A total of 40 male Sprague-Dawley rats (8 weeks old, 180–200 g) were obtained from the Laboratory Animal Center of Nanjing Medical University (Nanjing, China). Animals were housed at a constant temperature (22±1°C) in a 12:12-h light-dark cycle, with ad libitum access to a standard diet and water. The rats were cared for and treated in accordance with the Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation. The present study received ethical approval from the Animal Ethics Committee of Nanjing Medical University.

Mangiferin was purchased from Nanjing Zelang Medical Technological Co., Ltd. (Nanjing, China) and palmitic acid (PA) was from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (cat. no. KGA108) was obtained from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Matrigel was purchased from Corning, Inc. (Corning, NY, USA). Antibodies against phosphorylated (p)-NF-κB p65 (Ser536; 1:1,000; cat. no. 3033), NF-κB p65 (1:1,000; cat. no. 8242), NF-κB p50 (1:1,000; cat. no. 32360), p-NF-κB p50 (1:1,000; cat. no. 28849), cluster of differentiation (CD)9 (1:1,000; cat. no. 9276), CD63 (1:1,000; cat. no. 59479) and β-tubulin (1:1,000; cat. no. 2146) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Rat arterial endothelial cells were isolated according to a standard protocol as previously described (20). Briefly, the rats were anesthetized with diethyl ether by inhalation prior to conducting the experiments. After the rats were sacrificed by decapitation following anesthesia, the arteries were excised and immersed in 75% ethanol for 30 sec for disinfection. Next, the arteries were digested with 0.25 collagenase type II for 10 min and 0.25% trypsin for another 10 min at 37°C. Cells were then centrifuged at 300 × g for 6 min at 4°C, resuspended in endothelial cell growth basal medium-2 (EBM-2; cat. no. cc-4176; Lonza Group, Ltd., Basel, Switzerland) with 10% fetal bovine serum (FBS; cat. no. 10100154, Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and plated on laminin-coated dishes. Arterial endothelial cells at the third passage were used for further analyses.

Rats were anesthetized with diethyl ether and sacrificed by decapitation. PVAT samples were obtained from the rat thoracic aorta, cut into small pieces, weighed to an equal size (50 mg) and rinsed using PBS. PVAT samples were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% exosome-free FBS (cat. no. EXO-FBS500A-1; System Biosciences, Palo Alto, CA, USA). According to our previous study (21), the PVAT samples were cultured with PA (100 µM) in the presence or absence of mangiferin (0.1, 1 or 10 µM) for 2 h; samples cultured without PA or mangiferin were used as the control group. Subsequent to washing with PBS, PVAT samples were incubated in DMEM for a further 22 h. The supernatant was then collected as the conditioned medium (CM), and larger particles were removed by centrifugation at 10,000 × g for 5 min at 4°C and filtration through a 0.22-µm filter. Exosomes in the PVAT CM were separated using total exosome isolation reagent (cat. no. 4478359; Invitrogen; Thermo Fisher Scientific, Inc.), and were observed by transmission electron microscopy (JEM-2000EX; JEOL, Ltd., Tokyo, Japan). Finally, exosomes derived from control, PA and PA + mangiferin groups were added to endothelial cells.

Endothelial cells were seeded in 24-well plates, and then the cells were fixed with 4% paraformaldehyde for 30 min at 4°C. Triton X-100 (0.1%) was used to permeabilize endothelial cells prior to washing with PBS for three times. Endothelial cells were then blocked with 5% bovine serum albumin (BSA; Sangon Biotech Co., Ltd., Shanghai, China) for 20 min at room temperature. Cells were incubated overnight at 4°C with the primary antibodies (anti-CD63, anti-p65 and anti-p50) and then incubated at room temperature for 1 h with the secondary antibodies (Alexa Fluor^® 594-conjugated donkey anti-rabbit secondary antibody; 1:100; cat. no. 1869587, Invitrogen; Thermo Fisher Scientific, Inc.). The cells were washed with PBS and the nuclei were stained with DAPI (~20 µg/ml; Thermo Fisher Scientific, Inc.) for 15 min at room temperature.

Rats were anesthetized with diethyl ether and sacrificed by decapitation. The thoracic aorta was stripped, immediately placed in 4°C Krebs-Henseleit (K-H) solution and incubated with 95% O₂ and 5% CO₂. Following careful removal of adherent tissues, four to eight samples of 3-mm long rings were cut. Subsequently, the aortic samples were suspended in an organ bath (SV-4; Chengdu Taimeng Software Co., Ltd., Chengdu, China) containing 10 ml K-H solution, and were maintained at 37°C and pH 7.4, and continuously aerated with 95% O₂ and 5% CO₂. The aortic segments were exposed to 60 mM KCl to assess their functions following progressive stretching to a basal tension of 2.0 g, and then stabilized for ≥90 min. Exposure to 10 µM acetylcholine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to relax the segment, which was pre-contracted with 1 µM phenylephrine (Sigma-Aldrich; Merck KGaA). The integrity of the vascular endothelium was confirmed, while the diastolic rate reached >80%. The thoracic aorta was cultured with CM for 15 min in the organ bath, and the tone change was recorded and expressed in terms of tension.

