Kaempferol inhibits airway inflammation induced by allergic asthma through NOX4-Mediated autophagy

Abstract

Background

Kaempferol has important medicinal value in the treatment of asthma. However, its mechanism of action has not been fully understood and needs to be explored and studied.

Methods

A binding activity of kaempferol with nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) was analyzed by molecular docking. Human bronchial epithelial cells (BEAS-2B) were treated with different concentrations (0, 1, 5, 10, 20, 40 μg/mL) of kaempferol to select its suitable concentration. In the transforming growth factor (TGF)-β1-induced BEAS-2B, cells were treated with 20 μg/mL kaempferol or 20 μM GLX35132 (a NOX4 inhibitor) to analyze its effects on NOX4-mediated autophagy. In the ovalbumin (OVA)-induced mice, 20 mg/kg kaempferol or 3.8 mg/kg GLX351322 administration was performed to analyze the therapeutic effects of kaempferol on NOX4-mediated autophagy. An autophagy activator, rapamycin, was used to confirm the mechanism of kaempferol in treatment of allergic asthma.

Results

A good binding of kaempferol to NOX4 (score = −9.2 kcal/mol) was found. In the TGF-β1-induced BEAS-2B, the NOX4 expression was decreased with kaempferol dose increase. The secretions of IL-25 and IL-33, and the NOX4-mediated autophagy were significantly decreased by kaempferol treatment in the TGF-β1-induced BEAS-2B. In the OVA-challenged mice, kaempferol treatment improved airway inflammation and remodeling through suppressing NOX4-mediated autophagy. The rapamycin treatment clearly hampered the therapeutic effects of kaempferol in the TGF-β1-induced cells and OVA-induced mice.

Conclusions

This study identifies kaempferol binds NOX4 to perform its functions in the treatment of allergic asthma, providing an effective therapeutic strategy in the further treatment of asthma.

Introduction

Allergic asthma, a chronic inflammatory disease, is characterized by airway obstruction and bronchial hyperresponsiveness. ¹ Its clinical manifestations are recurrent wheezing, cough, chest tightness and shortness of breath. At present, the medicine treatment of asthma includes glucocorticoids, β2 receptor agonists, targeted biological agents, allergen immunotherapy, etc.^2,3 However, long-term use of these medicine could cause adverse reactions and drug resistance. ³ With the advances of phytochemistry and phytopharmacology, the roles of medicinal plant products for treating asthma are becoming more and more obvious. ⁴ Therefore, it is of great clinical significance for the development of asthma drugs to screen suitable effective herbal medicinal plant components.

Kaempferol (3,4′,5,7-tetrahydroxyflavone) is a natural flavonoid existing in many plants. It is one of the important effective components of traditional Chinese medicine for treating asthma, due to its antioxidant and anti-inflammatory effects.^5–9 Kaempferol has important medicinal value in the treatment of asthma. However, its mechanism of action has not been fully understood and needs to be continuously explored and studied. In a previous report, it has reported that kaempferol prevents pulmonary arterial hypertension by regulating abnormal autophagy and metabolic disorders. ¹⁰ In allergic asthma, allergens induce oxidative stress and mitochondrial dysfunction, leading to abnormal autophagy and metabolic disorders. ¹¹ However, the inhibitory effects of kaempferol on pulmonary autophagy induced by allergic asthma are not yet elucidated.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), containing NOX1-5, play an important role in the generation of reactive oxygen species (ROS). ¹² In asthma, increased NOX4 expression was found in airway smooth muscle. ¹³ Chan et al. reported that NAPDH oxidase (NOX)-4 was overexpressed in the house dust mites-induced bronchial endothelial cells, and the NOX4 expression was associated with increase of cellular nitrosation and mitochondrial oxidative stress. ¹⁴ Accumulating evidences suggest that autophagy is emerging as a major target of ROS and NOX enzymes, ¹⁵ especially NOX4. ¹⁶ However, the association of kaempferol and NOX4-mediated autophagy in allergic asthma remains unclear.

