Protection Mechanism Of Hydrogen Against Hyperoxi-induced Injury In Type Ⅱ Alveolar Epithelial Cells

PART I Hydrogen and PI3K/AKt/FOXO3 a signaling pathway in type Ⅱ alveolar epithelial cells with hyperoxia-induced apotosis and its mechanismObjective: This study sought to evaluate the effects of hydrogen on hyperoxia-induced lung injury and to investigate the underlying mechanism.Methods: Primary type Ⅱ alveolar epithelial cells(AECⅡs)were divided into four groups: control(21% oxygen),hyperoxia(95% oxygen),hyperoxia+hydrogen,and hyperoxia+hydrogen+LY294002(a PI3K/Akt specific inhibitor).Control group and hyperoxia group were exposued to 21% oxygen and 95% oxygen,respectively.Hydrogen was added to the hyperoxia+hydrogen group before hyperoxia exposure(The hydrogen is dissolved in a complete medium to make a hydrogen rich medium).Hydrogen and PI3K/Akt specific inhibitor LY294002 were added to hyperoxia+hydrogen+ LY294002 before hyperoxia exposure,and then placed into a closed box with 95% oxygen for 24 h.Proliferation,apoptosis and cycle of AECⅡs were assessed using MTS assay and flow cytometry(FCM),respectively.P-Akt、 PFox O3a、Foxo3a、Akt、Bim、Bax、cyclinD1 and Bcl-2 protein expression was detected by western blot analysis.Foxo3a、Bax、Caspase3 and Bcl-2 mRNA expression was detected by quantitative polymerase chain reaction(q-PCR)Results: Stimulation with hyperoxia decreased the expression of P-Akt,PFox O3 a,cyclinD1 and Bcl-2.Hyperoxic conditions increased levels of Bim,Bax,and Foxo3a(P<0.01),Foxo3a、Bax、Caspase3 mRNA expression were increased,while Bcl-2 mRNA expression is decreased,which induced proliferation restriction and apoptosis of AECⅡs.These effects of hyperoxia were reversed with hydrogen pretreatment(P<0.05).Furthermore,the protective effects of hydrogen were abrogated by PI3K/Akt inhibitor LY294002(P<0.01).The results of PCR is similar with Western blot.Conclusion: The results indicate that hydrogen protects AECⅡs from hyperoxia-induced apoptosis by inhibiting apoptosis factors and promoting the expression of anti-apoptosis factors.These effects were associated with activation of the PI3K/Akt/FoxO3 a pathway.PART II Hydrogen and MAPK-ERK1/2 signaling pathway in type II alveolar epithelial cells with hyperoxia-induced injury and its mechanismObjective: To investigate the protection mechanism of hydrogen against hyperoxia-induced injury in type II alveolar epithelial cells and whether it is related to MAPK-ERK1/2 signaling pathway.Methods: AECII were isolated and purified from premature rats and were divided into 4 groups: air group(21% oxygen),hyperoxia group(95% oxygen),hyperoxia+hydrogen group,hyperoxia+hydrogen+PD98059 group(a ERK1/2 specific inhibitor).The ability of cell proliferation was measured by CCK-8 colorimetric assay.The cell apoptosis was analyzed by flow cytometry.The protein levels of ERK1/2,pERK1/2,Bax were detected by Western blot.The m RNA levels of Bax and caspase-3 were assessed by qPCR.Results: Compared with air group,cell apoptosis rates and the m RNA levels of Bax and caspase-3 significantly increased,cells proliferation and the protein of p-ERK1/2 significantly decreased under hyperoxia exposure(P<0.01).While with hydrogen treatment,the ability of cells proliferation markedly increased,cell apoptosis rates,Bax,caspase-3 m RNA levels notably decreased and p-ERK1/2 protein levels increased(P<0.05).the protective effects of hydrogen was abolished by MAPK-ERK1/2 inhibitor PD98059(P<0.05).Conclusion: Hydrogen can inhibit the expression of apoptosis related-genes via activating the MAPK-ERK1/2 signaling pathway to improve the proliferation restriction of AECII cells induced by hyperoxia,and promote the survival of cells.