Demographic and Clinical Characteristics of the Study Subjects

The demographic and clinical characteristics of the study participants are summarized in Table 1. Of the 43 AR patients, 9 had a history of smoking and 6 reported a history of asthma. In contrast, the control group included 4 smokers, with no reported cases of asthma. Eosinophil counts were significantly higher in AR patients compared to controls (p < 0.0001). Additionally, 6 AR patients tested positive in skin prick tests, while the remaining 37 showed significantly higher blood Phadiatop Test results compared to the control group (p = 0.0006). Symptom severity of AR patients, assessed by the Visual Analog Scale (VAS), yielded an average score of 6.256 ± 1.989 out of 10.

Table 1 Basic demographic and clinical characteristics of subjects

Data were analyzed using an independent samples t-test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

In the "Allergic rhinitis" group, data for "Blood Phadiatop Test, kU/L" were obtained from 37 patients, while the remaining 6 patients tested positive for allergen skin prick tests

Gene Expression of Piezo1, ERK and TJs in Human Nasal Biopsies

To assess the gene expression of Piezo1 in AR, qRT-PCR was performed on nasal mucosal tissues from both groups. The results showed that Piezo1 mRNA level were significantly elevated in the AR group compared to the controls (p < 0.01, Fig. 1A). Given the established link between AR and epithelial barrier dysfunction, we also examined the expression of TJs in the nasal mucosa. The mRNA levels of ZO-1 (p < 0.001), Occludin (p < 0.01), and Claudin-1 (p < 0.01) were significantly lower in the AR group compared to the control group (Fig. 1D-F). Additionally, the AR group exhibited significantly lower mRNA expression levels ERK1 (p < 0.001) and ERK2 (p < 0.01) compared to the controls (Fig. 1B and C).

Fig. 1

mRNA expression levels of Piezo1, ERK1 and 2, and tight junctions components in human nasal mucosal tissues. (A-F) mRNA levels of Piezo1, ERK1 and 2, ZO-1, Occludin, and Claudin-1 as determined by qRT-PCR in the control group (n = 36) and AR group (n = 43). For clarity, the AR group samples include 1 sample from the ethmoid bullae, 20 from the middle turbinate, and 22 from the inferior turbinate, while the control group includes 2 samples from the ethmoid bullae, 17 from the middle turbinate, and 17 from the inferior turbinate. *p < 0.05, **p < 0.01, ***p < 0.001

Correlation Analysis of Piezo1, ERK1/2, and Tight Junction Genes

To further investigate the relationship between Piezo1 and TJs, a correlation analysis was conducted (Table 2). Piezo1 mRNA expression showed a negative correlation with Claudin-1 (r = −0.3514, p = 0.0209), Occludin (r = −0.5716, p < 0.0001), and ZO-1 (r = −0.524, p = 0.0003). Given the significant role of the ERK pathway in inflammation and allergic responses, we also analyzed the correlations between Piezo1 and ERK1/2, as well as between ERK1/2 and TJs. The results indicated that Piezo1 mRNA levels were inversely correlated with ERK1 (r = −0.5421, p = 0.0002) and ERK2 (r = −0.5609, p < 0.0001). Conversely, ERK1 mRNA levels were positively correlated with ERK2 (r = 0.9164, p < 0.0001), Claudin-1 (r = 0.612, p < 0.0001), Occludin (r = 0.8194, p < 0.0001), and ZO-1 (r = 0.876, p < 0.0001). Similarly, ERK2 mRNA levels were positively correlated with Claudin-1 (r = 0.6566, p < 0.0001), Occludin (r = 0.8304, p < 0.0001), and ZO-1 (r = 0.8982, p < 0.0001).

Table 2 Correlation analysis of genes expression in ARProtein Expression of Piezo1, ERK1/2, and Tight Junctions

To confirm the protein-level expression of Piezo1, ERK1/2 and TJs, we conducted Western blot analysis on nasal mucosal tissues from both groups (Fig. 2A). The results were consistent with the mRNA findings. Piezo1 protein levels was significantly elevated in the AR group compared to the controls (p < 0.05, Fig. 2B). In contrast, the protein levels of ZO-1 (p < 0.01), Occludin (p < 0.01), and Claudin-1 (p < 0.01) were markedly lower in the AR group (Fig. 2D-F). Similarly, ERK1/2 protein levels were significantly reduced in the AR group (p < 0.01) ( Fig. 2C).

