Titanium dioxide nanoparticles exacerbate allergic airway inflammation via TXNIP upregulation in a mouse model of asthma

Titanium dioxide nanoparticles exacerbate allergic airway inflammation via TXNIP upregulation in a mouse model of asthma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Titanium dioxide nanoparticles exacerbate allergic airway inflammation via TXNIP upregulation in a mouse model of asthma Je-Oh Lim, Se-Jin Lee, Woong-Il Kim, So-Won Pak, Changjong Moon, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-754255/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Sep, 2021 Read the published version in International Journal of Molecular Sciences → Version 1 posted You are reading this latest preprint version Abstract Background Titanium dioxide nanoparticles (TiO 2 NPs) are widely used in the fields of industry and medicine and in various consumer products. With the increasing use of TiO 2 NPs, there has been an increase in the number of toxicity studies; however, studies investigating the mechanism underlying its toxicity are very rare. In this study, we evaluated the potential toxic effects of TiO 2 NPs exposure on the lungs as well as the development of asthma in ovalbumin (OVA)-induced mouse model of asthma. We also investigated the related toxic mechanism. Results TiO 2 NPs caused pulmonary toxicity by exacerbating the inflammatory response, indicated by an increase in the number of inflammatory cells and levels of inflammatory mediators. Exposure of mice with OVA-induced asthma to TiO 2 NPs led to significant increases in inflammatory mediators, cytokines, and airway hyperresponsiveness compared with non-exposed mice with asthma. This was also accompanied by an increase in inflammatory cell infiltration and mucus production in the lung tissues. TiO 2 NPs also decreased the expression of B-cell lymphoma 2 (Bcl2) and increased the expression of thioredoxin-interacting protein (TXNIP), phospho-apoptosis signal-regulating kinase 1, Bcl2-associated X, and cleaved-caspase 3 in the lungs of asthmatic mice compared with those of non-exposed asthmatic mice. These responses were consistent with in vitro results obtained using human airway epithelial cells. TiO 2 NPs treated cells exhibited an increase in the mRNA and protein expression of IL-1β, IL-6, and TNF-α with an elevation of TXNIP signaling compared to non-treated cells. Moreover, pathophysiological changes induced by TiO 2 NPs treatment were significantly decreased by TXNIP knockdown in the airway epithelial cells. Conclusion Taken together, TiO 2 NPs exposure induced toxicological changes in the respiratory tract and exacerbated the development of asthma via activation of the TXNIP-apoptosis pathway. These results provide insights into the mechanism underlying TiO 2 NPs-mediated respiratory toxicity. Health Policy Toxicology Titanium dioxide nanoparticle Asthma Airway inflammation Thioredoxin-interacting protein Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Air pollutants, such as yellow dust and fine dust, have become a critical social issue and are life-threatening to patients with respiratory diseases. In particular, titanium dioxide nanoparticles (TiO 2 NPs), a component of Asian dust and air pollutants, have been reported to exacerbate respiratory distress in several studies [ 1 – 4 ]. Moreover, TiO 2 NPs induce an intensive inflammatory response by triggering inflammatory cell migration and pro-inflammatory cytokine secretion, and consequently, contributing to the development and exacerbation of respiratory diseases [ 5 , 6 ]. However, the mechanisms of action associated with the toxic effects of titanium on the respiratory system and its diseases have not been clearly identified. Of the known respiratory disease, asthma is an inflammatory disease of the respiratory airways that affects proximately 300 million people globally [ 7 ]. It is characterized by excessive inflammation of the bronchi and obstruction of the