Gyeji-tang water extract exerts anti-inflammatory activity through inhibition of ERK and NF-κB pathways in lipopolysaccharide-stimulated RAW 264.7 cells

Background Gyeji-tang (GJT, Guizhi Tang in Chinese, Keishi-to in Japanese) is a traditional herbal decoction composed of 5 medicinal herbs. GJT has been used to treat the common cold, headaches, and fever in Asian countries including Korea, China, and Japan. In the present study, we investigated the inhibitory effect of a water extract of GJT on inflammatory response using the murine macrophage cell line, RAW 264.7. Methods RAW 264.7 macrophages were treated with lipopolysaccharide (LPS) to upregulate inflammatory genes. Cells were pretreated with various concentrations of GJT for 4 h and stimulated with LPS for an additional 20 h. Productions of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) were measured by enzyme-linked immunosorbent assays (ELISAs). Protein expressions of heme oxygenase (HO)-1, extracellular signal-regulated kinase (ERK), and nuclear factor kappa-B (NF-κB) were analyzed by immunoblotting. Results Treatment with the GJT extract enhanced expression of HO-1 in macrophages without cytotoxicity. GJT extract significantly inhibited proinflammatory cytokines TNF-α and IL-6 in LPS-stimulated cells. GJT suppressed LPS-induced COX-2 expression, leading to a decrease in COX-2-derived PGE2 level. In addition, GJT extract prevented phosphorylation of ERK and NF-κB translocalization to the nucleus in LPS-treated RAW 264.7 cells. Conclusion These data suggest that GJT has anti-inflammatory possibly through blocking ERK and NF-κB signaling pathways.


Background
Inflammation is a tissue protective response against potentially harmful stimuli such as bacteria, damaged cells, or irritants [1,2]. Chronic inflammation is associated with pathogenesis of the various diseases such as cancer, atherosclerosis, rheumatoid arthritis, and type 2 diabetes.
Macrophages are significantly involved in the initiation and maintenance of the inflammatory process through secretion of inflammatory cytokines [3]. Blocking proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) or interleukin-6 (IL-6) is considered as an attractive therapeutic approach to inflammation [4,5]. In addition to inflammatory cytokines, prostaglandin E2 (PGE 2 ) as a proinflammatory mediators plays an important role in inflammatory response. PGE 2 is synthesized by cyclooxygenase-2 (COX-2) that is activated by proinflammatory stimuli such as cytokines, endotoxin, or growth factors [6]. These proinflammatory regulators can be controlled by several intracellular molecular pathways such as those involving mitogen-activated protein kinases (MAPKs) and nuclear factor kappa-B (NF-κB) [7]. Chronic inflammation and its related diseases are associated with the oxidative stress that generates proinflammatory mediators and cytokines. The antioxidative enzyme heme oxygenase 1 (HO-1) has anti-inflammatory properties and is therefore thought to be a potential molecular target for treating inflammatory diseases [8,9]. In macrophages, activation of HO-1 inhibits cytokine secretion [10,11] and suppresses COX-2 expression [12].
Gyeji-tang (GJT, Guizhi Tang in Chinese, Keishi-to in Japanese), a traditional Korean medicine, has been used to treat cold, headache, and fever in Asian countries including Korea, China and Japan [13]. GJT consists five herbs. However, the scientific evidence to support antiinflammatory effect of GJT is rare. To date, several groups reported effects of GJT on pancreatic acinar cell injury [14], diabetes mellitus [15], and Guizhi decoction syndrome [16]. In the present study, we investigated anti-inflammatory mechanisms of GJT water extract using lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. Inhibitory effects of GJT against the inflammatory response was elucidated by measuring production of TNF-α, IL-6 and PGE 2 , and analyzing MAPK and NF-κB pathways, and HO-1 expression.

Plant materials
The 5 medicinal herbs comprising GJT were purchased from Kwang Myung Dang Medicinal Herbs (Ulsan, Korea) as shown in Table 1. The taxonomic authenticity of these medicinal herbs was confirmed by Prof. Je Hyun Lee, Dongguk University, Gyeongju, Korea. Voucher specimen (2012-KE46-1 through KE46-5) have been deposited at the K-herb Research Center, Korea Institute of Oriental Medicine.

