Investigating the consequences of tape-stripping in ovalbumin-induced allergic mouse model: From physiology to gene regulation

TL;DR

This study shows that damaging the skin barrier worsens food allergies in mice by disrupting gut health and immune balance.

Contribution

The study identifies key genes and pathways linking skin barrier disruption to food allergy exacerbation via the skin-gut axis.

Findings

Tape-stripping worsens allergic symptoms and intestinal inflammation in mice.

Skin barrier damage elevates IgE, mast cells, and Tregs while reducing gut tight junction proteins.

Acta2, Kdr, and Cxcr4 are key genes linking barrier dysfunction to allergic inflammation.

Abstract

Food allergy is a growing global health concern, with increasing prevalence and significant impacts on patients quality of life. Epithelial barrier dysfunction, particularly through the skin-gut axis, has emerged as a critical factor in food allergy pathogenesis. However, the precise mechanisms linking epithelial barrier disruption to immune dysregulation remain poorly understood. This study investigates the effects of tape-stripping-induced epithelial barrier damage on the exacerbation of ovalbumin (OVA)-induced food allergy in a murine model, with a focus on histopathological, immunological, and transcriptomic changes.

Female BALB/c mice were randomly assigned to three experimental groups for comparison: Control, OVA (OVA-sensitized/challenged), and OVA + T (OVA-sensitized/challenged with additional tape-stripping). Mice in the OVA group were sensitized and challenged with ovalbumin, while the OVA + T group underwent the same OVA protocol plus repeated tape-stripping to simulate epithelial barrier disruption. Allergic responses were evaluated using a comprehensive approach, including histopathological analysis, quantification of serum biomarkers (IgE, histamine, IL-6, IgG1), immune cell profiling via flow cytometry, and Western blot analysis of tight junction proteins such as occludin, Zonula Occludens (ZO)-1, and claudin. RNA sequencing-based transcriptomic analysis of jejunum tissues identified dysregulated pathways through integrated bioinformatics analyses (differential expression, pathway enrichment, and PPI network), with subsequent qRT-PCR validation pinpointing key target genes.

Tape-stripping exacerbated allergic symptoms, including diarrhea and elevated rectal temperature, and induced significant histopathological changes in both skin and intestinal tissues, characterized by increased inflammatory cell infiltration and mast cell accumulation. Serum analysis revealed elevated levels of IgE, histamine, IL-6, and IgG1, indicating a heightened allergic state. Flow cytometry demonstrated an increase in CD4+ Foxp3+ regulatory T cells (Tregs) and IgE + cells in the spleen, suggesting systemic immune dysregulation. Western blot analysis confirmed the downregulation of tight junction proteins, indicating compromised intestinal barrier integrity. Transcriptomic analysis identified 444 differentially expressed genes (DEGs) in the jejunum, with significant enrichment in pathways related to calcium signaling, cytokine-cytokine receptor interactions, PI3K-Akt signaling, and MAPK signaling. PPI network analysis identified Acta2, Kdr, and Cxcr4 as key target genes mediating tape-stripping-induced food allergy exacerbation.

This study demonstrates that tape-stripping exacerbates food allergy by disrupting epithelial barrier integrity, inducing systemic immune dysregulation, and altering gene expression in intestinal tissues. The identification of key molecular pathways and target genes—particularly Acta2, Kdr, and Cxcr4, which are associated with angiogenesis and inflammatory responses—reveals novel mechanistic insights into food allergy exacerbation and proposes actionable therapeutic targets for simultaneous barrier repair and immune modulation. These findings underscore the importance of the skin-gut axis in food allergy pathogenesis and offer a foundation for developing targeted interventions to manage and prevent allergic diseases. Further research is needed to validate these findings and explore therapeutic strategies targeting the identified pathways.

•Tape-stripping-induced skin barrier damage exacerbates allergic symptoms and intestinal inflammation in a murine food allergy model.•Barrier disruption drives systemic immune dysregulation, elevating IgE, mast cells, and splenic Tregs, while reducing intestinal tight junction proteins.•PPI network analysis reveals Acta2, Kdr, and Cxcr4 as hub genes linking barrier dysfunction to allergic inflammation.•Findings underscore the skin-gut axis as a therapeutic target for restoring epithelial integrity and modulating immune response in food allergy.

Tape-stripping-induced skin barrier damage exacerbates allergic symptoms and intestinal inflammation in a murine food allergy model.

Barrier disruption drives systemic immune dysregulation, elevating IgE, mast cells, and splenic Tregs, while reducing intestinal tight junction proteins.

PPI network analysis reveals Acta2, Kdr, and Cxcr4 as hub genes linking barrier dysfunction to allergic inflammation.

Findings underscore the skin-gut axis as a therapeutic target for restoring epithelial integrity and modulating immune response in food allergy.

