Anti-Inﬂammatory Effects of Jakyakgamcho-Tang in IL-4-and TNF- α -Stimulated Lung Epithelial Cells and Lipopolysaccharide-Stimulated Macrophages

: Asthma is a chronic respiratory disease mediated by airway inﬂammation. Jakyakgamcho-tang (JGT), a traditional medicine, is widely subscribed to common diseases such as muscle pain and cramps in East Asian countries. Although the efﬁcacy of JGT on peripheral neuropathy, gouty arthritis, and colitis has been reported, the effect of JGT on airway inﬂammation related to asthma is not clearly investigated. In this study, we aimed to evaluate the effects of JGT water extract (JGTW) on factors related to airway inﬂammation using the human bronchial epithelial BEAS-2B and the mouse monocyte-macrophage RAW264.7 cell lines. Furthermore, the constituents in JGTW were quantitatively and qualitatively studied for future reference of JGTW standardization. JGTW reduced the generation of several airway inﬂammation mediators such as eotaxins, regulated on activation normal T-cell expressed and secreted (RANTES), and matrix metalloproteinase-9, and expressions of adhesion molecules (ICAM-1 and VCAM-1), which attracts leukocytes to the site of inﬂammation in interleukin-4 + tumor necrosis factor- α (IT)-stimulated BEAS-2B cells. In lipopolysaccharide-stimulated RAW264.7 cells, JGTW effectively suppressed inducible nitric oxide synthase (iNOS) induction by inhibiting the MAPK and NF- κ B signaling. In addition, JGTW treatment showed decreased inﬂammatory cells and Th2 cytokines in bronchoalveolar lavage ﬂuid and decreased IgE levels in plasma in the OVA-induced asthmatic mice model. In the ultra-performance liquid chromatography-diode array detector-tandem mass spectrometry analysis, 24 phytochemicals were identiﬁed in JGTW, and paeoniﬂorin (63.971 mg/g) and glycyrrhizin (11.853 mg/g) were found to be the most abundant. These ﬁndings suggest that JGTW has anti-inﬂammatory effects on airway inﬂammation by regulating inﬂammatory response-related factors, possibly through MAPK and NF- κ B in pulmonary epithelial cells and macrophages.


UPLC and MS Conditions
To identify chemical constituents in JGTW, a Dionex UltiMate 3000 system Thermo Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used. UPLC-DAD-MS/MS analysis was conducted using a modified method proposed in a previous study [20]. The compounds were separated using an Acquity BEH C 18 column (100 mm × 2.1 mm, 1.7 µm, Waters, Milford, MA, USA) at 40 • C by gradient elution of the two mobile phases, 0.1% (v/v) aqueous formic acid (A) and acetonitrile (B) as follows: 3% B for 0-1 min, 3-10% B for 1-2 min, 10-30% B for 2-10 min, 30-40% B for 10-15 min, 40-100% B for 15-20 min, 100% B for 20-23 min, and 3% B for 24-28 min. The injection volume and flow rates were 3 µL and 0.25 mL/min, respectively. Mass spectra were acquired in both positive and negative electrospray ionization (ESI) modes. The MS/MS conditions are described in a previous study [21]. The mass spectra were recorded in the range of 100-1500 m/z, with a collision energy of 25 eV. Data-dependent acquisition using a full MS-ddMS 2 setup was used. The acquired data were processed using Xcalibur v.3.0 and TraceFinder v.3.2 software (Thermo Fisher Scientific, Waltham, MA, USA).

Cell Culture
BEAS-2B and RAW264.7 cell lines were purchased from American Type Culture Collection (Rockville, MD, USA) and maintained in suggested media containing 10% fetal bovine serum (FBS; Gibco, New York, NY, USA) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, New York, NY, USA) at 37 • C in a humidified incubator with 5% CO 2 .