Preparation of GJT water extract
Five medicinal herbs was mixed as shown in Table 1 (total 5.0 kg; 30.0 g × 166.7) and extracted in a 10-fold mass of water at 100°C for 2 h under pressure (1 kgf/cm 2 ) using an electric extractor (COSMOS-660; Kyung Seo Machine Co., Incheon, Korea). The water extract was then filtered through a standard sieve (No. 270, 53 μm; Chung Gye Sang Gong Sa, Seoul, Korea). The solution was freezingdrying to give a powder using PVT100 freeze dryer (IlShinBioBase, Yangju, Korea). The yield of the GJT water extract was 9.75 % (487.5 g).

Cell culture
The murine macrophage cell line, RAW 264.7, was obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in Dulbecco's modified Eagle's medium (Gibco Inc., Grand Island, NY) supplemented with 5.5 % heat-inactivated fetal bovine serum (Gibco Inc.), penicillin (100 U/mL), and streptomycin (100 μg/mL) under an atmosphere of 5 % CO 2 in an incubator at 37°C.

Cytotoxicity assay
Cell viability assay was performed to determine the cytotoxicity of GJT using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). Cells were plated into a 96well microplates at 3 × 10 3 cells/well and treated with various concentrations of GJT for 24 h. After incubation with CCK-8 reagent for 4 h, optical density (OD) at 450 nm was measured by using a Benchmark plus microplate reader (Bio-Rad Laboratories, Hercules, CA). Cell viability was calculated using the following equation: Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted using Trizol reagent (Invitrogen Life Sciences, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was synthesized from 1 μg of total RNA using an iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) and subjected to PCR reactions with rTaq DNA polymerase (ELPIS Biotech Inc., Daejeon, South Korea). The relative expression of COX-2 was analyzed using β-actin as an internal control. The primer sequence for COX-1 was forward 5′-AGG AGA TGG CTG CTG AGT TGG-3′ and reverse 5′-AAT CTG ACT TTC TGA GTT GCC-3′, COX-2 was forward 5′-GTA TCA GAA CCG CAT TGC CTC TGA-3′ and reverse 5′-CGG CTT CCA GTA TTG AGG AGA ACA GAT-3′, and β-actin was forward 5′-ACC GTG AAA AGA TGA CCC AG-3′ and reverse 5′-TAC GGA TGA CAA CGT CAC AC-3′. The PCR conditions were 25 cycles of 94°C for 30 s, 57°C (β-actin) or 59°C for 1 min, and 72°C for 1.5 min. The amplification products were then separated by electrophoresis on 1 % agarose gels and detected using a Molecular Imager Gel Doc XR System (Bio-Rad Laboratories, Hercules, CA, USA).

Western blotting
Whole cell extract was prepared by suspending cells in an extraction lysis buffer (Sigma-Aldrich, St. Louis, MO) containing protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Nuclear extract was isolated using NE-PER Nuclear and Cytoplasmic Extraction reagents (Thermo Scientific, Rockford, IL) according to the manufacturer's protocol. Protein concentrations in the extracts were determined using a Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA). Equal amount of cell extract proteins (30 μg) were resolved by 4 %-20 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoridemembranes. The membrane was incubated with blocking solution (5 % skim milk in Tris-buffered saline containing Tween 20 (TBST), followed by an overnight incubation at 4°C with the appropriate primary antibodies; anti-phospho-p38 MAPK, anti-phospho-ERK, anti-phospho-JNK (Cell Signaling, Danvers, MA), HO-1 (Abcam, Boston, MA), NF-κB p65, HO-1, and β-actin (Santa Cruz Biotechnology, Dallas, TX). The membranes were washed three times with TBST, and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. The membranes were washed three times with TBST again, and then developed using an enhanced chemiluminescence kit (Thermo Scientific). Image capture was performed using a Chemi-Doc XRS system (Bio-Rad).

Immunofluorescence staining
Cells were plated onto poly-l-lysine coated glass slides and fixed in 4 % (v/v) methanol free formaldehyde solution (pH 7.4) at 4°C for 25 min. The cells were permeabilized in 0.2 % (w/v) Triton X-100, blocked in 5 % (w/v) bovine serum albumin (BSA) in humidified chamber, followed by immunostaining with NF-κB p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and Texas Red-conjugated secondary antibody. The cells were mounted with mounting medium with coverslips with a mounting medium containing DAPI (Vector Laboratories, Inc, Burlingame, CA) and visualized under an Flou-viewFV10i confocal microscope (Olympus, Tokyo, Japan).

