Molecular Medicine Reports Molecular Medicine Reports 1791-2997 1791-3004 D.A. Spandidos 10.3892/mmr.2021.12472 MMR-0-0-12472 Articles Formononetin ameliorates IL-13-induced inflammation and mucus formation in human nasal epithelial cells by activating the SIRT1/Nrf2 signaling pathway HuangJuanjuan ChenXianfeng XieAihua Department of Traditional Chinese Medicine, The Affiliated People's Hospital of Ningbo University, Ningbo, Zhejiang 315040, P.R. China Correspondence to: Dr Juanjuan Huang, Department of Traditional Chinese Medicine, The Affiliated People's Hospital of Ningbo University, 251 Baizhang East Road, Room 202, Building 6, Yinzhou, Ningbo, Zhejiang 315040, P.R. China, E-mail: huangjuanjuan20@163.com 12 2021 30 09 2021 24 6 832 21032021 28062021 Copyright: © Huang et al. 2021 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Formononetin has proven to be anti-inflammatory and able to alleviate symptoms of certain allergic diseases. The present study aimed to determine and elucidate the potential effects of formononetin in allergic rhinitis. JME/CF15 cells were pretreated with formononetin at different doses, followed by stimulation with IL-13. Cell Counting Kit-8 assay was performed to determine the cytotoxicity of formononetin. The expression levels of inflammation-related proteins, histamine, IgE, TNF-α, IL-1β, IL-6, granulocyte-macrophage colony-stimulating factor and eotaxin in IL-13-stimulated JME/CF15 cells were detected using ELISAs. The expression levels of phosphorylated-NF-κB p65, NF-κB p65 and cyclooxygenase-2 (Cox-2) were analyzed using western blotting. Reverse transcription-quantitative PCR, western blotting and immunofluorescence were performed to measure the levels of mucin 5AC oligomeric mucus/gel-forming. Expression levels of sirtuin 1 (SIRT1) and nuclear erythroid factor 2-related factor 2 (Nrf2) proteins were also measured using western blotting. The results of the present study revealed that formononetin exerted no cytotoxic effect on the viability of JME/CF15 cells. Following stimulation of JME/CF15 cells with IL-13, formononetin suppressed the upregulated expression levels of proinflammatory cytokines. IL-13-induced formation of mucus was also attenuated by formononetin treatment. Furthermore, it was found that the SIRT1/Nrf2 signaling pathway was activated in formononetin-treated JME/CF15 cells, whereas treatment with the SIRT1 inhibitor, EX527, reversed the effects of formononetin on IL-13-induced inflammation and mucus formation in JME/CF15 cells. In conclusion, the findings of the current study indicated that formononetin may activate the SIRT1/Nrf2 signaling pathway, thereby inhibiting IL-13-induced inflammation and mucus formation in JME/CF15 cells. These results suggested that formononetin may represent a promising agent for the treatment of allergic rhinitis.

 formononetin allergic rhinitis sirtuin 1/nuclear erythroid factor 2-related factor 2 inflammation mucus formation Introduction

Allergic rhinitis is a non-infectious disease of the nasal mucosa, which is characterized by paroxysmal sneezing, a runny nose, nasal itching and nasal congestion (1). Patients with allergic rhinitis may have an atopic constitution, usually showing familial aggregation with some associated genetic alterations (2). With regards to external factors, exposure to allergens, such as mites, pollen, fungal spores and animal dander, often serves as the trigger for the development of allergic rhinitis (3,4). Drug therapy, such as H1-antihistamines and intranasal corticosteroids, may control the symptoms of allergic rhinitis (5); however, there is currently no definitive cure (6). This disease although not fatal, does hinder general health, for example symptoms include sleep disruption and poor concentration, which can interfere with work and education (1,7). Hence, medical researchers continue to search for a cure for allergic rhinitis to improve the quality of life of patients.