Measurement of Chemokine Production, MMP Activity, and Adhesion Molecule Expression in BEAS-2B Cells
BEAS-2B cells (5 × 10 5 cells/well) were cultured in six-well plates overnight, then washed and incubated with of serum-free medium containing 50 ng/mL of IL-4 + TNF-α (IT; R&D Systems Inc., Minneapolis, MN, USA). After 48 h, culture supernatants were collected to measure the productions of eotaxin-3, eotaxin-1, and RANTES using and enzyme-linked immunosorbent assay (ELISA) and MMP-9 activity zymography as described previously [21]. The remaining cells were harvested to get the total RNA for measuring the expression levels of adhesion molecules (ICAM-1 and VCAM-1) and housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The detailed description of materials and experimental conditions for the RT-PCR have been described earlier [21].

Measurement of NO in LPS-Stimulated RAW264.7 Cells
Nitrite concentration was used as a measure of NO production in LPS-stimulated RAW264.7 cells. Cells were plated onto 96-swell plate at a density of 2 × 10 4 cells/well and pretreated with various concentrations of JGTW for 1 h and stimulated with 1 µg/mL LPS. After 24 h of incubation, the culture supernatants were collected for measuring nitrite concentrations using the Griess reagent (Promega, Madison, WI, USA), following the manufacturer's instructions.

Western Blot Analysis
RAW264.7 cells were pretreated with various concentrations of JGTW for 1 h, stimulated with 1 µg/mL LPS for 24 h, washed with cold PBS, and lysed in cold RIPA cell lysis buffer containing Halt protease and a phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). The total protein concentration was determined using a BCA assay kit (Thermo Scientific, Rockford, IL, USA), and 20 µg of total protein from each lysate was loaded and separated by SDS-PAGE and blotted onto PVDF membranes (ATTO Corporation, Tokyo, Japan). The membranes were blocked with EzBlockChemi (ATTO Corporation), probed with designated primary antibodies overnight, and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Bound antibodies were visualized with Supersignal West Femto Maximum sensitivity substrate (Thermo Scientific, Rockford, IL, USA).

OVA-Induced Asthmatic Mice
Specific pathogen-free (SPF) BALB/c female mice (six weeks of age) were purchased from Orientbio Inc. (Seongnam, Korea). Animals were maintained in environmentally controlled SPF conditions and were provided with a standard diet and water ad libitum. All experimental procedures were approved by the Animal Experimental Ethics Committee of Chungnam National University (Daejeon, Korea). Asthma is induced by ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO, USA). Mice were randomly divided into five groups (five animals per group): NC (normal control group), OVA (OVA-induction group), Dex (dexamethasone: OVA-induction + Dex at 1 mg/kg), JGTW-200 (OVA-induction + JGTW at 200 mg/kg), and JGTW-400 (OVA-induction + JGTW at 400 mg/kg). Dex (Sigma-Aldrich) was used as a positive control for relieving symptoms of allergic responses. BALB/c mice were sensitized on days 1, 8, and 15 via an intraperitoneal injection of OVA (50 µg) emulsified with 4 mg of Imject ® alum adjuvant (Thermo Fisher Scientific Inc., Waltham, MA, USA). Two weeks after the last sensitization, the mice were injected intranasally with OVA solution (1 mg/mL) on days 28, 31, 33. JGTW groups was administered by gavage to mice at doses of 200 or 400 mg/kg once daily from days 24 to 33. The NC, OVA, or Dex groups were administered distilled water or Dex orally, respectively. Two days after the last exposure, mice were sacrificed by intraperitoneal injection of pentobarbital, and tracheostomy was conducted to get bronchoalveolar lavage fluid (BALF). The total inflammatory cells in BALF were evaluated by counting in at least 4 squares of a hemocytometer (C-Chip, NanoEnTek, Seoul, Korea). Plasma obtained by cardiac puncture was used for subsequent analysis.

Measurement of IL-5, IL-13 and IgE Levels in BALF and Plasma
The levels of IL-5 and IL-13 (R&D Systems Inc.) in BALF; levels of total and OVAspecific immunoglobulin E (IgE; BioLegend, San Diego, CA, USA) in plasma were measured using ELISA kits following the manufacturer's instructions.

