Background
Cheongsangbangpung-tang (CBT; Qing-Shang-Fang-Feng-Tang in Chinese and Seijo-bofu-to in Japanese) is a traditional herbal formulation composed of 13 medicinal herbs:
Ledebouriellae radix,
Angelicae dahuricae radix,
Forsythiae fructus,
Platycodi radix,
Scutellariae radix,
Cnidii rhizoma,
Schizonepetae herba,
Gardeniae fructus,
Coptidis rhizoma,
Aurantii fructus,
Menthae herba,
Glycyrrhizae radix, and
Bambusae caulis in Liquamen. In Korean medicine, CBT has been used to treat inflammatory diseases, such as conjunctivitis, eczema, acne, furuncles on the face, rosacea, and ophthalmitis [
1,
2].
The immune system is one feature found in living organisms that acts as a defence against external pathogenic substances, such as bacteria, viruses and fungi. Once infected with pathogenic microorganisms, the skin and mucosal surfaces serve as primary physical barriers [
3]. An immune response (immunity) is launched to resist infection when a physical barrier has been compromised [
4,
5]. Homeostasis of the human body can be maintained with proper control of the immune response. In addition, an abnormal or overactive immune system can cause pathogenic conditions, such as asthma, autoimmunity and hypersensitivity [
6,
7].
Inflammation is one local defence reaction that is vital for protecting living tissue from injury. However, inflammation may have a negative impact on health if it is extreme or continuously repeats [
8]. Chronic inflammation can cause a variety of conditions, such as rheumatoid arthritis, cardiovascular disease and cancer [
9]. Various substances, such as cytokines and prostaglandins (PGs), are involved in the inflammatory response. Lipopolysaccharides (LPS) from gram-negative bacteria can trigger sepsis by activating macrophage cells and leading to the abnormal generation of cytokines and PGs [
10,
11]. Therefore, macrophage cells that have been stimulated by LPS present a good model system for studying the anti-inflammatory activities of potent candidate materials. In traditional Korean medicine, many herbal formulas have been prescribed to treat inflammatory diseases [
12]. CBT is expected to have anti-inflammatory effects in both in vitro and in vivo studies because it traditionally has been used to bring down the fever [
1,
2]. However, the mechanism of action of the anti-inflammatory activity of CBT has not yet been reported.
In this study, we evaluated the anti-inflammatory effects of CBT extract (CBTE) on both animal and cellular inflammation models. An LPS-inducible macrophage was used for the in vitro investigation of anti-inflammation. In addition, a carrageenan (CA)-induced paw oedema experiment was conducted to determine the effect on the acute-phase inflammation in vivo.
Methods
Preparation of the aqueous cheongsangbangpung-tang extract
CBT is composed of 13 herbs (Table
1), which were purchased from Daewon Pharmacy (Daegu, Korea). Ten folds the amount of CBT (284.6 g) of 12 herbs except
Bambusae caulis in Liquamen were extracted by boiling in 2 l of water for 3 h and then adding 187.5 g of
Bambusae caulis in Liquamen to the hot water extract; the solution was placed into a cotton bag and squeezed to obtain the final aqueous extract of CBT (CBTE), which was filtered through 0.2-μm filter paper (Nalgene, NY, USA), lyophilized and stored at −20 °C until its use.
Table 1
Composition of Cheongsangbangpung-tang
Ledebouriellae radix
| 3.75 |
Gardeniae fructus
| 1.87 |
Angelicae Dahuricae radix
| 3.0 |
Coptidis rhizoma
| 1.87 |
Forsythiae fructus
| 3.0 |
Aurantii fructus
| 1.87 |
Platycodi radix
| 3.0 |
Menthae herba
| 1.87 |
Scutellariae radix
| 2.62 |
Glycyrrhizae radix
| 1.12 |
Cnidii rhizoma
| 2.62 |
Bambusae caulis in Liquamen
| 18.75 |
Schizonepetae herba
| 1.87 | | |
Reagents
The LPS (E. coli 026:B6) and Griess reagent were obtained from Sigma (St. Louis, MO, USA). Glycyrrhizic acid, liquiritigenin, berberine, baicalin, baicalein, forsythiaside-A and poncirin were also purchased from Sigma. Enzyme-linked immunosorbent assay (ELISA) kits for tumour necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-1β were acquired from Pierce Endogen (Thermo Scientific, Waltham, MA, USA). The PGE2 ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA). The primary antibodies for the phospho (p)-inhibitor of NF-κB α (I-κBα), p-ERK, p-jun NH2-terminal kinase (JNK), p-p38, ERK, JNK and p38 antibodies were purchased from Cell Signaling (Danvers, MA, USA). COX-2 and iNOS antibodies were obtained from BD Biosciences (San Jose, CA, USA), while the NF-κB, IκBα, β-actin and lamin A/C antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Chromatographic analysis of CBTE
An ultra performance liquid chromatography (UPLC) system (Waters, USA) was used to identify the seven marker phytochemicals of CBTE: glycyrrhizic acid, liquiritigenin, berberine, baicalin, baicalein, forsythiaside-A, and poncirin. The system was equipped with an ACQUITYTM photodiode array detector (PDA) and a binary solvent delivery pump. The separation was executed on an ACQUITYTM BEH Shield RP 18 column (1.7 μm, 2.1 × 100 mm). The mobile phase was composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) with a gradient elution system at a flow rate of 1 ml/min. The injection volume was 2 μl. The column temperature was set at room temperature.