Formononetin is an active ingredient of a traditional Chinese herb, Radix Astragali, and may also be extracted from the inflorescence and flowered branches and leaves of the leguminous plant, Trifolium pratense L. and the whole leaf of Ononis spinosa L (8,9). The anticancer properties of formononetin have been investigated in numerous studies (10–12). An increasing number of studies have also begun to focus on its potential in treating other disease types. For example, formononetin was previously found to protect against airway inflammation and oxidative stress in a mouse model of murine allergic asthma (13). Formononetin was also shown to alleviate atopic dermatitis by activating G protein-coupled estrogen receptors and upregulating A20 expression (14). However, to the best of our knowledge, there has been no report to date on whether formononetin exerts therapeutic effects in allergic rhinitis. Sirtuin 1 (SIRT1) is an universal gene regulator that affects multiple inflammation-related signaling pathways and has also been reported to play a role in attenuating ovalbumin-induced allergic symptoms in vivo (15). Upregulation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase (HO)-1 signaling pathway by mangiferin was previously demonstrated to be associated with the improvement of the ovalbumin-induced inflammatory response in mice with allergic rhinitis (16). Moreover, the regulatory effect of formononetin on SIRT1 expression has already been identified in various types of disease. For example, formononetin was shown to ameliorate cholestasis by regulating hepatic SIRT1 and peroxisome proliferator-activated receptor α expression (17).

IL-13 is a typical T helper cell 2 cytokine and serves a prominent role in the regulation of numerous types of allergic disease by activating its receptor and the associated STAT6 (18). IL-13 is often used to simulate pathological changes observed in a number of airway allergic diseases, such as eosinophil recruitment, mucus cell metaplasia, subepithelial fibrosis and smooth muscle hypertrophy (19). On this basis, IL-13 induction was used in the present study to establish an in vitro allergic rhinitis model.

The present study hypothesized that formononetin may exert protective effects in an allergic rhinitis model established with IL-13 via regulating the SIRT1/Nrf2 signaling pathway to inhibit inflammatory cytokine secretion and mucus formation. The present study was undertaken to verify this hypothesis, with the aim of providing a novel agent for the effective treatment or alleviation of allergic rhinitis.

 Materials and methods <sec> <title>Cell culture and treatment

The JME/CF15 human nasal epithelial cell line was acquired from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. The cells were cultured in DMEM/F12 (cat. no. 11320033; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (cat. no. 12484010; Gibco; Thermo Fisher Scientific, Inc.), 2 mM L-glutamine (cat. no. A2916801; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin (cat. no. V900929; Sigma-Aldrich; Merck KGaA) and 100 mg/ml streptomycin (cat. no. HY-B0472; MedChemExpress). The cells were maintained in 95% air and 5% CO2 at 37°C.

The allergic rhinitis model was established by stimulating the JME/CF15 cells with 10 ng/ml IL-13 (cat. no. HY-P7033; MedChemExpress) at 37°C for 30 min as previously described (20), or pretreatment with formononetin [cat. no. CN00152; ChemeGen (Shanghai) Biotechnology Co., Ltd.] at doses of 0.1, 1 and 10 µM (14,21) or EX527 (SIRT1 inhibitor; cat. no. E7034; Sigma-Aldrich; Merck KGaA) at a dose of 10 µM at 37°C for 24 h (22). Normal JME/CF15 cells without any treatment were used as the model group.

 Cell Counting Kit-8 (CCK-8) assay

The cytotoxicity of formononetin was detected using a CCK-8 assay (cat. no. C0037; Beyotime Institute of Biotechnology) as previously described (23). Briefly, 100 µl cell suspension was added into a 96-well plate using a density of 2×103 cells/well, with incubation at 37°C for 24 h. Cells were then treated accordingly, and after 24 h, 10 µl CCK-8 solution was added to each well. Subsequently, the cells were incubated with CCK-8 solution at 37°C for 1.5 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader.

 ELISA

ELISAs were performed to examine the proinflammatory cytokine secretion in IL-13-stimulated JME/CF15 cells as previously described (24). The levels of histamine, IgE, TNF-α, IL-1β, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and eotaxin were detected using histamine (cat. no. ab213975; Abcam), IgE (cat. no. ab195216; Abcam), TNF-α (cat. no. PT518; Beyotime Institute of Biotechnology), IL-1β (cat. no. PI305; Beyotime Institute of Biotechnology), IL-6 (cat. no. PI330; Beyotime Institute of Biotechnology) ELISA kits, GM-CSF (cat. no. PG355; Beyotime Institute of Biotechnology) and eotaxin (cat. no. PC115; Beyotime Institute of Biotechnology), respectively. The optical density value was measured at a wavelength of 450 nm using an ELISA detector.

