Association of Insulin-like Growth Factors with Lung Development in Neonatal Rats

To explore the relationship between Insulin-like growth factor (IGF)- Ⅰ, -Ⅱ and lung development in neonatal rats. 80 timed pregnant Sprague-Dawley ( SD) rats were randomly divided into 4 groups (n=20): group A (Control group), group B (Dexamethasone (DEX) 1 group), group C (DEX 2 group), group D (retinoic acid (RA) group). 20 pregnant rats in group A, B and D were injected subcutaneously or intraperitoneally with vehicle (NS), DEX, or RA respectively during gestational day 16 to 18. All newborn rats in group C were subcutaneously injected with DEX at day 1 to 3 after birth. The lung tissue was obtained at the following times: fetuses at gestational ages of 18, 20 and 21 days, and 1, 3, 5, 7, 10, 14 and 21 days after birth. Lung tissues were used for histopathological study, the polypeptides analysis of IGF-Ⅰ, -Ⅱ (immunohistochemistry and Western blot) and mRNA analysis ( RT- PCR). The results showed that the strongest expression of IGF-Ⅰ in group A and D occurred at ages of 5-7 days (alveolar stage). The stronger their expressions, the better the alveolar develop. The peak stage of expression in group B occurred earlier, on the day 3 after birth. Compared with group A, the expression of IGF-Ⅰ during gestation age of 18 days to age of 3 days in group B were significantly higher (P<0.01), but significantly lower at other time points (P<0.01). The expression of IGF-Ⅰ was lower in group C all the time and always higher in group D than those in group A (P<0.01). The peak expression of IGF-Ⅱ took place at the gestation age of 18 days, then gradually dropped to trace. During 18 days of gestation to age of 3 days, the expression of IGF-Ⅱ in group B was significantly higher than that in group A (P<0.01). No difference was found among all other groups. The change in the expression of IGF-Ⅰ, -Ⅱ mRNA in all 4 groups was similar to that of their polypeptides. The results suggested that there is a close linking between IGF-Ⅰ, -Ⅱ and lung development in newborns. The IGF-Ⅱ works at early stage and the that of IGF-Ⅰ works at the stage of new septa formation and alveoli maturation. The stronger their expressions, the more mature the lung development.

Effects of Hyperoxia on the Dynamic Expression of Aquaporin5 in Premature Rats Lung Development

To explore the dynamic expression and role of Aquaporin5 ( AQP5) in lung development and hyperoxia lung injury, gestation 21-day Sprague-Dawley (SD) rats (term=22 days) were ran- domly assigned to air group and hyperoxia group within 12-24 h after birth. The rats in hypreoxia group were continuously exposed to about 85% oxygen and those in air group to room air. After 1 to 14 days of exposure, total lung RNA was extracted and the expression of AQP5 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry and west- ern-blot were used to detect the expression of AQP5 protein. The results showed that the expression of AQP5 in premature rats lung could be detected at various time points after birth, and the positive staining was restricted to the type Ⅰ alveolar epithelial cells. In air group, the AQP5 expression was detected in a very low level at day 1, but exhibited a persistent increase after birth. Compared with the air group, the expression of AQP5 in hyperoxia group was increased at day 1, and had significant difference in mRNA level (P<0.05), but decreased significantly in mRNA and protein levels after 4 to 14 days (P<0.01 or P<0.05 respectively). It was concluded that AQP5 might play a key role in the alveolar period of premature rats by regulating the lung water balance. Hyperoxia exposure leads to a down-regulation of the AQP5 expression, which may be an important factor for the development of hyperoxia lung injury.

Effect of Retinoic acid on Platelet-derived Growth Factorand Lung Development in Newborn Rats

Summary: The influence of platelet-derived growth factor (PDGF) on lung development in newborn rats and the effect of retinoic acid (RA) on PDGF in lung development were investigated. Newborn Sprague-Dawley (SD) rats were randomly assigned to two groups: control group and RA group. The rats in RA group was intraperitoneally injected with all trans-retinoic acid (500 μg/kg every day) for consecutive 3 days after birth, while those in the control group were not subjected to intervention. Immunohistochemical assay was performed to locate the expression of PDGF. mRNA levels of PDGF were measured by reverse transcription polymerase chain reaction (RT-PCR) at age of 1, 3, 5, 7, 10, 14, 21 days. The method of radial alveolar counts (RAC) was used to measure the amount of the alveoli of the lungs. It was found that with increasing days, levels of PDGF-A and PDGF-B changed to verying degrees. RA could elevate significantly the expression levels of PDGF-A mRNA and protein (P<0.01), but not affect the expression levels of PDGF-B mRNA and protein markedly (P>0.05). It is suggested that PDGF might play an important role in lung development. RA can stimulate lung development through increasing the expression levels of PDGF-A mRNA and protein.