Cell culture and cell viability test
The RAW264.7 macrophage cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium containing 10% foetal bovine serum made quiescent by serum starvation for 12 h. For all experiments, LPS was used at a final concentration of 1 μg/ml. Test samples were added to the culture medium 1 h prior to the addition of LPS and treated for the indicated time periods. For the cell viability experiment, the cells were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/ml) for 4 h. The dark blue crystals of formazan were dissolved into 200 μl of dimethyl sulphoxide, and the absorbance of the formazan solution was measured at 570 nm.
Determination of NO, PGE2 and cytokines
NO production was monitored by measuring the nitrite content in the culture media by mixing the cultured media with Griess reagent (1% sulphanilamide, 0.1% N-1-naphthylenediamine dihydrochloride and 2.5% phosphoric acid). The absorbance was measured at 540 nm after 10 min of incubation.
The levels of PGE2 and cytokines (TNF-α, IL-1β, and IL-6) were assessed by ELISA using anti-mouse PGE2, TNF- α, IL-1 β, or IL-6 antibodies and biotinylated secondary antibody according to the manufacturer’s instructions.
Assay of COX-2 activity
COX-2 enzyme activity was measured using an ELISA kit (Cayman Chemical Co., Ann Arbor, MI, USA). The cells were collected by centrifugation at 1500×g for 10 min at 4 °C and homogenized in a cold buffer (0.1 M Tris-HCl, pH: 7.8, 1 mM EDTA, 250 mM mannitol, and 0.3 mM diethyldithiocarbamic acid). The homogenized cells were centrifuged at 10,000×g for 15 min at 4 °C, and the supernatant (cell lysate) was removed for the COX-2 activity assay according to the manufacturer’s instructions.
Western blot analysis
Whole-cell lysates were prepared by lysing cells in an RIPA buffer, and the nuclear fractions for the NF-κB analysis were set up using a commercial kit (Chemicon International, Inc., Billerica, MA, USA). Equal amounts of proteins were resolved on 10% sodium dodecyl sulphate gel and then transferred to a nitrocellulose membrane. The membrane was incubated with primary antibodies for 2 h, followed by incubation with a secondary antibody for 1 h. The immunoreactivity was detected using a chemiluminescent detection kit (Amersham Bioscience, Piscataway, NJ, USA).
Statistical analysis for cell experiments
Statistical evaluations were performed using the Statistical Package for the Social Sciences (SPSS) software (version 18, IBM Corp., Armonk, NY, USA). A one-way analysis of variance followed by a Tukey’s test was used to assess the differences among treatment groups. Values are expressed as mean ± S.D. The criterion for statistical significance was set at P < 0.05 or P < 0.01.
Carrageenan-induced paw oedema
Sprague-Dawley rats at 6 weeks of age (male, 140–160 g) were provided by the Samtako Co. (Osan, Korea), allowed to acclimate to their new environment for 1 week and maintained in a clean room at the animal lab. The animals were caged under the supply of filtered, pathogen-free air and fed standard rat chow (Purina, Korea) and water ad libitum at a temperature between 20 and 23 °C with 12 h light and dark cycles and a relative humidity of 50%. All animal experiments were conducted in strict accordance with protocols approved by the Institutional Animal Care and Use Committee of Daegu Haany University (Approval number: DHU 2015-064). The rats were divided into five groups: normal; CA control; CA + dexamethasone (DEXA, 1 mg/kg) as a standard reference; CA + CBTE (0.3 g/kg); and CA + CBTE (1.0 g/kg). CBTE and DEXA were dissolved in saline and orally administered to the control rats for four consecutive days. Paw oedema was induced by injecting 0.1 ml of 1% w/v CA suspended in saline into the sub-plantar tissues of the right hind paw of each rat. The volume of the paw was measured with a Plethysmometer (LE 7500; LETICA Scientific Instruments, Spain) immediately prior to the CA injection and then again 1, 2, 3 and 4 h after the injection. The data were expressed as the variation in the paw volume (ml) and were compared to the pre-injection values.
