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

Salidroside, 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) and collagenase type I were obtained from Sigma (St Louis, MO, USA). Rapamycin was obtained from LC laboratories (Woburn, MA, USA). Dulbecco’s modified Eagle medium (DMEM, high glucose), streptomycin, penicillin G and fetal bovine serums (FBS) were obtained from Gibco BRL (Gaithersburg, MD, USA). The rabbit antibodies against LC3B, GAPDH, P-ULK1 (Ser 757 and Ser 317), mTOR, P-mTOR (Ser 2448) and p62 were obtained from Cell Signaling Technology (Beverly, MA, USA). The rabbit antibody against P-ULK1 (Ser 555) was obtained from US Biological (Swampscott, MA, USA). The rabbit antibody against P-AMPK (Thr 172) was obtained from Abways Technology (Shanghai, China). The rabbit antibody against AMPK was obtained from Abcam (Cambridge, UK). Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). The in-situ Cell Death Detection Kit was purchased from Roche Diagnostics (Penzberg, Germany). Tandem monomeric RFP-GFP-tagged LC3 (tfLC3) was purchased from Genechem (Shanghai, China).

Rat PASMCs were derived from pulmonary arteries as described previously [6, 7] and were cultured in DMEM supplemented with 100 μg/ml streptomycin, 100 IU/ml penicillin and 10% FBS. Then, cells were divided into five groups: the normoxia (N), hypoxia (H), hypoxia + MHY1485 (mTOR agonist, 2 μmol/L), hypoxia + rapamycin (mTOR inhibitor, 0.5 μmol/L), hypoxia + salidroside (499.5 μmol/L) groups. Hypoxia-treated PASMCs were treated as indicated for 24 h. All hypoxia groups were kept for 24 h in the hypoxia incubator at 37 °C with 5% CO₂, 5% O₂ and 90% N₂, whereas the normoxia group was kept in a normal incubator at 37 °C with 21% O₂, 5% CO₂ and 74% N₂.

Cell viability was determined by the CCK-8 assay. PASMCs were seeded in 96-well microplates at a concentration of 1 × 10⁴ cells/well. After they were preincubated in complete medium at 37 °C in 21% O₂ and 5%CO₂ for 24 h, PASMCs were pretreated with salidroside, rapamycin or MHY1485 before exposure to hypoxia. Cell growth was observed under a microscope before the CCK-8 assay. After 24 h of hypoxia, CCK-8 was added to the cells at a concentration of 10 μl/well for 2 h. A microplate reader was used to determine the absorbance at 450 nm.

After pretreatment, cells that adhered to the cover slips were fixed with fresh 4% paraformaldehyde for 1 h at 15 °C-25 °C. Cells were incubated with 3% H₂O₂ for 10 min at 15 °C-25 °C. Then, 0.1% Triton X-100 was incubated with the cells for 15 min at room temperature. Both anti-goat serum and 5% BSA were used to block non-specific binding. An in-situ Cell Death Detection Kit was used according to the manufacturer’s instructions. DAB and hematoxylin were used for cell staining. Light microscopy was used to observe the cover slips. Five randomly selected fields from each cover slip were analyzed to determine the percentage of terminal deoxynucleotidyl (TUNEL)-positive cells.

Cells were seeded onto 6-well microplates at a concentration of 5 × 10⁴ cells/well. After preincubation in complete medium at 37 °C in 21% O₂ and 5%CO₂ for 24 h, the cells were transfected with tfLC3 according to the manufacturer’s instructions to monitor autophagy flux. Eight hours after transfection, the cells were washed with PBS; next, complete culture medium was added to the cells. At 48 h after transfection, the cells were treated with salidroside, rapamycin or MHY1485. Cells were incubated in the hypoxia chamber for another 24 h. Finally, the samples were examined under a fluorescence microscope (Nikon, Tokyo, Japan).

Cells were fixed with both 2.5% glutaraldehyde and 1% osmic acid in sequence and then stained with 1% uranyl acetate. Acetone was used to dehydrate the cells, and then the cells were embedded in epoxy resin 812. Ultra-microtome slices V (Sweden) were used to cut the fixed cells into ultrathin sections. The number of autophagosomes in the cells was evaluated with Hitachi H-600 transmission electron microscopy (Hitachi, Japan).

