RESUMO
Objective:To study the dynamic changes of the localization and expression of nuclear transporter KPNA2 in the occurrence and development of bronchopulmonary dysplasia(BPD) in neonatal rats, and explore its role in the pathogenesis of BPD in premature infants.Methods:The BPD model of newborn SD rats was induced with 85% oxygen concentration( n=50), and the control group was inhaled with air( n=50). The lung tissue samples were collected on 1 d, 3 d, 7 d, 10 d, and 14 d, respectively, in the two groups and separated.Purification and culture of alveolar type Ⅱ epithelial cells.The distribution and expression of KPNA2 were detected by immunohistochemistry, immunofluorescence, western blot and RT-PCR. Results:Immunohistochemistry showed that KPNA2 mainly located in alveolar epithelial cells′ cytoplasm and nucleus, and BPD group was more expressive than control group.Cell immunofluorescence showed that KPNA2 in control group was mainly localized in the nucleus, and in BPD group, KPNA2 was mainly localized in the cytoplasm from 3 d to 14 d. The nuclear expression of KPNA2 was weaker than that in the control group, and the cytoplasmic expression was stronger than that in the control group.The expression trends of KPNA2 total protein, plasma protein and mRNA were basically the same.The BPD group began to increase on the 1st day ( P<0.05), and was still higher than the control group on the 14th day( P<0.05); in BPD group, KPNA2 nucleoprotein expression began to decrease on the 3rd day( P<0.01), continued to decrease to 14 days( P<0.05). Conclusion:The dysfunction of KPNA2 nuclear transport in neonatal rats exposed to hyperoxia may be an important mechanism that affects the early initiation of the DNA damage response of BPD alveolar epithelial cells.
RESUMO
Objective:To investigate the dynamic expression of DNA damage repair protein Nijmegen breakage syndrome protein 1(NBS1) in the neonatal rats with bronchopulmonary dysplasia(BPD), and its influence on the development and progression of BPD.Methods:Newborn rats were randomly divided into the BPD model group( n=50) and the control group( n=50) within 12 h after birth.The inhaled oxygen concentration was 80%-85% in the model group, and the control group inhaled air.In the two groups, lung tissue samples were collected on days 1, 3, 7, 10 and 14, and isolated, purified and cultured alveolar epithelial type Ⅱ cells(AEC Ⅱ). We observed pulmonary morphological changes under light microscope and evaluated alveolar development degree by radiate alveolar counts(RAC). Immunohistochemistry and cell immunofluorescence were used to observe the localization and expression of NBS1.Western blot and real-time quantitative PCR were used to detect the expression level of NBS1. Results:Compared with the control group, the RAC value in the model group was decreased significantly from 7 d after birth(control group 7.58±1.24, model group 5.42±1.24, P<0.01). Immunohistochemistry showed that NBS1 protein was mainly located in the nucleus of alveolar epithelial cells.In the model group, NBS1 was mainly expressed in the nucleus on the 1st day.With the prolonged exposure time, the number of cytoplasmic staining cells increased and the expression in the nucleus decreased.Cell immunofluorescence farther showed that NBS1 protein was mainly located in the nucleus in AEC Ⅱ.Compared with the control group, cytoplasmic staining in model group was enhanced from 3 d, while nuclear staining was gradually weakened, and was mainly located in the cytoplasm at 14 d. Western blot results showed that the expression of NBS1 protein in the model group peaked at 1 d compared to the control group(control group 0.72±0.29, model group 1.28±0.22, P<0.01), and then gradually decreased, with lower expression at 14 d compared to the control group(control group 0.73±0.19, model group 0.49±0.11, P<0.05). Similarly, the mRNA expression level of NBS1 in the model group peaked at 1 d compared to the control group(control group 1.00±0.00, model group 1.18±0.06, P<0.01), and then gradually decreased, with lower expression at 14 d than that in the control group(control group 1.07±0.13, model group 0.76±0.11, P<0.05). Conclusion:In the neonatal rats with BPD, the down-regulation expression and nuclear enrichment disorder of NBS1 may affect the DNA damage response and be one of the mechanisms mediating the onset of oxidative stress damage in BPD.