In this study, kaempferol showed a good binding activity of NOX4 by molecular docking. To confirm kaempferol suppresses airway inflammation by regulating NOX4-mediated autophagy in allergic asthma, we studied the effects of kaempferol on NOX4 expression in TGF-β1-induced BEAS-2B cells and ovalbumin (OVA)-induced mice. Furthermore, the interconnection between kaempferol and NOX4-mediated autophagy was explored to emphasize a role underlying kaempferol in asthmatic autophagy-associated airway inflammation.

Methods

Molecular docking

The three-dimensional (3D) structure of NOX4 was downloaded from PDB database (https://www.rcsb.org/) and saved as PDB format. The 3D structures of kaempferol (CAS:520-18-3) were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and saved as PDB format. According to previous procedure, ¹⁷ mechanical optimization, hydrogenation and charging of the NOX4 were carried out by UCSF chimera software (https://www.cgl.ucsf.edu/chimera/), and the molecular docking score was performed by AutoDock Vina tool. Docking score indicates the inter molecular energy (kcal/mol).

BEAS-2B cells culture

Human bronchial epithelial BEAS-2B cells (^#CL-0496, Procell, Wuhan, China) were cultured in DMEM containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin at 37°C, 5% CO₂. The medium was changed every 3 days. For all experiments, the same cell line was used to avoid changes in cell activity and viability that could result from repeated thawing and passage.

Toxicity of kaempferol for cells

The BEAS-2B cells were seeded in 96-well plates and treated with different dose (0, 1, 5, 10, 20, 40 μg/mL) of kaempferol (^#60010, Sigma-Aldrich) for 24 h. The kaempferol was solved in 0.1% dimethyl sulfoxide (DMSO). Cells viability was measured using CCK8 kit.

Cells groups and treatment

The BEAS-2B cells were cultured in the medium with 10 ng/mL TGF-β1 for 24 h, then changed to fresh medium with kaempferol (0, 5, 10, 20 μg/mL) for 24 h, respectively. The NOX4 expression was measured by western blot.

Further to confirm the kaempferol suppresses inflammation by regulating NOX4-mediated autophagy, the BEAS-2B cells were divided into following six groups: control group, 0.1% DMSO group, kaempferol group, GLX351322 (a NOX4 inhibitor) group, kaempferol plus rapamycin (an autophagy activator) group, and GLX351322 plus rapamycin group.

In the control group, the BEAS-2B cells were cultured in normal condition for 48 h.

In the 0.1% DMSO group, kaempferol group and GLX351322 group, the BEAS-2B cells were cultured in the medium with 10 ng/mL TGF-β1 for 24 h, then correspondingly changed to fresh medium with 0.1% DMSO, 20 μg/mL kaempferol or 20 μM GLX351322 ¹⁸ for 24 h.

In the kaempferol plus Rapamycin group and GLX351322 plus rapamycin group, the BEAS-2B cells were cultured in the medium with 10 ng/mL TGF-β1 for 24 h, then correspondingly changed to fresh medium with 20 μg/mL kaempferol plus 100 nM rapamycin ¹⁹ or 20 μM GLX351322 plus 100 nM rapamycin for 24 h.

Ovalbumin (OVA)-induced mice

The OVA-challenged asthma model was established according to previous procedures. ²⁰ Briefly, mice were intraperitoneally (i.p.) sensitized by 20 μg OVA and 1 mg Alum in 200 μL saline on days 1 and days 8, then challenged by inhalation with 5% (V/V) OVA for 30 min on days 15, 16, 17 after initial sensitization with OVA.

Mice treatment

Forty-eight C57BL/6J male mice, 6–8 weeks, were housed under specific pathogen free conditions. After one week of adaptation, the mice were randomly divided into following six groups (8 mice in each group): control group, 0.1% DMSO group, kaempferol group, GLX351322 group, kaempferol plus rapamycin group, and GLX351322 plus rapamycin group.

