Berberine, dimethyl sulfoxide, and compound C were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzol^® reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The Lactate Dehydrogenase (LDH) Assay kit was obtained from Beyotime Institute of Biotechnology (Jiangsu, China). The ImProm-II™ Reverse Transcription system was purchased from Promega Corporation (Madison, WI, USA). The SYBR master mix for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was purchased from Applied Biosystems Life Technologies (Foster City, CA, USA). Antibodies against NF-κB p65, phosphorylated- and total-adenosine monophosphate-activated protein kinase (AMPK), phosphorylated- and total-acetyl-CoA carboxylase (ACC), and histone were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-GAPDH, AMPKα1/2 specific small interfering (si)RNA, control scrambled siRNA and the siRNA reagent system were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Recombinant human TNF-α, and ELISA kits for ICAM-1 and MCP-1 were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).

The HAECs were obtained from Sciencell Research Laboratories (Carlsbad, CA, USA) and were maintained in endothelial cell medium supplemented with growth supplements (cat. no. 1052; Sciencell Research Laboratories) and fetal bovine serum (5% vol/vol; Invitrogen Life Technologies) at 37°C in an atmosphere containing 5% CO₂. Once the cells had reached 70–80% confluence and prior to initiation of the experiments, the cells were serum starved for 24 h in serum-free medium. Subsequently, the HAECs were incubated with berberine at various concentrations, and/or treated with recombinant human TNF-α (10 ng/ml). The cells were cultured for various time periods, and the supernatants were subsequently collected and the cells were harvested. To investigate the potential contribution of the AMPK pathway on the effects of berberine, the HAECs were pretreated with chemical inhibitor compound C (10 µM) for 30 min, or were transfected with AMPK siRNA (100 µM). Briefly, cells were transfected with AMPK siRNA or control scrambled siRNA (Santa Cruz Biotechnology, Inc.) using the siRNA reagent system for 6 h; the medium was then replaced with normal culture medium prior to treatment with berberine and TNF-α.

To evaluate the effects of berberine on the viability of the HAECs, an LDH assay was performed, which is based on the release of LDH. The HAECs were seeded into 96-well plates at a density of 5×10³ cells/well, and following 3 days incubation at 37°C the medium was replaced with serum-free medium, and the HAECs were incubated at 37°C for a further 24 h. Berberine was then added to the cells at final concentrations of 2, 5, 10 or 25 µM), and cultured for 24 h at 37°C. Subsequently, the cell culture supernatants were collected and the LDH content was analyzed, according to the manufacturer's instructions. The toxicity of berberine on the cells was also investigated using a trypan blue dye exclusion assay (0.4%; Sigma-Aldrich), as previously reported (18).

Immediately following termination of the experiment involving treatment with berberine, the cell culture supernatants were collected and stored at −80°C for subsequent analysis of the expression of inflammatory molecule proteins. The concentrations of ICAM-1 and MCP-1 were determined using ELISA kits, according to the manufacturer's instructions. Absorbance was measured at 450 nm using an ELISA reader (model 3550; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Total RNA was extracted from the cells using TRIzol^® reagent, according to the manufacturer's instructions. The isolated RNA was then reverse transcribed into cDNA using the reverse transcription system. mRNA expression levels were analyzed using an ABI Prism 7500 system (Applied Biosystems Life Technologies) and SYBR Green master mix. The PCR reaction mix (20 µl) consisted of 1 µl cDNA (50 ng), 10 µl 2X SYBR Green master mix and 5 pmol each of the forward (1 µl) and reverse (1 µl) primer (Beijing Sunbiotech Co., Ltd., Beijing) made up with sterile water. PCR cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 40 sec. Data were subsequently collected and quantitatively analyzed. GAPDH was used as an endogenous control for sample normalization. The results are presented as fold increases, compared with the expression of GAPDH. The primer sequences were as follows: ICAM-1, forward 5′-AATGCCCAGACATCTGTGTCCC-3′ and reverse 5′-GGCAGCGTAGGGTAAGGTTCTT-3′; MCP-1, forward 5′-TTCCATGGACCACCTGGACA-3′ and reverse 5′-TGTCTGGGGAAAGCTAGGGG-3′; and GAPDH, forward 5′-CTCCCCACACACATGCACTTA-3′ and reverse 5′-CCTAGTCCCAGGGCTTTGATT-3′. Relative quantification was performed using the 2^−ΔΔCt method (19).

