Animals and experimental design
Healthy 6 to 8 week old female Sprague-Dawley rats (170-190 g) were purchased from the animal center of the Hebei Medical University, China. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Hebei Medical University and complied with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals.
The rats underwent bilateral ovariectomy and were randomized into four groups (n = 8/group): normoxia group, normoxia+E2 group, hypoxia group, and hypoxia+E2 group. Previous studies showed that hypoxia exposure for 2-4 weeks can establish HPH models (21, 22). One week after surgery, the animals received 17β-estradiol (20 μg/kg/d, Sigma, St Louis, MO, USA) or saline (0.1 ml/d) subcutaneously for 8 weeks, as described (23). An additional file shows this in more detail (see Additional file 1).
Hemodynamics, hormone assays, and tissue preparation
Hemodynamics were recorded using a Med lab electrophysiolograph (BL-420S, Thaimeng Technology Co., Ltd., Chengdu, China). A commercially available estradiol ELISA kit (DRG International Inc., Springfield Township, NJ, USA) was used to detect the hormone levels. The lungs and heart were harvested. An additional file shows this in more detail (see Additional file 1).
Pathology and immunochemical staining
Immunohistochemistry was routinely performed for SIRT1 (1:400; ab110304, Abcam, Cambridge, MA, USA), FOXO3a (1:400; ab109629, Abcam, Cambridge, MA, USA), and PCNA (1:400; 610664, BD Biosciences, Franklin Lake, NJ, USA). An additional file shows this in more detail (see Additional file 1).
Cell culture and treatment
HPASMCs (3110, Sciencell Research Laboratories, Carlsbad, CA, USA) were routinely cultured at 37°C in smooth muscle cell medium (Sciencell Research Laboratories, Carlsbad, CA, USA) supplemented with 2% fetal bovine serum (Sciencell Research Laboratories, Carlsbad, CA, USA), 0.5% smooth muscle cell growth supplement (Sciencell Research Laboratories, Carlsbad, CA, USA), and 0.5% penicillin-streptomycin solution (Sciencell Research Laboratories, Carlsbad, CA, USA). The cells were divided into six groups: one normoxia group, four hypoxia groups treated with four different concentrations of E2 (10-6, 10-7, 10-8, and 10-9 mol/L), and one hypoxia alone group. An additional file shows this in more detail (see Additional file 1).
Cell proliferation assay
HPASMC proliferation was determined routinely using the MTT assay (MTT; D0801, Sigma, St Louis, MO, USA). An additional file shows this in more detail (see Additional file 1).
Western blotting
Western blotting was performed routinely for SIRT1 (ab110304, Abcam, Cambridge, MA, USA), FOXO3a (ab109629, Abcam, Cambridge, MA, USA), PCNA (610664, BD Biosciences, Franklin Lake, NJ, USA), and GAPDH (Abcam, Cambridge, MA, USA). An additional file shows this in more detail (see Additional file 1).
RT-PCR analysis
RT-PCR was performed routinely for SIRT1, FOXO3a, and GAPDH (172 bp). The expression levels of the target genes were calculated using the 2−ΔΔCt method (24). An additional file shows this in more detail (see Additional file 1).
SIRT1 deacetylase activity assay
SIRT1 deacetylase activity was assayed using the Universal SIRT Activity Assay Kit (Abcam, Cambridge, MA, USA), according to the manufacturer’s instructions.
Statistical analysis
All data were expressed as means ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA, followed by the least significant difference (LSD) test for post hoc multiple comparisons. Significant difference was accepted at P<0.05. Analyses were performed using SPSS 21.0 (IBM, Armonk, NY, USA).