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Article

Evaluation of Yukmijihwang-Tang as a Novel Preventive Agent in Ovalbumin-Induced Murine Asthma

by
Jun-Yeop Song
1,
Eun-Ju Hong
1,
Eun-Bok Baek
1,
Poornima Kumbukgahadeniya
1,
Yu-Jin Kim
2,
Mee-Young Lee
2 and
Hyo-Jung Kwun
1,*
1
Department of Veterinary Pathology, College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea
2
KM Convergence Research Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 34054, Republic of Korea
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(13), 5815; https://doi.org/10.3390/app14135815
Submission received: 24 May 2024 / Revised: 2 July 2024 / Accepted: 2 July 2024 / Published: 3 July 2024
(This article belongs to the Special Issue Asthma and Respiratory Disease: Prediction, Diagnosis and Treatment, Volume II)
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Versions Notes

Abstract

:
Yukmijihwang-tang (YJT) is a blend of six herbal ingredients that has long been used in Asia for various diseases, including diabetes mellitus and mental decline. Here, we assessed the prophylactic efficacy of YJT in a murine model of asthma induced by ovalbumin (OVA). Male C57BL/6 mice underwent sensitization followed by an airway challenge with OVA for 1 h. YJT (100 or 300 mg/kg once daily) was administered orally for 6 days. Our analyses revealed that YJT significantly reduced inflammatory cell counts in bronchoalveolar lavage fluid (BALF), decreased the concentrations of interleukin (IL)-4, IL-5, and IL-13 in BALF, and lowered the relative mRNA levels of thymic stromal lymphopoietin (TSLP) and tumor necrosis factor α (TNFα) in lung tissues. Histological analyses showed that YJT substantially decreased inflammation and mucus hypersecretion in the bronchial airway. YJT also effectively reduced oxidative stress, as evidenced by improved levels of malondialdehyde (MDA), total glutathione, glutathione reductase (GR), and reduced glutathione (GSH). YJT also markedly suppressed the phosphorylation of nuclear factor-kappa B (NF-κB) in lung tissues. Together, these results show that YJT effectively inhibits respiratory inflammation, mucus hypersecretion, and oxidative stress induced by OVA, suggesting its potential for asthma treatment.

1. Introduction

Asthma is a prevalent chronic respiratory condition that affects millions worldwide and is a focus of extensive research and medical advancements. This complex disease is characterized by airway hypersensitivity to diverse triggers, leading to recurrent symptoms such as coughing, breathlessness, wheezing, and chest tightness [1]. Despite the variability in its presentation among individuals, asthma shares common underlying mechanisms [2]. The many features of asthma must be understood to enable the development of innovative treatment approaches targeting both symptomatic relief and the root causes of the condition. Pathophysiologically, asthma is acknowledged as an inflammatory disease characterized by an inflammatory cell proliferation that is driven by T-helper type 2 (Th2) cell-produced cytokines and results in the infiltration of these cells into the airways [3]. The inflammatory response also induces an excessive production of mucus, exacerbating airway obstruction and presenting considerable challenges to efficient airflow [4]. The combination of inflammatory cell infiltration and mucus hypersecretion creates a hostile environment within the airways, exacerbating respiratory symptoms and contributing to the characteristic features of asthma [4]. The pathological mechanism of asthma also involves oxidative stress due to a disproportion between reactive oxygen species (ROS) and antioxidants [5,6]. Lungs that have been exposed to environmental triggers are particularly vulnerable to oxidative stress-induced damage [6]. Oxidative stress is implicated in airway inflammation, bronchial hyperresponsiveness, and tissue remodeling; it not only exacerbates acute symptoms but may also contribute to chronicity [7]. Indeed, oxidative stress is considered a key determinant of asthma manifestation.
While asthma may benefit by avoiding triggers, such as allergens and respiratory irritants, the incorporation of long-acting beta agonists (LABAs) or anti-leukotriene agents alongside inhaled corticosteroids is advised if symptoms persist [8]. The current therapeutic approaches rely heavily on corticosteroids and generally aim to alleviate symptoms rather than directly addressing the underlying causes. Despite the emergence of diverse therapeutic options, the increasing prevalence of asthma and its associated mortality emphasize the lack of a fundamental treatment addressing the pathogenesis of this condition [9]. It is increasingly acknowledged that new treatments must be developed to address the mechanisms underlying asthma.
Yukmijihwang-tang (YJT; also called Liu wei di huang tang in China) is an herbal remedy that is widely utilized in Asia for nourishing the body and preventing/managing diverse diseases [10]. YJT is composed of the extracts of six herbs: Rehmannia radix, Dioscoreae radix, Corni fructus, Hoelen, Mountain cortex radices, and Alismatis radix [11]. Numerous investigations have explored the pharmacological impacts of YJT, including its effects on xerostomia [12], Alzheimer’s disease [13], and type 2 diabetes mellitus [14]. In vivo, YJT was shown to mitigate bone loss in a rat model of ovariectomy-induced osteoporosis [15]. In the context of benign prostatic hyperplasia, YJT significantly reduced both prostate weights and dihydrotestosterone levels in the serum and prostates of experimental rats [16]. However, no previous study has examined the effectiveness of YJT in treating asthma. Here, we evaluated the curative impact of YJT in a murine model of asthma caused by ovalbumin and explored possible fundamental mechanisms.

