Fructose‐1,6‐bisphosphatase aggravates oxidative stress‐induced apoptosis in asthma by suppressing the Nrf2 pathway

Abstract Asthma is a chronic airway disease that causes excessive inflammation, oxidative stress, mucus production and bronchial epithelial cell apoptosis. Fructose‐1,6‐bisphosphatase (Fbp1) is one of the rate‐limiting enzymes in gluconeogenesis and plays a critical role in several cancers. However, its role in inflammatory diseases, such as asthma, is unclear. Here, we examined the expression, function and mechanism of action of Fbp1 in asthma. Gene Expression Omnibus (GEO) data sets revealed that Fbp1 was overexpressed in a murine model of asthma and in interleukin (IL)‐4‐ or IL‐13‐stimulated bronchial epithelial cells. We confirmed the findings in an animal model as well as Beas‐2B and 16HBE cells. In vitro investigations revealed that silencing of Fbp1 reduced apoptosis and the proportion of cells in the G2/M phase, whereas overexpression led to increases. Fbp1 knock‐down inhibited oxidative stress by activating the nuclear factor erythroid 2‐related factor 2 (Nrf2) pathway, whereas Fbp1 overexpression aggravated oxidative stress by suppressingthe Nrf2 pathway. Moreover, the Nrf2 pathway inhibitor ML385 reversed the changes caused by Fbp1 inhibition in Beas‐2B and 16HBE cells. Collectively, our data indicate that Fbp1 aggravates oxidative stress‐induced apoptosis by suppressing Nrf2 signalling, substantiating its potential as a novel therapeutic target in asthma.


| INTRODUC TI ON
Bronchial asthma is a common chronic disease of the airway, with clinical signs that include dyspnoea, chest tightness, wheezing and coughing. Asthma is a major public health concern and affects approximately 334 million individuals worldwide, with evidence of an increasing prevalence. 1 Allergic asthma is characterized by Th2-mediated inflammation, typically initiated by antigen-presenting and bronchial epithelial cells. 2 Previous studies have shown that bronchial epithelial injury is a cardinal component of asthma pathogenesis and is correlated with disease severity. 3 As asthma progresses, bronchial epithelial cells undergo aberrant apoptosis under allergen challenge. An increased rate of apoptosis has been reported in patients with severe asthma. 4 The delayed or defective clearance of apoptotic cells in individuals with asthma exacerbates airway inflammation and airway hyperresponsiveness (AHR). 5 Additionally, the occurrence of oxidative stress caused by an imbalance between oxidation and antioxidation plays a critical role in asthma via activation of inflammatory signalling and bronchial epithelial cell apoptosis. 6,7 However, the apoptosis and oxidative stress mechanisms of bronchial epithelial cells in asthma are largely uncharacterized.
Fructose-1,6-bisphosphatase (Fbp1) is a rate-limiting enzyme of gluconeogenesis that catalyses the splitting of fructose-1,6bisphosphate into fructose 6-phosphate and inorganic phosphate. 8 Fbp1 reportedly plays a critical role in several diseases, including type 2 diabetes mellitus, 9 cancers 10-12 and acute liver failure. 13 Kaur et al 9 reported that Fbp1 can be considered a potential target for the treatment of type 2 diabetes. In cholangiocarcinoma cells, overexpression of Fbp1 induces apoptosis and suppresses cell proliferation, migration and invasion. 14 Loss of Fbp1 also represses reactive oxygen species (ROS) production in breast cancer. 15 Wang et al 13 suggested that Fbp1 is a promising biomarker of acute liver failure, as high serum levels of Fbp1 were found to be correlated with high mortality. However, the role of Fbp1 in inflammatory diseases, such as asthma, has not been elucidated.
Studies have shown that nuclear factor erythroid-derived 2-related factor 2 (Nrf2) is an important endogenous antioxidant and anti-apoptotic transcription factor. The Nrf2 pathway is considered a vital mechanism of protection in individuals with asthma, and Nrf2 signalling is crucial for maintaining bronchial epithelial barrier integrity. 16 Moreover, Nrf2 activators have been shown to ameliorate airway inflammation, AHR and oxidative stress in mice. 17,18 However, the relationship between Fbp1 and the Nrf2 pathway has not been identified.
In the current study, we aimed to investigate the expression of Fbp1 in a murine model of asthma and interleukin (IL)-4-stimulated or IL-13-stimulated airway bronchial cell lines. Furthermore, we sought to examine the mechanisms underlying the effects of Fbp1 on oxidative stress-induced apoptosis in asthma.

