The Role Of SENP1-SIRT1-P53 Signaling Pathway In Hyperoxia-induced Lung Injury

Abstract:Objective:In this experimental study,hyperoxia exposure was used to construct a model of hyperoxia-induced lung injury in neonatal SD rats.By detecting the expression of key Proteins in the SENP1-SIRT1-P53 Pathway in lung tissue at different time points of hyperoxia exposure,to explore the possible mechanism of its action in preterm infants with hyperoxia-induced lung injury.It is hoped that it will provide new directions and possibilities for the concept and method of clinical prevention and treatment of bronchopulmonary dysplasia?BPD?.Methods:An animal model of hyperoxia-induced lung injury in premature infants was constructed using neonatal SD rats as subjects.The newborn SD rats within 12 hours after birth were divided into hyperoxia exposure group and air control group by randomized comparison table method.Air control treatment:treat by FiO₂=21%continuous air ventilation;treatment in the hyperoxia exposure group:treat by FiO₂=60%�3%continuous hyperoxia ventilation.The exposure time of the two groups was 14 days,and the mothers were exchanged once a day,the litter was replaced,and the environmental humidity and carbon dioxide levels were monitored.The daily weight changes and general conditions of the newborn SD rats in the two groups were observed and the growth and development were observed.The newborn SD rats were sacrificed at 1st day,7th day and 14th day after birth.At the same time,after obtaining lung tissue of each group,the indexes were as follows:1)Histopathological findings of lung tissue of each group,including pathological characterization of lung tissue after HE staining,and calculation of mean linear intercept?MLI?,and radial alveolar counts?RAC?,and used to assess the damage and development of lung tissue;2)TUNEL staining was used to detect the apoptosis rate in the lung tissue of each group of rats;3)Fluorescent microplate reader was used to quantitatively detect the amount of reactive oxygen species?ROS?produced in the lung tissue of each group of rats;4)Real-time quantitative PCR was used to detect the transcriptional expression of SENP1-SIRT1-P53 in the lung tissue of each group of rats;5)Western-blot was used to detect the protein expression levels of SENP1-SIRT1-P53 and phosphorylated P53?AC-P53?in the lung tissues of each group;6)Co-Immunoprecipitation?Co-IP?was used to detect the protein expression level of SUMO-modified SIRT1?SUMO-SIRT1?in lung tissue of each group.Results:1)Growth and development and general records showed that the body weight of neonatal rats in the hyperoxia-exposed group began to be significantly lower than that in the air control group on the 7th day after birth?P<0.05,the difference was statistically significant?,and the hair gloss and vitality were significantly lower than the air control group;2)After HE staining,the pathological results of lung tissue in each group were observed under light microscope.Compared with the air control group,the hyperoxia exposed group showed obvious structural disorder and simplification of alveolar structure in the lung tissue from the 7th day.At the same time,MLI results showed that the average alveolar diameter of neonatal SD rats in the hyperoxia exposed group was significantly higher than that in the air control group from the 7th day,and the results of RAC evaluation showed that the number of alveolar cells in the lung tissue of neonatal SD rats in the hyperoxia exposed group was significantly lower than that in the air control group from 7th day?P<0.05,the difference was statistically significant?.3)TUNEL staining results showed that the apoptosis rate in the lung tissue of neonatal rats in the hyperoxia exposed group was significantly higher than that in the air control group on the 7th day after continuous hyperoxia exposure?P<0.05,the difference was statistically significant?,and showing a trend of continuous increase;4)ROS quantitative results suggest that the production of ROS in the high-oxygen exposure group was significantly higher than that in the air control group at each time point of continuous high-oxygen exposure?P<0.05,the difference was statistically significant?;5)Real-time PCR results showed that the RNA transcription level of SENP1 in the lung tissue of the hyperoxia exposed group began to be higher than that of the air control group on the 7th day and continued to the 14th day?P<0.05,the difference was statistically significant?.The RNA transcription of SIRT1 in the lung tissue of the hyperoxia exposed group showed lower than that of the air control group on the first day and the 14th day?P<0.05,the difference was statistically significant?.At the same time,the semi-quantitative results of P53 RNA showed that the RNA transcription level of P53 continued to increase with the prolonged duration of hyperoxia exposure,and was significantly higher on the 7th and 14th day than the air control group?P<0.05,the difference was statistically significant?.6)Western-blot results indicated that the expression of SENP1 protein in lung tissue of hyperoxia exposed group was higher than that of air control group on the 7th and 14th day?P<0.05,the difference was statistically significant?.The expression of SIRT1protein in lung tissue of hyperoxia exposed group was lower than that of air control group on 1st and 14th day?P<0.05,the difference was statistically significant?.The protein expression of P53 in the lung tissue of hyperoxia exposure group continued to increase during the exposure period,and was higher than that of the air control group on the 7th and 14th day?P<0.05,the difference was statistically significant?.Compared with the air control group,the protein expression of acetylated P53?AC-P53?in the lung tissue of the hyperoxia exposed group continued to increase,and it was significantly higher than the air control group from the first day?P<0.05,The difference was statistically significant?;7)Co-IP results showed that the protein expression of SUMO-SIRT1 in the hyperoxia-exposed group was lower than that in the air control group on the 1st and 14th day?P<0.05,the difference was statistically significant?.Conclusion:In the animal model of neonatal SD rats,it was found that the SENP1-SIRT1-P53 signaling pathway may be involved in the mechanism of hyperoxia-induced lung injury in preterm infants.During hyperoxia-induced lung injury,hyperoxia exposure can induce oxidative stress and ROS bursting,and accompanied by an increase in the expression of SENP1 protein,the increased SENP1 protein can induce loss of SUMO protein modification,decreased expression,and decreased activity.Thereby,the biological activity of SIRT1 is decreased,the deacetylation is weakened,the acetylation modification of the downstream protein P53 is increased,the structure tends to be stable,and the biological activity is enhanced.It is speculated that it may eventually participate in the lung injury and lung development arrest process of neonatal SD rats induced by persistent hyperoxia exposure.