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. 2025 Jun 14;398(12):17595–17607. doi: 10.1007/s00210-025-04338-x

Alpha-asarone relieves nasal inflammation, epithelial barrier damage, and mitochondrial damage in allergic rhinitis by inhibiting mitochondrial ROS via the SIRT1/PGC-1α pathway

Bin Xu 1, Weimin Gao 1, Haitong Li 1, Xiaojuan Cao 1, Xiaohong Chen 1, Nannan Dong 1, Lingfang Wu 1, Yunzhen Luo 1,✉
PMCID: PMC12678613  PMID: 40514518

Abstract

Allergic rhinitis (AR), a chronic inflammatory disease characterized by nasal congestion, sneezing, itching, and rhinorrhea, significantly impairs the quality of life for those affected. Current treatments have limitations due to adverse effects, highlighting the urgent need for novel therapeutic alternatives. This study investigates the protective effects of α-asarone (ASA) on nasal inflammation and epithelial barrier damage in AR, focusing on its modulation of mitochondrial reactive oxygen species (mtROS) via the SIRT1/PGC-1α pathway. Herein, a murine model of AR was established using ovalbumin (OVA) sensitization. ASA ameliorated AR symptoms, reduced IgE, histamine, and nasal mucosal inflammation in mice. It restored tight junction proteins and mitochondrial function markers in the nasal mucosa. In vitro, ASA pretreatment of IL-4/IL-13 challenged human nasal epithelial cells (HNEpCs) suppressed pro-inflammatory cytokines, preserved epithelial barrier integrity, mtROS, and maintained mitochondrial function. Mechanistically, ASA’s protective effects were mediated by mtROS inhibition. Using a SIRT1 inhibitor (EX527) and a PGC-1α activator (ZLN005), it was demonstrated that ASA upregulates SIRT1 to promote PGC-1α deacetylation, thereby suppressing mtROS, restoring mitochondrial function, and alleviating nasal inflammation and epithelial barrier damage. SIRT1 inhibition markedly reduced ASA therapeutic effects, highlighting the critical role of the SIRT1/PGC-1α pathway. These results indicate that ASA mitigates nasal inflammation and epithelial barrier damage in AR by suppressing mtROS via the SIRT1/PGC-1α pathway. As a natural agent, ASA presents a promising AR treatment alternative with potentially fewer side effects than conventional therapies.

Keywords: Alpha-asarone, Inflammation, Epithelial barrier damage, Mitochondrial damage, Mitochondrial ROS, SIRT1/PGC-1α

Introduction

Allergic rhinitis (AR) is a prevalent chronic inflammatory disorder marked by symptoms including nasal obstruction, sneezing, itching, and rhinorrhea, which notably affects the quality of life for AR patients (Howarth et al. 2000). This condition affects approximately 10–30% of the global population, with increasing incidence over recent decades (Busold et al. 2023). Current treatments for AR include antihistamines, corticosteroids, and immunotherapy; however, long-term application of these drugs would induce undesirable side effects (May and Dolen 2017). As a result, there is a growing need for potential products that exhibit reduced toxicity and fewer adverse effects.

In AR, a Th2-dominant immune response was induced (Zhao et al. 2022). Consequently, Th2-associated cytokines, such as IL-4 and IL-13, trigger IgE activation, leading to the infiltration of inflammatory cells within the nasal mucosa (Ding et al. 2023). The epithelial barrier serves as the primary defense against various risk factors, ensuring homeostasis in the nasal mucosa (Gohy et al. 2020). Prior research has shown that a compromised nasal epithelial barrier is involved in AR pathogenesis (Steelant, et al. 2018). The disruption of the epithelial barrier results in increased permeability, exposure to allergens, and the production of pro-inflammatory cytokines, further aggravating the epithelial barrier damage and inflammation (Sun et al. 2022). Mitochondria, energy-generating organelles of the cell, are essential for cellular homeostasis by maintaining energy production, cellular metabolism, and the generation of reactive oxygen species (ROS) (Komaragiri et al. 2022). The overproduction of mitochondrial ROS (mtROS) has been reported to induce mitochondrial dysfunction, disrupting epithelial barrier function and inducing inflammation (Kim et al. 2022). In AR, excessive mtROS production has been implicated in the inflammation of the nasal mucosa (Shi et al. 2018). Therefore, targeting mtROS could help restore nasal epithelial barrier function and reduce inflammation in AR.

Sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, is a key regulator of cellular metabolism (Haigis and Sinclair 2010). Studies have shown that SIRT1 can promote mitochondrial biogenesis by activating peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α), a key regulator of mitochondrial biogenesis and function (Liang and Ward 2006), through its deacetylation (Gurd 2011). The SIRT1/PGC-1α pathway is crucial in maintaining epithelial barrier integrity and mitigating inflammation by inhibiting mtROS overproduction and preserving mitochondrial function (Zheng et al. 2023). Emerging evidence has demonstrated the protective role of SIRT1 in AR by attenuating AR symptoms, reducing inflammation, and inhibiting mucus formation (Li et al. 2020; Huang et al. 2021). In addition, PGC-1α has been reported to improve allergic asthma-relevant epithelial barrier dysfunction by promoting mitochondrial biogenesis (Saito et al. 2021). However, whether the SIRT1/PGC-1α pathway is involved in nasal inflammation, epithelial barrier damage, and mitochondrial dysfunction in AR remains unclear.

Alpha-asarone (ASA), a bioactive natural compound that can be found in Asarum and Acorus species, has garnered attention for its potent anti-inflammatory, antioxidant, and neuroprotective properties (Shin et al. 2014; Sutariya and Saraf 2018; Park et al. 2023). Besides, ASA also ameliorates allergic asthma by inhibiting mast cell activation and degranulation via the ERK/JAK2-STAT3 pathway (Bai et al. 2023), highlighting its anti-allergic effects. Interestingly, a recent study suggests that ASA can inhibit oxidative stress to protect mitochondrial integrity by promoting PGC-1α activity (Yan et al. 2023). Thus, ASA may exert therapeutic effects in AR by targeting the SIRT1/PGC-1α pathway.

The current investigation aims to investigate the effects of ASA on nasal epithelial barrier dysfunction and inflammation in AR, focusing on its role in modulating mitochondrial ROS and the SIRT1-dependent deacetylation of PGC-1α. By elucidating the mechanisms through which ASA exerts its protective effects, this research seeks to identify novel therapeutic strategies for AR management.

Materials and methods

Chemicals

The manufacturers and catalog numbers of all drugs and reagents used in this study are as follows: ovalbumin (OVA, Sigma, A5503), aluminum hydroxide (Sigma, A8222), ASA (Beyotime, Y256023), EX527 (Sigma, E7034), anti-ovalbumin IgE (mouse), ELISA kit (Cayman Chemical, 500,840), histamine ELISA kit (Abcam, ab213975), human TNF-α ELISA kit (Abcam, ab181421), mouse TNF-α ELISA kit (Abcam, ab208348), human IL-6 ELISA kit (Abcam, ab178013), mouse IL-6 ELISA kit (Abcam, ab222503), human IL-1β ELISA kit (Abcam, ab214025), mouse IL-1β ELISA kit (Abcam, ab197742), human IL-4 recombinant protein (PeproTech, 200–04), human IL-13 recombinant protein (PeproTech, 200–13), anti-Occludin antibody (Abcam, ab216327), anti-ZO-1 antibody (Abcam, ab276131), anti-E-cadherin antibody (Abcam, ab231303), anti-TOM20 antibody (Abcam, ab186735), anti-DRP1 antibody (Abcam, ab184247), anti-MFN2 antibody (Abcam, ab124773), anti-SIRT1 antibody (Abcam, ab189494), anti-PGC-1α antibody (Abcam, ab191838), IgG antibody (Abcam, ab172730), anti-acetylated lysine antibody (Abcam, ab190479), anti-GAPDH antibody (Abcam, ab9485), goat anti-rabbit IgG H&L (HRP) (Abcam, ab205718), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719), rotenone (Sigma, R8875), Mito-TEMPO (Sigma, SML0737), ZLN005 (Sigma, SML0802), CCK-8 kit (Beyotime, C0038), SR-18292 (Sigma, SML2146), MitoSOX™ (Invitrogen, M36007), and Diff-Quick Stain Kit (Solarbio, G1540).

