Kechuanting Acupoint Sticking Therapy Alleviates Type 2 Airway Inflammation in Mice with Allergic Asthma by Modulating the Number of ILC2s

Summary

This protocol describes establishing an ovalbumen (OVA)-induced allergic asthma (AA) mouse model to evaluate the therapeutic mechanisms of Kechuanting Acupoint Sticking Therapy (KAST) on type 2 airway inflammation by modulating pulmonary Group 2 innate lymphoid cells (ILC2s) via the calcitonin gene-related peptide (CGRP)/receptor activity-modifying protein 1 (RAMP1) pathway.

Abstract

Kechuanting Acupoint Sticking Therapy (KAST) has been shown to effectively improve airway inflammation in patients with allergic asthma (AA); however, its underlying mechanisms remain unclear. This study aimed to investigate the therapeutic mechanisms of KAST in an AA mouse model from a neuro-immune perspective. An ovalbumen (OVA)-induced allergic asthma mouse model was established, and the mice were treated with KAST. The effects of KAST on pulmonary Group 2 innate lymphoid cells (ILC2s) and type 2 airway inflammation were evaluated using flow cytometry, hematoxylin-eosin (HE) staining, and immunohistochemistry. Western blotting (WB) and ELISA were used to assess calcitonin gene-related peptide (CGRP) protein levels in lung tissues. Intranasal administration of CGRP or the CGRP receptor inhibitor BIBN-4096 was performed in mice with allergic asthma to investigate the regulatory role of the CGRP/receptor activity-modifying protein 1 (RAMP1) pathway on ILC2s and type 2 airway inflammation. KAST reduced the number of ILC2s and alleviated type 2 airway inflammation, accompanied by decreased CGRP expression in lung tissues. Intranasal CGRP increased RAMP1⁺ILC2s and total ILC2s in the lungs and exacerbated type 2 airway inflammation. Conversely, intranasal BIBN-4096 decreased pulmonary ILC2s and mitigated type 2 airway inflammation. KAST reduces airway inflammation in allergic asthma, which is closely associated with downregulation of the CGRP/RAMP1 pathway and subsequent suppression of pulmonary ILC2s, as supported by pharmacologic inhibition.

Introduction

Allergic asthma (AA) is the most common asthma phenotype of asthma, accounting for 60–80% of all asthma cases in China¹. It is characterized by a high prevalence and suboptimal disease control that severely impairs patients’ quality of life and imposes significant economic burdens^2,3,4,5. Although inhaled corticosteroids (ICS) remain the mainstay of clinical treatment⁶, some patients show limited responsiveness, and long-term usage is often associated with adverse effects and an inability to halt disease progression⁷. Thus, the development of safe and effective novel therapeutic strategies remains a priority in both clinical and research settings. Kechuanting Acupoint Sticking Therapy (KAST), a traditional Chinese medicine external application, delivers active compounds transdermally, which potentially avoids the hepatic first-pass effect and minimizes systemic adverse reactions^8,9. Clinical studies have demonstrated that KAST can relieve asthma symptoms, restore partial pulmonary function, and enhance patients’ quality of life^10,11,12. Concurrently, serum metabolomics, network pharmacology, and animal studies using asthma models suggest that KAST attenuates airway inflammation by reducing serum and bronchoalveolar lavage fluid (BALF) levels of inflammatory mediators, including downregulating IgE, IL-4, and IL-13 expression^9,13,14. Collectively, these findings provide supportive evidence for the therapeutic efficacy of KAST in asthma. However, its precise mechanisms of action remain unclear.

One core pathological mechanism driving AA recurrence is type 2 airway inflammation, in which group 2 innate lymphoid cells (ILC2s) play a key role¹⁵. Upon allergen stimulation, ILC2s secrete cytokines such as IL-5 and IL-13, thereby driving airway inflammation^16,17. Studies have shown that depletion of ILC2s significantly alleviates airway inflammation in asthmatic mice, underscoring their central role in disease progression¹⁸. Notably, the activation and function of ILC2s are tightly regulated by multiple mechanisms, with neuro-immune interactions drawing increasing attention in recent years. Emerging evidence indicates that calcitonin gene-related peptide (CGRP) can bind to receptor activity-modifying protein 1 (RAMP1) on the surface of ILC2s, inducing their activation and subsequent release of type 2 cytokines, which exacerbates airway inflammation in AA^19,20,21. In the present study, our high-throughput RNA-sequencing (RNA-seq) screening identified CGRP as a critically responsive neuropeptide heavily modulated by KAST intervention. Therefore, focusing on the CGRP/RAMP1 pathway, we employed KAST as an intervention in AA mice to investigate its potential mechanisms in modulating ILC2 activity and alleviating type 2 airway inflammation.

Protocol

All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Academies of Sciences. The protocol was approved by the Animal Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine (approval No. 2023DW-059-01). A comprehensive list of all materials, reagents, tools, and software used in this study is detailed in the Table of Materials.

Experimental animals and grouping

Healthy female BALB/c mice (SPF grade, 8–10 weeks old) were obtained from the Experimental Animal Center of Nanjing University of Chinese Medicine (license No. SYXK (Su) 2018-0049). Animals were housed under standard conditions with a 12 h light/dark cycle at 24–26 °C. Sterile food and water were provided ad libitum, bedding was replaced regularly, and cages were cleaned routinely. After 7 days of adaptation, the mice were randomly divided into six groups (n = 7/group): Normal group, model group, CGRP group, BIBN-4096 group, KAST group, and KAST + CGRP group. Animals were randomly assigned to groups using a computer-generated random number table. Group allocation was concealed from investigators until the interventions were assigned. To optimize tissue utilization, lung samples from the randomized cohort (n = 7 per group) were systematically allocated to different processing pathways, with the exact analyzed sample sizes (n = 5, 4, or 3) for specific histological and molecular assays explicitly indicated in the respective figure legends. No data were excluded from the analysis.

