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Introduction

Inflammation is an orchestrated biological process, induced by tissue injury or microbial infection, which protects the body from these inflammatory stimuli. However, persistent or excessive inflammation is associated with a variety of pathological conditions, including rheumatoid arthritis, bacterial sepsis and skin inflammation (1,2). Macrophages play a key role in the host defense against noxious substances and are involved in numerous inflammatory diseases (3). The activation of macrophages by inflammatory stimuli can generate reactive oxygen species, such as H2O2 and superoxide, and induce the expression of various genes such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, in addition to other inflammatory mediators, including nitric oxide (NO) and prostaglandin E2 (PGE2), which are synthesized by inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, respectively. Inflammatory cytokines and mediators contribute to the pathogenesis of numerous inflammation-associated human diseases (4). Lipopolysaccharide (LPS) from gram-negative bacteria induces inflammation and is frequently used to stimulate macrophages in order to study inflammation and the mechanisms of action of potential anti-inflammatory agents.

The anti-inflammatory actions of various phytochemicals have been found to be mediated through suppression of the NF-κB pathway (5). NF-κB is a key regulator of a various genes involved in immune and inflammatory responses (6). In resting cells, NF-κB is complexed with the inhibitor of κB (IκB) protein in the cytoplasm, and is thereby inactivated. When cells receive pathological stimuli, IκB kinase phosphorylates IκB, causing it to break away from NF-κB. The uncomplexed NF-κB then translocates to the nucleus, where it binds to DNA and activates the transcription of various genes including iNOS, COX-2, IL-6 and TNF-α (7). Therefore, NF-κB is regarded as a target molecule for anti-inflammatory drug development (8).

In addition to activating NF-κB, LPS also activates mitogen-activated protein kinases (MAPKs) (9). There are three major subgroups of MAPK, namely extracellular signal-regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK) and p38. These kinases play key roles in the regulation of numerous cellular functions, including cell survival, apoptosis and cellular responses to inflammation (10).

Hedyotis diffusa Willd (HDW), which is a herb of the Rubiaceae family, has a long history of use in Chinese medicine, and is widely distributed in northeast Asia. According to the theories of traditional Chinese medicine (TCM), it has heat-clearing, detoxification, blood circulation promoting and blood stasis eliminating effects (11,12). Pharmacological studies have shown that it contains compounds with anticancer, anti-inflammatory, antibacterial and immunomodulatory activities, which include flavonoids, anthraquinones, hemiterpenes, polyphenols, organic acids and polysaccharides (13–16).

The anti-inflammatory effects of total flavonoids of HDW (TFHDW) have been investigated in various inflammatory models, including ulcerative colitis induced by dextran sulfate sodium in mice (17,18), ear edema induced by dimethylbenzene in mice, granuloma pouch induced by turpentine in rats, and paw edema caused by egg white in rats (19). However, few studies on its anti-inflammatory activity in vitro and its mechanisms have been carried out. In the present study, the cell-based anti-inflammatory activities and the mechanisms of action of TFHDW in LPS-stimulated RAW 264.7 macrophages were investigated. In order to elucidate the mechanisms underlying the anti-inflammatory actions, the effect of TFHDW on the expression of iNOS, TNF-α, IL-6 and IL-1β at the mRNA and protein levels, as well as on NF-κB and MAPK signaling pathways were also studied.

Materials and methods

Materials and reagents

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).

Preparation of TFHDW from Hedyotis diffusa Willd

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).

Cell culture and treatment

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.

Cytotoxicity assay

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.

Determination of NO production

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.

Measurement of inflammatory cytokines

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.

RT-PCR

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.

Western blot analysis

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.

Bio-Plex phosphoprotein assay

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.

Statistical analysis

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.

Results

TFHDW did not exhibit cytotoxicity against RAW 264.7 cells

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).

Figure 1. Effect of TFHDW on the viability of RAW 264.7 cells. RAW 264.7 cells were treated with LPS in the presence and absence of TFHDW at concentrations of 0–800 µg/ml for 24 h. Cell viability was determined by MTT assay. The data were normalized to the viability of the untreated control cells. Each value indicates the mean ± standard deviation from at least three independent experiments. *P<0.05 vs. control group. TFHDW, total flavonoids of Hedyotis diffusa Willd; LPS, lipopolysaccharide; MTT, methyl thiazolyl tetrazolium assay.

TFHDW inhibits the LPS-induced inflammatory response in RAW 264.7 cells

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.

Figure 2. Effect of TFHDW treatment on the release of pro-inflammatory factors in LPS-stimulated RAW 264.7 cells. Cells were treated with LPS and/or the indicated concentrations of TFHDW. (A) Content of NO was measured using the Griess reaction. The secretion levels of TNF-α (B), IL-6 (C) and IL-1β (D) were measured using respective ELISA kits. Each value indicates the mean ± standard deviation from three independent experiments. *P<0.05 vs. untreated control cells; #P<0.05 vs. cells treated with LPS alone. TFHDW, total flavonoids of Hedyotis diffusa Willd; LPS, lipopolysaccharide; NO, nitric oxide; TNF, tumor necrosis factor; IL, interleukin; ELISA, enzyme-linked immunosorbent assay.