Cells were harvested and incubated with ice-cold RIPA buffer containing PMSF. Then, the samples were centrifuged at 4 °C at 12000 rpm for 15 min. The Bradford method was used to quantify the protein concentrations. A total of 40 μg of protein from each group was separated using 12% SDS-PAGE. The proteins were transferred to PVDF membranes and blocked with 5% skimmed milk. Specific primary antibodies against LC3B (1:1000, 2775S) and p62 (1:1000, 5114S) were used to detect the proteins. GAPDH (1:1000, 5174S) was used as an internal control. Horseradish peroxidase-conjugated secondary antibodies were incubated with the proteins at a 1:10,000 dilution. Quantity one-4.6.2 software (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify the density of immunoblots after the detection of immunoreactive bands with BeyoECL Plus reagents (Beyotime, China).

A total of 40 μg of protein from each group was separated using 8% SDS-PAGE. Specific primary antibodies against AMPKα1 (1:2000, ab32047), phosphorylated AMPKα1 (1:2000, CY5556), phosphorylated ULK1 (Ser 555, 1:1000, U1500-70F) and phosphorylated ULK1 (Ser 317, 1:1000, 12753S) were used to detect the proteins. The experimental procedures for western blotting analysis were performed as described above.

A total of 40 μg of protein from each group was separated using 8% SDS-PAGE. Specific primary antibodies against mTOR (1:1000, 2983S), phosphorylated mTOR (1:1000, 5536S), ULK1 (1:1000, 8054 s) and phosphorylated ULK1 (Ser 757, 1:1000, 14202S) were used to detect the proteins. The experimental procedures for western blotting analysis were performed as described above.

PASMCs were treated with hypoxia with or without rapamycin or MHY1485 to demonstrate the role of autophagy in hypoxia-induced PASMC proliferation and apoptosis resistance. As shown in Fig. 1a and b, the density of PASMCs under the microscope in the H group was greater than that in the N group. Additionally, the viability of hypoxia-treated PASMCs was obviously increased (P < 0.05). The density and viability of cells treated with rapamycin were reduced (P < 0.05), whereas MHY1485 had no effect on cell density and viability (P > 0.05). As shown in Fig. 2a and b, the apoptosis index of PASMCs under hypoxic conditions was obviously decreased (P < 0.05). The apoptosis index of PASMCs treated with rapamycin or MHY1485 was increased (P < 0.05). Meanwhile, the number of autophagosomes, the ratio of LC3II to LC3I and autophagic flux were increased and the expression of p62 was decreased in cells treated with hypoxia. These changes were further enhanced in PASMCs treated with rapamycin (Figs. 3, 4 and 5). Therefore, we infer that hypoxia could enhance autophagy, which can further be enhanced by rapamycin. In PASMCs treated with MHY1485, although the number of autophagosomes and the ratio of LC3II to LC3I were increased, the autophagic flux of PASMCs was decreased, and the expression of p62 was increased (Figs. 3, 4 and 5). Therefore, we concluded that MHY1485 could inhibit autophagy. These results indicate that the increase in autophagy under hypoxic conditions might compensatory, and further enhanced autophagy may decrease PASMC proliferation and increase PASMCs apoptosis. The detailed mechanisms underlying the decrease in autophagy, increase in the apoptosis index, and lack of effects on proliferation following MHY1485 treatment in hypoxic PASMCs were investigated next.

To confirm autophagy activation following different interventions, we transfected tfLC3 into PASMCs (Fig. 3). Since the GFP signal is sensitive to acidic conditions, this form of LC3 displays only red fluorescence when located in autolysosomes. Increased red spots were observed in hypoxic cells. Furthermore, an increased number of red spots were observed in cells treated with rapamycin and salidroside. However, both green and red spots were observed in cells treated with MHY1485. These data revealed that hypoxia enhanced the autophagy flux of cells, which was further enhanced by rapamycin and salidroside. However, MHY1485 weakened the autophagy flux of PASMCs.