Chemicals
The reference standards, gallic acid, benzoic acid, and coumarin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Albiflorin, paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde, glycyrrhizin, and 6-gingerol were purchased from Wako (Osaka, Japan). Benzoylpaeoniflorin and liquiritin apioside were purchased from Biopurify Phytochemicals (Chengdu, China) and Shanghai Sunny Biotech (Shanghai, China). A standard stock solution of these components were dissolved in methanol at concentrations of 1.0 mg/mL. For HPLC analysis of the GJT extract, 100 mg of lyophilized GJT extract was dissolved in 20 mL of distilled water and then the solution was diluted to 10-fold for quantitative analysis of paeoniflorin. Solutions were filtered through a SmartPor GHP 0.2 μm syringe filter (Woong Ki Science Co., Seoul, Korea) before application to the column.

HPLC analysis of GJT
Quantitative analysis of 12 compounds present in the GJT extract was performed using a Shimadzu LC-20A HPLC system (Shimadzu Co., Kyoto, Japan) consisting of a solvent delivery unit, an on-line degasser, a column oven, an autosampler, and a PDA detector. The data were acquired and processed by LabSolution software (version 5.54 SP3l Shimadzu Co.). The analytical column used was a SunFire C 18 (250 × 4.6 mm; particle size 5 μm, Waters, Milford, MA, USA) and was maintained at 40°C. The mobile phases consisted of water (A) and acetonitrile (B), which were both containing 0.1 % (v/v) formic acid. The gradient flow was as follows: 5 % -60 % B for 0-30 min, 60 % -100 % B for 30-40 min, 100 % B for 40-45 min, and 100-5 % B for 45-50 min. The flow-rate was 1.0 mL/min and injection volume was 10 μL.

Statistical analysis
The data are expressed as the mean ± SEM. Data were analyzed using one-way analysis of variance and Dunnett's multiple comparisons test. P < 0.05 was considered significant.

Results
GJT enhances HO-1 expression in RAW264.7 cells Macrophages play an important role in the process of inflammation including its initiation, maintenance, and resolution through the production of pro-or antiinflammatory cytokines [3]. In the present study, RAW 264.7 murine macrophages were used to investigate anti-inflammatory effects of the GJT extract. To investigate the inhibitory effects of GJT extract on inflammation, we initially analyzed HO-1 expression in RAW 264.7 cells. Treatment with GJT extract induced HO-1 expression in a dose-dependent manner at noncytotoxic concentrations (Fig. 1b).
GJT inhibits PGE 2 production and COX-2 expression in LPS-stimulated RAW264.7 cells PGE 2 is a key inflammatory mediator synthesized by COX-2 [6]. To support the observed inhibitory effect of GJT on inflammatory response, a PGE 2 assay was conducted using LPS-stimulated RAW 264.7 cells with or without GJT extract. As shown in Fig. 3a (left panel), LPS stimulation significantly increased production of PGE 2 by 15149.2 ± 768.01 pg/mL compared with untreated control (552.16 ± 58.82 pg/mL). By contrast, LPS-stimulated PGE 2 increase was significantly inhibited by GJT at 62.5 and 125 μg/mL by 12032.07 ± 423.16 and 9410.4 ± 315.41 pg/mL, respectively. Treatment with GJT alone showed a marginal increase in the production of PGE 2 compared with untreated cells (Fig. 3a, right  panel). Indomethacin used as a positive control also significantly decreased the level of PGE 2 production by LPS-treated RAW 264.7 cells. Treatment with GJT extract consistently suppressed COX-2, but not COX-1, mRNA expression induced by LPS stimulation (Fig. 3b), indicating the specificity of GJT as a COX-2 inhibitor.
GJT abrogates ERK phosphorylation in LPS-stimulated RAW264.7 cells MAPK signaling pathways are considered as molecular targets for anti-inflammatory therapy [17]. Therefore, we investigated the effect of GJT on the LPS-mediated phosphorylation of MAPK family members JNK, ERK, and p38 MAPK in RAW 264.7 cells. The cells were pretreated with GJT (0, 62.5, or 125 μg/mL) for 4 h and stimulated with LPS for 15 min. As shown in Fig. 4, LPS stimulation markedly increased levels of phosphorylated p38 MAPK, ERK, and JNK compared with untreated GJT inhibits NF-κB activation in LPS-stimulated RAW264.7 cells NF-κB signaling is involved in the development of inflammatory disorders [18]. We observed that GJT extract at 125 μg/mL markedly blocked LPS-stimulated nuclear expression of NF-κB p65 (Fig. 5a). Consistently, immunocytochemistry showed that LPS stimulation induced translocalization of NF-κB p65 from the cytosol into the nucleus whereas with GJT extract blocked the LPS-mediated nuclear translocalization of NF-κB p65 in RAW 264.7 cells (Fig. 5b).

