Anti-inflammatory effect of Sosihotang via inhibition of nuclear factor-jB and mitogen-activated protein kinases signaling pathways in
lipopolysaccharide-stimulated RAW 264.7 macrophage cells

Abstract

Sosihotang (SO) is an herbal medication, which has been widely used to treat fever, chill and vomiting due to common cold in east-Asian countries. In this study, to provide insight into the effects of SO on inflammation, we investigated its effect on pro-inflammatory mediator production in RAW 264.7 cells and mouse peritoneal macrophages using lipopolysaccharide (LPS) stimulation. SO significantly inhibited the production of nitric oxide (NO), tumor necrosis factor (TNF)-a and interleukin (IL)-6 as well as gene expression of inducible nitric oxide synthase (iNOS), its synthesizing enzyme. In addition, SO inhibited nuclear factor (NF)-jB activation and suppressed extracellular signal-regulated kinase (ERK), p38 and c- Jun NH2-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) phosphorylation. Further- more, we found SO suppresses the production of NO and IL-6 in LPS-stimulated peritoneal macrophage cells. High performance liquid chromatography (HPLC) analysis showed SO contains many active anti-inflammatory constituents such as liquiritigenin, baicalin, baicalein, glycyrrhizin and wogonin. We first elucidated the inhibitory mechanism of SO on inflammation induced by LPS in macrophage cells. Our results suggest SO has potential to be developed as a therapeutic agent for various inflammatory diseases.

1. Introduction

Inflammation is one of normal physiological and immune re- sponse and initiated by pathogens invasion, cell or tissue injury. Commonly, inflammation was accompanied by the activation of various immune cells such as macrophages, neutrophils and lym- phocytes. Particularly, macrophages play an important role in the regulaton of inflammation and immune response and are involved in various disease processes including autoimmune diseases, inflammatory disorders and infections (Pierce, 1990; Wadleigh et al., 2000; Erwig and Rees, 1999). Activated macrophages secrete lots of inflammatory mediators such as NO, prostaglandin (PG)E2, which are produced by iNOS and cyclooxygenase (COX)-2 proteins, respectively, as well as inflammatory cytokines such as TNF-a and IL-6 (Becker et al., 2005; Ronis et al., 2008; Kim et al., 2007; Jung et al., 2009). These inflammatory mediators and cytokines are essential for host survival following infection and are also required for the repair of tissue injury (Becker et al., 2005). Among these cytokines, TNF-a and IL-6 are known as important mediators in- volved in the progress of many inflammatory diseases.

These cytokines and their genes can be modulated by activa- tion of transcription factor NF-jB. NF-jB plays a critical role in expression of inflammatory genes and is composed of p65/p50 subunit in mammalian cells. Also, NF-jB is crucially involved in the pathogenesis of rheumatism and other chronic inflamma- tory diseases (Makarov, 2000). In unstimulated state, NF-jB is present in the cytosol, combined with inhibitory protein IjB. Specific stimuli such as LPS give rise to free NF-jB via the deg- radation of IjB through phosphorylation by IjB kinase (IKK) (de Martin et al., 1993). Activated NF-jB is translocated from cyto- plasm to nucleus and binds to promoter and modulates the expression of inflammatory genes including iNOS, COX-2, inflam- matory cytokines and chemokines (Pahl, 1999; Baeuerle and Baltimore, 1996; Chao et al., 2010). Most anti-inflammatory drugs have been shown to repress the expression of inflamma- tory mediator genes by inhibiting NF-jB activation pathway (Gilroy et al., 2004).

MAPK signaling pathways play an important role in conveying information from extracellular environment into cytoplasm and fi- nally into nucleus (Robinson and Cobb, 1997). There are three pathways of MAPKs (ERK, p38 and JNK MAPK). In ERK pathway, activated ERK can phosphorylate various transcription factors. An- other two classes of MAPKs, p38 and JNK constitute a part of stress response pathways that are activated by various stimuli induced by specific factors (Wang et al., 1998). MAPKs activated by phosphorylation induce NF-jB activation and activate iNOS gene expression. Previous reports demonstrated specific MAPK inhibitors could repress the expression of iNOS gene (Chan and Riches, 1998; Chen et al., 1999; Kim et al., 2004).