| Bioinformatics analyses
We downloaded seven gene expression data sets associated with asthma from the Gene Expression Omnibus (GEO) database (https:// www.ncbi.nlm.nih.gov/geo/). The GSE41667, GSE6858, GSE41665 and GSE79156 data sets were relevant to the murine model of ovalbumin-induced asthma, and the GSE19182, GSE37693 and GSE78914 data sets were relevant to IL-4-stimulated or IL-13stimulated bronchial epithelial cells. The GSE19182 data set was divided into two data sets because both IL-4 and IL-13 were used to stimulate the bronchial epithelial cells. Data pre-processing was carried out using a robust multi-array average derived using the Affy package in R (v3.5.1; http://www.r-proje ct.prg/). 19 Differentially expressed genes (DEGs) were identified using the limma package in R. 20 GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/) was also used to screen for DEGs in the asthmatic and control groups. 21

Significant
DEGs were selected using the criteria of |log fold change| > 1 and P < .05 and visualized as volcano plots constructed using the R package ggplot2. 22 Additional DEG data for the asthmatic mouse and control groups were selected from a previous study. 23 A plot of the overlapping up-regulated gene sets was created using the R package UpSetR. 24 The overlapping gene expression of Fbp1 in each data set was presented as a bar graph.

| Murine model of ovalbumin-induced asthma
Female BALB/c mice, 6-7 weeks old (weight 18-19 g), were purchased from Beijing Hfk Bioscience Co., Ltd and housed for 7 days to acclimate to the laboratory environment. All mice were housed under standard pathogen-free laboratory conditions with a 12-hour dark/light cycle, temperature of 22 ± 2°C and humidity of 55 ± 5% and provided ad libitum access to food and water. All animal experiments were conducted in accordance with the Chinese National The mice were sensitized with 50 µg ovalbumin (OVA; Grade V, Sigma-Aldrich) mixed with 0.8 mg aluminium hydroxide (Imject Alum Adjuvant, Thermo Fisher Scientific) in sterile saline by intraperitoneal (i.p.) injection on days 0, 7 and 14, as described previously. 25 On days 21-28, the mice were challenged for 30 minutes with 2% OVA using a nebulizer (DeVilbiss). Control mice were treated according to the same protocol but received saline only in the sensitization and challenge phases.

| Histological examination of lung tissue
Lung tissue specimens were fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin blocks and sliced into 4μm-thick sections. The sections were stained with haematoxylin and eosin (HE; Solarbio) to detect inflammatory cell infiltration or Periodic acid-Schiff (PAS; Wanleibio Group, Inc) to measure mucus production in the goblet cells.

| Bronchoalveolar lavage fluid (BALF) and cell counts
Mice were sacrificed by i.p. injection of overdose sodium pentobarbital 24 hours post-challenge, and BALF was collected as previously described. 26 Briefly, mouse tracheae were intubated with a catheter after the ligation of the right main bronchus, and BALF was collected by rinsing the left lung three times with 0.5 mL standard saline. The

| Immunohistochemistry
After fixation in 4% formalin and subsequent embedding in paraffin, lung tissues were sectioned at a 3μm thickness and stained with anti-Fbp1 antibody (1:200, Abcam). ImageJ software (National Institutes of Health) was used to assess protein expression.

| Cell culture transfection and treatment
The human bronchial epithelial cell line Beas-2B was purchased

| Western blot analysis
Lung samples and culture cells were homogenized with cold radioimmunoprecipitation buffer (Beyotime Biotechnology) together with protease inhibitor (Meilunbio) and phenylmethanesulfonyl fluoride (Beyotime Biotechnology). Nuclear protein was extracted from cells using a nuclear protein extraction kit according to the manufacturer's instructions (Beyotime Biotechnology). Protein concentration was measured using an enhanced BCA protein assay kit (Beyotime Biotechnology). Protein samples (25 μg) were electrophoresed and separated on 10% SDS polyacrylamide gels and then transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% skim milk or 5% bovine serum albumin (BSA) at room temperature for 2 hours and then incubated with primary antibodies at 4°C overnight.