Statistical Analyses
The values are presented as mean ± standard error of the mean (SEM) or standard deviation (SD). Differences among means were analyzed using one-way analysis of variance (ANOVA) followed by a Dunnett's post-hoc test. p-values of <0.05 were considered to be statistically significant. ESI modes were operated to acquire the MS spectra. The characteristics of all identified compounds in JGTW are summarized in Table 1. UV and base peak chromatograms of JGTW are presented in Figure 1A, and the extracted ion chromatograms for each phytochemical are shown in Figure 1B. Most compounds were identified in negative ion mode. The precursor ions and MS/MS fragment patterns of the phytochemicals were identified by comparisons with the mass spectra and retention times of reference standards (Supplementary Materials Figure S1). The contents of the 20 phytochemicals, except for schaftoside, isoschaftoside, neoliquiritin, and liquiritin, ranged from 0.001 to 63.971 mg/g. In EICs of several constituents (e.g., catechin, luteolin, echinatin, glycyrrhizin) of JGTW, multiple peaks with the same m/z that have different retention times were observed, considering their isomers and derivatives. Further study is required for the structural confirmation of identified constituents isolated from JGTW in detail. considered to be statistically significant.

UPLC-DAD-MS/MS Analysis of JGTW
UPLC-DAD-MS/MS analysis was conducted to identify and quantify the phytochemicals in JGTW. The 24 compounds were separated on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm, Waters) at 40 °C within 22 min. The both (+)-ESI and (−)-ESI modes were operated to acquire the MS spectra. The characteristics of all identified compounds in JGTW are summarized in Table 1. UV and base peak chromatograms of JGTW are presented in Figure 1A, and the extracted ion chromatograms for each phytochemical are shown in Figure 1B. Most compounds were identified in negative ion mode. The precursor ions and MS/MS fragment patterns of the phytochemicals were identified by comparisons with the mass spectra and retention times of reference standards (Supplementary Materials Figure S1). The contents of the 20 phytochemicals, except for schaftoside, isoschaftoside, neoliquiritin, and liquiritin, ranged from 0.001 to 63.971 mg/g. In EICs of several constituents (e.g., catechin, luteolin, echinatin, glycyrrhizin) of JGTW, multiple peaks with the same m/z that have different retention times were observed, considering their isomers and derivatives. Further study is required for the structural confirmation of identified constituents isolated from JGTW in detail.

Cytotoxicity of JGTW in BEAS-2B and RAW264.7 Cells
Next, we tested to see if JGTW have any effect on the viability and proliferation of pulmonary structural and effector cells. The human bronchial epithelial BEAS-2B cell was treated with various concentrations of the JGTW for 24 h, then the proliferation and viability of these cells were measured. The JGTW did not show any significant cytotoxic effects even at the highest concentration ( Figure 2). RAW264.7 cells were also treated with various concentrations of JGTW with or without 1 µg/mL LPS for 48 h, then the viability of cells was measured. In accordance with the results of BEAS-2B, JGTW showed neither cytotoxic property nor cell proliferative activity to naïve RAW264.7 cells and LPSstimulated 264.7 cells up to 400 µg/mL (data not shown). treated with various concentrations of the JGTW for 24 h, then the proliferation and viability of these cells were measured. The JGTW did not show any significant cytotoxic effects even at the highest concentration ( Figure 2). RAW264.7 cells were also treated with various concentrations of JGTW with or without 1 μg/mL LPS for 48 h, then the viability of cells was measured. In accordance with the results of BEAS-2B, JGTW showed neither cytotoxic property nor cell proliferative activity to naïve RAW264.7 cells and LPSstimulated 264.7 cells up to 400 μg/mL (data not shown).

