Anti-inflammatory effect and action mechanisms of traditional herbal formula Gamisoyo-san in RAW 264.7 macrophages

Background Gamisoyo-san (GMSYS) is a traditional herbal formula used to treat insomnia, dysmenorrhea, and infertility in Korea. The purpose of this study was to investigate the anti-inflammatory effect and action mechanisms of GMSYS in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Methods The anti-inflammatory effects of GMSYS were investigated using nitric oxide (NO) assay and ELISAs for prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). The anti-inflammatory action mechanisms of GMSYS were evaluated using Western blotting for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and activation of nuclear transcription factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs). Results GMSYS significantly inhibited the LPS-induced production of NO, PGE2, TNF-α, and IL-6 compared with the vehicle-treated cells. GMSYS consistently downregulated the expression of iNOS and COX-2 mRNA induced by LPS. In addition, pretreatment with GMSYS suppressed the LPS-induced activation of NF-κB and MAPKs such as p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Conclusions Our results indicate that the anti-inflammatory effects of GMSYS in RAW 264.7 macrophages are associated with inhibition of the release of inflammatory mediators and cytokines through the suppression of MAPK and NF-κB activation. These findings suggest that GMSYS may be a useful therapeutic candidate for the prevention or treatment of inflammatory diseases.

NO is generated from L-arginine by nitric oxide synthase (NOS) and plays an important role in the regulation of physiological responses [3]. Among the isoforms of NOS, the expression of inducible NOS (iNOS) is specifically stimulated by cytokines and bacterial lipopolysaccharide (LPS) [4]. Prostaglandins are key inflammatory mediators that are produced from arachidonic acid by cyclooxygenase (COX). Among the isoforms of COX, COX-2 is induced after exposure to specific stimulants including cytokines and LPS. The induction of COX-2 produces a large amount of PGE 2 that causes inflammatory reactions [5]. These inflammatory mediators and cytokines are critically associated with pain, fever, edema, and recruitment of additional immune cells to the site of inflammation [6]. However, overproduction of inflammatory mediators and cytokines is associated with tissue damage [7]. Therefore, inhibiting the release of inflammatory mediators and cytokines could be beneficial in attenuating the damage caused by inflammatory diseases.
Mitogen-activated protein kinases (MAPKs) such as p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) regulate inflammatory and immune responses, and their signaling pathways are involved in LPS-induced iNOS and COX-2 expression in macrophages [8,9]. In addition, nuclear transcription factor kappa B (NF-κB) is crucial during inflammation [10]. The regulatory free NF-κB translocates to the nucleus during inflammation, where it binds to κB-binding sites in the promotor regions of target genes and induces the transcription of inflammatory mediators and cytokines such as iNOS, COX-2, TNF-α, and IL-6 [11]. Thus, there may be an opportunity to improve the development of the inflammatory response by regulating the activation of the MAPK and NF-κB pathways.
In Korea, Gamisoyo-san (GMSYS) has been widely used for the treatment of dysmenorrhea, insomnia, and anxiety [12]. GMSYS is effective for treatment of sleep disturbances, headache, dizziness in postmenopausal women, depressive symptoms in premenstrual dysphoric disorder, and for tardive dyskinesia related to the use of antipsychotic drugs [13][14][15]. In addition, GMSYS has been reported to exert antistress, antidepressive, and antioxidant activities [12,[16][17][18][19][20]. Despite previous studies, there have been no reports on the effects and molecular mechanisms of GMSYS on inflammatory responses.
In the present study, we investigated the effect of GMSYS on the expression and release of inflammatory mediators and cytokines including NO, iNOS, PGE 2 , COX-2, TNF-α, and IL-6 from LPS-stimulated RAW 264.7 macrophages. In addition, we studied the molecular mechanisms possibly involved in the regulation of inflammatory responses by GMSYS.