Fig. 2

Protein expression levels of Piezo1, ERK1/2, and TJs in human tissues. (A-F) Protein levels of Piezo1, ERK1/2, ZO-1, Occludin, and Claudin-1 as analyzed and quantified by Western blotting (n = 6). All samples were collected from the middle turbinate. *p < 0.05, **p < 0.01

Subcellular Localization of Piezo1 in Nasal Mucosal Tissues

The subcellular localization of Piezo1 in nasal mucosal tissues was analyzed using immunofluorescence staining (Fig. 3). Piezo1, a mechanosensitive channel protein, was predominantly localized to the apical layer of nasal epithelial cells. The results showed that Piezo1 expression was significantly higher in the AR group compared to the control group, as indicated by an increased green fluorescence intensity (p < 0.01, Fig. 3D). Immunofluorescence staining for TJs proteins, including Claudin-1 (p < 0.001, Fig. 3A and E), ZO-1 (p < 0.001, Fig. 3B and F), and Occludin (p < 0.01, Fig. 3C and G) showed significantly reduced fluorescence intensities in the AR group relative to the controls. Furthermore, colocalization analysis demonstrated that Piezo1 was colocalized with TJs proteins in human nasal epithelial tissues.

Fig. 3

Distribution of Piezo1 and TJs in human tissues. Immunofluorescence staining of the subcellular localization of Piezo1 (green) in human tissues. (A) Expression of Claudin-1 (red) fluorescence in AR patients and the control group. Scale bar = 40 µm. (B) Expression of ZO-1 (red) fluorescence in AR patients and the control group. Scale bar = 40 µm. (C) Expression of Occludin (red) fluorescence in AR patients and the control group. Scale bar = 40 µm. Quantitative fluorescence analysis showed that the expression of Piezo1 was upregulated in the nasal mucosal tissues of AR patients compared to the control group, while Claudin-1, ZO-1 and Occludin were dowregulated (D-G, control group n = 15, AR group n = 18)

Relationship Between AR Symptoms (VAS) and Piezo1, ERK1/2, and Tight Junctions

The relationship between AR symptom severity, assessed by the Visual Analog Scale (VAS), and the expression levels of Piezo1, ERK1/2, and tight junction (TJ) genes was further analyzed. Interestingly, a significant positive correlation was observed between AR VAS scores and Piezo1 mRNA levels (r = 0.6229, p < 0.0001). In contrast, AR VAS scores were significantly negatively correlated with ERK2 mRNA levels (r = −0.3158, p = 0.0391), Occludin (r = −0.3909, p = 0.0095), and ZO-1 (r = −0.3038, p = 0.0477) (Table 3).

Table 3 Correlation analysis between VAS score and genes expressions of Piezo1, ERK1/2, and TJs in ARGene and Protein Expression of Piezo1, ERK, and Tight Junctions in Mouse

To further explore the role of Piezo1,ERK and TJs in the context of allergic rhinitis (AR), we evaluated their expression in nasal mucosal tissues from AR mouse models. The models were established based on validated methods [22]. At the transcriptional level, qRT-PCR revealed a significant increase in Piezo1 mRNA expression in the AR group compared to the controls (p < 0.01, Fig. 4A). Conversely, the mRNA levels of ZO-1 (p < 0.01, Fig. 4D), Occludin (p < 0.05, Fig. 4E), and Claudin-1 (p < 0.05, Fig. 4F) were significantly lower in the AR group. Additionally, both ERK1 (p < 0.01, Fig. 4B) and ERK2 (p < 0.05, Fig. 4C) mRNA levels were markedly reduced in the AR group.