2. Materials and Methods

2.1. Plant Materials and Preparation of Sample Solution

A water extract of YJT was provided by the Korea Institute of Oriental Medicine (Daejeon, Republic of Korea). This YJT extract was dissolved in methanol at 20 mg/mL, 0.2 μm-filtered, and applied for ultra-performance liquid chromatography–diode array detector–tandem mass spectrometry (UPLC-DAD-MS/MS) analysis.

2.2. UPLC-DAD-MS/MS Analysis

The 13 standard compounds (purities > 95%) used to identify the phytochemicals in YJT were purchased from ChemFaces Biochemical Co., Ltd. (Wuhan, China). MS-grade solutions of water, acetonitrile, methanol, and formic acid were obtained from Thermo Fisher Scientific (Bremen, Germany). UPLC-DAD-MS/MS analysis of YJT was performed on a Dionex UltiMate 3000 system connected to a Thermo Q-Exactive mass spectrometer with an electrospray ionization (ESI) source (Thermo Fisher Scientific, Bremen, Germany). The UPLC and MS/MS conditions used in the analysis were as previously reported [17,18]. The MS spectra were acquired in full MS-ddMS2 mode, and all data were processed using the Thermo Xcalibur v.3.0 and Tracefinder v.3.2 software (Thermo Fisher Scientific, Bremen, Germany).

2.3. Cell Viability Assay

The cell viability assay was performed using the EZ-Cytox assay (DoGen Bio, Seoul, Republic of Korea). The human pulmonary epithelial cell line NCI-H292 at a concentration of 2 × 105/mL was seeded into a 96-well tissue culture plate. After 24 h, NCI-H292 cells were treated with YJT at various concentrations (500, 125, 31.25, 7.81 μg/mL) for 24 h. Subsequently, cells were incubated for 2 h in 10 μL of EZ-Cytox solution at 37 °C in a humidified 5% CO2 atmosphere. Before measuring absorbance, the plate was gently shaken for 1 min. Absorbance was then measured at 450 nm using a Tecan Infinite m200pro reader (Tecan Life Sciences, Männedorf, Switzerland). The percentage of cell viability was determined by comparing the results to the control group, which consisted of medium alone, and was set as the 100% viability baseline.

2.4. Animals

Six-week-old male C57BL/6 mice from a laboratory animal supplier were acclimated for 1 week with free access to solid food and water under conditions of 20 ± 2 °C with a relative humidity of 50 ± 10% and a 12 h light and dark cycle. All animal experiments were conducted in accordance with the guidelines established by the Institutional Animal Care and Use Committee of Chungnam National University.

2.5. Induction of Asthma

Ovalbumin (OVA, 20 μg) was dissolved in phosphate-buffered saline (PBS, 200 μL) and then combined with 2 mg of aluminum hydroxide (Alum). The resulting OVA/Alum (200 μL) was intraperitoneally administered on days 0 and 14 for systemic sensitization. Additionally, local sensitization was induced on days 21 and 23 by exposing mice to 1% OVA for 1 h using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan) to establish an asthmatic mouse model. The animals were assigned to five groups without a particular pattern (n = 7 per group): (i) negative control group treated with PBS (NC), (ii) OVA administration group (OVA), (iii) OVA administration and dexamethasone (DEX, 200 μg per mouse) treatment group (OVA+DEX), and (iv, v) OVA administration and YJT treatment (100, 300 mg/kg treatment, respectively) groups (OVA+YJT100, 300). YJT and DEX were dissolved in PBS and orally administered via gavage at 0.125 mL per animal using a 1 mL syringe fitted with a metal zonde from days 18 to 23 of the experiment. All mice were sacrificed on day 25.