In the control group, mice were intraperitoneally received with 20 μL PBS in 200 μL saline at days 1 and 8, then were nebulized with 5% PBS for 30 min at days 15, 16 and 17 days after sensitization.

In the 0.1% DMSO group, kaempferol group and GLX351322 group, mice were sensitized with OVA and Alum, and correspondingly treated with 0.1 mL drug solution (0.1% DMSO, 20 mg/kg kaempferol ⁹ or 3.8 mg/kg GLX351322 ²¹ ) by orally administration 1 h before OVA inhalation.

In the kaempferol plus rapamycin group and GLX351322 plus rapamycin group, mice were sensitized with OVA and Alum, and correspondingly orally treated with 0.1 mL drug solution (20 mg/kg kaempferol or 3.8 mg/kg GLX351322) and intraperitoneally treated (i.p.) 4 mg/kg rapamycin ²² 1 h before OVA inhalation.

Airway hyperreactivity

At 24 hours after the last challenge with OVA, airway hyperreactivity of mice was assessed as previous description. ²³ A tracheostomy tube was placed in the trachea of mice, and the mice were ventilated using a Sci-Req FlexiVent machine under 150 breaths/min and a tidal volume of 2 mL. Airway resistance was determined at baseline and after administration of nebulized methacholine (0, 12.5, 25, 50, 100 mg/mL).

Samples collection

After airway hyperreactivity, bronchioalveolar lavage fluids (BLAFs) were obtained through injection of 1 mL PBS into the mice lung via the tracheal tube. Then, all mice were sacrificed, and the right lung tissues were injected with small amount of 4% paraformaldehyde and fixed in 4% paraformaldehyde, and the left lungs were kept at −80°C.

Numbers of eosinophils, lymphocytes and neutrophils

Total numbers cells in the BALFs were measured by trypan blue dye exclusion, then the numbers of eosinophils, lymphocytes and neutrophils were measured by Wright stain solution.

Enzyme-Linked immunosorbent assay (ELISA)

After centrifugation (×800 g, 15 min), the supernatants of cells and BLAFs were collected. The secretions of interleukin (IL)-25 (^#ab272200, Abcam) and IL-33 (^#PI631, Beyotime) in the BEAS-2B cells, and the levels of IL-4 (^#PI612, Beyotime), IL-5 (^#PI620, Beyotime), and IL-13 (^#PI539, Beyotime) in the BLAFs were measured by ELISA kits.

Western blot

According to previous protocols, ²⁴ proteins were collected from frozen cells and lungs by a RIPA buffer, then separated with 12% SDS-PAGE. The separated proteins were transferred to PVDF membranes, and incubated with appropriate primary antibodies overnight at 4°C. The antibodies included NOX4 (^#ab154244, 1:1000, Abcam), ATG5 (^#DF6010, 1:1000, Affinity), Beclin-1 (^#AF5128, 1:1000, Affinity) and GAPDH (^#AF7021, 1:1000, Affinity). The second antibody Goat anti-Rabbit IgG (H+L) HRP (^#S001, 1:1000, Affinity) was cultured with the washed membranes for 60 min. An enhanced chemiluminescence reagent kit was used to visualize the signals.

Immunofluorescence

As reported protocols, ²⁵ the fixed cells and lung sections (3 μm) were washed and blocked with BS-T containing 1% BSA, then incubated with rabbit NOX4 polyclonal antibody (^#ab154244, 1:500, Abcam) and mouse LC3B monoclonal antibody (^#MA5-37,852, 1:200, ThermoFisher Scientific) overnight at 4°C. After washing, the cells and sections were incubated with FITC-conjugated goat anti-rabbit IgG (H+L) antibody (#BA1105, 1:500, Boster) and Cy3-conjugated goat anti-mouse IgG (H+L) antibody (#BA1031, 1:500, Boster) for 60 min. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 60 s. After washing and sealing, the results were obtained under a confocal microscope.