airflow due to increased immune responses, resulting in varying respiratory symptoms, mainly difficulty breathing, wheezing, coughing and tightness in the chest [ 3 , 8 , 9 ]. Proinflammatory cytokines are a key player in the development and progression of asthma, and they induce elevated immune responses, resulting in characteristic responses in asthma, such as eosinophilia, airway hyperresponsiveness and mucus production [ 10 ]. Previous studies have shown that fine dust and air pollutants contribute to asthma exacerbation; however, the mechanism underlying the toxicity is not well established. Thioredoxin-interacting protein (TXNIP), a critical regulator of pathological responses, is induced by various of stressors, including inflammation, metabolic dysfunction, apoptosis, and lung dysfunction [ 11 – 13 ]. TXNIP is expressed in the lungs of experimental animals exposed to lipopolysaccharide and ovalbumin (OVA), which induce increased inflammatory responses via the activation of inflammasomes [ 14 – 16 ]. In contrast, TXNIP is involved in the apoptotic response of lung tissues. Elevation of TXNIP expression by various stimuli results in the activation of apoptotic signaling molecules, such as mitochondrial apoptosis signal-regulating kinase 1 (ASK1), B-cell lymphoma 2 associated X (Bax), p38 mitogen-activated protein kinase and cleaved-caspase 3 (Cas3), which eventually trigger the apoptosis of lung tissues [ 17 , 18 ]. The association between TiO 2 NPs and apoptosis under asthmatic conditions has not been well established. Therefore, an in-depth study is needed to understand the apoptosis mechanisms triggered via TXNIP in OVA-induced mice and how TiO 2 NPs pathologically exacerbate the development of asthma. In this study, we investigated the exacerbation of asthma in response to TiO 2 NPs exposure in OVA-induced asthmatic mice and explored the underlying mechanisms involving TXNIP and apoptosis. Results Physicochemical characterization of TiO 2 NPs The morphology, primary size, and hydrodynamic size of TiO 2 NPs are shown in Fig. 1 . The primary size and hydrodynamic size in phosphate-buffered saline (PBS) were 48.89 ± 15.49 and 238.94 ± 57.94 nm, respectively. The specific surface area of TiO 2 NPs was 40.45 and 39.38 m 2 /g as determined using Brunauer–Emmett–Teller (BET) and single point methods, respectively (Table 1 ). The zeta potential of the TiO 2 NPs was − 31.01 mV (Fig. 1 c). Purity of TiO 2 NPs was measured as 21.35% of Ti and 78.65% of O using energy-dispersive X-ray spectroscopy (Fig. 1 d). TiO 2 NPs suspensions did not show detectable endotoxin levels (data not shown). TiO 2 NPs concentrations in the lung tissues were determined using inductively coupled plasma mass spectrometry (ICP-MS) (Table 1 ); the amount of TiO 2 NPs in TiO 2 NPs-treated groups was markedly increased in a dose-dependent manner compared with that in the vehicle control (VC) group. Table 1 Measurement of surface area and ICP-MS of TiO 2 NPs. BET Surface area (m 2 /g) ICP-MS (mg/g) 40.45 VC TiO 2 NPs 5 TiO 2 NPs 10 TiO 2 NPs 20 0.38 ± 0.032 2.88 ± 0.311 5.68 ± 0.597 6.69 ± 0.613 Single point Surface area (m 2 /g) VC OVA OVA+ TiO 2 NPs 5 OVA+ TiO 2 NPs 10 OVA+ TiO 2 NPs 20 39.38 0.34 ± 0.056 0.32 ± 0.042 2.53 ± 0.397 6.43 ± 0.617 7.94 ± 0.673 Effects of TiO 2 NPs on hyperresponsiveness (AHR) and inflammatory cell counts In pulmonary toxicity study, exposure to TiO 2 NPs significantly increased inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) of mice compared with the BALF of VC mice. In particular, a marked increase in the number of neutrophils and macrophages was observed (Fig. S1). BALF of the OVA group showed increased inflammatory cell counts, especially that of eosinophils, which was significantly increased compared with the VC group (Fig. 2 a). OVA-induced mice exposed to TiO 2 NPs exhibited elevated counts of eosinophils, macrophages, and neutrophils compared with the OVA group, and this increase occurred