Celastrol, N-acetyl-L-cysteine (NAC), 3-methyladenine (3Ma), bafilomycin A1 (Baf A1), rapamycin (Rapa) and dimethyl sulfoxide (DMSO) were provided by Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Angiotensin II (Ang II) was obtained from EMD Millipore (Billerica, MA, USA). A Senescence β-galactosidase staining kit (cat. no. 9860), polyclonal rabbit antibodies against LC3A/B (cat. no. 4108), phosphorylated (p)-protein kinase B (Akt; cat. no. 9271), Akt (cat. no. 9272), p-mechanistic target of rapamycin (mTOR; cat. no. 2971), mTOR (cat. no. 2972), p-ribosomal protein S6 kinase β1 (p70S6K; cat. no. 9205), p70S6K (cat. no. 9202) and β-Actin (cat. no. 4967), and the monoclonal rabbit antibody against GAPDH (cat. no. 2118) were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Monoclonal mouse antibodies against p21 (cat. no. sc-6246) and p53 (cat. no. sc-99) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Polyclonal rabbit antibodies against p-phosphoinositide 3 kinase (PI3K; cat. no. ab182651) and PI3K (cat. no. ab40755) were obtained from Abcam (Shanghai, China). A polyclonal goat CY5-conjugated secondary antibody (cat. no. A10523) was from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). An ROS assay kit and a Cell Counting kit-8 (CCK-8) were from the Beyotime Institute of Biotechnology (Haimen, China). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were provided by Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

A total of 20 Sprague-Dawley rats (150–180 g, 6 weeks of age) were obtained from the Laboratory Animal Center of the Third Military Medical University (Chongqing, China). The animals had free access to food and water and were housed at a temperature of 25°C with 45–70% humidity, under a 12-h light/dark cycle in the specific pathogen free facility at Laboratory Animal Center of Third Military Medical University (Chongqing, China). All procedures were conducted in accordance with the animal care guidelines approved by the Animal Ethics Committee of the Third Military Medical University. The animal care and procedures were in accordance with the guidelines of the National Institutes of Health (Bethesda, MD, USA). Thoracic aortas were isolated from euthanized rats, and all efforts were made to minimize any pain to the animals.

Primary rat VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats by an explant method as previously described (21). Briefly, male Sprague-Dawley rats were anaesthetized with 5% chloral hydrate (1 ml/100 g body weight) and sacrificed to obtain VSMCs from the thoracic aorta. VSMCs were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO₂. The medium was changed every 2–3 days, and VSMCs at passages 3–8 were used for all experiments.

Senescent cells were identified using a Senescence β-Galactosidase Staining kit. Briefly, VSMCs were seeded into 6-well plates (NEST, Wuxi, China) at 2×10⁵/well. At 80% confluence, VSMCs were pretreated with celastrol at 10, 50 or 100 nM for 12 h before the addition of 100 nM Ang II for 2 days. Subsequently, cells were fixed with the fixative solution, rinsed twice with PBS and incubated with the β-galactosidase staining solution at 37°C overnight in a dry incubator (without CO₂). For 3Ma or Baf A1 stimulation, cells were pretreated with 3 mM 3Ma or 50 nM Baf A1 for 2 h.

The cell cycle analysis was performed according to the manufacturer's protocol. Briefly, after serum starvation for 12 h, VSMCs were treated with 50 nM celastrol for 12 h before incubation with Ang II for 2 days at 37°C. Subsequently, cells were harvested and fixed in 70% ethanol, stained for DNA content with propidium iodide for 30 min at room temperature and detected using a flow cytometer (BD FACScan, BD Biosciences, Franklin Lakes, NJ, USA). Fluorescence dot plots were analyzed and reconstructed with FlowJo version 7.2.5 software (FlowJo LLC, Ashland, OR, USA).

Western blot assays were performed as previously described (22). Briefly, VSMCs were harvested and lysed in RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China), which contained a cocktail of both protease and phosphatase inhibitors (Roche Diagnostics, Basel, Switzerland). Following extraction from VSMCs, the protein concentration was determined by the BCA protein assay (Beyotime Institute of Biotechnology). Samples containing 30 µg of protein were subjected to 10% SDS-PAGE and separated. Following protein transfer to nitrocellulose membranes (EMD Millipore), the membranes were blocked and incubated with primary antibodies overnight (12 h) at 4°C, then washed three times with TBST (Tris-buffered saline; 10 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.1% Tween-20) for 30 min and incubated with secondary antibodies for 1 h at room temperature. Membranes were washed four times with TBST for 40 min. The protein signals were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.). The relative protein levels were analyzed by Image J software version 1.6.0_24 (National Institutes of Health, Bethesda, MD, USA).

A ROS assay kit was used to determine the intracellular ROS generation. In brief, quiescent cells were incubated with 50 nM celastrol for 12 h before stimulation with 100 nM Ang II for 12 h, and then incubated with 2, 7-DCFH-DA in DMEM without FBS at 37°C for 30 min and rinsed three times with PBS. ROS levels were measured by using a flow cytometer.

