Ma Huang Tang Suppresses the Production and Expression of Inflammatory Chemokines via Downregulating STAT1 Phosphorylation in HaCaT Keratinocytes

Ma huang tang (MHT) is a traditional herbal medicine comprising six medicinal herbs and is used to treat influenza-like illness. However, the effects of MHT on inflammatory skin diseases have not been verified scientifically. We investigated determining the inhibitory effects of MHT against inflammation responses in skin using HaCaT human keratinocyte cells. We found that MHT suppressed production of thymus and activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22), regulated on activation of normal T-cell expressed and secreted (RANTES/CCL5), and interleukin-8 (IL-8) in tumor necrosis factor-α (TNF-α) and interferon-γ- (IFN-γ-) stimulated HaCaT cells. Consistently, MHT suppressed the mRNA expression of TARC, MDC, RANTES, and IL-8 in TNF-α and IFN-γ-stimulated cells. Additionally, MHT inhibited TNF-α and IFN-γ-stimulated signal transducer and activator of transcription 1 (STAT1) phosphorylation in a dose-dependent manner and nuclear translocation in HaCaT cells. Our finding indicates that MHT inhibits production and expression of inflammatory chemokines in the stimulated keratinocytes by downregulating STAT1 phosphorylation, suggesting that MHT may be a possible therapeutic agent for inflammatory skin diseases.


Introduction
Atopic dermatitis (AD) is a frequently occurring inflammatory skin disease associated with severe itching and allergies to environmental factors [1]. AD is characterized by the predominant infiltration of macrophages, mast cells, eosinophils, and other inflammatory cells and increased secretion of Th2related response factors by the production of tumor necrosis factor-(TNF-) and interferon-(IFN-) [2].
Keratinocytes play a pivotal role in the development of inflammatory skin diseases including AD. The cells produced different chemokines and cytokines, especially thymus and activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22), regulated on activation of normal T-cell expressed and secreted (RANTES/CCL5), and interleukin-8 (IL-8) in response to stimulation by TNF-/IFN- [3,4]. These mediators are thought to be crucial regulators of the pathogenesis of AD. Additionally, the stimulation of keratinocytes by TNF-/IFN-leads to the activation of various signaling pathways that involve caspases, mitogen activated protein kinases, nuclear factor-kappa B, and signal transducer and activator of transcription 1 (STAT1) which subsequently increase the expression of inflammatory mediators [5].
Ma huang tang (MHT) comprises six herbal medicines and has traditionally been used in the treatment of sweating, asthma, and febrile disease, such as influenza-like illness (high fever, headache, and cough) [6,7]. Previous research reported that MHT has antipyretic effect in pediatric patients [8]. Other studies showed that MHT has antiasthmatic effects [9]. However, there has been no investigation focusing on the skin inflammatory effects of MHT. Therefore, we investigated 2 Evidence-Based Complementary and Alternative Medicine