in a dose-dependent manner. As shown in Fig. 2 b, the mean Penh value was augmented in the OVA group compared with the VC group. In addition, Penh values increased in OVA + TiO 2 NPs mice compared with OVA mice, and this increase was dose-dependent (Fig. 2 b). Effects of TiO 2 NPs on cytokine production and OVA-specific IgE levels in serum To determine whether TiO 2 NPs affect the production of inflammatory cytokines in addition to increasing the number of inflammatory cells, the cytokine levels in BALF samples were measured. In pulmonary toxicity study, the levels of proinflammatory cytokines, namely tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, were significantly increased in TiO 2 NPs-treated groups compared with the VC group (Fig. S2). The levels of TNF-α, IL-6, and IL-1β in BALF were significantly increased in OVA group compared with the VC group. Exposure to TiO 2 NPs increased the levels of TNF-α, IL-6, and IL-1β compared with the OVA group, and this increase was dose-dependent (Fig. 3 a-c). Similar to the levels of TNF-α, IL-6, and IL-1β, the levels of IL-5 and IL-13 in BALF were increased in the OVA group. Moreover, the levels of IL-5 and IL-13 were increased dose-dependently in TiO 2 NPs-treated groups compared with the OVA group (Fig. 3 d and e). The OVA-specific IgE level in serum was elevated in the TiO 2 NPs-treated groups compared with the OVA group (Fig. 3 f). Effects of TiO 2 NPs on airway inflammation and mucous secretion in OVA-induced mice Mice exposed to TiO 2 NPs showed an accumulation of inflammatory cells around the alveoli and bronchi as well as increased mucus production (Fig. S3). OVA-induced mice showed a significantly higher degree of airway inflammation then VC mice. When the OVA-induced mice were treated with TiO 2 NPs, airway inflammation worsened in a dose-dependent manner compared to that in OVA mice (Fig. 4 a). Changes in the mucus production index exhibited a pattern similar to changes in airway inflammation wherein mucus production in TiO 2 NPs-treated groups was markedly increased in a dose-dependent manner compared with that in the OVA group (Fig. 4 b). Effects Of Tionps On Txnip And Apoptotic Protein Expression Immunohistochemistry (IHC) was used to estimate the expression levels of TXNIP and cleaved- Cas3 in lung tissues in response to OVA and TiO 2 NPs treatment. The lungs of normal mice exposed to TiO 2 NPs showed a dose-dependent increase in the expression of TXNIP and cleaved-Cas3 compared with those of VC mice. Similarly, exposure to TiO 2 NPs led to increases in the expression of TXNIP, phospho-ASK1 (p-ASK1), Bax, and cleaved-Cas3 and a decrease in the expression of B-cell lymphoma 2 (Bcl2) compared with that in VC mice (Fig. S4). TXNIP expression in the OVA group was increased compared with that in the VC group. TXNIP expression increased in a dose-dependent manner in TiO 2 NPs-treated groups compared with the OVA group (Fig. 5 a). Likewise, cleaved-Cas3 expression in the OVA group increased compared with that in the VC group and in the TiO 2 NPs-treated groups compared with that in the OVA group (Fig. 5 b). The effects of TiO 2 NPs on TXNIP activation were determined using immunoblotting. As shown in Fig. 6 a and b, TXNIP expression in the lungs was increased in the OVA group compared with the VC group. The expression of p-ASK1, Bax, and cleaved-Cas3 in the OVA group was also increased compared with that in the VC group. However, Bcl2 expression decreased in OVA group compared with the VC group. Compared with the OVA group, exposure to TiO 2 NPs induced a marked increase in the expression of TXNIP, p-ASK1, Bax, and cleaved-Cas3 in a dose-dependent manner. The Bcl2 level in TiO 2 NPs-treated groups was lower than that in the OVA group. Effects of TiO 2 NPs on the production of proinflammatory mediators in NCI-H292 cells For the in vitro experiments, concentrations of TiO 2 NPs for treatment groups were decided based on the results of the cell