Fetal bovine serum (FBS), RPMI-1640, penicillin, streptomycin, 0.05% (w/v) trypsin-ethylenediamine tetraacetic acid (EDTA) and phosphate-buffered saline (PBS) were HyClone products (GE Healthcare, Logan, UT, USA). Cytokine (IL-6, TNF-α and IL-1β) enzyme-linked immunosorbent (ELISA) kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). TRIzol reagent and SuperScript II reverse transcriptase were Invitrogen products (Thermo Fisher Scientific, Waltham, MA, USA). Anti-phosphorylated IκBα (anti-p-IκBα; #2859), anti-IκBα (#4812), anti-NF-κB p65 (#6956), anti-p-NF-κB p65 (#3033)and anti-β-actin monoclonal mouse or rabbit (#4970)antibodies and anto-rabbit IgG (#7074) and anti-mouse IgG (#7076) horseradish peroxidase (HRP)-conjugated secondary antibodies were from Cell Signaling Technology (Danvers, MA, USA). Bio-Plex phosphoprotein assay kits were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). All other chemicals, unless otherwise stated, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Dried plant materials of Hedyotis diffusa Willd. were purchased from Guo Yi Tang Chinese Herbal Medicine Store (Fujian, China). The original herb was identified as Hedyotis diffusa Willd by Dr Wei Xu at the Department of Pharmacology, Fujian University of Traditional Chinese Medicine (Fuzhou, China). The material was coarsely ground prior to extraction. A total of 300 g of the material was extracted three times with 80% ethanol for 3 h at 50°C. The fluid was filtered through a filter with a 1-mm pore-size. The filtrate was then evaporated, and the crude extract was isolated using a column containing AB-8 macroporous adsorption resin (CangZhou Bon Chemical Co., Ltd., Hebei, China) with the application of 80% aqueous ethanol to elute the flavonoids. The ethanol solvent was then evaporated using a rotary evaporator (Model RE-2000; Shanghai Yarong Biochemical Instrument Factory, Shanghai, China).

Cells of the RAW 264.7 mouse monocyte-macrophage cell line (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI-1640 supplemented with FBS (10%), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Cells were plated in 96-, 24- and 6-well plates at densities of 8×10⁴, 1×10⁵ and 5×10⁵ cells/well. When cell treatments were conducted, the cells were incubated in serum-free medium for 4 h, then treated with LPS (1 µg/ml) and/or TFHDW for 24 h (for ELISA), or for 12 h [for reverse transcription-polymerase chain reaction (RT-PCR)] or for 20 min (for western blotting) for the detection of protein or mRNA expression.

RAW 264.7 cells were grown in 96-well plates at a density of 8×10⁴ cells/ml. TFHDW was added at various concentrations (0, 25, 50, 100, 200, 400 and 800 µg/ml). A methyl thiazolyl tetrazolium assay (MTT) assay was used to measure the viability of the cells. Briefly, after 24 h incubation with or without TFHDW, MTT solution (0.05 mg/ml) was added and the cells were incubated for another 4 h at 37°C. Then, the supernatant was removed and 100 µl dimethylsulfoxide was added to dissolve the formazan. The absorbance of the cells was measured using a microplate reader (ELx800; BioTek, Winooski, VT, USA) at wavelength of 570 nm. The control group, which consisted of untreated cells, was considered to comprise 100% viable cells. Results are expressed as a percentage of viable cells compared with the control group.

The release of NO by iNOS is one of the major factors contributing to the inflammatory process (20). The production of nitrite, a metabolite of NO, was assessed by the Griess reaction. Cells were plated in 96-well plates (8×10⁴ cells/ml) and treated with LPS (1 µg/ml) in the presence or absence of TFHDW (50, 100 and 200 µg/ml). After incubation for 24 h, suspended media were collected for measurement of the nitrite concentrations using the Griess reaction. This involved taking a 50-µl aliquot of the culture supernatant, mixing it with an equal volume of Griess reagent [0.1% N-(1-naphthyl)-ethylenediamine, 1% sulfanilamide in 5% phosphoric acid] and incubating the mixture at room temperature (RT) for 10 min. The absorbance at 540 nm was measured using a microplate absorbance reader. The concentration of nitrite was determined from a sodium nitrite standard curve.

