Effects Of FTY720 Pretreatment On Vascular Endothelial Cell In Rats With Ventilator-induced Lung Injury

Part I Preparation of Ventilator-induced lung injury model in ratsObjective: To establish the rat model of ventilator-induced lung injury,observe the pathophysiological changes and investigate the mechanisms of lung injury induced by ventilation.Methods:.Thirty healthy adult male SD rats weighing 300-350 g were randomly divided into 3 groups(n=10 each): Group C: tracheotomy was performed without ventilation after anesthesia;Group TV,the rats were ventilated with traditional tidal volume(Vt 8ml/kg,40 breathes/min,PEEP 0 and Fi O2 21%);Group HV,the rats were subjected to injurious ventilation strategy(40ml/kg,40 breathes/min,PEEP=0 and Fi O2=21%).Arterial partial pressure of oxygen(Pa O2)was decected in femoral artery before ventilation or sham operation and 1h,2h,4h after ventilation or sham operation.After 4h of ventilation,the rats were killed by exanguination and the lung samples were obtained to measure MDA level and SOD activity.The levels of tumor necrosis factor-?(TNF-?),interleukine-6(IL-6)and interleukine-1?(IL-1?)were measured in the bronchoalveolar lavage fluid(BALF),and lung wet/dry weight ratio(W/D)and pulmonary permeability index(PPI)were calculated.Additionally,the histopathology changes of lung tissue section were observed under light microscope with HE staining and lung injury score was evaluated.Results:(1)Changes in Pa O2: No significant differences of baseline Pa O2 were found among three groups and among the different timepoints in Group C(P<0.05).As compared to baseline values,Pa O2 values were increased at 1h and 2h after ventilation in Group TV(P<0.05),no significant changes were found at 4h after ventilation(P>0.05);Pa O2 values were enhanced at 1h after ventilation and decreased at 4h after ventilation in Group HV(P<0.05).As compared to Group C,Pa O2 values in Group TV were increased at 1h and 2h after ventilation(P<0.05),with no significant changes at 4h after ventilation(P>0.05);Pa O2 values were increased at 1h and 2h after ventilation,and decreased at 4h after ventilation in Group HV(P<0.05).As compared to Group TV,Pa O2 values were increased at 1h after ventilation but were decreased at 4h after ventilation in Group HV(P<0.05).(2)Comparison of MDA level,SOD activity and lung injury score in lung tissue: As compared to Group C,both the MDA level and lung injury score were higher in Group TV and HV(P<0.05),SOD activity was decreased in Group HV(P<0.05),with no siginifcant change in SOD activity in Group TV(P>0.05).As compared to Group TV,MDA level and lung injury score were higher,and SOD activity was lower in Group HV(P<0.05).(3)Comparison of PPI and W/D: As compared to Group C,PPI was increased(P<0.05),no significant change in W/D was found(P>0.05),while both PPI and W/D were increased in Group HV(P<0.05).PPI and W/D values were higher in Group HV than those in Group TV(P<0.05).(4)Comparison of TNF-?,IL-6 and IL-1? levels in BALF: As compared to Group C,the levels of IL-6 and IL-1? in BALF were increased in Group TV after 4h-ventilation(P<0.05),no significant change in TNF-? was found(P>0.05),while TNF-?,IL-6 and IL-1? levels in BALF were increased in Group HV(P<0.05).TNF-?,IL-6 and IL-1? levels were higher in Group HV than those in Group TV(P<0.05).(5)Microstructural change under light microscope: clear alveolar cavity,no edema and inflammation in interstitial tissue were found in Group C.Scattered inflammatory cells in alveolar cavity were observed in Group TV,the remaining were similar to Group C.In Group HV,microstructures of lung tissue were damaged,a large number of inflammatory cells,mainly macrophagocyte and leukomonocyte,infiltrated in the perivascular,peribronchial and alveolar.Thickened alveolar septum,excessive effusion in alveolar cavity,formation of hyaline membrane,pulmonary interstitial hemorrhage and edema were demonstrated.Conclusion: In addition to physical factors,the release of pro-inflammatory cytokines,aggregation of inflammatory cells and lipid peroxidation might also contribute to VILI.Ventilation with tidal volume 40ml/kg for 4 hours could duplicate classic VILI in vivo model in rats.Part II Effects of FTY720 pretreatment on vascular endothelial cell in rats withventilator-induced lung injuryObjective: To investigate the effects of FTY720 pretreatment on vascular endothelial cell in rats with ventilator-induced lung injury and the role of sphingosine-1-phosphate receptor 1(S1P1).Methods: Forty healthy adult male SD rats were randomly divided into 4 groups(n=10 each): Group TV(traditional tidal volume Vt=8ml/kg),Group HV(high tidal volume Vt= 40ml/kg),Group HF and Group HFW.The rats in group HF receive gavage administration of FTY720 10 mg·kg-1·d-1 for continuous 7 days,while additional dose of W146(S1P1 antagonist)1 mg·kg-1·d-1 was administrated in group HFW before high tidal volume ventilation.After 4-hour mechanical ventilation,femoral artery blood samples were obtained for Pa O2 and serum total protein detection before the animals were sacrificed.The lungs were removed for histopathologic observation under light microscope and electron microscope,W/D and PPI values were caculated.The levels of MDA,NO,activities of i NOS,e NOS,SOD and Na+-K+-ATPase in lung tissue were measured.Real-time PCR method was used to detect i NOS and e NOS m RNA expression,while Na+-K+-ATPase ?1 and Aquaporin1(AQP1)protein expression were detected using Western-blot method.TNF-? and IL-1? levels in BALF were detected by ELISA.Results:(1)Comparison of Pa O2,W/D,PPI and serum total protein: as compared to Group TV,Pa O2 values were decreased,and the levels of W/D,PPI and total protein were increased in Group HV,HF and HFW(P<0.05).As compared to Group HV,Pa O2 was increased,while W/D,PPI and total protein were decreased in Group HF(P<0.05);Pa O2 was increased,W/D and PPI values were decreased in Group HFW(P<0.05),with no significant change in total protein level(P>0.05).As compared to Group HF,W/D and PPI values were higher(P<0.05),the differences of Pa O2 and total protein were not statistically significant(P>0.05).