Effect of JGTW on CHEMOKINE Production
ELISA was used to measure the contents of key inflammatory mediators in the pulmonary epithelial cell culture supernatant. As shown in Figure 3A,B, the concentration of eotaxin-1, eotaxin-3, and RANTES in the supernatant of IT-stimulated BEAS-2B cells were significantly higher than that in vehicle-treated cells (p < 0.01). However, treatment with 125, 250, and 500 μg/mL of JGTW significantly reduced the concentration of these chemokines dose dependently (p < 0.01).

Effect of JGTW on CHEMOKINE Production
ELISA was used to measure the contents of key inflammatory mediators in the pulmonary epithelial cell culture supernatant. As shown in Figure 3A,B, the concentration of eotaxin-1, eotaxin-3, and RANTES in the supernatant of IT-stimulated BEAS-2B cells were significantly higher than that in vehicle-treated cells (p < 0.01). However, treatment with 125, 250, and 500 µg/mL of JGTW significantly reduced the concentration of these chemokines dose dependently (p < 0.01).
viability of these cells were measured. The JGTW did not show any significant cytotoxic effects even at the highest concentration ( Figure 2). RAW264.7 cells were also treated with various concentrations of JGTW with or without 1 μg/mL LPS for 48 h, then the viability of cells was measured. In accordance with the results of BEAS-2B, JGTW showed neither cytotoxic property nor cell proliferative activity to naïve RAW264.7 cells and LPSstimulated 264.7 cells up to 400 μg/mL (data not shown).

Effect of JGTW on CHEMOKINE Production
ELISA was used to measure the contents of key inflammatory mediators in the pulmonary epithelial cell culture supernatant. As shown in Figure 3A,B, the concentration of eotaxin-1, eotaxin-3, and RANTES in the supernatant of IT-stimulated BEAS-2B cells were significantly higher than that in vehicle-treated cells (p < 0.01). However, treatment with 125, 250, and 500 μg/mL of JGTW significantly reduced the concentration of these chemokines dose dependently (p < 0.01).

Effect of JGTW on MMP-9 Activity
BEAS-2B cells were pretreated with various concentrations of JGTW and then stimulated with IT. The activities of MMP-9 and MMP-2 in cells increased after treatment with increased concentrations of IT ( Figure 4A). Pretreatment with JGTW markedly reduced MMP-9 activity in a dose-proportional manner. Again, the ratio of MMP-9/MMP-2 was much higher in IT-stimulated cells as compared to that in vehicle-treated cells (p < 0.01) and JGTW pretreatment effectively decrease the ratio of MMP9/MMP-2 in IT-treated cells (p < 0.01, Figure 4B). stimulated with IT. The activities of MMP-9 and MMP-2 in cells increased after treatment with increased concentrations of IT ( Figure 4A). Pretreatment with JGTW markedly reduced MMP-9 activity in a dose-proportional manner. Again, the ratio of MMP-9/MMP-2 was much higher in IT-stimulated cells as compared to that in vehicle-treated cells (p < 0.01) and JGTW pretreatment effectively decrease the ratio of MMP9/MMP-2 in IT-treated cells (p < 0.01, Figure 4B).

Effect of JGTW on Adhesion Molecules Expression
After being stimulated with IT, the mRNA expression of ICAM-1 and VCAM-1 in BEAS-2B cells was markedly increased ( Figure 5A,B). JGTW significantly suppressed IT-stimulated ICAM-1 and VCAM-1 expression proportionally over the doses (p < 0.01, Figure 5C,D).

