BM leaves (100 g) were cut into small pieces and immersed in distilled water. The mixture was treated with ultrasound for 1 h followed by heating at 100°C for 30 min twice. The supernatant was concentrated by rotary evaporation and kept at −80°C overnight. The freezing supernatant was then placed in a freeze dryer for 48 h to obtain the raw aqueous extract powder. The production ratio of BM was 12.3–13.6%. High-performance liquid chromatography (HPLC) analysis of BM aqueous extract was conducted on an Ultimate AQ-C18 column (250×4.6 mm; 5 μm). The mobile phase consisted of acetonitrile (A) and water (B), using a gradient elution of 5–8% A at 0–5 min, 8–14% A at 5–30 min, 14–50% A at 30–55 min and 50–90% A at 55–60 min. The solvent flow rate was 1.0 ml/min and the column temperature was ambient. The detection wavelength was set at 250 nm. The berberine concentration in the extracts was 0.014–0.018 mg/ml [berberine was recommended as a representative compound in BM by Chinese Pharmacopeia (13)] based on the absorbance at 250 nm assessed by ultraviolet-visible spectrophotometry (Fig. 1A). The powder was dissolved in phosphate-buffered solution (PBS) and passed through a 0.45-μm filter for later use.

Mouse macrophage RAW264.7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified incubator with 5% CO₂. Human GLM samples were collected and cut into small pieces. The shredded tissues were filtered with 200-μm nylon mesh and suspended in PBS solution. The filtered cells were centrifuged at 800 rpm for 5 min at room temperature and washed three times with PBS, and the precipitates were suspended and cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% FBS (Gibco, Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin.

The effect of BM on cell proliferation was studied by cell growth assay using 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described previously (14). Briefly, the cells were seeded onto a 96-well plate at a density of 4×10³ cells/well. Following serum starvation, different concentrations of BM (2.5, 5, 10, 20, 40, 60, 80 and 100 μg/ml) were added to the wells, with 6 repeats for each concentration. LPS was added at 1 μg/ml to simulate the inflammation status (15). The cell growth rate was detected using a cell proliferation MTT kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions. The purple formazan was dissovled with Dimethyl sulfoxide (Sigma, St. Louis, MO, USA) and detected at a wavelength of 540 nm. Triplicate independent experiments were performed.

RAW264.7 cells (1×10⁶/well) were synchronized in G1 phase by serum deprivation, and different concentrations of BM (25, 50 and 100 μg/ml) and 1 μg/ml LPS were then added. Cells were harvested after 48 h. Propidium iodide (PI)-stained single-cell suspension was analyzed on a FC5000 cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using CXP software version 2.0 (Beckman Coulter, Fullerton, CA, USA) for data analysis. The experiment was repeated three times. For apoptosis analysis, following BM treatment (25, 50 and 100 μg/ml) for 48 h, cells were stained with Annexin V and PI (Clontech Laboratories, Inc., Mountainview, CA, USA) for 15 min at room temperature. The apoptotic index was determined by flow cytometry with Flowjo software. 2′7′-Dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich; Merck KGaA) was used to measure ROS formation. After exposure to different concentrations of BM (25, 50 and 100 μg/ml) for 48 h, the cells were then incubated in DCFH-DA-containing medium (final concentration, 10 μM) at 37°C for 20 min. The cells were washed with PBS three times to remove DCFH-DA that had not entered into the cells. The intracellular concentration of DCFH-DA was then measured by flow cytometry.

NO levels in the culture media were determined by the Griess Reagent system according to the manufacturer's protocols (Promega Corporation, Madison, WI, USA). Briefly, 50 μl cell culture supernatant was transferred into a 96-well plate and mixed with 100 μl Griess reagent (equal volumes of 1% w/v sulfanilamide in 5% v/v phosphoric acid and 0.1% w/v naphthylethylenediamine-HCl). Next, the plate was incubated for 5–10 min at room temperature, and absorbance was measured at 550 nm using a microplate reader. The amount of NO was calculated according to the standard sodium nitrite curve.