TEMPORAL EXPRESSION OF NOTCH RECEPTORS DURING LUNG DEVELOPMENT IN RAT

Objective To investigate the temporal expression of Notch receptors in developing lungs of rats and to explore the regulating role of Notch in lung development. Methods We studied the expression of Notch1,2,3 isforms in embryonic days 18,20,21 and postnatal days 1,4,7,14, 21 rat lungs. Six rats of each group were used to assess lung histologic changes by HE staining and expression of Notch in lungs by immunohistochemistry. Total RNA was extracted by Trizol reagent from the frozen lung tissues. mRNA levels of Notch were measured by reverse transcription polymerase chain reaction (RT-PCR). Results It is showed that Notch_ 1-3 mainly localized in the airway surface epithelium、alveolar epithelium during the psdueoglandular stage, and reached the peaks at canalicular period. The expression patterns of Notch_ 1-3 were changed with the fetal age. Conclusion These results support multiple roles for Notch1,2,and 3 receptor activation during lung development, probably not only modulating the process of branching morphogenesis but also involved in determining the cell differentiation fate in fetal alveolar epithelium.

Emerging role of cellular senescence in normal lung development and perinatal lung injury

Cellular senescence is a status of irreversible growth arrest,which can be triggered by the p53/p21cip1 and p16INK4/Rb pathways via intrinsic and external factors.Senescent cells are typically enlarged and flattened,and characterized by numerous molecular features.The latter consists of increased surfaceome,increased resid-ual lysosomal activity at pH 6.0(manifested by increased activity of senescence-associated beta-galactosidase[SA-𝛽-gal]),senescence-associated mitochondrial dysfunction,cytoplasmic chromatin fragment,nuclear lamin b1 exclusion,telomere-associated foci,and the senescence-associated secretory phenotype.These features vary depending on the stressor leading to senescence and the type of senescence.Cellular senescence plays pivotal roles in organismal aging and in the pathogenesis of aging-related diseases.Interestingly,senescence can also both promote and inhibit wound healing processes.We recently report that senescence as a programmed pro-cess contributes to normal lung development.Lung senescence is also observed in Down Syndrome,as well as in premature infants with bronchopulmonary dysplasia and in a hyperoxia-induced rodent model of this disease.Furthermore,this senescence results in neonatal lung injury.In this review,we briefly discuss the molecular features of senescence.We then focus on the emerging role of senescence in normal lung development and in the pathogenesis of bronchopulmonary dysplasia as well as putative signaling pathways driving senescence.Finally,we discuss potential therapeutic approaches targeting senescent cells to prevent perinatal lung diseases.

Lung development and regeneration:newly defined cell types and progenitor status

The lung is the most critical organ of the respiratory system supporting gas exchange.Constant interaction with the external environment makes the lung vulnerable to injury.Thus,a deeper understanding of cellular and molecular processes underlying lung development programs and evaluation of progenitor status within the lung is an essential part of lung regenerative medicine.In this review,we aim to discuss the current understanding of lung development process and regenerative capability.We highlight the advances brought by multi-omics approaches,single-cell transcriptome,in particular,that can help us further dissect the cellular player and molecular signaling underlying those processes.

Comparison of protective effects of electroacupuncture and moxibustion at Zusanli(ST 36)on perinatal nicotine exposure-induced lung phenotype in rat offspring

