Mechanism Of Edaravone Protect Against Hyperoxia Induced Acute Lung Injury

BackgroundUnder certain circumstances,such as hypoxemia or acute respiratory failure,prolonged treatment of O2(hyperoxia)may be essential.However,hyperoxia treatment may induce hyperoxia acute lung injury(HALI),as the lung is particularly vulnerable to oxidative inhalants because of its large epithelial surface and anatomic location.HALI is a clinical syndrome characterized by extensive inflammation,accumulation of pleural fluid,disruption of the alveolar membrane,impaired gas exchange and respiratory failure as a result of prolonged supplement of high concentrations of oxygen.It is accepted that HALI is closely related to increase generation of reactive oxygen species(ROS),including superoxide(O2-),hydrogen peroxide(H-2O2)and hydroxyl radical(.OH).Excessive ROS could react with DNA,lipids and protein,leading to DNA breakage,lipid peroxidation and protein inactivation.As yet,no specific treatments are available for HALILEdaravone has been shown to be neuro-protective in cerebral ischemia and approved for treatment of cerebral infraction.Some investigators found that edaravone could eliminate.OH and other ROS,such as O2-and NO radicals and inhibit hyperoxia-induced lipid peroxidation.Edaravone could also activate antioxidative enzymes,including superoxide dismutase(SOD),catalase(CAT),oand guaiacol peroxidase(GPx),or upregulate heme-oxygenase-1(HO-1)expression,an important protectant for hyperoxic injury.These findings suggested that edaravone might be an effective treatment for HALI,considering ROS and antioxidative enzymes play an important role in the pathology of HALI.However,the potential of edaravone in the prevention or treatment of HALI was seldom explored,except a recent study carried by Wang etal.which indicated that edaravone inhibited inflammasome activation in a 2.5ATA hyperbaric oxygen(HBO)induced lung injury.In the present study,we exposed rats and human pulmonary alveolar epithelial cells(HPAEpiC)to 95%O2,then investigated whether edaravone could prevent rats and HPAEpiC against hyperoxia-induced damage.Survival rate,total cell count,leukocytes,total protein and LDH in bronchoalveolar lavage fluid(BALF),Lung wet/dry weight ratio,oxidative products and HO-lin lung tissues were measured in rats.Methyl thiazolyl tetrazolium(MTT)and lactate dehydrogrenase(LDH)leakage assays were preformed for cell mortality and injury analyses.Malondialdehyde(MDA)and 8-hydroxy-2-deoxyguanosine(8-OHdG)levels were examined for cellular lipid and DNA oxidative damage.The role of HO-1 was explored because:(1)HO-1 has protective effects in lung hyperoxia injuries;(2)edaravone was reported to be able to upregulate HO-1 expression.The involvement of phospgatidylinositol 3-kinase(PI3K)/Akt kinase pathway was investigated to explore the underlying mechanism.ObjectivesTo investigate the protective effects of edaravone in hyperoxic-induced lung injury,to explore the mechanism of edaravone protect against hyperoxic-induced lung injury.To determine a molecular target for prevention of HALI,to provide a theoretical basis for the clinical application and development adaptation of edaravona in HALI.Methods1.Construction of HALI rat models and protective effects of edaravone in HALI(1)Construction of HALI rat models①Adult male Sprague-Dawley rats,weighing 230±10g,were used to construct HALI models in the study,Rats were placed in cages in a 17-L pressure glass chamber(Zhongbin Co.,Ltd,Beijing,China)and exposed to 95%O2 for 72 hours.