The HAECs were harvested and lysed with lysis buffer containing 50 µM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA and 100 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology). The cell lysate was then centrifuged at 14,000 × g at 4°C for 5 min, and the supernatant was collected. Nuclear protein extracts were isolated using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Protein concentrations were determined using a bicinchoninic acid method (Beyotime Institute of Biotechnology). Equal quantities of protein (50 µg) from each sample were separated by 12% SDS-PAGE (Beyotime Institute of Biotechnology). The proteins were then transferred onto nitrocellulose membranes (Beyotime Institute of Biotechnology), blocked with 5% skim milk in tris-buffered saline supplemented with 0.1% Tween 20 (TBST; Beyotime Institute of Biotechnology), and incubated overnight at 4°C with the following polyclonal rabbit anti-human primary antibodies: Anti-NF-κB p65 (1:1,000; cat. no. 3039), anti-phosphorylated-AMPK (1:1,000; cat. no. 2531), anti-AMPK (1:1,000; cat. no. 2532), anti-phosphorylated-ACC (1:1,000; cat. no. 3661), anti-ACC (1:1,000; cat. no. 3662) and anti-histone (1:1,000; cat. no. 2578). The membranes were then washed three times with TBST, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2,000; cat. no. 7074) for 2 h at room temperature. The blots were visualized using enhanced chemiluminescence reagent (Pierce Biotechnology, Inc.). Relative protein expression levels were determined by densitometric analysis using ImageJ software, version 1.45 (National Institutes of Health, Bethesda, MD, USA).

Data are presented as the mean ± standard error of the mean. Statistical analyses were performed using GraphPad Prism 5.0 sofrware (GraphPad Software, Inc., La Jolla, CA, USA), and the statistical significance of differences was determined using one-way analysis of variance and Student's t-test. P<0.05 was considered to indicate a statistically significant difference. All experiments were performed at least three times.

The HAECs were cultured with various concentrations of berberine, in order to evaluate its toxic effects. Treatment with 2, 5, 10 or 25 µM berberine for 24 h did not lead to HAEC toxicity, compared with the controls (Fig. 1A). A trypan blue dye exclusion assay was also used to confirm the effects of berberine on HAEC toxicity, and similar results were observed (Fig. 1B).

To investigate the effects of berberine on the production of inflammatory molecules, the protein expression levels of ICAM-1 and MCP-1 was detected in the HAECs following co-treatment with TNF-α for various durations. TNF-α significantly increased the protein secretion of ICAM-1 and MCP-1 after 24 h (Fig. 2A and B), and the mRNA expression levels of ICAM-1 and MCP-1 were also increased 6 h following TNF-α stimulation (Fig. 2C and D). By contrast, berberine significantly attenuated TNF-α stimulated protein secretion and the mRNA expression levels of ICAM-1 and MCP-1 in HAECs in a concentration-dependent manner (Fig 2A–D).

Further experiments were performed to examine whether NF-κB activation was affected by treatment with berberine. As shown in Fig. 3, the expression of nuclear NF-κB p65 increased following stimulation of the HAECs with TNF-α, whereas berberine significantly downregulated the nuclear expression levels of TNF-α-stimulated NF-κB p65.

Treatment of the HAECs with berberine induced the activation of AMPK, which was determined by measuring the phosphorylation of AMPK. Berberine increased the activation of AMPK and its downstream molecule, ACC, in a time-dependent manner (Fig. 4).

The present study investigated the role of AMPK activation in the inhibitory effects of berberine on the TNF-α-induced expression of inflammatory molecules and NF-κB-p65 activation. The inhibitory effects of berberine on TNF-α induced expression of ICAM-1 and MCP-1 was attenuated when the HAECs were treated with AMPK inhibitor compound C, or were transfected with AMPK-specific siRNA (Fig. 5A and B). In addition, the inhibitory effects of berberine on TNF-α-induced NF-κB p65 activation were attenuated in the cells, which were treated with compound C or transfected with AMPK siRNA. These results indicated that AMPK activation may mediate the inhibitory effects of berberine on the TNF-α-induced expression of inflammatory molecules and NF-κB activation (Fig. 5C and D).