(2)MDA level and SOD activity in lung tissue: As compared to Group TV,MDA leves were increased and SOD activites were lower in Group HV,HF and HFW(P<0.05).As compared to Group HV,MDA level was lower in Group HF(P<0.05),with no significant change in SOD activity;the changes in MDA level and SOD activity were not significant in Group HFW(P>0.05).As compared to Group HF,MDA level in lung tissue was higher(P<0.05),and SOD activity were not significantly changed in Group HFW(P>0.05).(3)Comparison level of NO activity and gene expressions of i NOS and e NOS: as compared to Group TV,NO levels,i NOS activities and gene expressions in lung tissue in Group HV,HF and HFW were increased,while e NOS activities and gene expressions were decreased(P<0.05).As compared to Group HV,NO level,i NOS activity and gene expression were decreased,and e NOS activity and gene expression were increased in Group HF;NO level and i NOS gene expression were decreased(P<0.05),but i NOS activity,e NOS activity and gene expression had no significant changes in Group HFW(P>0.05).As compared to Group HF,NO contents and i NOS activity and gene expression were increased,activity and gene expression of e NOS were decreased in Group HFW(P<0.05).(4)Comparison of Na+-K+-ATPase activity and Na+-K+-ATPase ?1 protein expression in lung tissue: as compared to Group TV,Na+-K+-ATPase activities were decreased in Group HV,HF and HFW,and Na+-K+-ATPase ?1 protein expressions were downregulated in Group HV and HFW(P<0.05).As compared to Group HV,both Na+-K+-ATPase activity and Na+-K+-ATPase ?1 protein expression were increased in Group HF,and Na+-K+-ATPase ?1 protein expression was upregulated in Group HFW(P<0.05),without change in Na+-K+-ATPase activity(P>0.05).As compared to Group HF,both Na+-K+-ATPase activity and Na+-K+-ATPase ?1 protein expression were decreased in Group HFW(P<0.05).(5)Comparison of AQP1 protein expression in lung tissue: as compared to Group TV,AQP1 protein expressions were decreased in Group HV,HF and HFW(P<0.05).As compared to Group HV,AQP1 protein expression were increased in Group HF(P<0.05),without change in Group HFW(P>0.05).As compared to Group HF,AQP1 protein expression was decreased in Group HFW(P<0.05).(6)Comparison of TNF-? and IL-1? levels in BALF: as compared to Group TV,TNF-? and IL-1? levels in BALF were increased in Group HV,HF and HFW(P<0.05).As compared to Group HV,TNF-? and IL-1? levels were decreased in Group HF(P<0.05),and only IL-1? level was decreased in Group HFW(P>0.05).As compared to Group HF,TNF-? and IL-1? levels in BALF were increased in Group HFW(P<0.05).(7)Comparison of apoptosis rate of pneumonocyte: as compared to Group TV,apoptosis rates of pneumonocyte were increased in Group HV,HF and HFW(P<0.05).Ascompared to Group HV,apoptosis rate of pneumonocyte was decreased in Group HF(P<0.05),no significant changes were found in Group HFW(P>0.05).As compared to Group HF,apoptosis rate of pneumonocyte was increased in Group HFW(P<0.05).(8)Microstructural change under light microscope: microstructures of lung tissue were intact,no interstitial edema with scattered inflammatory cells infiltration was observed in Group TV.In Group HV,microstructures of lung tissue were damaged,thickened alveolar septum,remarkable interstitial hemorrhage and edema,a large number of inflammatory cells infiltration,excessive effusion in alveolar cavity,formation of hyaline membrane were demonstrated.The lung tissue damage was attenuated in Group HF as compared to Group HV,and the level of lung tissue damage in Group HFW was between Group HF and HV.(9)Ultrastructural change under electron microscope: ultrastructure of pulmonary epithelial cells was almost normal,intercellular joint was tight,and the arrangement of superficial microvillus of type II pulmonary epithelial cells was almost regular,abundant onion-like lamellar bodies and mitochondria were observed in cytoplasm.Ultrastructure of pulmonary tissue was severely damaged,the arrangement of superficial microvillus of type II pulmonary epithelial cells were ruptured,karyopyknosis,chromatin margination,increased vacuolation of lamellar bodies,and damaged mitochondrial structure were demonstrated in Group HV.Except for mitochondrial edema,the remaining ultrastruture damage was attenuated in Group HF,and the level of ultrastruture damage in Group HFW was between Group HF and HV.Conclusion: FTY720 pretreatment could protect against ventilation-induced lung injury in rats,one of the potential mechanism may be that,by activating S1P1,FTY720 pretreatment reduced inflammation response,decrease lipid peroxidation,improve water metabolism and vascular endothelial cell function.