Figures

Experimental schedule of OVA-induced mice with tape stripping. 27 mice were devided into three groups (n = 9 per group), Control group, OVA group, OVA + T group. Mice in all groups except the Control group were intraperitoneally (i.p.) immunized with 200 μg OVA in 1 mg alum adjuvant dissolved in PBS 200ul on the days 0, 5 and 10, on days 15,17,19, 21, 23, 25, mice with oral gavage (i.g.) were challeged with 50 mg of OVA in 200ul PBS every other day for 6 times in the OVA group and OVA + T group. On days 15, 17, 19, 21, mice were taped stripping the skin in OVA + T group. Mice in the control gr

Tape-stripping effectively exergerated (a) rectal temperature changes at the 30-min time point following the sixth oral challenge and (b) anaphylactic diarrhea in OVA-induced mice (##*P* < 0.01, OVA + T group *vs.* OVA group, ∗∗∗*P* < 0.001,∗∗∗∗*P = *0.000, n = 9). Observation in the (c) jejunal and (d) skin tissue by hematoxylin and eosin staining with the tape-stripping (magnification, × 200). Mast cell filtration by Toluidine blue staining in the (e) jejunal and (f) skin tissue with the tape-stripping (magnification, × 200). (g) Histopathological scores in the jejunum across different group

Inflammatory cytokines levels in serum measured by ELISA. Results of (a) IgE, (b) histamine, (c) IL-6, and (d) IgG1 of mice in different groups. Each value indicates mean ± standard error of the mean. n = 9/group. (e) CD4^+^ Foxp3^+^ Tregs in CD45^+^ T cells proportion in the spleen. (f) Proportion of IgE + cells in the spleen. n = 3/group. ∗*P* < 0.05, ∗∗*P* < 0.01, ∗∗∗*P* < 0.001. IL, interleukin. CD, the cluster of differentiation; Fox, forkhead box.Fig. 3

Results of RNA seq in the intestinal tissue with the tape-stripping compared to the OVA-induced mice in the OVA + T *vs.* OVA group. (a) The Volcano plot of 444 differential gene expression, top 10 upregulated genes and top 10 downregulated genes. (b) The top KEGG pathways with significantly enrichment of differentially expressed genes (DEGs). (c) Gene counts of Gene ontology (GO) terms distribution in each level. (d) The top KEGG pathways with significantly upregulated enrichment of DEGs. (e) Gene counts of GO terms distribution in each level in the upregulated enrichment of DEGs. (f) The top

qRT-PCR validation of tape-stripping-enhanced gene expression in OVA-induced allergic mice. n = 4. ∗*P* < 0.05,∗∗∗∗*P* < 0.0001 OVA + T group *vs* OVA group, ^####^*P* < 0.0001 OVA group *vs* Control group. OVA, ovalbumin.Fig. 6

Full text

Introduction

1

Food allergy has emerged as a significant global health challenge, with its prevalence exhibiting a concerning upward trajectory worldwide [1,2]. This condition not only severely compromises patients' quality of life but also poses substantial risks of life-threatening anaphylactic reactions. While research efforts have yielded important insights into food allergy mechanisms, the precise pathogenesis remains incompletely elucidated. The skin and mucosal barriers, serving as the primary interface between the host and external environment, play a pivotal role in allergen sensitization and immune regulation. Emerging evidence suggests that disruption of these epithelial barriers significantly increases susceptibility to food allergy development and exacerbation of existing conditions [3,4]. Various pathological conditions, including mechanical trauma, thermal injury, microbial infections, and chronic inflammatory disorders such as atopic dermatitis, can compromise epithelial barrier integrity [5]. These insults not only impair the physical barrier function but also induce profound alterations in the local immune microenvironment, potentially modulating the host's immune response to food allergens through mechanisms involving inflammatory cell infiltration and mediator release [6].

Recent advancements have particularly highlighted the critical role of epithelial barrier dysfunction in food allergy pathogenesis, with growing evidence supporting the skin-gut axis hypothesis [7,8]. Among experimental approaches to investigate this phenomenon, tape-stripping has emerged as an exceptionally valuable and clinically relevant model. This technique, widely employed in medical practice, effectively replicates key aspects of epithelial barrier disruption while maintaining high translational relevance. Preclinical studies utilizing repeated tape-stripping protocols have demonstrated its capacity to exacerbate ovalbumin (OVA)-induced food allergy, manifesting as localized epithelial remodeling, behavioral changes, and robust type 2 immune responses [[9], [10], [11], [12]]. These findings not only validate clinical observations but also establish tape-stripping as an indispensable experimental paradigm for elucidating the mechanisms underlying food allergy exacerbation. However, despite these advances, the precise molecular and immunological mechanisms linking epithelial barrier damage to food allergy exacerbation remain incompletely understood. This is particularly true for the relative importance of key genes and the temporal sequence of signaling pathways activated in the skin versus the gut, which warrant further investigation.

