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Anti-inflammatory effects of Hwang-Heuk-San, a traditional Korean herbal formulation, on lipopolysaccharide-stimulated murine macrophages
© Kang et al. 2015
Received: 16 June 2015
Accepted: 14 December 2015
Published: 23 December 2015
Hwang-Heuk-San (HHS), a Korean traditional herbal formula comprising four medicinal herbs, has been used to treat patients with inflammation syndromes and digestive tract cancer for hundreds of years; however, its anti-inflammatory potential is poorly understood. The aim of the present study was to investigate the anti-inflammatory effects of HHS using a lipopolysaccharide (LPS)-activated RAW 264.7 macrophage model.
The inhibitory effects of HHS on LPS-induced nitric oxide (NO), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) production were examined using Griess reagent and enzyme-linked immunosorbent assay (ELISA) detection kits. The effects of HHS on the expression of inducible NO synthase (iNOS), IL-1β and TNF-α, their upstream signal proteins, including nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and activator protein (AP-1), were also investigated.
A noncytotoxic concentration of HHS significantly reduced the production of NO, IL-1β and TNF-α in LPS-stimulated RAW 264.7 cells, which was correlated with reduced expression of iNOS, IL-1β and TNF-α at the mRNA and protein levels. HHS efficiently blocked the phosphorylation of MAPKs, especially that of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) but not that of the p38 MAPK. The reduced production of inflammatory molecules by HHS was followed by decreased activity of NF-κB and AP-1.
These results suggest that HHS may offer therapeutic potential for treating inflammatory diseases accompanied by macrophage activation.
There is increasing awareness that inflammation is a natural defense system found in the human body and that it plays a major role in the pathogenesis of many inflammatory disorders [1, 2]. Although macrophages are important in the host-defense mechanism, pathogen-induced overproduction of inflammatory factors from macrophages and cellular damage-derived inflammation-inducing molecules have been implicated in inflammation-related diseases, such as arthritis, inflammatory bowel diseases, and asthma .
Accumulating evidence indicates that specific stimuli, such as the endotoxin lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, give rise to the activation of macrophages and result in the secretion of a number of different proinflammatory mediators and cytokines, including nitric oxide (NO), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) [3–5]. LPS can bind to toll-like receptor 4 (TLR4), which is expressed on macrophages. This complex activates various cellular signaling events, including mitogen-activated protein kinases (MAPKs), and subsequently induces the activation of various transcription factors, such as nuclear factor (NF)-κB and activator protein (AP-1) [6, 7].
MAPKs, extracellular signal regulated kinase (ERK), c-Jin NH2-terminal kinase (JNK), and p38 MAPK are a group of signaling molecules that also play an important role in relaying inflammatory information from the extracellular space to the cytoplasm and nucleus to modulate inflammatory responses [7, 8]. Phosphorylation-induced activation of MAPKs is known to be a critical component in the production of proinflammatory molecules in activated macrophages. Previous studies have shown that TLR4-induced activation of MAPKs resulted in the activation of the nuclear translocation of NF-κB and AP-1 and, finally, the initiation of proinflammatory responses [9, 10]. Under normal physiological conditions, NF-κB dimmers of p50 and p65 subunits are present in the cytoplasm and attached to the suppressor protein inhibitor of NF-κB (IκB). NF-κB activation occurs via phosphorylation and subsequent activation of MAPKs, followed by degradation of IκB bound to NF-κB, resulting in the translocation of NF-κB from the cytoplasm to the nucleus to promote the expression of various proinflammatory genes [6, 11]. AP-1 is an important regulator of gene expression involved in inflammation activation, and it forms heterodimer complexes with c-Jun and c-Fos. Various factors, including growth factors, cytokines, and stress, induce AP-1 [7, 8]. AP-1 is activated by TLR agonists and cytokines, dependent on the activation of MAPKs. Therefore, treatments aimed at inhibiting MAPKs and NF-κB, as well as AP-1, may have potential therapeutic advantages as anti-inflammatory agents [12, 13].