Objective:To compare the effects of electroacupuncture(EA)and moxibustion at Zusanli(ST 36)on the lung phenotype of rat offspring exposed to nicotine during the perinatal period.Methods:Pregnant Sprague-Dawley rats were randomly divided into 4 groups:the control group(saline only),the model group(nicotine only),the EA group(nicotine+EA at ST 36 acupoints bilaterally),and the moxibustion group(nicotine+moxibustion at ST 36 acupoints bilaterally).n=6 rats per group.On postnatal day 21,the body weight,lung weight,and pulmonary function were determined and lung morphometry was performed.Peroxisome proliferator-activated receptor gamma andβ-catenin levels in the lung tissue of offspring were also determined.Results:Perinatal nicotine exposure(PNE)results in decreased body and lung weights of offspring rats,abnormal lung tissue morphology,and significantly altered pulmonary function,showing an increase in total airway resistance and a decrease in tidal volume,minute ventilation,total airway compliance,and peak expiratory flow.Bilateral EA at ST 36 acupoints could block all of these perinatal nicotine-induced effects.Although moxibustion also had protective effects in nicotine-induced offspring lungs,some of these effects did not reach statistical significance,e.g.,protection against the upregulation ofβ-catenin,the downregulation of PPARγsignaling,and the increase in peak expiratory flow.Conclusion:Maternal EA at ST 36 blocked the PNE-induced changes in key developmental signaling pathways,prevented the PNE-induced changes in lung morphology,and protected pulmonary function.Moxibustion at ST 36 showed similar but weaker protective effects against the PNE-induced changes in the exposed offspring.It is important to note that the mechanism underlying the protective effects of moxibustion at ST 36 may be different from those of EA at ST 36,and further research is needed to understand these differences.

Expression of Lung Surfactant Proteins SP-B and SP-C and Their Modulating Factors in Fetal Lung of FGR Rats

This study investigated the expression of lung surfactant proteins SP-B and SP-C, and their modulating factors TTF-1 and PLAGL2 in the fetal lung of rats with fetal growth restriction（FGR）. The rat FGR model was established by prenatal hypoxia in the first stage of pregnancy, 180 rats for experiment served as hypoxia group, and 197 healthy rats served as normal control group. The FGR incidence in hypoxia was compared with that in normal control group. The histological changes in the fetal lung were observed under the light microscope and electronic microscope in two groups. The SP-B, SP-C, TTF-1 and PLAGL2 proteins were determined in the fetal lung of two groups immunohistochemically. The expression levels of SP-B, SP-C, TTF-1 and PLAGL2 protein and m RNA in the fetal lung of two groups were detected by using Western blotting and RT-PCR respectively. The FGR rat model was successfully established by using hypoxia. Pathologically the fetal lung developed slowly, and the expression levels of SP-B, SP-C, TTF-1 and PLAGL2 protein and mR NA in the fetal lung were significantly reduced in hypoxia group as compared with those in normal control group. It was suggested that maternal hypoxia in the first stage of pregnancy could induce FGR, and reduce the expression of SP-B and SP-C, resulting in the disorder of fetal lung development and maturation.

Effect of Retinoic Acid on Lung Injury in Hyperoxia-Exposed Newborn Rats

To investigate whether treatment with retinoic acid (RA) could improve level of lung alveolarization and influence lung collagen in newborn rats exposed to hyperoxia, newborn Sprague-Dawley rats aged 2 days were randomly assigned to 8 groups:(1) air, (2) O 2, (3) air+NS, (4) O 2+NS, (5) air+dex, (6) O 2+dex, (7) air+RA and (8) O 2+RA. Group 2, 4 6 and 8 were kept in chambers containing 85 % oxygen, the values were checked 3 times a day. The other 4 groups were exposed to room air. Level of alveolarization and lung collagen were analyzed at age of 14 or 21 days through radial alveolar counts, alveolar airspace measurements, type Ⅰ, Ⅲ collagen immunohistochemical methods (SP method) and image processing system. Transforming growth factor-β receptors and procollagen mRNA accumulation were examined at age of 14 days through immunohistochemical methods and in situ hybridization. Our results showed that radial alveolar counts were increased and distal airspace was enlarged in group 8. TypeⅠcollagen was markedly increased, and transforming growth factor-β receptors and procollagen mRNA were decreased by retinoic acid in bronchial epithelial cells, alveolar epithelial cells and alveolar intersitium. It is concluded that retinoic acid can partially reverse lung development arrest during exposure to hyperoxia by increasing lung collagen.

