Gan-Pi-Tong-Zhi Decoction Ameliorates OVA-Induced Asthma Through MAPK and Nrf2 Signaling Pathways and Regulating Gut Microbiota

Introduction

Asthma is a highly prevalent chronic respiratory disease, affecting approximately 300 million individuals worldwide. Over the past two decades, both its incidence and associated mortality rates have steadily increased.^1,2 Upon exposure to allergens, air pollution, viral infections, cigarette smoke, excessive sensitivity of the airways leads to uncontrolled bronchoconstriction, resulting in reversible airflow obstruction, which can cause hypoxia and even death.³ Despite advances in conventional treatments, including short-acting β₂-agonists (SABA) for rapid relief and inhaled corticosteroids (ICS) for inflammation control, a significant number of patients continue to experience suboptimal symptom control and drug-related side effects.^4–6 Asthma symptoms and adverse events reduce treatment adherence, leading to a vicious cycle of poorer disease control, therapeutic failure, and higher costs.⁷ Therefore, developing effective and safe alternative therapies to alleviate symptoms of asthma has become an urgent priority.

Inflammatory responses and oxidative stress are intricately linked pathophysiological processes. Sustained local inflammation not only induces direct cellular and tissue damage but also weakens endogenous antioxidant capacity, further perpetuating a self-amplifying cycle. In asthma pathogenesis, crosstalk between oxidative stress and inflammatory pathways plays a critical role. As a typical proinflammatory pathway, the excessive activation of mitogen-activated protein kinase (MAPK) pathway has been proved to be closely related to respiratory diseases, such as asthma and COPD.⁸ Induced by IL-6 and IL-1β, MAPKs enhance the expression of RORγt, promoting the secretion of IL-17 and IL-10, which results in Treg/Th17 dysregulation, causing the initiation, persistence, and exacerbation of airflow limitation in asthma.⁹ Inflammatory cells generate reactive oxygen species (ROS), leading to lipid peroxidation and airway remodeling. Nrf2 plays a critical role in activating cytoprotective enzymes in response to oxidative insults. It is involved in the upregulation of numerous antioxidant genes (SOD, CAT, GSH) and is critical for modulating airway inflammation associated with exposure to particulate matter.¹⁰ Studies have shown that exposure to PM2.5 from biodiesel sources leads to increased inflammation, marked by elevated expression of TNF-α, ROS production, and MDA levels, resulting in lung tissue injury. This damage can be alleviated by activating the protective Nrf2 pathway.¹¹

The human gut harbors a complex and diverse microbiota. An increasing number of studies have revealed profound connections between alterations in the gut microbial community and the pathogenesis of asthma, establishing the microbiota as a key mediator in lung–gut tissue crosstalk during the progression of asthma.¹² The maintenance of a balanced gut microbiota may play a key role in simultaneously regulating the inflammatory and oxidative stress responses associated with asthma. Bacteroides, Bifidobacterium, and Faecalibacterium prausnitzii, ameliorate asthma by producing metabolites that enhance regulatory T cell activity, thereby suppressing airway inflammation, reducing eosinophil accumulation, and inhibiting abnormal mucus production.¹³ Notably, the gut microbiota and its derived metabolites, such as short-chain fatty acids (SCFAs), have been identified as upstream regulators that can concurrently modulate the MAPK signaling pathway to mediate inflammatory responses and activate the Nrf2/Keap1 axis to alleviate oxidative stress.^14,15

Traditional Chinese Medicine (TCM) offers therapeutic benefits for asthma by targeting multiple pathways and restoring systemic equilibrium. Gan-Pi-Tong-Zhi Decoction (GPTZ) was developed by the TCM expert Professor Li Xu based on years of clinical experience. It has shown favorable effects in alleviating clinical symptoms and reducing the frequency of acute asthma attacks. GPTZ is designed to harmonize the liver and spleen and facilitate the flow of lung qi. Previous studies have indicated that GPTZ can treat asthma by modulating the Th17/Treg balance, increasing Foxp3 mRNA expression in tissues, and beneficially regulating CD4⁺ T cell subsets, thereby ameliorating chronic airway inflammation.¹⁶ However, the mechanism by which GPTZ alleviates asthma by regulating oxidative stress and inflammation through the gut-lung axis is still unclear.

Asthma is now recognized as a heterogeneous syndrome comprising multiple phenotypes and distinct mechanistic endotypes.^17,18 Accordingly, the “intrinsic” phenotype is increasingly defined by non-IgE-mediated immunoreactivity, revealing a pathophysiology beyond classical allergic responses.¹⁹ This study utilized the ovalbumin (OVA)-sensitized rat model to establish such a specific endotype, and then elucidated how GPTZ ameliorates asthma by modulating inflammation, oxidative stress, and the gut microbiota. The findings provided theoretical support for GPTZ as a potential intervention method for preventing asthma.

Materials and Methods

Materials and Reagents

The eight TCMs contained in GPTZ were products of Anhui Puren Chinese Herbal Pieces Co., Ltd. (Anhui, China). The herbs were formally identified by the Affiliated Hospital of Liaoning University of TCM’s Pharmacy Department following standard protocols. Ovalbumin (OVA, A5503) was obtained from Sigma (California, USA). Dexamethasone (DEX, 141023) was provided by Tianjin Pharmaceutical Co., Ltd (Tianjin, China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6 (1.56 pg/mL, ml064292), IL-1β (15.63 pg/mL, ml037361), and TNF-α (15.63 pg/mL, ml002859) were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. Primary antibodies including β-actin (20536-1-AP), P38 (14064-1-AP), and p-P38 (28796-1-AP), HO-1 (10701-1-AP) were from Proteintech (Wuhan, China). Antibodies against Nrf2 (A21176), JNK1/2/3 (A4867), p-JNK (AP0631), Erk1/2 (A4782), and p-Erk1/2 (AP0234) were acquired from Abclonal (Wuhan, China).