Establishment of OVA-induced AR model and ASA treatment

To establish an OVA-induced AR model, BALB/C mice were sensitized via intraperitoneal injection of 200 µL saline containing 50 µg of OVA and 1 mg of aluminum hydroxide on days 0, 7, and 14. Then, intranasal OVA challenge (400 µg OVA/40 µL PBS) was administered on days 21, 23, 25, and 27. Mice in the Control group were administered only an equal amount of PBS. Mice in ASA groups were administered ASA (5, 10, and 20 mg/kg) once daily via intraperitoneal injection from days 21 to 27. Mice in the Control and OVA groups were only administered an equal amount of PBS. EX527 (10 mg/kg) was administered via intraperitoneal injection 1 h prior to intranasal OVA challenge. All mice were euthanized on day 28 after the last OVA challenge.

Observation of AR symptoms

The nose-rubbing and sneezing numbers of the mice were observed and recorded within 30 min of the last stimulation.

Hematoxylin and eosin (H&E) staining

Histopathological morphology of mouse nasal mucosa was observed by H&E staining.

ELISA

OVA-specific IgE, histamine, and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) levels in mouse serum were determined using specific commercial ELISA kits.

Nasal lavage fluid (NALF) collection and analysis

After sacrifice, 1 mL ice-cold PBS was gently instilled into the mouse nasopharynx three times to collect NALF. The total cell counts in NALF were evaluated using the Countess II Cell Counter (Thermo Fisher Scientific). To determine the number of differential inflammatory cells, NALF was centrifuged, mounted on a slide, and stained with the Diff-Quick Stain Kit. Then, the cells were observed under a microscope.

Western blotting

The total protein was extracted from mouse nasal mucosa and cells using RIPA lysis buffer, measured for concentration using a BCA assay kit, separated into equal quantities by SDS-PAGE, and then transferred onto the PVDF membrane. The membranes were blocked in a blocking solution (5% dry nonfat milk), followed by incubation with specific primary antibodies and secondary antibodies. Finally, the bands were visualized by ECL and quantified using ImageJ software.

Cell culture and treatment

Human nasal epithelial cells (HNEpCs) (BeNa Culture Collection, China) were incubated in EMEM containing 10% FBS in a humidified incubator (5% CO2, 37 °C).

To mimic AR in vitro, HNEpCs were treated with 15 ng/mL recombinant human IL-4/IL-13 (7.5 ng/mL of each) for 24 h. For ASA pretreatment, HNEpCs were treated with ASA at 25, 50, and 100 µM for 30 min before IL-4/IL-13 induction. To induce or scavenge mtROS, HNEpCs were treated with rotenone (5 µM) or Mito-TEMPO (Mito-T, 100 µM) for 1 h before IL-4/IL-13 induction. For SIRT1 inhibition and PGC-1α activation, HNEpCs were treated with SIRT1 inhibitor (EX527, 1 µM) and PGC-1α activator (ZLN005, 1 µM) for 1 h before IL-4/IL-13 induction.

CCK-8

The cytotoxic effects of ASA on HNEpCs were evaluated using the CCK-8 assay. Briefly, HNEpCs were placed in a 96-well plate (1 × 104 cells/well) and treated with ASA at various concentrations (0, 25, 50, 100, and 200 µM) for 24 h. Then, CCK-8 solution (Promega, USA) was added (10 µL/well). After an additional 2-h incubation, the absorbance was recorded at 450 nm with a microplate reader.