RAW 264.7 cells were plated in a 24-well cell culture plate (1×10⁵ cells/ml) and incubated with TFHDW (50, 100 and 200 µg/ml) in the presence or absence of LPS (1 µg/ml) for 24 h. A 1-ml volume of culture-medium supernatant was then collected for measurement of the levels of IL-6, TNF-α and IL-1β using the relevant ELISA kit according to the manufacturer's instructions.

Total RNA was extracted from the cells using TRIzol reagent following the manufacturer's protocol. The purity and integrity of the RNA were assessed using a NanoDrop spectrophotometer (ND-2000C; Thermo Fisher Scientific). Subsequently, first-strand cDNA synthesis was performed with 2 µg total RNA using SuperScript II reverse transcriptase kit (Fermentas; Thermo Fisher Scientific) according to the manufacturer's protocol. The obtained cDNA was used to determine the mRNA levels of TNF-α, IL-6, IL-1β and iNOS using a DreamTaq Green PCR Master Mix (2X) PCR kit (Fermentas; Thermo Fisher Scientific). The primers used were as follows: TNF-α forward, 5′-CTCAAGGACAACAGCCAGTTC-3′ and reverse, 5′-GGCACTAAGGGCTCAGTCAG-3′; IL-6 forward, 5′-GGATACCACCCACAACAGACC-3′ and reverse, 5′-AATCAGAATTGCCATTGCAC-3′; IL-1β forward, 5′-ATCACTCATTGTGGCTGTGG-3′ and reverse, 5′-GTCGTTGCTTGGTTCTCCT-3′; iNOS forward, 5′-CAGATCGAGCCCTGGAAGAC-3′ and reverse, 5′-CTGGTCCATGCAGACAACCT-3′; and GADPH forward, 5′-CACTCACGGCAAATTCAACGGCA-3′ and reverse, 5′-GACTCCACGACATACTCAGCAC-3′. The PCR cycling reaction was performed using an S1000 thermocycler (Bio-Rad Laboratories, Inc.). Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as an internal control.

Total cells were harvested after treatment, washed twice with ice-cold PBS, and gently lysed in radioimmunoprecipitation assay buffer containing phosSTOP phosphatase inhibitor cocktail and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Lysates were centrifuged for 15 min at 12,000 × g to obtain a supernatant for further analysis. The protein concentration of the lysate was measured using a bicinchoninic acid (BCA) quantification assay (Pierce, Rockford, IL, USA). Proteins (50 µg) were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (0.45-µm pore size IPVH00010; Millipore, Billerica, MA, USA). The membranes were incubated with primary antibody overnight at 4°C. The primary antibodies were monoclonal antibodies targeting IκBα, p-IκBα, NF-κB p65, p-NF-κB p65 and β-actin (1:1,000) diluted in immunoblot buffer (TBS containing 0.05% Tween-20 and 5% non-fat dry milk). Following washing with TBS and Tween 20 three times, membranes were incubated with the secondary HRP-conjugated anti-mouse (or rabbit) IgG antibody (1:1,000) for 1 h at RT. After washing, the blots were detected with Clarity ECL Western Blotting Substrate (Bio-Rad Laboratories, Inc.) for 1 min using a camera with the ChemiDoc XRS⁺ System (Bio-Rad Laboratories, Inc.). The pixel intensities of the immunoreactive bands were quantified using the percentage adjusted volume feature of Image Lab software (Bio-Rad Laboratories, Inc.). β-actin served as an internal control.