Preparation of GPTZ

GPTZ is composed of eight herbal medicines, including 6g of Ephedra (Ma Huang), 10g of Fritillaria cirrhosa (Chuan Bei), 15g of Apricot Seed (Xing Ren), 15g of Pinellia (Ban Xia), 10g of Citrus Peel (Chen Pi), 15g of Gastrodia (Tian Ma), 30g of Poria (Fu Ling), and 20g of Licorice (Gan Cao). First, these herbal medicines were soaked in 500mL of distilled water at room temperature (approximately 25°C) for 30 minutes. The mixture was then heated vigorously until reaching the boiling point (100°C). After the heat reduced to a low level, the process was continued for another 30 minutes. Next, the solution was filtered through a two-layered gauze. The residue was mixed with an additional 500mL water, and the above steps were repeated. Subsequently, the two decoction solutions were mixed and concentrated to achieve concentration of 0.97 g/mL.

Animals

SPF grade male Sprague-Dawley (SD) rats, aged 6–8 weeks, were raised under standardized environmental conditions. SD rats were selected for its well-established utility in modeling allergic asthma and its suitability for the comprehensive biochemical and histological analyses required in this study.^20,21 To ensure stable experimental conditions, the room temperature was strictly regulated at 22 ± 2 °C. Before the experiments began, all the animals underwent a 7-day adaptation period which they were allowed to freely drink water and receive standard laboratory feed. All procedures were conducted in accordance with the ARRIVE guidelines for animal and were approved by the Animal Ethics Committee of the Liaoning Academy of TCM (Ethics Approval Code: NO.LZYY240405).

Analysis of Chemical Components in GPTZ Extract Based on UPLC-Q-TOF-MS

A 200μL aliquot of the GPTZ decoction was diluted and dissolved in 800 μL of methanol, followed by thorough mixing and collection of the supernatant. The chemical constituents of GPTZ were extracted and analyzed using QExactive high-resolution mass spectrometer (Thermo Fisher Scientific, China) equipped with an AQ-C18 column (150 × 2.1 mm, 1.8 μm; Welch). The column temperature was maintained at 35 °C. Data were acquired in two modes. For positive ion mode detection, the mobile phase consisted of 0.1% formic acid in water (eluent A) and acetonitrile (eluent B), with a linear gradient increasing from 5% to 100% B over 45 minutes. An identical gradient program was applied in negative ion mode, using water (A) and acetonitrile (B) as the mobile phase. Electrospray ionization (ESI) was employed with a spray voltage of 3.2 kV. Raw data acquired from the high-resolution LC–MS system were processed using Compound Discoverer 3.3 (Thermo Fisher Scientific) and queried against the mzCloud database for compound identification.

Network Pharmacological Analysis

The SMILES of GPTZ determined by UPLC-Q-TOF-MS was retrieved from PubChem. The Swiss Target Prediction database was exploited to predict the targets of the potential ingredients. Meanwhile, the related targets were retrieved from multiple databases, including OMIM, TTD, and GeneCards. Venny 2.1.0 was employed to identify the overlapping targets between the ingredients of GPTZ and asthma. These overlapping targets were further analyzed in STRING to construct a protein - protein interaction (PPI) network, and key targets were screened using Cytoscape 3.10.2. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of these overlapping targets using Metascape. Subsequently, Cytoscape 3.10.2 was used to construct an “active ingredient-target-disease” interaction network, and core components were identified on the basis of degree values.

Molecular Docking Analysis

The active components in GPTZ were combined with the key targets through molecular docking methods, and the binding energy was obtained.

Establishment of OVA-Induced Asthma Rat Model and Treatment

All animal experiments and drug protocols were based on our previous studies.¹⁶ 32 rats were randomly divided into four groups (n = 8 per group) using a random number generator. On days 0 and 7, the control group received intraperitoneal injections of 1 mL saline. Whereas the OVA (the model), OVA + DEX, and OVA + GPTZ groups were injected with 1 mL of an OVA/aluminum hydroxide mixture (1% OVA + 10% aluminum hydroxide in 1 mL saline). Starting from day 14, sensitized rats were placed in a nebulization chamber, then exposed them to 1% OVA aerosol for 30 minutes per day over 14 consecutive days to establish the asthma model. One hour prior to each OVA challenge, DEX (1.35 × 10⁻⁴ g/kg) was given to the OVA + DEX group, GPTZ (10.89 g/kg) was given to the OVA + GPTZ groups. The control and OVA groups were received same volume of saline. The dose administered to rats was determined using the body surface area conversion factor from humans to rats. Animal euthanasia was performed on day 28 by administering a lethal dose of 3% sodium pentobarbital to all animals. Lung tissues, colon tissues, BALF, serum, and fecal samples were collected for subsequent analysis.