2.6. Necropsy and Histopathologic Examination

Lung specimens were fixed in 10% buffered neutral formalin, sliced, paraffin-embedded, and sectioned at 5 μm using standard tissue processing techniques. Finally, hematoxylin and eosin (H&E) staining was performed. Inflammatory cell infiltration was assessed, and inflammation was scored as previously outlined [19]. Periodic acid–Schiff (PAS) staining was performed, tissue images were randomly selected, and the positive area (%) was quantified relative to the total bronchial epithelium area.

2.7. Microscopy of Inflammatory Cells in Bronchoalveolar Lavage Fluid (BALF)

BALF was obtained through a tracheally inserted cannula, with aspiration of 0.5 mL performed three times. Bronchoalveolar fluid (100 μL) was placed on a slide, and cells were fixed by centrifugation using a cytospin and stained with trypan blue. The total cell count, excluding dead cells, was calculated using a hemocytometer with measurements performed in triplicate. Inflammatory cells were specifically stained to quantify the populations of neutrophils, eosinophils, lymphocytes, and macrophages.

2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

The concentrations of IL-4, IL-5, and IL-13 in BALF and malondialdehyde (MDA), total glutathione, and glutathione reductase (GR) in lung tissues were quantified with commercial ELISA kits (IL-4, IL-5, IL-13: R&D Systems, MN, USA; MDA, total glutathione, GR: Cell Biolabs, CA, USA) following the manufacturer’s instructions. The absorbance was measured with a Tecan Infinite m200pro reader.

2.9. RNA Extraction and Real-Time PCR Analysis

The homogenization of lung tissues was followed by RNA extraction with TRIzol (Invitrogen, Carlsbad, CA, USA). Absorbance at 260 nm was measured to assess the RNA concentration, while its purity was evaluated by the 260 nm/280 nm ratio. Complementary DNA (cDNA) was synthesized utilizing a commercially available kit (Toyobo, Osaka, Japan). PCR amplification was conducted on a real-time PCR machine (A30299; Applied Biosystems, Foster City, CA, USA) using SYBR Green (Applied Biosystems, Foster City, CA, USA). The utilized PCR primer pairs are shown in Table S1. The obtained data were analyzed utilizing the software v1.5.3 (Applied Biosystems, Foster City, CA, USA). The fold change in the expression of the target genes relative to the control gene (GAPDH) was determined using the 2−ΔΔCt method as previously outlined [20].

2.10. Western Blot Analysis

Lung tissue specimens were homogenized using RIPA lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Equal amounts of lung proteins (30 μg) were quantified and resolved by 8% SDS-PAGE at 60 V. The resolved proteins were transferred to a polyvinylidene fluoride (PVDF) membrane at 250 V for 2 h. Membranes were incubated in a blocking solution composed of 1% Tween 20 in PBS (PBST) and 5% skim milk for 1 h and incubated overnight at 4 °C with primary antibodies, including anti-NF-κB, anti-phospho-NF-κB (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were applied for 2 h at room temperature, and the final expression was envisioned with a luminograph machine (ATTO, Tokyo, Japan).

2.11. Immunohistochemistry Assay

Immunohistochemistry (IHC) was conducted using anti-MUC5AC (Sigma-Aldrich, St. Louis, MO, USA) followed by a 2 h incubation with a biotinylated secondary antibody. Immune complexes were detected using diaminobenzidine (DAB; Vector Laboratories, Newark, CA, USA), and counter-staining was performed with hematoxylin. The positive areas were analyzed with Image J software (v.1.53k).

2.12. Statistical Analysis

The data are expressed as mean ± standard deviation (SD). Statistical analysis was conducted utilizing one-way analysis of variance (ANOVA) as applied with the GraphPad Prism 6 software (GraphPad, San Diego, CA, USA), followed by Tukey’s multiple-comparison test. p < 0.05 was considered statistically significant.

3. Results

3.1. Identification of Phytochemicals in YJT

In this study, our UPLC-DAD-MS/MS analysis identified 12 phytochemicals in YJT: geniposidic acid, loganic acid, morroniside, paeonolide, loganin, sweroside, paeoniflorin, isoquercitrin, hyperoside, isoacteoside, mudanpioside C, quercetin, and benzoylpaeoniflorin (Table 1). Their precursor ions and MS/MS fragments were compared with those reported in previous studies [21,22,23,24,25,26,27]. The UV (254 nm) pattern, base peak chromatogram of YJT in negative ion mode, and the extracted ion chromatograms for each phytochemical are shown in Figure 1.