Effect of JGTW on the iNOS Expression and NO Production
To investigate the effect of JGTW on LPS-induced inflammatory responses, RAW264.7 cells were pretreated with various amounts of JGTW (6.25 to 100 µg/mL) for 1 h and stimulated with 1 µg/mL LPS for 24 h. Expression of iNOS was visualized using Western blot analysis. As shown in Figure 6A, iNOS expression was elevated with LPS stimulation, and pretreatment with JGTW suppressed iNOS overexpression in a dose-dependent manner. We also measured NO production from LPS-stimulated RAW264.7 cells with or without JGTW pretreatment. Furthermore, JGTW pretreatment had a suppressive effect on NO production induced by LPS showing statistically significant decrease at doses over 50 µg/mL ( Figure 6B). Appl. Sci. 2021, 11, x FOR PEER REVIEW 9 of 16

Effect of JGTW on the iNOS Expression and NO Production
To investigate the effect of JGTW on LPS-induced inflammatory responses, RAW264.7 cells were pretreated with various amounts of JGTW (6.25 to 100 μg/mL) for 1 h and stimulated with 1 μg/mL LPS for 24 h. Expression of iNOS was visualized using Western blot analysis. As shown in Figure 6A, iNOS expression was elevated with LPS stimulation, and pretreatment with JGTW suppressed iNOS overexpression in a dosedependent manner. We also measured NO production from LPS-stimulated RAW264.7 cells with or without JGTW pretreatment. Furthermore, JGTW pretreatment had a suppressive effect on NO production induced by LPS showing statistically significant decrease at doses over 50 μg/mL ( Figure 6B).

Effect of JGTW on the LPS-Induced MAPK Activation and NF-κB Signaling
To test whether this anti-inflammatory response of JGTW was mediated by the MAPK-responsive mechanism, the levels of p38 and ERK phosphorylation were

Effect of JGTW on the iNOS Expression and NO Production
To investigate the effect of JGTW on LPS-induced inflammatory responses, RAW264.7 cells were pretreated with various amounts of JGTW (6.25 to 100 μg/mL) for 1 h and stimulated with 1 μg/mL LPS for 24 h. Expression of iNOS was visualized using Western blot analysis. As shown in Figure 6A, iNOS expression was elevated with LPS stimulation, and pretreatment with JGTW suppressed iNOS overexpression in a dosedependent manner. We also measured NO production from LPS-stimulated RAW264.7 cells with or without JGTW pretreatment. Furthermore, JGTW pretreatment had a suppressive effect on NO production induced by LPS showing statistically significant decrease at doses over 50 μg/mL ( Figure 6B).

Effect of JGTW on the LPS-Induced MAPK Activation and NF-κB Signaling
To test whether this anti-inflammatory response of JGTW was mediated by the MAPK-responsive mechanism, the levels of p38 and ERK phosphorylation were

Effect of JGTW on the LPS-Induced MAPK Activation and NF-κB Signaling
To test whether this anti-inflammatory response of JGTW was mediated by the MAPKresponsive mechanism, the levels of p38 and ERK phosphorylation were evaluated in JGTW-treated and LPS-stimulated RAW264.7 cells. As presented in Figure 7A, RAW264.7 cells treated with LPS showed a significant increase in the phosphorylation of p38 and ERK, which was reduced by pretreatment of JGTW dose-dependently. We also examined the phosphorylation of JNK; however, the suppression of JNK phosphorylation by JGTW treatment was less significant compared to those of p38 and ERK (data not shown). by JGTW treatment was less significant compared to those of p38 and ERK (data not shown).
It is well known that iNOS expression is mediated by the transcription factor NF-κB. Consequently, the effect of JGTW on the phosphorylation of NF-κB subunit p65 and IκBα was investigated. LPS-stimulated RAW264.7 cells showed a marked increase in phosphorylated NF-κB subunit p65 and IκB-α; however, pretreatment with JGTW attenuated the increase of NFκB and IκB phosphorylation ( Figure 7B).

Effect of JGTW on Number of Inflammatory Cells
We next evaluate the effect of JGTW on the pulmonary inflammation and allergic response in the OVA-induced asthmatic mouse model. To investigate the effect of JGTW on recruitment of inflammatory cells, we counted the total cell numbers in BALF. As shown in Figure 8, the number of inflammatory cells in BALF of OVA-treated group significantly increased compared with NC group (p < 0.01). In contrast, mice treated with JGTW (200 and 400 mg/kg, p < 0.01) and Dex (p < 0.01) showed a significantly decreased number of inflammatory cells in BALF compared with OVA-treated mice (Figure 8).  It is well known that iNOS expression is mediated by the transcription factor NF-κB. Consequently, the effect of JGTW on the phosphorylation of NF-κB subunit p65 and IκB-α was investigated. LPS-stimulated RAW264.7 cells showed a marked increase in phosphorylated NF-κB subunit p65 and IκB-α; however, pretreatment with JGTW attenuated the increase of NFκB and IκB phosphorylation ( Figure 7B).