Transcriptomic analysis has revolutionized our ability to investigate complex biological processes, providing comprehensive insights into gene expression dynamics, post-transcriptional modifications, and non-coding RNA regulation [13]. The spatiotemporal specificity of transcriptomic profiles offers unique advantages over genomic approaches, enabling detailed analysis of gene expression patterns and functional networks within specific biological contexts [14,15]. In this study, we employed a sophisticated transcriptomic approach to investigate the mechanisms underlying tape-stripping-induced exacerbation of OVA-induced food allergy. Through comparative analysis of transcriptomes from tape-stripped and non-stripped food-allergic mice, we aimed to elucidate the complex interplay between epithelial barrier disruption and immune dysregulation in food allergy pathogenesis. Our investigation focused on identifying key molecular pathways and regulatory factors across different tissues, with particular emphasis on immune cell activation and mediator release following epithelial barrier damage. This comprehensive approach not only provides critical insights into the pathogenesis of food allergy but also establishes a robust theoretical framework for developing novel therapeutic strategies targeting barrier restoration and immune modulation in clinical practice.

Materials and methods

2

Animals

2.1

Four-week-old female BALB/c mice (weighing 18±2 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd, under standard pathogen-free conditions. All animal experiments were performed in accordance with ARRIVE guidelines and the National Institutes of Health Animal Research Guidelines (NIH Publications No.8023, revised 1978).

The food allergy model experiment involved dividing 27 mice into three groups (n = 9 per group), OVA group, OVA + T group and Control group. 18 mice in two OVA-challenged groups received intraperitoneal injections of 200ug OVA (No. A5503, Sigma) in 1 mg alum adjuvant (No. 239186, Sigma), dissolved in PBS to make it a volume of 200ul on days 0, 5 and 10. On days 15, 17, 19, 21, 23, 25, mice were challenged with intragastric gavage (i.g.) administration of 50 mg of OVA in 200ul PBS every other day for 6 times.To strengthen the food allergy model, the skin of mice in the OVA + T group was tape-stripped once on days 15, 17, 19, and 21, half an hour before each oral challenge. Mice were sensitizated and challenged with the same amount of PBS in the Control group (Fig. 1).Fig. 1. Experimental schedule of OVA-induced mice with tape stripping. 27 mice were devided into three groups (n = 9 per group), Control group, OVA group, OVA + T group. Mice in all groups except the Control group were intraperitoneally (i.p.) immunized with 200 μg OVA in 1 mg alum adjuvant dissolved in PBS 200ul on the days 0, 5 and 10, on days 15,17,19, 21, 23, 25, mice with oral gavage (i.g.) were challeged with 50 mg of OVA in 200ul PBS every other day for 6 times in the OVA group and OVA + T group. On days 15, 17, 19, 21, mice were taped stripping the skin in OVA + T group. Mice in the control group were i.p. injected and challenged with same amount of PBS. OVA, ovalbumin.Fig. 1

Diarrhea severity was assessed using a standardized 4-point scoring system:

0: Normal stool consistency, 1: Mild diarrhea (occasional wet, unformed stools),

2: Moderate diarrhea (multiple wet, unformed stools with visible perianal fur staining), 3: Severe diarrhea (watery stools with extensive perianal fur staining) [9,16]. The allergic response of the rectal temperature in each group was measured prior to the final challenge, and subsequent changes in rectal temperature were recorded and diarrhea was evaluated for a duration of up to 60 min following the last challenge. After the intervention was completed, mice were anesthetized via intraperitoneal injection of sodium pentobarbital, following which tissue samples were collected.

Isolation of single cells from the spleen

2.2

One hour subsequent to the final challenge, the spleen was removed and rinsed with ice-cold PBS. Single cells from the spleen were isolated by employing Lymphocyte Separation Medium kits (No. P8870, Solarbio) as per the manufacturer's guidelines. In brief, short pieces of the dissected spleen were obtained through a 40-μm mesh filter and delicately placed onto the surface of lymphocyte separation medium. After centrifugation at 900g for 30 min and two washes with wash buffer followed by centrifugation at 250g for 10 min, single cells of the spleen were successfully separated and readied for further experiments [17].

