The Effect Of Moderate Hyperoxia On Normal Human Lung Bronchial Epithelial Cells

Objective:Hypoxia is the most common clinical manifestation in the critically ill of neonates and preterm infants.Hypoxia is more likely to cause cellular metabolism and organ dysfunction,even threaten the life of newborns.Oxygen therapy can provide sufficient oxygen concentration to improve the oxygen pressure and blood carrying capacity,thus ensuring the supply of tissue oxygen.Although oxygen is one of the most common"drugs"in clinical,oxygen therapy can also cause problems.Premature infants are very sensitive to oxygen and extremely susceptible to oxygen poisoning.Oxygen poisoning can directly lead to the damage of alveolar epithelial and capillary endothelial cells which increase the permeability of alveolar capillaries leading to a consequence of a series of inflammatory reactions.Tumor necrosis factor alpha?TNF-a?is a cytokine that regulates programmed cell death and apoptosis.It is expressed in different cells of human skin and lung which can induce inflammatory response.It can mediate many kinds of acute and chronic lung diseases,including hyperoxia induced lung injury.TNF-a can start the tumor necrosis factor receptor associated apoptosis signal-regulating kinase-1?ASK1?interaction protain to release ASK1 and then start the process of apoptosis endothelial cells.Studies have shown that the synthesis of nuclear factor-kappa B?NF-?B?is closely related to the synthesis of TNF-a.It includes five family members of Rel A,Rel B,REL,NF-?B1?P50?and NF-?B2?p52?.There are two main ways to activate NF-?B:the classical pathway and the non-classical pathway.RelA mediates the activation of the classical pathway and RelB mediates the activation of the non-classical pathway.A large number of hyper physiological stimuli,such as inflammatory reactions,can active the two pathways of NF-?B.TNF-a can promote the secretion of secretory component?SC?.Some studies have shown that SC can protect the acute lung injury induced hyperoxia and the up-regulation of cytokines also affects the expression of SC.The most commonly used in clinical treatment of neonatal non-invasive oxygen supply methods is nasal catheter oxygen inhalation,mask oxygen inhalation and so on.The common concentration of oxygen is generally 40%-60%?moderate concentration?.In our study,we choose 40%oxygen concentration.40%oxygen concentrations can also be used to improve the survival of the organs;WHO has also recommended that 40%hyperoxia can be used to treat some lung diseases.Our goal is to compare the influence of moderate hyperoxia?40%O₂?and extreme hyperoxia?85%O₂?on pneumonia pathway,exploring the mechanism of moderate hyperoxia on pulmonary bronchial epithelial cells and provide a theoretical basis for the clinical use of oxygen therapy.Methods:Normal human tracheobronchial epithelium cells?NHBE cells?were obtained from the Cell Biological Institute of Shanghai,Chinese Academy of Sciences.The cells were cultured in sterile basal medium/DMEM-H containing 10%fetal bovine serum,100U penicillin/mL,100 mg streptomycin/mL,1%L-glutamine and 0.25 mg amphotericin B/mL into incubator?37oC and 5%CO₂?.Before treatment,cells?1×10⁶ cells/m L?were well plated and cultured for 24 h.Then the cells were put into incubator?40%O₂ or 85%O₂?for 24h.The control cells were no treatment.Finally,the protein and RNA of cells were extracted.The effect of 40%O₂ on the viability of NHBE cells was investigated by MTT experiment.Immunohistochemical staining was used to detect the distribution and expression of RelA,Rel B and SC.Western Blot analysis was performed to determine the quantitative of RelA,RelB,ASK1,TNF-?and SC.Real-time PCR was used to analysis the mRNA of Rel A,RelB,ASK1,TNF-?and SC.All experiments were repeated at least6 times.The experimental data were analyzed by Prism Graph 6.0 software and expressed by Mean±SD.The non paired t test?unpaired t-test?was used to compare the differences between every two groups.The results showed that P<0.05 was statistically significant.Results:1.Moderate hyperoxia has a protective effect on NHBE cells.The results of MTT cell proliferation test showed that the cell survival rate of 40%O₂ group was higher than that of the control group?P<0.0001?.85%O₂ group compared with the control group,the cell survival rate was decreased?P<0.0001?.2.The effect of moderate hyperoxia on the expression of TNF-a in NHBE cells.40%O₂ group compared with the control group,the protein levels of TNF-?were significantly increased?P<0.0001?,the TNF-?mRNA was up-regulated?P<0.001?;40%O₂ group compared with 85%O₂group,the protein levels of TNF-?were significantly deduced?P<0.05?,the transcription of TNF-?was deduced?P<0.0001?.3.Moderate hyperoxia up-regulated the expression of ASK1 in NHBE cells.The expression of ASK1 in protein and gene levels were detected by Western Blot and Real-time PCR.40%O₂ group compared with the control group,the protein levels of ASK1 were significantly increased?P<0.01?,the ASK1 mRNA was up-regulated?P<0.0001?;40%O₂ group compared with 85%O₂group,the protein levels of ASK1 were significantly deduced?P<0.0001?.4.Moderate hyperoxia regulates the expression of RelA and RelB.4.1 Immunohistochemical method was used to locate the expression of RelA and RelB.The nucleus and cytoplasm are brown and yellow is the positive reaction.After the activation of the NF-?B pathway,RelA and RelB enter the nucleus.The positive expression of Rel A and RelB in 40%O₂group were increased in the nucleus,indicating that the NF-?B pathway was activated.40%O₂ group compared with the control group,the expression of Rel A and RelB were increased?P<0.0001,P<0.01?;Compared with 85%O₂ group,the expression of RelA and RelB were decreased?P<0.001,P<0.0001?.4.2 The protein expressions of RelA and RelB in cells by Western Blot.40%O₂ group compared with the control group,the protein levels of RelA and RelB were increased?P<0.01,P<0.0001?;Compared with85%O₂ group,the protein levels of RelA were decreased?P<0.01?,the protein levels of RelB were no significant statistical difference.4.3 The mRNA expressions of RelA and RelB in cells.40%O₂ group compared with the control group,the mRNA levels of RelA and RelB were increased?P<0.05,P<0.001?;40%O₂ group compared with 85%O₂group,the mRNA levels of RelA were decreased?P<0.001?,the mRNA levels of RelB were no significant statistical difference.5.Moderate hyperoxia affects the expression of SC in NHBE cells.The expression of SC was detected by immunohistochemical method,and the expression of SC was in the cytoplasm.SC did not increase in cell cytoplasm of40%O₂ group compared with the control group;Compared with 85%O₂ group,the expression of SC was decreased?P<0.0001?.40%O₂ group compared with the control group,the protein levels was not significantly difference,the SC m RNA was up-regulated?P<0.0001?;40%O₂ group compared with 85%O₂ group,the protein levels of SC were significantly deduced?P<0.0001?,the SC mRNA was deduced?P<0.0001?.Conclusions:1.Moderate hyperoxia can promote the proliferation of NHBE.2.Moderate hyperoxia can activate the ASK1,NF-?B pathways and SC by promoting the secretion of TNF-?,finally play a protective role in the lung bronchial epithelial cells.