Cells were lysed in RIPA buffer (Sigma) containing a protease inhibitor mixture (Roche Diagnostics, GmbH, Mannheim, Germany). The protein concentration was determined with the bicinchoninic acid assay (Thermo Fisher Scientific, Inc., Bonn, Germany). Quantified protein lysates (15 μg) were resolved by 10% sodium dodecyl sulfate - 12% polyacrylamide gel electrophoresis. The proteins were then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Each membrane was blocked for 2 h with 2% bovine serum albumin (Sigma) and then incubated overnight at 4°C with 1 μg/ml primary antibody (dilution, 1:1,000). The antibodies COX-2 (A5787), NF-κB (A2574), p-NF-κB (AP0475), IκB kinase (IKK)α (A2062), IKKβ (A2087), ERK (A0229), p-ERK (AP0472), JNK (A6117), p-JNK (AP0276), p38 (A0227), p-p38 (AP0056) were obtained from ABclonal (Boston, MA, USA). The antibodies p-IKKα/β (2697), IKBα (4814) p-IKBα (2859) and β-actin (4970s) were purchased from Cell Signaling Technology (Danvers, MA, USA). iNOS (bs-0162R) was provided by BIOSS (Beijing, China). After 3 washes with Tris-buffered saline with 0.05% Tween-20, the membranes were incubated with HRP-conjugated anti-rabbit (7074s) or anti-mouse (7076) secondary antibodies (dilution, 1:2,000; Cell Signaling Technology) for 2 h at room temperature. The signals were visualized using the ECL Advance reagent (GE Healthcare) and quantified using Quantity One software (version 4.5; Bio-Rad Laboratories, Hercules, CA, USA).

Total RNA from the cells was extracted using TRIzol reagent (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and RT was performed using a first strand CDNA synthesis kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer's protocols. PCR amplification conditions consisted of an initial denaturation step at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 50°C for 30 sec and extension at 72°C for 45 sec; a final extension period at 72°C for 5 min completed the amplification. qPCR analysis was performed using a SYBR-Green kit (Roche Diagnostics, Basel, Switzerland) on a Roche lightcycler 480 detector (Roche Diagnostics, GmbH, Manheim, Germany). The primers for iNOS, COX-2, IL-1β, IL-6, CCL-2, CCL-3, CCL-5, secreted tumor necrosis factor receptor 1 (sTNFR1) and glyceraldehyde 3-phosphate dehydrogenase are listed in Table I. Cq value was measured during the exponential amplification phase. The relative expression level (defined as fold-change) of the target gene was given by 2^−ΔΔCq and normalized to the internal control.

The pathway reporter plasmids of phosphorylated (p)NF-κB-Luc and phosphorylated activated protein 1 (pAP-1)-Luc, which contain NF-κB or AP-1 binding sites in the promoter region, were purchased from Clontech Laboratories, Inc. Overnight cultured RAW264.7 cells in 96-well plates were transfected with 100 ng pNFκB-Luc or pAP-1-Luc reporter vector and 5 ng phRL-TK Renilla luciferase reporter (Promega Corporation) using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). At 16 h post-transfection, the cells were treated with various concentrations of BM and LPS (for NF-κB reporter). After another 24 h, the cells were lysed for Firefly/Renilla luciferase activity assay using a dual-luciferase assay kit (Promega Corporation). The relative luciferase activities were normalized by LPS/IFN-c or PMA control.

RAW264.7 cells (3×10⁵/well) were seeded in 24-well plates containing coverslips. Following LPS or BM treatment for 24 h, coverslips were then fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100. After blocking in 10% goat serum (DAKO, Glostrup, Denmark) for 1 h at room temperature, the coverslips were incubated with primary NF-κB antibody (1:100; A2574; ABclonal) overnight at 4°C and then with secondary Alexa Fluor 555-conjugated antibody (1:200; 4413s; Cell Signaling Technology) for 2 h at room temperature. Finally, the samples were incubated with 1 μg/ml 4′,6-diamidino-2-phenylindole DAPI (Sigma) for 15 min at room temperature for nuclear staining and detected under a confocal microscope (LSM710; Carl Zeiss, Jena, Germany).

Mouse and human inflammatory antibody array C1 kits were purchased from RayBiotech (Norcross, GA, USA). Briefly, cell supernatants from RAW264.7 cells or primary cultured GLM samples prior to and subsequent to BM treatment were collected. Once the antibody array membranes had been blocked in 5% BSA for 30 min at room temperature, cell supernatants were cultured with antibody arrays overnight at 4°C and washed three times. A biotinylated antibody cocktail was then incubated with the membranes for 2 h, followed by signal amplification with horseradish peroxidase-streptavidin. Finally, the signals were detected by chemiluminescence method with an ECL kit purchased from GE Healthcare Life Sciences (Little Chalfont, UK).