Herbal components and amount of HHS decoction
Herbal medicine (pharmacognostic nomenclature)
Raw material amount (g/%)
Rheum palmatum L. (Rhei Radix et Rhizoma)
Psoralea corylifolia L. (Psoraleae Fructus)
Pharbitis nil Chois. (Pharbitidis Semen)
Arctium lappa L. (Arctii Fructus)
Materials and reagents
Dulbecco’s modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from WelGENE Inc. (Daegu, Republic of Korea). LPS (Escherichia coli Serotype 055:B5), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and Griess reagent were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Mouse IL-1β and TNF-α enzyme-linked immunosorbent assay (ELISA) detection kits, and enhanced chemiluminescence (ECL) detection system were purchased from R&D Systems (Minneapolis, MN, USA) and Amersham Co. (Arlington Heights, IL, USA), respectively. Various primary and secondary antibodies for Western blot analysis were purchased from Cell Signaling Technology, Inc. (Boston, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from Sigma-Aldrich Chemical Co.
Preparation of the HHS
The four medicinal herbs forming HHS were obtained from Dongeui Oriental Hospital, Dongeui University College of Korean Medicine (Busan, Republic of Korea). The origin of the medicinal herbs was confirmed taxonomically by Professor Su Hyun Hong, Dongeui University College of Korean Medicine (Busan, Republic of Korea). Each of the four herbs in HHS was cut into small pieces and then mixed together to obtain a total amount of 76 g in the ratios shown in Table 1. The mixture was boiled with distilled water (76 g/500 ml) for 3 h. The extract solution was filtered using a 0.45-mM filter to remove insoluble materials, and the blended supernatants were lyophilized and then crushed into a thin powder (yield = 21 % w/w, dried extract/crude herb). The dried extract was dissolved to a 100 mg/ml concentration with distilled water before use, and the stock solution was then diluted with medium to the desired concentration prior to use.
Cell culture and LPS stimulation
A murine macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in DMEM containing 10 % FBS, 100 U/ml of penicillin, and 100 mg/ml of streptomycin in an incubator at 37 °C, 5 % CO2, and 95 % humidity. To stimulate the cells, the medium was exchanged with fresh DMEM, and LPS (100 ng/ml) was added in the presence or absence of HHS for the indicated periods.
Assessment of cell viability
The effects of HHS on cell viability were evaluated using a colorimetric MTT assay. In brief, the RAW 264.7 cells were seeded at a density of 1 × 104 cells/well in a 96 well-plate, incubated at 37 °C for 24 h, and treated with various concentrations of HHS alone or with LPS (100 ng/ml). After incubation for 24 h, the medium was discarded, and MTT solution was added to each well and incubated for another 3 h at 37 °C. The medium was discarded, and dimethyl sulfoxide was added to dissolve the formazan dye. The optical density was the read at 450 nm using an ELISA reader (Infinite M200, Tecan, Männedorf, Switzerland).
Measurement of NO production
The accumulation of NO was assayed using Griess reagent. In brief, the cells were pretreated with different concentrations of HHS for 1 h and stimulated with LPS for 24 h. Then, 100 μl of the Griess reagent were mixed with an equal volume of cell supernatant and incubated at room temperature for 5 min. The optical density at 540 nm was measured, and the concentration of nitrite was calculated according to the standard curve generated from known concentrations of sodium nitrite .
IL-1β and TNF-α immunoassay
After treatment with HHS in the presence or absence of LPS, the levels of IL-1β and TNF-α in the culture media were quantified using ELISA kits according to the manufacturer’s instructions. The absorbance was read at a wavelength of 450 nm using a microplate reader .
RNA isolation and reverse transcriptase polymerase chain reaction (RTPCR)
Primers of targeted genes
Western blot analysis
The cells were washed and scraped into cold phosphate-buffered saline (PBS) and centrifuged at 500 × g at 4 °C. The cell pellets were resuspended in lysis buffer (20 mM sucrose, 1 mM ethylenediaminetetraacetic acid, 20 μM Tris-HCl, pH 7.2, 1 mM dithiothreitol, 10 mM KCl, 1.5 mM MgCl2, and 5 μg/ml aprotinin). After cell debris was discarded following centrifugation at 13,000 g for 15 min, the protein concentration was determined using a Bio-Rad kit (Bio-Rad Laboratories, Hercules, CA, USA). In a parallel experiment, nuclear and cytosolic proteins were prepared using nuclear extraction reagents (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s protocol. Equal amounts of protein from each sample were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). Nonspecific sites were blocked by the incubating membranes for 1 h at room temperature in 5 % (w/v) nonfat milk powder in Tris-buffered saline containing 0.05 % (v/v) Tween-20 (TBS-T). Thereafter, the membranes were incubated overnight at 4 °C with the corresponding primary antibodies and subsequently incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase. The specific proteins were detected using an ECL detection system.