Histological process
The dorsum and ventrum pedis skins of the hind paws were separated and fixed in 10% neutral buffered formalin then embedded in paraffin, sectioned (3 ~ 4 μm) and stained with haematoxylin and eosin (HE) to determine their general histopathological profiles and the immunohistochemistry for COX-2 and iNOS.
Immunohistochemistry
The changes in the immunoreactivities of COX-2 and iNOS were observed by purified primary antibodies with avidin-biotin-peroxidase (ABC) and peroxidase substrate kits (Vector Labs, Burlingame, CA, USA). Briefly, the endogenous peroxidase activity was blocked by incubation in methanol and 0.3% H2O2 for 30 min, while non-specific binding of immunoglobulin was blocked with normal horse serum blocking solution for 1 h in a humidity chamber after heating (95–100 °C)-based epitope retrievals in 10 mM citrate buffers (pH: 6.0). The primary antisera were treated overnight at 4 °C in a humidity chamber and then incubated with biotinylated universal secondary antibody and ABC reagents for 1 h at room temperature in a humidity chamber. Finally, the sections were reacted with a peroxidase substrate kit for 3 min at room temperature.
Histomorphometry
To observe more detailed changes induced by CA either with or without CBTE treatment, the thicknesses of the dorsum and ventrum pedis skins (from the epidermis to the dermis; keratin layers were excluded) were measured with an automated image analyzer (iSolution FL ver 9.1, IMT i-solution Inc., Vancouver, Quebec, Canada) and microscopy at 40× magnification (Model Eclipse 80i, Nikon, Tokyo, Japan) to prepare skin histological samples as μm/paw; the number of infiltrated inflammatory cells in the dermis was also counted using an automated image analyser as cells/mm2 of dermis under 200× magnification. In addition, the cells occupied by over 10% of COX-2 and iNOS immunoreactivities were regarded as positive. In the present study, the number of COX-2- and iNOS-positive cells were separately calculated on the epithelial lining as cells/100 epithelial cells and on the mm2 of dermis (cells/mm2 of dermis) using a digital image analyser. The histopathologist was blinded to the group distribution when this analysis was carried out.
Statistical analysis for animal experiments
Multiple comparison tests for different dose groups were conducted. The variance homogeneity was examined using the Levene test. If the Levene test indicated no significant deviations from the variance homogeneity, the obtained data were analysed by a one-way analysis of variance (ANOVA) test followed by a least-significant differences (LSD) multi-comparison test to determine which pairs of group comparisons were significantly different. Differences were considered significant at P < 0.05. In addition, the per cent-point changes between normal and CA groups were calculated to monitor the severities of acute inflammations induced in this study, and the per cent-point changes between CA and test material, CBTE or DEXA-treated skins were also determined to further the understanding of the efficacy as follows: Per cent-point Changes Compared with Normal (%) = ((Data of CA–Data of Normal)/Data of Normal) ×100; and Per cent-point Changes Compared with CA (%) = {(Data of test material treated rats-Data of CA)/Data of CA} ×100.
Discussion
Removing heat from the upper body is a characteristic action of CBT in Korean medicine; therefore, CBT has been prescribed in traditional Korean medicine to resolve pimples on the face and neck or to treat eye and ear diseases. The biological activity of CBT has not been extensively studied, although several publications about the antibacterial and anti-acne activity of CBT have produced valid evidence that explains the clinical use of CBT for the treatment of pimples [
2,
14]. In the present study, we investigated the effects of CBTE on in vivo and in vitro inflammation. CBTE showed anti-inflammatory properties by regulating markers of inflammation in RAW264.7 murine macrophage treated with LPS and also by relieving CA-induced acute paw swelling in rats.
Inflammation occurs due to infection, physical and chemical injury, ischemia and excessive immune response as part of the host’s attempt to remove damaged tissue and foreign substances, such as invading bacteria [
9]. A variety of factors and cells are involved in the inflammatory process. Macrophages are immune cells that primarily trigger the production of inflammatory mediators such as NO and PGE
2 when activated by bacterial endotoxins like LPS [
10,
11]. The increased production of NO and PGE
2 in LPS-stimulated macrophages can be led by inducible enzymes iNOS and COX-2, respectively [
15]. It was reported that the genetic inhibition of iNOS reduced the degree of macrophage inflammation caused by LPS [
16]. It has also been shown that selective PGE
2 synthesis inhibitors can relieve pyrexia and chronic inflammation in rats [
17]. We observed that the up-regulated expression of iNOS and COX-2 and the excessive production of NO and PGE
2 were abolished in the presence of CBTE. Moreover, CBTE had an inhibitory effect on the LPS-induced production of cytokines, such as TNF-α, IL-1β and IL-6, which play important roles in inflammation by producing septic shock syndrome at high levels. TNF-α stimulates the production of interleukins that participate in the response to infection or injury, and IL-1β acts on macrophages to release IL-6 and IL-8 related to the pathogenesis of various inflammatory diseases [
18,
19].