Hematoxylin and eosin (HE) and Masson

According to previous protocols, ²⁶ the fixed lung sections (3 μm) were respectively stained with HE and Masson solution. The sections were separated and dehydrated with ethanol, cleared with xylene, and sealed with neutral gum. The results were obtained under a light microscope. According to previous reports,^27,28 the quantitative results of HE and Masson staining were measured by Image J software. For results of HE staining, inflammatory cell infiltration area (%) = inflammatory cell infiltration area/total area. For results of Masson staining, collagen fiber area (%) = collagen fiber area/total area.

Statistical analysis

Data were analyzed by SPSS20.0 software (National Institutes of Health, US), and showed as mean ± standard deviation by GraphPad Prism 6.0 software (GraphPad Software Inc., CA). Difference among multiple groups was performed by one-way analysis of variance following Tukey test. Statistical significance was considered as a value of p < 0.05.

Results

Kaempferol inhibited NOX4 expression in TGF-β1-induced BEAS-2B cells

As shown in Figure 1(a), a good binding activity of kaempferol (score = −9.2 kcal/mol) with NOX4 was found through molecular docking. In order to choose appropriate dose of kaempferol, the BEAS-2B cells were treated with different dose of kaempferol (0, 1, 5, 10, 20, 40 μg/mL) for 24 h. Through observing the cell viability (Figure 1(b)), we found 40 μg/mL kaempferol treatment significantly suppressed cell viability when compared to the other doses. Additionally, the expression levels of NOX4 in the 10 ng/mL-induced BEAS-2B were downregulated with kaempferol dose increase (Figure 1(c)). To further study the mechanism of kaempferol in the treatment of allergic asthma, we selected 20 μg/mL kaempferol in the following cells experiment.

Figure 1.

Effects of kaempferol on cell viability and NOX4 expression. (a) The binding of kaempferol to NOX4 was analyzed by molecular docking, and a good activity of kaempferol to NOX4 was showed (score = −9.2 kcal/mol); (b) Toxicity of kaempferol to BEAS-2B was analyzed by CCK-8 kit, and 40 μg/mL kaempferol significantly downregulated the cells viability; (c) Effects of kaempferol on NOX4 expression in BEAS-2B. The NOX4 expressions were measured by western blot. ^**p < 0.01.

Kaempferol suppressed inflammation through NOX4-mediated autophagy in TGF-β1-induced BEAS-2B cells

After different treatment, the secretions of IL-25 and IL-33 in the BEAS-2B cells were measured (Figure 2(a)). The IL-25 and IL-33 levels were significantly upregulated in the TGF-β1-induced BEAS-2B cells contrasted to the control cells. After treatment with 20 μg/mL kaempferol or 20 μM GLX351322, their contentions were clearly decreased. However, the 100 nM rapamycin treatment hampered the effects of kaempferol or GLX351322 on the secretions of IL-25 and IL-33. The expressions of NOX4 and LC3B in cells were measured by immunofluorescence (Figure 2(b)). After stimulation with 10 ng/mL TGF-β1, the NOX4 and LC3B levels were significantly increased in the BEAS-2B cells. Compared with the vehicle treated cells, the NOX4 levels were decreased after administration with kaempferol or GLX351322. The application of rapamycin clearly suppressed the roles of kaempferol or GLX351322 on the NOX4 expression.

Figure 2.