Cells were fixed with 4% paraformaldehyde for 20 min, rinsed three times with PBS, blocked with 1% bovine serum albumin (Beyotime Institute of Biotechnology) for 1 h, and incubated with a rabbit anti-LC3 A/B antibody for 1 h at room temperature. Subsequently, the cells were washed and incubated with a CY5-conjugated secondary antibody for 1 h at room temperature. Immunofluorescence was detected by using a confocal microscope (TCS-SP5, Leica Microsystems GmbH, Wetzlar, Germany).

Data analysis was performed using GraphPad Prism 5.0 software package (GraphPad Software, Inc., La Jolla, CA, USA). All experimental data are expressed as the mean ± standard error with each experiment repeated at least three times. The statistical analysis was performed with SPSS software version 20.0 (IBM Corp., Armonk, NY, USA). The data comparisons were analyzed using one-way analysis of variance followed by Bonferroni's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

Senescent cells are characterized by stable growth arrest (2) and increased expression of SA-β-gal, p53, p21 and p16 (3,4). Treatment with celastrol alone had little effect on VSMC senescence compared with treatment with the vehicle control (DMSO). A significant increase in VSMC senescence upon stimulation with Ang II was evidenced by the increase in the number of SA-β-gal-positive cells. In this setting, celastrol demonstrated a dose-dependent inhibition of VSMC senescence with its maximal effect achieved at 50 nM (Fig. 1A). Protein expression levels of p53 and p21 were significantly increased in VSMCs after a 2-day period of stimulation with Ang II. Pretreatment with celastrol significantly counteracted this effect of Ang II on (Fig. 1B). Flow cytometry was also used to investigate the effect of celastrol on the cell cycle of VSMCs. The senescent cells were defined as arrested primarily in the G0/G1 phase but occasionally at the S or G2 phase. After stimulation with 100 nM Ang II for 2 days, the percentage of G0/G1 phase cells was significantly increased compared with that of the DMSO control group. In contrast, in VSMCs treated with Ang II, the number of G0/G1-arrested cells was decreased while the number of cells in S/G2 phase was increased by celastrol (50 nM)-pretreatment (Fig. 1C), indicating that the proliferative capacity of senescent VSMCs induced by Ang II was restored by celastrol.

Intracellular ROS serves a major role in the development of Ang II-induced cellular senescence (23), which can be alleviated by the specific ROS scavenger NAC in VSMCs (8). A ROS assay kit was used to investigate whether celastrol attenuated the Ang II-induced ROS activity in VSMCs. Treatment with celastrol alone had little effect on basal ROS generation, similar to that of the DMSO control (Fig. 1D). ROS production was increased in the Ang II-treated group and this was significantly decreased in the celastrol-pretreated group, although such inhibitory efficacy was less potent compared with cells treated with NAC (Fig. 1D). These results suggested that the effects of celastrol on cellular senescence in VSMCs are potentially mediated through the inhibition of ROS production.

Immunofluorescence assay demonstrated that the level of total LC3 was significantly increased in the celastrol-pretreated group compared with the control group (Fig. 2A). Western blot assay further demonstrated that compared with the DMSO control, the protein expression level of LC3 II was increased in the celastrol-treated group, which could be further increased by bafilomycin A1, or decreased by 3Ma (Fig. 2B). Furthermore, the p62 protein level was decreased in the celastrol-treated group, and treatment with the two autophagy inhibitors reduced these effects (Fig. 2B). Ang II stimulation significantly increased cellular ROS levels in VSMCs, and celastrol reduced the Ang II-stimulated ROS levels, similar to the effects of the positive control rapamycin; however, pretreating with the autophagy inhibitor 3Ma reversed the effect of celastrol (Fig. 2C), suggesting that celastrol at least partially inhibits intracellular ROS by activating autophagy in VSMCs.

To further confirm whether celastrol-induced autophagy can reduce the Ang II-induced senescence of VSMCs, senescence β-galactosidase staining and western blot analysis were performed. The results demonstrated that, just like in the autophagy inducer rapamycin-pretreated group, the SA-β-gal positive cell counts were significantly decreased in the celastrol-pretreated group, which was reversed by the autophagy inhibitors 3Ma and Baf A1 (Fig. 3A). Western blot assay further demonstrated that the autophagy inhibitor 3Ma largely abolished the effects of celastrol on p53 and p21 expression (Fig. 3B).

The present study then assessed tested whether celastrol could suppress the PI3K/Akt/mTOR signaling pathway. The results demonstrated that, compared with the control group, the phosphorylation levels of PI3K, Akt, mTOR and p70S6K in the celastrol group were downregulated (Fig. 4), indicating that celastrol treatment suppressed the PI3K/Akt/mTOR signaling pathway.