Effect of JGTW on Number of Inflammatory Cells
We next evaluate the effect of JGTW on the pulmonary inflammation and allergic response in the OVA-induced asthmatic mouse model. To investigate the effect of JGTW on recruitment of inflammatory cells, we counted the total cell numbers in BALF. As shown in Figure 8, the number of inflammatory cells in BALF of OVA-treated group significantly increased compared with NC group (p < 0.01). In contrast, mice treated with JGTW (200 and 400 mg/kg, p < 0.01) and Dex (p < 0.01) showed a significantly decreased number of inflammatory cells in BALF compared with OVA-treated mice (Figure 8). evaluated in JGTW-treated and LPS-stimulated RAW264.7 cells. As presented in Figure  7A, RAW264.7 cells treated with LPS showed a significant increase in the phosphorylation of p38 and ERK, which was reduced by pretreatment of JGTW dose-dependently. We also examined the phosphorylation of JNK; however, the suppression of JNK phosphorylation by JGTW treatment was less significant compared to those of p38 and ERK (data not shown).
It is well known that iNOS expression is mediated by the transcription factor NF-κB. Consequently, the effect of JGTW on the phosphorylation of NF-κB subunit p65 and IκBα was investigated. LPS-stimulated RAW264.7 cells showed a marked increase in phosphorylated NF-κB subunit p65 and IκB-α; however, pretreatment with JGTW attenuated the increase of NFκB and IκB phosphorylation ( Figure 7B).

Effect of JGTW on Number of Inflammatory Cells
We next evaluate the effect of JGTW on the pulmonary inflammation and allergic response in the OVA-induced asthmatic mouse model. To investigate the effect of JGTW on recruitment of inflammatory cells, we counted the total cell numbers in BALF. As shown in Figure 8, the number of inflammatory cells in BALF of OVA-treated group significantly increased compared with NC group (p < 0.01). In contrast, mice treated with JGTW (200 and 400 mg/kg, p < 0.01) and Dex (p < 0.01) showed a significantly decreased number of inflammatory cells in BALF compared with OVA-treated mice (Figure 8).

Effect of JGTW on Th2 Cytokines and IgE Levels
To examine the effect of JGTW on Th2-type immune response, we measured the levels of Th2 cytokines and total IgE, and OVA-specific IgE in mouse models of asthma induced by OVA. The level of IL-5 and IL-13 in BALF were greatly elevated in the OVA group compared with NC group (IL-5; p < 0.05, IL-13; not significant (ns)). However, JGTW (200 and 400 mg/kg, ns) and Dex (ns) groups showed a tendency to decrease the levels of IL-5 ( Figure 9A) and IL-13 ( Figure 9B). levels of Th2 cytokines and total IgE, and OVA-specific IgE in mouse models of asthma induced by OVA. The level of IL-5 and IL-13 in BALF were greatly elevated in the OVA group compared with NC group (IL-5; p < 0.05, IL-13; not significant (ns)). However, JGTW (200 and 400 mg/kg, ns) and Dex (ns) groups showed a tendency to decrease the levels of IL-5 ( Figure 9A) and IL-13 ( Figure 9B).
As shown in Figure 9C,D, the levels of total and OVA-specific IgE in plasma were significantly increased in the OVA group compared with NC group (p < 0.01). However, the JGTW (200 and 400 mg/kg, p < 0.01) and Dex (p < 0.01) groups significantly reduced the levels of total IgE. In addition, JGTW (200 mg/kg, p < 0.01) and Dex (p < 0.05) groups significantly inhibited the levels of OVA-specific IgE. Figure 9. Effect of JGTW on Th2 cytokines and IgE levels in OVA-induced mice. The levels of Th2 cytokines and IgE were determined by ELISA kits. Th2 cytokines: IL-5 (A) and IL-13 (B). IgE: total IgE (C) and OVA-specific IgE (D). The values are presented as mean ± SEM. # p < 0.05 or ## p < 0.01 versus NC group; and * p < 0.05 or ** p < 0.01 versus OVA-induced group.