In Northeast Asia (Korea, China and Japan), Sosihotang (SO) is a traditionally used herbal medication since ancient times. SO is a prescription described in the Sanghanron, which is a medical book in ancient China, and had been used for the treatment of cold-re- lated symptom. Currently, it is usually prescribed as an herbal medicine for the treatment of fever and throat pain. Previous stud- ies demonstrated that SO inhibits anaphylactic reaction in the mast cells (Kim et al., 1998) and has the repressive effect on chronic hep- atitis B (Qin et al., 2010). In addition, recent studies have revealed SO contains the inhibitory effect on H22 liver cancer growth in mice through the enhancement of immune function (Li et al., 2008) and also has an effect on the treatment of endometriosis (Zheng and Zuo, 2006). However, the effect of SO on inflammation still remains unknown.
In this study, we evaluated the inhibitory effect of SO on LPS-induced inflammation in RAW 264.7 macrophage cells. Furthermore, we investigated whether SO regulates NF-jB/IjBa and MAPK sig- nal pathways to elucidate the mechanism of anti-inflammatory ef- fect of SO.

2. Materials and methods

2.1. Materials and reagents

Materials for cell culture were obtained from Lonza (Basel, Switzerland). LPS, Bovine serum albumin (BSA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylthiazo- lium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Various pri- mary and secondary antibodies for Western blot analysis were purchased from cell signaling technology, Inc. (Boston, MA, USA). Antibodies for ELISA were obtained from eBioscience (San Diego, CA, USA). RNA extraction kit was obtained from iN- tRON (Sungnam, Korea). The oligonucleotide primers were synthesized from Bio- neer (Daejeon, Korea). The standard compound of liquiritin and liquiritigenin were purchased from Wako Pure Chemical Industries, Ltd (Japan) and Chengdu Bio- purify phytochemicals, Ltd (China), respectively. Baicalin, baicalein, glycyrrhizin and wogonin were purchased from Korea Food and Drug Administration (KFDA) (Cheongwon, Korea). Purity (%) of all standard compounds was above 97.0%. HPLC-grade acetonitrile was purchased from J.T. Baker (Austin, TX, USA). Analyti- cal-grade Trifluoroacetic acid (TFA) was obtained from Sigma (St. Louis, MO, USA). The third distilled water was filtered through a 0.45 lm membrane filter (ADVANTEC, Japan) before analysis.

2.2. Preparation of herbal extract, SO

SO is composed of Bupleurum Root, Scutellaria Root, Pinellia Tuber, Zingiberis Rhizoma Crudus, Ginseng Radix, Zizyphi Fructus and Glycyrrhizae Radix et Rhi- zoma. All herb were obtained from Yeongcheon Herbal Market (Yeongcheon, Kor- ea). All voucher specimens were deposited in herbal tank, placed in distilled water and then extracted by heating for 3 h at 115 °C (Gyeongseo Extractor Cos- mos-600, Inchon, Korea). After extraction, the solution was filtered out, freeze-dried and kept in desiccators at 4 °C before use.

2.3. Cell culture and drug treatment

RAW 264.7 cells were obtained from Korea Cell Line Bank (Seoul, Korea) and grown in RPMI 1640 medium containing 10% FBS and 100 U/mL of antibiotics sul- fate. The cells were incubated in humidified 5% CO2 atmosphere at 37 °C. To stim- ulate the cells, the medium was changed with fresh RPMI 1640 medium and LPS (200 ng/mL) was added in the presence or absence of SO (10, 50 and 100 lg/mL) for the indicated periods.

2.4. Peritoneal macrophage isolation and cell culture

Female ICR mice were inoculated with 1 mL of sterile 3% sodium thioglycollate (Sigma, St. Louis, MO, USA). All mice were housed, 4 per cage at a room temperature (RT) and 12 h:12 h light/dark cycle. Three days later, macrophages were harvested by washing their peritoneal cavity with 5 mL of ice-cold PBS. The cell suspension was centrifuged at 250g for 5 min at 4 °C and the supernatant was discarded (Biten- court et al., 2011). After removal of red blood cells (RBC) using RBC lysis buffer, the cell pellet was suspended in RPMI 1640 medium with 10% FBS and 100 U/mL anti- biotics. Then, the cells were incubated for 18 h to be attached to plate. To stimulate the cells, the medium was changed with fresh RPMI 1640 medium and LPS (1 lg/ mL) (Lee et al., 2011; Mogi et al., 2009) was added in the presence or absence of SO (10, 50 and 100 lg/mL) for the indicated periods.

2.5. MTT assay for cell viability

Cytotoxicity was analyzed using the MTT assay. SO was added to the cells and incubated for 48 h at 37 °C with 5% CO2. MTT solutions were added to each well and the cells were incubated for another 4 h. The formazan was melting in dimethyl sulfoxide (DMSO) and then the optical density was read at 570 nm using ELISA reader (infinite M200, TECAN, Männedorf, Switzerland).

