Traditional Herbal Formula Banhasasim-tang Exerts Anti-Inflammatory Effects in RAW 264.7 Macrophages and HaCaT Keratinocytes

Banhasasim-tang (BHSST) is a Korean traditional herbal formula comprising eight medicinal herbs. The aim of the present study was to investigate the anti-inflammatory effect of BHSST using macrophage and keratinocyte cell lines. First, we evaluated the effects of BHSST on inflammatory mediator and cytokine production in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. BHSST markedly inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin- (IL-) 6. BHSST significantly suppressed the protein expression of toll-like receptor 4 (TLR4) and phosphorylated nuclear factor-kappa B (NF-κB) p65 in RAW 264.7 cells. Second, we examined whether BHSST influences the production of chemokines and STAT1 phosphorylation in tumor necrosis factor-α/interferon-γ TI-stimulated HaCaT keratinocytes. BHSST significantly suppressed the production of RANTES/CCL5, TARC/CCL17, MDC/CCL22, and IL-8 in TI-stimulated HaCaT cells. BHSST also suppressed TI-induced phosphorylation of STAT1 in HaCaT cells. These results suggest that BHSST may be useful as an anti-inflammatory agent, especially for inflammatory skin diseases.


Introduction
Inflammation is a protective response to pathogens such as bacteria, viruses, and parasites. Coordinate regulation of inflammatory mediator and cytokine release is necessary for optimal host defense. Nitric oxide (NO) and prostaglandin E 2 (PGE 2 ), important inflammatory mediators, are secreted by activated immune cells, such as macrophages. Under pathological conditions, NO production is increased by inducible NOS (iNOS) and subsequently leads to cytotoxicity and tissue damage. PGE 2 is produced at inflammatory sites by cyclooxygenase-2 (COX-2), which is therefore implicated as an important mediator in the processes of inflammation. In addition, tumor necrosis factor-(TNF-) and interleukin-(IL-) 6 are proinflammatory cytokines that act as signaling molecules for immune cells and coordinate inflammatory responses.
Toll-like receptors (TLRs) mediate signal transduction cascades that ultimately promote the nuclear translocation of nuclear factor-kappa B (NF-B). In particular, TLR4 is a pattern recognition receptor for lipopolysaccharide (LPS) [1]. NF-B signaling is considered a pivotal mechanism for the regulation of immune and inflammatory responses by controlling the transcription of inflammatory cytokines [2].
However, overproduction of these inflammatory mediators and cytokines can result in various pathological conditions [3,4]. In particular, atopic dermatitis-(AD-) like inflammatory skin diseases are aggravated by inflammatory mediators and cytokines in chronic states of inflammation [5]. Migration of inflammatory cells into lesioned skin is 2 Evidence-Based Complementary and Alternative Medicine regulated by inflammatory chemokines, a group of small cytokines produced by various cell types. In particular, Th2type cell-specific chemokines, such as regulated on activation, normal T-cell expressed and secreted (RANTES/CCL5), thymus-and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), are produced from keratinocytes, and may be effective targets for the treatment of inflammatory skin diseases [6]. In addition, IL-8 is also an important mediator in inflammatory skin diseases [7]. Traditional herbal formulas have been widely used over several thousand years to prevent and treat various diseases in East Asia, including Korea, China, and Japan [8]. Among them, Banhasasim-tang (BHSST, Hange-shashin-to, and Ban xia xie xin tang), comprising eight medicinal herbs (Table 1), has been used for treating gastritis, diarrhea and gastric ulcer [9,10]. Despite previous reports, there have been no reported studies on the effects or action mechanisms of BHSST on inflammatory responses.
In this study, we investigated the anti-inflammatory effects of BHSST in RAW 264.7 macrophages and HaCaT keratinocytes in vitro.
2.6. Cytotoxicity Assay. Cell viability was assessed using a CCK-8 kit according to the manufacturer's instructions. RAW 264.7 cells (3 × 10 3 cells/well) and HaCaT cells (1 × 10 3 cells/well) were incubated in 96-well plates with various concentrations of BHSST in the absence or presence of respective LPS (1 g/mL) and TNF-and IFN-(TI, each 10 ng/mL) for 24 h. The CCK-8 reagent was added to each well, followed by incubation for an additional 4 h. Absorbance by well contents was measured at 450 nm using a Benchmark plus microplate reader (Bio-Rad Laboratories). The percentage of viable cells was calculated using the following formula: cell viability (%) = (mean absorbance in test wells/mean absorbance in control wells) × 100.

