Study On The Mechanism Of Fbp1 Regulating Nrf2 Pathway In Apoptosis Induced By Oxidative Stress Of Airway Epithelium In Asthma

Objective:Bronchial asthma is a common chronic disease of the airway,with clinical signs that include dyspnea,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.Asthma is typically initiated by antigen-presenting and bronchial epithelial cells.Previous studies have shown that bronchial epithelial injury is a cardinal component of asthma pathogenesis and is correlated with disease severity.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.The delayed or defective clearance of apoptotic cells in individuals with asthma exacerbates airway inflammation and airway hyper-responsiveness(AHR).Additionally,the occurrence of oxidative stress caused by an imbalance between oxidation and anti oxidation plays a critical role in asthma via activation of inflammatory signaling and bronchial epithelial cell apoptosis.However,the apoptosis and oxidative stress mechanisms of bronchial epithelial cells in asthma are largely uncharacterized.To explore the possible mechanisms,we selected asthma related datasets from the GEO database and found that the fructose-1,6-bisphosphatase(FBP1)was highly expressed in asthma.Fructose-1,6-bisphosphatase(Fbpl)is a rate-limiting enzyme of gluconeogenesis that catalyzes the splitting of fructose-1,6-bisphosphate into fructose 6-phosphate and inorganic phosphate.Fbpl reportedly plays a critical role in several diseases,including type 2 diabetes mellitus,cancers,and acute liver failure.It has been reported that Fbpl can be considered a potential target for the treatment of type 2 diabetes.In cholangiocarcinoma cells,overexpression of Fbpl induces apoptosis and suppresses cell proliferation,migration,and invasion.Loss of Fbpl also represses reactive oxygen species(ROS)production in breast cancer.Meanwhile,it’s suggested that Fbpl is a promising biomarker of acute liver failure,as high serum levels of Fbpl were found to be correlated with high mortality.However,the role of Fbpl 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 signaling is crucial for maintaining bronchial epithelial barrier integrity.Moreover,Nrf2 activators have been shown to ameliorate airway inflammation,AHR,and oxidative stress in mice.However,the relationship between Fbp1 and the Nrf2 pathway has not been identified.Based on the above,this study intends to find and summarize the value of FBP1 in asthma from Bioinformatics analyses.Meanwhile,the expression and localization of FBP1 in asthma were further investigated in animal and cell experiments and whether FBP1 affects oxidative stress and apoptosis of airway epithelial cells was further explored.Finally,the potential mechanism of FBP1 affecting airway epithelial oxidative stress and apoptosis was clarified.Methods:1.Asthma-related datasets was selected from the public database,an animal model of asthma and an experimental model in vitro was built to analyze the expression levels of Fbpl.(1)Asthma-related datasets selection.We downloaded gene expression datasets associated with asthma from the Gene Expression Omnibus(GEO)database.The GSE41667,GSE6858,GSE41665,and GSE79165 datasets were relevant to the murine model of ovalbumin-induced asthma,and the GSE19182,GSE37693,and GSE78914 datasets were relevant to IL-4-stimulated or IL-13-stimulated bronchial epithelial cells.The GSE19182 dataset was divided into two datasets because both IL-4 and IL-13 were used to stimulate the bronchial epithelial cells.The R language was used for data quality supervision,and the R language combined with GEO2R method was used for data processing.The gene expression in the datasets was visualized as volcano plots.At the same time,additional DEG data for the asthmatic mouse and control groups were selected from a previous study.(2)A plot of the overlapping upregulated gene sets from above 9 datasets was created using the R package UpSetR to analyze the core genes.The overlapping gene expression of Fbpl in each dataset was presented as a bar graph.