2.6. Measurement of NO production

NO production was analyzed by measuring the nitrite in the supernatants of cultured macrophage cells. The cells were pretreated with SO and stimulated with LPS for 24 h. The supernatant was mixed with a same volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2.5% phosphoric acid) and incubated at room temperature (RT) for 5 min (Choi et al., 2007). The absorbance at 570 nm was read.

2.7. Determination of TNF-a and IL-6 cytokine production

The inhibitory effect of SO on the level of TNF-a and IL-6 was determined by an ELISA antibody set (eBioscience, San Diego, CA, USA) according to the manufac- turer’s instructions.

2.8. Western blot analysis

Various protein expressions were evaluated by Western blot analysis according to standard procedures. The cells were pretreated with SO and stimulated with LPS for at 37 °C indicated periods. After incubation, the cells were harvested and resus- pended in protein lysis buffer (PRO-PREP, iNtRON, Sungnam, Korea). After cell deb- ris was discarded by centrifugation, the concentration of protein present in the supernatant was determined by Bradford’s method and equal amounts of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE). After transferring the proteins onto a nitrocellulose membrane (Milli- pore, Bedford, MA, USA), the membrane was blocked in Tris-buffered saline with 0.1% Tween 20 (TBS-T) and 3% BSA. And then, the membrane was incubated with each primary antibody at 4 °C for overnight and subsequently incubated with HRP-conjugated secondary antibodies. The specific proteins were detected using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).

2.9. Preparation of cytosolic and nuclear extracts for NF-jB and IjBa detection

Cytosolic and nuclear fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA) according to the procedure described by the manufacturer. The fractions were stored at
—80 °C before use.

2.10. RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated using easy-BLUE™ RNA extraction kit (iNtRON, Daej- eon, Korea) according to the procedure described by the manufacturer. The total RNA was transformed to cDNA using AccuPower® CycleScript RT PreMix from Bioneer (Bioneer, Daejeon, Korea). Specific primers for amplified by polymerase chain reaction were described in Table 1. The following PCR conditions were ap- plied: iNOS, TNF-a, IL-6 and b-actin, 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C (iNOS), 65 °C (TNF-a), 57 °C (IL-6) and 57 °C (b-actin) for 30 s, and extension at 72 °C for 30 s (Choi et al., 2007; Jo et al., 2008; Kim et al., 2010).

2.11. Preparation of standard and sample

The standard solutions of six components, liquiritin, baicalin, liquiritigenin, baicalein, glycyrrhizin and wogonin were prepared by dissolving 2 mg of each com- pound in methanol at the concentration to 200 ppm. The powdered Sosihotang was dissolved at a concentration of 50 mg/mL in water. Then, suspension was filtered through a 0.45 lm PVDF membrane filter before analysis.

2.12. Chromatographic conditions

The experiments were performed using an Elite Lachrom HPLC-DAD system equipped with L-2130 pump, L-2200 auto sampler, L-2350 column oven, L-2455 photodiode array UV/VIS detector (Hitachi High-Technologies Co., Tokyo, Japan). The output signal of the detector was recorded using an EZchrom Elite software (Version 3.3.1a). The chromatographic separation was conducted with RS tech (Optimapak C18, 4.6 × 250 mm, 5 lm, Daejeon, Korea). The column oven temperature was kept at 40 °C and the injection volume was 10 lL. The wavelength of the UV detector was set at 254 nm. The mobile phase composed of water containing 0.1% trifluoroacetic acid (A) and acetonitrile (B). The run time was 70 min and the mobile phase program was the gradient elution as follows; 10% B (0–5 min), 10–40% B (5–40 min), 40% B (40–60 min) and 40–100% B (60–70 min). The flow rate of the mobile phase was 1.0 mL/min. The chromatographic conditions were pre- sented in Table 2.

2.13. Statistical analysis

The results are expressed as mean ± SE values for the number of experiments. Statistical significance was compared each treated group with the control and determined by Student’s t tests. Each experiment was repeated at least three times to yield comparable results. Values with P < 0.05 and P < 0.005 were considered significant.

3. Results

3.1. SO treatment inhibits NO production and iNOS expression

NO is endogenously synthesized by iNOS through activated NF- jB and MAPK and take a central role in inflammatory response. Be- cause the inhibition of NO could relieve of inflammation, we first checked whether SO represses NO generation in LPS-stimulated RAW 264.7 cells. We used dexamethasone, which is widely used for the treatment of inflammation-related diseases, as a positive control. As presented in Fig. 1A, SO dose-dependently repressed NO generation. In particular, the extent of inhibition reached to 63% at a concentration of 100 lg/mL. Next, when we checked the effect of SO on the expression of iNOS, which is synthase of NO, SO significantly reduced both protein and mRNA expression in a dose-dependent manner (Fig. 1B). These results indicate that the inhibitory effect of SO on NO production is related with the sup- pressive effect of SO on iNOS gene expression.