3.2. Toxicity Analysis of YJT

The cytotoxic effect of various concentrations of YJT (7.81 to 500 μg/mL) was examined in NCI-H292 cells. As shown in Figure 2, YJT had no cytotoxicity at any of the concentrations tested.

3.3. Histopathologic Analysis in Lung Tissue

Histopathological YJT treatment-related pulmonary alterations were histopathologically evaluated through H&E staining. The OVA group exhibited a noticeable increase in inflammatory cells surrounding the bronchi and bronchioles compared to the NC group. Importantly, the infiltration of inflammatory cells was diminished in the tissues of both OVA+DEX and OVA+YJT animals (Figure 3A,B). However, there was no difference observed based on YJT concentration. These findings indicate that YJT effectively mitigated OVA-induced inflammation.

3.4. Analysis of Inflammatory Cells in BALF

We evaluated the influence of YJT on OVA-induced inflammatory responses by examining the counts of inflammatory cells in BALF. The OVA group displayed substantial increases in the numbers of total cells, lymphocytes, macrophages, neutrophils, and eosinophils in BALF compared to the NC group. In contrast, a notable decrease was observed in the OVA+DEX and OVA+YJT groups. The effect of YJT did not exhibit concentration dependence (Figure 4).

3.5. Analysis of Cytokine Production in BALF

We quantified Th2 cytokine levels in BALF through enzyme-linked immunosorbent assays (ELISAs) and investigated the relative mRNA levels of TSLP and TNFα using real-time PCR [28]. The OVA group exhibited elevated levels of IL-4, IL-5, and IL-13 in BALF compared to the NC group, and these levels were significantly reduced in the OVA+DEX and OVA+YJT groups (Figure 5A–C). The mRNA levels of TSLP and TNFα were also elevated in the OVA group but decreased in the OVA+DEX and OVA+YJT groups (Figure 5D,E). These effects of YJT did not appear to be concentration-dependent.

3.6. Analysis of Mucus Production in Lung Tissues

Comparisons of glycoproteins in bronchiolar epithelial cells via PAS staining revealed that the PAS-positive area was increased in the OVA group compared to the NC group, and this increase was notably reduced in the OVA+DEX and OVA+YJT groups (Figure 6A,C). When assessed by immunohistochemistry, the MUC5AC-positive area was found to be higher in the OVA group compared to the NC group, but this increase was markedly attenuated in the OVA+DEX and OVA+YJT groups (Figure 6B,D). Consistently, the relative mRNA expression level of MUC5AC in lung tissues was higher in the OVA group than in the NC group, and this enhancement was significantly decreased in the OVA+DEX and OVA+YJT groups (Figure 6E). Although these effects did not depend on the YJT concentration, our findings indicate that YJT effectively mitigated OVA-induced mucus secretion.

3.7. Analysis of Oxidative Stress in Lung Tissues

The MDA concentration was decreased in the OVA+YJT group compared to the OVA group (Figure 7A). Both total glutathione and GR were considerably increased in the OVA+DEX and OVA+YJT groups compared to the OVA group (Figure 7B,C). The mRNA levels of GR and GSH were substantially decreased in the OVA group compared to the NC group and markedly increased in the OVA+DEX and OVA+YJT groups compared to the OVA group (Figure 7D,E). The effects did not depend on the concentration of YJT. These results suggest that YJT treatment decreases oxidative stress.

3.8. Analysis of NF-κB Expression

Given that NF-κB phosphorylation is a critical trigger of inflammatory responses in the lungs [29,30], we assessed the potential effect of YJT treatment on phosphorylated NF-κB expression. Western blot analysis revealed that phosphorylated NF-κB was elevated in the OVA group compared to the NC group, and this enhancement was significantly reduced in the OVA+DEX and OVA+YJT groups (Figure 8).