Cigarette Smoke Induces Apoptosis by Activation of Caspase-3 in Isolated Fetal Rat Lung Type II Alveolar Ep-ithelial Cells <i>in Vitro</i>

Smoking during pregnancy is a major source of fetal exposure to numerous harmful agents present in tobacco smoke. Lung development involves complex biochemical processes resulting in dramatic changes which continue even after birth. In addition to type I cells which form the blood-air barrier, type II alveolar epithelial (AE) cells have important and diverse functions related to immunological protection and stabilization of the alveolus through synthesis and secretion of the pulmonary surfactant. Apoptosis or programmed cells death is an important physiological process during lung embryogenesis and for the proper maintenance of homeostasis. Caspases are proteases that play important roles in regulating apoptosis. Caspase-3 is the key executioner caspase in the cascade of events leading to cell death by apoptosis. We explored the hypothesis that cigarette smoke extract (CSE) induces apoptosis in fetal rat lung type II AE cells by activation of caspase-3. To analyze these factors, isolated fetal rat lung type II AE cells were used. The cells were exposed to different concentrations of CSE (5%, 10% or 15%) (v/v) for 60 min. The results of the present study showed that CSE induced apoptosis in fetal rat lung type II AE cells with a significant increase (p 0.05) in caspase-3 activity and decrease in cell proliferation at CSE concentrations of 10% and 15% (v/v). These observations indicate that cigarette smoke extract induces apoptosis by activation of caspase-3 in fetal rat lung type II AE cells in a dose-dependent manner and may potentially alter the regulated development of the lung and the appearance of the surfactant-producing type II alveolar cells which are critical for the establishment of adequate gas exchange at birth.

Fetal lung growth predicts the risk for early-life respiratory infections and childhood asthma

Background Early-life respiratory infections and asthma are major health burdens during childhood.Markers predicting an increased risk for early-life respiratory diseases are sparse.Here,we identified the predictive value of ultrasound-monitored fetal lung growth for the risk of early-life respiratory infections and asthma.Methods Fetal lung size was serially assessed at standardized time points by transabdominal ultrasound in pregnant women participating in a pregnancy cohort.Correlations between fetal lung growth and respiratory infections in infancy or early-onset asthma at five years were examined.Machine-learning models relying on extreme gradient boosting regressor or classifier algorithms were developed to predict respiratory infection or asthma risk based on fetal lung growth.For model development and validation,study participants were randomly divided into a training and a testing group,respectively,by the employed algorithm.Results Enhanced fetal lung growth throughout pregnancy predicted a lower early-life respiratory infection risk.Male sex was associated with a higher risk for respiratory infections in infancy.Fetal lung growth could also predict the risk of asthma at five years of age.We designed three machine-learning models to predict the risk and number of infections in infancy as well as the risk of early-onset asthma.The models’R2 values were 0.92,0.90 and 0.93,respectively,underscoring a high accuracy and agreement between the actual and predicted values.Influential variables included known risk factors and novel predictors,such as ultrasound-monitored fetal lung growth.Conclusion Sonographic monitoring of fetal lung growth allows to predict the risk for early-life respiratory infections and asthma.

Loss of Hox5 function results in myofibroblast mislocalization and distal lung matrix defects during postnatal development

Alveologenesis is the final stage of lung development and is responsible for the formation of the principle gas exchange units called alveoli. The lung mesenchyme, in particular the alveolar myofibroblasts, are drivers of alveolar development, however,few key regulators that govern the proper distribution and behavior of these cells in the distal lung during alveologenesis have been identified. While Hox5 triple mutants(Hox5 aabbcc) exhibit neonatal lethality, four-allele, compound mutant mice(Hox5 AabbCc) are born in Mendelian ratios and are phenotypically normal at birth. However, they exhibit defects in alveologenesis characterized by a BPD-like phenotype by early postnatal stages that becomes more pronounced at adult stages. Invasive pulmonary functional analyses demonstrate significant increases in total lung volume and compliance and a decrease in elastance in Hox5 compound mutants. SMA+ myofibroblasts in the distal lung are distributed abnormally during peak stages of alveologenesis and aggregate, resulting in the formation of a disrupted elastin network. Examination of other key components of the distal lung ECM, as well as other epithelial cells and lipofibroblasts reveal no differences in distribution. Collectively, these data indicate that Hox5 genes play a critical role in alveolar development by governing the proper cellular behavior of myofibroblasts during alveologenesis.