Preparation of GMSYS water extract
The 12 raw herbal medicines composing the GMSYS formula were purchased from a traditional herb market, Kwangmyungdang Medicinal Herbs (Ulsan, Republic of Korea). The 12 herbal medicines were authenticated by an expert taxonomist, Prof. Je-Hyun Lee, Dongguk University, Gyeongju, Republic of Korea. Voucher specimens were deposited at the K-herb Research Center, Korea Institute of Oriental Medicine (2012-KE45-1~KE45-11). To obtain a water decoction of GMSYS, the 12 herbal medicines were mixed as shown in Table 1  High-performance liquid chromatography (HPLC) analysis of GMSYS Quantitative analysis of the GMSYS sample was performed using an LC-20A Prominence HPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a solvent delivery unit, an on-line degasser, a column oven, an autosampler, and a photo diode array (PDA) detector. Data were acquired and processed using LabSolution software (version 5.54, SP3; Shimadzu Corp.). Separation was achieved on a SunFire C 18 analytical column (250 × 4.6 mm; particle size 5 μm, Waters, Milford, MA, USA) as the stationary phase at a column temperature set to 40°C. The mobile phases con-  water and then the solution diluted 10-fold for quantitative analysis of geniposide and paeoniflorin. Samples were filtered through a SmartPor GHP 0.2 μm syringe filter (Woongki Science, Seoul, Korea) before application onto the HPLC column.

Cytotoxicity assay
A cell viability assay was performed to determine the cytotoxicity of GMSYS using a Cell Counting NO assay RAW 264.7 macrophages were pretreated with various concentrations of GMSYS (0, 250, 500, or 1000 μg/mL) for 4 h and stimulated with LPS (1 μg/mL) for an additional 20 h. NO synthesis was determined by measuring the accumulation of nitrite in the culture supernatant using a Griess Reagent System (Promega, Madison, WI). Briefly, an equal volume of supernatant and sulfanilamide solution was mixed and incubated for 10 min at room temperature, and then naphthylethylenediamine dihydrochloride solution was added. The mixture was incubated for an additional 5 min, and its absorbance was measured at 540 nm using a Benchmark plus microplate reader (Bio-Rad Laboratories). The nitrite concentration in the supernatants was determined from a standard curve generated with sodium nitrite.
RNA extraction and quantitative real-time polymerase chain reaction (RT-qPCR) Total RNA was extracted using Trizol reagent (Invitrogen Life Sciences, Carlsbad, CA) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using an iScript cDNA synthesis kit (Bio-Rad Laboratories). RT-qPCR was performed by using a Rotor Gene Q system (Qiagen, Hilden, Germany) and a reaction mixture that consisted of SYBR Green 2 × PCR Master Mix, cDNA template, and forward and reverse primers. The PCR protocol consisted of 35 cycles of denaturation at 95°C for 15 sec, followed by 55°C for 30 s to allow for extension and amplification of the target sequence. The relative levels of iNOS and COX-2 mRNA expression were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the 2-ΔΔCT method. The primer sequences used in this study are shown in Table 2. appropriate primary antibodies; anti-phospho-p38 MAPK, anti-phospho-ERK, anti-phospho-JNK (Cell Signaling, Danvers, MA), anti-NF-κB p65, anti-β-actin, anti-αtubulin and anti-Lamin B (Santa Cruz Biotechnology, Dallas, TX). The membranes were washed three times with TBST, and then incubated with a corresponding horseradish peroxidase-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 immunereactivity visualized using an enhanced chemiluminescence (ECL) (Thermo Fisher Scientific). Images were captured using Chemi-Doc (Bio-Rad Laboratories).

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

Identification and quantification of the marker components of GMSYS
We selected 11 components-including gallic acid, neomangiferin, chlorogenic acid, mangiferin, geniposide, paeoniflorin, berberine, liquiritin, nodakenin, glycyrrhizin, and atractylenolide III-as marker compounds of GMSYS. Calibration curves for the 11 marker components showed good linearity with correlation coefficients (r 2 ) ≥ 0.9996 in their various concentration ranges. Using optimized chromatography conditions, a three-dimensional chromatogram was obtained using HPLC-PDA detector, and the 11 compounds were eluted within 35 min (Fig. 1)

Cytotoxicity of GMSYS in RAW 264.7 macrophages
To determine the cytotoxicity of GMSYS, RAW 264.7 macrophages were treated with GMSYS at various concentrations ranging from 62.25 to 1000 μg/mL for 24 h, and a viability assay was conducted. As shown in Fig. 2, GMSYS did not produce any significant cytotoxicity up to 1000 μg/mL. Subsequent experiments were performed at nontoxic concentrations.

