| IntroductionPulmonary ischemia-reperfusion(I/R)injury was frequently found during the operations of lung transplantation or cardiopulmonary bypass,shock and after the thrombolysis of pulmonary vessels.Pulmonary dysfunction from I/R injury is the main cause of mortality and prognosis after lung transplantation and cardiopulmonary bypass. Impaired pulmonary function is also induced by I/R injury during shock,cardiopulmo-nary resuscitation and other organ ischemia.Therefore,the research about the mechanism and treatment of pulmonary I/R injury becomes popular.Many studies found that inhaled anesthetics,such as halothane,enflurane, isoflurane and sevoflurane,had protective effect on I/R injury in heart,brain,liver and kidney.But the literatures are less and still imcompatible in the relationship of anesthetics and pulmonary I/R injury.Nielsen and his colleague found desflurane increased permeability of alveolar-capillary membrane after the reperfusion of aorta occlusion.The study by Chen showed that isoflurane decreased pulmonary edema in isolated rabbit lung model.Sevoflurane has many features,such as rapid anesthesia induction,analepsia and steady hemodynamics,so it is widely used in operations. Sevoflurane has no effect on lung in ordinary ventilation,but its effect on pulmonary I/R injury is unknown.Pulmonary I/R injury means increased pulmonary vascular permeability and vascular resistance.In recent years,we have more recognition about the mechanism of vascular permeability with the development of molecular biology.The integrity of constitution and function of cell junctions,especially tight junction,is important to pulmonary vascular permeability.In this study,we established a rat model of lung I/R injury to observe the changes of pulmonary vascular permeability,the content of TNF-α, the gene expression and location of tight junction proteins(occluding and ZO-1),the expression of PKC-αand histology at different time point in rat,in order to explore the effect and mechanism of sevoflurane administration before lung I/R,to offer the experimental and theoretical evidence for the choice of anesthetics in clinical practice. This study includes three fractions;1,the effect of sevoflurane on pulmonary tissue permeability in I/R rats;2,the effect of sevoflurane on TNF-αand expression of PKC-αduring pulmonary I/R injury in rats;3,the effect of sevoflurane on expression of tight junction proteins,occluding and ZO-1,during pulmonary I/R injury in rats.Materials1.Experiment animals96 male wistar rats,provided by the laboratory animal center of China Medical University,weight 250～350g.2.Chemical and reagentsTNF-αkits(Nanjing)Sevoflurane(Baxter,USA)PKC-αantibody(Sigma,USA)occluding antibody(Santa-Cruz,USA)ZO-1 antibody(Santa-Cruz,USA)protein extraction kit(Nanjing)RT-PCR kits and DNA Taq enzyme(TaKaRa,Japan)Primers(TaKaRa,Japan)RNA extraction kits and other reagents(Shanghai).3.Experiment instrumentsSiemens monitor(Sirecust730,Germany)Microinfusion bump(Braun,Germany)Datex anesthetic gas monitor(Finland)Drager anesthesia machine(USA)Animal ventilator(Zhejiang)Transmission electron microscope(CM-10,USA)Ultrathin slicing machine(8800,Sweden)ultraviolet spectrophotometer(UV-260,Japan)electrophoresis apparatus(BIO-RAD-PAC300,USA)electrophoresis chamber(BIO-RAD mini,USA) histology ultrasonic homogenate apparatus(UP200H,USA)low temperature and high speed centrifuger(Heraeus-Biofuge-PrimoR,Germany)PCR amplification device(TP-600,Japan)Digitized gel scanning analyzing system(Tanon GIS-2020,Tianjin)Methods1.Animal modelSimulating Eppinger method to establish a rat model of lung I/R injury.After male wistar rat was anesthetized intraperitoneally with 20% urethane,the left pulmonary hilum was occluded for 45 min and unclamped for continuous perfusion and ventilation.2.Experiment groupsNinety six male wistar rats were randomly divided into four groups.Group C(n=24);continuous perfusion without ischemia; group IR(n=24);occlusion of the left pulmonary hilum for 45 min,followed by reperfusion; group Sev-C(n=24);1MAC sevoflurane inhalation for 30min before ischemia,followed by continuous perfusion; group Sev-IR(n=24);lMAC sevoflurane inhalation for 30min before ischemia, followed by ischemia-reperfusion.In every group,the indexes were measured at 45min after lung ischemia and at 60min,120min of reperfusion.Moreover,another six rats were performed for bronchial lavage at 120min of reperfusion.3.Indexes and methods①the change of hemodynamics②the change of pulmonary histology at all time points;light microscope and electron microscope③the lung wet-to-dry weight ratio(W/D)④the total protein of serum and left bronchalveolar