②Lung tissue histological changes were assessed by HE staining,the total protein content in BALF was quantified with a BAC protein assay kit(Pierce，Rockford,IL,USA).Total cell number was counted using a hemocytometer.Slides were prepared by cytocentrifugation(Cytospin 4,Thermo)and stained with Diff-Quick(Dade Behring,Dudingen,Switzerland),then the leukocytes counts were determined in randomly chosen fields using morphological criteria.The LDH activity was measured with a LDHdetection kit(Roche Molecular Biochemicals,Mannheim,Germany)according to manufacturer’s instructions.LDHactivity was expressed as U/L using a LDH standard provided by the manufacturer.③ Measurement of malondialdehyde(MDA),protein carbonyl and 8-hydroxy-2-deoxyguanosine(8-OHdG)in lung tissues;Measurement of SOD、catalase(CAT)and GSH-px activities in lung tissues to evaluate oxidative damage.2.Protective effects of edaravone in HALI①Adult male Sprague-Dawley rats,weighing 230±10g,were used in the study,and then divided into four groups:Normoxia(21%oxygen)group,Hyperoxia(exposed to 95%O2 for 72 hours)group,Edaravone(edaravone was given intraperitoneally at a daily dose of 6ml/kg body weight)group,Hyperoxia+ Edaravone(edaravone was given intraperitoneally at a daily dose of 6ml/kg body weight during hyperoxia exposure)group②Total cell number and total protein and LDH measurement in BALF in different groups.⑧ Measurement of malondialdehyde(MDA),protein carbonyl and 8-hydroxy-2-deoxyguanosine(8-OHdG)in lung tissues;Measurement of SOD、catalase(CAT)and GSH-px activities in lung tissues among different groups 2.Intrevention in different groups of HALI models,to demonstrate the protective effects of edaravone through upregulating HO-1 expression.①Adult male Sprague-Dawley rats,weighing 230±10g,were used in the study,and then divided into four groups:Normoxia(21%oxygen)group,Hyperoxia(exposed to 95%O2 for 72 hours)group,Hyperoxia+ Edaravone(edaravone was given intraperitoneally at a daily dose of 6ml/kg body weight during hyperoxia exposure)group,Hyperoxia+Edaravone+ZnPP-9(edaravone was given intraperitoneally at a daily dose of 6ml/kg body weight together with ZnPP-9 was given intraperitoneally at a daily dose of 3ml/kg body weight during hyperoxia exposure)group.②RT-PCR shows the HO-1 mRNA expression changes,HO-1 activity changes were investigated in different groups.3.To examine the protective effect of edaravone against hyperoxia-induced damage in human pulmonary alveolar epithelial cells models(1)we treated human pulmonary alveolar epithelial cells with hyperoxia in vitro,different dose of edaravone were added into the culture before hyperoxia treatment,then the protective effect of edaravone against hyperoxia-induced damage were observed and the proper concentration of edaravone was examined.① HPAEpiC(Cat,ScienCell Research Laboratories,CA,USA)were cultured in Alveolar Epithelial Cell Medium(AEpiCM,Cat.ScienCell Research Laboratories,C A,US A)according to the supplier’s instructions.Before treatment,they were divided inti six groups:Normoxia(21%oxygen)group,N ormoxia+50 u M edaravone group,Hyperoxia(95%oxygen)+ 0 u M edaravone group,Hyperoxia+ 25 u M edaravone group,Hyperoxia+50 u M edaravone group,Hyperoxia+ 100uM edaravone group.Edaravone(purity≥98%)were purchased from Yuanye Biotech(Shanghai,China).For hyperoxia treatment,cells were exposed to hyperoxia for 24 hours.Different doses of edaravone(25,50,100 u M)were added to culture 30 minutes before hyperoxia treatment.② Cell viability was determined by a MTT assay kit(Beyotime,Haimen,China)similarly to the method of Bai et al.LDH leakage assay kit was a product of Promega Company(Cyto Tox 96 Non-Radioactive Cytotoxicity Assay),LDH assay was followed the kit manual.Cell viability and LDH leakage were estimated the hyperoxia damage of cell membranes.The MDA and 8-OHdG content in the supernatant were measured using MDA and 8-OHdG assay kits.they were evaluated lipld peroxidation and DNA breakage.2.Adiministration of different ways in human pulmonary alveolar epithelial cells(HPAEpiC)were probed the mechanism of edaravone protect against hyperoxia induced damage.