viability assay (Fig. 7 a). TiO 2 NPs treatment significantly elevated the levels of IL-1β, IL-6, and TNF-α in NCI-H292 cells in a dose-dependent manner compared to untreated cells (Fig. 7 b-d). As shown in Fig. 8 a-d, real-time reverse-transcription polymerase chain reaction (qRT-PCR) results revealed that the expression of IL-1β , IL-6 , IL-8 , and TNF-α was markedly increased in a dose-dependent manner in NCI-H292 cells treated with TiO 2 NPs compared with those in the control group. Effects of TiO 2 NPs on TXNIP and apoptosis protein expression in NCI-H292 cells Immunoblotting revealed that TXNIP expression increased in TiO 2 NPs-treated cells compared to untreated cells, and this increase occurred in a dose-dependent manner. TiO 2 NPs treatment also led to an increase in the levels of p-ASK1, Bax, and cleaved-Cas3 compared to untreated cell, this also occurred in a dose-dependent manner (Fig. 9 a and b). To determine the role of TXNIP in mediating the effects of TiO 2 NPs, we transfected NCI-H292 cells with TXNIP -specific small interfering RNA (siRNA). The control siRNA did not have any effect on the increased expression of p-ASK1, Bax and cleaved-Cas3 and decreased Bcl2 expression seen with TiO 2 NPs treatment; however, treatment with TXNIP -specific siRNA decreased the expression of Bax and cleaved-Cas3 and increased the expression of Bcl2 in TiO 2 NPs-treated cells, restoring them to levels similar to those of the control group (Fig. 10 a and b). Discussion There has been an increase in the number of patients with underlying respiratory diseases, and they are a vulnerable subpopulation that should be considered when evaluating the potential respiratory toxicity of various substances [ 19 ]. The aim of this study was to examine the effect of TiO 2 NPs on asthma exacerbation and elucidate the mechanism that underlies this aggravation. We showed that exposure to TiO 2 NPs aggravated asthma, increased TXNIP expression, and activated apoptosis in the lungs of OVA-induced mice. In addition, TiO 2 NPs treatment of NCI-H292 cells led to an upregulation of apoptotic machinery via upregulation in TXNIP. In this study, exposure to TiO 2 NPs was found to increase the inflammatory response in the respiratory tract and worsen the major symptoms of asthma, namely, airway inflammation, mucus overproduction, and AHR, in mice with OVA-induced asthma. Eosinophilic inflammatory response, which is characteristic of asthma, is known to be induced by IL-4, IL-5, and IL-13 produced by CD4 + T helper type 2 (Th2) cells [ 20 ]. In addition, proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, function as growth factors for B cells and play an important role in the differentiation of CD4 + Th2 cells [ 21 ]. These cytokines have been reported to increase the secretion of mucus by stimulating the goblet cells of bronchi, resulting in AHR [ 22 , 23 ]. Asthmatic mice exposed to TiO 2 NPs showed a characteristic increase in cytokines, and the aforementioned major symptoms of asthma further worsened. In NCI-H292 cells treated with TiO 2 NPs, cytokine production was significantly increased, similar to our in vivo results. Thus, we demonstrated that TiO 2 NPs cause respiratory toxicity and exacerbate asthma; this is in conformity with previous reports [ 1 , 6 , 24 ]. TXNIP can directly bind to thioredoxin (TRX) and inhibit TRX function, leading to the activation of apoptotic signaling pathway. Under normal conditions, TRX inhibits the activation of ASK1 via formation of a complex with ASK1. However, activated TXNIP induces the dissociation of this TRX-ASK1 complex, resulting in the activation of ASK1 and, consequently, apoptosis [ 25 , 26 ]. The association between nanoparticles and TXNIP has been reported in several studies [ 14 , 15 ]. For example, exposure to silica dioxide nanoparticles has been shown to exacerbate asthma and increase pulmonary toxicity via upregulation of TXNIP. In this study, exposure to TiO 2 NPs increased