维生素D对高氧致新生鼠支气管肺发育不良的肺保护作用
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摘要
目的探讨维生素D对支气管肺发育不良的保护作用及其作用机制。方法应用高氧诱导的支气管肺发育不良新生小鼠模型,将36只幼鼠出生后立即给予实验处理,依据不同处理随机分为4组:空气+维生素D组,空气+生理盐水组,高氧+维生素D组,高氧+生理盐水组。将各组实验鼠称重后处死,同时抽取心室内血液,采用ELISA方法测定血清维生素D水平。分离肺组织病理切片制作和组织细胞形态学检查取不同处理组小鼠的肺组织,光镜下观察并分析肺终末呼吸单位的组织形态;用图像分析软件对辐射状肺泡计数、肺泡次级间隔体积密度,比较不同处理组的肺终末呼吸单位损伤参数;取不同处理组实验小鼠的肺组织,提取总蛋白,采用Western blot法检测血管内皮生长因子(VEGF)和VEGF-R2水平。结果空气+生理盐水组和空气+维生素D组的体质量增长速率大于高氧+生理盐水组;空气+生理盐水组和空气+维生素D组的肺组织重量大于高氧+生理盐水组(P<0.05)。辐射状肺泡计数,高氧+生理盐水组的RAC降低(P<0.001),而高氧+维生素D(1250倍)组的辐射状肺泡计数升高(P<0.001),且当稀释倍数降低,高氧+维生素D(125倍)组的辐射状肺泡计数比高氧+维生素D(1250倍)组升高(P>0.01)。与高氧+生理盐水组相比,高氧+维生素D(1250倍)组的次级突起计数显著升高(P<0.001),且当稀释倍数降低,高氧+维生素D(125倍)组的次级突起比高氧+维生素D(1250倍)组升高(P>0.01)。高氧+生理盐水组的VEGFR2表达量低于空气+生理盐水组(P<0.05),空气+维生素D组的VEGFR2表达量低于空气+生理盐水组(P<0.01);高氧+维生素D(1250倍)组的VEGFR2表达量高于高氧+生理盐水组(P<0.001)和高氧+维生素D(125倍)组(P<0.001),高氧+维生素D(125倍)组的表达量也高于高氧+生理盐水组(P<0.05)。但是,高氧+维生素D(1250倍)组的VEGFR2表达量高于高氧+生理盐水组与HE染色、辐射状肺泡计数以及次级突起计数的趋势相反。结论新生小鼠在支气管肺发育不良状态下增加维生素D可增加肺组织重量,为肺成熟提供物质基础,高浓度维生素D更有助于对肺的保护作用,有助于肺血管的生长。
Objective To investigate the protective effect of vitamin D(VD) against hyperoxia-induced bronchopulmonary dysplasia(BPD) in newborn mice and explore the mechanism. Methods Thirty-six newborn mice were randomly divided into air + VD group, air + saline group, hyperoxia + VD group, and hyperoxia + saline group. In all the groups, saline or VD was administered on a daily basis via intramuscular injection. After 3 weeks of treatment, the mice were weighed and cardiac blood was collected for measurement of serum VD level using ELISA, and histological examination of the lungs was performed.Radial alveolar counting(RAC) and alveolar secondary interval volume density were measured using image analysis software.The expression levels of vascular endothelial cell growth factor(VEGF) and VEGF receptor 2(VEGFR2) in the lung tissues were detected using Western blotting. Results The weight gain rate of the mice and the weight of the lungs were significantly higher in air + saline group and air + VD group than in the hyperoxia + saline group. The RAC was significantly lower in hyperoxic+saline group than that in hyperoxia+VD group(P<0.001), and was significantly higher in hyperoxic+VD(125 times)than in hyperoxia + VD(1250 times) group(P<0.01). The alveolar secondary protrusion count was significantly higher in hyperoxic + VD(1250 times) group than in hyperoxic + saline group(P<0.001), and was significantly higher in hyperoxia + VD(125 times) group than in hyperoxia + VD(1250 times) group(P<0.01). Compared with that in air + saline group, VEGFR2 expression was significantly lowered in hyperoxia + saline group(P<0.05) and in air + VD group(P<0.05); VEGFR2 expression was significantly higher in hyperoxia+VD(1250 times) group than in hyperoxia+saline group(P<0.001) and hyperoxia+VD(125 times) group(P<0.001); VEGFR2 expression was significantly higher in hyperoxia + VD(125 times) group than in hyperoxia +saline group(P<0.05). Conclusion In newborn mice with BPD, VD supplement can increase the weight of the lungs and promote lung maturation, and a higher concentration of VD can better protect the lungs and promote the growth of pulmonary blood vessels.