4. Discussion

In this study, we explored the potential effects of YJT against an OVA-induced murine model of asthma. YJT exhibited inhibitory effects on inflammatory cell infiltration and cytokine production in BALF and lung tissues, significantly mitigated the mucus production and oxidative stress caused by OVA, and downregulated NF-κB phosphorylation in lung tissues.
Asthma, which is characterized by persistent airway inflammation, is a chronic respiratory disease. Its etiology involves the multifaceted interplay of diverse factors that generate intricate pathogenic mechanisms [31]. When introduced into the respiratory system, ovalbumin serves as a specific allergen, stimulating the bronchial epithelium to produce TSLP via the NF-κB signaling pathway [32]. TSLP elicits immune responses, including T-cell sensitization [33], and the T cells initiate a Th2-cell response characterized by GATA3-mediated NF-κB signaling [34]. This cascade prompts the inflammation-provoking secretion of various cytokines, including IL-4, IL-5, and IL-13, which are pivotal for the initiation of airway inflammation [35]. IL-4 triggers the upregulation of vascular cell adhesion molecule (VCAM)-1 on vascular endothelial cells to promote the migration of lymphocytes, monocytes, and eosinophils to inflammatory sites. It additionally enhances eosinophilic inflammation by amplifying chemotaxis and elevating eotaxin expression [35,36]. IL-5 also regulates the migration and survival of eosinophils. Upon activation by IL-5, eosinophils undergo degranulation and release cytokines with antimicrobial effects that induce damage to adjacent cells and tissues [37]. IL-13 critically influences IgE synthesis, leading to excessive mucus production and airway hyperresponsiveness [38]. Meanwhile, the inflammatory cytokine TNFα is secreted by antigen-activated M1 macrophages [39]. TNFα induces mast cells to produce histamine and serves as a chemoattractant for neutrophils and eosinophils, making it a crucial factor in inflammation [40]. Direct stimulation by IL-13 and TNFα increases the production of TSLP by epithelial cells [28,41]. This enhanced TSLP prompts Th2 cells to increase cytokine secretion, thereby exacerbating asthma symptoms [28,42]. Here, we show that the administration of YJT significantly decreased inflammatory cell infiltration into lung tissues, inflammatory cell counts in BALF, and the production of Th2 cytokines and other related cytokines in BALF. These findings strongly suggest that YJT can suppress the inflammatory response associated with asthma.
The Th2 inflammatory response contributes to increasing mucus secretion by upregulating glycoprotein, which is the main component of mucin [35]. Th2 interleukins also contribute to MUC5AC expression via NF-κB signaling within goblet cells [43]. IL-4 stimulates mucin production by goblet cells and airway epithelial cells [44], while IL-5 enhances eosinophil recruitment to further stimulate goblet cells [37]. IL-13 not only induces goblet cell hyperplasia but also impairs their ciliary functions [45]. Together, these cytokines create a situation that enhances mucus production, which exacerbates respiratory symptoms by causing airway obstruction, persistent coughing, and breathing difficulty. The excess mucus also traps irritants, leading to further inflammation and contributing to the severity of asthma [46]. Therefore, managing mucus production is a crucial step in alleviating symptoms and improving overall respiratory function in asthma. Here, we show that the administration of YJT markedly downregulated glycoprotein and MUC5AC in lung tissues and may, therefore, effectively attenuate asthma-associated mucus production.