TXNIP expression and activated TXNIP downstream signaling in the lungs of normal and asthmatic mice. Furthermore, we found that the TiO 2 NPs-activated apoptosis was suppressed by down-regulation of the TXNIP gene in human airway epithelial cells. In contrast, upregulation of TXNIP promotes apoptosis by increasing the Bax/Bcl2 ratio and cleaved-caspase 3 expression [ 18 ]. Taken together, exposure of mice to TiO 2 NPs increases the expression of TXNIP in the lungs, demonstrating that TXNIP may be involved in the molecular pathogenesis of asthma. This suggests that TXNIP may be responsible for the aggravating effect of TiO 2 NPs-induced apoptosis in asthmatic lungs. Conclusion In light of the corona virus disease of 2019 pandemic, interest in respiratory-related diseases is increasing, and the prevention and management of underlying respiratory diseases has become more important. In this study, we showed that TiO 2 NPs induced respiratory toxicity and exacerbated asthma via TXNIP upregulation. Underlying diseases of the respiratory system can be further exacerbated by various environmental allergens such as viruses, house dust mites, and air pollutants. Thus, not only patients, but also normal people may suffer health disorders. As mentioned above, TiO 2 NPs may contribute to the development of respiratory diseases and is life-threatening as it reduces resistance to environmental allergens by exacerbating any underlying respiratory disease. Therefore, this study presents toxicological information about TiO 2 NPs, which has not been previously reported, and provides evidence for the toxicological mechanism underlying TiO 2 NPs-mediated respiratory toxicity and diseases. Methods Characterization of TiO 2 NPs TiO 2 NPs were purchased from Sigma-Aldrich (particle size < 25 nm, 637254, St. Louis, MO, USA). We quantified the morphology and size of TiO 2 NPs using transmission electron microscopy (JEM-1210, JEOL, Tokyo, Japan) at an accelerating voltage of 200 kV and scanning electron microscopy (Zeiss EVO-MA10; Carl Zeiss Meditec AG, Jena, Germany) at an accelerating voltage of 15 kV. The specific surface area of TiO 2 NPs was measured by nitrogen absorption methods based on the multipoint BET method (ASAP2020; Micromeritics, Norcross, GA, USA). The hydrodynamic size and zeta potential of TiO 2 NPs were determined by ELS-8000 (Otsuka Electronic, Tokyo, Japan). The purity of TiO 2 NPs used in the experiment were determined by energy-dispensive X-ray spectoroscopy (Rayny EDX-700, Shimadzu). The endotoxin levels in TiO 2 NPs suspension were determined using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, USA). After completion of treatment procedures, lung tissue was harvested, weighed, and digested overnight with concentrated nitric acid, and the resultant samples were analyzed for elemental TiO 2 NPs using ICP-MS (Perkin Elmer, Waltham, MA, USA). Experimental procedure for allergic asthma induction Specific pathogen-free female BALB/c mice (6 weeks old) were purchased from Samtako Co. (Osan, Republic of Korea) quarantined and acclimatized for seven days. The animals were maintained at 22 ± 2 °C in a room with a relative humidity of 50 ± 5%, artificial lighting from 08:00–20:00, and 13–18 air changes per hour. Animals were provided with ad libitum access to a standard laboratory diet and water. All experimental procedures were carried out in accordance with the National Institute Health Guidelines for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of Chonnam National University approved experimental protocols involving animals (CNU IACUC-YB-2020-19). To investigate pulmonary toxicity of TiO 2 NPs, twenty-four healthy female mice were randomly assigned to four experimental groups (n = 6 per group); VC group and three TiO 2 NPs-treated (5, 10, and 20 mg/kg, respectively) groups. On day 1, 3, and 5, animals of the TiO 2 NPs treated groups (5, 10, and 20 mg/kg doses in 50 μL of PBS, respectively) received