All human GLM patients provided written informed consent for the use of clinical specimens for medical research. Studies using human tissue were reviewed and approved by the committee for the ethical review of research involving human subjects of Guangzhou University of Chinese Medicine (Guangzhou, Guangdong, China). The methods for the collection and application of human specimens were in accordance with International Ethical Guidelines for Biomedical Research Involving Human Subjects. Clinical specimens were obtained based on the diagnosis of clinical syndromes, ultrasound findings and pathological characteristics. A total of 10 GLM specimens were sliced into small pieces and put into digestion buffers (StemCell Technologies, Vancouver, Canada) containing collagenase and hyaluronidase for 2–3 h with agitation. The cell suspension was then filtered through a 100-μm cell strainer and the filtered content was then centrifuged at 200 × g for 8 min at 4°C. The cell pellets were then suspended in 1 ml RPMI-1640 medium above 5 ml 45% Percoll (GE Healthcare, Uppsala, Sweden) in the middle and 5 ml 60% Percoll at the bottom in a 15-ml tube. Mononuclear cells were collected from the cell layer in the interphase between 45 and 60% Percoll. The cells were then cultured in RPMI-1640 medium at 37°C and treated with BM. The cells supernatants were then collected for the cytokine array detection and the cellular protein lysates were subjected to western blot assay as stated above.

Data are presented as the mean ± standard error of the mean (SEM). The results were analyzed by SPSS 20.0 software (IBM Corp., Armonk, NY, USA). Mean values were derived from at least three independent experiments. Statistical comparisons between experimental groups were performed by one-way analysis of variance followed by post hoc Dunnett's test. The level of statistical significance was set at P<0.05.

As with the majority of TCM formulae, a decoction of BM used clinically. The pharmacological activity of BM and the repeatability of the experiments performed using it are dependent on the controlled quality of the decoction. In the present study, in order to maintain a consistent quality, BM was extracted with water and prepared carefully. The preparation procedure is summarized in Fig. 1B. HPLC analysis further validated that the chemical fingerprints were in accordance in three different batches (Fig. 1A). Meanwhile, quality analysis of BM was performed with LC-MS. As shown in Fig. 1C, the MS spectra revealed the presence of chlorogenic acid, jateorhizine, magnoflorine and berberine in the BM extract.

To study whether BM could inhibit the proliferation of RAW264.7 cells, LPS was added as positive inducer of inflammation and three batches of BM were tested in a dose- and time-dependent manner, respectively. The results showed that BM exerted few inhibitory effects on the proliferation of RAW264.7 cells, which not only validated the bioactivity consistency between three different batches of BM, but also implied that BM had few cytotoxic effects (Fig. 2A). Similarly, cell cycle analysis also indicated that BM had limited influence on the distribution of the cell cycle (Fig. 2B). Meanwhile, Annexin V-FITC/PI assay demonstrated that following BM administration, limited changes in the apoptotic ratio of RAW264.7 cells occurred (Fig. 2C). All these results indicated that the anti-inflammation activity of BM was not due to its survival and proliferation inhibitory effects.

During inflammation, a significant increase in oxygen consumption would result in a massive elevation of intracellular ROS, which acts as an upstream signal to promote the inflammation process and even destroys cells to induce abscess formation (16). To determine whether the LPS-induced intracellular redox state could be suppressed by BM, ROS production was detected prior to and following BM treatment. The results showed that the level of ROS in response to LPS was significantly higher than that of unstimulated cells. However, with the increasing dose of BM in the medium, the ROS production was significantly inhibited in a dose-dependent manner (Fig. 3A and B). Furthermore, since excessive NO production is a critical marker of inflammatory diseases, such as rheumatoid arthritis and autoimmune diseases, the present study detected whether BM also led to a decrease in NO levels. RAW264.7 cells were treated with different doses of BM for 2 h prior to stimulation with LPS (1 μg/ml) (17). NO production is reflected in the accumulation of nitrite in the cell culture medium. The results revealed that LPS stimulation caused a significant increase of nitrite concentration, while BM administration inhibited NO release in a dose-dependent manner, with >50% inhibition at a concentration of 100 μg/ml (Fig. 3C). These results suggested that BM was capable of scavenging ROS level and thereby alleviating inflammation severity.

The iNOS gene is known to be the primary regulator of NO production in macrophages, and the COX-2 gene is usually increased under inflammation and malignant conditions (18). To determine the anti-inflammatory effects of BM, the expression levels of iNOS and COX-2 were measured by western blotting and qPCR analysis. As shown in Fig. 4A, the protein expression levels of iNOS and COX-2 were significantly elevated following LPS stimulation, but the two proteins displayed a marked downregulated tendency in response to increasing concentration of BM treatment. Consistently, the mRNA expression levels of iNOS and COX-2 were also suppressed by BM (Fig. 4B). Since NF-κB acts as the common transcription factor of the two genes, the results indicated that BM may inhibit inflammation via suppressing NF-κB signaling.