Collection of Bronchoalveolar Lavage Fluid (BALF)

Following euthanasia, the rats were immobilized and surgically incised the chest cavity to expose the trachea. To avoid contamination, the left bronchus was isolated and ligated. The right lung was flushed with 2 mL saline, held for 30 seconds, before withdrawn to collect BALF, resulting in total BALF volume of approximately 3 mL per rat. The BALF was centrifuged at 3000 rpm for 15 min using a refrigerated centrifuge at 4°C, and store the supernatant for subsequent analysis at −80°C.

Measurement of Cytokine Levels

Levels of IL-6, IL-1β, and TNF-α in BALF were quantified using ELISA following the manufacturer’s instructions.

Behavioral Scoring of Asthma

Asthma symptoms in rats were observed and scored during the 30-minute period following nebulization initiation, based on an established evaluation method.²²

Histopathological Evaluation

To evaluate inflammatory responses and mucus production, lung and colon tissues were fixed with 4% paraformaldehyde for over 24 h, embedded in paraffin, and sectioned. Tissue sections were stained with hematoxylin and eosin (H&E) for assessment of general morphology and inflammation, stained with periodic acid–Schiff (PAS) for detection of mucin secretion. Histopathological changes were examined under microscope, and semiquantitative scoring of inflammation and PAS-positive areas was performed according to established criteria.²³

Immunohistochemical Analysis of the Lung

Tissue sections (4μm thickness) prepared from paraffin-embedded samples were deparaffinized, rehydrated, and heat-induced epitope retrieval in 0.01M citrate buffer. Sections were incubated overnight at 4 °C with a primary antibody, then washed with PBS, and incubated with secondary antibody. Color development was carried out with DAB substrate, and counterstaining was performed with hematoxylin. Stained sections were imaged and quantitatively evaluated with ImageJ software.

Quantitative PCR (qPCR)

The levels of mRNA extracted from lung tissue were quantitatively analyzed using qPCR. Briefly, 50 mg of lung tissue was homogenized with a 10-fold volume of Trizol reagent. After determining the concentration and purity of the RNA, single-stranded cDNA was synthesized according to the standard protocol. Subsequently, a 25 μL PCR reaction mixture was prepared and amplified using a CFX 96 Real-Time PCR Detection System. Relative quantification of mRNA was conducted using the 2^−ΔΔCT method, using β-actin expression as the reference. All primer sequences are listed in Table 1

Table 1 The Sequences of Primers

Western Blot Analysis

Total protein was extracted from lung tissue using RIPA buffer according to standard procedures. Protein concentration was quantified using a BCA protein assay kit. Under constant pressure of 90V, an equal amount of protein extract was separated by SDS-PAGE, and then the protein was transferred to a PVDF membrane at low temperature (400 mA, 1 kDa/min). After blocking treatment with 5% skim milk for 1 h at room temperature, the membrane was subsequently incubated overnight at 4 °C with primary antibodies, including JNK1/2/3 (1:2000 dilution), p-JNK (1:3000 dilution), Erk1/2 (1:4000 dilution), p-Erk1/2 (1:1000 dilution), P38 (1:6000 dilution), p-P38 (1:2000 dilution), Nrf2 (1:2000 dilution), HO-1 (1:20000 dilution). The next day, the membrane was incubated with fluorescence labeled secondary antibodies (1:1000 dilution). Protein levels were finally detected using enhanced chemiluminescence (ECL) and visualized with a chemiluminescence detection system. Quantification was performed using ImageJ software.

16S rRNA Genes Sequencing

DNA samples collected from the colonic contents of SD rats in different experimental groups. The hypervariable V3–V4 region of the bacterial 16S rRNA gene was amplified via PCR using the primers 338F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GACTACHVGGGTATCTAATCC-3′). The PCR products were purified, quantified, and mixed in equimolar amounts. Paired-end sequencing (2×250 bp) was subsequently performed on the Illumina NovaSeq 6000 platform, followed by bioinformatic analysis of the sequencing data.

Statistical Analysis

Data are presented as the mean ± standard deviation (mean ± SD). Statistical analyses were performed using GraphPad Prism 10.0. The assumption of homogeneity of variances was assessed using Levene’s test. Differences among groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. P-value < 0.05 was considered statistically significant.

Result

Chemical Components Contained in GPTZ

Following analysis by UPLC-Q-TOF-MS, the active constituents were observed to elute within the retention time range of 0 to 30 minutes. A total of 25 components in GPTZ were unequivocally identified, as illustrated in Figure 1 and Table 2.

Table 2 The Major Chemical Components in GPTZ

Figure 1 GPTZ in positive electrospray ionization (ESI+) and negative electrospray ionization (ESI−) mode.

Network Pharmacology Reveals the Potential Targets of GPTZ in the Treatment of Asthma Through the Gut-Lung Axis

A total of 25 active components were identified, which are capable of modulating 506 distinct biological targets. After duplicate removal, 2297 asthma-related targets and 1745 gut–lung axis-related targets with a relevance score ≥ 1.0 were screened from databases including OMIM, TTD, and GeneCards. Furthermore, it revealed that 150 core targets of GPTZ were shared between the treatment of asthma and the gut–lung axis (Figure 2A). An “active component–target–disease” network was constructed (Figure 2B). The PPI network indicated that core targets of GPTZ in treating asthma and the gut–lung axis include IL6, TNF, IL-1β, MAPKs, HMOX1 (HO-1), and NFE2L2 (Nrf2) (Figure 2C).

