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.
Methods
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.
Table 1
Herbal composition of GJT
Cinnamomi Ramulus |
Cinnamomum cassia
| Vietnam | 11.25 |
Paeoniae Radix |
Paeonia lactiflora
| Uiseong, Korea | 7.50 |
Glycyrrhizae Radix et Rhizoma |
Glycyrrhiza uralensis
| China | 3.75 |
Zingiberis Rhizoma Crudus |
Zingiber officinale
| Yeongcheon, Korea | 3.75 |
Zizyphi Fructus |
Zizyphus jujuba
| Yeongcheon, Korea | 3.75 |
Total amount | | 30.00 |
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 freezing-drying 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 % CO2 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 96-well 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:
$$ \mathrm{Cell}\ \mathrm{viability}\ \left(\%\right)=\frac{\mathrm{Mean}\ \mathrm{O}\mathrm{D}\ \mathrm{in}\ \mathrm{G}\mathrm{J}\mathrm{T}\ \mathrm{treated}\ \mathrm{cells}}{\mathrm{Mean}\ \mathrm{O}\mathrm{D}\ \mathrm{in}\ \mathrm{untreated}\ \mathrm{cells}}\times 100 $$
ELISAs for TNF-α, IL-6, and PGE2
Cells were pretreated with various concentrations of GJT for 4 h and stimulated with LPS (1 μg/mL) for an additional 20 h. Production of TNF-α, IL-6, and PGE2 in the culture supernatants was measured using commercial ELISA kits from R&D systems (Minneapolis, MN), BD Biosciences (Mountain View, CA), and Cayman Chemical Co. (Ann Arbor, MI), respectively.
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 \( \mu \)g 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 FlouviewFV10i 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 C18 (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.
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. Anti-inflammatory 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 anti-inflammatory 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 LPS-stimulated 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.
As mentioned above, GJT consists of 5 different herbal medicines
Cinnamomum cassia,
Paeonia lactiflora,
Glycyrrhiza uralensis, Zingiber officinale, and
Zizyphus jujube with certain ratio based on ‘Sang han lon’. Of interest, it has been reported anti-inflammatory effects of individual herbs of GJT [
25‐
29]. In addition, the major components of the 5 medicinal herbs are known as follows: coumarins (e.g. coumarin) and phenylpropanoids (e.g. cinnamic acid and cinnamaldehyde) from
C. cassia [
30,
31], monoterpenoids (e.g. albiflorin and paeoniflorin) from
P. lactiflora, triterpene saponin (e.g. glycyrrhizin) and flavonoids (e.g. liquiritin and liquiritigenin), from
G. uralensis [
32], phenols (e.g. 6-, 8-, and 10-gingerol) from
Z. officinale [
33], and flavonoids (e.g. spinosin and 6ʹʹʹ-feruloylspinosin) from
Z. jujube [
34]. Among those constituents, we analyzed 12 compounds, such as galiic acid, albiflorin, paeoniflorin, liquiritin apioside, liquiritin, benzoic acid, coumarin, cinnamic acid, benzoyl paeoniflorin, cinnamaldehyde, glycyrrhizin, and 6-gingerol using HPLC–PDA. An optimized HPLC–PDA method was applied for quantitative analysis of these 12 compounds in the GJT water extract. Consequently, paeoniflorin (40.29 ± 0.12 mg/g), which is marker compound of
P. lactiflora, was detected as the most abundant component in GJT extract. Anti-inflammatory activities of several GJT component compounds have been reported, including gallic acid [
35], paeoniflorin [
36], benzoic acid [
37], cinnamaldehyde [
38], glycyrrhizin [
39], and 6-gingerol [
40]. Further studies, including in animal models, will be required to elucidate the precise pharmacological mechanisms of the active compounds from GJT extract and their pharmacokinetics/pharmacodynamics.
Acknowledgement
This research was supported by a grant for “Construction of Scientific Evidence for Herbal Medicine Formulas (K16251)”, from the Korea Institute of Oriental Medicine (KIOM).