| BackgroundsBronchopulmonary Dysplasia (BPD) was first reported and defined by Northway in1967as the most common and difficult chronic lung damage in premature infants including barotrauma, volutrauma, and oxygen toxicity. BPD has traditionally been defined as the presence of persistent respiratory signs and symptoms, the need for supplemental oxygen to treat hypoxemia, and an abnormal chest radiograph at36weeks postmenstrual age. In contrast to classic BPD, the new BPD develops in preterm newborns who generally require minimal ventilator support and relatively low FiO2(fraction of inspired oxygen) during the early postnatal days. Pathologic signs of severe lung injury with striking fibroproliferative changes have become rare. At autopsy, lung histology now displays more uniform and milder regions of injury, and signs of impaired alveolar and vascular growth are more prominent. These features include a pattern of alveolar simplification with enlarged distal airspaces and reduced growth of the capillary bed, with vessels that are often described as dysmorphic because of their centralized location in the thickened mesenchyme. It is the cause of prolonged hospitalization with serious social and economic consequences. Deeper understanding of the disease such as future preventative measures, aiming at reducing its incidence, minimizing complications, diminishing hospital costs and improve infant health had become the prime objective for the public health.Although its exact etiology and pathogenesis has yet to be fully ascertained, many researches suggest that it results from the complex interplay between impairments in the premature lung, perinatal insults and resulting from supportive care of the infant.Several factors increase the susceptibility of the premature newborn to the development of BPD, including surfactant deficiency, decreased antioxidant defenses, impaired epithelial ion and water transport function, and lung structural immaturity. Lung injury after premature birth and the subsequent arrest of lung growth results from complex interactions between multiple adverse stimuli, including inflammation, hyperoxia, mechanical ventilation, and infection, of the poorly defended developing lung. Most recently, prenatal exposure to proinflammatory cytokines, such as TNF-a, IL-6, IL-8, and others, due to maternal chorioamnionitis, have been shown to enhance lung maturation in utero, but increase the risk for BPD. In conclusion, hyperoxia and oxidant stress are critical factors in the development of BPD.Few therapies are known to effectively prevent or treat BPD and corticosteroid therapy may decrease lung injury through a variety of mechanisms, such as decreasing inflammatory response. Majority of serious short-term and long-term side effects were found, such as gastrointestinal bleeding, gastrointestinal perforation, hyperglycemia and hypertension that give clinicians reason to severely limit exposure to treatment. The survival rate of premature neonatal had significantly increased, but the morbidity of BPD also showed an increasing trend. Epidermal growth factor-like domain7(EGFL7) is a protein secreted from endothelial cells which plays an important role in vascular tubulogenesis. Study found that EGFL7gene expression was significantly decreased in the neonatal rat lungs after hyperoxic exposure and was important for cell survival. It was identified as a potential therapeutic target for lung injury. Thus antioxidant therapy has been considered as a potential preventive or treatment option for BPD.Astragalus polysaccharides (APS), an extract from a kind of Chinese traditional herb Astragalus membranaceus, has attracted people attention by its outstanding anti-oxidant and anti-inflammatory effects. R. CHEN et al. verified that Astragalus could reduce oxidative damage by reduced Malondialdehyde(MDA) concentration and increased the activities of SOD and GSHPX, which include scavenging free oxygen radicals. Besides, the antioxidative effect of Astragalus in brain and kidney tissue after ischemia-reperfusion injury has also been demonstratedIt was also proved to have strong immunoregulatory properties. However, the effects of APS in hyperoxia-induced newborn BPD model rats are unknown. So, we hypothesis that in the hyperoxide induced BPD, APS can reduce airway remodeling and alveolar damage by up-regulate EGFL7expression, down-regulate the expression of NF-KBp65and some other preinflammations and has some protective effects on BPD of neonatal rats. We use an oxygen chamber, into which oxygen was continuously delivered (FiO2=0.7-0.8) to build BPD rat model and investigate the preventive effects of Astragalus polysaccharides (APS) on BPD, and explored its potential molecular mechanisms.Objectives1、To determine APS can reduce airway remodeling and alveolar damage and promote the development of vascular in lung tissue by up-regulate EGFL7expression and has some protective effects on BPD of neonatal rats. 2、To investigate the preventive effects of Astragalus polysaccharides (APS) on BPD, and explored its potential molecular mechanisms.Methods1、One hundred and forty-four Sprague-Dawley (SD) newborn rats (7.77±0.56g) and fungible mother rats were obtained from the Experimental Animal Center of Southern Medical University (Guangzhou, China). These rats were randomly divided into six groups (n=24):Control group, room air plus low-dose APS group(RA+LD APS group), room air plus high-dose APS group(RA+HD APS group), BPD model group, low-dose APS group (20mg/kg-d)(SaiNuo, Pharmaceutical Co. Ltd, Tianjin, China) and high-dose APS group (40mg/kg·d). Rat model of BPD was induced by hyperoxia. Ten hours after birth, pups in the control group and room air plus APS groups were kept in room air containing21%O2, and the latter received daily injections of APS (i.p.) according to the predicted dose throughout postnatal day, while those in hyperoxia and hyperoxia plus APS groups were placed in an oxygen chamber, into which oxygen was continuously delivered (FiO2=0.85±0.03) and received daily injections of saline (i.p.) and APS (i.p.) according to the predicted dose throughout postnatal day, respectively. Temperature and humidity were maintained at22℃-25℃and60%-70%respectively. The chamber was opened for1hour daily to switch dams between air and O2environment to protect the dams from oxygen toxicity. These pups were then sacrificed at the4th,10th,14th day after experiments and the lung tissues were collected.2、Following anesthesia with pentobarbital (60mg/kg, i.p.), the lungs of pups were fixed with an intratracheal injection of4%paraformaldehyde and post-fixed overnight at room temperature. Tissues were paraffin embedded, sectioned to5μm thickness and stained with hematoxylin and eosin. Quantitative analysis of pulmonary mean linear intercept (MLI), mean alveolar number (MAN) was measured according to previously described methods.3、The contents level of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by the assay kit.4、BALF was collected on day4th,10th and14th by using three consecutive instillations of PBS (0.5ml each) at room temperature. Approximately1.2ml of the PBS was consistently recovered with gentle handling and centrifuged at500g at4℃for5min. Cell pellets were resuspended in0.5ml PBS and smeared for analysis of cellular infiltration. Cellular infiltration in BALF was assessed on Wright’s stain.5、The protein and/or mRNA expression level of NF-κBp65, CD31, ICAM-1and TNF-α were also detected by corresponding method.6、The results were expressed as mean±D. Statistical analysis was subjected to One-way ANOVA and Student Newman Keuls Test with factors of treatment and disease or normal type by SPSS13.0statistical software (SPSS Inc. Chicago). A value of P<0.05was considered significantly different.Results1、From this study we can know that there was no damage in control groups at the10th and14th day. It was not significant different from both RA plus high and low-dose APS group compared with control group. However, the lung tissues showed cells infiltration, alveolar and vascular development arrested in BPD group at the10th and14th day. Lung histological type was as follow:such as fewer and larger simplified alveoli, negligible airway lesions, variable interstitial fibroproliferation, fewer and dysmorphic capillaries, and less severe arterial lesions. But in the APS treatment group, the extents of inflammation, the severity of lung damage and the alveolar and lung vascular development were significantly improved in a dose dependent manner at the10th and14th day.2、The mean linear intercept(MLI) in APS treatment group were significant low and the mean alveolar number(MAN) per square area were significant high compared with time matched BPD group (p<0.05or p<0.01).3、The activity of SOD significantly decreased and the content of MDA significantly increased compared with the control group at the14th day in the BPD group. For HD APS group, the activity of SOD was increased and the content of MDA was reduced compared with BPD group at any time point, while this result was only seen at the10th day on LD APS group (Table1). There was no significant difference for the activity of SOD and the content of MDA in RA plus high and low-dose APS group compared with control group at any time point.4、From this study, both the number of neutrophils and macrophages were significantly increased in the BPD group compared with control group at any time points (p<0.01, Table4). The counts of neutrophils and macrophages were significantly decreased in APS groups compared with the BPD group (p<0.05or p<0.01). No marked differences were apparent between control group and room air plus APS group at any time points.5、The western blotting and Real Time PCR results showed that the expressions of NF-κBp65, Bax, TNF-α and ICAM-1in the BPD model group were increased compared with control group at the4th,10th,14th day, respectively (p<0.05or p<0.01). In APS groups, the protein expressions of NF-κBp65, Bax, TNF-α and ICAM-1were significantly decreased in a dose-dependent manner compared with the BPD model group. The expression of EGF7、Bcl-2and CD31in BPD groups were decreased as compared with control group. Besides, vascular density in BPD group was decreased compared with control group. Protein expressions of NF-κBp65, Bax, TNF-α, ICAM-1, EGFL7, Bcl-2and CD31in RA plus high-dose APS groups and RA plus low-dose APS groups were not significantly different from control group at any time points.ConclusionsOur results not only contribute to the understanding of the molecular mechanism of APS function in the hyperoxia-induced BPD rats, but also indicate the possible mechanism scavenge oxidative-free radicals, down-regulate some of proinflammatory mediators and adhesion molecules involved in the immune and inflammatory responses, including TNF-α, NF-κBp65, ICAM-1, which could be a preferred strategy for molecular intervention of pathological process, such as preventive effects on lung injury. |
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