Tonggyu-tang, a traditional Korean medicine, suppresses pro-inflammatory cytokine production through inhibition of MAPK and NF-κB activation in human mast cells and keratinocytes

Background Allergic diseases including allergic rhinitis, asthma, and atopic dermatitis are increasing worldwide. Common medications used to treat these inflammatory disorders are anti-histamines and corticosteroids, but they have their own limitations such as short duration and severe side effects. Thus, interest in complementary and alternative medicine is continually growing. Here, we investigate the anti-inflammatory mechanisms of Tonggyu-tang (TGT), a traditional Korean medicine that has been used to treat patients with allergic nasal disorders. Methods We measured mRNA expressions and production of pro-inflammatory cytokines such as interleukin (IL)-4, IL-6, IL-8 and tumor necrosis factor alpha (TNF-α) by RT-PCR and ELISA assays in HMC-1 (human mast cell line-1) and HaCaT cells, immortalized human keratinocytes. Moreover, we evaluated the effect of TGT on two major inflammation-related pathways, mitogen activated protein kinase (MAPK) and NF-κB signaling pathway in these two cells. Results Our results revealed that that TGT significantly reduced the expression and production of inflammatory cytokines such as IL-4, IL-6, IL-8, and TNF-α in the agonist-treated HMC-1 and HaCaT cells. We also found that TGT suppressed MAPK signaling pathway including extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), and c-Jun N-terminal kinase (JNK) as well as NF-κB pathway, which are known to regulate inflammatory cytokine expression. Conclusion Taken together, our results demonstrate that TGT inhibits expression of pro-inflammatory cytokines by suppressing MAPK and NF-kB pathway in both mast cells and keratinocytes, suggesting the potential use of TGT in treating allergic inflammatory diseases. Electronic supplementary material The online version of this article (doi:10.1186/s12906-017-1704-5) contains supplementary material, which is available to authorized users.


Background
Allergic diseases are often caused by numerous inflammatory mediators such as histamine, chemokines, and cytokines from immune cells, which are affected by inflammatory cytokines from T cells and immunoglobulin E (IgE) from B cells [1,2]. When the allergens enter the human body, phagocytes such as dendritic cells and macrophages process and present them to T cells, resulting in differentiation of naïve T cells into cytotoxic T (Tc) cells or helper T (Th) cells. While activated Tc cells kill infected cells, Th cells, especially Th2 cells, release interleukin (IL)-4, IL-6 and IL-10 to induce B cells to produce IgE, which in turn activates mast cells for histamine secretion [3,4].
Current medications commonly used to relieve symptoms of allergic reactions are anti-histamines and corticosteroids, but they have their own limitations such as short duration and severe side effects [5,6]. Thus, the use of complementary and alternative medicine to treat allergic diseases is largely gaining an interest. In traditional Korean Medicine, several herbal medicines have been used for the treatment of allergic inflammation. Especially, Tonggyu-tang (TGT) composed of 12 herbs (Table 1), similarly Pyeongwee-San [7], Biyeom-Tang [8], Hyeonggaeyeongyo-Tang [9] and So-Cheon-Ryon-Tang [10] have shown anti-inflammatory effects on patients with allergic nasal disorder.
Although TGT has been commonly used for the treatment of allergic diseases, its underlying molecular mechanism of anti-inflammatory effect is unknown yet. Therefore, we hereby investigate the anti-inflammatory effect and the detailed molecular mechanism of TGT in HMC-1 (human mast cell line-1) and HaCaT cells which both participate in allergic disorders.

Statistical analysis
Results are expressed as the mean ± standard error (S.E.) of independent experiments, and statistical analyses were performed using ONEWAY ANOVA to determine differences between groups. All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Values with * p < 0.05 and # p < 0.05 are considered to indicate statistical significance.

Effect of TGT on expression and production of inflammatory cytokines in HMC-1 cells
In order to examine the anti-inflammatory effects of TGT, we used the human mast cell line-1, HMC-1 cells, one of the most representative cells for studying inflammatory response. First, to evaluate the effect of TGT on HMC-1 cell viability, an MTS assay was performed. As a result, TGT did not affect HMC-1 viability up to concentration of 1000 μg/ml (Additional file 1: Fig. S1a). Next, to evaluate the anti-inflammatory effect of TGT, HMC-1 cells were stimulated with both PMA and ionomycin (PI), followed by treatment with various concentrations of TGT (100, 200, 500, 1000 μg/ml). As in Fig. 1a and b, PI treatment of HMC-1 cells increased mRNA levels of IL-4, IL-6 and TNF-α. Adding of TGT significantly reduced the mRNA expression of these cytokines. Note that in the PI-treated HMC-1 cells, TGT did not cause significant cell cytotoxicity up to 1000 μg/ml as well (Additional file 1: Fig. S1b). Furthermore, we assessed the effect of TGT on cytokine production by ELISA. As shown in Fig. 2, TGT treatment significantly reduced production of IL-4, IL-6 and TNF-α in the PI-treated HMC-1 cells.