Preparation of KAST formulation

The formulation was prepared by the Pharmacy Department of the Affiliated Hospital of Nanjing University of Chinese Medicine²². Nine traditional Chinese medicinal herbs were used: Semen Brassicae (2 parts), Rhizoma Corydalis (2 parts), Radix Kansui processed with vinegar (1 part), Asarum (1 part), Ephedra (1 part), Semen Lepidii (1 part), Clove (1 part), Cinnamon bark (1 part), and Gleditsia sinensis (1 part). Each herb was ground into fine powder and passed through an 80-mesh sieve. For every 100 g of mixed powder, 70 mL of fresh ginger juice and 30 mL of liquid paraffin were added, thoroughly mixed, and processed into 0.3 g ± 5% patches with a diameter of 0.5 cm. Specifically, all herbal ingredients and fresh ginger were authenticated by Chief Pharmacist Lin-gang Zhao and stored at 4 °C in a dark, dry environment prior to use⁹. To guarantee intervention reproducibility and batch-to-batch stability, a rigorous quality control standard was implemented based on our team's established UPLC-Q-TOF-MS and HPLC methods, confirming the consistent presence of major active markers such as sinapine thiocyanate²³. In practice, the 0.5 cm patches were secured using 2.5 cm × 5.5 cm medical breathable hypoallergenic adhesive tape to ensure optimal transdermal delivery.

Establishment of the AA mouse model

The AA model was induced according to an established protocol from the literature using OVA sensitization and intranasal challenge²⁴. Except for the Normal group, all mice were sensitized by intraperitoneal injection of 200 µL saline containing 100 µg OVA and 2 mg aluminum hydroxide on days 0 and 12. From days 18 to 23, mice were challenged intranasally once daily with 50 µg OVA in 50 µL saline. From days 24 to 53, OVA was administered intranasally every other day. On day 54, after weighing the mice, euthanasia was performed via cervical dislocation, and tissues were collected. Subsequently, the lung coefficient was determined by dividing the total lung weight by body weight.

Interventions

From days 40 to 53, mice in the KAST group and KAST + CGRP group were shaved over a 3 cm × 6 cm dorsal area. Patches were applied over bilateral Feishu (BL13), Geshu (BL17), Pishu (BL20), and Shenshu (BL23) acupoints, fixed with adhesive tape for 1.5 h once daily for 14 consecutive days. Mice in the CGRP and KAST + CGRP groups received intranasal administration of 10 µL Calcitonin/CALCA solution (1 pg/µL) every other day from day 40 to 53. Mice in the BIBN-4096 group received intranasal administration of 10 µL BIBN-4096 solution (0.3 mg/mL, CGRP receptor antagonist) every other day. Normal and model groups received standard feeding without intervention. KAST was administered from day 40 to day 53, after the establishment of allergic airway inflammation (starting at day 40, which is 22 days after the first OVA challenge on day 18). Therefore, KAST in this study served as a therapeutic rather than a prophylactic intervention.

For skin preparation, hair removal over the designated dorsal region was performed using an electric clipper. Acupoint localization strictly adhered to the standard Nomenclature and Location of Acupoints for Laboratory Animals - Part 3: Mice (T/CAAM 0002-2020) issued by the Chinese Acupuncture-Moxibustion Society. The four pairs of bilateral dorsal sites were precisely marked and defined as follows: Feishu (BL13: between the ribs below the 3rd thoracic vertebra spinous process, 3 mm lateral to the posterior midline), Geshu (BL17: below the 7th thoracic vertebra spinous process, 3 mm lateral to the midline), Pishu (BL20: below the 12th thoracic vertebra spinous process, 3 mm lateral to the midline), and Shenshu (BL23: below the 2nd lumbar vertebra spinous process, 3 mm lateral to the midline). The 3M tape was tightly wrapped to achieve stable adhesion, ensuring no patch displacement or secondary skin erosion during the 1.5 h retention period.

The doses of CGRP/CALCA (1 pg/µL, 10 µL per mouse) and BIBN-4096 (0.3 mg/mL, 10 µL per mouse) were selected based on published studies²⁵. Specifically, for the CGRP intervention, a 0.1% BSA solution was prepared by dissolving 1 mg of BSA in 1 mL of ddH₂O, followed by vortexing to achieve a final concentration of 1 mg/mL. Prior to reconstitution, the lyophilized Calcitonin/CALCA protein powder was centrifuged at 12,000 × g for 30 s at 4 °C. Then, 20 µL of the prepared 0.1% BSA solution was added to achieve a 1 µg/µL stock solution. The vial was gently inverted several times (without vortexing), spun down for a few seconds, and left at room temperature for several minutes to ensure complete protein reconstitution. To prepare the 1 pg/µL working solution without generating an unmanageably large single-step volume, a standard two-step serial dilution was implemented: 1 µL of the 1 µg/µL stock solution was first diluted in 999 µL of sterile saline to formulate a 1 ng/µL intermediate solution, and then 1 µL of this intermediate solution was further mixed with another 999 µL of sterile saline to achieve the final 1 pg/µL working solution. The working solution was kept at 4 °C, while remaining aliquots were stored at -20 °C for up to 2–3 months. For the BIBN-4096 group, 1 mg of BIBN-4096 powder was dissolved in 333 µL of ddH₂O to obtain a 3 mg/mL stock solution, which was aliquoted into 50 µL per vial and stored at -80 °C. To prepare the 0.3 mg/mL working solution, each 50 µL aliquot of stock solution was thoroughly mixed with 450 µL of sterile saline. From days 40 to 53, a 10 µL volume of either the 1 pg/µL CGRP solution or the 0.3 mg/mL BIBN-4096 solution was administered intranasally to each mouse every other day under gentle restraint without anesthesia.

Outcome measures and detection methods

Behavioral testing

On day 53, following the final intranasal challenge, the frequencies of nasal rubbing and sneezing episodes were monitored and recorded using a digital video system for a duration of 10 min. The behavior frequencies in the videos were independently counted by two researchers in a double-blind manner. In cases of a significant discrepancy between their counts, a third researcher re-evaluated the video, and the data with the highest consistency were ultimately selected for statistical analysis.

