Role Of OGG1 In The Hyperoxia-Induced Bronchopulmonary Dysplasia Model And The Underlying Mechanism

Bronchopulmonary dysplasia is a common respiratory complication of premature infants.Oxygen inhalation is applied in order to correct the hyoxemia of premature infants.However,consistent inhalation of high-concentration oxygen leads to chronic pulmonary disease which mainly manifested as the block of alveolar development and fibrosis.7,8-dihydro-8-oxoguanine（8-oxo G）is the primary product of oxidative damage bases,and was considered the biomarker of DNA oxidative damage.8-oxoguanine DNA glycosylase-1（OGG1）,the initiator of DNA base excision repair pathway,could recognize and excise 8-oxo G from genome,preventing the massive accumulation of DNA oxidative damage which might leads to pathological changes such as tumor genesis.It is reported that the accumulation of 8-oxo G and elevated OGG1 expression were observed in a BPD experimental model,which suggests that OGG1 might participates in the development of BPD.In order to evaluate the enrichment of OGG1 in genome,we visualized the OGG1 Ch IP-Seq data of a oxidative stress model,and observed that the binding sites of OGG1 distributed widely through the gene regulatory regions of TTF1,suggesting that OGG1 might influence pulmonary development through its regulation of TTF1 expression.Objective:To investigate whether OGG1 participates in the generation and development of BPD,and to confirm the mechanism through which OGG1 influences pulmonary development.Whether it’s achieved by correcting oxidative stress or regulating the expression of related-gene,and have a preliminary exploration of the underlying mechanism.Methods:To explore the ideal condition of BPD modeling,wildtype/OGG1 knock out mice were kept in a self-made oxygen chamber for oxygen inhalation within 12 h after birth,inhaling oxygen of60%or 80%concentration.The hyperoxia stress was given 24 h/day,and last for 7 or 14 days.Mice were transferred to room air after hyperoxia stress and feed till day 21 to sacrifice.H&E staining was used for observation of inflammation infiltration and pulmonary developmentary condition.Images were processed by image pro plus 6.0 to count the mean linear intercept and area proportion of alveoli,evaluating alveoli development.Malondialdehyde（MDA）assay kit and Superoxide Dismutase（SOD）assay kit were used for evaluation of the oxidant/antioxidant balance of lung.Oxy Blot TM Protein Oxidation Detection Kit was used to detect the oxidative modification level of total protein.At transcriptional level,Real-time PCR was used for m RNA expression of SPA、SPB、SPC、SOX2、TTF1,and the protein was detected by Western Blot and Immunohistochemical.Self-made oxygen chamber was used for hyperoxia stress on MLE-12 cells,functional disturbance of OGG1 was achieved by TH5487,the inhibitor of OGG1.Detection of pulmonary development related genes was performed by Real-time PCR and Western-Blot,Chromatin immunoprecipitation was used for detecting the binding of OGG1 and the gene regions of TTF1after hyperoxia stress.Result:1.Lung of mice received hyperoxia stress of 60%oxygen for7 days and transferred to room air till day 21 did not show BPD-like manifestation.While lung of mice received hyperoxia stress of 80%oxygen for 14 days and transferred to room air till day 21 showed classic BPD-like manifestation:massive alveolar fusion and thickened alveolar septum in hyperoxia stressed mice,the alveolar area proportion and mean linear intercept of hyperoxia stressed mice were also significantly higher than control group,suggesting that we successfully built the hyperoxia-induced BPD model on mice.However,the alveolar development of wildtype mice and ogg1^-/-mice showed no significant difference.2.To eliminate the influence of the period during which mice were feed in room air,we changed the modeling condition:treat mice with80%oxygen 24 h/day for 14 days,and sacrifice within 12 h after hyperoxia stress.We observed classic BPD-like manifestation,and ogg1^-/-mice shows severer alveoli development retardation comparing to wildtype mice,manifesting as severer alveoli fusion and thickened alveoli septum,while OGG1 knockout has no impact on normal characters of mice.Besides,the total protein oxidation level of the lung tissue from ogg1^-/-mice is significantly lower than wildtype,and its MDA level is higher.The m RNA and protein expression of SPA、SPC、SOX2、TTF1 were significantly lower than wildtype.We built hyperoxia stress model on MLE-12 cells,and the expression of TTF1 was downregulated after hyperoxia,and combined treatment with TH5487 further decreased the expression of TTF1.Ch IP assay found that the binding of OGG1 to the intron regions of TTF1 increased after hyperoxia stress.Conclusion:More OGG1 bound to the intron of TTF1 after hyperoxia stress,which might regulate the expression of TTF1 and further affects the expression of downstream genes related to pulmonary development,by which OGG1 participates in the development of hyperoxia-induced BPD,causing severer pathological changes such as alveoli development retardation and thickened alveolar septum.Figures:23,Tables:12,References:62...