Trans-epithelial electrical resistance (TEER) determination

HNEpCs were cultured on Transwell inserts (0.4-µm polyester membrane, 24-mm diameter) within 6-well plates (Corning, USA) (1 × 105 cells/insert). When the cells grew to complete confluence, the TEER level was assessed with a Millicell® ERS-2 voltmeter (Merck Millipore, Germany).

MitoSOX staining

The measurement of mtROS in HNEpCs was performed through MitoSOX staining. Following a 24-h induction with IL-4/IL-13, HNEpCs were treated with 5 µM MitoSOX dye for 10 min at 37 °C, rinsed with PBS, and then imaged at a wavelength of 594 nm.

Co-immunoprecipitation (Co-IP)

As previously described, HNEpCs were subjected to lysis using a specialized lysis buffer. Protein samples of equal quantity from each condition were then incubated overnight at 4 °C with either an anti-PGC-1α antibody or a control IgG antibody. Then, the resulting immunocomplexes were precipitated with Protein G magnetic beads. The precipitated complexes were subsequently analyzed through western blotting, employing an anti-acetylated lysine antibody to assess the acetylation level of PGC-1α.

Statistical analysis

Results were presented using the mean ± standard deviation (SD). SPSS 25.0 software was utilized for data analysis. One-way ANOVA followed by Tukey’s post hoc test was employed to assess statistical significance across multiple groups. The difference with a p-value less than 0.05 was regarded as statistically significant.

Results

ASA ameliorates OVA-induced AR-like symptoms and allergic responses in vivo

To investigate the impact of ASA in AR, an OVA-induced AR mouse model was established (Fig. 1A). Compared with the Control group, mice in the OVA group exhibited higher nose-rubbing and sneezing frequencies; however, treatment with ASA significantly improved such nasal symptoms (Fig. 1B and C). To assess the effects of ASA on OVA-induced allergic responses, ELISA kits were used to measure the IgE and histamine levels in mouse serum. As shown in Fig. 1D and E, IgE and histamine levels significantly increased in the OVA group, relative to the Control group; after ASA administration, OVA-specific IgE and histamine levels significantly reduced. Therefore, ASA ameliorates OVA-induced AR-like symptoms and allergic responses in vivo.

Fig. 1.

Fig. 1

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ASA ameliorates OVA-induced AR-like symptoms and allergic responsesin vivo. A OVA-induced AR mouse model and treatment protocol. B, C The nose-rubbing and sneezing frequencies of mice from each group. D, E IgE and histamine levels in mouse serum were evaluated by ELISA. n = 6 for each group; **p < 0.01; ***p < 0.001

ASA mitigates OVA-induced nasal inflammation, epithelial barrier injury, and mitochondrial damage in vivo

Histopathological alterations in the nasal mucosa of the mice were assessed using H&E staining. In comparison to the Control group, H&E staining (Fig. 2A) revealed that the OVA group showed disordered nasal mucosal epithelial cells and marked inflammatory cell infiltration, which was reversed by ASA administration in a dose-dependent manner. To determine the anti-inflammatory effects of ASA on AR, inflammatory cells in NALF were measured. As shown in Fig. 2B, the overall count of inflammatory cells significantly increased in the AR group, which was dose-dependently reduced by ASA. Notably, the infiltration of immune cells, such as eosinophils, macrophages, neutrophils, and lymphocytes, markedly increased in the NALF of AR mice; however, ASA treatment significantly attenuated the infiltration of these cells (Fig. 2B and C). Consistently, ASA treatment also reversed the OVA-induced increase in the production of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) (Fig. 2D). Nasal epithelial barrier dysfunction is an important indicator of AR. To evaluate the effects of ASA on OVA-induced nasal epithelial barrier dysfunction in mice, the expression levels of tight junction proteins (Occludin and ZO-1) and adherence junction protein (E-cadherin) in the nasal mucosa were detected. The results showed that mice in the OVA group manifested a significant decrease in Occludin, ZO-1, and E-cadherin protein levels in the nasal mucosa; however, ASA treatment effectively restored Occludin, ZO-1, and E-cadherin protein levels (Fig. 2E). To evaluate mitochondrial function in mouse nasal mucosa, the expression levels of mitochondrial membrane protein (TOM20), mitochondrial fission protein (DRP1), and mitochondrial fusion protein (MFN2) were detected. As shown in Fig. 2F, OVA-treated mice showed a significant decrease in TOM20 and MFN2 protein levels, as well as a significant increase in DRP1 protein level in the nasal mucosa, which was significantly abated by ASA treatment. Therefore, ASA may reduce OVA-induced nasal inflammation, epithelial barrier damage, and mitochondrial dysfunction in vivo.