Western blot analysis

2.3

Total proteins were isolated from mouse intestinal jejunum tissue using RIPA buffer (HX1862-1, Huaxingbio) containing protease and phosphatase inhibitors. The homogenates were centrifuged at 15,000×g for 10 min at 4 °C, and the supernatants were collected. Protein concentration was determined using a BCA assay kit (B5001, LABLEAD). For each sample, an equal amount of 30 μg of total protein was resolved by SDS-PAGE (Ba1012, Baiqiandu). The protein was electroporated onto a PVDF membrane. The separated proteins were transferred to PVDF membranes (IPVH00010, Millipore), blocked with 5% milk (HX1866, Huaxingbio) for 1h, and then incubated with primary antibodies (1:1000), including occludin (Ab216327, Abcam), Zonula Occludens (ZO)-1 (221547, Abcam) and claudin (15098, Abcam), along with β-actin as a loading control, at 4 °C overnight. After 3 washes with TBST (Ba1024, Baiqiandu) for 5 min each, the membranes were incubated with secondary antibody, goat anti-rabbit IgG H&L (ab205718, Abcam) for 60 min at room temperature. Subsequently, the membranes were imaged using an EPSON scanner (L1548, Nagano Prefecture). The optical density of the membranes was analyzed by the Image J software processing system.

Histology and immunohistochemistry

2.4

Following euthanasia, the same tissue section from all mice was promptly fixed in 4% paraformaldehyde and subsequently processed for both paraffin and frozen sections. Hematoxylin and eosin staining were performed, along with toluidine blue (TB) staining to visualize mast cells in the jejunum [18]. Intestinal mucosal damage was assessed using a modified Sidhu-Harry scoring system (0: normal villus architecture; 1: mild epithelial injury [villus tip swelling]; 2: moderate damage [villus shortening/partial fusion]; 3: severe lesions [flattened villi/crypt hyperplasia]; 4: ulceration/necrosis).

Enzyme-linked immunosorbent assay

2.5

The levels of IgE, mucosal mast cell protease-1 (mMCP-1), histamine, IL-6 and IgG1 in the serum were quantified using commercially available ELISA kits following the manufacturer's instructions. IgE (No. 555248 & No. 550534, BD), mMCP-1 (No. 446207, Biolegend), histamine (No. CEA927Ge, USCN), IgG1 (No. EK0101, BOSTER). The procedure is as follows: First, pre-coat or coat 96-well plates with the capture antibody overnight at 4 °C. Then, add 100 μl of serum sample per well and incubate at 37 °C for 90 min. After five washes with wash buffer, add 100 μl of biotinylated detection antibody per well and incubate at 37 °C for 1 h. After another five washes, add streptavidin-horseradish peroxidase (100 μl/well) and incubate at about 37 °C for half an hour. Wash the wells five more times before adding substrate solution (100μl/well), followed by an incubation of 15 min at around 37 °C. Finally, add a stop solution and measure the optical density values at a wavelength of 450 nm.

RNA isolation, cDNA library preparation, and RNA-seq

2.6

RNA sequencing was performed on jejunum tissue samples derived from three randomly selected mice per group (OVA and OVA + T), resulting in a total of six samples analyzed. Agarose gel electrophoresis was used to assess the integrity of RNA and detect the presence of DNA contamination in skin and jejunum samples. Subsequently, the constructed library was sequenced using Novaseq 6000 (Illumina, USA).

The preferred sequencing fragments were subjected to high-throughput testing. The image data acquired from the sequencing instrument was converted into base calls using CASAVA software. To ensure the quality and reliability of data analysis, the sequence data (reads) underwent stringent quality control measures. Raw data was filtered to obtain clean data, upon which all subsequent analyses were based. High-quality clean data was then utilized for further processing.

First, the reference genome and gene model annotation files were downloaded using HISAT2 v2.0.5. An index of the reference genome was then constructed, and paired-end clean reads were aligned to the reference genome using HISAT2 v2.0.5. StringTie (version 1.3.3b) was employed for preliminary testing of new etiologies. FeatureCounts (version 1.5.0-p3) was subsequently used to calculate the number of reads mapped to each gene, and the FPKM values of each gene were calculated based on read counts and gene length to quantify gene expression levels. Differential expression analysis among biological replicates was conducted using DESeq2 (version 1.20.0), and differentially expressed genes were identified. GO and KEGG pathway enrichment analyses were performed using clusterProfiler (version 3.8.1) to screen for potential signal transducers. Finally, protein-protein interaction (PPI) networks of differentially expressed genes were analyzed using the STRING database, and core targets were identified.

qRT-PCR validation

2.7

Following the 6th oral challenge, jejunal tissue samples from the Control group, OVAgroup, and OVA + T group were immediately collected, rinsed with RNase-free PBS, and snap-frozen in liquid nitrogen. Total RNA was isolated using the RNAprep Pure Tissue Kit (No. DP451, TIANGEN) with on-column DNase I treatment. RNA integrity was verified by agarose gel electrophoresis (RIN >7.0) and quantified by Nanodrop spectrophotometry (A260/A280 ratio≥1.8). First-strand cDNA synthesis was performed with 1 μg total RNA using the Exscript RT Reagent Kit (No. RR036A, TAKARA) with random hexamer under the following conditions: 37 °C for 15 min, 85 °C for 5 s. Quantitative real-time PCR was conducted in triplicate using the Mx3000P system (Stratagene) with TB Green Premix Ex Taq II (No. RR820A,TAKARA Bio) under optimized cycling parameters: Initial denaturation: 95 °C for 30 s, Amplification: 40 cycles of: 95 °C for 5 s (denaturation), 60 °C for 30 s (combined annealing/extension). Gene expression levels of target genes (Acta2, Kdr, Cxcr4) were normalized to the reference gene β-actin and calculated via the 2−ΔΔCt method. The following primer pairs were used.