In asthma, inflammatory cell infiltration and decreases in antioxidant defense lead to the excess generation of ROS, contributing to the onset of oxidative stress [5], which damages tissues and thereby fosters airway inflammation and hyperresponsiveness [5,47]. The principal antioxidant mechanisms responsible for regulating intracellular ROS involve glutathione [47]. Glutathione predominantly exists in a reduced form (GSH); it provides antioxidant protection by reducing oxidative species, such as ROS, and then undergoing oxidation to GSSG via the enzymatic activity of glutathione peroxidase [5,47]. Glutathione reductase (GR) employs NADPH to enzymatically convert GSSG back to GSH, thereby initiating a redox cycle. Accordingly, the GSH/GSSG ratio is critical for cellular homeostasis [48]. Under profound oxidative stress, the rate of GSH depletion may surpass that of GSSG generation, resulting in a decrease in the total glutathione level [48]. Under such circumstances, cells endeavor to sustain the GSH/GSSG ratio by increasing GSH synthesis or decreasing GSSG via the action of GR [49]. Here, we report that YJT treatment can reduce the oxidative stress marker MDA while upregulating total glutathione, GSH, and GR. This indicates that YJT effectively attenuates oxidative stress in the context of asthma.
The activation of the NF-κB pathway is very important for the pathophysiology of asthma [30,50,51], as this pathway orchestrates many fundamental reactions, including induction of the Th2 response, expression of MUC5AC in goblet cells, secretion of TNFα by macrophages, and release of TSLP by endothelial cells. This intricate involvement of NF-κB signaling underscores its significance as a central regulatory mechanism in the development of asthma. Given a previous report that YJT inhibited the receptor activator of NF-κB ligand-induced differentiation of osteoclasts [17], we assessed the phosphorylation of NF-κB in our system. Indeed, our results revealed that YJT significantly reduced the phosphorylation of NF-κB in the lung tissues of OVA-induced asthmatic mice.
Among the component herbal plants of the YJT, geniposidic acid has been shown to reduce inflammation [52], and loganic acid and morroniside are effective in mitigating both inflammation and oxidative stress [53,54]. These findings suggest that the therapeutic efficacy of YJT is likely due to the synergistic effects of its individual components. However, the other components of YJT, quercetin and hyperoside, have been reported to have neurotoxicity and nephrotoxicity, respectively [55,56]. Here, we showed that YJT had no cytotoxicity at concentrations of 0~500 μg/mL. This is consistent with previous studies showing that YJT has no cytotoxicity at any concentration (~200 μg/mL) in human urinary bladder cancer cell line T24 and bone marrow macrophages [15,56]. Furthermore, we previously reported that a 13-week repeated oral administration of aqueous YJT extract in SD rats indicated no toxicity, and the no-observed-adverse-effect level (NOAEL) was found to be 2000 mg/kg/day [57]. However, further research is necessary to elucidate the specific contributions and potential toxic side effects of each component of YJT in vivo.
Another limitation of our study is that although YJT significantly reduced the development of OVA-induced asthma in mice, there were no dose-dependent effects of YJT. Shin et al. showed that YJT (200 or 400 mg/kg) effectively inhibited testosterone-induced benign prostatic hyperplasia in rats [16]. Lee et al. also demonstrated that YJT (100, 200, or 500 mg/kg) suppresses the production of proinflammatory cytokines in the intravesical hydrochloric acid-induced cystitis rat model [56]. However, no dose-dependent effects of YJT were observed in these studies. Since phytochemicals can have both beneficial and adverse effects depending on the concentration, it is essential to administer an appropriate dose [58]. Consequently, our future objective is to identify the optimal concentration that maximizes the inhibitory effect on asthma while minimizing potential toxic effects.
In sum, our investigation revealed that YJT exerts a protective effect against asthma in an OVA-induced mouse model by mitigating inflammation, mucus production, and oxidative stress, at least partly by inhibiting the NF-κB pathway. Our results suggest that YJT could be a promising candidate for the treatment of asthma.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app14135815/s1, Table S1: Primer sequences.