Figure 2 Network pharmacology analysis. (A) Venny diagram of targets shared by GPTZ, asthma and gut-lung axis. (B) Active component–target–disease network. (C) The PPI network of intersection targets. (D) GO function enrichment analysis. (E) KEGG enrichment analysis.

As illustrated in Figure 2D, GO enrichment analysis related to component–target interactions revealed several key findings. In the biological process (BP) category, enriched terms primarily included response to bacteria, inflammatory response and cellular response to cytokine stimulus. In the category of cellular component (CC), significant enrichment was observed in vesicle lumen, cytoplasmic vesicle lumen and secretory granule lumen. For molecular function (MF), protein homodimerization activity, receptor ligand activity, and signaling receptor activator activity were notably enriched. KEGG enrichment analysis identified approximately 194 significantly enriched pathways (p < 0.05) relevant to this study (Figure 2E). Major pathways included MAPK signaling pathway, TNF signaling pathway, and asthma. This study primarily focused on biological mechanisms such as inflammatory response and oxidative stress. The MAPK and the Nrf2 signaling pathways, which is closely associated with asthma, were constituted central focuses of the investigation.

Docking Scores Analysis

Molecular docking was used to verify the interactions between GPTZ components and key targets within the MAPK and Nrf2 signaling pathways. A binding energy lower than –5.0 kcal/mol is generally considered indicative of a strong ligand–receptor interaction, as a more favorable binding affinity corresponds to a lower binding energy. The results demonstrated that obacunone and limonin exhibited the strongest binding affinities to the core targets (Figure 3).

Figure 3 Result of molecular docking. (A) Docking analysis of bioactive compounds with key target proteins. (B) The binding energy of active ingredients to target proteins. Darker red indicates a lower binding energy. (Unit: kcal/mol).

GPTZ Alleviates Airway Inflammation in Asthmatic Rats

We first established an asthma model, as shown in Figure 4A. The behavioral scores of asthmatic rats were significantly increased but decreased after treatment with DEX and GPTZ (Figure 4B). ELISA results showed that the concentrations of IL-6, IL-1β, and TNF-α in the BALF of the OVA group were significantly elevated (p < 0.001, p < 0.001, p < 0.001). However, compared with the OVA group, the levels of IL-6, IL-1β, and TNF-α in OVA + GPTZ groups were significantly reduced (p < 0.001, p < 0.001, p=0.0015) (Figure 4C–E). Histological analysis via H&E and PAS staining revealed no significant pathological changes in the lungs of the control group, while the OVA group exhibited marked peribronchial inflammatory cell infiltration, degeneration and necrosis of bronchial epithelial cells, and thickening of the bronchial wall. Furthermore, PAS staining results indicated excessive mucus secretion in the bronchial lumen of OVA-treated mice (Figure 4F). Intervention with GPTZ significantly ameliorated the inflammatory cell infiltration, degeneration and necrosis of bronchial epithelial cells, and OVA-induced bronchial wall thickening, and markedly suppressed excessive mucus secretion, with effects comparable to DEX. The inflammation score and PAS score were significantly decreased in these two groups (Figure 4G and H). These results indicate that GPTZ significantly alleviates OVA-induced airway inflammation in asthmatic rats.

Figure 4 GPTZ alleviates airway inflammation and oxidative stress in asthmatic rats. (A) Construction of asthmatic rat model. (B) Behavioral score. (C–E) IL-6, IL-1β, TNF-α level in BALF. (F) H&E and PAS staining of lung tissue (scale bar: 200 μm). (G) The inflammation score. (H) PAS score. (I–L) MDA, CAT, GSH, SOD level in serum. Data are presented as mean ± SD. (n = 6). ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs OVA group.

GPTZ Alleviates Oxidative Stress in Asthmatic Rats

Compared with the OVA group, rats treated with GPTZ showed notably elevated levels of GSH (p=0.0125), SOD (p=0.0026), and CAT (p = 0.0201), along with a marked reduction in MDA (p=0.0089). These results indicate that GPTZ alleviates oxidative stress injury by enhancing antioxidant enzyme activity (Figure 4I–L).

GPTZ Effects the Histopathology of the Colon Tissue in Asthmatic Rats

H&E staining showed normal colon structure in the control group. The intestinal villi were neatly arranged, no edema or inflammatory cell infiltration. In the OVA group, inflammatory cells proliferated and infiltrated the colonic mucosa, congestion was also observed. However, no significant exudation or necrosis was found. The OVA+GPTZ group showed only mild inflammatory cell proliferation and infiltration. The abnormal colon tissue structure was improved. Mucous secretion by mast cells was observed via AB-PAS staining. Compared with the control group, the number of goblet cells decreased in the OVA group. In the OVA+GPTZ group, the number of mast cells increased significantly. And the mucus secretion volume showed the same trend (Figure 5).

Figure 5 GPTZ effects the histopathology of the colon tissue in asthmatic rats. (A) H&E staining of colon tissue (scale bar: 200 μm). (B) AB-PAS staining of colon tissue (scale bar: 200 μm).

GPTZ Regulates the Diversity of the Gut Microbiota

Given the growing body of evidence supporting the gut–lung axis theory, it was hypothesized that GPTZ might modulate the structure of the gut microbial community while treating asthma in rats. Compared with the control group, the Chao1, Shannon and ACE indices in the OVA group were significantly decreased (p=0.0063, p=0.0057, p=0.0062). This indicates a decrease in microbial diversity. After treatment with GPTZ, the Chao1, Shannon, and ACE indices increased (p=0.0159, p=0.0197, p=0.0159). The differences were statistically significant. These results suggest that GPTZ effectively enhances both the diversity and species richness of the gut microbiota (Figure 6A–D).