• Acta2 forward 5′-CAGCCAAGCACTGTCAGGAAT-3′
• reverse 5′-CCATCACCCCCTGATGTCTG-3′
• Kdr forward 5′-TTCTTGGCTGTGCAAAAGTG-3′
• reverse 5′-TCTTCAGTTCCCCTCCATTG-3′
• Cxcr4 forward 5′-CAGTGGCCGACCTCCTCTT-3′
• reverse 5′-GGACTGCCTTGCATAGGAAGTT-3′

Statistical analysis

3

Data were analyzed by GraphPad Prism v8 (GraphPad Software, San Diego,

CA, USA, www.graphpad.com) and SPSS version 21.0 (IBM SPSS, Chicago, USA). Normally distributed data are expressed as the mean ± standard deviation (SD). Two groups of parametric variables were compared by a t-test, while one-way analysis of variance (ANOVA) was applied for multi-group comparisons. For non-normally distributed data, the Mann-Whitney U test was used for two-group comparisons, and the Kruskal-Wallis H test was employed for comparisons among multiple groups. P < 0.05 was considered statistically significant.

Results

4

Tape-stripping aggravated histological pathological changes of skin and intestinal tissue

4.1

Tape stripping significantly exacerbated the hypothermic response (measured by rectal temperature drop) at the 30-min time point following the sixth oral challenge, while also intensifying anaphylactic diarrhea in OVA-sensitized mice (All P < 0.01) (Fig. 2a and b). HE staining was conducted, and it was found that, in comparison with the food allergy mice, tape-stripping led to an exacerbation of local injury in the skin and an increase in the number of inflammatory cells within the intestinal tissues (Fig. 2c and d). Similarly, TB staining demonstrated that tape-stripping resulted in a growing infiltration of mast cells in both the skin and intestinal tissues (Fig. 2e and f). Strikingly, tape stripping induced pronounced jejunal histopathological alterations in food-allergic mice, with a significant increase in overall pathology scores (P = 0.0000) (Fig. 2g).Fig. 2. Tape-stripping effectively exergerated (a) rectal temperature changes at the 30-min time point following the sixth oral challenge and (b) anaphylactic diarrhea in OVA-induced mice (##P < 0.01, OVA + T group vs. OVA group, ∗∗∗P < 0.001,∗∗∗∗*P = *0.000, n = 9). Observation in the (c) jejunal and (d) skin tissue by hematoxylin and eosin staining with the tape-stripping (magnification, × 200). Mast cell filtration by Toluidine blue staining in the (e) jejunal and (f) skin tissue with the tape-stripping (magnification, × 200). (g) Histopathological scores in the jejunum across different groups (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001,∗∗∗∗P = 0.0000. n = 9). Edema, inflammational cells infiltration (red and black arrows), showed thickening of the acanthoderm (blue arrows), multifocal collagen fibroplasia of the dermis (green arrow).Fig. 2

Tape-stripping aggravated inflammation in the serum of food allergy mice

4.2

Serum samples were collected an hour after the final challenge on day 25 and stored at −80 °C before being analyzed using ELISA. Significant alterations were observed in the levels of IgE, histamine, IL-6, IgG1 expression (Fig. 3a–d). The level were aggravated by administration of tape-stripping.Fig. 3. Inflammatory cytokines levels in serum measured by ELISA. Results of (a) IgE, (b) histamine, (c) IL-6, and (d) IgG1 of mice in different groups. Each value indicates mean ± standard error of the mean. n = 9/group. (e) CD4^+^ Foxp3^+^ Tregs in CD45^+^ T cells proportion in the spleen. (f) Proportion of IgE + cells in the spleen. n = 3/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. IL, interleukin. CD, the cluster of differentiation; Fox, forkhead box.Fig. 3

Tape-stripping increased CD4+ Foxp3+ Tregs in CD45+ T cells proportion and released IgE + cells population in the spleen

4.3

We investigated the impact of tape-stripping on the expression of surface markers on CD45^+^ T cells in the spleen. Flow cytometry analysis revealed a significant decrease in the proportion of CD4^+^ Foxp3+ Tregs within CD45^+^ T cells in the food allergy mice, tape-stripping exeragerated the reduction of Tregs proliferation (Fig. 3e). Furthermore, tape-stripping elevated the population ratio of IgE + cells in the spleen compared to food allergy mice (Fig. 3f).