Bronchial provocation test

Within 24 h after the final challenge, airway responsiveness was assessed using nebulized acetylcholine (ACh) at concentrations of 0, 5, 10, 20, and 40 mg/mL (300 µL per concentration, nebulized for 1 min). Enhanced pause (Penh) values, a dimensionless index used to empirically indicate airway constriction and evaluate airway hyperresponsiveness (AHR), were recorded for 5 min after each nebulization using a non-invasive whole-body plethysmography. Mice were acclimated to the chamber for 10 min prior to recording. Penh was automatically calculated using the integrated manual storage/streaming system software according to the empirical formula balancing expiratory/inspiratory time and peak flows.

Histopathology (HE staining)

Lung tissues fixed in 4% paraformaldehyde were dehydrated through a graded alcohol series, cleared in xylene, and embedded in paraffin blocks using an automated processor. The blocks were sectioned at a thickness of 4 µm, floated in a 42 °C water bath, and mounted onto glass slides. Slides were baked at 80 °C for 2 h and dried overnight before cooling to room temperature. For staining, sections were deparaffinized in xylene (I and II, 20 min each), rehydrated through a graded ethanol series (100% I/II and 75%, 5 min each), and rinsed with tap water. Slides were stained with hematoxylin for 3–5 min, differentiated with 1% acid alcohol, and blued in a bluing solution. After washing, sections were dehydrated via 85% and 95% ethanol (5 min each) and counterstained with eosin for 5 min. Finally, slides were dehydrated in absolute ethanol (I, II, and III, 5 min each), cleared in xylene (I and II, 5 min each), mounted with neutral balsam, and air-dried. Images were captured from random fields at 200× magnification using a light microscope and the CaseViewer system. Lung inflammation and epithelial thickening were graded blindly on a 0–4 scale: 0, no inflammation; 1, occasional inflammatory cells; 2, few (<3) inflammatory foci; 3, multiple (≥3) inflammatory foci; 4, diffuse infiltration throughout the lung. Epithelial thickening was scored as: 0, normal; 1, visible thickening; 2, partial airway thickening; 3, near-complete obstruction; 4, absent normal structure. For each experimental group, a total of 20 random microscopic fields were imaged and evaluated to obtain the raw histopathological scores. The data from every four fields were averaged to yield a single independent data point, resulting in a final sample size of n = 5 replicates per group for statistical analysis. The scoring was performed in a blinded manner.

RNA-seq

Total RNA was extracted from murine lung tissues using TRIzol reagent according to the manufacturer's protocol. RNA purity, concentration, and integrity were evaluated using a spectrophotometer and a microfluidics-based platform. Transcriptome libraries were constructed using a commercial library preparation kit and sequenced on a high-throughput sequencing platform to generate 150 bp paired-end reads. Raw data were quality-controlled using appropriate filtering software to obtain clean reads, which were mapped to the reference genome using a splice-aware alignment tool. Gene expression levels were calculated as FPKM, and read counts were compiled via a read-counting utility. Differential expression analysis was performed using a specialized statistical package, with thresholds set at |log2 Fold Change| ≥ 1 and adjusted p < 0.05.

ELISA

Fresh murine lung tissues were precisely weighed and homogenized in PBS buffer at a strict ratio of 20 mg tissue to 200 µL buffer. The homogenates were centrifuged at approximately 13,400 × g for 10 min at 4 °C to harvest the protein supernatants. For normalization, the raw protein content of the supernatants was calibrated using the BCA method. The ELISA procedure was performed in technical triplicate using commercial kits according to the manufacturer's protocol. Briefly, 50 µL of standard dilutions or 50 µL of pre-diluted samples (10 µL protein sample mixed with 40 µL sample diluent, achieving a 5-fold final dilution) were added to the antibody-coated 96-well plates. The plates were sealed and incubated at 37 °C for 30 min. After washing five times with a 1:30 diluted wash buffer, 50 µL of enzyme-linked conjugate reagent was added to each well (except the blank wells), and the mixture was incubated at 37 °C for 30 min. After another five washes, 50 µL of chromogen solution A and 50 µL of chromogen solution B were added to each well, and the plate was incubated in the dark at 37 °C for 10 min. The reaction was stopped by adding 50 µL of the stop solution, which shifted the color from blue to yellow. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader within 15 min. Standard curves were plotted using a four-parameter logistic or linear regression model (ensuring R² > 0.99), from which the target protein concentrations in the lung tissue homogenates were calculated.

Flow cytometry

Lung tissues were excised, minced, and digested in 5 mL of RPMI-1640 containing 10% FBS, 50 µL Liberase TM, and 5 µL DNase I at 37 °C for 45 min with shaking at 80 rpm. The digested suspension was passed through a 400-mesh cell strainer and centrifuged at 300 × g for 10 min at 4 °C. The pellet was resuspended in 2 mL of erythrocyte lysis buffer for 10 min at room temperature in the dark. After washing, cells were resuspended and stained with Ghost Dye Red 780 (1.25 µL per sample) for 20 min at 4 °C in the dark for dead cell exclusion. Following a wash, cells were blocked with 10 µL of FcR Blocking Reagent for 10 min at 4 °C. Cells were then surface-stained with the following fluorochrome-conjugated antibodies for 30 min at 4 °C: FITC anti-mouse CD45 (0.5 µL), PE anti-mouse/human KLRG1 (1.25 µL), BB700 rat anti-mouse CD90.2 (0.5 µL), and a biotinylated lineage mixture containing anti-mouse CD3ε, CD4, CD8a, CD11b, CD11c, CD49b, TER-119, Ly-6G/Ly-6C (Gr-1) (all 0.5 µL each), and anti-mouse CD19 (2 µL). After washing, cells were incubated with Brilliant Violet 605 Streptavidin (2 µL) for 30 min at 4 °C. Data were acquired on a BD flow cytometer platform and analyzed using FlowJo software. Fluorescence spillover compensation matrices were automatically monitored and adjusted by the instrument's calibrated acquisition software. The hierarchical gating strategy for identifying pulmonary ILC2s was sequentially implemented as follows: total lymphocytes were first isolated via forward/side scatter (FSC-A/SSC-A) morphology, followed by doublet discrimination through singlet gating (FSC-H/FSC-A). Viable cells were subsequently identified by dead cell exclusion (Ghost Dye^-), and then standard gates were progressively applied to select the CD45⁺ and Lineage^- populations. Within the viable CD45⁺ Lineage^- gating window, pulmonary ILC2s were explicitly defined and quantified based on the subsequent positive sequential cascading gates of CD90.2⁺ and KLRG1⁺ clusters.