(3)Murine model of ovalbumin-induced asthma.Sixteen Balb/c mice were divided into two groups:normal saline control group and OVA asthma group.The mice were sensitized with 50 μg ovalbumin mixed with 0.8 mg aluminum hydroxide in sterile saline by intraperitoneal injection on days 0,7,and 14.On days 21-28,the mice were challenged for 30 min with 2%OVA using a nebulizer.Control mice were treated according to the same protocol but received saline only in the sensitization and challenge phases.(4)The bronchoalveolar lavage fluid was collected from asthma group and control group.Then the total and classified counts of leukocytes in the alveolar lavage fluid were carried out.(5)Lung tissue samples of asthma group and control group were collected for HE staining and PAS staining to evaluate airway inflammation and mucus secretion.(6).Immunohistochemistry was used to detect the expression of FBP1 in the airway epithelial cells of the lung tissues in asthma group and control group.Western blot and qPCR were used to detect the protein and mRNA expression levels of FBP1 in the lung tissues of asthma group and control group.(7)The human bronchial epithelial cell line 16HBE and Beas-2B were treated with human recombinant IL-4 or IL-13.The protein expression levels of p-Stat6,Stat6 and FBP1 in the 16HBE and BEAS-2B stimulated with IL-4 or IL-13 were detected by Western blot.2.The effects of FBP1 on oxidative stress and apoptosis of bronchial epithelial cells.(1)FBP1 was overexpressed and knockdown in 16HBE and BEAS-2B cells.Then different group of cells were stained with propidium iodide and Annexin V-PE/7AAD to evaluate whether FBP1 could affect cell cycle and apoptosis by flow cytometry.The expression levels of Bax,Bcl-2,cleaved caspase-3 and caspase-3 were detected by Western Blot to determine whether FBP1 could affect the biomarker of apoptosis.(2)FBP1 was overexpressed and knockdown in 16HBE and BEAS-2B cells,and the content of reactive oxygen species(ROS)in different groups was detected by 2’,7’-dichlorofluorescein diacetate(DCFH-DA)probe.Cellular malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione(GSH)levels were quantified using an MDA Assay Kit,GSH Assay Kit,and total-SOD Assay Kit respectively,according to the manufacturers’ instructions.3.The mechanism of FBP1 aggravating apoptosis induced by oxidative stress in airway epithelial cells.(1)FBP1 was overexpressed and knocked down in 16HBE and BEAS-2B cells,and the protein expression levels of nuclear Nrf2,Keapl,total Nrf2 and HO-1 were detected by Western Blot to determine whether FBP1 could affect the Nrf2 pathway.(2)In 16HBE and BEAS-2B cells,the Nrf2 inhibitor ML385 was simultaneously added to the cells being transfected with Fbpl siRNA or negative control siRNA.Annexin V-PE/7AAD flow cytometry and Western Blot were used to detect the changes of apoptosis and the protein expression levels of Bax,Bcl-2,cleaved caspase-3 and caspase-3 respectively to determine whether the effect of FBP1 on apoptosis was partially dependent on the Nrf2 pathway.(3)In 16HBE and BEAS-2B,he Nrf2 inhibitor ML385 was simultaneously added to the cells being transfected with Fbpl siRNA or negative control siRNA,and the content of ROS was detected by DCFH-DA probe to determine whether the effect of FBP1 on oxidative stress was partly dependent on the Nrf2 pathway.Results:1.Fbpl was overexpressed in a murine model of asthma and IL-4-stimulated or IL-13-stimulated bronchial epithelial cells(1)The quality of the datasets GSE41667,GSE6858,GSE41665 and GSE79156 related to a murine model of asthma and the datasets GSE19182,GSE37693 and GSE78914 related to IL-4-stimulated or IL-13-stimulated bronchial epithelial cells were of acceptable quality and could be included in the present study.R language combined with GEO2R was used to analyze differentially expressed genes in the datasets.We then considered the intersection of the upregulated genes from the nine datasets using UpSetR of the R package,and a single gene,Fbp1,was selected.(2)In the murine model of asthma,the number of leukocytes in BALF was increased,and the eosinophils were the mainly increased leukocytes in BALF.Pathologically,there were lots of inflammatory cells infiltrating around the trachea and blood vessels,airway goblet cell hyper-proliferation,and mucus hypersecretion.