| RNA preparation and quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted from lung tissues and cells using 5′-ATGCCACAGGATTCCATACC-3′ (reverse). The 2 −ΔΔct method was used to assess the relative mRNA fold changes with β-actin serving as the reference gene. All data shown represent the average results from three replicates.

| ROS production analysis
Beas-2B and 16HBE cells were stimulated with IL-13 for 48 hours.

tions. Assay results were measured using a Multi-Mode Microplate
Reader (BioTek Synergy HT). All data were normalized to protein concentrations.

| Cell cycle assay
Cells were harvested and fixed in 70% ethanol for 12 hours at −20°C.
The cells were then treated with RNase A (20 mg/mL) for 30 minutes at 37°C followed by treatment with 50 mg/mL propidium iodide (Solarbio) for 30 minutes in the dark at room temperature. The cell cycle was assessed by flow cytometry using a FACSCalibur (BD Biosciences).

| Statistical analysis
All data are expressed as the mean ± standard deviation. A t test was performed when assessing two groups, while one-way analysis of variance (ANOVA) was used for comparing multiple groups from a minimum of three independent experiments. SPSS 22.0 software (SPSS) and GraphPad Prism software Version 8.0 (GraphPad Software) were used to perform statistical analysis. P < .05 was considered statistically significant.

| Fbp1 expression levels in asthma were upregulated based on bioinformatics analyses
The GEO database was used to examine DEGs in the asthmatic and control groups. Ovalbumin is commonly employed in murine models of asthma for sensitization and challenge. 28 Accordingly, we selected four GEO data sets of the murine model of ovalbumin-induced asthma to identify the core genes in asthma: GSE6858, GSE41665, GSE41667 and GSE79156. In addition to these four data sets, another data set was obtained from a previously published study that used microarray to evaluate DEGs in asthmatic mice compared to those in control mice. 23 Furthermore, because allergic asthma is considered a Th2-type disease, Th2 cytokines such as IL-4 and IL-13 are widely applied in vitro to mimic the asthmatic environment. 29,30 In the current study, we selected three IL-4-stimulated or IL-13-stimulated bronchial epithelial cell GEO data sets: GSE19182, GSE37693 and GSE78914. As both IL-4 and IL-13 were used in data set GSE19182, we performed separate analyses regarding IL-4 and IL-13 for this database. Overall, a total of eight GEO data sets and one previously published data set were selected.
A bioinformatics approach was used to identify the DEGs in the data sets, which were visualized as volcano plots ( Figure 1A-H). We then considered the intersection of the up-regulated genes from the nine data sets using UpSetR of the R package and a single gene, Fbp1, was selected ( Figure 1I). As shown in Figure 1J-Q, the Fbp1 relative values were substantially higher in asthmatic mice than in control mice ( Figure 1J-M). Furthermore, the Fbp1 relative values were significantly increased in IL-4-stimulated or IL-13-stimulated bronchial epithelial cells compared to those in control cells ( Figure 1N-Q).
These results suggest Fbp1 expression is increased in asthma and may play an important role in disease progression.

| Fbp1 was overexpressed in a murine model of asthma and IL-4-stimulated or IL-13-stimulated bronchial epithelial cells
To verify the bioinformatics analyses results, we first established an

| Fbp1 induced cell apoptosis and G2/M arrest in vitro
To investigate the biological function and mechanism of action These results demonstrate that Fbp1 was able to induce apoptosis in 16HBE and Beas-2B cells.
Cell cycle arrest is closely associated with apoptosis, and block-  We then evaluated the levels of GSH, SOD and MDA, which are closely associated with oxidative stress. As indicated in Figure 4D-I, the levels of GSH and SOD were significantly increased in the Fbp1-siRNA-transfected group compared to those in the control group, while the level of MDA was significantly decreased. In contrast, Fbp1 overexpression significantly reduced GSH and SOD levels but resulted in elevated MDA levels. Taken together, the data suggest that the Fbp1 could aggravate oxidative stress in 16HBE and

| Fbp1 induced apoptosis and aggravated oxidative stress by inhibiting the Nrf2 pathway
To investigate the mechanism underlying the effect of Fbp1 on apop-

| Aggravation of oxidative stress-induced apoptosis by Fbp1 was partially reversed by Nrf2 inhibitor ML385
To explore whether the Nrf2 pathway plays a role in the mechanism by which Fbp1 exacerbates apoptosis induced by oxidative stress, we administered the Nrf2 inhibitor ML385 during transfection of the 16HBE and Beas-2B cells. As shown in Figure 6A-D, the expres-