Discussion
Asthma is one of the most common and complex chronic respiratory disorder with airway inflammation as a main cause. Various types of tissues and cells are involved in the pathophysiology of asthma. Airway epithelial cells are the first barrier in the lung as opposed to foreign antigens inducing inflammation and when it fails, sustained inflammation can worsen the symptoms of asthma and result in exacerbation. Chemokines from airway epithelial cells, such as eotaxins and RANTES, have been . The values are presented as mean ± SEM. # p < 0.05 or ## p < 0.01 versus NC group; and * p < 0.05 or ** p < 0.01 versus OVA-induced group.
As shown in Figure 9C,D, the levels of total and OVA-specific IgE in plasma were significantly increased in the OVA group compared with NC group (p < 0.01). However, the JGTW (200 and 400 mg/kg, p < 0.01) and Dex (p < 0.01) groups significantly reduced the levels of total IgE. In addition, JGTW (200 mg/kg, p < 0.01) and Dex (p < 0.05) groups significantly inhibited the levels of OVA-specific IgE.

Discussion
Asthma is one of the most common and complex chronic respiratory disorder with airway inflammation as a main cause. Various types of tissues and cells are involved in the pathophysiology of asthma. Airway epithelial cells are the first barrier in the lung as opposed to foreign antigens inducing inflammation and when it fails, sustained inflammation can worsen the symptoms of asthma and result in exacerbation. Chemokines from airway epithelial cells, such as eotaxins and RANTES, have been described to contribute to chronic airway inflammation by recruiting macrophages, eosinophils, and Th cells at the site of inflammation [24][25][26][27][28][29].
In our study, JGTW demonstrated potent inhibitory effects on the generation of inflammatory chemokines in IT-stimulated BEAS-2B cells, a widely used in vitro model of airway epithelial cells. In addition, the JGTW treatment effectively suppressed MMP-9 activity, an enzyme that degrades extracellular matrix proteins, and infiltrating inflammatory effector cells [30,31], in IT-stimulated BEAS-2B cells. It is a well-known fact that the increased expression of ICAM-1 and VCAM-1 on the respiratory epithelial cells is an important foundation of inflammatory cell infiltration into the inflamed airways [32], resulting in the development of asthma. JGTW treatment significantly suppressed the increased expression of these mediators in BEAS-2B cells stimulated with IT. Collectively, these findings demonstrated that JGTW had anti-inflammatory function acting through downregulation of pro-inflammatory soluble mediator expression, decreasing MMP-9 activity, resulting in lowered expression of adhesion molecules in a pulmonary epithelial cell model.
It is well known that eosinophils and mast cells are key players in the pathophysiology of asthma; however, evidence for the importance of macrophage-mediated inflammation in asthma is emerging [8,9,33]. High levels of macrophages are found in lung tissues in both asthma patients and mice with experimental asthma [33,34]. In our study, LPSstimulated RAW264.7 cells are used to mimic the inflammatory responses of macrophages. LPS treatment markedly increased iNOS expression, a widely used marker for inflammation, in RAW264.7 cells and pretreatment with JGTW for 1 h efficiently blocked this elevated expression of iNOS. In line with this result, JGTW also suppressed nitrite production in LPS-stimulate RAW264.7 cells. It is also reported that JGTW significantly decreased prostaglandin E2 production via suppressing COX-2, IL-1β, and IL-6, which are known mediators of airway inflammation in asthma [13]. Collectively, our data and others suggested that JGTW alleviates the inflammatory response by reducing various mediators such as iNOS, COX2, and cytokine expression.