Objective To investigate the protective effect of vitamin D(VD) against hyperoxia-induced bronchopulmonary dysplasia(BPD) in newborn mice and explore the mechanism. Methods Thirty-six newborn mice were randomly divided into air + VD group, air + saline group, hyperoxia + VD group, and hyperoxia + saline group. In all the groups, saline or VD was administered on a daily basis via intramuscular injection. After 3 weeks of treatment, the mice were weighed and cardiac blood was collected for measurement of serum VD level using ELISA, and histological examination of the lungs was performed.Radial alveolar counting(RAC) and alveolar secondary interval volume density were measured using image analysis software.The expression levels of vascular endothelial cell growth factor(VEGF) and VEGF receptor 2(VEGFR2) in the lung tissues were detected using Western blotting. Results The weight gain rate of the mice and the weight of the lungs were significantly higher in air + saline group and air + VD group than in the hyperoxia + saline group. The RAC was significantly lower in hyperoxic+saline group than that in hyperoxia+VD group(P<0.001), and was significantly higher in hyperoxic+VD(125 times)than in hyperoxia + VD(1250 times) group(P<0.01). The alveolar secondary protrusion count was significantly higher in hyperoxic + VD(1250 times) group than in hyperoxic + saline group(P<0.001), and was significantly higher in hyperoxia + VD(125 times) group than in hyperoxia + VD(1250 times) group(P<0.01). Compared with that in air + saline group, VEGFR2 expression was significantly lowered in hyperoxia + saline group(P<0.05) and in air + VD group(P<0.05); VEGFR2 expression was significantly higher in hyperoxia+VD(1250 times) group than in hyperoxia+saline group(P<0.001) and hyperoxia+VD(125 times) group(P<0.001); VEGFR2 expression was significantly higher in hyperoxia + VD(125 times) group than in hyperoxia +saline group(P<0.05). Conclusion In newborn mice with BPD, VD supplement can increase the weight of the lungs and promote lung maturation, and a higher concentration of VD can better protect the lungs and promote the growth of pulmonary blood vessels.
引文
[1]Lykkedegn S,Sorensen GL,Beck-Nielsen SS,et al.Vitamin Ddepletion in pregnancy decreases survival time,Oxygen saturation,lung weight and body weight in preterm rat offspring[J].PLoS One,2016,86(1):102-13.
[2]HalaykoAJ,Tran T,Gosens R.Phenotype and functional plasticity of airway smooth muscle:role of caveolae and caveolins[J].Proc Am Thorac Soc,2008,5(1):80-8.
[3]Sakurai R,Shin E,Fonseca S,et al.1 alpha,25(OH)(2)D-3 and its 3-epimer promote rat lung alveolar epithelial-mesenchymal interactions and inhibit lipofibroblast apoptosis[J].Am J Physiol Lung Cell Mol Physiol,2009,297(3):L496-505.
[4]Liu CB,Chen Z,Li W,et al.Vitamin D enhances alveolar development in antenatal lipopolysaccharide-treated rats through the suppression of interferon-gamma production[J].Front Immunol,2018,8(12):1923-35.
[5]Lykkedegn S,Sorensen GL,Beck-Nielsen SS,et al.The impact of vitamin D on fetal and neonatal lung maturation.a systematic review[J].Am J Physiol Lung CellMol Physiol,2015,308(7):L587-602.
[6]黄铃沂,段江,封志纯.支气管肺发育不良小鼠模型的建立[J].中国实验动物学报,2009,17(3):186-8,247.
[7]Nadeau K,Montermini L,Mandeville I,et al.Modulation of Lgl1 by steroid,retinoic acid,and vitamin D models complex transcriptional regulation during alveolarization[J].Pediatr Res,2010,67(4):375-81.
[8]Saadoon A,Ambalavanan N,Zinn K,et al.Effect of prenatal versus postnatal vitamin D deficiency on pulmonary structure and function in mice[J].Am J Respir CellMol Biol,2017,56(3):383-92.
[9]Cetinkaya M,Cekmez F,Erener-Ercan T,et al.Maternal/neonatal vitamin D deficiency:a risk factor for bronchopulmonary dysplasia in preterms[J].J Perinatol,2015,35(10):813-7.
