Sprague-Dawley male rats (n=24; 8-weeks old; weight, 200–250 g) were supplied by the Laboratory Animal Center of Nanjing Qinglongshan (Nanjing, China). The care and treatment of these rats was performed in accordance with the Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation. All animals were housed in a room with a constant temperature (22±1°C) and a humidity of 50±%, allowed access to a standard diet and water ad libitum, and exposed to a 12-h light/dark cycle. The present study was approved by the Ethics Committee of Ninth Hospital of Xi'an (Xi'an, China).

Sprague-Dawley rats were anesthetized with diethyl ether and sacrificed by cervical dislocation. PVAT (located around the thoracic aorta) was isolated, sectioned into small pieces and rinsed in phosphate buffered saline (PBS). Equal quantities (40 mg) of PVAT were individually pretreated with MTX (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) (0.1, 0.25 and 0.5 µM) or salicylate (5 mM; Tianjin Kemiou Chemical Reagent Co., Ltd., Tianjin, China) or aminoimidazole-4-carboxamide ribonucleotide (AICAR; 500 µM; Sigma-Aldrich, Merck Millipore) in the presence or absence of 25 µM AMPK inhibitor compound C (Sigma-Aldrich; Merck Millipore) and stimulated with 100 µM palmitic acid (PA; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 2 h. Following treatment, the PVAT was washed twice with PBS to remove the reagents, and cultured in fresh Dulbecco's modified Eagle's medium (DMEM, Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) for 22 h at 37°C. The medium was collected as CM. The supernatant was then collected as conditioned CM and stored at −70°C.

Endothelium-dependent relaxation was assayed as previously described (7). Briefly, the prepared aortic ring was suspended in an organ bath containing 10 ml K-H solution (118.3 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH2PO₄, 2.5 mM CaCl₂, 25 mM NaHCO₃, 0.026 mM calcium disodium EDTA and 5.0 mM glucose, pH 7.4) maintained at 37°C pH 7.4, and continuously aerated with 95% O₂ and 5% CO₂. Following a 60 min stabilization period, the contractive ability of the vessel was examined by contractive response to 60 mM KCl, and the functionality of vascular endothelium was confirmed by relaxation to 10 µM acetylcholine (ACh; Sigma-Aldrich; Merck Millipore). The aortic ring (relaxation, ≥80%) was treated with CM for 30 min. Following washing, the aortic ring was pre-contracted with phenylephrine and the endothelium-dependent relaxation was induced by cumulative addition of ACh (0.001–10 µM). Relaxation was expressed as a percentage of the phenylephrine-induced contraction.

3T3-L1 pre-adipocytes were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). A total of 2×105/ml cells were cultured in 6-well plates in DMEM containing 10% FBS and 25 mM glucose at 37°C in a humidified atmosphere containing 5% CO₂.

PVAT from rats were pretreated with MTX in the presence of PA (100 µM) for 2 h. Following washing with PBS, PVAT was cultured in fresh DMEM supplemented with 10% (v/v) FBS for 22 h. PVAT was then collected and homogenized with 500 µl TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) on ice. Total RNA was obtained using Total RNA Extraction Reagent (Nanjing Sunshine Biotechnology Co., Ltd., Nanjing, China). cDNA was synthesized with the TransScript First-strand cDNA Synthesis SuperMix kit (Beijing Transgen Biotech Co., Ltd., Beijing, China). qPCR was performed using SsoFast™ EvaGreen Supermix (Bio-Rad Laboratories Inc., Hercules, CA, USA). Amplification was performed with the Bio-Rad iQ5 sequence detection system (Bio-Rad Laboratories Inc.) with the following conditions: 95°C for 10 min, followed by 40 cycles at 95°C for 30 sec, and 52°C for 15 sec, and a final extension at 72°C for 5 min. The following primers were used: Adiponectin forward, 5′-AAGGGGACAACAATGGACTCTA-3′, and reverse, 5′-CTACGGGCTGCTCTGAATTAGT-3′; β-actin forward, 5′-GACGTTGACATCCGTAAAGACC-3′, and reverse, 5′-TGCTAGGAGCCAGGGCAGTA-3′. The mRNA expression level of individual genes was normalized and presented as a ratio to β-actin, and calculated using the 2-^ΔΔCq method (11).