Tape-stripping released intestinal barrier function in food allergy mice

4.4

The effect of tape-stripping on intestinal barrier function was assessed by examining tight junction proteins occludin, claudin, and ZO-1 in the intestinal epithelium. Western blot analysis revealed that tape-stripping significantly reduced the protein expression levels of occludin, claudin-1, and ZO-1 (p < 0.001), suggesting a substantial disruption of intestinal epithelial barrier integrity (Fig. 4a and b).Fig. 4(a) Occludin, Claudin, ZO-1 protein levels in the small intestine in different groups. (b) The protein quantified as band density in different groups. This experiment was performed independently three times. Each value represents the means ± SD (n = 3). ∗∗∗P < 0.001 vs. OVA group; ###P < 0.001 vs. OVA + T group.Fig. 4

Global analysis of the RNA-Seq data

4.5

To determine the effects of tape-stripping in OVA-induced mice, we identified the expressed genes in the OVA and OVA + T groups based on the threshold criteria of a log 2 fold change≥ 1 and FDR≤ 0.05. In total, we detected 2790 expressed genes in the samples of two groups. Compared with the skin tissues of the OVA group, we discovered that five differential genes were identified in the skin tissues after percutaneous sensitization, among which there were four up-regulated genes, namely Angpt4, Fos, Krt6b, and Draxin, and one down-regulated gene, Tcf15. However, 444 differential genes were identified in the intestinal tissues, including 110 up-regulated differential genes and 334 down-regulated differential genes, as well as top 10 DEGs (Fig. 5a). In the 444 different genes, compared to the OVA group, the three most significantly DEG-enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the OVA + T group were Calcium signaling pathway, Adrenergic signaling in cardiomyocytes, and Vascular smooth muscle contraction (Fig. 5b). Gene counts of Gene ontology (GO) terms distribution in each level were conducted, related to the 444 genes, The three most significantly upregulated DEG-enriched Biological process (BP) pathways in jejunum were angiogenesis, ion transport, negative regulation of apoptotic process. The three most significantly DEG-enriched Cellular Component (CC) pathways in jejunum were membrane, cytoplasm, and plasma membrane. The three most significantly upregulated DEG-enriched Molecular function (MF) pathways in jejunum were protein binding, metal ion binding, and identical protein binding (Fig. 5c).Fig. 5. Results of RNA seq in the intestinal tissue with the tape-stripping compared to the OVA-induced mice in the OVA + T vs. OVA group. (a) The Volcano plot of 444 differential gene expression, top 10 upregulated genes and top 10 downregulated genes. (b) The top KEGG pathways with significantly enrichment of differentially expressed genes (DEGs). (c) Gene counts of Gene ontology (GO) terms distribution in each level. (d) The top KEGG pathways with significantly upregulated enrichment of DEGs. (e) Gene counts of GO terms distribution in each level in the upregulated enrichment of DEGs. (f) The top KEGG pathways with significantly downregulated enrichment of DEGs. (g) Gene counts of GO terms distribution in each level in the downregulated enrichment of DEGs. (h) PPI network core targeting analysis. KEGG, Kyoto Encyclopedia of Genes and Genomes; DEG, differentially expressed genes; GO, gene ontology; BP, Biological process; CC, Cellular Component; MF, Molecular function.Fig. 5

We respectively conducted enrichment analysis of the up-regulated and down-regulated differential genes. The results showed that compared to the OVA group, in the 110 significantly upregulated genes, compared to the OVA group, the top three significantly DEG-enriched KEGG pathways in the OVA + T group were Cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, and MAPK signaling pathway (Fig. 5d). Related to the 110 upregulated genes, the three most significantly upregulated DEG-enriched BP pathways in jejunum were positive regulation of transcription by RNA polymerase II, angiogenesis, inflammatory response. The three most significantly upregulated DEG-enriched CC pathways in jejunum were plasma membrane, extracellular space, extracellular region. The three most significantly upregulated DEG-enriched MF pathways in jejunum were protein binding, RNA polymerase II cis-regulatory region sequence-specific DNA binding, sequence-specific DNA binding (Fig. 5e).

Compared to the OVA group, related to the 334 significantly downregulated genes, the three most significantly downregulated DEG-enriched KEGG pathways in the OVA + T group were Vascular smooth muscle contraction, cGMP-PKG signaling pathway, Calcium signaling pathway (Fig. 5f). The three most significantly downregulated DEG-enriched BP pathways in jejunum were ion transport, transmembrane, potassium ion transmembrane transport. The three most significantly downregulated DEG-enriched CC pathways in jejunum were Membrane, Cytoplasm, Plasma membrane. The three most significantly downregulated DEG-enriched MF pathways in jejunum were protein binding, metal ion binding, identical protein binding (Fig. 5g).