 Western blotting

Whole cell protein extracts were extracted using RIPA lysis buffer, while nuclear and cytoplasmic proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (cat. no. P0027; Beyotime Institute of Biotechnology), and all protein samples were quantified using the BCA method. The sample proteins (20 µg/lane) were separated via 10% SDS-PAGE and subsequently transferred onto PVDF membranes. The membrane was removed from the transfer apparatus and soaked in TBS with 10% Tween-20 (TBST) washing buffer (Shanghai Aladdin Biochemical Technology Co., Ltd.) twice for 15 min each time. The membrane was then blocked with 10% non-fat dried milk freshly diluted in TBST and rocked on a rotating shaker for 15 min at room temperature. After rinsing with TBST, the membranes were incubated with the following primary antibodies diluted in 1% non-fat dried milk at 4°C overnight: Anti-phosphorylated (p)-NF-κB p65 (1:1,000; cat. no. ab239882; Abcam), anti-NF-κB p65 (1:1,000; cat. no. ab207297; Abcam), anti-cyclooxygenase 2 (Cox-2; 1:1,000; cat. no. 12282; Cell Signaling Technology, Inc.), anti-mucin 5AC oligomeric mucus/gel-forming (MUC5AC; 1:20,000; cat. no. ab198294; Abcam), anti-Nrf2 (1:1,000; cat. no. ab62352; Abcam), anti-SIRT1 (1:1,000; cat. no. ab189494; Abcam), anti-β-actin (1:1,000; cat. no. ab8227; Abcam) and anti-lamin B1 (1:1,000; cat. no. ab229025; Abcam). Following the primary antibody incubation, the membranes were rinsed three times in TBST and incubated with a HRP-conjugated secondary antibody (1:2,000; cat. no. ab97051; Abcam) diluted in 1% non-fat dried milk for 30 min at room temperature. Excess secondary antibody was then removed from the membrane with three rinses in 20 ml TBST for 5 min each time. Protein bands were analyzed using a standard chemiluminescence detection reagent (cat. no. P0018S; Beyotime Institute of Biotechnology) and ImageJ software (version 1.48v; National Institutes of Health) was used for semi-quantification as previously described (23).

 Reverse transcription-quantitative PCR (RT-qPCR)

mRNA expression levels of MUC5AC in IL-13-stimulated JME/CF15 cells were detected using RT-qPCR as previously described (23). Total RNA from JME/CF15 cells was extracted using TRIzol® reagent (cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.) and the quality was assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using a Sensiscript RT kit (cat. no. 205213; Qiagen, Inc.) according to the manufacturer's protocol. qPCR was subsequently performed using a TaqMan Universal Master mix II (cat. no. 4440048; Applied Biosystems; Thermo Fisher Scientific, Inc.) on an ABI Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the qPCR: Initial denaturation for 3 min at 95°C; followed by 40 cycles of denaturation for 15 sec at 95°C, and annealing and extension for 30 sec at 60°C. The primers used for the qPCR were as follows: MUC5AC forward, 5′-GCTTCCTGCTCCGAGATGT-3′ and reverse, 5′-AAGACGCAGCCCTCATAGAA-3′; and GAPDH forward, 5′-CACCCACTCCTCCACCTTTG-3′ and reverse, 5′-CCACCACCCTGTTGCTGTAG-3′. The relative expression levels were calculated using the 2−ΔΔCq method (25).

 Immunofluorescence (IF)

IF was employed to further verify the expression of MUC5AC in IL-13-stimulated JME/CF15 cells as previously described (26). Briefly, following fixation with 4% paraformaldehyde (Shanghai Aladdin Biochemical Technology Co., Ltd.) for 10 min at room temperature, the cells were incubated with 0.5% Triton X-100 (cat. no. HFH10; Invitrogen; Thermo Fisher Scientific, Inc.) at 25°C for 20 min. Subsequently, 5% BSA (cat. no. B265993; Shanghai Aladdin Biochemical Technology Co., Ltd.) diluted in PBS (Beyotime Institute of Biotechnology) was used to block the cells at room temperature for 1 h before incubation with an anti-MUC5AC antibody (1:250) overnight at 4°C. Following the primary antibody incubation, the cells were washed with PBS and incubated with a FITC-conjugated goat anti-rabbit IgG secondary antibody (1:200; cat. no. P0186; Beyotime Institute of Biotechnology) at 25°C for 1 h, followed by staining with DAPI (MedChemExpress) for 15 min at room temperature. Stained cells were visualized using a fluorescence microscope (magnification, ×200).