It is known that the MAPK and NF-κB signaling pathways are an important part in mediating the pathophysiology of asthma, and these signaling pathways are also associated with the activation of effector cells during inflammation [35,36]. Hence, the inhibitory effect of JGWT on aberrant MAPK activation in LPS-stimulated RAW264.7 cells was assessed. JGTW efficiently suppressed the phosphorylation of p38 and ERK. Furthermore, JGTW exerted an inhibitory effect on the activation of NF-κB signaling, showing decreased expression of phosphorylated-NF-κB p65 subunit and IκBα. These results suggest that the inhibition of iNOS expression and anti-inflammatory properties by JGTW is mediated via suppression of NF-κB activation at least partly through p38 and ERK suppression. In our data, suppression of ERK is more effective in lower doses of JGTW treatment, which is occasionally observed result that the lower dose of herbal extract is more effective in regulating signaling pathway. This might be due to the combinational effect of various compounds of JGTW; hence, further studies regarding the active compound responsible for inhibiting MAPK pathway should be done.
OVA-sensitized and challenged mice represented typical asthmatic features such as increased inflammatory cell recruitment and elevated IgE level. In the present study, JGTW treatment after OVA sensitization effectively reduced the number of inflammatory cells in BALF, suggesting the effectiveness of anti-inflammatory response by JGTW shown in vitro on relieving symptoms of asthma in animal model. Additionally, the levels of IL-5 and IL-13 in BALF, a hallmark of Th2 response in asthma, which were elevated in response to the OVA treatment, showed a tendency to decrease by JGTW treatment. Furthermore, plasma levels of IgE, both total and OVA-specific, in JGTW treated mice were significantly lower than those of OVA-induced mice. Collectively, these results suggest that JGTW suppresses the inflammatory and allergic responses associated with asthma.
The individual herbal compounds that constitute JGT have been reported to be effective against airway inflammation-related diseases, such as asthma. Paeoniflorin, a major component of P. lactiflora, exerts anti-asthmatic effects by modulating the Th1/Th2 equilibrium [37] and inhibiting the growth and migration of airway smooth muscle cells [38]. Total glucosides of P. lactiflora improve ovalbumin-induced allergic asthma in mice by suppressing mast cell degranulation [39]. Glycyrrhizin, a major component of G. uralensis, alleviates asthmatic symptoms in mice [40] and ameliorates the chronic histopathological changes in the lungs of mice suffering from asthma [41] and asthmatic progression through modulation of Th1/Th2 cytokines and enhancement of CD4 + CD25 + Foxp3 + Tregs [42]. In addition, components of G. uralensis, isoliquiritigenin and licochalcone A, relax guinea pig tracheal smooth muscle in vitro and in vivo [43] and alleviate oxidative stress and hyper-responsiveness in asthmatic mice [44], respectively. These previous studies and our quantitative analysis data for the phytochemicals in JGTW suggest that the anti-inflammatory activity of JGTW in IT-stimulated BEAS-2B and LPS-stimulated RAW264.7 cells may be attributed to paeoniflorin and glycyrrhizin.
In summary, our results discovered that JGTW might exert anti-inflammatory effects by suppressing the airway inflammation-related factors in pulmonary epithelial cells and macrophages. JGTW also demonstrates potency in reducing inflammatory cell recruitment and allergic response in the OVA-induced asthmatic mouse model, suggesting the clinical effectiveness of JGT on relieving symptoms of asthma. In addition, paeoniflorin and glycyrrhizin were abundantly present in JGTW, which might contribute to its antiinflammatory effects. Follow-up studies on identifying the bioactive compounds that inhibit airway inflammation would be necessary to develop a new therapeutic option for asthma utilizing traditionally used herbal medicine.

Data Availability Statement:
The data presented in this study are available within the article.