Author Contributions

Conceptualization, H.-J.K.; Methodology, E.-J.H., P.K., Y.-J.K. and M.-Y.L.; Formal analysis, E.-B.B. and E.-J.H.; Investigation, J.-Y.S. and E.-B.B.; Writing—Original draft, J.-Y.S. and H.-J.K.; Writing—Review and editing, H.-J.K. and E.-B.B.; Supervision, H.-J.K.; Project administration, H.-J.K.; Funding acquisition, H.-J.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by grants from the Korea Institute of Oriental Medicine and Chungnam National University (grant numbers KSN1823221).

Institutional Review Board Statement

The animal study protocol was approved by the Animal Experimental Ethics Committee of Chungnam National University.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. UPLC-DAD-MS/MS analysis of YJT: the ultraviolet (UV) (254 nm) base peak chromatogram in negative ion mode and the extracted ion chromatogram (EIC) of the identified phytochemicals in YJT. In the EIC, the peaks are labeled as follows: 1: geniposidic acid, 2: loganic acid, 3: morroniside, 4: paeonolide, 5: loganin, 6: sweroside, 7: paeoniflorin, 8: isoquercitrin/hyperoside, 9: isoacteoside, 10: mudanpioside C, 11: quercetin, and 12: benzoylpaeoniflorin.
Figure 1. UPLC-DAD-MS/MS analysis of YJT: the ultraviolet (UV) (254 nm) base peak chromatogram in negative ion mode and the extracted ion chromatogram (EIC) of the identified phytochemicals in YJT. In the EIC, the peaks are labeled as follows: 1: geniposidic acid, 2: loganic acid, 3: morroniside, 4: paeonolide, 5: loganin, 6: sweroside, 7: paeoniflorin, 8: isoquercitrin/hyperoside, 9: isoacteoside, 10: mudanpioside C, 11: quercetin, and 12: benzoylpaeoniflorin.
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Figure 2. Cell viability of YJT-pretreated NCI-H292 cells. CON, control (medium alone); results are presented as means ± SD (ns, not significant).
Figure 2. Cell viability of YJT-pretreated NCI-H292 cells. CON, control (medium alone); results are presented as means ± SD (ns, not significant).
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Figure 3. Effect of YJT on inflammatory cell recruitment to the airway. (A) Representative images of H&E staining in lung tissues. Scale bar, 100 μm; magnification, 200×. (B) Assessment of inflammation score graded from 0 (absent) to 4 (severe). NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone-administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (## p < 0.01 compared with the NC group; ** p < 0.01 compared with the OVA group).
Figure 3. Effect of YJT on inflammatory cell recruitment to the airway. (A) Representative images of H&E staining in lung tissues. Scale bar, 100 μm; magnification, 200×. (B) Assessment of inflammation score graded from 0 (absent) to 4 (severe). NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone-administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (## p < 0.01 compared with the NC group; ** p < 0.01 compared with the OVA group).
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Figure 4. Effect of YJT on inflammatory cell counts in BALF. (A–E) Counts of total cells (A), lymphocytes (B), macrophages (C), neutrophils (D), and eosinophils (E). NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone-administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (## p < 0.01 compared with the NC group; * p < 0.05, ** p < 0.01 compared with the OVA group).
Figure 4. Effect of YJT on inflammatory cell counts in BALF. (A–E) Counts of total cells (A), lymphocytes (B), macrophages (C), neutrophils (D), and eosinophils (E). NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone-administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (## p < 0.01 compared with the NC group; * p < 0.05, ** p < 0.01 compared with the OVA group).
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Figure 5. Effect of YJT on pro-inflammatory cytokines in BALF and lung tissues. (A–C) Levels of IL-5 (A), IL-13 (B), and IL-4 (C) in BALF, as assessed by ELISA. (D,E) mRNA expression levels of TSLP (D) and TNFα (E) in lung tissues. NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone-administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (## p < 0.01 compared with the NC group; * p < 0.05, ** p < 0.01 compared with the OVA group).
Figure 5. Effect of YJT on pro-inflammatory cytokines in BALF and lung tissues. (A–C) Levels of IL-5 (A), IL-13 (B), and IL-4 (C) in BALF, as assessed by ELISA. (D,E) mRNA expression levels of TSLP (D) and TNFα (E) in lung tissues. NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone-administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (## p < 0.01 compared with the NC group; * p < 0.05, ** p < 0.01 compared with the OVA group).
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Figure 6. Effect of YJT on airway mucus production. (A) Representative images of PAS staining in lung tissues. Black arrow indicates a notable increase in the PAS-positive area. Scale bar, 100 μm; magnification, 200×. (B) Representative images of immunohistochemical staining of MUC5AC in lung tissues. White arrow indicates a notable increase in the MUC5AC-positive area. Scale bar, 100 μm; magnification, 200×. (C,D) PAS-positive area (C) and MUC5AC-positive area (D) were calculated based on the total area of airway epithelium. (E) mRNA expression levels of MUC5AC in lung tissues. NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone-administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (## p < 0.01 compared with the NC group; * p < 0.05, ** p < 0.01 compared with the OVA group).