From the comparison between the OVA and OVA + T groups, 444 DEGs were identified. These genes were then uploaded to the STRING website to establish a Protein-Protein Interaction (PPI) network. Using the Cytoscape software, the top three differential genes were selected based on node degree values (Fig. 5h). The results suggested that Acta2, Kdr, and Cxcr4 could be the pivotal target genes by which tape-stripping potentiates food allergy.

Validation of transcriptomic findings by qRT-PCR

4.6

To validate our RNA-seq results, we quantified the mRNA expression levels of three key candidate genes (Acta2, Kdr, and Cxcr4) identified through transcriptomic analysis using quantitative real-time PCR. Compared to the Control group, the OVA group showed baseline elevation of Acta2, Kdr, and Cxcr4, which was further exacerbated by tape-stripping (all P < 0.05, Fig. 6).Fig. 6qRT-PCR validation of tape-stripping-enhanced gene expression in OVA-induced allergic mice. n = 4. ∗P < 0.05,∗∗∗∗P < 0.0001 OVA + T group vs OVA group, ^####^P < 0.0001 OVA group vs Control group. OVA, ovalbumin.Fig. 6

Discussion

5

Our study demonstrates that tape-stripping exacerbates allergic diarrhea, induces body temperature changes, and causes significant histopathological alterations in both the skin and intestinal tissues of OVA-induced food allergy mice. Histological analyses revealed increased local skin damage, inflammatory cell infiltration in the intestine, and elevated mast cell infiltration in both tissues. These findings align with previous studies [19] and suggest that tape-stripping disrupts the structural and immune homeostasis of the skin and intestine, potentially exacerbating inflammatory responses. This highlights the role of the skin-gut axis in food allergy pathogenesis.

Immune dysregulation and barrier disruption

5.1

In our study tape-stripping significantly impacted regulatory T cell (Treg) populations and IgE + cell populations in the spleen. IgE + cells, critical mediators of allergic reactions, were elevated, indicating a heightened allergic state. Histamine release, closely associated with allergic symptoms such as vasodilation and pruritus, and IL-6, a key inflammatory cytokine, were also significantly increased. The skin, as the body's primary barrier, may facilitate allergen penetration upon damage, subsequently affecting distal tissues such as the intestine [[20], [21], [22], [23]]. Tregs play a crucial role in immune regulation by suppressing type 2 immune cells, including Th2 cells, type 2 innate lymphoid cells, and IgE-producing B cells, while promoting tolerogenic dendritic cells, regulatory B cells, and IgG4-producing B cells [24,25]. The observed increase in Tregs following tape-stripping may represent a compensatory mechanism to counteract excessive immune activation. These findings suggest that tape-stripping disrupts the skin barrier, induces inflammatory mediator release, and triggers systemic immune responses, ultimately exacerbating food allergy.

Experimentally, tape-stripping in OVA-sensitized mice induced robust, quantifiable signs of neural hyperexcitability, most notably increased vocalization (squeaking) and compulsive scratching. These behavioral phenotypes thus implicate the activation of peripheral sensory neurons within our skin-gut axis model. We propose that this neurogenic component constitutes a plausible and testable mechanism underlying the observed crosstalk, although the precise molecular signaling pathways merit further investigation.

Intestinal barrier dysfunction

5.2

Tight junction proteins, such as occludin, ZO-1, and claudin, are critical for maintaining intestinal barrier integrity. Their downregulation, as observed in our study, is associated with increased intestinal permeability, facilitating the translocation of bacteria, toxins, and other harmful substances into systemic circulation, thereby exacerbating inflammation [[26], [27], [28], [29], [30], [31]]. Western blot analysis confirmed a significant reduction in these proteins following tape-stripping, likely due to systemic inflammatory responses triggered by the procedure. The upregulation of genes involved in protein binding, metal ion binding, and identical protein binding in the jejunum further supports the link between barrier dysfunction and systemic inflammation (Fig. 5c).

Transcriptomic insights and molecular mechanisms

5.3

Transcriptomic analysis revealed that tape-stripping induces significant gene expression changes in both skin and intestinal tissues. In the skin, Angpt4, Fos, Krt6b, and Draxin were upregulated, while Tcf15 was downregulated. These genes are implicated in vascular stability, immune cell maturation, and skin immune responses, which are closely associated with inflammatory disease pathogenesis [[32], [33], [34], [35], [36], [37]].

