钙敏感受体可影响持续性肺动脉高压新生模型小鼠的肺血管重塑

Calcium-sensitive receptors influence pulmonary vascular remodeling in neonatal mice with persistent pulmonary hypertension

背景:课题组早期研究结果显示在新生小鼠持续性肺动脉高压模型中钙敏感受体的表达上调,在钙敏感受体激动剂组该上调趋势更加明显,而在钙敏感受体抑制剂组有所下降。目的:探讨钙敏感受体在低氧诱导的持续肺动脉高压新生小鼠肺血管重塑中的机制。方法:将80只刚出生的C57BL/6小鼠随机平均分成4个组。其中,低氧组、低氧+激动剂组(以下简称激动剂组)和低氧+抑制剂组(以下简称抑制剂组)同时放置于低氧箱中(氧浓度为12%),后2组分别每天1次腹腔内注射钙敏感受体激动剂Gd Cl3(16 mg/kg)或抑制剂NPS2390(1 mg/kg);常氧组小鼠暴露在空气中,与低氧组一样每天注射等量的生理盐水,共注射14 d,第15天麻醉后取出小鼠心肺组织。显微镜下测量各组小鼠心室壁及肺血管壁的厚度、观察肺泡形态学变化;使用酶联免疫吸附实验检测B型钠尿肽水平;使用Western bolt和免疫组化检测小鼠肺组织中小凹蛋白1蛋白的表达和定位,并使用qRT-PCR检测小凹蛋白1 mRNA的表达。实验方案经石河子大学医学院第一附属医院实验动物伦理委员会批准(批准号为2018-172-01)。结果与结论:(1)与常氧组比较,低氧14 d后低氧组小鼠肺小动脉血管壁增厚、右心室与左心室壁厚度比及B型钠尿肽水平增加,平均肺泡内衬间隔变宽、径向肺泡计数减少,Western bolt和qRT-PCR显示小凹蛋白1蛋白和mRNA表达上调(P<0.05);激动剂组变化更为显著(P<0.05);抑制剂组可逆转低氧所致的变化(P<0.05);(2)结果提示,钙敏感受体影响持续肺动脉高压新生小鼠肺组织血管的重塑,其作用机制可能与改变了小凹蛋白1的表达有关。

BACKGROUND:Previous studies have shown the up-regulated expression of calcium-sensitive receptors in the neonatal mouse model of persistent pulmonary hypertension.The up-regulation trend is more obvious in the calcium-sensitive receptor agonist group,while the expression is declined somewhat in the calcium-sensitive receptor inhibitor group.OBJECTIVE:To explore the mechanism of calcium-sensitive receptors in pulmonary remodeling in neonatal mice with hypoxia-induced persistent pulmonary hypertension.METHODS:Eighty newborn C57BL/6 mice were randomly divided into four groups.Mice in the hypoxic group,the hypoxic+agonist group(hereinafter referred to as agonist group),and the hypoxic+antagonist group(hereinafter referred to as antagonist group)were placed in a hypoxic chamber simultaneously(oxygen concentration 12%).In addition,the agonist and antagonist groups were given intraperitoneal injection of calcium-sensitive receptor agonist Gd Cl3(16 mg/kg)and inhibitor NPS2390(1 mg/kg)once a day respectively.Mice in the normoxia group were exposed to air and injected with the same amount of normal saline daily as that in the hypoxic group.After 14 days,heart and lung tissues were removed.The thickness of ventricular wall and pulmonary vascular wall was measured and alveolar morphology was observed.The level of B-type brain natriuretic peptide was determined by enzyme linked immunosorbent assay.Western blot and immunohistochemistry were used to detect the expression and localization of caveolin-1 protein in mouse lung tissue,and qRT-PCR was used to detect the expression of caveolin-1 mRNA.The study protocol was approved by the Experimental Animal Ethic Committee of the First Affiliated Hospital of Shihezi University School of Medicine(approval No.2018-172-01).RESULTS AND CONCLUSION:After 14 days of hypoxia,the wall thickness of small pulmonary arterioles in mice increased,and the thickness ratio of right ventricle to left ventricle and B-type brain natriuretic peptide level increased in the hypoxic group compared with the normoxia group.The average alveolar lining interval was widened and the radial alveolar count decreased in the hypoxic group compared with the normoxia group.Western blot and qRT-PCR results showed that the expression levels of caveolin-1 protein and mRNA were up-regulated hypoxic group compared with the normoxia group(P<0.05).These changes were more significant in the agonist group(P<0.05),while the inhibitor group reversed the changes caused by hypoxia(P<0.05).To conclude,calcium-sensitive receptor affects the vascular remodeling in the lung of neonatal mice with persistent pulmonary hypertension,and the mechanism may be related to the changes in the caveolin-1 expression.