Measurement of Inflammatory Mediator
Levels. RAW 264.7 cells (2.5 × 10 5 cells/well) were cultured in 48-well plates. After reaching confluence, the cells were treated with various concentrations of BHSST for 4 h, and then stimulated with 1 g/mL of LPS for 20 h. The culture supernatants were used to determine levels of secreted nitrite, PGE 2 , TNFand IL-6. The nitrite production was measured by Griess reagent as an indicator of NO, and the PGE 2 , TNF-, and IL-6 production were determined using ELISA kits. l-NMMA and indomethacin were used as positive controls for NO and PGE 2 , respectively.

Measurement of Chemokine Production.
HaCaT cells (1 × 10 6 cells/well) were cultured in 6-well plates. After reaching confluence, the cells were washed and treated with various concentrations of BHSST in 1 mL of serum-free medium that contained TI (each 10 ng/mL) for 24 h. The supernatants were harvested, and production of RANTES, TARC, MDC, and IL-8 were determined using ELISA kits.

Western Blotting.
To determine the effect of BHSST on expression of COX-2, TLR4, and phosphorylated NF-B in RAW 264.7 cells, the cells were pretreated with various  (4), berberine (5), wogonoside (6), baicalein (7), glycyrrhizin (8), and wogonin (9). After washing three times with TBST, the membranes were incubated with a horseradish peroxidase-(HRP-) conjugated secondary antibody (Jackson Immuno Research, PA, USA) for 1 h at room temperature. The membranes were washed three times with TBST, and then developed using an ECL kit. The membranes were photographed using a ChemiDoc XRS + imaging system (Bio-Rad Laboratories).

Immunofluorescence
Staining. Cells were seeded onto glass coverslips and incubated with TI (each 10 ng/mL) in the absence or presence of BHSST (500 g/mL) for 30 min. The cells were fixed in 4% paraformaldehyde and 100% acetone, blocked in 0.5% bovine serum albumin, and incubated with anti-STAT1 antibody (Cell Signaling Tech.) for 1 h at room temperature. Then, FITC-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Invitrogen, Carlsbad, CA, USA) was used as a secondary antibody. The immunostained cells were mounted with medium containing DAPI and visualized by use of an Olympus FLUOVIEW FV10i confocal microscope (Tokyo, Japan).
2.11. Statistics. All data are presented as the mean ± SEM. One-way analysis of variance was used to detect significant differences between the control and treatment groups. Dunnett's test was used for multiple comparisons. The differences were considered significant at < 0.05 and < 0.01.

Quantitative Determination of the Nine Compounds in BHSST.
The established HPLC analytical method was applied for the simultaneous quantification of nine compounds in BHSST. The typical chromatogram patterns for standard compounds and the BHSST decoction are shown in Figure 2.  Table 2.

Effects of BHSST on Inflammatory Mediators in RAW 264.7 Cells.
The cytotoxicity of BHSST in the absence or presence of LPS (1 g/mL) for 24 h was determined in RAW 264.7 cells (Figures 3(a) and 3(b)), and subsequent experiments were performed at nontoxic concentrations. To investigate the anti-inflammatory effect of BHSST, we measured NO, PGE 2 , and IL-6 levels in LPS-stimulated RAW 264.7 cells. As shown in Figure 4, LPS-treated RAW 264.7 cells significantly increased NO, PGE 2 , TNF-and IL-6 levels compared with the vehicle-treated cells ( < 0.01). By contrast, BHSST (250, 500 or 1000 g/mL) suppressed LPS-induced NO, PGE 2 , and IL-6 production in a dose-dependent manner compared with cells treated with LPS alone ( < 0.01) (Figure 4). However, BHSST had no effect on LPS-induced TNF-production in RAW 264.7 cells (data not shown). Positive controls using l-NMMA and indomethacin showed significant decreases in LPS-stimulated NO and PGE 2 production, respectively (Figures 4(a) and 4(b)).  (Figures 5(a) and 5(b)). By contrast, BHSST reduced LPSstimulated TLR4 expression at a dose of 1000 g/mL ( < 0.01) (Figures 5(a) and 5(c)). To determine whether BHSST mediates the inhibition of inflammatory responses through NF-B pathway, phosphorylation of NF-B p65 was analyzed by Western blotting. As shown in Figure 6, phosphorylation of NF-B p65 was increased by LPS stimulation. By contrast, BHSST significantly suppressed LPS-stimulated phosphorylation of NF-B p65 at a dose of 1000 g/mL ( < 0.01). . By contrast, BHSST suppressed TI-stimulated RANTES and TARC production at a dose of 500 g/mL ( < 0.01) (Figures 7(a) and 7(b)). The increased MDC and IL-8 production brought about by TI stimulation were significantly decreased by BHSST in a dose-dependent manner (Figures 7(c) and 7(d)). A positive control, silymarin showed a significant decrease in TI-stimulated chemokine production.