① For zinc protoporphyrin-Ⅸ(ZnPP-Ⅸ)treatment,ZnPP-Ⅸ(Alexis,San Diego,CA,USA)was added into the medium to reach a final concentration of 10u M.For specifically blocking PI3K/Akt signaling,LY249002 purchased from Sigma-Aldrich(St.Louis,MO,USA)was added into the medium to reach a final concentration of 20 u M.②Cell viability was determined by a MTT assay kit similarly to the method of Bai et al.Western-Blotting analysis was evaluated the expression of HO-1 and p-Akt/Akt.The HO-I activity assay was performed by the method of Velislava et al.HO-I activity was expressed as pmolbilirubin min-1 mg protein-1.Results1.Construction of HALI rat models and protective effects of edaravone in HALI① After exposure to hyperoxia for 24 hours,mice in hyperoxia group showed moveless,irritability,shortness of breath and so on.Lung tissue hist extensive inflammationological changes was assessed by HE staining,changes were observed obvious hameorrhage,effusion,alveolar septal thickness and transparent blot formation.Hyperoxia group mice showed significantly elevated BALF protein concentration(37.53±4.46 vs.95.63±6.44μg/ml,n=10)and BALF total cell number(19.33±2.08 vs.69.86±6.78 ×104/ml,n=10)as compared with controlled group mice.Meanwhile,LDH leakage(10.53±0.75 vs.66.23±3.75 U/ml,n=10)was significantly higher in Hyperoxia group mice(P<0.05).②Measurement of malondialdehyde(MDA),protein carbonyl and 8-hydroxy-2-deoxyguanosine(8-OHdG)in lung tissues of the hyperoxia group mice were significantly increased as compared with controls,however,they were significantly decreased after edaravone administration which indicated edaravone protecting lung tissues against hyperoxia-induced injury.③Measurement of SOD、catalase(CAT)and GSH-px activities in lung tissues of the hyperoxia group mice were significantly decreased as compared with controls,however,they were significantly increased after edaravone administration which indicated edaravone protecting lung tissues against hyperoxia-induced injury.2.Intrevention in different groups of HALI models,to demonstrate the protective effects of edaravone through upregulating HO-1 expression.RT-PCR shows the HO-1 mRNA expression changes,HO-1 activity changes were investigated in different groups.Gray scan statistical results confirm:the HO-1 mRNA expression in lung tissues of the hyperoxia group mice were significantly elevated as compared with controls,meanwhile,they were further significantly increased after edaravone pretreatment.it shows both hyperoxia and edaravone significantly stimulate HO-1 activity,but the HO-1 inhibitor ZnPP-Ⅸ reversed this result.Moreover,we can find the same trend because the HO-1 inhibitor ZnPP-Ⅸ treatment almost abolished the HO-1 acitivity completely.3.To examine the protective effect of edaravone against hyperoxia-induced damage in human pulmonary alveolar epithelial cells models① We determined the influences of different concentration of edaravone on hyperoxia-treated HPAEpiC viability,the results showed that edaravone alone did not alter the cell viability,but it dose-dependently increased cell viability of hyperoxia-treat HPAEpiC.②The LDH leakage results showed that edaravone alone did not alter the LDH leakage,25 u M edaravone did not significantly decreased the LDH level of hyperoxia-treated HPAEpiC,but 50 u M and 100 u M edaravone did not significantly prevented the LDH leakage.③To evaluate the peroxidation of lipid in HPAEpiC,we examined the MDA level in cells,50 uM and 100 uM edaravone alone did not alter the cellular MDA content.After hyperoxia treatment,MDA level was dramatically increased.But with the inclusion of increased amount of edaravone,MDA were gradually decreased to basal level.