3.2. Inhibitory effect of SO on LPS-induced inflammatory cytokines production

Next, we investigated the inhibitory effect of SO on the expres- sion of inflammatory cytokines, which is other parameter of inflammation. In this study, we examined the effect of SO on TNF-a and IL-6 expressions. The expression of TNF-a and IL-6 in protein and mRNA was analyzed using ELISA and RT-PCR analysis, respectively. The levels of protein and mRNA of TNF-a were inhib- ited by SO treatment in a dose-dependent fashion (Fig. 2A and B). Consistent with TNF-a result, SO significantly inhibited IL-6 gene expression, dose-dependently (Fig. 2C and D).

3.3. SO strongly blocks LPS-induced NF-jB activation

NF-jB pathway is closely related with the expression of iNOS and inflammatory cytokine. We investigated the effect of SO on NF-jB activation through detecting the level of translocated p65 into nucleus and the extent of IjBa in the cytosol. Western blot analysis in Fig. 3A shows SO at concentrations of 50 and 100 lg/ mL repressed significantly translocation of p65 into nucleus. Consistently, less amount of IjBa was found in the cytoplasm in the presence of same concentrations of SO. These results suggest SO effectively inhibits LPS-induced NF-jB activation through blocking nuclear translocation of NF-jB and IjBa degradation.

3.4. Inhibitory effect of SO on phosphorylation of MAPKs in LPS- stimulated RAW 264.7 cells

Since MAPKs activated by phosphorylation make an important role in NF-jB stimulation, we examined whether MAPKs activity is inhibited by SO treatment. Here, we checked the phosphoryla- tion levels of MAP kinases including ERK 1/2, p38 and JNK. When RAW 264.7 cells were stimulated with LPS, in the presence of SO, the levels of phosphorylated ERK 1/2 and p38 MAPK were signifi- cantly decreased, in a dose-dependent manner (Fig. 4A and B). But, SO did little affect JNK activity. We also checked that total pro- tein levels of MAPKs were not changed by SO treatment at all.

3.5. Effect of SO on RAW 264.7 cell viability

We showed that SO at concentrations of 50 and 100 lg/mL sig- nificantly inhibits inflammatory mediators including cytokines and inflammatory signal pathway. From that results, we used MTT as- say to assess the cytotoxicity of SO at those concentrations. As shown in Fig. 5, SO did not affect cell viability up to 100 lg/mL, indicating SO is not toxic to cells.

3.6. SO inhibits NO and IL-6 production by LPS stimulation in mouse peritoneal macrophages

From the inhibitory effect of SO on inflammatory mediator pro- duction in RAW 264.7 cells by LPS stimulation, we further investi- gated the inhibitory effect of SO in mouse peritoneal macrophage cells. We examined the effect of SO on NO secretion by LPS stimu- lation in mouse peritoneal macrophages. Consistent with the re- sults in RAW 264.7 cells, SO significantly inhibited NO generation by LPS stimulation, dose-dependently (Fig. 6A). Specifically, at a concentration of 100 lg/mL, SO reduced NO generation up to 53%. In the test of inhibitory effect on inflammatory cytokine secre- tion, SO exhibited the inhibitory effect on IL-6 production in peri- toneal macrophages (Fig. 6B). Also, SO did not show cytotoxic effect on peritoneal macrophage cells. These results imply that 100 lg/mL of SO significantly suppresses inflammatory reaction in mouse peritoneal macrophage cells without toxicity.

3.7. HPLC analysis

The HPLC analysis was optimized and performed for the identi- fication of six main components in SO (Fig. 7). The analysis was carried out using a C18 column and flow rate of mobile phase was fixed at 1.0 mL/min. The gradient elution proportions of water and acetonitrile were tested to achieve desired separation. Trifluo- roacetic acid was added to water in order to improve a peak shape and inhibit peak tailing. The UV wavelength of six components was set based on maximum UV spectra absorption of each component.

Liquiritin, Liquiritigenin, baicalin, baicalein and wogonin were de- tected at 280 nm and glycyrrhizin was detected at 254 nm. The amount of six components was calculated using a calibration curve of each component. From the results, Table 3 shows main compo- nents identified in SO. The characterization of each component was conducted by comparing retention time and UV spectrum of target peaks in SO. The profiles of main components were shown in Fig. 8.

