钙敏感受体在κ-阿片受体兴奋抗低氧性肺动脉高压中的作用及其机制

It has been suggested that calcium sensing receptor (CaSR) is involved in the pathogenesis of hypoxic pulmonary hypertension (HPH), such as pulmonary artery constriction, remodeling and inflammation. Our previous study suggests that the anti-HPH activity induced by κ-opioid receptor (κ-OR) stimulation is associated with inhibiting pulmonary artery constriction and remodeling. Recently, the preliminary experiment suggests that U50,488H, a κ-opioid receptor agonist, not only inhibits pulmonary artery smooth muscle cell proliferation, pulmonary artery constriction and remodeling, which are triggered by hypoxia, but inhibits CaSR expression in pulmonary artery and p38MAPK phosphorylation, suggesting that CaSR and its signaling pathway may be involved in the anti-HPH activity induced by κ-OR stimulation, but its underlying mechanism remains unclear. This project, therefore, aims to investigate the role of CaSR in the anti-HPH activity induced by κ-OR stimulation and its underlying mechanism in vivo and in vitro (vascular level and cellular level). The animals and methods include CaSR-knockout mice, CaSR siRNA, vascular function experiment, laser confocal and Western-Blot, etc. The expected results will provide new insights for HPH, and provide scientific basis for the clinical use of κ-OR agonist in the prevention and treatment of HPH.

已明确钙敏感受体（CaSR）参与了肺动脉收缩、重构和炎症等低氧性肺动脉高压（HPH）发生的环节。我们前期的研究表明k-阿片受体（k-OR）介导的抗HPH的作用与抑制肺动脉收缩和重构有关。近期的预实验结果显示k-OR激动剂U50,488H在抑制低氧诱导的肺动脉平滑肌细胞增殖、肺动脉收缩和重构的同时，还可抑制肺动脉上CaSR的蛋白表达及抑制p38MAPK蛋白的磷酸化，结果提示CaSR及其相关信号可能参与了k-OR介导的抗HPH的作用，但其潜在的机制尚不清楚。本研究拟采用CaSR基因敲除小鼠、针对CaSR的siRNA干扰、血管功能实验、激光共聚焦及Western-Blot等技术，从整体、血管和细胞水平，探讨CaSR在k-OR介导的抗低氧性肺血管收缩、肺血管重构以及抗炎症反应中的作用及其机制。预期结果可为HPH的研究提供新的思路和切入点，也可为临床使用k-阿片受体激动剂防治HPH提供科学依据。

低氧性肺高压（hypoxic pulmonary hypertension, HPH）主要表现为肺血管收缩和肺血管重构和炎症反应。研究表明，钙敏感受体（calcium sensing receptor，CaSR）在HPH形成过程中发挥了重要作用，可能会成为防治HPH的重要靶点。我们预实验表明，兴奋κ-阿片受体（κ-OR）可抑制肺动脉平滑肌细胞（pulmonary artery smooth muscle cells，PASMCs )上的CaSR的表达，但对其功能的影响尚不清楚。.结果表明：1.常氧时，κ-OR和CaSR在PASMCs均有表达，两受体存在共定位和相互作用关系。2.κ-OR对CaSR蛋白具有负性调节作用。3.血管张力测定实验，CaSR激动剂Spermine可收缩SD大鼠的肺动脉。Spermine对HPH大鼠肺动脉的收缩效应较正常时显著增加，且作用持续的时间也显著增长。U50,488H可显著抑制Spermine这一效应。并且发现激活CaSR诱导的肺血管收缩效应是通过兴奋MAPK信号介导的。4.CaSR另一激动剂新霉素可上调常氧和低氧组大鼠的RVP，且低氧组较常氧组升高的更为明显，持续时间更长。而预先给予U50,488H处理可抑制新霉素的这一作用。5.在低氧模型制备过程中，低氧前给予U50,488H和CaSR阻断剂NPS2143均可显著下调低氧诱导的SD大鼠的RVP和mPAP，而U50,488H的这一作用可被nor-BNI所阻断。此外，电镜结果显示，提前给予U50,488H和NPS2143均可显著改善低氧诱导的肺血管内皮结构紊乱和心脏结构紊乱。6.与常氧组相比，低氧组SD大鼠对内皮依赖性舒张剂ACh的反应性显著下降，而低氧前给予U50,488H和NPS2143均可显著增加肺血管对ACh的反应性。7.在细胞水平，激活CaSR可促进PASMCs增殖，这一作用通过MAPK信号介导的。另外，激活CaSR可显著促进正常和低氧PASMCs的PCNA和P-Erk的蛋白表达，U50,488H可显著抑制该作用，而U50,488H的这一作用又可被nor-BNI所阻断；8.κ-OR兴奋可显著抑制低氧诱导的炎症反应。.结果表明：κ-OR兴奋通过负性调节CaSR/MAPKs信号通路起到防治HPH。为临床应用阿片类药物防治HPH提供新的实验依据。