Figure 6 GPTZ regulates the changes in the gut microbiota of asthma rats. α-diversity analysis: (A) Chao1 index analysis, (B) Shannon index analysis, (C) Simpson index analysis and (D) ACE. β-diversity analysis: (E) PCoA and (F) NMDS. Data were shown as means ± SD. **p < 0.01 vs Control group; #p < 0.05, ##p < 0.01 vs OVA group.

GPTZ Improves the Composition of the Gut Microbiota

Principal coordinates analysis (PCoA) demonstrated a clear separation between the sample clusters of the OVA group and the control group, whereas samples from the OVA + DEX and OVA + GPTZ groups clustered more closely with the control group. These results indicate that GPTZ can ameliorate OVA-induced gut microbiota dysbiosis and restore the microbial composition to a state closer to that of the normal gut microbiota (Figure 6E and F).

The composition of species richness across groups was compared among different groups. At the phylum level (Figure 7A), Firmicutes, Bacteroidota, Thermodesulfobacteriota, Actinobacteria, and Proteobacteria were the most abundant taxa in each group. Compared with the control group, the OVA showed a decrease in the relative abundances of Bacteroidota and Thermodesulfobacteriota, while the abundances of Actinobacteria and Campylobacterota were increased. More specifically, compared to the OVA group, the proportions of Actinobacteria decreased, while Bacteroidota increased in the OVA + DEX and OVA + GPTZ groups. In addition, the Firmicutes/Bacteroidota (F/B) ratio was significantly reduced in OVA rats compared to the OVA + GPTZ group (Figure 7C). At the genus level (Figure 7B), compared with the control group, the OVA group exhibited decreased relative abundances of Muribaculaceae, Clostridia_UCG-014, Lactobacillus, and Litchfieldia, while the abundances of Ligilactobacillus, Lachnospiraceae_NK4A136_group, and Ruminococcus were increased. Compared with the OVA group, treatment with DEX and GPTZ increased the abundances of Lactobacillus, Clostridia_UCG-014, and Litchfieldia, and reduced the abundances of Muribaculaceae and Prevotella. DEX showed the most pronounced effect in increasing the abundance of Ligilactobacillus (Figure 7D–F).

Figure 7 GPTZ alters the relative abundance of the gut microbiota. Microbial community structure at the (A) phylum and (B) genus levels. (C) F/B ratio. (D-F) Representative differentially enriched species at the genus level: Ligilactobacillus, Lactobacillus, and Litchfieldia. Data are presented as mean ± SD. #p < 0.05, ##p < 0.01, ###p < 0.001 vs OVA group.

LEfSe Analysis

LEfSe was used to analyze biomarker species, and the results revealed a score plot of differentially abundant species; bars in different colors represent distinct groups, with their length indicating the effect size (Figure 8A). By incorporating these specifically enriched species into an LEfSe cladogram, Figure 8B was constructed. The results showed that the OVA + DEX group contained the greatest number of specific highly abundant species. At the genus level, the control group exhibited a higher relative abundance of differentially enriched species, including [Eubacterium]_coprostanoligenes_group, Intestinimonas, and Prevotellaceae_Ga6A1_group; Dubosiella, Allobaculum, and Alistipes in the OVA group; Ligilactobacillus, Lactobacillus, and Litchfieldia in the OVA + DEX group; and Romboutsia, NK4A214_group, [Eubacterium]_xylanophilum_group, and Candidatus_Saccharimonas in the OVA + GPTZ group. The LEfSe analysis identified biomarker signatures of the gut microbiota across groups, suggesting that GPTZ may alleviate asthma symptoms by modulating the gut microbial community.

Figure 8 LEfSe analysis of the gut microbiota. (A) Histogram of LDA scores. (B) Cladogram from LEfSe analysis.

GPTZ Inhibits the MAPK Signaling Pathway in Asthma

MAPK represents a key target in inflammatory processes and contributes significantly to asthmatic pathology. Network pharmacology analysis also suggested the potential influence of MAPK pathway in the treatment of asthma with GPTZ. The expression of p-Erk1/2, p-JNK, and p-P38 proteins was significantly elevated in the OVA group, indicating activation of the MAPK signaling pathway in asthma (p <0.001, p <0.001, p =0.0015). Compared with OVA group, treatment with GPTZ significantly suppressed the p-Erk1/2, p-JNK, and p-P38 (p<0.001, p<0.001, p=0.0130) (Figure 9E–H). On the other hand, treatment with DEX and GPTZ led to a significant reduction in the mRNA expression of Erk1, Erk2, JNK, and P38 (all p <0.05) (Figure 9A–D).

Figure 9 GPTZ modulated the MAPK signaling pathways. (A–D) Relative mRNA expression of Erk1, Erk2, P38, and JNK in lung tissue. (E–H) Protein expression of P38, p-P38, Erk, p- Erk, JNK, and p-JNK in lung. Data are expressed as mean ± SD (N = 3). *p < 0.001 vs Control group; #p < 0.05, ###p <0.001 vs OVA group.

To further validate these findings, immunohistochemical analysis showed that compared with the OVA group, GPTZ was found to significantly regulate the expression of p- Erk1/2, p-JNK and p-P38 proteins in lung tissues (p<0.001, p=0.0149, p<0.001) (Figure 10).