Endothelial cells were incubated with exosomes obtained from the different treatment groups for 48 h. Cells were subsequently collected using 0.25% trypsin without EDTA. Following three washes in PBS, cells were incubated with 500 µl binding buffer containing 5 µl Annexin V/FITC and 5 µl PI (Nanjing KeyGen Biotech Co., Ltd.) for 30 min in the dark. Subsequently, apoptosis was measured using a BD FACSCalibur flow cytometer and data were analyzed using BD CellQuest™ (both from BD Biosciences, Franklin, Lakes, NJ, USA).

Endothelial cells were incubated in EBM-2 at 37°C for 48 with exosomes (10 µg/ml) obtained from the different PVAT treatment groups. Cells were subsequently collected using 0.25% trypsin for 1 min at 37°C without EDTA, seeded in the upper chambers 6-well Transwell inserts with an 8-µm pore size at a density of 2×10⁴ cells/well in normal EBM-2, and incubated at 37°C for 24 h; normal EBM-2 with 10% FBS was added to the lower chambers. Non-migrated cells were gently removed with cotton swabs from the upper chamber, whereas cells that had migrated to the lower chamber were fixed using 4% paraformaldehyde at 4°C for 24 h and stained with 0.1% crystal violet for 30 min at room temperature. The filters were washed with distilled water, and images were obtained using an inverted microscope (magnification, ×100; BX41; Olympus Corporation, Tokyo, Japan).

Matrigel was thawed at 4°C for 24 h and then polymerized at 37°C for 1 h. Following incubation of endothelial cells with exosomes derived from different PVAT groups for 48 h as aforementioned, cells were collected using 0.25% trypsin and resuspended to obtain a concentration of 2×10⁵ cells/ml. Cells were then seeded on Matrigel-coated dishes for 8 h. Tube formation was examined using a microscope (magnification, ×200; IX51; Olympus Corporation).

Endothelial cells were incubated with exosomes from different PVAT treatment groups for 48 h as aforementioned, and the supernatants were subsequently collected. Then, the supernatants were concentrated with ultrafiltration tubes (Amicon Ultra-3K; Merck KGaA) by centrifugation at 3,000 × g for 30 min at 4°C. The concentrations of interleukin-6 (IL-6; cat. no. PR6000B) and tumor necrosis factor-α (TNF-α; cat. no. PRTA00) were then determined using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA).

Endothelial cells were incubated with exosomes obtained from PVAT treated with PA and mangiferin for 24 h as aforementioned. Total RNA was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The extracted RNA was dissolved in diethyl pyrocarbonate (DEPC)-treated water, and the RNA concentration was determined by optical density measurement at 260 nm using a spectrophotometer. RT was subsequently performed on RNA samples (2 µg) using the PrimeScript™ RT reagent (Takara Bio, Inc., Otsu, Japan), following the manufacturer's protocol. cDNA (1 µl) mixed with Ssofast™ EvaGreen^® Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), DEPC-treated water, forward primer and reverse primer (Sangon Biotech Co., Ltd.) to obtain a 20-µl reaction mixture was used for qPCR (22). qPCR was conducted as follows: 95°C for 30 sec, then 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The primers used in qPCR are presented in Table I. Expression was quantified using the 2^−ΔΔCq method and normalized to the endogenous control β-actin (23).

Cells were cultured in 6-well plates and treated with exosomes from the various groups as aforementioned upon reaching 70% confluence. The secretion of NO was determined 48 h later using an NO test kit (cat. no. A013-2, Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China) according to the manufacturer's protocol.

Briefly, the total protein was extracted from the samples using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentrations were quantified using a Bicinchoninic Acid Protein Assay kit (Biosky Biotechnology, Nanjing, China) according to the manufacturer's protocol. Next, protein samples (30 µg/lane) were separated by SDS-PAGE on 10% gels and transferred to polyvinylidene difluoride membranes (0.45 µm; EMD Millipore, Billerica, MA, USA). Membranes were blocked for 2 h at room temperature with 1–5% BSA (Sangon Biotech Co., Ltd.) in TBS-Tween-20 [containing 5 mmol/l Tris-HCl (pH 7.6), 136 mmol/l NaCl and 0.05% Tween-20] and then incubated with the aforementioned antibodies against p65, p-p65, p50, p-p50, CD9, CD63 and tubulin overnight at 4°C. Subsequently, the membranes were incubated with secondary antibody [horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; cat. no. A0208) or anti-mouse IgG (cat. no. A0216); 1:5,000; Beyotime Institute of Biotechnology] at 37°C for 2 h. Signals were detected using enhanced chemiluminescence western detection reagents (Thermo Fisher Scientific, Inc.) and quantified using Image-Lab version 5.2.1 software (Bio-Rad Laboratories, Inc.).

