Protein Kinase C-beta / The 66-kDa Isoform Of The Growth Factor Adaptor Shc Oxidative Stress Pathway In Mediating The Production Of The Reactive Oxygen Species In Hyperoxia-exposed Premature Infant

Objective To study the possible mitochondria oxidative stress signal transduction pathway,which probably mediated the production of reactive oxygen species in hyperoxia-exposed premature infants.Methods Control group :twenty cases of premature infants whose gestational age was below 34 weeks were diagnosed as no respiratory distress syndrome and respiratory failure[exposed to air over 48 h, pulse oxygen saturation(Sp O2) > 85%) ] in Department of Neonatology, the Affiliated Hospital of Si Chuan Medical University, from July 2012 to December 2012.The control group’s main reason for in hospital was premature infants. Hyperoxia group: twenty cases of premature infants with the same gestational age were diagnosed as respiratory failure(treated with exposure to over 400 m L/L oxygen within 48h-69 h in the same room simultaneously). The respiratory failures include two types: Ⅰ type failure: arterial blood oxygen partial pressure(Pa O2) < 6.67 k Pa; Ⅱ type failure: Pa O2 < 6.67 k Pa, arterial blood co2 partial pressure(Pa CO2)>6.67 k Pa. The main reason of hospitalization was with varying degrees of respiratory distress syndrome with respiratory failure in hyperoxia group. Two groups of infants were taken to hospital for 0.25 ~ 10.00 h after birth. And with the following disease were excluded:(1)ABO hemolysis and glucose- 6- phosphate dehydrogenase(G6PD) deficient;(2) Congenital heart disease;(3)Convulsions,intracranial and gastrointestinal bleeding;(4) Infection.After authorized by the ethics committee of the Affiliated Hospital of Lu Zhou Medical College and families after informed consent, two groups of preterm infants were sampled blood 2 ml which was putted in heparin anticoagulant tub 72 h after admission from radial artery blood after sterilization. Blend fresh diluted anticoagulant 2 ml with Hank’s liquid According to the proportion of 1:1, absorb the fresh diluted blood 4ml through a straw, add to on the surface of the lymphocyte separation liquid along the centrifugal tube wall slowly, horizontal centrifugal, extract Annular milky white layer which was peripheral blood mono-nuclear cells(PBMC), drop on the cover glass, fix PBMC on the cover glass with 40g/L paraformaldehyde. The expression of Protein kinase C-beta(PKC β)?the 66-k Da isoform of the growth factor adaptor Shc(p66Shc) ?prolyl isomerase1(Pin 1)?phosphorylated the 66-k Da isoform of the growth factor adaptor Shc-Ser36(phosphorylated p66Shc-Ser36)were detected by immunohistochemistry(PV) in the PBMC of hyperoxia group and control group. The positive staining was yellow or tan particles observed under inverted phase contrast microscope. The production of mitochondrial reactive oxygen species(ROS) were detected by fluorescence microscope in the PBMC of hyperoxia group and control group.The positive color was red fluorescence.Results 1.The expression of PKC β from premature infants of control group and hyperoxia group: staining for the yellow and the tan is located in the cytoplasm. Comparing with the control group, the expression of PKC β in hyperoxia group was significantly increased( t=21.139,P<0.001); 2.The expression of p66 Shc from premature infants of control group and hyperoxia group: dyeing for most of the yellow and the tan is located in the cytoplasm. Comparing with the control group, the expression of p66 Shc in hyperoxia group was significantly increased(t=5.592,P<0.001);3.The expression of Pin1 from premature infants of control group and hyperoxia group:Staining for the yellow and the tan is located in the cytoplasm or nucleus. Comparing with the control group, the expression of Pin1 in hyperoxia group was significantly increased(t=7.476,P<0.001); 4.The expression of phosphorylation p66 Shc Ser36 from premature infants of control group and hyperoxia group: dyeing for most of the yellow and the tan is located in the cytoplasm.Comparing with the control group, the expression of phosphorylation p66 Shc Ser36 in hyperoxia group was significantly increased(t=16.895,P<0.001);5.The mitochondrial ROS present red fluorescence.The red fluorescence of normal control group is weak. Comparing with the control group, the production of ROS increased and the red fluorescence of the huperoxia group was obviously enhanced(t=10.586,P<0.001);6.There are positive correlations between the production of reactive oxygen species and the expression of PKCβ/p66Shc/Pin1/phosphorylated p66 Shc Ser36 in hyperoxia group(r=0.893,0.795,0.681,0.663,all P<0.001).Conclusions The expression of PKC β?p66Shc ?Pin 1?phosphorylated p66Shc-Ser36 and the production of ROS in premature infants’ PBMC were significantly increased induced by hyperoxia.This proved that PKC β/p66 Shc oxidative stress pathway might mediate the production of ROS in hyperoxia-exposed premature infants.