HPLC analysis of GJT
All calibration curves were obtained by plotting peak areas versus the concentration of standard solutions in seven different concentration ranges. The calibration curves of the 12 components showed good linearity with correlation coefficients (r 2 ) ≥ 0.9997. The limit of detection (LOD) and quantitation (LOQ) were 9.15-258.61 ng/mL and 27.33-783.66 ng/mL, respectively. These results are summarized in Table 2. Using optimized chromatography conditions, three-dimensional chromatograms were obtained using HPLC-PDA detector and 12 compounds were eluted within 35 min (Fig. 6). The amounts of the 12 marker compounds were 0.16-40.29 mg/g and are shown in Table 3.

Discussion
Inflammation is part of the abnormal body response caused by physical, biological or chemical stimuli [3]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used for treating inflammatory disorders. NSAIDs target the COX enzyme and include aspirin, ibuprofen, and naproxen. However, long-term use of NSAIDs can trigger severe side effects such as gastric erosion, renal damage, myocardial infarction and asthma exacerbation [19]. Thus, novel therapeutic drugs with higher efficacy and fewer side effects are necessary for the treatment of inflammatory disorders.
In the present study, we investigated whether a traditional herbal formula GJT has anti-inflammatory effects in RAW 264.7 murine macrophages. An inflammatory reaction was induced by LPS treatment according to the previous reports [20,21]. Consistently, our data revealed that LPS stimulation significantly increased levels of proinflammatory cytokines TNF-α and IL-6 in RAW 264. 7 cells. By contrast, GJT treatment inhibited LPS-induced secretion of TNF-α and IL-6 without cytotoxicity. Antiinflammatory activity of GJT extract was further confirmed by analyzing the expressions of PGE 2 and COX-2. Inflammatory stimulators such as LPS can induce COX-2 expression that produces PGE 2 , a key inflammatory mediator [22]. In the present study, GJT extract significantly decreased LPS-stimulated PGE 2 production and COX-2 mRNA expression in RAW 264.7 cells. HO-1 overexpression inhibits proinflammatory cytokine production and suppresses proinflammatory enzymes [23]. Thus, we investigated whether GJT influence on the expression of HO-1. We found that GJT treatment enhanced protein expression of HO-1 in RAW 264.7 macrophages. However, dose-dependency of HO-1 expression by GJT was consistent with the production of TNF-α, but not IL-6 and PGE 2 . Further experiments will be required to determine the relationship of antiinflammatory and antioxidant activities of GJP in macrophages.
Molecular regulation of inflammatory responses is closely associated with several signaling pathways such as those of MAPK and NF-κB. Macrophage stimulation with LPS induces phosphorylation of MAPK family proteins ERK1/2, JNK, and p38 MAPK [24]. We observed that LPS stimulation clearly increased levels of phosphorylated ERK1/2, JNK, and p38 MAPK in RAW 264.7 cells. By contrast, GJT extract suppressed phosphorylation of ERK1/2, but not JNK or p38 MAPK, in LPSstimulated cells, indicating the importance of ERK1/2 to the anti-inflammatory regulation of GJT extract. However, we cannot exclude the possible inhibition of NF-κB activation. Indeed, our immunoblotting and immunofluorescence staining data showed that GJT treatment clearly reduced the nuclear level of NF-κB p65. Overall, GJT potentially controls inflammatory markers by blocking ERK and NF-κB signaling pathways in macrophages.

Conclusions
Our results show that an water extract of the traditional herbal formula GJT inhibits LPS-stimulated production of proinflammatory cytokines TNF-α and IL-6, and proinflammatory mediator PGE 2 in macrophages. Antiinflammatory activity of GJT is associated with enhancement of HO-1 expression and regulated via targeting  ERK and NF-κB pathways. These findings suggest the prophylactic effects if GJT decoction on inflammatory diseases. Further studies will be considered in the near future to determine the effects of GJT for treating inflammatory diseases by the concurrent or post LPS treatment with GJT decoction.