④To investigate the protective effect of edaravone on DNA damage,we detected the 8-OHdG level in cells,a typical marker for oxidative DNA damage.The results showed that similiarly to MDA results,the edaravone alone did not alter the cellular 8-OHdG content.But hyperoxia induced elevation of 8-OHdG level in cells,which was remarkably prohibited by edaravone.⑤ Western-blotting analysis shows the HO-1 mRNA expression changes,HO-1 activity changes were investigated in different groups.Gray scan statistical results confirm:we found that after HPAEpiC were exposed to hyperoxia for 24 hours,the protein expression of HO-1 was not significantly changed.Pretreatment with 50 uM edaravone significantly increased HO-1 in cells exposed to hyperoxia.Co-treatment of 10uM ZnPP-IX,the inhibitor of HO-1,and 20 uM LY249002,the inhibitor of PI3K/Akt pathway,both decreased the expression of HO-1 to some extent in protein levels.⑥Western-blotting analysis shows the p-Akt expression changes,Gray scan statistical results confirm:for p-Akt expression,p-Akt/Akt was not influenced by the hyperoxia treatment,but significantly enhanced by edaravone.⑦ Different treatments show the HO-1 activity changes.Both hyperoxia and edaravone significantly stimulate HO-1 activity,but the HO-1 inhibitor ZnPP-Ⅸ and PI3K/Akt pathway inhibitor LY249002 reversed it.Moreover,the inhibitor ZnPP-Ⅸtreatment almost abolished the HO-1 activity completely.⑧As shown in different treatment,edaravone significantly increased the cell viability to 80%,which was completely or partly abolished by the co-treatment of 10 u M ZnPP-Ⅸor 20 u M LY249002(P<0.05,compared to edaravone group).In addition,the inhibitor ZnPP-IX treatment significantly decreased the viability of HPAEpiC exposed to hyperoxia(P<0.05,compared to Hyperoxia group).Summary1.To observe edaravone protects against hyperoxia-induced lung injury in rats①Edaravone was determined to alleviate total inflammatory in BALF and to decreased lung tissue histological injury.② Edaravone was significantly decreased the levels of malondialdehyde(MDA)protein carbonyl and 8-hydroxy-2-deoxyguanosine(8-OHdG)in lung tissues together with increased activities of SOD、catalase(CAT)and GSH-px in lung tissues.The protective effects may be related to balanced oxidation and antioxidation.2.Edaravone protects rats against hyperoxia-induced injury and the antioxidant effect may be related to upregulation of HO-1①the HO-1 mRNA expression in lung tissues of the hyperoxia group mice were significantly elevated as compared with controls,meanwhile,they were further significantly increased after edaravone pretreatment.it shows both hyperoxia and edaravone significantly stimulate HO-1 activity.②the HO-1 inhibitor ZnPP-Ⅸ reversed edaravone upregulation of HO-1,it revealed HO-1 may be involved the process.3.Edaravone protects human pulmonary alveolar epithelial cells against hyperoxia-induced injury and the antioxidant effect may be related to upregulation of HO-1,which is regulated by PI3K/Akt pathway.①edaravone dose-dependently increased cell viability and decreased cell injury of hyperoxia-treat HPAEpiC.②MDA and 8-OHdG were gradually decreased to basal level by edaravone treatment in hyperoxia-treat HPAEpiC.It clearify the protective effect of edaravone on the peroxidation of lipid and DNA damage in HPAEpiC.③Earavone upregulate expression of HO-1 and p-Akt/Akt by the hyperoxia treatment in HPAEpiC.④Earavone significantly stimulate HO-1 activity,but the HO-1 inhibitor ZnPP-Ⅸand PI3K/Akt pathway inhibitor LY249002 reversed it.Moreover,the inhibitor ZnPP-Ⅸtreatment almost abolished the HO-1 activity completely.ConclusionsThis study indicates that edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia-induced injury and the antioxidant effect may be related to upregulation of HO-1,which is regulated by PI3K/Akt pathway.