lavage fluid(BALF)⑤the content of TNF-αin rat lungs⑥The expression of PKC-αin cytoplasm and cell membrane; the expression of occluding and ZO-1 in rat lungs⑦The gene expression of occluding and ZO-1 in lungs ⑧The location of occluding and ZO-1 in cells4.Statistical treatmentAll of the data was expressed as mean±standard deviation.Statistical analysis was performed with the SPSS11.5 software.One-sample ANOVA analysis was used in the inter-group comparisons of the measurement data,the Student-Newman-Keuls test was used in the intra-group comparison.P＜0.05 was considered as the significant difference.Results1.the effect of sevoflurane on pulmonary tissue permeability in I/R rats①Homodynamics;MAP kept stable at the time of ischemia and reperfusion in groups.In IR and Sev-IR groups,PaO2 decreased gradually and was obviously lower than that in C and Sev-C groups during reperfusion(P＜0.05).But there was no significant difference in PaO2 between IR and Sev-IR groups(P＞0.05).②The change of pulmonary vascular permeability;During reperfusion the lung W/D and pulmonary permeability index(LPI)in IR and Sev-IR groups increased progressively(P＜0.05).Moreover,compared with IR group,preadministration of sevoflurane could inhibit the increase in the lung W/D and LPI after reperfusion (P＜0.05).③Light microscope;In IR group,the lung injury was aggravated gradually after reperfusion and there was marked pulmonary capillary congestion,interstitial edema and leukocyte infiltration,intra-alveolar hemorrhage.Compared with IR group,the degree of injury in Sev-IR group was significantly ameliorated.④Electron microscope;In IR group,the tight junctions between adjacent pulmonary microvascular endothelial cells were disrupted and not dense at 120min of reperfusion.While endothelial TJs in Sev-IR group and TJs between adjacent alveolar epithelial cells in all groups remained integrity.2.The effect of sevoflurane on expression of TNF-αand PKC-αduring pulmonary I/R in rats①In IR group,the content of TNF-αincreased progressively after reperfusion in rat lung,and reached the peak value at 120min of reperfusion(P＜0.05).Compared with IR group,the increase of TNF-αwas inhibited in Sev-IR group(P＜0.05).②After reperfusion,the density ratio of PKC-αin IR group to C group in cellular membrane increased obviously(P＜0.05),while the density ratio in cytoplasm decreased accordingly in IR group(P＜0.05).The inhalation of sevoflurane could inhibit the change of the density ratio of PKC-αin cellular membrane and cytoplasm afer reperfusion in rat lung(P＜0.05).3.The effect of sevoflurane on protein levels of occluding and ZO-1 during pulmonary I/R in rats①The protein levels of TJ molecules;In IR group,the density ratioes of occluding and ZO-1 in IR group to C group decreased progressively after reperfusion (P＜0.05),and reached their lest values at 120min of reperfusion.Compared with IR group,the decrease of density ratio of TJ proteins was inhibited in Sev-IR group (P＜0.05).②The mRNA levels of TJ molecules;In IR group,the density ratio of occluding toβ-actin and ratio of ZO-1 toβ-actin decreased obviously after reperfusion,and reached their lest values at 120min of reperfusion(P＜0.05).Inhalation of sevoflurane could inhibit the decrease of density ratio of TJ molecules after reperfusion(P＜0.05).③The location of TJ proteins in cells;The degree of brown stain of occluding or ZO-1 was weaker in IR group and Sev-IR group than C group after reperfusion (P＜0.05),while the stain of TJ molecules in IR group was marked weaker than in Sev-IR group(P＜0.05).The occluding and ZO-1 were located at plasma membrane or plasma closer to membrane.Both pulmonary capillary endothelia and alveolar epithelia were stained,especially intense in cell border between adjacent cells.Conclusions1,The pulmonary tissue permeability increases after rats are reperfused with blood. Sevoflurane protects the pulmonary function and decreases pulmonary permeability in I/R rats.2,The release of TNF-αafter reperfusion induces the lung injury in rat,while sevoflurane protects the pulmonary function possibly through inhibiting the production of TNF-α. 3,The translocation and activation of PKC-αcan be found in injured lung after reperfusion.Sevoflurane given before the hilum is occluded can inhibit the translocation and activation of PKC-α.4,The decreased levels of mRNA and protein of TJ molecules are responsible for the increased pulmonary vascular permeability in I/R lung.The protective effects of sevoflurane might be fulfilled by inhibiting the translocation and attenuating the down-regulation of TJ molecules at mRNA and protein levels.
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