Measurement of Chemokine Production.
HaCaT cells (1 × 10 6 cells/well) were cultured in 6-well plates. After reaching confluency, the cells were washed and treated with MHT in 1 mL of serum-free medium containing TNF-and IFN-  TARC  ACT GCT CCA GGG ATG CCA TCG TTT TT  ACA AGG GGA TGG GAT CTC CCT CAC TG  270  MDC  AGG ACA GAG CAT GGC TCG CCT ACA GA  TAA TGG CAG GGA GCT AGG GCT CCT GA  362  RANTES  CCC CGT GCC GAG ATC AAG GAG TAT TT  CGT CCA GCC TGG GGA AGG TTT TTG TA  313  IL-8  GTG GCT CTC TTG GCA GCC TTC CTG AT  TCT CCA CAA CCC TCT GCA CCC AGT TT  253  GAPDH  GTG ATG GCA TGG ACT GTG GT  AAG GGT CAT CAT CTC TGC CC  204 (TI, each 10 ng/mL; R&D Systems Inc., Minneapolis, MN, USA) for 24 h. The supernatant fractions were harvested, and production of TARC, MDC, RANTES, and IL-8 was determined using a sandwich immunoassay, performed according to the protocols provided by R&D Systems.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
Total RNA was isolated using TRIzol reagent according to the manufacturer's instructions (Invitrogen Life Technologies, Inc.). HaCaT cells (1 × 10 6 cells/well) were cultured to 80-90% confluency in 6-well plates. When the cells reached confluence, the cells were washed and treated with MHT in 1 mL serum-free medium (Gibco-BRL) supplemented with TI for 24 h. Silymarin (Sigma-Aldrich Inc., St. Louis, MO) was used as a positive control drug. Total RNA (1 g/mL) was then converted into cDNA using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.), containing oligo-dT primers. Diethylpyrocarbonate-treated water was added to a final volume 20 L followed by incubation at 42 ∘ C for 30 min using a Bio-Rad iCycler apparatus (Bio-Rad Laboratories, Inc.). For PCR amplification, gene-specific primers sequences are listed in the 5 to 3 orientation in Table 2. The PCR reaction mixture contained 1 L cDNA and 1.56 L Taq PCR master mix (EBT-1014; Elpis Biotech, Inc., Daejeon, Korea), which contained 1.5 mM MgCl 2 , 0.1 M of each forward and reverse primer and 7.44 L water in a final volume of 10 L. The thermocycling program comprised initial denaturation at 94 ∘ C for 5 min, followed by 25 cycles of denaturation at 94 ∘ C for 30 sec, annealing at 64 ∘ C for 1 min, extension at 72 ∘ C for 1 min 30 sec for all of the chemokines, and 25 cycles of denaturation at 94 ∘ C for 30 sec, annealing at 52 ∘ C for 1 min, and extension at 72 ∘ C for 1 min 30 sec for GAPDH. A final extension step was conducted at 72 ∘ C for 7 min. The amplified products were separated by 1.5% agarose gel and visualized using Loading STAR staining (A750; DYNE Bio, Seongnam, Korea). The relative expression levels of TARC, MDC, RANTES, and IL-8 mRNA were normalized to those of GAPDH mRNA using a Chemi-Doc Band Analysis system (Bio-Rad Laboratories, Inc.).

Western Blotting.
HaCaT cells were treated with various concentrations of MHT for 1 h and then incubated in the presence of TI for 30 min. The cells were collected by centrifugation, washed twice with PBS, and suspended in the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL) containing protease inhibitors. The protein concentration was determined using a protein assay reagent (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. Nuclear protein (30 g) was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. The membrane was incubated with blocking solution [5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST)], followed by an overnight incubation at 4 ∘ C with the appropriate primary antibody. The following primary antibodies and dilution were used: anti-STAT1 and antiphospho-STAT1 (1 : 1000 dilution; Abcam, Cambridge, UK). The membranes were washed three times with TBST and then incubated with a 1 : 3000 dilution of a horseradish peroxidase-(HRP-) conjugated secondary antibody (Jackson ImmunoResearch, PA) for 1 h at room temperature. The membranes were again washed three times with TBST and then developed using an enhanced chemiluminescence kit (Thermo scientific, Rockford, IL). Image capture was performed using Chemi-Doc XRS + system (Bio-Rad Laboratories, Inc.).

Immunofluorescence Staining.
HaCaT cells were seeded onto glass coverslips and incubated with TI in the absence or presence of MHT (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, Danvers, MA) for 1 h at room temperature. Then, fluorescein isothiocyanate-conjugated antirabbit immunoglobulin G antibody (Invitrogen, Carlsbad, CA, USA) was used as a secondary antibody. The immunostained cells were mounted with medium containing 4 6diamidino-2-phenylindole (DAPI) and visualized using an Olympus FLUOVIEW FV10i confocal microscope (Tokyo, Japan).

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. Results with a value < 0.05 were considered to be statistically significant.