Immunofluorescence (IF)

Frozen lung sections were thawed at room temperature for 15–30 min and washed three times with PBST (PBS containing 0.05% Tween-20) for 6 min each to remove the OCT compound. After blotting excess liquid, a hydrophobic barrier was drawn around the tissue using an immunohistochemistry pen. Sections were incubated in a blocking and permeabilization buffer containing 0.3% Triton X-100 and 5% goat serum at 37 °C for 100 min, followed by overnight incubation (16–20 h) at 4 °C with the primary antibody. The next day, sections were equilibrated at room temperature for 30 min, washed three times with PBST, and incubated with a species-matched fluorophore-conjugated secondary antibody at 37 °C in the dark for 60–90 min. After three additional PBST washes, nuclei were counterstained with DAPI (diluted 1:1 or 2:1 in PBS) for 8 min at room temperature in the dark. Following a final round of PBST washes (3 × 6 min), sections were mounted with an anti-fade mounting medium, sealed to prevent bubble formation, and stored in the dark. Images were captured using a confocal microscope at 400× magnification. The number of positive cells per field was counted manually using Leica LAS X software. For each experimental group, a total of 15 random microscopic fields were imaged to obtain the raw cell counts. The data from every three fields were averaged to yield a single independent data point, resulting in a final sample size of n = 5 replicates per group for statistical analysis. The counting was performed in a blinded manner.

Immunohistochemistry (IHC)

Paraffin sections were deparaffinized in three changes of xylene (15 min each), rehydrated through a graded ethanol series (100% twice, 85%, and 75%; 5 min each), and rinsed with distilled water. Antigen retrieval was performed in citrate buffer (pH 6.0) using a pressure cooker (3 min at high pressure after boiling), followed by cooling and three 5-min washes in PBS (pH 7.4). Endogenous peroxidase was blocked with 3% H₂O₂ for 15 min at room temperature in the dark. After washing, sections were encircled with a hydrophobic pen and blocked with 3% goat serum for 30 min. Sections were then incubated overnight at 4 °C with primary antibodies diluted in PBS: anti-IL-4 (1:400), anti-IL-5 (1:300), or anti-IL-13 (1:150). The following day, sections were washed and incubated with HRP-conjugated secondary antibodies (anti-mouse for IL-4; anti-rabbit for IL-5 and IL-13) for 50 min at room temperature. Signals were visualized using a freshly prepared DAB solution under microscopic control and terminated with tap water. Finally, sections were counterstained with hematoxylin, differentiated, blued, dehydrated through graded alcohols and xylene, and mounted with neutral balsam for microscopic examination. Mean optical density (IOD/Area) was quantified using an Image analysis software platform at 200× magnification. For each experimental group, a total of 15 random microscopic fields were imaged to obtain the raw values. The data from every three fields were averaged to yield a single independent data point, resulting in a final sample size of n = 5 replicates per group for statistical analysis. The scoring was performed by two blinded observers.

Western blot (WB)

For sample preparation, murine lung tissues (approximately 30 mg) were finely minced and homogenized in 300 µL of pre-cooled RIPA lysis buffer supplemented with 1% protease/phosphatase inhibitor cocktail. Homogenization was performed using a tissue homogenizer at 60 Hz for four cycles (60 s per cycle). The lysates were then centrifuged at approximately 13,400 × g for 15 min at 4 °C to collect the supernatant. Total protein concentrations were determined via a standard BCA protein assay kit. Samples were then normalized, mixed with 5x loading buffer at a 4:1 volume ratio, and denatured at 95 °C for 7 min. For SDS-PAGE, protein samples (20 µg per lane) were loaded alongside a pre-stained protein molecular weight marker, resolved at a constant voltage of 80 V for 20 min, and then adjusted to 120 V for 70 min. Proteins were then wet-transferred to 0.22 µm PVDF membranes under a constant current of 300 mA for 120 min in an ice bath. The membranes were blocked with 5% BSA in TBST for 1 h at room temperature and incubated overnight (up to 18 h) at 4 °C with primary antibodies against CGRP (1:500) and GAPDH (1:2000). After washing with TBST three times (10 min each), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000) for 1–2 h at room temperature. Following another three washes, the protein bands were visualized using an enhanced chemiluminescent (ECL) substrate within a VILBER Fusion FX imaging system. Band intensities were quantified using ImageJ 1.8, and target protein expression was normalized to the GAPDH internal control.

Statistical analysis

All experimental data are expressed as the mean ± standard deviation (SD). Data analysis and statistical plotting were performed using a specialized statistical software platform. Prior to inter-group comparisons, the datasets were systematically subjected to normality testing. For normally distributed data with homogeneous variances, ordinary one-way or two-way Analysis of Variance (ANOVA) was implemented, followed by Tukey’s, Šídák’s, Holm-Šídák’s, or Fisher’s LSD post hoc tests for multiple pairwise comparisons. When comparing multiple groups against a single control group, Dunnett's test was applied. For datasets with heterogeneous variances, the Brown-Forsythe ANOVA was used, followed by Dunnett's T3 post-hoc test. For datasets that did not conform to normality, the nonparametric Kruskal-Wallis test was used, followed by Dunn's post hoc test for multiple comparisons. A P value < 0.05 was considered to indicate statistically significant differences. Different significance levels are dynamically denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All experimental outcome assessments, including histological scoring, flow cytometry analysis, and image quantification, were conducted in a strictly blinded manner.