TiO 2 NPs via intranasal instillation under light anesthesia using Zoletil 50 ® (Virbac Laboratories, Carros, France). The VC group received 50 μL of PBS via intranasal instillation. TiO 2 NPs were prepared in PBS and sonicated in an ultrasonicator (VCX-130, Sonics and Materials, Newtown, CT) for 3 min (130 W, 20 kHz, pulse 59/1) before intranasal instillation. To investigate the effect of TiO 2 NPs on the development of asthma, 30 animals were randomly assigned to five experimental groups (each group, n = 6); VC group, OVA group, and three OVA+ TiO 2 NPs (5, 10, and 20 mg/kg) groups. On day 1 and 15, mice were sensitized with an intraperitoneal injection of 20 μg of OVA (Sigma-Aldrich) emulsified with 2 mg of aluminum hydroxide (Thermo Scientific) in 200 μL of PBS (pH 7.4). On day 22, 24, and 26, the mice received a 1 h airway challenge with 1% (w/v) OVA solution aerosolized using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan). On day 21, 23, and 25, animals of the TiO 2 NPs treatment groups (5, 10, and 20 mg/kg doses in 50 μL of PBS, respectively) received TiO 2 NPs via intranasal instillation under light anesthesia using Zoletil 50 ® (Virbac Laboratories). The VC and OVA groups received 50 μL of PBS via intranasal instillation. TiO 2 NPs were prepared in PBS and sonicated in an ultrasonicator for 3 min before intranasal instillation. Measurement of airway AHR Penh values were indirectly assessed at 24 h after the final intranasal instillation via single-chamber whole body plethysmography (Allmedicus, Seoul, Republic of Korea). Briefly, mice were anesthetized with an intraperitoneal injection of a mixture of Zoletil and Xylazine (40 mg/kg and 10 mg/kg, respectively), placed in a chamber, and nebulized with aerosolized PBS or methacholine in increasing concentrations (10, 20, and 40 mg/mL). Collection of BALF and cell counting Mice were sacrificed at 24 h after measurement of AHR via an intraperitoneal injection of Zoletil 50 ® (Virbac Laboratories), and a tracheostomy was performed. To obtain BALF, ice-cold PBS (0.7 mL) was infused into the lungs twice and was withdrawn each time using a tracheal cannula (a total volume of 1.4 mL). The BALF samples were centrifuged, and its supernatant was collected for biochemical analysis. Collected cells were resuspended in ice-cold PBS (0.5 mL), and 200 μL of the resuspended solution was centrifuged (200 g, 4 °C, 10 min) onto slides using a Cytospin (Hanil Science Industrial Co., Ltd., Seoul, Republic of Korea). The slides were dried, and the cells were fixed and stained. Differential cell counts were performed using the Diff-Quik® staining reagent (Sysmex Corporation, Kobe, Japan) according to the manufacturer's instructions. Cytokines assay The levels of several cytokines, namely TNF-α, IL-6, IL-1β, IL-5, and IL-13, were measured in BALF using commercial enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. The serum level of OVA-specific IgE was measured using an ELISA kit (BioLegend, San Diego, CA, USA). Absorbance was measured at 450 nm using an ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA). Histopathology and IHC After BALF samples were collected, the lung tissue was fixed with 4% (v/v) paraformaldehyde for 48 h. The tissues were paraffin-embedded, sectioned at a thickness of 4 μm and stained using hematoxylin and eosin (Sigma-Aldrich) or periodic acid-Schiff solution (IMEB Inc., San Marcos, CA, USA) to evaluate airway inflammation and mucus production, respectively. In addition, the sectioned tissues were processed for IHC, as previously described (Lim et al., 2020b). Primary antibodies used for detection of protein expression were anti-TXNIP (NBP1-54578; 1:200 dilution; Novus Biologicals, Littleton, CO, USA) and anti-cleaved-Cas3 (#9661; 1:200 dilution; Cell signaling, Danvers, MA, USA). Each slide was examined manually by investigators blind to the treatment groups using a light microscope (Leica, Wetzlar, Germany) with 10 and 20x