mRNA(2-ΔΔCt):4.45±2.26 vs.1,1.22±0.39,both P<0.05]。CONCLUSION: Rac1 protein may through the activation of Rac1 / MAPK/ERK signaling pathway in lung tissue of rats caused by F-actin expression,skeleton reconstruction in alveolar epithelial cells and cell membrane permeability change thus mediated or aggravating VILI.