Protective Effects Of Hypoxic Preconditioning On Ischemia Reperfusion Lung Injury In Rats

Partâ… Establishment of Protective model of hypoxic preconditioning on ischemia reperfusion lung injuryObjective To establish animal model of hypoxic preconditioning in rat and to explore its protective effects and general mechanisms on lung ischemia reperfusion.Methods 24 males adult Sprague-Dawley rats were randomly divided into three groups:control group,lung ischemia reperfusion group(I/R) and hypoxic preconditioning group(WHPC).The left pulmonary hilum of rats from I/R group was occluded for 60 minuts and then reperfused for 120 minutes so that to establish the modal of lung ischemia reperfusion injury.Rats from WHPC group were subjected to 2h of whole-body hypoxia in a custom-made ambience chamber and then to suffered the same lung ischemia reperfusion injury as I/R group rats.Control animals under-went the same procedure,except the microvscular clamp was not applied to the hilum (sham thoracotomy alone).After that,the changes of lung Histomorphology,lung wet-to-dry weight and MPO activity of all animals were determined.MDA and SOD levels in rats plasma were also detected. Results(1) compared to the control,the lung wet-to-dry weight,white blood cell count in the alveolar lavage fluid and the MPO activity in lung of I/R group rats were significantly elevated(p＜0.01);the plasma MDA level of I/R group rats gradually increased with the extension of reperfusion time and all above control group at every time point(p＜0.01);while the plasma SOD level of I/R group rats gradually decreased with the extension of reperfusion time and all below control group at every time point(p＜0.01).(2) Compared with I/R group,the lung wet-to-dry weight, white blood cell count in the alveolar lavage fluid and the MPO activity in lung of WHPC group rats were significantly improved(p＜0.01);the plasma MDA level of WHPC group rats obviously decreased and the SOD level of WHPC group rats obviously increased(p＜0.01).Conclusion Hypoxic preconditioning has some protective effects on lung ischemia reperfusion in rats.The mechanisms involve the inhibition of lipid peroxidation, inflammation and nertrophil activation.Partâ…¡Effects of hypoxic preconditioning on apoptosis in ischemia reperfusion lung injuryObjective To study the effects of hypoxic preconditioning on apoptosis in ischemia reperfusion lung injury in rats,especially the apoptosis of alveolar typeâ…¡epithelial cells.Methods The models of hypoxic preconditioning and lung ischemia reperfusion were made as the same as partâ… .54 males adult Sprague-Dawley rats were randomly divided into three groups:control group,lung ischemia reperfusion group(I/R) and hypoxic pre- conditioning group(WHPC).The latter two were further divided into three subgroups separately according to the time of reperfusion:reperfusion for 30 minutes(P₁,R₁),reperfusion for 60 minutes(P₂,R₂) and for 120 minutes(P₃,R₃).4 rats were placed in control group and 6 in each other subgroups.After all the treatment,cell apoptosis in lung of all groups was detected by TUNEL and apoptosis index of each group was counted,alveolar typeâ…¡epithelial cells were isolated after lung ischemia reperfusion and the apoptosis rate was detected by flow cytometry.Results(1) The apoptosis index in I/R group was significantly increased than control group(p＜0.05),and the number of apoptotic cells gradually increased with the extension of reperfusion time.Cell apoptosis also existed in WHPC group,but the number of apoptotic cells was significantly decreased than I/R group(p＜0.01).(2) The apoptosis rate of alveolar typeâ…¡epithelial cells in the I/R and WHPC group were both obviously increased compared with control group,especially I/R group (p＜0.01).The apoptosis rate of alveolar typeâ…¡epithelial cells in WHPC group was lower than I/R group(p＜0.01).Conclusions(1) Hypoxic preconditioning could extenuate cell apoptosis in ischemia reperfusion lung injury in rats.(2) Hypoxic preconditioning could extenuate alveolar typeâ…¡epithelial cell apoptosis in ischemia reperfusion lung injury and play a protective role.Partâ…¢The effect and mechanism of HIF-1 in the protection of hypoxic preconditioning on ischemia reperfusion lung injuryObjective To explore the effect and the mechanism of HIF-1 in the protection of hypoxic preconditioning on ischemia reperfusion lung injury.To determine whether DMOG,an inhibitor of proline hydroxylase,could imitate the delayed protective effect of hypoxic preconditioning on ischemia reperfusion lung injury.Methods 40 male adult Sprague-Dawley rats were randomly divided into five groups:control group,saline group,hypoxic preconditioning group(WHPC),NS398 group(WHPC+NS) and DMOG group.The models of hypoxic preconditioning and lung ischemia reperfusion were made as the same as partâ… .Normal saline 0.5ml was injected into the abdominal cavity 24 hours before the lung ischemia reperfusion model was made in saline group;NS-398(10 mg/kg) was injected into the abdominal cavity 15 minutes before the lung ischemia reperfusion model was made in WHPC+NS group;DMOG(20 mg/kg) was injected into the abdominal cavity 24 hours before the lung ischemia reperfusion model was made in DMOG group.After all the treatment,the expression of HIF-1αmRNA and HO-1 mRNA in lung was detected by RT-PCR;the expression of caspase-3 protein was detected by western blot;the expression level and position of HIF-1α,HO-1,and caspase-3 protein was observed by immunohistochemical method.Further more,the cell apoptosis in lung of all groups was detected by TUNEL,and lung wet-to-dry weight was also calculated.Results(1) HIF-1αmRNA was expressed in lung in all groups,there were no significant difference.HO-1 mRNA was also expressed in lung in all groups,but compared with the control,the expression level were higher in other groups(p＜0.01). Among these,WHPC and DMOG group were higher than Saline group,and WHPC+NS group was lower than WHPC group(p＜0.05).(2) The HIF-1αand HO-1 protein expression level was significantly higher in WHPC and DMOG group than Saline group,while caspase-3 protein expression level was significantly lower than Saline group.The HIF-1αand HO-1 protein expression level was lower in WHPC+NS group than WHPC group,while caspase-3 protein expression level was higher than WHPC group.(3) The apoptosis index and lung wet-to-dry weight significantly decreased in WHPC and DMOG group compared with Saline group,but increased in WHPC+NS group compared with WHPC group.Conclusions(1) HIF-1αcould play an important role in the protection of hypoxic preconditioning on ischemia reperfusion lung injury.(2) Hypoxic preconditioning could extenuate cell apoptosis in ischemia reper- fusion lung injury via inducing HIF-1αexpression,increasing HO-1 expression level and decreasing caspase-3 expression.(3) DMOG,an inhibitor of proline hydroxylase,could imitate the delayed protective effect of hypoxic preconditioning on ischemia reperfusion lung injury,and NS-398 could cancel those effects.