PMID- 29233105 OWN - NLM STAT- MEDLINE DCOM- 20180730 LR - 20211204 IS - 1471-2466 (Electronic) IS - 1471-2466 (Linking) VI - 17 IP - 1 DP - 2017 Dec 12 TI - Salidroside attenuates hypoxia-induced pulmonary arterial smooth muscle cell proliferation and apoptosis resistance by upregulating autophagy through the AMPK-mTOR-ULK1 pathway. PG - 191 LID - 10.1186/s12890-017-0477-4 [doi] LID - 191 AB - 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 AMPKalpha1-ULK1 and AMPKalpha1-mTOR-ULK1 pathways. FAU - Gui, Di AU - Gui D AD - 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. FAU - Cui, Zhimin AU - Cui Z AD - 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. FAU - Zhang, Lin AU - Zhang L AD - 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. FAU - Yu, Chang AU - Yu C AD - Department of Invasive Technology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325000, People's Republic of China. FAU - Yao, Dan AU - Yao D AD - 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. FAU - Xu, Min AU - Xu M AD - 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. FAU - Chen, Mayun AU - Chen M AD - 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. FAU - Wu, Peiliang AU - Wu P AD - 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. FAU - Li, Guoping AU - Li G AD - Department of Respiratory Medicine, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang, 310013, People's Republic of China. FAU - Wang, Liangxing AU - Wang L AD - 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. wzyxywlx@163.com. FAU - Huang, Xiaoying AU - Huang X AD - 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. zjwzhxy@126.com. LA - eng PT - Journal Article DEP - 20171212 PL - England TA - BMC Pulm Med JT - BMC pulmonary medicine JID - 100968563 RN - 0 (Glucosides) RN - 0 (Phenols) RN - EC 2.7.11.1 (Autophagy-Related Protein-1 Homolog) RN - EC 2.7.11.31 (AMP-Activated Protein Kinases) RN - EC 2.7.11.31 (Prkaa1 protein, rat) RN - M983H6N1S9 (rhodioloside) SB - IM MH - AMP-Activated Protein Kinases/*metabolism MH - Animals MH - Apoptosis/drug effects MH - Autophagy/drug effects MH - Autophagy-Related Protein-1 Homolog/*metabolism MH - *Cell Proliferation/drug effects/physiology MH - Glucosides/pharmacology MH - Hypoxia/*metabolism MH - Male MH - *Myocytes, Smooth Muscle/drug effects/metabolism MH - Phenols/pharmacology MH - Rats PMC - PMC5726034 OTO - NOTNLM OT - Ampk OT - Autophagy OT - Hypoxia OT - PASMCs OT - ULK1 OT - mTOR COIS- ETHICS APPROVAL: All experimental protocols were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health and were approved by the Animal Ethics Committee of Wenzhou Medical University. Additionally, all animals were handled humanely during the study protocol and during euthanasia. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EDAT- 2017/12/14 06:00 MHDA- 2018/07/31 06:00 PMCR- 2017/12/12 CRDT- 2017/12/14 06:00 PHST- 2017/05/16 00:00 [received] PHST- 2017/10/06 00:00 [accepted] PHST- 2017/12/14 06:00 [entrez] PHST- 2017/12/14 06:00 [pubmed] PHST- 2018/07/31 06:00 [medline] PHST- 2017/12/12 00:00 [pmc-release] AID - 10.1186/s12890-017-0477-4 [pii] AID - 477 [pii] AID - 10.1186/s12890-017-0477-4 [doi] PST - epublish SO - BMC Pulm Med. 2017 Dec 12;17(1):191. doi: 10.1186/s12890-017-0477-4.