All values are presented as the mean ± standard deviation. The statistical significance between mean values was evaluated using one-way analysis of variance, followed by Tukey's test. Statistical analyses were performed using SPSS software, version 23.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Initially, the effect of mangiferin on the release of exosomes was determined. As demonstrated in Fig. 1A, a large number of exosomes were present in the CM supernatant. Exosomes were extracted and concentrated from PVAT CM following stimulation with PA and mangiferin (0.1, 1 and 10 µM), or control. PA evidently inhibited the release of exosomes from PVAT compared with the control group. However, the number of PVAT-released exosomes was markedly increased by mangiferin under PA-induced inflammation conditions in a dose-dependent manner. Subsequently, exosomes were extracted from CM using total exosome isolation reagents. A total of 10 µg exosomes was selected in all groups to ensure that the same concentration of exosomes was added to the endothelial cell culture. As displayed in Fig. 1B, the results revealed that the expression of CD9 and CD63 proteins, which are specific markers of exosomes, was similar in all groups. The uptake of exosomes by endothelial cells was similar when CM that was obtained from PVAT stimulated with mangiferin and PA was used, indicating that the intake of exosomes by endothelial cells was not influenced by the pre-treatment with mangiferin or PA. Immunofluorescence further demonstrated the uptake of PVAT-derived exosomes by endothelial cells (Fig. 1C).

Further experiments were performed to determine whether exosomes from mangiferin-stimulated PVAT influence the endothelial function. Preliminary results indicated that the effect of exosomes on endothelial cells in vitro was not altered following 24-h exposure of the cells to exosomes (data not shown). Thus, 48-h exposure of endothelial cells to exosomes was conducted in the current study in order to observe the regeneration and apoptosis levels. Exosomes produced following mangiferin treatment (1 and 10 µM) markedly promoted the tube formation ability of endothelial cells compared with the PA-induced exosomes (Fig. 2A). In addition, the migration of endothelial cells was inhibited by PA-induced exosomes, whereas migration was clearly increased by mangiferin (10 µM)-induced exosomes compared with PA-induced exosomes (Fig. 2B). The apoptosis level was also determined in these treatment groups, and the data revealed that mangiferin (1 and 10 µM)-induced exosomes significantly reduced the apoptosis of endothelial cells compared with PA-induced exosomes (Fig. 2C). Organ bath analysis identified that PA-induced PVAT-derived exosomes significantly stimulated the constriction of normal aortic rings compared with the control, whereas mangiferin (1 and 10 µM)-induced exosomes significantly promoted the relaxation of aortic rings compared with PA-induced exosomes (Fig. 2D), indicating that the relaxation of aortic rings was blocked by exosomes produced from PA-stimulated PVAT. However, this effect was reversed upon the use of exosomes produced by mangiferin-treated PVAT, ameliorating the endothelial dysfunction caused by PA-induced adipose tissue inflammation.

Endothelial inflammation induced by PA treatment was further analyzed using RT-qPCR and ELISA. The mRNA expression levels of the inflammatory factors IL-6 and TNF-α were significantly increased in endothelial cells treated with PA-induced PVAT-derived exosomes (PA alone group) compared with the control group (Fig. 3A); however, these effects were significantly reversed in the cells incubated with mangiferin-induced exosomes. In addition, the levels of ARG1 mRNA in endothelial cells were significantly upregulated upon incubation with mangiferin-induced PVAT-derived exosomes compared with PA-induced exosomes (Fig. 3A). As compared with the PA-stimulated alone group, exosomes produced by mangiferin-stimulated PVAT (0.1, 1 and 10 µM) significantly inhibited the release of IL-6 and TNF-α proteins (Fig. 3B), and promoted NO generation in endothelial cells (Fig. 3C). These results demonstrated that exosomes from mangiferin-stimulated PVAT reduced endothelial inflammation and increased endothelial relaxation.

As mangiferin-induced PVAT-derived exosomes ameliorated endothelial inflammation, the mechanisms underlying the effect of mangiferin-induced exosomes on endothelial cells were investigated. Mangiferin (10 µM)-induced PVAT-derived exosomes significantly inhibited p65 phosphorylation in endothelial cells compared with the effect of PA-induced exosomes (Fig. 4A). Furthermore, mangiferin (10 µM)-induced PVAT-derived exosomes also markedly reduced p50 phosphorylation in endothelial cells (Fig. 4B). The translocation of p65 and p50 in endothelial cells treated with PVAT-derived exosomes was also detected. PA-induced PVAT-derived exosomes evidently promoted the translocation of p65 and p50, whereas the effects were reversed when exosomes from mangiferin (10 µM)-treated PVAT were used (Fig. 4C and D). These results demonstrated that exosomes produced from mangiferin-treated PVAT reduced the activation of NF-κB signaling.