| Part 1:Construction of isolated LIRI modelObjectlive We use rat isolated lung perfusion machine to establish a stable lung ischemia-reperfusion injuiy(LIRI)model,which provides a basis for the study of the effects of drugs on LIRI.Method Thirty-two healthy male SD rats,250-300g,were randomly divided into four groups:blank control group(group Control),ischenia reperfusion 45min group(group IR-45),ischemia reperftusion 60min group(group IR-60),ischemia reperfusion 75min group(group IR-75).After Thoracotomy,lung was put into the IL-2 ex-vivo lung perfusion system to maintain ventilation and perfusion.When isolated lung was adapted for 1smin and get a stable statue in EVLP,the control group continued to maintain ventilation and perfusion for 135 min,and the rest of the three groups were treated with stopping ventilation and perfusion for 45min,60min,75min and than kept ventilation and perfusion for 60min.Recorded tidal volume(TV),pulmonary arterial partial pressure of oxygen(PaO2),lung compliance(CompI)and airway resistance(Res)values at the end of the balance(T0),immediately prior to ischemia and hypoxia(T1),reperfusion 30min(T2)and reperfusion 60min(T3).Measured lung wet/dry weight ratio(W/D)and observed lung tissue by HE staining.Results There were no significant differences in the values of lung function between groups at TO and T1 moment.TV,Comp1 and Res indexes of IR-60 group were close to 0 or unable to be shown at T4 moment,while TV,Comp1 and Res indexes of IR-75 group were also unable to be shown at T3,T4 moment.Compared with T0 moment,TV,Compl and PaO2 of control group and IR-45 group decresed sigificatly at T4 moment(P<0.05),while Res increased(P<0.05).Compared with the control group,the TV,PaO2 and Compl indexes decreased(P<0.05)and Res increased(P<0.05)in the IR-45 group at T3 and T4 moment;the TV,PaO2 and Compl indexes decreased(P<0.05)and Res icreased(P<0.05)in IR-60 group at T2 and T3 moment;the TV,PaO2 and Compl indexes decreased(P<0.05)and Res icreased(P<0.05)in IR-75 group at T2 moment.The lung function indexs of IR-60 group were changed higher than that of IR-60 group.Compared with the control group,the W/D ratio of the other three groups increased(P<0.05).The W/D ratio of IR-75 group increased most,followed by IR-60 group.Pulmonary morphology showed that three ischemic groups had varying degrees of pulmonary edema and tracheal perfusion fluid.Pathological changes showed that IR-75 group had the most serious injury and IR-45 group was lighter than IR-60 group but worse than control group.The histopathological score showed that IR-75 group had the most pathological score and IR-60 group was higher than that of IR-45 group,which the score of pathology in control group was the lowest.Conclusion The model of lung ischemia reperfusion injury was successfully established,and a stable and sensitive model was obtained in the condition of 60 min ischemia and 60 min reperfusion.Part 2:Effect of emulsified isoflurane on lung ischemia-reperfusion in ratsObjective To investigate the effect of emulsified isoflurane(El)on LIRI by establishing an isolated lung ischemia-reperfusion injury(LIRI)model,and to investigate whether post-treatment of emulsified isoflurane has a protective effect on ischemia-reperfusion injury effect.Method Thirty-two healthy male SD rats,250-300g,were randomly divided into four groups:blank control group(group C),ischemia-reperfusion group(group IR),emulsified isoflurane post-conditioning group(group El),intralipid post conditioning group(group IL).Four groups were added in the modified K-H solution with physiological saline,physiological saline,8%emulsified isoflurane and 30%fat emulsion to make the concentration of 1.68mmol/L in lung perfusate.After thoracotomy,put isolated lung into IL-2 ex-vivo lung perfusion system and kept ventilation and perfusion,and then establish a rat model of isolated lung ischemia-reperfusion injury.Recorded tidal volume(TV),pulmonary venous partial pressure of oxygen(PaO2),lung compliance(CompI)and airway resistance(Res)values at the end of the balance(To),immediately prior to ischemia and hypoxia(T1),reperfusion 