 Statistical analysis

Independent experiments were performed at least three times. The data are presented as the mean ± SD and were analyzed using a one-way ANOVA followed by a Tukey's post hoc test. GraphPad Prism 6 software (GraphPad Software, Inc.) was used for the statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Formononetin does not affect JME/CF15 cell viability

The cytotoxicity of formononetin was examined using a CCK-8 assay, which demonstrated that the viability of JME/CF15 cells was not significantly altered following formononetin treatment at doses of 0.1, 1 or 10 µM (Fig. 1). These results suggested that formononetin may exert no cytotoxic effect on JME/CF15 cells.

 Formononetin inhibits inflammatory cytokine secretion in IL-13-stimulated JME/CF15 cells

According to the results of the ELISAs (Fig. 2A and B), the levels of the immunoregulatory autacoid histamine, the allergy index IgE and the proinflammatory cytokines, TNF-α, IL-1β and IL-6, were significantly upregulated in IL-13-stimulated JME/CF15 cells compared with the control group, and were subsequently downregulated in a dose-dependent manner following pretreatment with 0.1, 1 or 10 µM formononetin. Furthermore, IL-13 stimulation significantly elevated the levels of GM-CSF and eotaxin in JME/CF15 cells compared with the control group, which were then dose-dependently reduced by formononetin treatment at all doses (Fig. 2C). Western blotting revealed that the expression levels of inflammation-related Cox-2 and p-NF-κB p65/NF-κB p65 were significantly upregulated in IL-13-stimulated JME/CF15 cells compared with the control group, while their expression levels were subsequently downregulated following formononetin treatment (Fig. 2D). These results suggested an inhibitory effect of formononetin on the secretion of inflammatory cytokines in IL-13-stimulated JME/CF15 cells.

 Formononetin inhibits mucus formation in IL-13-stimulated JME/CF15 cells

Overexpression of human MUC5AC has been reported to contribute to the formation of mucus in airway inflammation (27,28); therefore, its protein expression levels were detected in the allergic rhinitis model in the present study. It was observed that, while IL-13 induced high expression of MUC5AC, pretreatment with formononetin effectively reduced MUC5AC expression levels in IL-13-stimulated JME/CF15 cells (Fig. 3A and B). Formononetin at 10 µM was associated with the most significant reduction in MUC5AC expression and was thus selected for use in subsequent experiments. As shown in Fig. 3C, the fluorescence intensity of MUC5AC was increased in the IL-13 group compared with the control group, while it was markedly weakened in the IL-13 + formononetin group. These results indicated that formononetin may prevent the formation of mucus in IL-13-stimulated JME/CF15 cells.

 Formononetin activates the SIRT1/Nrf2 signaling pathway

To understand the mechanism of action of formononetin, it was investigated whether there was an interaction between formononetin and the SIRT1/Nrf2 signaling pathway. Western blotting revealed that the expression levels of SIRT1 and nucleic Nrf2 were downregulated in IL-13-stimulated JME/CF15 cells compared with the control group, and were subsequently dose-dependently upregulated following treatment with formononetin (Fig. 4). Conversely, the expression levels of cytoplasmic Nrf2 exhibited the opposite trends. These findings indicated that formononetin may activate the SIRT1/Nrf2 signaling pathway.