Fig. 2.

Fig. 2

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ASA mitigates OVA-induced nasal inflammation, epithelial barrier injury, and mitochondrial damage in vivo. A H&E staining of nasal mucosa from each group. B Total counts of inflammatory cells. C Differential cell counts in NALF. D Pro-inflammatory cytokine (TNF-α, IL-6, and IL-1β) levels in mouse serum were evaluated by ELISA. E The protein levels of tight junction proteins (Occludin and ZO-1) and adherence junction protein (E-cadherin) in nasal mucosa from each group were detected by western blotting. F The protein levels of mitochondrial membrane protein (TOM20) and mitochondrial fusion proteins (DRP1 and MFN2) in nasal mucosa from each group were detected by western blotting. n = 6 for each group; **p < 0.01; ***p < 0.001

ASA attenuates IL-4/IL-13-induced inflammatory responses, epithelial barrier damage, and mitochondrial damage in HNEpCs

To further explore the role and regulatory mechanism of ASA in AR in vitro, a cellular model of AR was established by exposing HNEpCs to IL-4/IL-13 treatment. First, the cytotoxicity of ASA on HNEpCs was determined. CCK-8 assay results showed that ASA had no significant effects on HNEpC viability at or below 100 µM (Fig. 3A). As shown in Fig. 3B and C, IL-4/IL-13 challenge led to a significant increase in the production of pro-inflammatory cytokines, which was abrogated by ASA treatment. Besides, ASA partially abolished IL-4/IL-13-triggered decrease in the TEER level of HNEpCs (Fig. 3D) and the decrease in ZO-1, claudin-1, and E-cadherin protein levels in HNEpCs (Fig. 3E). Moreover, ASA pretreatment mitigated IL-4/IL-13-induced alteration of TOM20, DRP1, and MFN2 protein levels (Fig. 3F). These results indicate the beneficial effect of ASA on IL-4/IL-13-induced HNEpCs.

Fig. 3.

Fig. 3

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ASA attenuates IL-4/IL-13-induced inflammatory responses, epithelial barrier damage, and mitochondrial damage in HNEpCs. A The viability of HNEpCs after treatment with ASA at increasing concentrations (0, 25, 50, 100, 200 µM) for 24 h was assessed by CCK-8 assay. B, C TNF-α and IL-6 levels in cell supernatant from each group were assessed by ELISA. D TEER level of HNEpCs from each group. E Occludin, ZO-1, and E-cadherin protein levels in HNEpCs from each group. F TOM20, DRP1, and MFN2 protein levels in HNEpCs from each group. n = 3 for each group; *p < 0.05; **p < 0.01; ***p < 0.001