Immunofluorescent staining for NF-κB p65
RAW 264.7 cells were seeded on glass coverslips in 6-well plates for 24 h, and the cells were treated with HHS for 1 h and then stimulated with LPS for 30 min. Then, the cells were fixed with 3.7 % paraformaldehyde in PBS for 10 min at 4 °C. The cells were incubated with 0.4 % Triton X-100 for 10 min and blocked with 5 % bovine serum albumin for 1 h, followed by probing with rabbit anti-p65 NF-κB antibody overnight at 4 °C. They were then incubated with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 2 h at room temperature. After washing with PBS, nuclei were counterstained with 4,6-diamidino-2-phenyllindile (DAPI) solution (1 mg/ml) for 15 min in the dark, and fluorescence was visualized using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) .
Electrophoretic mobility assay (EMSA)
DNA-protein binding assays were carried out with nuclear extract. Briefly, the preparation of nuclear extracts was conducted using NE-PER nuclear extraction reagents (Pierce, Rockford, IL, USA). Synthetic complementary NF-κB (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and AP-1 (5'-CGC TTG ATG ACT CAG CCG GAA-3') binding oligonucleotides (Santa Cruz Biotechnology) were 30-biotinylated using a biotin 30-end DNA labeling kit (Pierce) according to the manufacturer’s instructions and annealed for 30 min at room temperature. The reaction mixtures were electrophoretically separated on 4 % polyacryl-amide gels in 0.5× Tris-borate buffer and transferred to nylon membranes. The transferred DNAs were cross-linked to the membranes at 120 mJ/cm2 and detected using horseradish peroxidase-conjugated streptavidin (LightShift™ chemiluminescent EMSA kit, Pierce) according to the manufacturer’s instructions.
Data from at least three independent experiments were expressed as the mean ± standard deviation (SD). Statistical comparisons between different groups were performed using a one-way ANOVA, followed by Student’s t-tests after comparing each treated group to the negative control. Values of p < 0.01 were considered statistically significant.
HHS inhibited the release of NO and expression of iNOS in LPS-stimulated RAW 264.7 cells
HHS inhibited the release and expression of IL-1β and TNF-α in LPS-stimulated RAW 264.7 cells
HHS inhibited the activation of the NF-κB pathway in RAW 264.7 macrophages upon LPS stimulation
HHS attenuated LPS-induced AP-1 activation in RAW 264.7 macrophages
Effects of HHS on the cell viability of LPS-stimulated RAW 264.7 macrophages
HHS suppressed LPS-induced phosphorylation of ERK and JNK in RAW 264.7 macrophages
In this study, we evaluated the anti-inflammatory activities of HHS, a Korean traditional herbal formula, in LPS-stimulated RAW 264.7 murine macrophages in an attempt to source an anti-inflammatory agent from traditional medicinal resources with more effectiveness than current agents. Our results indicated that i) HHS significantly inhibited LPS-induced production of NO through the downregulation of iNOS expression; ii) HHS markedly suppressed LPS-induced release and expression of IL-1β and TNF-α; iii) HHS inhibited LPS-induced phosphorylation of ERK and JNK, but not p38 MAPK; iv) HHS attenuated the activation of NF-κB by blocking the nuclear translocation NF-κB and degradation of IkB; and v) HHS markedly inhibited LSP-induced activation AP-1 by suppressing the phosphorylation of c-Jun and c-Fos. The results presented in this study demonstrate that the underlying anti-inflammatory mechanisms of HHS are due, at least in part, to inhibition of LPS-induced activation of NF-κB, MAPK, and AP-1 signaling pathways.
