Zhou Yaguang, Wang Yuanbo, Wang Xu, Tian Xin, Zhang Shumiao, Yang Fan, Guo Haitao, Fan Rong, Feng Na, Jia Min, Gu Xiaoming, Wang Yuemin, Li Juan, Pei Jianming
Department of Physiology, National Key Discipline of Cell Biology, Fourth Military Medical University, Xi'an, China.
Student brigade, Fourth Military Medical University, Xi'an, China.
Cell Physiol Biochem. 2017;44(5):1965-1979. doi: 10.1159/000485886. Epub 2017 Dec 8.
BACKGROUND/AIMS: In a previous study, we showed that κ-opioid receptor stimulation with the selective agonist U50,488H ameliorated hypoxic pulmonary hypertension (HPH). However, the roles that pulmonary arterial smooth muscle cell (PASMC) proliferation, apoptosis, and autophagy play in κ-opioid receptor-mediated protection against HPH are still unknown. The goal of the present study was to investigate the role of autophagy in U50,488H-induced HPH protection and the underlying mechanisms.
Rats were exposed to 10% oxygen for three weeks to induce HPH. After hypoxia, the mean pulmonary arterial pressure (mPAP) and the right ventricular pressure (RVP) were measured. Cell viability was monitored using the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected by flow cytometry and Western blot. Autophagy was assessed by means of the mRFP-GFP-LC3 adenovirus transfection assay and by Western blot.
Inhibition of autophagy by the administration of chloroquine prevented the development of HPH in the rat model, as evidenced by significantly reduced mPAP and RVP, as well as decreased autophagy. U50,488H mimicked the effects of chloroquine, and the effects of U50,488H were blocked by nor-BNI, a selective κ-opioid receptor antagonist. In vitro experiments showed that the inhibition of autophagy by chloroquine was associated with decreased proliferation and increased apoptosis of PASMCs. Under hypoxia, U50,488H also significantly inhibited autophagy, reduced proliferation and increased apoptosis of PASMCs. These effects of U50,488H were blocked by nor-BNI. Moreover, exposure to hypoxic conditions significantly increased AMPK phosphorylation and reduced mTOR phosphorylation, and these effects were abrogated by U50,488H. The effects of U50,488H on PASMC autophagy were inhibited by AICAR, a selective AMPK agonist, or by rapamycin, a selective mTOR inhibitor.
Our data provide evidence for the first time that κ-opioid receptor stimulation protects against HPH by inhibiting PASMCs autophagy via the AMPK-mTOR pathway.
背景/目的：在之前的一项研究中，我们发现用选择性激动剂U50,488H刺激κ-阿片受体可改善缺氧性肺动脉高压（HPH）。然而，肺动脉平滑肌细胞（PASMC）增殖、凋亡和自噬在κ-阿片受体介导的HPH保护作用中所起的作用仍不清楚。本研究的目的是探讨自噬在U50,488H诱导的HPH保护作用中的作用及其潜在机制。
将大鼠置于10%氧气环境中3周以诱导HPH。缺氧后，测量平均肺动脉压（mPAP）和右心室压力（RVP）。使用细胞计数试剂盒-8（CCK-8）检测法监测细胞活力。通过流式细胞术和蛋白质印迹法检测细胞凋亡。通过mRFP-GFP-LC3腺病毒转染检测法和蛋白质印迹法评估自噬。
给予氯喹抑制自噬可预防大鼠模型中HPH的发展，mPAP和RVP显著降低以及自噬减少证明了这一点。U50,488H模拟了氯喹的作用，且U50,488H的作用被选择性κ-阿片受体拮抗剂nor-BNI阻断。体外实验表明，氯喹抑制自噬与PASMC增殖减少和凋亡增加有关。在缺氧条件下，U50,488H也显著抑制自噬，减少PASMC增殖并增加其凋亡。U50,488H的这些作用被nor-BNI阻断。此外，暴露于缺氧条件下显著增加了AMPK磷酸化并降低了mTOR磷酸化，而U50,488H消除了这些作用。U50,488H对PASMC自噬的作用被选择性AMPK激动剂AICAR或选择性mTOR抑制剂雷帕霉素抑制。
我们的数据首次提供了证据，表明κ-阿片受体刺激通过AMPK-mTOR途径抑制PASMC自噬来预防HPH。