To confirm the number of autophagosomes in PASMCs after different interventions, transmission electron microscopy was performed (Fig. 4). The number of autophagosomes were increased in cells under hypoxic conditions. The number of autophagosomes were further increased in cells treated with salidroside, rapamycin and MHY1485. These results together with the autophagy flux analysis indicated that salidroside increased the number of autophagosomes by activating autophagy. However, MHY1485 increased the number of autophagosomes through suppressing lysosomal fusion.

To confirm autophagy activation following different interventions, protein levels of LC3 and p62 were detected by western blotting analysis (Fig. 5). The ratio of LC3II to LC3I was increased and p62 expression was diminished in the hypoxia group (P < 0.05). In both the rapamycin and salidroside groups, changes in the ratio of LC3II to LC3I and p62 expression were further enhanced (P < 0.05). However, both the ratio of LC3II to LC3I and p62 expression were increased in the MHY1485 group compared to the hypoxia group (P < 0.05). These data together with autophagy flux and autophagosome detection revealed that rapamycin and salidroside further increases autophagy compared to hypoxia, which reverses PASMC proliferation and apoptosis resistance (Figs. 1a, b and 2a, b). However, increased levels of autophagosomes in the MHY1485 group resulted in increased LC3II expression, which may be related to the reversal of PASMC proliferation and apoptosis resistance.

To confirm whether the increase in autophagy induced by salidroside is dependent on the regulation of the AMPK-ULK1 pathways, cells were treated with hypoxia and salidroside. The levels of AMPK, phosphorylated AMPK and phosphorylated ULK1 (Ser 555) were upregulated in the hypoxia group. The changes in AMPK, phosphorylated AMPK and phosphorylated ULK1 (Ser 555) expression were further enhanced in the salidroside group compared to the hypoxia group (P < 0.05). To further confirm whether the role of salidroside is mTOR dependent, cells were treated with the mTOR inhibitor rapamycin and the mTOR agonist MHY1485. The expression of these proteins in cells treated by MHY1485 and rapamycin was similar to that in the hypoxia group (P > 0.05). The phosphorylation of ULK1 at serine 317 had no significant effect on the five groups (P > 0.05) (Fig. 6a). These data revealed that salidroside can exert a protective effect through the AMPK-ULK1 (Ser 555) pathway (Fig. 7).

To confirm whether the increase in autophagy induced by salidroside was also dependent on the regulation of the mTOR-ULK1 pathway, mTOR, P-mTOR, ULK1 and P-ULK1 (S757) protein levels were detected (Fig. 6b). Protein expression of mTOR, phosphorylated mTOR and phosphorylated ULK1 (Ser 757) was decreased in the hypoxia group (P < 0.05). Changes in mTOR, phosphorylated mTOR and phosphorylated ULK1 (Ser 757) expression were further enhanced in the salidroside and rapamycin groups compared to the hypoxia group (P < 0.05). Protein expression levels of mTOR, phosphorylated mTOR and phosphorylated ULK1 (Ser 757) were increased in the MHY1485 group compared to the hypoxia group (P < 0.05). The expression of ULK1 contrasts that of P-ULK1 (Ser 757). These data revealed that salidroside can exert a protective effect through the mTOR-ULK1 (Ser 757) pathway (Fig. 7).

In our research, autophagy flux, autophagosome levels and the ratio of LC3II to LC3I were increased and p62 expression was decreased in PASMCs under hypoxic conditions; additionally, salidroside further enhanced the abovementioned changes and attenuated hypoxia-induced pulmonary arterial smooth muscle cell proliferation and apoptosis resistance. We concluded that the increase in autophagy in hypoxic PASMCs was a compensatory response and that salidroside could further enhance autophagy. Enhanced autophagy induced by salidroside through both the AMPKα1-ULK1 (Ser 555) and AMPKα1-mTOR-ULK1 (Ser 757) pathways could decrease cell proliferation and increase cell apoptosis in PASMCs under hypoxic conditions.