The overproduction of proinflammatory mediators and cytokines by activated macrophages causes various inflammatory diseases [3, 26]. Therefore, identifying new agents capable of lowering the production of proinflammatory agents is regarded as an essential requirement for the alleviation of a number of inflammation-related disorders attributed to macrophage activation [12, 27]. In the present study, the production of NO was markedly elevated in response to LPS. However, the application of HHS inhibited the production of NO by LPS in a concentration-dependent manner (Fig. 1a). The results from the RT-PCR and Western blot analysis showed that pretreatment with HHS concentration-dependently reduced iNOS mRNA levels, with correlated reductions in the corresponding protein level (Figs. 1b and c). In addition, HHS was highly effective at inhibiting IL-1β and TNF-α production, which was associated with a reduction of mRNA and protein expression of IL-1β and TNF-α in the LPS-treated RAW 264.7 cells (Figs. 2 and 3). The present data indicated that HHS reduced mRNA levels of iNOS, IL-1β and TNF-α, which led to decreased protein levels of iNOS, IL-1β and TNF-α, and consequently reduced the quantities of NO, IL-1β and TNF-α that were produced by these enzymes.
The accumulated data demonstrates that specific transcription factors are mainly responsible for the transcriptional regulation of a variety of proinflammatory mediators and cytokines from activated macrophages [7, 10]. Among these, the NF-κB transcription factor family is a critical mediator of inflammatory processes. Thus, the inactivation of NF-κB in the immune system is a major therapeutic target for the downregulation of inflammatory responses. Along with NF-kB activation, AP-1 is able to regulate the expression of a large number of proinflammatory genes, which attract or activate immune cells [8, 11]. NF-κB and AP-1 is composed mainly of two proteins: p65 and p50 or c-Jun and c-Fos, respectively. In unstimulated cells, they exist in the cytosol in a quiescent form. Upon stimulation with LPS, they are activated and translocate to the nucleus where they activate their target genes by binding to their consensus sequences in their promoter regions. In addition to the NF-κB and AP-1 signaling pathways, MAPKs are a major group of signaling molecules that appear to play key roles in inflammatory processes because the phosphorylation of MAPK can stimulate the activation of NF-κB and AP-1 [27, 28]. Therefore, the MAPK signaling cascade is also an attractive therapeutic target for the development of treatments for inflammatory disorders.
In this study, Western blotting revealed that HHS was able to inhibit the LPS-evoked degradation of IkB and the nuclear translocation of NF-κB p65 (Fig. 4). Based on these results, we tested whether HHS inhibited NF-κB activity in RAW 264.7 macrophages by using an EMSA and found that HHS inhibited LPS-induced DNA-binding of NF-κB. To investigate whether the inhibition of NF-κB activation by HHS was associated with the MAPK pathway, the LPS-induced phosphorylation of various MAPKs family proteins, particularly ERK, JNK, and p38 MAPK, was assessed. The immunoblotting results revealed that HHS strikingly induced p38 MAPK phosphorylation, but it had no effect on the activity of ERK and JNK. In addition, the HHS pretreatment abolished the LPS-induced phosphorylation of ERK and JNK (Fig. 6). However, HHS failed to inhibit LPS-induced p38 MAPK phosphorylation, indicating that the anti-inflammatory responses by HHS may be independent on the p38 MAPK signaling pathway. In subsequent studies, the results from Western blotting of nuclear extracts indicated that HHS inhibited the expression and phosphorylation of c-Jun and c-Fos in LPS-challenged RAW 264.7 cells. Furthermore, the EMSA results demonstrated that the DNA-binding activity of AP-1 was significantly reduced in nuclear extracts obtained from LPS-activated RAW 264.7 cells that had been pretreated with HHS (Fig. 5). These results suggested that HHS blocked the binding of NF-κB and AP-1 to specific sequences of DNA, thereby preventing the formation of DNA/NF-κB and DNA/AP-1 complexes. In addition, changes in the phosphorylation of ERK and JNK might mediate HHS-induced inhibition of NF-κB and AP-1 transcriptional activities. In particular, deficiencies in the phosphorylation of these kinases could lead to decreases in the expression levels of inflammation-related genes.
In conclusion, our findings showed that HHS effectively inhibited the LPS-induced production of proinflammatory factors, such as NO, IL-1β and TNF-α, in RAW 264.7 macrophages without causing cytotoxicity. A possible mechanism for this effect involves the ability of HHS to activate a signaling cascade, which results in the repression of NF-κB, ERK, and JNK and the activation of AP-1 in LPS-challenged macrophages. Although further investigation is needed to clarify the precise mechanisms by which HHS inhibits NF-κB and AP-1 activation and to identify the biologically active compounds of HHS responsible for the observed effects, HHS may be considered a potential therapeutic agent for the treatment of inflammation-related disease.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea government (2012046358 and 2015R1A2A2A01004633).
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