During the progression of inflammation, IL-1β and IL-6 are known to be pro-inflammatory cytokines, which are constitutively secreted and activated, mediating macrophage chemotaxis, angiogenesis and sustained tissue destruction (19,20). qPCR analysis was also applied to determine the levels of IL-1β and IL-6 prior to and following BM administration. When the cells were induced by LPS, the expression levels of IL-1β and IL-6 mRNA were markedly increased. However, when BM was added to the media, the mRNA levels of the two cytokines were significantly decreased in a dose-dependent manner (Fig. 4C). All these results indicated that BM is able to block the secretion of pro-inflammatory cytokines.

Since NF-κB and AP-1 control the transcription of iNOS/COX-2 and a variety of pro-inflammatory cytokines, the present study investigated the effect of BM on these signaling pathways using a luciferase reporter assay. As demonstrated in Fig. 5, the transcriptional activities of NF-κB and AP-1 were inhibited dose-dependently following BM treatment, but the inhibition ratio of NF-κB was higher. These results indicated that BM blocked LPS-induced NF-κB and AP-1 signaling, leading to the inhibition of LPS-induced production of NO and COX-2.

Considering the nuclear translocation of the p65 subunit is a critical step determining its downstream transcriptional activity, the present study investigated whether BM affects the subcellular compartmentalization of p65. RAW264.7 cells were pretreated with BM for 12 h and stimulated with LPS for another 1 h, and the intracellular p65 distribution was then evaluated by confocal microscopy. As shown in Fig. 6A, the p65 subunit was mainly distributed in the nucleus following BM treatment. However, pre-treatment with BM significantly reduced the p65 translocation compared with that in the control cells with LPS stimulation. In order to identify the upstream target of BM-mediated NF-κB suppression, the expression status of IKK and IκBα was investigated. RAW264.7 cells were stimulated with LPS for 1 h in the presence or absence of BM. As shown in Fig. 6B–D, the phosphorylated level of IKKα/β was significantly elevated following LPS stimulation, but began to decrease gradually with the increasing dose of BM. However, the expression status of IKKα and IKKβ exhibited limited changes in response to BM treatment. Notably, the phosphorylation of IκBα and p65 was also inhibited following BM administration in the presence of LPS stimulation, respectively. All these results indicated that BM inhibited NF-κB activation by blocking the phosphorylation of IκBα and its upstream kinase IKKα/β.

Since the MAPK pathway is also involved in regulating NF-κB and AP-1 signaling transduction, the phosphorylated levels of ERK1/2, JNK and p38 prior to and following BM treatment were evaluated. As expected, LPS significantly induced the phosphorylation of all three after 1 h of incubation. Notably, BM inhibited the phosphorylation of JNK and p38 in a dose-dependent manner, but had little effect on the phosphorylation status of ERK1/2 (Fig. 6C and D). These findings suggested the ability of BM to suppress JNK and p38 activation, which may be responsible for the inhibition of NF-κB signaling and pro-inflammatory cytokine expression.

Although it was demonstrated that BM inhibited the inflammation via the NF-κB and MAPK pathways, the precise molecular mechanisms accounting for its anti-inflammation effects remained unclear. In order to determine the molecular target of BM in RAW264.7 cells, an inflammation array was applied. The supernatants of RAW264.7 cells prior to and following BM treatment were collected and subjected to array test. As shown in Fig. 7A, a total of 40 inflammatory-related cytokines were detected. Following BM administration, the expression levels of CCL-2 (also known as monocyte chemotactic protein-1), CCL-3 (also known as macrophage inflammatory protein-1α), CCL-5 (also known as regulated on activation, normal T cell expressed and secreted) and sTNFR1 were significantly inhibited. The results indicated that BM could inhibit the inflammation via interacting with multiple cytokines. qPCR further validated that among the 4 cytokines, the inhibited ratios of CCL-3 and CCL-5 were the most significant, implying that the anti-inflammatory molecular target of BM may be attributed to their downregulation (Fig. 7B). Importantly, western blotting results further validated that the inhibitory effects of BM on p65 phosphorylation were relieved after adding CCL-5, indicating that the anti-inflammatory effects of BM may be partly attributed to CCL-5 suppression (Fig. 7C).

Meanwhile, human GLM samples were also collected to validate the molecular mechanisms of BM (Fig. 8A). Consistent with the in vitro findings, cytokine array results indicated that the expression of CCL-2, CCL-3, CCL-5 and sTNFR was inhibited following BM treatment; the results further confirmed the anti-inflammatory effects of BM ex vivo (Fig. 8B and Table II). Similar to the results in in vitro studies, CCL-5 also exhibited the highest inhibition ratio following BM treatment, and western blotting results also indicated that the expression of phosphorylated p65, iNOS and COX-2 was inhibited by BM on GLM samples (Fig. 8C). All these results indicated that BM could inhibit inflammation via suppressing CCL-5 in in vitro and ex vivo models.