Effects of BHSST on STAT1 Phosphorylation.
Previous studies have reported that STAT1 is an important regulator of TI-induced inflammatory and immune responses [11,12]. Therefore, we examined the effect of BHSST on the phosphorylation of STAT1 in TI-treated HaCaT cells. As shown in Figure 8, phosphorylation of STAT1 was increased by TI treatment. BHSST clearly inhibited TI-induced phosphorylation  of STAT1. Treatment of silymarin reduced STAT1 phosphorylation (Figure 8(a)). Consistently, immunofluorescent staining revealed that BHSST blocked TI-induced nuclear localization of STAT1 in HaCaT cells (Figure 8(b)).
Inflammatory responses play a central role in the pathogenesis of many diseases, initiated by the invasion of pathogens or by tissue injury, followed by a series of vascular and cellular reactions. Macrophages are principal immune cells, which are activated by inflammatory mediators including NO and PGE 2 , as well as inflammatory cytokines such as TNF-, IL-6, and IL-1 in inflammatory responses. High levels of these inflammatory mediators and cytokines in states of chronic inflammation can result in various pathological conditions [3,4]. Therefore, these inflammatory mediators and cytokines are important targets for treating inflammatory diseases. In the present study, we observed that LPS stimulated the production of NO, PGE 2 , TNF-, and IL-6 as well as expression of COX-2 in RAW 264.7 cells. By contrast, BHSST markedly reduced the production of NO, PGE 2 , and IL-6 in LPS-stimulated RAW 264.7 cells (Figure 4). These findings demonstrate that BHSST possesses anti-inflammatory effects by inhibiting production of NO, PGE 2 , and IL-6, whereas it is not controlled by TNF-production and COX-2 expression in macrophages. Although BHSST had the inhibitory effect on PGE 2 production, it did not affect COX-2 expression level. However, it may be possible that COX-2 enzymatic activity, but not expression, is involved in anti-inflammatory action of BHSST.
LPS activates the TLR4-mediated signaling pathway, and leading to the activation of NF-B to regulate the release of cytokines [34,35]. To detect the inhibitory mechanism on inflammatory action of BHSST, we carried out the effect of BHSST on TLR4 and phosphorylation of NF-B. The results showed that BHSST pretreatment significantly suppressed LPS-stimulated TLR4 and NF-B p65 phosphorylation in RAW 264.7 cells (Figures 5 and 6). These finding suggested that BHSST exerts anti-inflammatory effect through the suppression of TLR4/NF-B pathway. It seems to be related to inhibitory effect of bioactive components of BHSST, such as baicalin [33].
Inflammatory mediators and cytokines aggravate inflammatory responses to skin lesions, which are an important feature of inflammation skin diseases such as AD [5]. Skin inflammation including AD develops on allergic reaction and is characterized by the overexpression of inflammatory cytokines. Furthermore, AD changed the expression of various inflammatory chemokines [36]. Chemokines are basic proteins that are secreted and critically important in allergic inflammation via leukocyte trafficking and activation. In particular, it has been reported that the serum level of RANTES, TARC, and MDC, which are Th2-type chemokines, is associated with skin inflammation [5,6]. Moreover, IL-8, a member of the chemokine family, is produced by various types of cells upon stimulation with inflammatory stimuli [7]. Therefore, we investigated the effect of BHSST on RANTES, TARC, MDC, and IL-8 levels in TI-stimulated HaCaT cells, and found that BHSST inhibited the production of RANTES, TARC, MDC, and IL-8 in the cells (Figure 7). These findings suggest that BHSST regulates the recruitment of Th2-type cells into inflammatory skin lesions by suppressing production of inflammatory chemokines.
IFN-activates an inflammatory transcriptional program through the canonical Janus-kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway [12]. TARC production from TI-stimulated HaCaT cells is decreased by treatment with JAK/STAT inhibitors [11]. To confirm the mechanism of action of BHSST, we examined the effect of BHSST on STAT1 activation by TI stimulation in HaCaT cells. BHSST suppressed STAT1 phosphorylation and nuclear translocalization in TI-stimulated HaCaT cells (Figure 8).
In summary, our results demonstrate that BHSST exerts anti-inflammatory effects by reducing the production of NO, PGE 2 , and IL-6, as well as TLR4 and the phosphorylation of NF-B p65 in LPS-stimulated RAW 264.7 cells. BHSST exerts suppressive effects on the production of inflammatory chemokines RANTES, TARC, MDC, and IL-8, and the phosphorylation of STAT1 in TI-stimulated HaCaT cells. Overall, these findings provide evidence that BHSST can act as a preventive and therapeutic agent for inflammationrelated skin diseases.