Fig. 4

mRNA expression levels of Piezo1, ERK1/2, and TJs in mouse nasal mucosal tissues. (A-F) The mRNA levels of Piezo1, ERK1/2, ZO-1, Occludin, and Claudin-1 were determined by qRT-PCR (n = 6). *p < 0.05, **p < 0.01

At the protein level, Western blot analysis corroborated these findings (Fig. 5A). Piezo1 protein expression was significantly higher in the AR group compared to controls (p < 0.01, Fig. 5B). In contrast, the protein levels of ZO-1 (p < 0.01, Fig. 5D), Occludin (p < 0.01, Fig. 5E), Claudin-1 (p < 0.01, Fig. 5F), and ERK1/2 (p < 0.01, Fig. 5C) were significantly reduced in the AR group, which is consistent with the mRNA results.

Fig. 5

Protein expression levels of Piezo1, ERK1/2, and TJs in mouse nasal mucosal tissues. (A-F) Protein levels of Piezo1, ERK1/2, ZO-1, Occludin, and Claudin-1 as analyzed and quantified by Western blotting (n = 6). *p < 0.05, **p < 0.01

Regulation of TJs by Piezo1 in HNEpCs

To determine whether Piezo1 directly regulates TJs, HNEpCs were transfected with shRNA lentivirus targeting Piezo1 at two distinct sites. After establishing a stable knockdown (KD) group, the cells were stimulated with IL-13 to mimic an inflammatory response and assess the interaction between Piezo1 and TJs.

qRT-PCR analysis revealed that Piezo1 mRNA levels were significantly reduced in both KD1 and KD2 groups compared to the negative control (NC) group (p < 0.05 and p < 0.01, respectively, Fig. 6A). In the KD1 group, mRNA levels of ZO-1 (p < 0.01, Fig. 6D), Occludin (p < 0.01, Fig. 6E), Claudin-1 (p < 0.05, Fig. 6F), and ERK1/2 (p < 0.05, Fig. 6B, C) were significantly upregulated. The KD2 group exhibited a similar trend in ZO-1, Claudin-1, ERK1, and ERK2 mRNA levels; however, these differences were not statistically significant compared to the NC group (p > 0.05, Fig. 6B-D, F).

Fig. 6

Expression levels of ERK1/2 and TJs following Piezo1 knockdown in HNEpCs..MRNA levels of Piezo1, ZO-1, Occludin, Claudin-1, and ERK1/2 in two knockdown groups (KD1, KD2) as determined by qRT-PCR (n = 6)(A-F).Protein levels of Piezo1, ZO-1, Occludin, Claudin-1, and ERK1/2 in two knockdown groups (KD1, KD2) as analyzed and quantified by Western blotting (n = 3)(G-L). *p < 0.05, **p < 0.01, ***p < 0.001

Western blot analysis further confirmed the decrease in Piezo1 protein levels in both KD1 and KD2 groups (p < 0.05, Fig. 6H). In the KD1 group, ZO-1 (p < 0.01, Fig. 6J), Occludin (p < 0.05, Fig. 6K), Claudin-1 (p < 0.01, Fig. 6L), and ERK1/2 (p < 0.05, Fig. 6I) protein levels were significantly increased. In the KD2 group, ZO-1 and Claudin-1 protein expression showed an upward trend but did not reach statistical significance (p > 0.05, Fig. 6J, L).

Given the greater stability and effectiveness of KD1 in regulating gene expression, the KD1 group was selected for further experiments. Following IL-13 stimulation for 24 h, Piezo1 protein expression was significantly elevated in the NC group (p < 0.01). Occludin (p <0.05) and Claudin-1 (p < 0.01) protein levels, which were downregulated by IL-13 stimulation, were restored following Piezo1 knockdown (p > 0.05, Fig. 7D-E). Additionally, Piezo1 knockdown also influenced ERK1/2 protein expression (p < 0.05, Fig. 7C). Notably, even under inflammatory conditions, Claudin-1 expression remained significantly higher in the Piezo1 knockdown group compared to the NC group (p < 0.01, Fig. 7E).

Fig. 7

Expression and regulation of TJs by Piezo1 in HNEpCs. (A-E) Protein expression levels of Piezo1, ERK1/2, Occludin, and Claudin-1 after IL-13 stimulation, as analyzed and quantified by Western blotting (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001