 SIRT1 inhibitor EX527 reverses the effects of formononetin on IL-13-stimulated JME/CF15 cells

To verify the role of the SIRT1/Nrf2 axis in the mechanism of action of formononetin, the expression levels of SIRT1 in IL-13-stimulated JME/CF15 cells were inhibited using EX527. A significant rise in the levels of histamine, IgE, TNF-α, IL-1β and IL-6 was observed in IL-13-stimulated formononetin-treated JME/CF15 cells following SIRT1 inhibition (Fig. 5A and B). EX527 treatment also increased the levels of GM-CSF and eotaxin, which were suppressed by formononetin treatment in IL-13-stimulated JME/CF15 cells (Fig. 5C). Furthermore, while the expression levels of Cox-2 and p-NF-κB p65/NF-κB p65 were downregulated by formononetin treatment in IL-13-stimulated JME/CF15 cells, inhibiting SIRT1 with EX527 partially reversed their expression levels (Fig. 5D). The expression levels of nucleic Nrf2 were downregulated, while the expression levels of cytoplasmic Nrf2 were upregulated in IL-13-stimulated JME/CF15 cells. After pretreatment with formononetin, the expression level of nucleic Nrf2 was upregulated and cytoplasmic Nrf2 expression was downregulated, which was partially reversed following EX527 treatment (Fig. 5E). Similarly, EX527 treatment rescued the expression levels of MUC5AC, which were downregulated by formononetin treatment in IL-13-stimulated JME/CF15 cells (Fig. 5F and G). These results indicated that formononetin may inhibit the secretion of proinflammatory cytokines and mucus formation in IL-13-stimulated JME/CF15 cells by activating the SIRT1/Nrf2 signaling pathway.

 Discussion

Allergic rhinitis, which currently affects ~40% of the world population, is a global health concern that is associated with a notable socioeconomic burden and may lead to serious complications, including asthma and otitis media (29). Allergic rhinitis is an atopic disorder of the nasal mucosa that is mainly characterized by IgE-mediated histamine release and involves a variety of immunocompetent cells and cytokines, such as pathogenic memory T helper 2 cells, IL-4, IL-5 and IL-13, which are triggered following exposure of the atopic individual to allergens (30,31). Risk factors for allergic rhinitis are present at all ages (32), and include genetic factors (33,34), environmental pollution (35) and environmental allergen exposure (36). The symptoms of the disease may be effectively controlled through standardized treatment (examples include treatment with intranasal corticosteroids, H1-antihistamine and leukotriene receptor antagonist) (5) and care (37); however, long-term treatment poses a heavy economic burden (38).

Formononetin, a type of phytoestrogen derived from the medicinal plant Trifolium pretense L., has been proven to be an active component with promising anticancer properties, as evidenced by previous research (39,40). In addition to its anticancer properties, formononetin has also been found to exert antioxidant and anti-inflammatory effects in various types of disease. For example, previous studies have reported that formononetin played a role in preventing allergic diseases and that it could effectively attenuate atopic dermatitis through regulation of G-protein-coupled estrogen receptor and A20 expression (14,41). It was also shown that formononetin regulated NF-κB signaling to inhibit the inflammatory response and promote the angiogenesis of the gastric mucosa in mice with gastric ulcers (42). However, to the best of our knowledge, whether formononetin has a beneficial effect on allergic rhinitis has yet to be reported. Excessive release of histamine induced by allergen exposure is commonly known to be an active contributor to the occurrence of allergic rhinitis (43). IgE is an important mediator of the endogenous release of histamine, the level of which is also closely associated with asthma, food allergies and allergic rhinitis (44–47). It was previously demonstrated that formononetin decreased the levels of histamine and proinflammatory cytokines to alleviate allergic inflammation (21). In the present study, a cellular model of allergic rhinitis was established using IL-13 stimulation, in which high levels of histamine, IgE and proinflammatory cytokines, TNF-α, IL-1β and IL-6, were detected. However, treatment with formononetin markedly decreased these levels in a dose-dependent manner. Previous studies have reported high concentrations of GM-CSF in patients with nasal allergy, which was also found to be associated with aggravated rhinorrhea (48,49). A more recent study also demonstrated the value of GM-CSF in assessing chronic rhinitis due to its positive correlation with eosinophilic inflammation (50). The results of the present study revealed high levels of both GM-CSF and eotaxin in IL-13-stimulated JME/CF15 cells, while treatment with formononetin was demonstrated to effectively suppress GM-CSF and eotaxin levels. Upregulated Cox-2 expression has been proven to be a cancer-related biomarker and is also closely associated with inflammation (51,52). Some of the anti-inflammatory effects of formononetin were discovered to be mediated through its suppression of Cox-2 expression in neurological lesions and kidney injury, among others (53,54). NF-κB p65 is a member of the NF-κB family that acts as a master regulator of inflammation and is widely found in animal cells (55). Formononetin was reported to inactivate NF-κB p65 to obstruct osteoclastogenesis and protect against liver injury (56,57). Consistent with previous findings, downregulated expression levels of Cox-2 and p-NF-κB p65 were also found in the present study following formononetin treatment in IL-13-stimulated JME/CF15 cells. Taken together, these changes suggested that formononetin may inhibit IL-13-induced proinflammatory cytokine secretion in JME/CF15 cells.