NF-κB is a functional transcription factor implicated in inflammation. The activation of NF-κB is critical to induce the expression of various inflammatory enzymes and cytokines, including iNOS, COX-2, TNF-α, IL-1β and IL-6 [
20,
21]. In resting macrophages, NF-κB is inactive and placed in the cytosol through interaction with inhibitory protein I-κBα. NF-κB is activated by the phosphorylation and subsequent proteolysis of I-κBα in response to various stimuli, including LPS [
22]. We found that LPS potently induced the expression of p-I-κBα, which was restored by CBTE treatment. In line with changes in I-κBα phosphorylation, CBTE inhibited NF-κB activity by decreasing the expression level in the nuclear fraction. These findings suggest that CBTE exerts anti-inflammatory activity through suppressing NF-κB activation in LPS-stimulated RAW264.7 macrophage cells.
Phosphorylation of MAPKs by LPS-stimulation has been shown to initiate inflammatory responses by inducing NF-κB activation [
23]. Several reports have indicated that the p38 MAPK pathway is associated with the regulation of inflammatory cytokines [
24,
25]. In our study, LPS induced the phosphorylation of three MAPKs (ERK, JNK and p38) that correspond to the increased cytokine level and NF-κB activation. However, CBTE reduced the expression level of p-ERK, p-JNK and p38 protein. These results indicated that the inhibition of MAPK phosphorylation by CBTE was closely associated with the suppression of macrophage inflammation mediated by NF-κB signalling.
Local treatment with CA induces severe oedematous acute inflammations, and a CA-induced hind paw acute oedematous inflammation animal model has been widely used to detect the anti-inflammatory effects of various drugs [
26,
27]. Histopathologically, a loosening of connective tissues and inflammatory cell infiltrations mainly involving neutrophils were observed near the CA-treated sites [
28,
29]. In the present study, marked rises in the number of infiltrated inflammatory cells with increases of skin thicknesses in both the dorsum and ventrum pedis were detected following treatment with CA as compared with normal rat paw skin. However, these CA-induced acute oedematous inflammatory changes were inhibited by CBTE and also DEXA, respectively. These results are considered direct evidence that CBTE has favourable anti-inflammatory activities.
COX-2, a key enzyme for the synthesis of prostaglandins, which are chemical mediators of inflammation, is also involved in angiogenesis and cancer progression [
30,
31]. Therefore, decreases in COX-2 immunoreactivities have been used to predict the favourable effects of test materials on inflammation [
32,
33]. In addition, increases in iNOS activities related to proinflammatory agents, such as endotoxin, IL-1β, TNF-α and interferon-γ, can be induced by shock and also occur following inflammatory responses in the body [
34]. iNOS is involved in the development of inflammation early after CA administration, and the NO produced by iNOS is responsible for the subsequent maintenance of the inflammatory response. These CA-activated inflammatory cascades are related not only to innate immunity but also to the generation of reactive oxygen species [
35]. In the present study, marked increases of epithelia and dermis COX-2 and iNOS-immunoreactive cells were detected on the dorsum pedis and ventrum pedis skin in CA compared with normal rats, which corresponded well with our previous CA rat studies [
36,
37]. However, these increases in inflammatory mediator immunolabelled cells were decreased by treatment with both different dosages of CBTE in a dose-dependent manner and also by DEXA. Therefore, CBT should favourably inhibit the acute inflammation mediated by the modulation of inflammatory mediators, such as COX-2 and iNOS expression.
Conclusions
This study demonstrated the immunoregulatory activity of CBTE via the suppression of transcription factor, NF-κB, which is critical for the expression of cytokines, iNOS and COX-2. In macrophage cells, CBTE decreased the production of LPS-inducible inflammatory mediators, including NO, PGE2 and inflammatory cytokines, and inhibited NF-κB activation and MAPK phosphorylation. We also confirmed that CBTE inhibits acute oedematous inflammation through the modulation of COX-2 and iNOS expression in our in vivo study. In conclusion, our results provide scientific evidence to support the clinical efficacy of CBTE in Korean medicine and also contribute to better understanding the molecular mechanism of CBTE in regulating inflammatory responses.