大通牦牛与枣北黄牛肺组织发育学特点比较

本试验选取不同发育阶段大通牦牛和枣北黄牛作为研究对象,运用苏木精-伊红(H.E.)染色、Verhoeff's Van Gieson(EVG)染色等方法,观测其肺组织的显微结构。结果显示:随年龄增长,大通牦牛肺泡隔厚度(MAST)逐渐增厚,与枣北黄牛相比1日龄和成年时差异显著(P<0.05),大通牦牛单位面积内肺泡数(MAN)先增长后减少,随年龄增长,肺泡壁弹性纤维占比和血管弹性纤维占比先降低后升高再降低,血管中膜占比呈现先升高再降低的特点;与枣北黄牛相比,大通牦牛具有出生时肺泡隔较薄,成年后肺泡隔较厚,单位面积内肺泡数较多,肺实质占比较多,幼龄时肺泡壁弹性纤维较少,成年后较多,血管弹性纤维含量较多的特点。结果表明,大通牦牛在不同发育阶段肺组织的结构特点是其适应高原低氧环境的组织学基础。本试验结果为高原牦牛低氧适应的研究提供了基础数据。

敲减NEDD1表达抑制肺腺癌细胞增殖与迁移

目的:探讨神经前体细胞表达发育下调蛋白1(NEDD1)在肺癌发生和发展中的作用。方法:采用免疫组化实验检测NEDD1在肺癌组织中的表达情况,结合数据库资料分析NEDD1在肺癌组织与癌旁正常组织中的表达有无差异,并分析NEDD1的表达水平与肺癌预后的相关性;敲减NEDD1在A549细胞的表达后用平板集落形成实验检测集落形成能力的变化,流式细胞术检测细胞凋亡和细胞周期分布的情况,Transwell小室实验检测对细胞的迁移能力的影响,Western blot检测细胞周期相关蛋白的表达,并分析NEDD1与周期蛋白依赖性激酶2(CDK2)表达的相关性。结果:与正常肺组织相比,肺癌组织中NEDD1表达水平升高,Kaplan-Meier曲线预后分析显示NEDD1高表达者预后差,NEDD1低表达者预后较好;光镜下观察可见敲减NEDD1表达的A549细胞增殖速度明显减慢,平板集落形成能力降低(P<0.05);与对照组相比,敲减NEDD1表达促进A549细胞凋亡,抑制细胞的迁移;与对照组相比,敲减NEDD1表达后G 1期细胞的比例增加,细胞阻滞于G 1/S期,Western blot检测周期相关蛋白水平可见p-Rb和cyclinD1蛋白表达水平降低,p-Chk1、p-Chk2和p-p53蛋白表达水平升高(P<0.05);数据库分析结果示NEDD1与CDK2的表达呈正相关。结论:NEDD1具有抑制肺癌细胞凋亡、加速细胞周期进程从而促进细胞增殖的能力,且其在肺腺癌组织中的高表达与患者预后不良相关,因此可以作为肺腺癌诊断和预后新的标志分子。

长链非编码RNA TDRG1表达对非小细胞肺癌预后判断的价值

目的:探讨长链非编码RNA睾丸发育相关基因1(lncRNA TDRG1)对非小细胞肺癌(NSCLC)预后判断的价值。方法:采用实时荧光定量聚合酶链反应(qRT-PCR)检测60例NSCLC组织和癌旁组织中lncRNA TDRG1的表达水平。Kaplan-Meier方法评估总生存期,Cox比例风险模型进行单因素和多因素分析。结果:NSCLC组织中lncRNA TDRG1的表达显著高于癌旁组织。高表达lncRNA TDRG1与TNM分期及淋巴结转移密切相关。lncRNA TDRG1高表达患者总体生存时间较低表达患者明显缩短。单因素和多因素分析进一步确定高表达lncRNA TDRG1可作为患者总体生存时间的独立预测因子。结论:lncRNA TDRG1在NSCLC组织中高表达与总体生存时间相关,可作为NSCLC进展的潜在预后标志物。

Chronic lung disease, lung regeneration and future therapeutic strategies

Chronic lung diseases have been recognized as one of the world's leading causes of death in recent decades. Lacking effective treatments brings the patients not only bad quality of life but also higher risk for lung cancer development. By increasing the understanding of deeper mechanism of how lung develops and regenerates, researchers now focus on studying lung regenerative medicine, aiming to apply different and more efficient therapies to treat chronic lung diseases. This review will provide a wide picture of both basic lung developmental, regeneration mechanism and different designed strategies for treating chronic lung diseases in the future decades.