| D ISCUSS I ON
Allergic asthma is a widespread chronic respiratory disease that results in significant morbidity and mortality worldwide and has attracted substantial attention from the scientific community.
However, in order to develop more effective treatment strategies, there is a need for improved understanding of the underlying mechanisms of asthma. Towards this end, we retrieved four data sets of a murine model of ovalbumin-induced asthma and four data sets of IL-4-stimulated or IL-13-stimulated bronchial epithelial cells from the GEO database as well as one data set from a previously published murine asthma model. These data sets were then used to analyse the DEGs in asthma. Core genes were screened by the intersection of the nine data sets, and a single gene, Fbp1, was ul- and Beas-2B cells. This is consistent with our results suggesting that Fbp1 may be involved in the pathogenesis of asthma and that its elevated level in epithelial cells following ovalbumin challenge or IL-4 or IL-13 treatment may be a detrimental mechanism inducing injury in bronchial epithelial cells.
Apoptosis is a form of programmed cell death that plays an essential role in lung disease, such as asthma. We then investigated oxidative stress as a potential mechanism of Fbp1-induced apoptosis. It is well-known that cell cycle arrest and apoptosis can be induced by excessive oxidative stress. [44][45][46] It is also known that oxidative stress plays a key role in the development of chronic diseases, including diabetes, cardiovascular disorders, cancer and asthma. 47 Allergic asthma is a respiratory disorder involving allergic reactions, airway inflammation and apoptosis.
Excessive oxidative stress in asthma results in exacerbated bronchospasms, aggravates airway inflammation and enhances apoptosis that may worsen lung damage. 48,49 Notably, it has been reported that natural antioxidants can alleviate the pathological progression of asthma by suppressing oxidative stress. 50,51 It has been reported that Fbp1 exhibits pro-oxidative effects on basal-like breast cancer and that Fbp1-expressing cells show a marked increase in ROS levels. 15 Forced Fbp1 expression also restores ROS generation in cancer stem-like cells. 42 In the current study, Fbp1 knock-down attenuated ROS and MDA levels and increased the expression of antioxidant enzymes SOD and GSH. Meanwhile, Fbp1 overexpression increased ROS and MDA levels and decreased SOD and GSH levels.
These results indicate that Fbp1 induced abnormal oxidative stress accumulation, which could subsequently lead to apoptosis and cell arrest. Thus, Fbp1 may be a potential target for improving asthma treatment.
Apoptosis related to oxidative stress is often accompanied by Nrf2 pathway inactivation. 52 Nrf2, a protective transcription factor closely associated with asthma, is associated with antioxidant and anti-apoptotic activity. In an animal model of asthma, Nrf2 knockout not only resulted in increased levels of eosinophils and neutrophils in BALF and lung tissues, which generate more ROS, but also aggravated AHR, epithelial cell apoptosis and goblet cell hyperplasia. 53 Under normal conditions, Nrf2 exists in an inactive state by binding to the inhibitory protein Keap1. In response to stress, such as oxidative stress, endoplasmic reticulum stress or hyperglycaemia, Nrf2 is phosphorylated and released from Keap1 and then translocates to the nucleus where it promotes the expression of antioxidant genes such as HO-1. [54][55][56][57] However, the relationship between the Nrf2 pathway and Fbp1 remains unclear. In an attempt to gain further insight into the molecular processes underlying asthma, we evaluated the protein expression of this pathway. Fbp1 knock-down by siRNA increased protein levels of total Nrf2, p-Nrf2, nuclear this mechanism, the Nrf2 inhibitor ML385 was used to investigate whether Fbp1 performed pro-apoptosis and pro-oxidative functions through the Nrf2 pathway. We found that co-treatment of cells with ML385 and Fbp1 siRNA partially reversed the decreased apoptosis and oxidative stress induced by Fbp1 knock-down. Therefore, the Nrf2 pathway appears to be integral to Fbp1-related oxidative stress-induced apoptosis. The potential mechanisms remain to be further explored.
In conclusion, we showed that Fbp1 expression was increased in the bronchial epithelial cells of a murine model of allergic asthma.

CO N FLI C T O F I NTE R E S T
There are no potential conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data that support the findings of this study are available from the corresponding author upon reasonable request.