ASA improves IL-4/IL-13-induced damage to HNEpCs by scavenging mtROS

Previous studies have proven that mtROS inhibition is crucial for inhibiting inflammatory responses, epithelial barrier damage, and mitochondrial dysfunction (Hong et al. 2022; Kumar et al. 2020). As shown in Fig. 4A, mtROS production was significantly increased in IL-4/IL-13-challenged HNEpCs, whereas ASA significantly inhibited excessive mtROS production. To determine whether ASA exerts protective effects depending on mtROS, a mtROS stimulant (rotenone) and a mtROS scavenger (Mito-T) were applied to pretreat HNEpCs. The results showed that rotenone significantly reversed ASA-mediated protective effects against IL-4/IL-13-induced inflammatory responses (Fig. 4B), epithelial barrier damage (Fig. 4C and D), and mitochondrial damage (Fig. 4E); on the contrary, Mito-T reinforced the protective effects of ASA. Therefore, ASA may inhibit IL-4/IL-13-induced damage to HNEpCs by inhibiting mtROS.

Fig. 4.

Fig. 4

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ASA improves IL-4/IL-13-induced damage to HNEpCs by scavenging mtROS. A The mtROS of HNEpCs from each group was detected using MitoSOX staining. Next, HNEpCs were assigned to Control, IL-4/IL-13, IL-4/IL-13 + ASA, IL-4/IL-13 + ASA + rotenone, and IL-4/IL-13 + ASA + Mito-T groups. B TNF-α and IL-6 levels in cell supernatant from each group. C TEER level of HNEpCs from each group. D Occludin, ZO-1, and E-cadherin protein levels in HNEpCs from each group. E TOM20, DRP1, and MFN2 protein levels in HNEpCs from each group. n = 3 for each group; *p < 0.05; **p < 0.01; ***p < 0.001

ASA restores PGC-1α expression by promoting PGC-1α deacetylation via SIRT1 upregulation

As illustrated in Fig. 5A and B, decreased SIRT1 and PGC-1α protein levels and increased PGC-1α acetylation levels were observed in the nasal mucosa of mice from the OVA group, compared with the Control group; however, ASA pretreatment significantly reversed such effects. Similarly, ASA mitigated IL-4/IL-13-induced inhibition of SIRT1 and PGC-1α expression, as well as PGC-1α acetylation in HNEpCs (Fig. 5C and D). To investigate whether ASA regulated PGC-1α expression and acetylation in AR via SIRT1, the SIRT1 inhibitor (EX527) and PGC-1α activator (ZLN005) were applied. The results showed that EX527 significantly reversed ASA-mediated SIRT1 and PGC-1α upregulation (Fig. 5E) and PGC-1α deacetylation (Fig. 5F), which was partially offset by ZLN005. Therefore, ASA may ameliorate AR by upregulating PGC-1α expression via SIRT1-dependent PGC-1α deacetylation.

Fig. 5.

Fig. 5

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ASA restores PGC-1α expression by promoting PGC-1αdeacetylation via SIRT1 upregulation. A SIRT1 and PGC-1α protein levels in mouse nasal mucosa from each group, n = 6. B PGC-1α acetylation level in mouse nasal mucosa from each group was assessed using Co-IP, n = 6. C SIRT1 and PGC-1α protein levels in HNEpCs from each group, n = 3. D PGC-1α acetylation level in HNEpCs from each group, n = 3. E SIRT1 and PGC-1α protein levels in HNEpCs from each group, n = 3. F PGC-1α acetylation level in HNEpCs from each group, n = 3; *p < 0.05; **p < 0.01; ***p < 0.001

ASA exerts beneficial effects on IL-4/IL-13-treated HNEpCs via the SIRT1/PGC-1α pathway

Next, the role of the SIRT1/PGC-1α pathway in ASA-mediated protective effects on IL-4/IL-13-challenged HNEpCs was evaluated. The results showed that EX527 treatment remarkably blocked the ameliorative effects of ASA on IL-4/IL-13-triggered inflammatory responses (Fig. 6A), epithelial barrier dysfunction (Fig. 6B and C), and mitochondrial damage (Fig. 6D and E) in HNEpCs, which were partly neutralized by ZLN005 treatment. Therefore, ASA may exert protective effects on IL-4/IL-13-treated HNEpCs by regulating the SIRT1/PGC-1α pathway.