(3)The expression levels of FBP1 protein and mRNA in lung tissues of asthmatic mice were higher than those in control group.While Fbpl expression was observed in bronchial epithelial cells of both asthmatic and control mice,immunohistochemical analyses revealed that the expression of Fbpl in asthmatic epithelial cells was significantly higher than that in the control group.(4)IL-4 and IL-13 were then used individually to stimulate 16HBE and Beas-2B cells.Stat6 phosphorylation was observed following incubation of 16HBE and Beas-2B cells with IL-4 or IL-13.Accordingly,both cytokines were considered active components in this experiment.Western blot analysis demonstrated that the protein levels of Fbpl in IL-4-stimulated or IL-13-stimulated 16HBE and Beas-2B cells were higher than those in control cells.2.Fbpl induced apoptosis and aggravated oxidative stress in bronchial epithelial cells(1)The protein levels of Fbp1 were significantly decreased after transfection with Fbp1 siRNAl and Fbp1 siRNA2 and increased after transfection with Fbp1 plasmid in 16HBE cells and BEAS-2B cells.Fbpl knockdown significantly reduced the proportion of cells in the G2/M phase for both cell lines,and Fbpl overexpression significantly increased the proportion of cells in the G2/M phase detected by propidium iodide flow cytometry.Knockdown of Fbpl resulted in decreased apoptosis,while overexpression of Fbpl 1ed to increased apoptosis detected by Annexin V-PE/7AAD flow cytometry.Western blot analysis demonstrated that Fbpl silencing reduced the levels of Bax and cleaved-caspase-3 and increased the levels of Bcl-2 in 16HBE and Beas-2B cells;however,the level of caspase-3 did not differ between the Fbpl-knockdown cells and Si-nc-transfected cells.In addition,overexpression of Fbpl 1ed to increased levels of Bax and cleaved-caspase-3 and reduced levels of Bcl-2 in both 16HBE and Beas-2B cells.The level of caspase-3 did not change in either 16HBE or Beas-2B cells.(2)Fbpl knockdown reduced the levels of ROS in the 16HBE and Beas-2B cells,while Fbpl overexpression increased the ROS levels.Similarly,the levels of antioxidant markers GSH and SOD were significantly increased in the Fbpl-siRNA-transfected group compared to those in the control group,while the level of the lipid peroxidation marker MDA was significantly decreased.In contrast,Fbpl overexpression significantly reduced GSH and SOD levels but resulted in elevated MDA levels.3.Study on the mechanism of FBP1 aggravating oxidative stress-induced apoptosis by regulating Nrf2 signaling pathway.(1)The protein levels of total-Nrf2,HO-1,and nuclear Nrf2 were significantly higher in the knockdown groups than in the control group,whereas the protein levels of Keap1 were significantly reduced in 16HBE and Beas-2B cells.In comparison,the expression of total-Nrf2,HO-1 and nuclear Nrf2 was significantly lower in the overexpression group than in the control group,whereas the expression of Keapl was significantly elevated.(2)The expression levels of Nrf2 were markedly decreased after the addition of ML385 in 16HBE and Beas-2B cells.The downregulation of apoptosis was greater in the ML385-treated group than in the untreated knockdown group,whereas apoptosis was lower than that in the negative control group detected by Annexin V-PE/7AAD flow cytometry.Knockdown of Fbp1 resulted in decreased Bax and cleaved-caspase-3 expression in 16HBE and Beas-2B cells and increased Bcl-2 expression.However,ML385 treatment during transfection partially reversed the effect of Fbp1 knockdown on the expression of Bax and cleaved-caspase-3 in both cell lines.Meanwhile,Bcl-2 levels were significantly lower in the ML385 treatment groups than in the knockdown groups,except for siRNA2 in 16HBE cells.(3)In 16HBE and BEAS-2B cells,compared with the Fbpl knockdown group,ML385 treatment during Fbpl knockdown increased ROS,but it was lower than the transfection control group detected by DCFH-DA probe.Conclusions:1.Fbpl was highly expressed in airway epithelial cells of asthmatic mice and 16HBE and BEAS-2B cells stimulated by IL-4 or IL-13.2.In 16HBE and Beas-2B cells,overexpression of Fbpl aggravated cell apoptosis and oxidative stress,while Fbpl knockdown reduced cell apoptosis and oxidative stress.3.Fbpl aggravated oxidative stress-induced apoptosis by suppressing Nrf2 signaling pathway.Nrf2 inhibitors could partially restore oxidative stress and apoptosis induced by Fbp1.