红景天苷通过 AMPK-mTOR-ULK1 通路上调自噬来减轻低氧诱导的肺动脉平滑肌细胞增殖和抗凋亡。

Salidroside attenuates hypoxia-induced pulmonary arterial smooth muscle cell proliferation and apoptosis resistance by upregulating autophagy through the AMPK-mTOR-ULK1 pathway.

机构信息

Division of Pulmonary Medicine, The First Affiliated Hospital of Wenzhou Medical University, Key Laboratory of Heart and Lung, Wenzhou, Zhejiang, 325000, People's Republic of China.

Department of Invasive Technology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325000, People's Republic of China.

出版信息

BMC Pulm Med. 2017 Dec 12;17(1):191. doi: 10.1186/s12890-017-0477-4.

DOI:10.1186/s12890-017-0477-4

PMID:29233105

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5726034/

Abstract

BACKGROUND

Recent studies have shown that both adenosine monophosphate activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) are energy sensors and are related to autophagy. Our recent reports have shown that salidroside can exert protective effects against hypoxia-induced pulmonary arterial smooth muscle cell (PASMC) proliferation and apoptosis resistance through the AMPK pathway. This study aims to explore the relationship among AMPK, mTOR and ULK1 in PASMCs under hypoxic conditions and to investigate whether the protective effects of salidroside are related to the autophagic cell death pathway.

METHODS

Rat PASMCs were cultured and divided into five groups: the normoxia, hypoxia, hypoxia + MHY1485 (mTOR agonist), hypoxia + rapamycin (mTOR inhibitor) and hypoxia + salidroside groups. Hypoxic cells were treated as indicated for 24 h. Cell viability was evaluated by the CCK-8 assay. Cell apoptosis was measured by the TUNEL assay. The autophagy flux of PASMCs was evaluated with tandem mRFP-GFP fluorescence microscopy. Autophagosomes were detected by electron microscopy. Protein expression of LC3, p62, AMPK, P-AMPK (Thr 172), P-ULK1 (Ser 555 and Ser 317), mTOR, P-mTOR (Ser 2448), ULK1 and P-ULK1 (Ser 757) was detected by western blot assay.

RESULTS

PASMC proliferation and apoptosis resistance were observed under hypoxic conditions. Autophagy flux, the number of autophagosomes and the LC3II/LC3I ratio were increased in the hypoxia group compared with the normoxia group, whereas p62 expression was decreased. Treatment with rapamycin or salidroside reversed hypoxia-induced PASMC proliferation and apoptosis resistance and further increased autophagy flux, autophagosome levels and the LC3II/LC3I ratio but decreased p62 expression. Treatment with MHY1485 reversed hypoxia-induced PASMC apoptosis resistance and decreased autophagy flux as well as increased autophagosome levels, the LC3II/LC3I ratio and p62 expression. P-AMPK (Thr 172) and P-ULK1 (Ser 555) of the AMPK-ULK1 pathway were increased in the hypoxia group and were further increased in the salidroside group. Rapamycin and MHY1485 had no effect on either P-AMPK (Thr 172) or P-ULK1 (Ser 555). Phosphorylation of ULK1 at serine 317 did not significantly affect the five groups. Furthermore, P-mTOR (Ser 2448) and P-ULK1 (Ser 757) of the AMPK-mTOR-ULK1 pathway were decreased in the hypoxia group and were further decreased in the salidroside group. MHY1485 increased the expression of both P-mTOR(Ser 2448) and P-ULK1(Ser 757), whereas rapamycin had the opposite effect.

CONCLUSIONS

Salidroside might inhibit hypoxia-induced PASMC proliferation and reverse apoptosis resistance via the upregulation of autophagy through both the AMPKα1-ULK1 and AMPKα1-mTOR-ULK1 pathways.

摘要

背景

最近的研究表明，腺苷单磷酸激活蛋白激酶（AMPK）和哺乳动物雷帕霉素靶蛋白（mTOR）都是能量感受器，与自噬有关。我们最近的研究报告表明，红景天苷可以通过 AMPK 途径发挥对低氧诱导的肺动脉平滑肌细胞（PASMC）增殖和抗凋亡的保护作用。本研究旨在探讨低氧条件下 PASMC 中 AMPK、mTOR 和 ULK1 之间的关系，并研究红景天苷的保护作用是否与自噬性细胞死亡途径有关。

方法

将大鼠 PASMC 培养并分为五组：常氧组、缺氧组、缺氧+MHY1485（mTOR 激动剂）组、缺氧+雷帕霉素（mTOR 抑制剂）组和缺氧+红景天苷组。用相应的药物处理缺氧细胞 24 小时。用 CCK-8 法评估细胞活力。用 TUNEL 法测定细胞凋亡。用串联 mRFP-GFP 荧光显微镜评估 PASMC 的自噬流。用电子显微镜检测自噬体。用 Western blot 法检测 LC3、p62、AMPK、P-AMPK（Thr 172）、P-ULK1（Ser 555 和 Ser 317）、mTOR、P-mTOR（Ser 2448）、ULK1 和 P-ULK1（Ser 757）的蛋白表达。

结果

在低氧条件下观察到 PASMC 增殖和抗凋亡。与常氧组相比，缺氧组的自噬流、自噬体数量和 LC3II/LC3I 比值增加，而 p62 的表达减少。用雷帕霉素或红景天苷处理可逆转低氧诱导的 PASMC 增殖和抗凋亡，并进一步增加自噬流、自噬体水平和 LC3II/LC3I 比值，减少 p62 的表达。用 MHY1485 处理可逆转低氧诱导的 PASMC 抗凋亡，并减少自噬流，增加自噬体水平、LC3II/LC3I 比值和 p62 的表达。AMPK-ULK1 通路中的 P-AMPK（Thr 172）和 P-ULK1（Ser 555）在缺氧组中增加，在红景天苷组中进一步增加。雷帕霉素和 MHY1485 对 P-AMPK（Thr 172）或 P-ULK1（Ser 555）均无影响。ULK1 丝氨酸 317 的磷酸化在五组中均无明显变化。此外，AMPK-mTOR-ULK1 通路中的 P-mTOR（Ser 2448）和 P-ULK1（Ser 757）在缺氧组中减少，在红景天苷组中进一步减少。MHY1485 增加了 P-mTOR（Ser 2448）和 P-ULK1（Ser 757）的表达，而雷帕霉素则有相反的作用。