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

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.

Keywords: Ampk; Autophagy; Hypoxia; PASMCs; ULK1; mTOR.

Fig. 1

Autophagy is involved in hypoxia-induced PASMC proliferation. a Cell density in each group as viewed under a microscope. b CCK-8 values in each group. *p < 0.05 vs. the N group; and #p < 0.05 vs. the H group

Fig. 2

Autophagy is involved in hypoxia-induced PASMC apoptosis resistance. a Images of cell apoptosis in each group as determined by the TUNEL assay. b Quantitative analysis of the cell apoptosis index by TUNEL assay. *p < 0.05 vs. the N group; and #p < 0.05 vs. the H group

Fig. 3

Enhanced autophagy flux may be involved in the effects of salidroside on PASMC proliferation and apoptosis resistance under hypoxic conditions. After transfection with tfLC3, PASMCs were exposed to hypoxia and treated with MHY1485, rapamycin or salidroside; next, the cells were observed under a fluorescence microscope. *p < 0.05 vs. the N group; and #p < 0.05 vs. the H group

Fig. 4

Increased numbers of autophagosomes may be related to salidroside-induced decreases in the proliferation and increases in the apoptosis of PASMCs under hypoxic conditions. Images of autophagosomes in PASMCs of each group acquired by Hitachi H-600 transmission electron microscopy. The control group exhibited less autophagosomes than the hypoxia group. The salidroside group exhibit increased numbers of autophagosomes than the hypoxia group. Red arrow: autophagosomes

Fig. 5

Increased autophagy may be related to the salidroside-induced attenuation of PASMC proliferation and apoptosis resistance under hypoxic conditions. Images and quantitative analysis of p62 expression and the LC3II/LC3I ratio in PASMCs by western blot assay. GAPDH was used as an internal control. *p < 0.05 vs. the N group; and #p < 0.05 vs. the H group

Fig. 6

The AMPK-ULK1 (Ser555) pathway is upregulated and The mTOR-ULK1 (Ser757) pathway is downregulated by salidroside in PASMCs. a Images and quantitative analysis of AMPKα1, phosphorylated AMPKα1, and phosphorylated ULK1 (Ser555, 317) in PASMCs by western blot assay. b Images and quantitative analysis of mTOR, phosphorylated mTOR, ULK1 and phosphorylated ULK1 (Ser 757) in PASMCs by western blot assay. GAPDH was used as an internal control. *p < 0.05 vs. the N group; and #p < 0.05 vs. the H group

Fig. 7

The signaling pathways of this experiment. Salidroside exerted protective effects against hypoxic PASMCs via the upregulation of autophagy through both the AMPK-ULK1 (Ser 555) and AMPK-mTOR-ULK1 (Ser 757) pathways