Figure 3. Effect of TFHDW on the transcription of pro-inflammatory factors in LPS-stimulated RAW 264.7 cells. (A) The mRNA expression of iNOS, TNF-α, IL-6 and IL-1β was determined by reverse transcription-polymerase chain reaction. GADPH was used as the internal control. Images are representatives of three independent experiments. (B) Densitometric analysis. The data were normalized to the mean mRNA expression of the untreated control. *P<0.05 vs. untreated control cells; #P<0.05 vs. cells treated with LPS alone. TFHDW, total flavonoids of Hedyotis diffusa Willd; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; TNF, tumor necrosis factor; IL, interleukin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

TFHDW prevents LPS-induced activation of the NF-κB pathway in RAW 264.7 cells

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).

Figure 4. NF-κB pathway is involved in the inflammatory effect of TFHDW in LPS-induced RAW 264.7 cells. Cells were treated with LPS alone or in the presence of different concentrations of TFHDW for 24 h. The levels of proteins associated with NF-κB pathway were measured by western blotting analysis. Representative images for (A) p-p65 and p65 and (B) p-IκBα were from one of three repeated experiments. The expression of all proteins was quantified by densitometry analysis. Data shown re the mean ± standard deviation. *P<0.05 vs. untreated control cells; #P<0.05 vs. cells treated with LPS alone. NF-κB, nuclear factor-κB; TFHDW, total flavonoids of Hedyotis diffusa Willd; LPS, lipopolysaccharide; p, phospho; IκBα, inhibitor of κBα.

TFHDW suppresses the phosphorylation of p38, JNK and ERK 1/2

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).

Figure 5. Effects of TFHDW on LPS-induced phosphorylation of MAPKs in RAW 264.7 cells. The phosphorylation levels of (A) p38, (B) JNK and (C) ERK 1/2 in RAW 264.7 cells were determined by Bio-Plex phosphoprotein assay. The data were normalized to the phosphorylation level within controls and represented as fold of control. Data were shown as the mean ± SD. *P<0.05 vs. untreated control cells; #P<0.05 vs. cells treated with LPS alone. TFHDW, total flavonoids of Hedyotis diffusa Willd; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal protein kinase; ERK, extracellular signal-regulated kinase; p, phospho.

Discussion

Inflammation is a biological protective response to tissue injury or microbial invasion capable of causing cell injury, under which pro-inflammatory mediators are released (21). Inflammatory factors are important elements in the chronic inflammation associated with diseases such as arteriosclerosis, obesity, diabetes, neurodegenerative diseases and cancer. Steroidal and non-steroidal anti-inflammatory drugs are currently used to treat acute inflammation. However, these drugs are not entirely successful in curing chronic inflammatory disorders, and often have side effects. Therefore, the identification of new, safer and more effective anti-inflammatory compounds is necessary (22). TCM, which plays an important role in primary health care in China, involves the use of extracts of various plants for the treatment of pathological disturbances, including acute and chronic inflammation. Flavonoids, which are an active constituent of TCM have been found to have notable biological properties, including anticancer, antimicrobial, antiviral, anti-inflammatory, immunomodulatory and antithrombotic activities (23–25). TFHDW, the flavonoid compounds of the traditional Chinese herb HDW, has been demonstrated to be effective for the treatment of inflammation in vivo (17–19). The purpose of the present study was to further investigate the anti-inflammatory effects of TFHDW on pro-inflammatory mediators (NO, TNF-α, IL-6, IL-1β) and pro-inflammatory enzyme (iNOS) expression in LPS-activated RAW 264.7 cells and to explore the mechanism. It was found that TFHDW inhibited LPS-induced iNOS, TNF-α, IL-6 and IL-1β expression at the protein and transcription levels, confirming that TFHDW exhibits an anti-inflammatory effect.

The NF-κB signaling pathway is an important LPS-activated pathway. NF-κB is present as a complex with IκB located in the cytoplasm in unstimulated cells. When stimulated, IκB is phosphorylated, ubiquitinated and rapidly degraded, which releases the p65 subunit of NF-κB from IκB, resulting in p65 translocation into the nucleus where it regulates gene transcription (26,27). To explore the mechanism of NF-κB inactivation by TFHDW, the effects of the extract on the LPS-induced phosphorylation of p65 and IκB were examined by western blotting. The levels of phosphorylation were increased upon exposure to LPS, and treatment with TFHDW reduced the phosphorylation of p65 and IκB without affecting the total cellular levels of these proteins.

Inhibition of the MAPK pathway is also known to disrupt the synthesis of proinflammatory mediators (28). Several studies have shown that MAPKs are involved in the activation of NF-κB (28–30). The effects of TFHDW on the LPS-induced phosphorylation of p38, JNK and ERK1/2 were examined in the present study in order to further elucidate its anti-inflammatory mechanism. TFHDW inhibited the phosphorylation of p38, JNK and ERK1/2 in LPS-stimulated cells in a concentration-dependent manner. The attenuation of MAPK activation may be a mechanism by which TFHDW reduces cytokine production. The results of the current study suggest that the anti-inflammatory activity of TFHDW is mediated by the inhibition of iNOS, TNF-α, IL-6 and IL-1β expression via the downregulation of the NF-κB and MAPK signaling pathways.

In conclusion, in the present study, it was demonstrated that TFHDW negatively modulates the production of NO and cytokines (TNF-α, IL-6 and IL-1β) in LPS-treated RAW 264.7 cells in a concentration-dependent manner. It was also found that TFHDW blocks the activation of NF-κB and MAPK signaling pathways. These data suggest that TFHDW has notable anti-inflammatory activity and is worthy of further development as a herbal remedy for the treatment of various inflammatory diseases.

Acknowledgements

This study was supported as Project No. 81173203 by the National Natural Science Foundation of China.

Glossary

Abbreviations

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References