Fig. 6.

Fig. 6

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ASA exerts beneficial effects on IL-4/IL-13-treated HNEpCs via the SIRT1/PGC-1α pathway. A TNF-α and IL-6 levels in cell supernatant from each group. B TEER level of HNEpCs from each group. C Occludin, ZO-1, and E-cadherin protein levels in HNEpCs from each group. D The mtROS of HNEpCs from each group. E TOM20, DRP1, and MFN2 protein levels in HNEpCs from each group. n = 3 for each group; *p < 0.05; **p < 0.01; ***p < 0.001

Inhibition of the SIRT1/PGC-1α pathway eliminates the protective effects of ASA in AR mice

The role of the SIRT1/PGC-1α pathway in the ameliorative effects of ASA in AR was further validated in vivo. As illustrated in Fig. 7A and B, EX527 treatment significantly reversed the effects of ASA on SIRT1, PGC-1α, and PGC-1α acetylation levels in mouse nasal mucosa from the OVA group. The results showed that SR-18292 (a PGC-1α inhibitor) partly reversed the inhibitory effects of ASA on nose-rubbing and sneezing frequencies (Fig. 7C and D), serum IgE and histamine levels (Fig. 7E and F), inflammatory cell infiltration in nasal mucosa (Fig. 7G–I), inflammatory responses (Fig. 7J), nasal epithelial barrier dysfunction (Fig. 7K), and mitochondrial damage (Fig. 7L) in AR mice. Therefore, ASA may reduce OVA-induced nasal inflammation, epithelial barrier damage, and mitochondrial dysfunction in vivo via the SIRT1/PGC-1α pathway.

Fig. 7.

Fig. 7

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Inhibition of the SIRT1/PGC-1α pathway eliminates the protective effects of ASA in AR mice. A SIRT1 and PGC-1α protein levels in mouse nasal mucosa from each group. B PGC-1α acetylation level in mouse nasal mucosa from each group was assessed using Co-IP. C, D Nose-rubbing and sneezing frequencies of mice from each group. E, F IgE and histamine levels in mouse serum were evaluated by ELISA. G H&E staining of mouse nasal mucosa from each group. H Total counts of inflammatory cells. I Differential cell counts in NLF. ND, none detected. J TNF-α, IL-6, and IL-1β levels in mouse serum from each group. K Occludin, ZO-1, and E-cadherin protein levels in nasal mucosa from each group. L TOM20, DRP1, and MFN2 protein levels in mouse nasal mucosa from each group. n = 6 for each group; *p < 0.05; **p < 0.01; ***p < 0.001

Discussion

ASA demonstrates anti-inflammatory and antioxidant properties across various conditions (Pages et al. 2010). Nevertheless, its function and the mechanisms behind it in AR remain unexplored. The present study investigates the therapeutic efficacy of ASA in mitigating AR by targeting mtROS via the SIRT1/PGC-1α pathway. Our findings provide compelling evidence that ASA significantly alleviates key pathological features of AR, such as nasal inflammation, epithelial barrier dysfunction, and mitochondrial damage. These findings hold substantial implications, suggesting a promising therapeutic approach for AR.

In this study, an OVA-induced mouse model of AR was established. Besides, HNEpCs were subject to IL-4/IL-13 stimulation to imitate allergic inflammation in AR in vitro. Our results showed that the administration of ASA in OVA-induced AR mice resulted in a marked reduction in nose-rubbing and sneezing frequencies, which are key indicators of AR symptoms, suggesting that ASA effectively alleviates the symptomatic burden of AR. Furthermore, the significant decrease in serum IgE and histamine levels following ASA treatment highlights its role in modulating the allergic response in AR. These observations align with previous research demonstrating ASA’s anti-allergic properties through the inhibition of mast cell activation and degranulation (Bai et al. 2023).