MUC5AC is a gel-forming mucin that is normally present in human airways, the high expression of which leads to the oversecretion of mucus and was recently identified in the airway mucus of patients with severe coronavirus-19 disease (58). In the present study, IL-13 significantly upregulated the protein expression levels of MUC5AC in JME/CF15 cells, which were markedly suppressed following formononetin treatment, as evidenced by the results of the RT-qPCR, western blotting and IF experiments. These findings indicated that formononetin may inhibit mucus formation in allergic rhinitis.

In addition to the effects of formononetin on allergic rhinitis, the mechanism underlying its effects was also investigated. SIRT1 is a well-known regulator of chronic inflammatory responses, which has been investigated in the context of the alleviation of asthma as well as allergic rhinitis (15,59). Wu et al (60) found that SIRT1 activation upregulated Nrf2 expression and inhibited the NF-κB signaling pathway to relieve sepsis-induced inflammation and apoptosis. Furthermore, it was demonstrated in a previous study that formononetin served as a SIRT1 activator and improved diabetic neuropathy (61). In addition, formononetin was found to upregulate SIRT1 expression and inhibit lipopolysaccharide-induced high mobility group box 1 release, thereby inhibiting the cellular inflammatory response (62). SIRT1 activated by resveratrol could also alleviate ovalbumin-induced allergic rhinitis in mice (63). SIRT1 knockdown alleviated the symptoms and inflammatory indicators of allergic rhinitis in HIF1αCD11c−/− mice (64). Activation of the Nrf2/HO-1 signaling pathway could suppress allergic rhinitis activity (65). In addition, mangiferin attenuated allergic rhinitis by enhancing the Nrf2/HO-1 signaling pathways (16). The present study revealed that formononetin may activate the SIRT1/Nrf2 signaling pathway. Normally, Nrf2 and Kelch like ECH associated protein (Keap1) bind as a dimer in the cytoplasm, and when stimulated, Nrf2 dissociates from Keap1 and translocates into the nucleus, thereby inducing the expression of encoded related enzymes, such as HO-1 (66). In the present study, formononetin was shown to upregulate the expression of Nrf2 in the nucleus and promote the nuclear translocation of Nrf2, and thus exert an effect. Moreover, inhibiting SIRT1 with EX527 largely reversed the effects of formononetin on IL-13-induced inflammation and mucus secretion in JME/CF15 cells, and reduced the nuclear translocation of Nrf2, further verifying the role of SIRT1/Nrf2 signaling in the mechanism of action of formononetin. However, the present conclusion is based solely on cell experiments and further animal experiments should be conducted to validate the current study findings.

In conclusion, the findings of the present study provided evidence to suggest that formononetin may ameliorate IL-13-induced proinflammatory cytokine secretion and mucus formation in JME/CF15 cells by activating the SIRT1/Nrf2 signaling pathway. These findings are significant as they indicate the curative potential of formononetin in the treatment of allergic rhinitis, and it may be considered as a novel therapeutic strategy for this disease.

 Acknowledgements

Not applicable.

 Funding

No funding was received.

 Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

 Authors' contributions

JH conceived and designed the experiments in the present study. JH, XC and AX performed the experiments. JH and XC analyzed the data. JH and AX wrote the manuscript. All authors read and approved the final manuscript. JH and XC confirm the authenticity of all the raw data.

 Ethics approval and consent to participate

Not applicable.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

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Formononetin has no impact on JME/CF15 cell viability. Cytotoxicity of formononetin in JME/CF15 cells was detected using a Cell Counting Kit-8 assay.