Kaempferol suppressed inflammation through NOX4-mediated autophagy in TGF-β1-induced BEAS-2B cells. (a) The secretions of IL-25 and IL-33 in cells were analyzed by ELISA; (b) The NOX4 and LC3B expression in cells was measured by immunofluorescence (Scale = 20 μm). The TGF-β1-induced BEAS-2B cells were treated with 20 μg/mL kaempferol or 20 μM GLX35132 (a NOX4 inhibitor) for 24 h. The 100 nM rapamycin (an autophagy activator) treatment significantly weakened the effects of kaempferol or GLX35132. vs. control group, ^**p < 0.01; vs. 0.1% DMSO group, ^#p < 0.05, ^##p < 0.01; vs. kaempferol group, ^&p < 0.05, ^&&p < 0.01; vs. GLX351322group, ^p < 0.05, ^^p < 0.01.

Furthermore, the expressions of NOX4, ATG5 and Beclin-1 proteins in the BEAS-2B cells were measured by western blot (Figure 3(a)). After TGF-β1 induction, the expressions of NOX4, ATG5 and Beclin-1 were significantly increased. Compared with the 0.1% DMSO group, the expressions of NOX4, ATG5 and Beclin-1 were clearly decreased in the kaempferol group or GLX351322 group. However, the roles of kaempferol or GLX351322 were obviously weakened by rapamycin treatment.

Figure 3.

Kaempferol suppressed NOX4-mediated autophagy in TGF-β1-induced BEAS-2B cells. (a) The expressions of NOX4, ATG5 and Beclin-1 proteins were analyzed by western blot; The relative expressions of NOX4 (b), ATG5 (c) and Beclin-1 (d) were normalized by GAPDH. The expressions of proteins were quantified by Image J software. vs. control group, ^**p < 0.01; vs. 0.1% DMSO group, ^#p < 0.05, ^##p < 0.01; vs. kaempferol group, ^&p < 0.05, ^&&p < 0.01; vs. GLX351322 group, ^p < 0.05, ^^p < 0.01.

Kaempferol suppressed airway inflammation through NOX4-mediated autophagy in OVA-induced mice

In order to further confirm our findings, the OVA-induced allergic asthma mice were treated with kaempferol or GLX351322 (Figure 4(a)). After OVA challenge, the airway resistance significantly upregulated compared with the control mice (Figure 4(b)). When mice were treated with 20 mg/kg kaempferol or 3.8 mg/kg GLX351322, the airway resistance of mice was clearly downregulated. However, the airway resistance of mice, who treated with 20 mg/kg kaempferol plus 4 mg/kg rapamycin or 3.8 mg/kg GLX351322 plus 4 mg/kg rapamycin, was obviously upregulated when compared with the kaempferol group or GLX351322 group (Figure 4(b)).

Figure 4.

Kaempferol inhibited Th2 inflammation in OVA-induced mice. (a) Diagram of experimental protocols in OVA challenged mice. 20 mg/kg kaempferol or 3.8 mg/kg GLX351322 solutions were orally treated, and 4 mg/kg rapamycin intraperitoneally (i.p.) treated to analyze the mechanism of kaempferol. (b) Airway hyperreactivity was analyzed by airway resistance to increasing concentrations of nebulized methacholine. ^**p < 0.01. (c) Numbers of eosinophils, lymphocytes and neutrophils in bronchioalveolar lavage fluids (BALFs) were analyzed by Wright stain solution. (d) Levels of IL-4, IL-5 and IL-13 in BALFs were analyzed by ELISA. vs. control group, ^**p < 0.01; vs. 0.1% DMSO group, ^#p < 0.05, ^##p < 0.01; vs. kaempferol group, ^&p < 0.05, ^&&p < 0.01; vs. GLX351322 group, ^^p < 0.01.

The of eosinophils, lymphocytes and neutrophils in BALFs were clearly increased in OVA-induced mice (Figure 4(c)). In the kaempferol group or GLX351322 group, the numbers of eosinophils, lymphocytes and neutrophils were notably decreased compared with the 0.1% DMSO group. However, the rapamycin intraperitoneal treatment prevented the effects of kaempferol or GLX351322. The levels of IL-4, IL-5 and IL-13 in BALFs were also measured in different groups (Figure 4(d)). A similar trend of results was showed in these inflammatory cytokines that kaempferol or GLX351322 treatment clearly suppressed the secretion of these inflammatory cytokines induced by OVA. The application of rapamycin blocked the roles of kaempferol or GLX351322 on these inflammatory cytokines.