A bead-based multiplex assay for phosphoproteins (Bio-Plex Phosphoprotein assay) was used to detect p-ERK1/2, p-JNK and p-p38. Cells were lysed using a lysis kit (Bio-Rad Laboratories, Inc.) and were then centrifuged at 15,000 × g for 15 min. Protein concentrations were determined by BCA protein assay. Then, 25 µl protein extract and 25 µl testing assay buffer were transferred into a 96-well filter plate coated with antibodies against p-ERK1/2, p-JNK and p-p38 and the plate was incubated overnight on a platform shaker at room temperature. Following a series of washes to remove the unbound proteins, a mixture of biotinylated detection antibodies, each specific for a different epitope, was added to the reaction. Streptavidin-phycoerythrin was then added to bind to the biotinylated detection antibodies. Data acquisition and analysis was conducted using the Bio-Plex 200 suspension array system (Bio-Rad Laboratories, Inc.). The total proteins for ERK1/2, JNK and p38 were also quantified using the Bio-Plex total protein assay kit (Bio-Rad Laboratories, Inc.). The phosphorylation level was expressed as the ratio of phosphoprotein to total protein.

Data are expressed as mean ± standard deviation. One-way analysis of variance was used when comparing the data obtained under different experimental conditions. In vitro experiments were conducted in triplicates; representative results are shown. A P-value of <0.05 was considered to indicate a statistically significant result.

RAW 264.7 cells were treated with various concentrations of TFHDW for 24 h, and the viability and cytotoxicity were determined by MTT assay. TFHDW did not exhibit cytotoxicity to RAW 264.7 cells in the absence and presence of LPS at concentrations of 50, 100 and 200 µg/ml, and these concentrations were used in the following experiments. However, when the concentrations reached 800 µg/ml, TFHDW appeared to inhibit cell viability (Fig. 1).

The effect of TFHDW on LPS-induced inflammation in RAW 264.7 cells was evaluated by measuring the production of NO and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β). As shown in Fig. 2, stimulation with LPS for 24 h significantly induced the release of NO, TNF-α, IL-6 and IL-1β, indicating that an inflammatory response was induced in the RAW 264.7 cells. However, the LPS-induced release of inflammatory mediators was significantly inhibited by TFHDW treatment in a concentration-dependent manner. To further verify these observations, the effect of TFHDW on the mRNA expression of these pro-inflammatory factors was determined. As shown in Fig. 3, stimulation with LPS significantly and concentration-dependently increased the mRNA expression levels of iNOS, TNF-α, IL-6 and IL-1β in RAW 264.7 cells and these increases were markedly suppressed by TFHDW treatment.

NF-κB is an important upstream transcription factor inducing the expression of iNOS, TNF-α, IL-6 and IL-1β mRNA following stimulation by LPS. As p65 is the functional subunit of NF-κB activated by LPS in macrophages, the phosphorylation of p65 was analyzed by western blot analysis. As shown in Fig. 4, treatment with LPS significantly increased the phosphorylation of p65 in the RAW 264.7 cells; However, pretreatment with TFHDW notably inhibited this excessive phosphorylation. During the p65 phosphorylation process, phosphorylation of IκBα is essential to release NF-κB from the NF-κB/IκBα complex. Therefore, the effect of TFHDW on the LPS-induced phosphorylation of IκBα was also investigated. The results showed that TFHDW strongly suppressed the phosphorylation of IκBα, suggesting that TFHDW inhibited NF-κB activation and the phosphorylation of p65 by reducing the phosphorylation of IκBα (Fig. 4).

In order to investigate whether the anti-inflammatory effects of TFHDW are mediated via MAPK signaling pathways, the phosphorylation of the MAPK signaling molecules p38, JNK and ERK 1/2 was analyzed using a Bio-Plex phosphoprotein assay. The results suggest that LPS significantly induced the phosphorylation of p38, JNK and ERK 1/2; however, the phosphorylation levels of the MAPK isoforms were markedly decreased in the cells treated with TFHDW and LPS compared with the cells treated with LPS alone. These results indicate that TFHDW effectively blocks MAPK signaling pathways in activated macrophages (Fig. 5).