Effect of TGT on MAPK signaling pathway in HMC-1 cells
To understand the molecular mechanism underlying the inhibitory effects of TGT on cytokine expression, we examined the mitogen activated protein kinase (MAPK) signaling pathway that is known to be closely related to allergy diseases by regulating the expressions of inflammatory cytokines such as TNF-α and IL-6 [17,18]. We observed that TGT treatment of HMC-1 cells suppressed the PI-induced activation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), and c-Jun Nterminal kinase (JNK) in a dose-dependent manner (Fig. 3a). IκB, NF-κB are translocated into the nucleus, then initiate inflammatory-related gene transcription [19,20]. We found that increased phosphorylation levels of IKKα/β, IκBα/β, as well as STAT3 by PI treatment were inhibited by TGT co-treatment in a dose-dependent manner in HMC-1 cells (Fig. 3b).

Effect of TGT on inflammatory cytokines in HaCaT cells
Given that TGT suppresses the cytokine expression in HMC-1 cells, we next investigated the inhibitory effect of TGT on cytokine expression in HaCaT cells, an immortalized human keratinocytes. Of note, TGT did not show significant cytotoxicity in mock or LPS-treated HaCaT cells (Additional file 2: Fig. S2). Similar to HMC-1 cells, treatment of HaCaT cells with TGT markedly reduced the LPS-induced expression and production of inflammatory cytokines including IL-4, IL-6, IL-8 and TNF-α, as measured by RT-PCR and ELISA, respectively (Figs. 4 and 5).

Discussion
Allergic diseases such as allergic rhinitis, atopic dermatitis and asthma share a number of pathogenic and epidemiological features. Although detailed mechanisms of these diseases at the cellular level are still unclear, recent studies have focused on the roles of immune cells, especially Th2 cells in the pathogenesis of these allergic disorders [21]. TGT is an herbal medicine composed of 12 different herbs that are frequently used for treatment of patients with nasal disorder [11]. In this study, we added 4 additional anti-inflammatory herbs including Astragalus membranaceus, Xanthium strumarium, Magnolia denudate, and Mentha arvensis, to the original constituents to increase TGT's anti-allergic effects. Among these 16 herbs in the formulation of TGT, a number of studies  have showed that extracts or an active compound from individual herbs have anti-inflammatory activities (Table 1) [12][13][14][15][16][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]. Especially, six herbs including Ephedra sinica and Asarum sieboldii have been reported to have anti-histamine actions like inhibition of histamine release from mast cells or several histamine-mediated biological processes [37][38][39][40][41][42].
Since no studies have been performed to examine cellular and molecular actions of TGT, we sought to investigate the effects of TGT on human mast cells and keratinocytes. Mast cells play a key role in the development of allergic responses by releasing inflammatory mediators such as TNF-α, IL-6, IL-8, IL-13, and histamine [43,44]. IL-6 stimulates the growths of neutrophils and B cells, and IL-13 influence the apoptosis and survival of eosinophils, respectively [45][46][47].
In this study, we showed that TGT significantly reduced the expression and production of inflammatorycytokines in both HMC-1 and HaCaT cells. MAPK such as ERK, p38, and JNK are closely involved in the synthesis of inflammation mediators. Therefore, inhibitors targeting MAPKs are developed to reduce inflammation [48]. Our findings showed that TGT inhibits agonistinduced MAPK activation in a dose-dependent manner. Furthermore, we found that TGT treatment suppressed the agonist-induced activation of NF-κB pathway which is another important cellular signaling pathway for production of inflammatory cytokines. Overall, our results suggest that TGT can be effective treatment option for patients with inflammatory disorders including allergic rhinitis, atopic dermatitis through suppressing MAPK and NF-κB mediated production of inflammatory cytokines.

Conclusions
Our results clearly indicate that TGT has suppressing activity on expression and production of pro-inflammatory cytokines through inhibition of MAPK and NF-κB signaling pathways in mast cells and keratinocytes. Therefore, TGT can be used to reduce inflammatory symptoms in allergic disorders including not only allergic rhinitis but also atopic dermatitis and asthma.

Additional files
Additional file 1: Figure S1. Effect of TGT on the viability of PMA and Ionomycin (PI)-stimulated HMC-1 cells. Various concentration of TGT was added to PI treated HMC-1 cells. Cell viability was measured by MTS assay. *; compared to control. #; compared to stimulated cells.