Results

KAST reduces pulmonary ILC2s and alleviates type 2 airway inflammation

To investigate the therapeutic effects of KAST, an OVA-induced AA mouse model was established and treated with patches applied at Feishu (BL13), Geshu (BL17), Pishu (BL20), and Shenshu (BL23) acupoints (Figure 1A). After modeling, AA mice showed decreased body weight, whereas KAST mice exhibited significantly increased body weight compared with the model group (Figure 1B). The lung coefficient, an indicator of pulmonary inflammation, was elevated in AA mice but markedly reduced after KAST (Figure 1C). Behavioral assessments revealed increased nasal scratching and sneezing in AA mice, which were significantly alleviated by KAST (Figure 1D–E). Whole-body plethysmography demonstrated that Penh values, indicative of airway hyperresponsiveness, were elevated in AA mice but decreased following treatment, suggesting improved lung function (Figure 1F). Histopathological analysis with HE staining revealed peribronchial and perialveolar inflammatory cell infiltration in the model group compared with normal controls. In contrast, inflammatory infiltration was reduced in the KAST group (Figure 2A–B). Immunohistochemistry showed that IL-4, IL-5, and IL-13 were primarily localized in the cytoplasm of infiltrating inflammatory cells and some epithelial cells, with potential accumulation in interstitial spaces. Compared with normal controls, the model group exhibited markedly increased positive signals and mean optical density values, which were significantly reduced in the KAST group (Figure 2C–H). Flow cytometry further demonstrated that ILC2 numbers were significantly increased in the lungs of AA mice compared with normal controls, whereas KAST markedly reduced ILC2 counts (Figure 2I–J).

KAST alleviates type 2 airway inflammation by suppressing the CGRP/RAMP1 axis

ILC2 hyperactivation is regulated by multiple factors. RNA-seq analysis revealed that CGRP expression was significantly upregulated in AA mice, and KAST markedly downregulated pulmonary CGRP levels (Figure 3A). Western blot and ELISA confirmed elevated CGRP protein levels in the model group compared with controls, which were reduced by KAST (Figure 3B–D). These results suggest that KAST attenuates CGRP expression in AA lungs, thereby modulating ILC2 immune responses. Together, these variations in high-throughput sequencing and protein screening suggest a strong association between the KAST therapeutic intervention and the downregulation of pulmonary CGRP levels in AA mice.

To further explore the role of CGRP, AA mice were treated with exogenous CGRP, the CGRP receptor antagonist BIBN-4096, or KAST combined with CGRP. ELISA results showed significantly reduced CGRP protein levels in the KAST and BIBN-4096 groups, elevated levels in the CGRP group, and higher levels in the KAST + CGRP group compared with KAST alone (Figure 4A). Flow cytometry revealed that ILC2 numbers were reduced in the KAST and BIBN-4096 groups but increased in the CGRP and KAST + CGRP groups relative to the model group (Figure 4B–C). HE staining showed reduced inflammatory cell infiltration around the airways and alveoli in the KAST and BIBN-4096 groups, while infiltration was aggravated in the CGRP and KAST + CGRP groups (Figure 4D–E). ELISA further confirmed decreased IL-4, IL-5, and IL-13 levels in the KAST and BIBN-4096 groups, but increased levels in the CGRP and KAST + CGRP groups (Figure 4F–H).

The number of pulmonary RAMP1⁺ILC2s across different groups was evaluated via multi-color immunofluorescence staining. The results demonstrated that, compared to the normal control group, the number of both total RAMP1⁺ILC2s in the lung tissues of the model group was significantly increased. Compared to the model group, the number of pulmonary RAMP1⁺ILC2s further significantly increased in the CGRP group, whereas they were prominently decreased in both the BIBN-4096 group and the KAST group. Notably, compared to the KAST group, the KAST + CGRP group exhibited a significant elevation in pulmonary RAMP1⁺ILC2s counts, indicating that exogenous CGRP replenishment successfully reversed the inhibitory effects of KAST on the populations of total RAMP1⁺ILC2s (Figure 5A–B).

DATA AVAILABILITY:

The full raw datasets generated and analyzed during the current study have been deposited in a publicly accessible repository. Specifically, these comprehensive materials include all full, original membrane scans for Western blot (WB) assays, all full, raw, high-resolution microscopic images for histology (HE and IHC) and immunofluorescence (IF) assays, as well as individual replication datasets and quantification Excel sheets. All raw data are openly available at the following persistent DOI: https://doi.org/10.6084/m9.figshare.32979065. The gating order for flow cytometry is provided in Supplementary Figure 1. Two full blot images are submitted as Supplementary Figures 2–3, and the quantitative data are submitted in Supplementary Table 1.

Figure 1: Effects of KAST on symptoms and lung function in AA mice. (A) Protocol for inducing allergic asthma using OVA. (B) Body weight statistics of mice in each group, n = 5. (C) Lung coefficient of mice in each group, n = 5. (D) Number of nasal rubs within 10 min after the last challenge, n = 5. (E)  Frequency of sneezing within 10 min after the last challenge, n = 5. (F) Penh values of mice in each group following stimulation with different concentrations of acetylcholine, n = 5. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical comparisons were performed using either one-way ANOVA or two-way ANOVA, followed by a post hoc multiple comparisons test where applicable. n = biological replicates. Please click here to view a larger version of this figure.

Figure 2: Regulation of ILC2 numbers and type 2 airway inflammation by KAST in AA mice. (A) Morphological changes of the lungs examined by HE staining. Scale bar = 50 µm. Magnification: 200×. The inflammation score was assessed in a blinded manner. (B) Statistical analysis of inflammation scores among different groups, n = 5. (C–H) Representative IHC images and mean optical density values of IL-4 (C, D), IL-5 (E, F), and IL-13 (G, H) protein expression in lung tissues of different groups, n = 5. Scale bar = 50 µm. Magnification: 200×. (I) Representative flow cytometry plots showing the final gating of pulmonary ILC2s across the Normal, Model, and KAST groups. Singlet, viable cells were gated sequentially as CD45⁺ Lineage^- (CD3ε, CD4, CD8a, CD11b, CD11c, CD49b, TER-119, Ly-6G, CD19), followed by CD90.2⁺, and pulmonary ILC2s were ultimately identified by the positive expression of KLRG1⁺. Data were acquired on a high-performance flow cytometry platform and analyzed using a specialized flow cytometry analysis software package. (J) Analysis of ILC2s indicated by the percentage of CD45⁺ Lineage⁻ CD90.2⁺ KLRG1⁺ cells among live cells, n = 5. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical comparisons were performed using one-way ANOVA followed by appropriate post hoc multiple comparison tests. n = biological replicates. Please click here to view a larger version of this figure.