Kaempferol improved airway inflammation and fibrosis through NOX4-mediated autophagy in OVA-induced mice

Pathological changes of lung tissue were observed by HE (Figure 5(a)) and Masson (Figure 5(b)). In Figure 5(a), OVA challenge induced inflammatory infiltration (red arrows) in the lung tissues of mice, and the inflammation responses were weakened after kaempferol or GLX351322 treatment. However, the administration of rapamycin aggravated the inflammatory infiltration and hampered the effects of kaempferol or GLX351322 treatment. In Figure 5(b), collagen fibers (red arrows) were significantly increased in the lung tissues after OVA challenge. Compared with the 0.1% DMSO group, the collagen fibers clearly decreased after the kaempferol or GLX351322 treatment. Rapamycin treatment significantly inhibited the effects of GLX351322 treatment when compared with the GLX351322 group.

Figure 5.

Kaempferol improved airway inflammation and fibrosis in the lung tissues of OVA-induced mice. (A) Inflammation infiltration (red arrows) in lung tissue was observed by HE staining, scale = 200 μm, 100 μm; (B) Collagen fibers (red arrows) in lung tissue were observed Masson staining, scale = 200 μm, 100 μm. The inflammatory infiltration area and collagen fiber area were analyzed using Image J software. vs. control group, ^**p < 0.01; vs. 0.1% DMSO group, ^#p < 0.05, ^##p < 0.01; vs. kaempferol group, ^&p < 0.05, ^&&p < 0.01; vs. GLX351322 group, ^^p < 0.01.

Kaempferol downregulated NOX4 and LC3B expression in the lung tissues of OVA-induced mice

The NOX4 and LC3B expressions in the lung tissues of each group were measured by immunofluorescence (Figure 6(a)). The NOX4 and LC3B expressions were notably upregulated in the lung tissue of OVA-challenged mice, and their levels were downregulated due to kaempferol or GLX351322 administration (Figure 6(b)). The treatment of rapamycin increased the kaempferol-inhibited NOX4 and LC3B expressions (Figure 6(b)).

Figure 6.

Kaempferol downregulated NOX4 and LC3B expressions in the lung tissues of OVA-induced mice. (a)The NOX4 and LC3B expressions were measured by immunofluorescence. (b) The mean gray value of NOX4 and LC3B expression was analyzed using Image J software. vs. control group, ^**p < 0.01; vs. 0.1% DMSO group, ^#p < 0.05, ^##p < 0.01; vs. kaempferol group, ^&p < 0.05, ^&&p < 0.01; vs. GLX351322 group, ^p < 0.05, ^^p < 0.01.

Kaempferol suppressed NOX4-mediated autophagy proteins in the lung tissues of OVA-induced mice

The NOX4, ATG5 and Beclin-1 expressions in the lung tissues of each group were observed by western blot (Figure 7(a)). Their relative expression levels were obviously increased in the OVA-induced lung tissues contrasted to the control lung tissues (Figures 7(b) to (d)). By contrast to the 0.1% DMSO treatment, the kaempferol or GLX351322 treatment clearly suppressed expressions of these proteins in the damaged lung tissues. The application of rapamycin significantly prevented the roles of kaempferol or GLX351322 on expressions of these proteins (Figures 7(b) to (d)).

Figure 7.

Kaempferol downregulated the NOX4, ATG5 and Beclin-1 expressions in the lung tissues of OVA-induced mice. (a) The NOX4, ATG5 and Beclin-1 expressions were measured by western blot; (b–d) The relative expressions of these proteins were normalized by GAPDH, and quantified by Image J software. vs. control group, ^**p < 0.01; vs. 0.1% DMSO group, ^#p < 0.05, ^##p < 0.01; vs. kaempferol group, ^&p < 0.05, ^&&p < 0.01; vs. GLX351322 group, ^p < 0.05, ^^p < 0.01.