Previous studies underscored the potent anti-inflammatory effects of ASA in various inflammatory conditions (Zhang et al. 2023; Saldanha et al. 2020). Herein, ASA significantly attenuated inflammatory cell infiltration in the nasal mucosa of AR mice. Pro-inflammatory cytokines play a critical role in the inflammatory cascade associated with AR, promoting the recruitment and activation of various immune cells (Zhang et al. 2022). The anti-inflammatory effects of ASA were further supported by the decreased levels of these cytokines observed both in vivo and in vitro.

A significant aspect of AR pathology involves the disruption of the epithelial barrier (Bernstein et al. 2024), which increases mucosal permeability and allows greater allergen penetration (Nur Husna et al. 2021). Our study showed that ASA restored the expression of key tight junction proteins (Occludin and ZO-1) and adherence junction protein (E-cadherin) in the nasal mucosa. This restoration is crucial for maintaining epithelial barrier integrity (Shi et al. 2024), which is often compromised in AR due to inflammation and allergen exposure (Ndika et al. 2017). The improvement in TEER in IL-4/IL-13-challenged HNEpCs by ASA treatment further validates its role in reinforcing the epithelial barrier integrity.

Mitochondrial dysfunction and elevated mtROS levels are known to exacerbate epithelial barrier damage and inflammation in AR (Liu et al. 2023). Our findings indicate that ASA treatment restored the expression of mitochondrial process proteins (TOM20, DRP1, and MFN2) both in vivo and in vitro, indicating the effects of ASA in mitigating mitochondrial damage in AR. Furthermore, ASA also reduced mtROS levels in HNEpCs stimulated by IL-4/IL-13. The role of mtROS in AR pathogenesis is well-documented (Shi et al. 2018), with excessive mtROS production contributing to epithelial barrier disruption and inflammation (Kim et al. 2022; Zhao et al. 2021). In addition, the beneficial effects of ASA against IL-4/IL-13-induced inflammation, epithelial barrier damage, and mitochondrial dysfunction were further enhanced by mtROS scavenging via Mito-T but partly offset by mtROS stimulation via rotenone. These findings are consistent with previous research highlighting the antioxidant and mitochondrial protective properties of ASA (Manikandan and Devi 2005; Gao et al. 2022).

Central to this study is the involvement of the SIRT1/PGC-1α pathway in the protective effects of ASA. Emerging evidence supports the importance of PGC-1α deacetylation by SIRT1 in the maintenance of mitochondrial integrity and function, as well as the regulation of cellular metabolism (Gurd 2011; Tang 2016). Notably, our results demonstrated that the beneficial effects of ASA were mediated by SIRT1-dependent PGC-1α deacetylation and upregulation. Upon SIRT1 inhibition with EX527, ASA-mediated activation of the SIRT1/PGC-1α pathway and its protective effects were significantly abrogated, while activation of PGC-1α with ZLN005 partially restored these effects, confirming the involvement of the SIRT1/PGC-1α pathway in the observed therapeutic outcomes.

In conclusion, this study provides compelling evidence that ASA effectively alleviates nasal inflammation, epithelial barrier damage, and mitochondrial dysfunction in AR by inhibiting mtROS through the SIRT1/PGC-1α pathway. These findings provide a foundation for the development of ASA as a potential therapeutic agent for AR, emphasizing the importance of targeting mitochondrial health and the SIRT1/PGC-1α axis in allergic diseases.

Author contributions

BX and YZL conceived and designed research. YZL and BX conducted experiments. YZL contributed new reagents. LFW and NND contributed analytical tools. WMG、HTL、XJL and XHC analyzed data. BX and YZL wrote the manuscript. All authors read and approved the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

Funding

This work was financially supported by the Zhejiang Provincial Medicine and Health Technology Project (2022 KY1247).

Data availability

All source data for this work (or generated in this study) are available upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Data Availability Statement

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