Formononetin inhibits inflammatory cytokine secretion in IL-13-stimulated JME/CF15 cells. Concentrations of (A) histamine and IgE, (B) TNF-α, IL-1β and IL-6 and (C) GM-CSF and eotaxin in IL-13-stimulated JME/CF15 cells treated with 0, 0.1, 1 or 10 µM formononetin were detected using ELISAs. (D) Relative expression levels of Cox-2 and p-NF-κB p65/NF-κB p65 in IL-13-stimulated JME/CF15 cells treated with 0, 0.1, 1 or 10 µM formononetin were detected using western blotting. *P<0.05, **P<0.01, ***P<0.001 vs. control; #P<0.05, ##P<0.01, ###P<0.001 vs. IL-13; @P<0.05, @@P<0.01 vs. IL-13 + 0.1 µM; ∆P<0.05, ∆∆∆P<0.001 vs. IL-13 + 1 µM. GM-CSF, granulocyte-macrophage colony-stimulating factor; Cox-2, cyclooxygenase 2; p-, phosphorylated.

Formononetin inhibits mucus formation in IL-13-stimulated JME/CF15 cells. Relative mRNA and protein expression levels of MUC5AC in IL-13-stimulated JME/CF15 cells treated with 0, 0.1, 1 or 10 µM formononetin were detected using (A) reverse transcription-quantitative PCR and (B) western blotting, respectively. **P<0.01, ***P<0.001 vs. control; #P<0.05, ###P<0.001 vs. IL-13; @@P<0.01 vs. IL-13 + 0.1 µM; ∆∆∆P<0.001 vs. IL-13 + 1 µM. (C) Fluorescence intensity of MUC5AC in JME/CF15 cells treated with control, IL-13 or IL-13 + formononetin (10 µM) was detected using immunofluorescence. MUC5AC is shown as the green fluorescence Scale bar, 50 µm. MUC5AC, mucin 5AC oligomeric mucus/gel-forming.

Formononetin activates the SIRT1/Nrf2 signaling pathway. Relative protein expression levels of SIRT1, and cytoplasmic and nucleic Nrf2 in IL-13-stimulated JME/CF15 cells treated with 0, 0.1, 1 or 10 µM formononetin were detected using western blotting. **P<0.01, ***P<0.001 vs. control; #P<0.05, ##P<0.01, ###P<0.001 vs. IL-13; ∆P<0.05, ∆∆P<0.01 vs. IL-13 + 1 µM. SIRT1, sirtuin 1; Nrf2, nuclear factor erythroid 2-related factor 2.

Sirtuin 1 inhibitor EX527 reverses the effects of formononetin on IL-13-stimulated JME/CF15 cells. Concentrations of (A) histamine and IgE, (B) TNF-α, IL-1β and IL-6 and (C) GM-CSF and eotaxin in JME/CF15 cells treated with IL-13, IL-13 + formononetin or IL-13 + formononetin + EX527 were detected using ELISAs. (D) Relative protein expression levels of Cox-2 and p-NF-κB p65/NF-κB p65 in JME/CF15 cells treated with IL-13, IL-13 + formononetin or IL-13 + formononetin + EX527 were detected using western blotting. (E) Relative protein expression levels of nucleic and cytoplasmic Nrf2 in JME/CF15 cells treated with IL-13, IL-13 + formononetin or IL-13 + formononetin + EX527 were detected using western blotting. Relative mRNA and protein expression levels of MUC5AC in JME/CF15 cells treated with IL-13, IL-13 + formononetin or IL-13 + formononetin + EX527 were detected using (F) reverse transcription-quantitative PCR and (G) western blotting, respectively. *P<0.05, **P<0.01, ***P<0.001 vs. control; #P<0.05, ##P<0.01, ###P<0.001 vs. IL-13; @P<0.05, @@P<0.01, @@@P<0.001 vs. IL-13 + formononetin. GM-CSF, granulocyte-macrophage colony-stimulating factor; Cox-2, cyclooxygenase 2; p-, phosphorylated; Nrf2, nuclear factor erythroid 2-related factor 2; MUC5AC, mucin 5AC oligomeric mucus/gel-forming.