钙敏感受体对持续性肺动脉高压新生小鼠内皮型一氧化氮合酶及一氧化氮的影响
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摘要
目的探讨钙敏感受体(CaSR)在持续性肺动脉高压(PPH)新生小鼠模型中对内皮型一氧化氮合酶(eNOS)表达及一氧化氮(NO)浓度的影响。方法将80只新生C57BL/6小鼠随机分为对照组、PPH组、激动剂组和抑制剂组。对照组小鼠暴露于空气中,PPH组、激动剂组和抑制剂组小鼠暴露于12%的氧浓度中。激动剂组和抑制剂组分别腹腔注射CaSR激动剂(GdCl_3)16 mg/kg、CaSR抑制剂(NPS2390)1 mg/kg,PPH组和对照组以生理盐水替代,共14 d。采用苏木精-伊红染色检测各组小鼠肺泡和肺血管变化;采用Western blot、qRT-PCR和免疫组化检测各组小鼠肺组织中e NOS蛋白、mRNA的表达;采用ELISA法分别检测肺组织匀浆中脑利钠肽(BNP)及NO的含量。结果与对照组相比,PPH组和激动剂组肺泡平均内衬间隔、肺小动脉血管壁厚度、右心室与左心室壁厚度比(RV/LV)及BNP浓度均明显增大,径向肺泡计数明显减少(P<0.05);除RV/LV外,上述指标在抑制剂组均较PPH组和激动剂组有所改善(P<0.05)。与对照组相比,eNOS蛋白、mRNA表达量及NO浓度在PPH组明显增高,在激动剂组中表达水平进一步增加,而在抑制剂组中表达减少(P<0.05)。结论 CaSR可能通过影响eNOS的表达和NO浓度在新生小鼠PPH发病中发挥重要作用。
Objective To study the effect of calcium-sensitive receptors(CaSR) on the expression of endothelial nitric oxide synthase(eNOS) and the concentration of nitric oxide(NO) in a neonatal mouse model of persistent pulmonary hypertension(PPH). Methods Eighty neonatal C57BL/6 mice were randomly divided into control, PPH, agonist and antagonist groups. The control group was exposed to air, and the other three groups were exposed to 12% oxygen. The agonist and antagonist groups were intraperitoneally injected with a CaSR agonist(GdCl_3 16 mg/kg) and a CaSR antagonist(NPS2390, 1 mg/kg), respectively, while the PPH and control groups were intraperitoneally injected with normal saline instead. All mice were treated for 14 days. Alveolar development and pulmonary vessels were assessed by hematoxylin-eosin staining. The protein and mRNA expression of eNOS and its localization in lung tissues were determined by Western blot, qRT-PCR and immunohistochemistry. The levels of brain natriuretic peptide(BNP) and NO in lung homogenate were determined using ELISA. Results Compared with the control group, the PPH and agonist groups showed significant increases in alveolar mean linear intercept, the percent wall thickness of pulmonary arterioles, right to left ventricular wall thickness ratio(RV/LV) and BNP concentration, but a significant reduction in radial alveolar count(P<0.05). The antagonist group had significant improvements in all the above indices except RV/LV compared with the PPH and agonist groups(P<0.05). Compared with those in the control group, the protein and m RNA expression of e NOS and NO concentration were significantly increased in the PPH group and increased more significantly in the agonist group, but were significantly reduced in the antagonist group(P<0.05). Conclusions Ca SR plays an important role in the development of PPH in neonatal mice, possibly by increasing e NOS expression and NO concentration.
Objective To study the effect of calcium-sensitive receptors(CaSR) on the expression of endothelial nitric oxide synthase(eNOS) and the concentration of nitric oxide(NO) in a neonatal mouse model of persistent pulmonary hypertension(PPH). Methods Eighty neonatal C57BL/6 mice were randomly divided into control, PPH, agonist and antagonist groups. The control group was exposed to air, and the other three groups were exposed to 12% oxygen. The agonist and antagonist groups were intraperitoneally injected with a CaSR agonist(GdCl_3 16 mg/kg) and a CaSR antagonist(NPS2390, 1 mg/kg), respectively, while the PPH and control groups were intraperitoneally injected with normal saline instead. All mice were treated for 14 days. Alveolar development and pulmonary vessels were assessed by hematoxylin-eosin staining. The protein and mRNA expression of eNOS and its localization in lung tissues were determined by Western blot, qRT-PCR and immunohistochemistry. The levels of brain natriuretic peptide(BNP) and NO in lung homogenate were determined using ELISA. Results Compared with the control group, the PPH and agonist groups showed significant increases in alveolar mean linear intercept, the percent wall thickness of pulmonary arterioles, right to left ventricular wall thickness ratio(RV/LV) and BNP concentration, but a significant reduction in radial alveolar count(P<0.05). The antagonist group had significant improvements in all the above indices except RV/LV compared with the PPH and agonist groups(P<0.05). Compared with those in the control group, the protein and m RNA expression of e NOS and NO concentration were significantly increased in the PPH group and increased more significantly in the agonist group, but were significantly reduced in the antagonist group(P<0.05). Conclusions Ca SR plays an important role in the development of PPH in neonatal mice, possibly by increasing e NOS expression and NO concentration.