Effect of GMSYS on NO production and iNOS expression in LPS-stimulated RAW 264.7 macrophages
The level of NO in culture supernatant was measured using a Griess reaction. As shown in Fig. 3a, LPS treatment significantly increased the level of NO compared with vehicle-treated macrophages (P < 0.01). By contrast, LPSinduced NO production was markedly reduced in macrophages pretreated with GMSYS (500 or 1000 μg/mL). A positive control using N-methyl-L-arginine (L-NMMA, 100 μM) showed a significant decrease in LPS-induced NO production.
To investigate the cause of reduced NO production by GMSYS, the expression of iNOS by the macrophages was determined by RT-qPCR. LPS treatment of the macrophages significantly increased the expression of iNOS compared with vehicle-treated macrophages (P < 0.01). By contrast, GMSYS (500 or 1000 μg/mL) pretreatment significantly decreased the expression of LPS-induced iNOS (P < 0.01, Fig. 3b).

Effect of GMSYS on PGE 2 production and COX-2 expression in LPS-stimulated RAW 264.7 macrophages
As shown in Fig. 4a, LPS-treated macrophages produced significantly increased levels of PGE 2 compared with the vehicle-treated macrophages. By contrast, pretreatment with GMSYS (250, 500, or 1000 μg/mL) significantly decreased the level of PGE 2 produced by the LPS-stimulated macrophages compared with macrophages treated with LPS alone (P < 0.01) (Fig. 3b). Indomethacin (2.5 ng/mL) was used as a positive control and markedly decreased PGE 2 production by the stimulated macrophages.
To investigate whether the inhibitory effect of GMSYS on PGE 2 production was related to the expression of COX-2, we measured the COX-2 mRNA expression using RT-qPCR. As shown in Fig. 4b, the expression of COX-2 was significantly induced by LPS stimulation (P < 0.01). By contrast, pretreatment of macrophages with GMSYS (250, 500, or 1000 μg/mL) markedly inhibited their LPS-induced COX-2 expression (P < 0.05, Fig. 4b).
Effect of GMSYS on the production of inflammatory cytokines in LPS-stimulated RAW 264.7 macrophages To determine the effect of GMSYS on the production of proinflammatory cytokines TNF-α and IL-6, RAW 264.7 macrophages were pretreated with various concentrations of GMSYS (0, 250, 500, or 1000 μg/mL) for 4 h and then stimulated with LPS (1 μg/mL) for 20 h. As shown in Fig. 5, the levels of TNF-α and IL-6 were significantly increased in LPS-stimulated RAW 264.7 macrophages compared with vehicle-treated macrophages (P < 0.01). By contrast, pretreatment of the macrophages with GMSYS (250, 500, or 1000 μg/ mL) significantly decreased the levels of TNF-α in a dose-dependent manner (P < 0.01) (Fig. 5a). LPSinduced IL-6 levels were also reduced by pretreatment of the macrophages with GMSYS (500 or 1000 μg/ mL) (P < 0.01, Fig. 5b).

Effect of GMSYS on MAPK phosphorylation in LPSstimulated RAW 264.7 macrophages
To clarify the molecular mechanism of the antiinflammatory effects of GMSYS, we analyzed the phosphorylation of p38, ERK, and JNK by Western blotting. As shown in Fig. 6a, GMSYS (250, 500, or 1000 μg/mL) decreased the expression of phosphorylated p38 in LPS-stimulated RAW 264.7 macrophages. In addition, the phosphorylation of ERK and JNK was