[10]Kazzi S,Karnati S,Puthuraya SA.Vitamin D deficiency and respiratory morbidity among African American very low birth weight infants[J].Early Hum Dev,2018,119(1):19-24.
[11]Hagaman JT,Panos RJ,Mccormack FX,et al.Vitamin D deficiency and reduced lung function in connective tissue-associated interstitial lung diseases[J].Chest,2011,139(2):353-60.
[12]Mandell E,Seedorf G,Gien J,et al.Vitamin D treatment improves survival and infant lung structure after intra-amniotic endotoxin exposure in rats:potential role for the prevention of bronchopulmonary dysplasia[J].Am J Physiol Lung Cell Mol Physiol,2014,306(5):L420-8.
[13]Kose M,Bastug O,Sonmez MF,et al.Protective effect of vitamin Dagainst hyperoxia-induced lung injury in newborn rats[J].Pediatr Pulmonol,2017,52(1):69-76.
[14]Zosky GR,Berry LJ,Elliot JG,et al.Vitamin D deficiency causes deficits in lung function and alters lung structure[J].Am J Respir Crit CareMed,2011,183(10):1336-43.
[15]Yang Y,Feng Y,Chen X,et al.Is there a potential Link between vitamin D and pulmonary morbidities in preterm infants[J].J Chin MedAssoc,2018,81(5):482-6.
[16]Konstantinopoulou S,Tapia IE.Vitamin D and the lung[J].Paediatr Respir Rev,2017,24(1):39-43.
[17]Joung KE,Burris HH,Van Marter LJ,et al.Vitamin D and bronchopulmonary dysplasia in preterm infants[J].J Perinatol,2016,36(10):878-82.
[18]Bozzetto S,Carraro S,Giordano G,et al.Asthma,allergy and respiratory infections:the vitamin D hypothesis[J].Allergy,2012,67(1):10-7.
[19]Wagner CL,Hollis BW.The implications of vitamin D status during pregnancy on mother and her developing child[J].Front Endocrinol(Lausanne),2018,9(4):500-12.
[20]Yang Y,Li Z,Yan G,et al.Effect of different doses of vitamin Dsupplementation on preterm infants-an updated meta-analysis[J].JMatern Fetal NeonatalMed,2018,31(22):3065-74.
[21]Mandell E,Powers KN,Harral JW,et al.Intrauterine endotoxininduced impairs pulmonary vascular function and right ventricular performance in infant rats and improvement with early vitamin Dtherapy[J].Am J Physiol Lung Cell Mol Physiol,2015,309(12):L1438-46.
[22]Schittny JC.Development of the lung[J].Cell Tissue Res,2017,367(3):427-44.
[23]Chen L,Wilson R,Bennett E,et al.Identification of vitamin Dsensitive pathways during lung development[J].Respir Res,2016,193(2):47-59.
[24]Karim T,MuhitM,Khandaker G.Interventions to prevent respiratory diseases-nutrition and the developing world[J].Paediatr Respir Rev,2017,22(1):31-7.
[25]Jobe AH.The new bronchopulmonary dysplasia[J].Curr Opin Pediatr,2011,23(2):167-72.
[26]Vasiliou JE,Lui S,Walker SA,et al.Vitamin D deficiency induces Th2 skewing and eosinophilia in neonatal allergic airways disease[J].Allergy,2014,69(10):1380-9.
[27]Reichetzeder C,Chen H,Foeller MA,et al.Maternal vitamin Ddeficiency and fetal programming-lessons learned from humans and mice[J].Kidney Blood Press Res,2014,39(4):315-29.
[2]HalaykoAJ,Tran T,Gosens R.Phenotype and functional plasticity of airway smooth muscle:role of caveolae and caveolins[J].Proc Am Thorac Soc,2008,5(1):80-8.
[3]Sakurai R,Shin E,Fonseca S,et al.1 alpha,25(OH)(2)D-3 and its 3-epimer promote rat lung alveolar epithelial-mesenchymal interactions and inhibit lipofibroblast apoptosis[J].Am J Physiol Lung Cell Mol Physiol,2009,297(3):L496-505.
[4]Liu CB,Chen Z,Li W,et al.Vitamin D enhances alveolar development in antenatal lipopolysaccharide-treated rats through the suppression of interferon-gamma production[J].Front Immunol,2018,8(12):1923-35.
[5]Lykkedegn S,Sorensen GL,Beck-Nielsen SS,et al.The impact of vitamin D on fetal and neonatal lung maturation.a systematic review[J].Am J Physiol Lung CellMol Physiol,2015,308(7):L587-602.