4. Discussion

Many previous studies on natural herb or herbal medicine have been conducted to find the potential natural anti-inflammatory products using in vitro and in vivo systems. SO is one of important medication of oriental herbal medicine and has been frequently used to treat various cold-related diseases in East Asia since an- cient times.
In the present study, we have demonstrated the anti-inflam- matory activity of SO in LPS-stimulated RAW 264.7 mouse macro- phage cells. Because overproduction of NO is known to relate with various inflammatory and autoimmune diseases (Guzik et al., 2003; Southan and Szabo, 1996), we examined the inhibi- tory effect of SO on LPS-stimulated NO production. We found that

SO strongly suppresses NO production. We further researched whether the reduced generation of NO by SO is occurred from the inhibition of its synthesis. We found SO exhibits mighty inhibitory effect on iNOS expression at both protein and mRNA level, dose-dependently. These results suggest that SO contains strong inhibitory activity on pro-inflammatory mediator production.

NF-jB is a main regulatory transcription factor involved in cellular responses to specific stimuli (Brasier, 2006; Gilmore, 1999, 2006; Perkins, 2007; Tian and Brasier, 2003). In addition, NF-jB plays an important role in adjustment of cell survival and expres- sion of diverse inflammatory mediators including nitrite, PGE2 and inflammatory cytokines (Chen et al., 1995; Roshak et al., 1996; Schmedtje et al., 1997; Xie et al., 1993, 1994). When we investi- gated whether SO inhibits NF-jB activation, we found that SO blocks nuclear translocation of p65 under LPS stimulation in a dose-dependent manner via the inhibition of IjBa degradation. These findings are consistent with other reports that NF-jB re- sponse elements are present on the promoter of iNOS, TNF-a and IL-6 genes (Ahn et al., 2005; Barnes and Karin, 1997; Chen et al., 2000; Kim et al., 1997).

Because activated MAPKs take part in LPS-induced iNOS expres- sion in mammalian cells (Caivano, 1999), we also investigated the inhibitory effect of SO on the MAPKs phosphorylation in RAW 264.7 macrophages. SO significantly suppressed the levels of phos- phorylation of ERK 1/2 and p38 MAPKs in a dose-dependent man- ner. These results suggest that the inhibitory effect of SO on phosphorylation of MAPKs is directly involved in the reduced pro- duction of pro-inflammatory mediators in RAW 264.7 cells.

We further investigated whether SO inhibits inflammatory mediator production by LPS stimulation in mouse primary macro- phage cells. Consistent with the results in RAW 264.7 cells, SO effectively inhibited the secretion of NO and IL-6 cytokine in mouse peritoneal macrophage cells without toxicity. These results indicate SO strongly inhibits inflammatory response in primary cells.

As shown in Fig. 8, we identified six main components (Liquir- itin, liquiritigenin, baicalin, baicalein, glycyrrhizin and wogonin) in SO. A previous study reported that liquiritigenin exerts anti- inflammatory effect by inhibiting NF-jB-dependent iNOS and pro-inflammatory cytokines expression in RAW 264.7 cells (Kim et al., 2008). Additionally, it was demonstrated that baicalin inhib- its the production of NO, iNOS, ROS, TNF-a, endothelin (ET)-1 and thromboxane A2 (TXA2) by LPS stimulation in macrophages (Liu et al., 2008). Another recent study demonstrated that baicalein contains anti-inflammatory properties and the inhibition of leuko- triene C4 biosynthesis (Butenko et al., 1993). Recent study showed that glycyrrhizin inhibits H5N1 influenza A virus-induced pro- inflammatory cytokine and chemokine expression in human mac- rophages (Michaelis et al., 2010). Also, other report demonstrated that wogonin inhibits NO production in RAW 264.7 cells (Kim et al., 1999). These facts suggest that the anti-inflammatory activ-
ity of SO might be related with active components contained in SO, including liquiritigenin, baicalin, baicalein, glycyrrhizin and wogonin.
In conclusion, SO exerts significant inhibitory effect on NO gen- eration, inflammatory cytokines and iNOS expressions in LPS-stim- ulated RAW 264.7 cells and these effect was mediated by the suppression of NF-jB activation through IjBa stabilization and the blockage of MAPKs phosphorylation. Furthermore, SO exhib- ited the inhibitory effect on inflammatory mediator production including NO and IL-6 cytokine in mouse primary cells. These re- sults imply that SO could be developed as a new anti-inflammatory NSC 167409 therapeutic herbal medicine without cytotoxicity after further in vivo studies.