HPLC Determination of the Nine Marker Components in MHT.
The optimized HPLC-PDA analytical method was successfully applied for the simultaneous determination of the 9 marker compounds in MHT extract. All compounds in MHT extract were identified based on the retention time and UV spectra of each reference standard. As a 4

Effects of MHT on the Cell Viability in HaCaT Keratinocytes.
To obtain a suitable concentration range for investigating the effects of MHT on the viability in HaCaT cells (Figure 2), we treated cells with the concentration ranging from 62.5 to 1000 g/mL of MHT for 24 h. We observed no significant alteration in the cell viability following MHT treatment up to 1000 g/mL. Therefore, nontoxic concentrations (125, 250, or 500 g/mL) of MHT were used in subsequent experiments. Additionally, MHT had no effect on cell death by pH level (Supplementary Figure 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/ 7831291).

Effects of MHT on the Inflammatory Chemokines in
HaCaT Keratinocytes. To assess the inhibitory effects of MHT, we cotreated HaCaT cells with TI in the absence or presence of MHT for 24 h, and the production of TARC,

Effects of MHT on TNF-and IFN--Activated STAT1
Phosphorylation in HaCaT Keratinocytes. We investigated the effect of MHT on phosphorylation of STAT1 in keratinocytes. HaCaT cells were pretreated with MHT for 1 h and then stimulated with TI for 30 min. MHT suppressed the TI-stimulated phosphorylation of STAT1 ( Figure 5(a)). We also confirmed the effect of MHT on nuclear translocation of STAT1 by immunofluorescence analysis. STAT1 protein at cytosol was translocated to nucleus after TI stimulation whereas it was inhibited by MHT treatment (Figure 5(b)).
These results indicate that MHT inhibits TI-stimulated STAT1 activation in HaCaT keratinocytes.
Chemokine products play important roles in the development of inflammatory cells in the skin. The secretion of these inflammatory chemokines is the first recruitment of inflammatory skin diseases and can be observed in immune cells, including lymphocytes, keratinocytes, and mast cells, which are activated by various stimuli [20]. Keratinocytes may promote an amplification on the inflammatory response with the production of TNF-and IFN-. Stimulated keratinocytes have been reported as important sources of proinflammatory chemokines, including TARC, MDC, RNATES, and IL-8, which affect T lymphocyte differentiation and the recruitment of leukocytes to skin inflammatory diseases such as AD [21,22]. Consistent with the results of previous studies, we found that stimulation with TI markedly increased the production and mRNA expression of TARC, MDC, RNATES, and IL-8 in HaCaT keratinocytes. By contrast, MHT treatment decreased both the production and mRNA expression of these chemokines compared with the TI-stimulated cells.
These results indicate that MHT may have the inhibitory activity against skin inflammation through regulation of chemokines expression in keratinocytes.
STAT1 is a crucial molecule for the IFN-/cytokine signaling pathways [22]. These activated pathways can modulate Evidence-Based Complementary and Alternative Medicine the secretion of inflammatory mediators including TARC, MDC, and RANTES. Inhibition of STAT1 phosphorylation is considered to be an important step in treating skin inflammatory diseases [23]. To confirm the action mechanisms of the effect of MHT on the STAT1 activated by TI-stimulated HaCaT keratinocytes, we performed immunoblotting and immunocytochemistry assays (using anti-STAT1 antibody). In our study, MHT inhibited TI-stimulated STAT1 phosphorylation and nuclear translocation in HaCaT cells. These results suggest that MHT might block the induction inflammatory chemokines production by inhibiting STAT1 phosphorylation in TI-stimulated keratinocytes.
In summary, our present study demonstrates that MHT inhibited the production and mRNA expression of inflammatory chemokines including TARC, MDC, RANTES, and IL-8 by suppressing the activation of STAT1 in TI-stimulated HaCaT keratinocytes. Further investigation is required to elucidate the detailed mechanisms involved in anti-skin