Figure 6. Effect of YJT on airway mucus production. (A) Representative images of PAS staining in lung tissues. Black arrow indicates a notable increase in the PAS-positive area. Scale bar, 100 μm; magnification, 200×. (B) Representative images of immunohistochemical staining of MUC5AC in lung tissues. White arrow indicates a notable increase in the MUC5AC-positive area. Scale bar, 100 μm; magnification, 200×. (C,D) PAS-positive area (C) and MUC5AC-positive area (D) were calculated based on the total area of airway epithelium. (E) mRNA expression levels of MUC5AC in lung tissues. NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone-administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (## p < 0.01 compared with the NC group; * p < 0.05, ** p < 0.01 compared with the OVA group).
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Figure 7. Effect of YJT on oxidative stress in lung tissues. (A–C) MDA (A), total glutathione (B), and GR (C) levels in lung tissues as assessed using ELISA. (D,E) mRNA expression levels of GR (D) and GSH (E) in lung tissues. NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone-administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (# p < 0.05, ## p < 0.01 compared with the NC group; * p < 0.05, ** p < 0.01 compared with the OVA group).
Figure 7. Effect of YJT on oxidative stress in lung tissues. (A–C) MDA (A), total glutathione (B), and GR (C) levels in lung tissues as assessed using ELISA. (D,E) mRNA expression levels of GR (D) and GSH (E) in lung tissues. NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone-administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (# p < 0.05, ## p < 0.01 compared with the NC group; * p < 0.05, ** p < 0.01 compared with the OVA group).
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Figure 8. Effect of YJT on phospho-NF-κB activity in lung tissues. Phospho-NF-κB, NF-κB, and β-actin expression in lung tissues was assessed by Western blotting. NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (# p < 0.05 compared with the NC group; * p < 0.05, ** p < 0.01 compared with the OVA group).
Figure 8. Effect of YJT on phospho-NF-κB activity in lung tissues. Phospho-NF-κB, NF-κB, and β-actin expression in lung tissues was assessed by Western blotting. NC, negative control; OVA, ovalbumin-exposed mice; OVA+DEX, dexamethasone administered and ovalbumin-exposed mice; OVA+YJT100, YJT (100 mg/kg) and ovalbumin-exposed mice; OVA+YJT300, YJT (300 mg/kg) and ovalbumin-exposed mice. Results are presented as means ± SD (# p < 0.05 compared with the NC group; * p < 0.05, ** p < 0.01 compared with the OVA group).
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Table 1. Phytochemicals identified from YJT by UPLC-DAD-MS/MS.
Table 1. Phytochemicals identified from YJT by UPLC-DAD-MS/MS.
No.RT (min)Precursor Ion (m/z)Error (ppm)FormulaMS/MS Fragments (m/z)Identifications
CalculatedEstimatedAdduct
14.62373.1140373.1140M-H2.8408C16H22O10-Geniposidic acid [21]
24.89375.1297375.1295M-H2.5146C16H24O10375.1292, 213.0761, 169.0858, 151.0752Loganic acid [22]
35.12451.1457451.1457M+HCO22.3800C17H26O11243.0871, 179.0547, 155.0338, 141.0544, 123.0439, 59.0122Morroniside [22]
45.63505.1557505.1564M+HCO22.4165C20H28O12293.0883, 165.0546Paeonolide [23]
55.68435.1508435.1506M+HCO22.1421C17H26O10227.0917, 127.0388, 101.0230Loganin [22]
65.91403.1246403.1245M+HCO22.6154C16H22O9195.0651, 125.0229, 81.0330Sweroside [22]
76.37525.1614525.1610M+HCO21.4157C23H28O11449.1473, 327.1082, 121.0280Paeoniflorin [24]
86.78463.0882463.0882M-H2.3612C21H20O12-Isoquercitrin [25], Hyperoside [25]
97.38623.1981623.1981M-H1.6246C29H36O15623.1980, 461.1662, 161.0232Isoacteoside [26]
108.86599.1770599.1769M-H1.7083C30H32O13599.1754, 447.1300, 137.0231, 121.0280Mudanpioside C [23]
119.55301.0354301.0352M-H3.1166C15H10O7301.0350, 178.9976, 151.0022Quercetin [27]
1210.89629.1876629.1875M+HCO21.6180C30H32O12431.1339, 165.0545Benzoylpaeoniflorin [23]
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Song, J.-Y.; Hong, E.-J.; Baek, E.-B.; Kumbukgahadeniya, P.; Kim, Y.-J.; Lee, M.-Y.; Kwun, H.-J. Evaluation of Yukmijihwang-Tang as a Novel Preventive Agent in Ovalbumin-Induced Murine Asthma. Appl. Sci. 2024, 14, 5815. https://doi.org/10.3390/app14135815

AMA Style

Song J-Y, Hong E-J, Baek E-B, Kumbukgahadeniya P, Kim Y-J, Lee M-Y, Kwun H-J. Evaluation of Yukmijihwang-Tang as a Novel Preventive Agent in Ovalbumin-Induced Murine Asthma. Applied Sciences. 2024; 14(13):5815. https://doi.org/10.3390/app14135815

Chicago/Turabian Style

Song, Jun-Yeop, Eun-Ju Hong, Eun-Bok Baek, Poornima Kumbukgahadeniya, Yu-Jin Kim, Mee-Young Lee, and Hyo-Jung Kwun. 2024. "Evaluation of Yukmijihwang-Tang as a Novel Preventive Agent in Ovalbumin-Induced Murine Asthma" Applied Sciences 14, no. 13: 5815. https://doi.org/10.3390/app14135815

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