[6]黄铃沂,段江,封志纯.支气管肺发育不良小鼠模型的建立[J].中国实验动物学报,2009,17(3):186-8,247.
[7]Nadeau K,Montermini L,Mandeville I,et al.Modulation of Lgl1 by steroid,retinoic acid,and vitamin D models complex transcriptional regulation during alveolarization[J].Pediatr Res,2010,67(4):375-81.
[8]Saadoon A,Ambalavanan N,Zinn K,et al.Effect of prenatal versus postnatal vitamin D deficiency on pulmonary structure and function in mice[J].Am J Respir CellMol Biol,2017,56(3):383-92.
[9]Cetinkaya M,Cekmez F,Erener-Ercan T,et al.Maternal/neonatal vitamin D deficiency:a risk factor for bronchopulmonary dysplasia in preterms[J].J Perinatol,2015,35(10):813-7.
[10]Kazzi S,Karnati S,Puthuraya SA.Vitamin D deficiency and respiratory morbidity among African American very low birth weight infants[J].Early Hum Dev,2018,119(1):19-24.
[11]Hagaman JT,Panos RJ,Mccormack FX,et al.Vitamin D deficiency and reduced lung function in connective tissue-associated interstitial lung diseases[J].Chest,2011,139(2):353-60.
[12]Mandell E,Seedorf G,Gien J,et al.Vitamin D treatment improves survival and infant lung structure after intra-amniotic endotoxin exposure in rats:potential role for the prevention of bronchopulmonary dysplasia[J].Am J Physiol Lung Cell Mol Physiol,2014,306(5):L420-8.
[13]Kose M,Bastug O,Sonmez MF,et al.Protective effect of vitamin Dagainst hyperoxia-induced lung injury in newborn rats[J].Pediatr Pulmonol,2017,52(1):69-76.
[14]Zosky GR,Berry LJ,Elliot JG,et al.Vitamin D deficiency causes deficits in lung function and alters lung structure[J].Am J Respir Crit CareMed,2011,183(10):1336-43.
[15]Yang Y,Feng Y,Chen X,et al.Is there a potential Link between vitamin D and pulmonary morbidities in preterm infants[J].J Chin MedAssoc,2018,81(5):482-6.
[16]Konstantinopoulou S,Tapia IE.Vitamin D and the lung[J].Paediatr Respir Rev,2017,24(1):39-43.
[17]Joung KE,Burris HH,Van Marter LJ,et al.Vitamin D and bronchopulmonary dysplasia in preterm infants[J].J Perinatol,2016,36(10):878-82.
[18]Bozzetto S,Carraro S,Giordano G,et al.Asthma,allergy and respiratory infections:the vitamin D hypothesis[J].Allergy,2012,67(1):10-7.
[19]Wagner CL,Hollis BW.The implications of vitamin D status during pregnancy on mother and her developing child[J].Front Endocrinol(Lausanne),2018,9(4):500-12.
[20]Yang Y,Li Z,Yan G,et al.Effect of different doses of vitamin Dsupplementation on preterm infants-an updated meta-analysis[J].JMatern Fetal NeonatalMed,2018,31(22):3065-74.
[21]Mandell E,Powers KN,Harral JW,et al.Intrauterine endotoxininduced impairs pulmonary vascular function and right ventricular performance in infant rats and improvement with early vitamin Dtherapy[J].Am J Physiol Lung Cell Mol Physiol,2015,309(12):L1438-46.
[22]Schittny JC.Development of the lung[J].Cell Tissue Res,2017,367(3):427-44.
[23]Chen L,Wilson R,Bennett E,et al.Identification of vitamin Dsensitive pathways during lung development[J].Respir Res,2016,193(2):47-59.
[24]Karim T,MuhitM,Khandaker G.Interventions to prevent respiratory diseases-nutrition and the developing world[J].Paediatr Respir Rev,2017,22(1):31-7.
[25]Jobe AH.The new bronchopulmonary dysplasia[J].Curr Opin Pediatr,2011,23(2):167-72.
[26]Vasiliou JE,Lui S,Walker SA,et al.Vitamin D deficiency induces Th2 skewing and eosinophilia in neonatal allergic airways disease[J].Allergy,2014,69(10):1380-9.
[27]Reichetzeder C,Chen H,Foeller MA,et al.Maternal vitamin Ddeficiency and fetal programming-lessons learned from humans and mice[J].Kidney Blood Press Res,2014,39(4):315-29.