引文
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[4]王萌萌,李贺,张方方,等.钙敏感受体在新生小鼠持续性肺动脉高压中的作用[J].中国当代儿科杂志,2017,19(2):208-214.
[5]李贺,谷强,王萌萌,等.钙敏感受体对低氧诱导的持续性肺动脉高压小鼠肺动脉平滑肌细胞内钙离子浓度的影响[J].中国新生儿科杂志,2018,33(1):59-64.
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[9]Xu XF,Gu WZ,Wu XL,et al.Fetal pulmonary vascular remodeling in a rat model induced by hypoxia and indomethacin[J].J Matern Fetal Neonatal Med,2011,24(1):172-182.
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[12]Rudic RD,Shesely EG,Maeda N,et al.Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling[J].J Clin Invest,1998,101(4):731-736.
[13]Kruzliak P,Maruyama J,Maruyama K.Role of nitric oxide in pathophysiology and treatment of pulmonary hypertension[J].Vitam Horm,2014,96:407-424.
[14]Komai H,Naito Y,Aimi Y,et al.Nitric oxide synthase expression in lungs of pulmonary hypertensive patients with heart disease[J].Cardiovasc Pathol,2001,10(1):29-32.
[15]曹静,朱艳萍,李明霞.缺氧诱导因子-1α及下游因子在缺氧性肺动脉高压新生大鼠肺内的表达研究[J].中国新生儿科杂志,2014,29(3):194-199.
[16]桑葵,周英,李明霞.缺氧诱导因子-1α及其调控因子在缺氧性肺动脉高压新生大鼠肺组织的蛋白质表达研究[J].中华儿科杂志,2012,50(12):919-924.
[2]Drummond WH.Neonatal pulmonary hypertension[J].Equine Vet J,1987,19(3):169-171.
[3]Godo S,Sawada A,Saito H,et al.Disruption of physiological balance between nitric oxide and endothelium-dependent hyperpolarization impairs cardiovascular homeostasis in mice[J].Arterioscler Thromb Vasc Biol,2016,36(1):97-107.
[4]王萌萌,李贺,张方方,等.钙敏感受体在新生小鼠持续性肺动脉高压中的作用[J].中国当代儿科杂志,2017,19(2):208-214.
[5]李贺,谷强,王萌萌,等.钙敏感受体对低氧诱导的持续性肺动脉高压小鼠肺动脉平滑肌细胞内钙离子浓度的影响[J].中国新生儿科杂志,2018,33(1):59-64.
[6]Ambalavanan N,Bulger A,Murphy-Ullrich J,et al.Endothelin-A receptor blockade prevents and partially reverses neonatal hypoxic pulmonary vascular remodeling[J].Pediatr Res,2005,57(5 Pt 1):631-636.
[7]Knudsen L,Weibel ER,Gundersen HJ,et al.Assessment of air space size characteristics by intercept(chord)measurement:An accurate and efficient stereological approach[J].J Appl Physiol(1985),2010,108(2):412-421.
[8]Cooney TP,Thurlbeck WM.The radial alveolar count method of emery and mithal:a reappraisal 1-postnatal lung growth[J].Thorax,1982,37(8):572-579.
[9]Xu XF,Gu WZ,Wu XL,et al.Fetal pulmonary vascular remodeling in a rat model induced by hypoxia and indomethacin[J].J Matern Fetal Neonatal Med,2011,24(1):172-182.
[10]Wedgwood S,Lakshminrusimha S,Schumacker PT,et al.Hypoxia inducible factor signaling and experimental persistent pulmonary hypertension of the newborn[J].Front pharmacol,2015,6:47.
[11]Siragusa M,Fleming I.The e NOS signalosome and its link to endothelial dysfunction[J].Pflugers Arch,2016,468(7):1125-1137.
[12]Rudic RD,Shesely EG,Maeda N,et al.Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling[J].J Clin Invest,1998,101(4):731-736.
[13]Kruzliak P,Maruyama J,Maruyama K.Role of nitric oxide in pathophysiology and treatment of pulmonary hypertension[J].Vitam Horm,2014,96:407-424.
[14]Komai H,Naito Y,Aimi Y,et al.Nitric oxide synthase expression in lungs of pulmonary hypertensive patients with heart disease[J].Cardiovasc Pathol,2001,10(1):29-32.
[15]曹静,朱艳萍,李明霞.缺氧诱导因子-1α及下游因子在缺氧性肺动脉高压新生大鼠肺内的表达研究[J].中国新生儿科杂志,2014,29(3):194-199.
[16]桑葵,周英,李明霞.缺氧诱导因子-1α及其调控因子在缺氧性肺动脉高压新生大鼠肺组织的蛋白质表达研究[J].中华儿科杂志,2012,50(12):919-924.