Figure 10 Immunohistochemical staining of MAPK pathway proteins in lung tissue. (A) Micrographs show immunohistochemical staining for p-P38, p- Erk, and p-JNK (scale bar: 200 μm). (B–D) Quantitative analysis of p-P38, p-Erk and p-JNK in lung tissues was scored by staining intensity. Data are expressed as mean ± SD (N = 5). ***p < 0.001 vs Control group; ##p < 0.01, ###p <0.001 vs OVA group.

GPTZ Promotes the Nrf2 Signaling Pathway in Asthma

Under oxidative stress conditions, Nrf2 enters the nucleus and mediates the transcriptional upregulation HO-1. The mRNA expression of Nrf2 and HO-1 in asthmatic rats was evaluated using qPCR, as shown in Figure 11A and B. The OVA group showed significantly decreased mRNA levels of Nrf2 and HO-1 (p < 0.001, p < 0.001), indicating that OVA treatment blocked the Nrf2 pathway. In contrast, treatment with DEX and GPTZ significantly upregulated their mRNA expression (Figure 11A and B). Furthermore, the protein levels of Nrf2 and HO-1 were assessed by Western blot and immunohistochemical analysis. Consistent with the qPCR results, the expression of Nrf2 and HO-1 was markedly suppressed in asthmatic rats, while GPTZ and DEX treatments activated the Nrf2 signaling pathway and increased HO-1 expression (Figures 11C–E and 12A-C).

Figure 11 GPTZ modulated the Nrf2 signaling pathways. (A and B) Relative mRNA expression of Nrf2 and HO-1 in lung tissue. (C–E) Protein expression of Nrf2 and HO-1 in lung tissue. Data are expressed as mean ± SD (N = 3). *p < 0.05, ***p < 0.001 vs Control group; ##p < 0.01, ###p <0.001 vs OVA group.

Figure 12 Immunohistochemical staining of Nrf2 pathway proteins in lung tissue. (A) Micrographs show immunohistochemical staining for Nrf2 and HO-1 (scale bar: 200 μm). (B and C) Quantitative analysis of Nrf2 and HO-1 in lung tissues was scored by staining intensity. Data are expressed as mean ± SD (N = 5). ##p < 0.01, ###p <0.001 vs OVA group.

Discussion

Asthma is a heterogeneous disease that can be subdivided into several categories on the basis of phenotypic and immunologic features, such as a higher proportion of eosinophils or neutrophils. The pathogenesis of asthma is complex and variable. Most patients have the allergic form of the disease, which is triggered by an adaptive immune response against inhaled antigens. Our study provides a systematic investigation into the mechanism of action of GPTZ, a TCM formulation for asthma. Consistent with the holistic principle of TCM, our findings demonstrate that GPTZ does not target a single molecule but exerts multi-tiered, integrated effects by remodeling the gut microbiota, and subsequently regulating the MAPK and Nrf2 signaling pathways. This orchestrated regulation across different biological tiers underscores the novelty of our findings.

This study utilized UPLC-QTOF-MS to preliminarily identify potential active components of GPTZ, revealing 25 compounds with potential bioactivity including obacunone, limonin, rhamnetin, and sanguinarine. Using a network pharmacology approach, this study predicted potential therapeutic targets of GPTZ for asthma treatment via the gut-lung axis and explored its pharmacological mechanisms. Through analysis of core targets, KEGG enrichment analysis, and GO enrichment analysis, pathways associated with relevant genes were identified, including the MAPK pathway, the Nrf2 pathway, and their roles in responding to bacterial molecules. Further molecular docking demonstrated that active components of GPTZ bind potently to critical nodes within the MAPK and Nrf2 signaling. These findings support the hypothesis that GPTZ exerts anti-asthmatic effects.

Our study demonstrated that OVA-induced asthmatic rats exhibited significant airway inflammation, with markedly elevated levels of IL-6, IL-1β, and TNF-α. GPTZ treatment markedly reduced the production of these pro-inflammatory cytokines (all p < 0.05). Meanwhile, histopathological analysis revealed that OVA challenge markedly elevated pathological scores relative to the control group, evidenced by obvious airway inflammatory cell infiltration, and enhanced mucus secretion. However, both DEX and GPTZ significantly reduced these scores and alleviated airway inflammation and mucus hypersecretion. During OVA inhalation challenge, treatment with DEX and GPTZ significantly reduced the behavioral scores of asthmatic rats. These results suggest that GPTZ can ameliorate airway inflammation in asthmatic rats, prompting further investigation into its underlying mechanisms.

The concept of the gut–lung axis describes the bidirectional communication network linking the digestive tract and the respiratory system. Accumulating evidence indicates active and multifaceted crosstalk between the gut–lung axis and its role in regulating immune responses.²⁴ The balance between commensal and pathogenic microorganisms, and the production of microbial metabolites collectively contribute to the regulation of lung health. Disruption of gut microbiota homeostasis can increase the risk of asthma attack and exacerbation.²⁵ Pulmonary diseases can also affect the gut microbiota through lymphocyte migration and inflammatory mediators, triggering intestinal disorders and disrupting intestinal barrier function and inducing structural remodeling of the mucosa.²⁶ Probiotics exert immune regulatory effects on the host through multiple pathways. Their administration reduces the infiltration of diseased mast cells and inhibits the Th2 response, effectively alleviating asthma.²⁷ This study confirmed this viewpoint by observing the effects of GPTZ on intestinal histopathology in asthmatic rats, suggesting that GPTZ may regulate asthma through the gut-lung axis. Therefore, maintaining a stable gut microbiota plays a critical role for both mitigating asthma risk and optimizing therapeutic efficacy. In this study, a multi-experimental approach was used to verify the effect of GPTZ on OVA-induced asthma and its effect on the gut microbiota.