PVAT from Sprague-Dawley rats was pretreated with MTX for 30 min and then incubated with PA for a further 30 min. The aorta was incubated with CM for 30 min and then exposed to ACh for another 30 min. PVAT was isolated directly following the sacrifice of the rats. For protein analysis, PVAT or the aorta was homogenized in radio immunoprecipitation assay lysis buffer with phenylmethane sulfonyl fluoride. Protein concentration in the supernatants was assayed using Bicinchoninic Acid Protein Assay kit (Nanjing Baxi Technology Co., Ltd. Nanjing, China). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and transferred to 0.45 µM polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) by semi-dry electrophoretic transfer (Bio-Rad Laboratories, Inc.). The PVDF membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) and then incubated with the appropriate primary antibodies overnight at 4°C: Rabbit monoclonal anti-phospho-AMPK (cat. no. 2531) and anti-AMPK (cat. no. 2532), rabbit monoclonal anti-eNOS (cat. no. 9586) and anti-p-eNOS (cat. no. 9571), anti-NF-κB p65 (cat. no. 4764) and anti-p-NF-kB p65 (cat. no. 3033; all at 1:1,000 and obtained from Cell Signaling Technology, Inc., Beverly, MA, USA). The PVDF membrane was washed three times with TBST buffer and then incubated with the HRP-conjugated secondary antibodies (cat. no. BS13278; 1:10,000, MyBioSource, Inc., San Diego, CA, USA) at room temperature for 2 h. The membranes were developed with an enhanced chemiluminescence detection reagent (Thermo Fisher Scientific, Inc.) and quantitated by densitometry with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

The levels of TNF-α and IL-6 in the supernatant were assayed with commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D, Minneapolis, MN, USA).

3T3-L1 pre-adipocytes were transfected with siRNA for AMPKα (cat. no. sc-45313) to knock down levels of endogenous AMPKα, and negative control siRNA (cat. no. sc-37007), using siRNA transfection reagent (cat. no. sc-29528) in transfection medium (cat. no. sc-36868) for 6 h, according to the manufacturer's protocol. All siRNAs were obtained from Santa Cruz Biotechnology, Inc., Dallas, TX, USA. Following an additional 48 h, the efficiency of siRNA-mediated AMPK knockdown was confirmed by western blot analysis as aforementioned.

Data were expressed as the mean ± standard deviation from at least three independent experiments. SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis. Individual group statistical comparisons were analyzed using an unpaired Student's t-test with Bonferroni correction and multiple-group comparisons were evaluated by one-way analysis of variance followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

AMPK is important in the regulation of lipid metabolism, therefore the present study first investigated the effect of MTX on AMPK activity in PVAT. As presented in Fig. 1A, MTX treatment increased basal AMPK activity at concentrations ranging from 0.1 to 0.5 µM, indicated by enhanced AMPK phosphorylation. In addition, it was observed that when PVAT was exposed to PA, a reduction of AMPK phosphorylation was observed, and this alteration was prevented by pretreatment with MTX (P<0.05, Fig. 1B). Furthermore, treatment with AICIR which is an AMPK agonist, exhibited similar effects (P<0.05). These results indicated that MTX enhanced AMPK activation under basal and inflammatory conditions.

It has previously been demonstrated that free fatty acids induce inflammation in the endothelium via the nuclear factor (NF)-κB pathway and stimulation with PA enhanced phosphorylation of NF-κB p65 in PVAT (12). As presented in Fig. 2A, MTX effectively reduced NF-κB p65 phosphorylation, indicative of its anti-inflammatory activity (P<0.05). In addition, the effect of MTX on the expression of adipocytokines implicated in inflammation was examined. As presented in Fig. 2B and C, the expression levels of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-6, were increased in cells treated with PA for 24 h and this was reversed by MTX pretreatment (P<0.05), demonstrating its anti-inflammatory activity. Furthermore, the expression of adiponectin was downregulated, however these alterations were reversed by treatment with MTX (P<0.05, Fig. 2D).

The AMPK inhibitor to confirm the role of AMPK in the anti-inflammatory effect of MTX, 3T3-L1 cells were transfected with AMPKa1/2-specific siRNA to knockdown AMPK expression. As presented in Fig. 3A, silencing AMPK significantly decreased the inhibitory effect of MTX on PA-mediated NF-κB p65 phosphorylation (P<0.05). MTX treatment altered the production of TNF-α (Fig. 3B), IL-6 (Fig. 3C) and adiponectin (Fig. 3D) upon PA challenge, however, this action was also blocked by knockdown of AMPK (P<0.05, Fig. 3B-D). These results indicated that MTX inhibited inflammation in a AMPK-dependent manner.

Endothelial dysfunction is characterized by the loss of endothelium-dependent vasodilation. To investigate the influence of PVAT on vessel function, PVAT was incubated with PA and collected the medium as CM to stimulate the rat aorta. Data presented in Fig. 4A indicated that ACh induced vasodilation, whereas stimulation with PA led to a significant loss in vessel relaxation (P<0.05). Pretreatment of PVAT with MTX prevented the alterations and effectively restored the loss of ACh-mediated vessel relaxation in a concentration-dependent manner, but this action was attenuated by co-treatment with AMPK inhibitor compound C (P<0.05, Fig. 4A). Furthermore, it was observed that treating PVAT with MTX effectively normalized eNOS phosphorylation in the aorta subjected to CM challenge (P<0.05, Fig. 4B). AMPK inhibitor compound C blocked the actions of MTX in the regulation of vasodilation and eNOS phosphorylation, suggesting the involvement of AMPK in the mechanism of MTX action. These results suggested that MTX improved vessel function via regulation of AMPK activity.