In the intestine, 444 differentially expressed genes (DEGs) were identified, with significant enrichment in pathways such as calcium signaling, adrenergic signaling in cardiomyocytes, and vascular smooth muscle contraction (Fig. 5b). Upregulated genes were enriched in cytokine-cytokine receptor interactions, PI3K-Akt signaling, and MAPK signaling pathways (Fig. 5d), suggesting their pivotal roles in immune dysregulation. Conversely, downregulated genes were associated with vascular smooth muscle contraction and calcium signaling, potentially reflecting compensatory anti-inflammatory mechanisms (Fig. 5f). Protein-protein interaction (PPI) network analysis identified Acta2, Kdr, and Cxcr4 as key target genes mediating tape-stripping-induced food allergy exacerbation (Fig. 5h).

Mechanistically, our findings indicate that tape-stripping induces marked inflammatory infiltration in the skin and is associated with the dysregulation of key genes, including Acta2, Kdr, and Cxcr4. These molecules may represent promising therapeutic targets for mitigating tape-stripping-induced food allergy. The angiopoietin pathway emerges as a key regulator, with Angpt4 demonstrating dual functionality in vascular homeostasis and inflammatory modulation [38,39]. Our experimental model reveals that repetitive mechanical injury (tape-stripping) initiates a multistep pathogenic cascade: (i) Angpt4 upregulation enhances cutaneous vascular permeability while paradoxically maintaining epithelial integrity through Akt-mediated signaling; (ii) Concurrent Krt6b overexpression induces keratinocyte dysfunction, triggering Fos-dependent proinflammatory mediator release that systemically activates intestinal Kdr expression; (iii) Persistent barrier disruption elevates Tcf15 expression, disrupting cutaneous immune regulation and establishing a feed-forward inflammatory loop through sustained Fos activation; (iv) This systemic inflammatory milieu promotes intestinal Acta2-mediated fibrotic remodeling and Cxcr4-dependent recruitment of eosinophils/neutrophils, collectively compromising intestinal barrier function [[40], [41], [42]]. Our data point toward a skin-gut axis as a potential mechanistic framework in food allergy. The association between skin inflammation and specific intestinal gene alterations, while requiring mechanistic validation, forms a credible hypothesis and identifies these interactions as targets meriting exploration for therapeutic intervention. Future studies could explore pharmacological or genetic interventions targeting these genes to restore immune balance and barrier function.

Additionally, the upregulation of PI3K-Akt and MAPK signaling pathways in the jejunum suggests that these pathways may play a central role in mediating the systemic immune dysregulation observed in tape-stripped mice. PI3K-Akt signaling is known to promote cell survival and inflammatory responses, while MAPK signaling regulates cytokine production and immune cell activation [43,44]. Future studies should investigate whether pharmacological inhibition of these pathways can alleviate tape-stripping-induced food allergy.

The findings of this study have important clinical implications for the management of patients with allergic conditions, particularly those with compromised skin barriers, such as individuals with atopic dermatitis or food allergies. Tape-stripping, a common procedure in clinical settings for securing medical devices or dressings, may inadvertently exacerbate allergic responses by disrupting the skin barrier and triggering systemic inflammation. Therefore, it is crucial to explore safer alternatives to traditional adhesive tapes for patients with sensitive or allergy-prone skin.

Despite these findings, this study has several limitations. First, the precise cascade linking tape-stripping-induced skin inflammation with the dysregulation of gene expression and signaling pathways remain unclear. Second, the temporal dynamics of immune cell infiltration and cytokine production following tape-stripping were not fully explored. Future studies should focus on the following aspects: (i) the role of Acta2, Kdr, and Cxcr4 in mediating immune dysregulation using knockout or overexpression models; (ii) the temporal dynamics of immune cell infiltration and cytokine production following tape-stripping; and (iii) the potential therapeutic effects of targeting tight junction proteins to restore intestinal barrier function.

In conclusion, this study demonstrates that tape-stripping exacerbates pathological changes, inflammatory responses, and immune cell redistribution in the spleen, while compromising intestinal barrier integrity and inducing widespread gene expression alterations in OVA-induced food allergy mice. These findings provide a theoretical foundation for understanding the interplay between skin barrier disruption and allergic reactions and offer novel insights for the prevention and treatment of allergic diseases. However, the precise mechanisms by which tape-stripping influences signaling pathways and gene expression remain unclear, warranting further investigation. Future studies should focus on validating these findings and exploring therapeutic strategies targeting the identified molecular pathways.

Ethics approval

This study was approved by the Animal Experiment Committee at Beijing Center for Physical&Chemical Analysis (No. 210520-SWDWF-005).

Author contributions

YZ and ZL designed the study. YZ and JZ performed experiments. YZ analyzed the data. XZ provided insightful discussions on the manuscript. ZW and QW confirmed the authenticity of all the raw data. All authors read and approved the final manuscript.

Funding sources

The study was supported by National Key Clinical Specialty Construction program (No.2023(09), Health commission of Shanxi Province) and "San Jin Ying Cai" program (No.SJYC2024200).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.