This study revealed that after GPTZ intervention, both alpha and beta diversity were significantly improved. However, in the OVA+DEX group, the Shannon index decreased while the Simpson index increased. This shift in alpha-diversity suggests a potential influence of the elevated abundance of Lactobacillus observed in this treatment group. Compared with the OVA group, GPTZ treatment elevated the representation of Firmicutes and the abundance of Bacteroidota and Proteobacteria were decreased, resulting in a significantly higher F/B ratio (p < 0.05). The F/B ratio increases may promote energy acquisition by enhancing fermentation to produce SCFAs, whereas an increase in Bacteroidota can inhibit fermentation efficiency or compete for substrates, thereby reducing SCFA production.²⁸ The studies have indicated an elevated prevalence of Proteobacteria in asthma patients relative to healthy control subjects.²⁹ An elevated abundance of Proteobacteria was correlated with worsened asthma control and more frequent severe exacerbations, which was paralleled by elevated sputum neutrophil levels and the induction of Th17 associated gene expression.³⁰ This study indicates that at the genus level, in OVA-induced asthmatic rats, the inflammation-associated Prevotellaceae_Ga6A1_group was increased, while beneficial bacteria was decreased such as Lactobacillus and Lachnospiraceae_NK4A136_group. After GPTZ treatment, the abundance of Lactobacillus and NK4A214_group (Lachnospiraceae) were significantly increased, the abundance of Prevotellaceae_Ga6A1_group was reduced, suggested microbial dysbiosis was improved. Lactobacillus is a typical probiotic and a major producer of SCFAs in the gut. It generates various SCFAs, such as butyrate and propionate, by hydrolyzing maltose during dietary fiber fermentation.³¹ Propionate regulates dendritic cell (DC) progenitor populations within the bone marrow via a mechanism dependent on GPCRs, consequently suppressing the development of Th2 immune responses in the lung.³¹ Butyrate and propionate have HDAC inhibitory activity, enhancing the histone acetylation state in the Foxp 3 gene locus, and inducing tolerogenic DCs to enhance Treg production, thereby reducing airway hyperresponsiveness.^32,33 Correlation analyses revealed a significant association between Lactobacillus abundance and the therapeutic efficacy of GPTZ in asthma, suggesting a potential role for this genus in mediating treatment outcomes. Prevotella forms specific microbial networks with bacteria such as Veillonella and Haemophilus, which may promote asthma by reducing community diversity or enhancing pathogenic functions.³⁴ In addition, previous studies have demonstrated that Lachnospiraceae encodes a PLP-dependent enzyme that uses cystine as a substrate to produce RSS, such as CysSS. CysSSH can directly scavenge reactive oxygen and nitrogen species (RONS), inhibit oxidative damage, and increase MDA levels. RSS can form sulfur-modified adducts that interfere with the Keap1-Nrf2 interaction, promoting the nucleus translocation of Nrf2.^35,36 This provides evidence supporting the speculation that GPTZ may promote the proliferation of Lachnospiraceae and enhance the expression of the Nrf2 and HO-1 by RSS. These results support the accumulating evidence corroborating the importance of GPTZ in modulating the gut microbiota in asthma.

In order to illustrate the regulatory mechanism of GPTZ on asthma, we further investigated its influence on both the MAPK and Nrf2 signaling pathways. The MAPK family comprises three distinct stress-activated protein kinase pathways: P38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (Erk). After activation, MAPKs undergo phosphorylation, which subsequently moves to the nucleus and binds to specific transcription factors, thereby triggering the synthesis of proinflammatory cytokines and chemokines.³⁷ In immune cells, MAPKs drive activation responses and modulate the differentiation and survival of these cells after exposure to inflammatory stimuli or pathogenic agents.³⁸ Immunostaining of asthma patients showed that p-Erk1/2 was significantly expressed in airway epithelium and smooth muscle cells. p-JNK is limited to staining airway smooth muscle cells.³⁹ P38 is likely to drive the basal metabolic process of this specific cell type. A significant correlation between the clinical severity of asthma and the immunostaining intensity of p-Erk1/2 and p-P38, as well as between p- Erk1/2 and the numbers of eosinophils and neutrophils in airway tissues.³⁹ The prototypical dual-specificity phosphatase DUSP1 inactivates MAPKs through dephosphorylation and serves as an essential negative regulator of the MAPK pathway, playing an crucial role in suppressing inflammatory processes.⁴⁰ In peripheral blood mononuclear cells (PBMCs) of asthmatic children, the gene expression of DUSP1 is significantly lower than in healthy children, accompanied by reduced histone H4 acetylation in its promoter region, leading to dysregulated pro-inflammatory MAPK signaling.⁴¹ The present study demonstrates that the MAPK signaling pathway is activated in asthmatic rats. GPTZ treatment could inhibit the inflammatory response by reducing the levels of p-Erk, p-JNK and p-P38 MAPK (p <0.001; p <0.001; p < 0.05), and reducing the downstream release of IL-6, IL-1β and TNF-α (p < 0.001, p < 0.001, p=0.0015).

