The Mechanism Of P47phox Induced Reactive Oxygen Species Increasing Within PBMCs After Oxygen Exposed

Objective:To observe p47 phox and reactive oxygen species(ROS) generate after oxygen therapy in premature infants whose gestational age less than 32 weeks. Beside, using NADPH oxidase inhibitors DPI and apocynin combined with high concentration oxygen treat PBMCs, then detect the change of p47 phox and reactive oxygen species,to explore the mechanism of NADPH oxidase subunit p47 phox regulating ROS increasing within PBMCs and the new targets for reducing oxidative stress injury in preterm infants.Methods:The experiment was conducted in two parts. The Firstly part of experiment, the premature infants less than 32 weeks were regarded as the research object in clinical. Those who were diagnosed with NRDS need oxygen as oxygen exposure groups. According to the concentration of oxygen,they were divided into three groups, Fi O2<30% is low concentration oxygen group, Fi O2 between(30-40)% is middle concentration oxygen group, Fi O2>40% is high concentration oxygen group. Each group of preterm infants are 10 cases, including 18 males,12 females. The gestational age is between(28-32) weeks, body weight is about(1350±138)g.The gestational age and birth weight have no significant differences among the exposure groups. At the same period, 10 cases of premature infants as air group who are 32 weeks following without oxygen exposed.Male 5 cases, female 5 cases.The gestational age of(30-32) weeks, body weight(1347±116) g.The gestational age and birth weight have no significant differences between exposure groups and air group. This experiment had been obtained the approval of the ethics committee of the affiliated hospital of Sichuan Medical University,and their parents had signed informed consents as well. Air group and those premature infants exposed to oxygen for 48 h,we gain blood 3ml via radial artery,separated PBMCs and serum for detection. The second part of experiment, the PBMCs from premature infants less than 32 weeks were regarded as the research object, those premature infants less than 32 weeks who have no oxygen exposure before blood were taken. Of course, we obtained parental consent. The separated cells were divided into four groups:1,routine culture group, PBMCs were put in a culture box with 37℃ and 5%CO2 for 48 hours. 2,High oxygen group, PBMCs were exposed to mixture gas contains 95%O2 and 5%CO2 with the speed of 3 L /min. After 10 min, those culture bottles were sealed immediately with a sealing adhesive.3,High oxygen and DPI group. According to reports in the literature and preliminary experimental results, after treated with 5μmol/L DPI, PBMCs were exposed to high oxygen group for 48 hours. 4,High oxygen and apocynin group.According to reports in the literature and preliminary experimental results, after treated with 100μmol/L apocynin, PBMCs were exposed to high oxygen group for 48 hours.After 48 hours,we use CYS-1 digital oxygen sensor to detection the concentration of O2 in culture bottle. If the concentration of O2 is less than 90%,the specimenwill be discardded.After treated according to the request, their PBMCs and medium were collected as samples. The ROS in all PBMCs were detected by Mitosox Red marker combined with confocal laser scanning microscopy,the content of MDA in serum or medium were measured by thiobarbituric acid colorimetric method, the locate p47 phox in all PBMCs were detected by immunofluorescence and then calculated their translocation rate,the expression of p47 phox protein in all PBMCs were tested by western blot.Results: Firstly,in those clinical samples,with the oxygen concentration increasing in premature infants,the level of ROS and MDA were increased significantly,the cell number of p47 phox from the cytoplasm to the cell membrane translocation and their translocation rates were significantly increased, the expression of p47 phox protein were up-regulated as well( P < 0.05). Secondly,after PBMCs were treated by high oxygen and corresponding inhibitors. Compared with the high oxygen group, the level of ROS and MDA were decreased significantly,the p47phox’s translocation rates and the protein expression were also reduced significantly(P<0.05) in the others three groups. Compared with the routine culture group,all indexes have no significant differences between the high oxygen and DPI group as well as high oxygen and apocynin group(P>0.05).Conclusion: After premature infants/PBMCs were exposed to oxygen, the translocation of the p47 phox were increased and it’s expression were also up-regulated. These changes are compatible with increasing of ROS and MDA.However, afer using the NADPH oxidase inhibitors DPI and apocynin to treat PBMCs,p47phox’s translocation rate and expression would be decreased, at the same time, the level of ROS and MDA were reduced accordingly. Those findings suggested that after oxygen exposure,p47 phox translocated to membrane and it’s expression increasing may induce reactive oxygen species increasing in PBMCs.While DPI and apocynin may reduce it’s translocation and expression of p47 phox to reduce the reactive oxygen species production.