Figure 3: KAST inhibits CGRP expression in the lung tissues of AA mice. (A) Heatmap of gene expression among different groups, n = 4. (B) Representative WB images of CGRP (10 kDa) and GAPDH (37 kDa) expression in lung tissues from each group. (C) WB analysis of CGRP protein expression in lung tissues, presented as relative CGRP expression (CGRP/GAPDH ratio), n = 3. (D) ELISA analysis of CGRP protein expression in lung tissues, n = 4. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical comparisons were performed using one-way ANOVA followed by a post hoc multiple comparisons test. n = biological replicates. Please click here to view a larger version of this figure.

Figure 4: Effects of KAST on ILC2 numbers and type 2 airway inflammation via CGRP in AA mice. (A) ELISA analysis of CGRP protein expression in lung tissues from different groups, n = 4. (B) Representative flow cytometry plots of ILC2s (CD45⁺ Lineage⁻ CD90.2⁺ KLRG1⁺) in each group. (C) Analysis of ILC2s indicated by the percentage of CD45⁺ Lineage⁻ CD90.2⁺ KLRG1⁺ cells among live cells, n = 5. (D) Morphological changes of the lungs examined by HE staining. Scale bar = 50 µm. Magnification: 200×. (E) Statistical analysis of inflammation scores among different groups, n = 5. (F–H) ELISA analysis of IL-4 (F), IL-5 (G), and IL-13 (H) protein expression in lung tissues from different groups, n = 4. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical comparisons were performed using one-way ANOVA followed by appropriate post hoc multiple comparison tests. n = biological replicates. Please click here to view a larger version of this figure.

Figure 5: Effects of KAST on ILC2 numbers in lung tissues via the CGRP/RAMP1 pathway. (A) Cell counts of RAMP1⁺ ILC2s (defined as KLRG1⁺GATA3⁺RAMP1⁺ triple-positive cells) per 400× field in lung tissues, n = 5. (B) Representative fluorescence staining of lung tissues. Nuclei were stained with DAPI (blue), and immunostaining was performed for KLRG1 (purple), GATA3 (red), and RAMP1 (green). Scale bar = 50 µm. Quantification: manual counting of RAMP1⁺ ILC2s (KLRG1⁺GATA3⁺RAMP1⁺ cells) in a blinded manner using a specialized image analysis platform. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical comparisons were performed using one-way ANOVA followed by a post hoc multiple comparisons test. n = biological replicates. Please click here to view a larger version of this figure.

Supplementary Figure 1: Representative flow cytometry plots showing the sequential gating strategy for the isolation of pulmonary ILC2s. First, total events were gated using forward scatter (FSC-A) and side scatter (SSC-A). Doublets were excluded via FSC-A vs. FSC-H to obtain single cells. Live cells were identified as Ghost Dye Red 780⁻ to eliminate dead cell populations. Leukocytes were then gated on CD45⁺, followed by selection of lineage-negative (Lin⁻) cells. Within the CD45⁺ Lin⁻ subset, cells were further gated on CD90.2⁺, and pulmonary ILC2s were ultimately defined by the positive expression of KLRG1 (KLRG1⁺).Please click here to download this file.

Supplementary Figure 2: Representative Western blot image of internal reference GAPDH (37 kDa) from mouse lung tissue samples. Band-integrated optical density was quantified by ImageJ software for subsequent normalization.Please click here to download this file.

Supplementary Figure 3: Representative Western blot image of target protein CGRP (10 kDa) from mouse lung tissue samples. Relative CGRP expression was calculated by dividing the integrated optical density of CGRP bands by the corresponding GAPDH integrated optical density values.Please click here to download this file.

Supplementary Table 1: Raw data values. Raw data values that were used to plot graphs.Please click here to download this file.

Discussion

AA treatment faces several limitations, including side effects associated with long-term medication use, incomplete symptom control, and frequent relapses. Although conventional pharmacotherapy is effective, it is often accompanied by adverse outcomes such as steroid dependence^26,27. In contrast, KAST offers a safe, economical, and convenient alternative for the management of AA. Clinical evidence has shown that KAST can effectively alleviate asthma symptoms by modulating pulmonary immune function²⁸. In this study, we further demonstrated that KAST attenuates type 2 airway inflammation in AA mice by suppressing the CGRP/RAMP1 pathway and reducing the number of ILC2s.

Innate immunity, particularly ILC2s, plays an indispensable role in the initiation, amplification, and maintenance of type 2 airway inflammation in AA²⁹. Upon allergen exposure, ILC2s rapidly respond to epithelial-derived alarmins, secreting large amounts of IL-5 and IL-13 within hours, thereby triggering type 2 inflammatory responses^30,31. Moreover, ILC2s promote Th2 differentiation and IgE synthesis, further amplifying adaptive immune responses and exacerbating airway inflammation³². Studies have shown that genetic ablation of ILC2s significantly shortens the duration of airway inflammation in AA models, highlighting their central role in sustaining type 2 inflammation¹⁸. ILC2s are regulated by cytokines (e.g., IL-25, IL-33, TSLP), inflammatory mediators (e.g., leukotrienes), and neuropeptides (e.g., CGRP, VIP, NMU)³³. Among these, neuropeptides play pivotal roles in innate immune regulation. Neuromedin U (NMU) directly enhances ILC2 activity, rapidly inducing IL-5 and IL-13 release³⁴. Vasoactive intestinal peptide (VIP) contributes to nutrient intake and circadian regulation, promoting IL-5 production by ILC2s and maintaining eosinophil homeostasis³³. CGRP markedly augments IL-5 release from ILC2s, thereby promoting airway inflammation in AA. Notably, RAMP1, the receptor subunit for CGRP, is expressed at much higher levels on ILC2s compared with receptors for other neuropeptides, suggesting that CGRP is the most potent ligand for ILC2 activation³⁵. In this study, transcriptomic analysis identified CGRP as one of the most differentially expressed neuropeptides in AA mice, and protein-level assays confirmed its upregulation in lung tissues. Exogenous administration of CGRP further increased ILC2 numbers and type 2 cytokine release, aggravating airway inflammation.