Accumulating evidence underscores the pivotal role of oxidative stress in asthma pathogenesis. The excessive ROS produced by various cells in the airways of asthma patients can exacerbate asthma attacks. The additional ROS accumulation creating a self-amplifying cycle, which further exacerbates tissue damage and dysfunction, ultimately leading to necrosis, apoptosis, inflammation, and fibrosis.⁴² Alveolar macrophages (AMs) serve as the primary effector cells responsible for generating reactive oxygen species (ROS) in lung tissue during acute asthma episodes. Furthermore, in individuals with allergic and mild asthma, AMs exhibit enhanced oxidative metabolic activity and produce greater levels of superoxide upon allergen challenge.⁴³ Disruption of redox homeostasis depletes endogenous antioxidants, including SOD and CAT, leading to lipid peroxidation, protein and DNA damage, and ultimately cellular oxidative injury and tissue damage.⁴⁴ MDA, which derived from the decomposition of peroxidized lipids, cyclooxygenase-mediated prostaglandin breakdown, or the metabolism of various amino acids and carbohydrates, is also widely recognized as a key biomarker of intracellular lipid peroxidation.⁴⁵ GSH is the major intracellular thiol antioxidant and acts as a direct scavenger of reactive oxygen species. Nrf2 serves as a central regulator of the cellular antioxidant response mechanism. Allergen challenge reduces catalase activity and downregulates the expression of the cytoprotective transcription factor Nrf2 in lung tissues.⁴⁶ Deficiency in Nrf2 increases vulnerability to tobacco smoke-induced lung injury, severe airway inflammation, and asthma. This deficiency is also correlated with diminished antioxidant gene expression, elevated type 2 cytokines, and impaired Treg cell differentiation.^47,48 Under physiological conditions, Nrf2 protein is bound to Keap1 and undergoes rapid degradation. During oxidative stress, Keap1 becomes oxidatively modified and loses its ability to target Nrf2 for degradation, allowing Nrf2 to translocate into the nucleus.⁴⁹ This process subsequently stimulates antioxidant genes expression, including HO-1, through the induction of genes containing antioxidant response elements (ARE). Subsequently, the HO-1 directly binds to the Neh4 transactivation domain of Nrf2. This interaction inhibits the β-TrCP-mediated ubiquitin–proteasome degradation pathway, significantly prolonging Nrf2 stability and nuclear retention, then enhancing its antioxidant function.⁵⁰ Consequently, Nrf2 is increasingly regarded as an attractive pharmacological target, and enhancing Nrf2 signaling offers a novel approach for alleviating asthma. GPTZ treatment alleviated OVA-induced oxidative stress and tissue damage by reversing key oxidative stress markers: it significantly elevated the levels of GSH (p < 0.05), SOD (p < 0.01), and CAT (p < 0.05), while reducing MDA (p < 0.01). This restorative effect was associated with potent activation of the Nrf2 signaling pathway, as demonstrated by both qPCR and Western blot analyses (all p < 0.01).

Moreover, studies have revealed a close relationship between the MAPK and Nrf2 pathways. ROS produced under hyperoxia can activate the Erk pathway and regulate Nrf2 expression. The combined effect of the two can activate the level of IL-17D. It concurrently promotes the expression and recruitment of IL-10, Zo-1, and occludin, which collectively contribute to the protection and reinforcement of the epithelial barrier.⁵¹ TGF-β1 mediates the EMT through activation of the MAPK and NRF2 signaling cascades. Consequently, reversal of MAPK and NRF2 pathways leads to a downregulation of EMT-associated proteins. This mechanism represents a critical therapeutic strategy for counteracting EGFR-TKI resistance and mitigating metastatic progression in NSCLC.⁵²

In summary, this study explored the potential mechanisms of GPTZ in asthma treatment. The results indicate that GPTZ reduces inflammatory responses and oxidative stress in asthmatic rats and regulates the MAPK and Nrf2 signaling pathways, thereby blocking further progression and exacerbation of the disease. The underlying mechanism for this effect may involve the capacity of GPTZ to modulate the gut-lung axis, thereby promoting microbial homeostasis. These findings may provide experimental support to the continued development of GPTZ as a promising candidate for treating asthma.

Limitations of the Present Study

Our study has several limitations, such as limitations related to individual differences, and we were only able to preliminarily confirm the associated anti-inflammatory and antioxidant mechanisms in vivo. Further causal validation using gene knockout models or specific inhibitors is required, along with more detailed investigation into the mechanisms of key pathways in cellular contexts. Future studies should also incorporate fecal microbiota transplantation (FMT) or antibiotic depletion models, integrated metagenomic sequencing, and metabolomic analyses to further explore the critical roles of metabolites in asthma, thereby elucidating the specific regulatory mechanisms between gut microbes and the gut–lung axis. This represents a key direction for our future research.

Conclusion

Our study identifies 25 compounds in GPTZ, with obacunone and limonin emerging as potential key contributors. Additionally, GPTZ can alleviate asthma symptoms by regulating the gut-lung axis, restoring microbial balance, and subsequently coordinating the inhibition of the MAPK signaling pathway and the activation of the Nrf2/HO-1 antioxidant axis. This multi-targeted action alleviates airway inflammation, mucus hypersecretion, and oxidative stress, thereby preserving lung tissue integrity. Overall, these findings lay a solid foundation for GPTZ as a promising therapeutic strategy, while clinical validation remains a critical objective for future research.