Discussion

In this study, we found a good binding of kaempferol to NOX4 (score = −9.2 kcal/mol) due to the binding of two hydrogen bonds between kaempferol and NOX4. The NOX4 expression was downregulated with kaempferol dose increase in TGF-β1-induced BEAS-2B cells, indicating that kaempferol might inhibit inflammation by regulating NOX4.

In order to confirm this hypothesis, 20 μg/mL kaempferol was selected to treat TGF-β1-induced BEAS-2B, and 20 μM GLX35132 (a NOX4 inhibitor) was as a positive control to compare the effects of kaempferol on NOX4-mediated inflammation in BEAS-2B cells. Many researchers have reported that kaempferol treatment could suppress type II inflammation in allergic asthma.^6–8 As known, allergic asthma is mediated by type II immune response. In type II immune response, helper type 2 (Th2) cells and their cytokines, IL-4, IL-5 and IL-13 play a central role. Airway epithelial cells would secrete IL-25 and IL-33 to activate natural immune cells, and further initiate type Ⅱ immune response.^29,30 In this study, we found that the secretions of IL-25 and IL-33 in the TGF-β1-induced BEAS-2B cells were significantly decreased by kaempferol treatment. At the same time, similar trends were found after the treatment of GLX35132 in the TGF-β1-induced BEAS-2B cells. These data indicated that kaempferol regulated NOX4 to play its anti-inflammation in the TGF-β1-induced BEAS-2B cells.

In this study, we also found the co-expressions of NOX4 and LC3B were significantly increased in the TGF-β1-induced BEAS-2B cells, and kaempferol treatment obviously decreased the NOX4 and LC3B expressions. Oxidant stress is an essential molecular mechanism of airway inflammation in the allergic asthma, and excess intercellular ROS, mainly generated by mitochondria and NADPH oxidase, contributes to airway inflammation.^31,32 In the epithelial cells, persistent TGF-β3 activation increased ROS levels in a NOX4-dependent pathway and subsequently induced autophagy. ³³ As a subtype of nonphagocytic NADPH oxidase, NOX4 derives ROS and contributes to the process of autophagy dysfunction in smoking-induced lung damage. ¹⁶ In mammals, the net amount of microtubule-associated protein one light chain 3B (LC3B) is a critical hallmark for monitoring autophagy, and other autophagic proteins including ATG5 and Beclin-1 are also involved in the formation of autophagosomes.^33,34 In order to confirm that kaempferol suppresses inflammation through NOX4-mediated autophagy, we used rapamycin, an autophagy activator, to stimulate autophagy. Rapamycin is an mTOR inhibitor, and mTOR inhibits autophagy by targeting the ULK1 complex, which is needed for the formation of the autophagosome. ¹⁹ Our results showed that rapamycin administration obviously weakened the roles of kaempferol or GLX35132 in the TGF-β1-induced BEAS-2B cells.

To confirm our findings, OVA-induced mice were used to observe the therapeutic mechanism of kaempferol treatment. The data showed that kaempferol treatment significantly improved Th2-driven inflammation and collagen deposition through suppressing NOX4-mediated autophagy. In the OVA-induced mice, the application of rapamycin was also significantly weakened the effects of kaempferol. All results of this study reveal that kaempferol suppresses airway inflammation through regulating NOX4-mediated autophagy in the OVA-induced mice.

Conclusions

This study identifies that kaempferol binds NOX4 to perform its functions in the treatment of asthma. Through downregulation of NOX4-mediated autophagy, kaempferol administration prevented Th2-driven inflammation and airway remodeling in TGF-β1-induced cells and OVA-induced mice.