Mechanistically, CGRP binds to RAMP1 on ILC2s to regulate their downstream immune responses. While some existing literature characterizes CGRP as a negative regulator that limits ILC2 expansion to protect against hyper-inflammation^20,21, counter-evidences indicate that CGRP can collaborate with alarmins to robustly provoke group 2 innate immunity within specific pathological microenvironments. Our experimental endpoints specifically realign with the latter cascade, demonstrating a CGRP-driven upregulation of pulmonary ILC2 infiltration in the OVA-induced allergic microenvironment. Conversely, blockade of the CGRP pathway with the receptor antagonist BIBN-4096 significantly ameliorates ILC2-mediated airway inflammation. Rather than providing direct genetic ablation evidence, our findings leverage associative transcriptomic variations and parallel pharmacologic intervention experiments (utilizing exogenous CGRP and the antagonist BIBN-4096) to demonstrate that the therapeutic protection of KAST is closely correlated with the suppression of CGRP/RAMP1 signaling clusters on ILC2s.

Despite these promising findings, several inherent limitations must be acknowledged. First, while the OVA-induced murine model provides valuable mechanistic insights, it may not fully recapitulate the complex pathophysiological and immunological landscape of human allergic asthma due to cross-species differences. Second, the relatively small sample size in certain exploratory assays dictates that larger cohorts are required to achieve broader statistical generalization. Third, this study primarily prioritized the CGRP/RAMP1/ILC2 signaling axis, meaning that other potentially relevant neuro-immune crosstalk pathways and overlapping cytokine networks warrant deeper exploration in the future. Most notably, a major technical limitation is the absence of a dedicated sham acupoint sticking control group utilizing non-medicated patches or non-acupoint locations. Consequently, potential non-specific confounding factors—such as mechanical stress from shaving, experimental handling, and the adhesive stimulation of the patch itself—cannot be entirely isolated from the specific therapeutic properties of KAST. To resolve these caveats, our future research pipelines will strictly incorporate rigorously designed sham controls and multi-pathway validations to further consolidate the specificity and reproducibility of these findings. Regarding the observed reduction of CGRP protein levels following BIBN-4096 intervention, it is critical to clarify that while BIBN-4096 primarily functions as a competitive antagonist targeting the RAMP1/CRLR receptor complex rather than a direct CGRP synthesis inhibitor, its in vivo administration can downregulate local ligand tissue accumulation. This secondary phenomenon is likely mediated through the disruption of the neuro-immune positive feedback loop. By blocking RAMP1 on target immune cells like ILC2s, BIBN-4096 suppresses the downstream release of effector cytokines, which in turn diminishes the retrograde inflammatory activation of sensory nerve terminals and pulmonary neuroendocrine cells (PNECs), thereby indirectly attenuating downstream CGRP synthesis and tissue retention.