Effect of GMSYS on NF-κB activation in LPS-stimulated RAW 264.7 macrophages
To investigate the mechanism by which GMSYS reduces LPS-induced inflammatory responses, the nuclear translocation of NF-κB p65 was assessed by Western blotting. As shown in Fig. 6b, nuclear NF-κB p65 was markedly increased in the nucleus of LPStreated macrophages compared with the vehicle alone. By contrast, pretreatment with GMSYS reduced the nuclear translocation of NF-κB p65 in LPS-treated macrophages compared with those without such pretreatment.
In the present study, we analyzed 11 marker compounds found in an GMSYS-including gallic acid, neomangiferin, chlorogenic acid, mangiferin, geniposide, paeoniflorin, berberine, liquiritin, nodakenin, glycyrrhizin, and atractylenolide III-using HPLC-PDA detector. Nine of these compounds have anti-inflammatory effects; namely, gallic acid [32], chlorogenic acid [33], mangiferin [34], geniposide [35], paeoniflorin [36], berberine [37], nodakenin [38], glycyrrhizin [39], and atractylenolide III [40]. For these reasons, we predict that GMSYS, which contains these anti-inflammatory herbal medicines and components, has a preventive and therapeutic effect on inflammatory diseases. However, the anti-inflammatory effect and mechanisms of its action has not been thoroughly elucidated. Therefore, we sought to investigate the anti-inflammatory effect and molecular mechanisms of an extract of GMSYS in LPSstimulated RAW 264.7 macrophages, to our knowledge for the first time. We found that GMSYS inhibited the release of inflammatory mediators such as NO and PGE 2 , and downregulated iNOS and COX-2 expression in LPSstimulated macrophages. These findings suggest that GMSYS decreases NO and PGE 2 levels by suppressing iNOS and COX-2 expression, respectively.
TNF-α and IL-6 have been implicated in autoimmune diseases including rheumatoid arthritis [41]. Therefore, the measurement of TNF-α and IL-6 production may Fig. 4 Effect of GMSYS on the production of PGE 2 and expression of COX-2 in LPS-stimulated RAW 264.7 macrophages. The macrophages were pretreated with GMSYS for 4 h and then treated with LPS (1 μg/mL) for 20 h. a PGE 2 levels in supernatants were measures by ELISA. b Total RNA was isolated from the cell pellets and subjected to RT-qPCR to detect the expression of COX-2 mRNA. Levels of COX-2 mRNA expression were normalized to the expression of GAPDH mRNA. Bar graphs represent the means from three independent experiments. ## P < 0.01 vs vehicle-treated cells and ** P < 0.01 vs LPS-treated cells suggest an anti-inflammatory effect. In the present study, we showed that GMSYS suppressed the production of TNF-α and IL-6 in LPS-stimulated RAW 264.7 macrophages.
MAPK represents a group of signaling molecules that appear to play critical roles in inflammatory processes. In particular, they control cellular responses to cytokines and play an important role in the modulation of NF-κB activity. LPS activates three molecules involved in MAPK cascades, p38, ERK, and JNK [42,43]. To identify the signaling pathways involved in the GMSYS-mediated anti-inflammatory responses, we investigated the phosphorylation of these molecules by Western blotting. GMSYS diminished the phosphorylation of p38, ERK, and JNK in LPS-stimulated RAW 264.7 macrophages.
NF-κB plays an important role in various pathological states as a transcription factor for iNOS, TNF-α, and IL-1β, and is translocated into the nucleus by LPS stimulation [44]. In the present study, we found that GMSYS attenuated the nuclear translocation of p65 in LPS-stimulated RAW 264.7 macrophages. These findings suggest that GMSYS has an anti-inflammatory effect by inhibiting NF-κB activation.
Although synthetic drugs have been used for treatment of inflammatory diseases such as atherosclerosis, many of them exhibit varying degrees of adverse effects [45]. By contrast, an examination of herbal medicines may contribute to the discovery of novel drugs as potential anti-inflammatory agents with fewer side effects. Some traditional herbal formulas produce their anti-inflammatory effects by inhibiting the production of inflammatory mediators by blocking NF-kB activation in RAW 264.7 macrophages [46,47]. These reports and our findings suggest that natural products Fig. 5 Effect of GMSYS on the production of TNF-α and IL-6 in LPS-stimulated RAW 264.7 macrophages. The macrophages were pretreated with GMSYS (0, 250, 500, or 1000 μg/mL) for 4 h and then treated with LPS (1 μg/mL) for 20 h. ELISAs were used to determined levels of (a) TNF-α and (b) IL-6 in collected supernatants. Bar graphs represent the means from three independent experiments. ## P < 0.01 vs vehicle-treated cells and ** P < 0.01 vs LPS-treated cells including GMSYS could be used as anti-inflammatory agents with fewer adverse effects than synthetic drugs.

Conclusions
In conclusion, the present study showed that GMSYS inhibits the production and expression of NO, iNOS, PGE 2 , COX-2, TNF-α, and IL-6 in LPS-stimulated RAW 264.7 macrophages. These effects of GMSYS are related to the suppression of MAPK and NF-κB activation. Our results suggest that GMSYS should be considered as a source of potent anti-inflammatory candidates for the treatment or prevention of inflammatory diseases.