30min(T2)and reperfusion 60min(T3);measured lung wet/dry weight ratio(W/D);observed the morphological manifestations of pulmonary organs after perfusion and lung tissue HE staining.Results Compared with C group,TV,Compl and PaO2 significantly decreased but Res obviously increased at T2-3 moment in IR group and IL group(P<0.05).Compared with IR group,TV,Compl and PaO2 increased but Res decreased at T2-3 moment in El group and IL group(P<0.05).Compared with El group,PaO2 decreased at T2-3 moment,TV and Compl decreased but Res increased at T3 moment in IL group(P<0.05).Compared with C group,the W/D ratio of IR and IL group increased(P<0.05).Compared with IR group,the W/D ratio of IL group and El group decreased(P<0.05).Pulmonary morphology showed that IR group and IL group had pulmonary left lower lobe edema,tissue transparency increased and white foam and perfusion fluid flow from the trachea,while the C group and the El group showed no pulmonary edema,increased transparency and perfusion flow from the trachea.Pathology showed that lung injury in IL group and El group was lighter than that in IR group,and lung injury in EI group was lighter than that in IL group.The histopathological score showed that IR group had the most pathological score and IL group was higher than that of EI group,which the score of pathology in C group was the lowest.Conclusions Emulsified isoflurane of 1.68 mmol/L concentration can improve the pulmonary function indexes of lung ischemia-reperfixsion injury,reduce the edema and histopathological changes of lung tissue,and protect the lungs from ischemia-reperfusion injury.Part 3:Molecular Mechanism of Protective Effect of emulsified isoflurane Postconditioning on Isolated Rat lung ischemia-reperfusion injuryObjective To detect the protective effect of emulsified isoflurane on lung ischemia reperfusion injury,which was studied from the aspects of oxygen free radical,iniflammatory reaction and endoplasmic reticulum stress.Method Lung tissues from the second part were taken from the-80?refrigerator,which were prepared 10%lung tissue homogenate for various molecular mechanisms.The activity of SOD and the content of MDA were measured by the kit method,which can reflect the oxidative stress level.The expression of TNF-?,IL-6 and IL-1? mRNA in lung tissue was detected by real-time fluorescence quantitative PCR,which can reflect degree of inflammation reaction.The expression of p-PERK,p-IF2a,GRP78 and CHOP protein in lung tissue were detected by Western Blot method,which reflect the stress level of endoplasmic reticulum.Results 1.Compared with the control group,the activity of SOD in the other three groups decreased and the MDA content in IR group increased(P<0.05).Compared with IR group,SOD activity increased and MDA content decreased in El group and IL group(P<0.05).Compared with El group,the activity of SOD in IL group decreased(P<0.05).2.Compared with the control group,the mRNA expression levels of IL-1?,IL-6 and TNF-a were increased in the other three groups increased(P<0.05).Compared with IR group,the mRNA expression of IL-1?,IL-6 and TNF-a mRNA in lung tissue of El and IL group decreased(P<0.05).Compared with El group,the expression of IL-6 and TNF-a mRNA in lung tissue of IL group decreased(P<0.05).3.Compared with control group,the protein expression of p-PERK,GRP78,p-eIF2a and CHOP in lung tissue of IR group increased(P<0.05),p-PERK in lung tissue of El group and p-PERK,GRP78 and CHOP in lung tissue of IL group increased(P<0.05).Compared with IR group,the protein expression of p-PERK,GRP78,p-eIF2a and CHOP in lung tissue of El group decreased(P<0.05),and the protein expression of GRP78 and p-eIF2a in lung tissue of IL group decreased(P<0.05).Compared with El group,the protein expression of CHOP in lung tissue of IL group increased(P<0.05).Conclusions The protective effect of 1.68 mmol/L emulsified isoflurane on lung ischemia-reperfusion injury may be related to the mechanism of reducing the formation of oxygen free radicals,reducing the inflammatory reaction and relieving endoplasmic reticulum stress. |
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