In summary, our empirical findings demonstrate that KAST effectively reshapes the pulmonary innate immune microenvironment by targeting the neural CGRP/RAMP1 axis. While we recognize that direct clinical extrapolation requires cautious optimization given the biological differences between rodents and humans, these endpoints provide a critical, previously missing mechanistic foundation that substantiates the traditional "Fei Wai He Pi Mao" (Lung combining with skin and hair) theory. By offering a refined neuro-immune perspective, this protocol establishes a reliable scientific baseline to guide and support future prospective, multi-center clinical translations of traditional herbal acupoint sticking therapies for allergic asthma management.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3M Medical Breathable Tape	3M, USA	1530C	Patch fixation
Acetylcholine chloride	Sigma-Aldrich, USA	A6625	Bronchial provocation
Agilent 2100 Bioanalyzer	Agilent Technologies, USA	G2939BA	RNA quality and integrity evaluation
Alexa Fluor 488 goat anti-rabbit IgG (H+L)	Thermo Fisher Scientific, USA	(custom order)	Immunofluorescence (secondary)
Aluminum hydroxide	Sigma-Aldrich, USA	239186	Adjuvant
Anti-CGRP antibody (rabbit)	Abcam, UK	ab81887	Western blot / IF
BALB/c mouse (female, SPF, 8–10 weeks, 18–22 g)	Sibelfu (Suzhou) Biotechnology Co., Ltd.	B202407260106	Animal model
BB700 rat anti-mouse CD90.2 (clone 53-2.1)	BD Biosciences, USA	566465	Flow cytometry
BD FACSCanto II flow cytometer	BD Biosciences, USA	FACSCanto II	Flow cytometry
BIBN-4096 (Olcegepant hydrochloride)	MedChemExpress LLC, USA	HY-10095A	CGRP receptor antagonist
Biotin-Lineage Cocktail	BD Biosciences, USA	(Customized, clones as described)	Flow cytometry
Brilliant Violet 605 Streptavidin	BD Biosciences, USA	563260	Flow cytometry
BSA Albumin Fraction V	BioFroxx (neoFroxx GmbH), Germany	4240GR100	Blocking
Calcitonin/CALCA protein	MedChemExpress LLC, USA	HY-P77587	CGRP intervention
CD11c/ITGAX antibody (rabbit)	Abmart, Shanghai, China	MU115408S	Immunofluorescence
Citrate antigen retrieval buffer	Boster, China	AR0024	Immunohistochemistry
DAPI Staining Solution	Beyotime, Shanghai, China	C1006	Nuclear staining
ddH2O (Double-distilled water)	Sigma-Aldrich, USA	10977015	Protein reconstitution and powder dissolution
DESeq2	Bioconductor (R package)	Version 1.42.0	Differential expression analysis software
DNase I	Roche Diagnostics, Germany	10104159001	Lung tissue digestion
Donkey Anti-Mouse IgG H&L (Alexa Fluor 555)	Abcam, UK	ab150106	Immunofluorescence (secondary)
ECL Substrate (Super Femto)	Nanjing Lanken Biotech, China	–	Western blot chemiluminescence
Enhanced BCA Protein Assay Kit	Beyotime, Shanghai, China	P0010	Protein quantification
Eosin Y Staining Solution	Sigma-Aldrich, USA	HT110216	Histology (HE staining)
Ethanol (Absolute, Analytical Grade)	Sinopharm Chemical Reagent, China	10009218	Tissue dehydration and histology
F4/80 antibody (mouse, clone C-7)	Santa Cruz Biotechnology, USA	sc-377009	Immunofluorescence
fastp	Open Source (GitHub)	Version 0.23.4	RNA-seq raw data quality control software
FITC anti-mouse CD45 (clone 30-F11)	BD Biosciences, USA	553080	Flow cytometry
FlowJo	BD Biosciences, USA	10	Flow cytometry analysis
GAPDH monoclonal antibody (mouse)	Proteintech, Wuhan, China	60004-1-Ig	Western blot
GATA3 antibody (mouse, clone L50-823)	Santa Cruz Biotechnology, USA	sc-268	Immunofluorescence
Ghost Dye Red 780	Tonbo Biosciences, USA	13-0865-T100	Dead cell exclusion
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody	Thermo Fisher Scientific, USA	A11008	Immunofluorescence (secondary)
GraphPad Prism	GraphPad Software, USA	10.1.2	Statistical analysis
Hematoxylin Solution (Harris)	Sigma-Aldrich, USA	HHS16	Histology (HE staining)
HISAT2	Johns Hopkins University (Open Source)	Version 2.2.1	RNA-seq reference genome alignment software
HRP-conjugated Goat Anti-Mouse IgG (H+L)	Proteintech, Wuhan, China	SA00001-1	Western blot (secondary)
HRP-conjugated Goat Anti-Rabbit IgG (H+L)	Proteintech, Wuhan, China	SA00001-2	Western blot (secondary)
HTSeq-count	EMBL (Python utility)	Version 2.0.3	RNA-seq genomic feature read-counting utility
Hydrogen Peroxide (H2O2, 3% Solution)	Sigma-Aldrich, USA	H1009	Endogenous peroxidase blocking
iFluor™ 488 Conjugated Goat anti-rabbit IgG Antibody	Huabio, China	HA1121	Immunofluorescence (secondary)
IL-4 Monoclonal antibody	Proteintech, Wuhan, China	66142-1-Ig	Western blot
IL13 Antibody	Affinity Biosciences, USA	DF6813	Western blot
IL5 Antibody	Affinity Biosciences, USA	AF5123	Western blot
Illumina Novaseq 6000	Illumina, USA	–	RNA sequencing
ImageJ	NIH, USA	1.8	Western blot quantification
Image-Pro Plus	Media Cybernetics, USA	Version 6.0	Image optical density quantification
KAST herbal formulation	Pharmacy Department, Affiliated Hospital of Nanjing University of Chinese Medicine	–	Intervention
KLRG1 Monoclonal Antibody (2F1)	Thermo Fisher Scientific, USA	58-5893-82	Immunofluorescence
Leica LAS X	Leica Microsystems, Germany	4.5	Immunofluorescence quantification
Leica STELLARIS 8 DIVE confocal microscope	Leica Microsystems, Germany	STELLARIS 8	Immunofluorescence imaging
Liberase TM	Roche Diagnostics, Germany	5401119001	Lung tissue digestion
Marker (Prestained Protein Ladder)	NCM Biotech, Suzhou, China	P9001	Western blot
Mouse CGRP ELISA kit	Aifang Biotechnology, Hunan, China	AF2491-A	ELISA
Mouse IL-13 ELISA kit	Aifang Biotechnology, Hunan, China	AF-02456M1	ELISA
Mouse IL-4 ELISA kit	Aifang Biotechnology, Hunan, China	AF-02448M1	ELISA
Mouse IL-5 ELISA kit	Aifang Biotechnology, Hunan, China	AF-11729M1	ELISA
Neutral balsam mounting medium	Sinopharm Chemical Reagent, China	10014061	Histology slide mounting
Normal goat serum (for blocking)	Abcam, UK	ab7481	Immunohistochemistry/IF blocking
OCT embedding compound	Sakura, USA	4583	Cryosection embedding
One-Step PAGE Gel Fast Preparation Kit	Vazyme, Nanjing, China	E303-01	SDS-PAGE gel preparation
Ovalbumin (OVA)	Sigma-Aldrich, USA	A5503	Sensitization/challenge
Paraformaldehyde (4% in PBS)	Biosharp, China	BL539A	Tissue fixation
PE anti-mouse/human KLRG1 (clone 2F1)	BD Biosciences, USA	564022	Flow cytometry
Phosphatase inhibitor cocktail (50×)	Beyotime, Shanghai, China	P1092	Protein extraction
Poly-L-lysine solution	Boster, China	AR0003	Histology slide coating
Proteinase and phosphatase inhibitor	NCM Biotech, Suzhou, China	P002	Protein extraction
PVDF membrane (0.22 μm)	Merck Millipore, USA	ISEQ00010	Western blot
RAMP1 polyclonal antibody (rabbit)	Proteintech, Wuhan, China	10327-1-AP	Immunofluorescence
Red blood cell (RBC) lysis buffer	Beyotime, Shanghai, China	C3702	Flow cytometry sample prep
RIPA lysis buffer (Strong)	Beyotime, Shanghai, China	P0013B	Protein extraction
RPMI-1640 medium	Thermo Fisher Scientific, USA	11875093	Tissue cell digestion base
SuperKine™ Enhanced Antifade Mounting Medium	Wuhan Yakeyin Biotechnology Co., Ltd.	BMU104	Immunofluorescence mounting
Triton X-100 (Detergent)	Sigma-Aldrich, USA	T8787	Immunofluorescence permeabilization
TRIzol reagent	Thermo Fisher Scientific, USA	15596026	RNA extraction
VAHTS Universal V10 RNA-seq Library Prep Kit	Vazyme, Nanjing, China	NR605-01	RNA-seq library construction
Whole-body plethysmography system	Guangyuanda, Beijing, China	WBP-4	Lung function (Penh)
Xylene (Analytical Grade)	Sinopharm Chemical Reagent, China	10023418	Deparaffinization and clearing