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.
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 (
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,
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 (
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.
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 (
The cytotoxicity of formononetin was detected using a CCK-8 assay (cat. no. C0037; Beyotime Institute of Biotechnology) as previously described (
ELISAs were performed to examine the proinflammatory cytokine secretion in IL-13-stimulated JME/CF15 cells as previously described (
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 (
mRNA expression levels of MUC5AC in IL-13-stimulated JME/CF15 cells were detected using RT-qPCR as previously described (
IF was employed to further verify the expression of MUC5AC in IL-13-stimulated JME/CF15 cells as previously described (
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.
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 (
According to the results of the ELISAs (
Overexpression of human MUC5AC has been reported to contribute to the formation of mucus in airway inflammation (
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 (
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 (
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 (
Formononetin, a type of phytoestrogen derived from the medicinal plant
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 (
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 (
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.
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No funding was received.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
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.