objective lenses and a 100x oil immersion lens. Ten randomly selected nonoverlapping areas per slide were captured with a digital camera (IMTcamCCD5; IMT Inc., Daejeon, Republic of Korea), and quantitative analyses of airway inflammation, mucus production, and protein expression were performed using an image analyzer (IMT i-Solution software, Vancouver, BC, Canada). Western blot analysis To quantify protein expression, we performed immunoblotting as previously described [27]. Primary antibodies used are as follows: anti-TXNIP (NBP1-54578; Novus Biologicals), anti-p-ASK1 (SAB4504337; Sigma-Aldrich), anti-total-ASK1 (t-ASK1, ab45178; Abcam, Cambridge, UK), anti-Bcl2 (#2876; Cell signaling), anti-Bax (#2772; Cell signaling), anti-cleaved-Cas3 (#9661; Cell signaling), and anti-β-actin (β-act, #4967; Cell signaling). Densitometric analysis of expression was performed using Chemi-Doc (Bio-Rad Laboratories). Cell culture The human airway epithelial cell line NCI-H292 was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in RPMI 1640 medium (WELGENE, South Korea) with 10% fetal bovine serum, streptomycin (100 μg/mL), and penicillin (100 U/mL) and incubated in a humidified chamber maintained at 37 °C with 5% CO 2 . The cells were serum-starved for 1 h before use. Cell viability assay Cell viability was performed using an EZ-Cytox cell viability assay kit (DAELIL lab, Seoul, Republic of Korea). Briefly, NCI-H292 cells were seeded in 96 well-plate (4×10 4 cells/well). After 24 h, the medium was replaced with fresh medium, and various concentrations of TiO 2 NPs (1.56, 3.13, 6.25, 12.5, 25 µg/mL) were added. The culture plate was incubated for another 24 h. Subsequently, the viable cells were determined by adding 10 µL of the kit solution to each well and incubating for 4 h. Absorbance was measured at 450 nm using an ELISA reader (Bio-Rad Laboratories). Measurement of mRNA expression of proinflammatory cytokines in NCI–H292 cells To quantify proinflammatory cytokines mRNA expression, we measured using qRT-PCR as described previously [28]. qRT-PCR experiments were performed using specific forward and reverse primers (Table S1). Small interfering RNA transfection of NCI-H292 cells TXNIP -specific siRNA (4392420) and scrambled siRNA (4390843) were purchased from Ambion (Waltham, MA, USA). Each siRNA (20 nM) was transfected into NCI-H292 cells using LipofectamineTM RNAiMAX reagent (Invitrogen, Waltham, MA, USA) following the forward transfection method, as prescribed by the manufacturer. After suppression of endogenous TXNIP expression, the cells were treated with 25 µg/mL TiO 2 NPs or PBS and harvested after 6 h. To investigate the protein expression involved in TXNIP-apoptosis signaling, western blot was performed as mentioned above. Statistical analysis Data are expressed as means ± standard deviation (SD). Statistical significance was determined using analysis of variance followed by Dunnett’s test for multiple comparisons. P values less than 0.05 were considered statistically significant. Abbreviations TiO 2 NPs: Titanium dioxide nanoparticles; TXNIP: Thioredoxin-interacting protein; OVA: Ovalbumin; ASK1: Apoptosis signal-regulating kinase 1; Bax: B-cell lymphoma 2 associated X; Cas3: Caspase 3; PBS: Phosphate-buffered saline; BET: Brunauer–Emmett–Teller; ICP-MS: Inductively coupled plasma mass spectrometry; VC: Vehicle control; AHR: Hyperresponsiveness; BALF: Bronchoalveolar lavage fluid; TNF-α: Tumor necrosis factor-α; IL: Interleukin; IHC: Immunohistochemistry; p-ASK1: Phospho-ASK1; t-ASK1: Total-ASK1; Bcl2: B-cell lymphoma 2; qRT-PCR: Real-time reverse-transcription polymerase chain reaction; siRNA: Small interfering RNA; Th2: T helper type 2; TRX: Thioredoxin; ELISA: Enzyme-linked immunosorbent assay; β-act: β-actin; SD: standard deviation. Declarations Acknowledgments Not applicable. Authors’ contributions J.O.L, S.J.L., W.I.K., S.W.P., and C.M. designed and performed the experiments, analyzed the data, and interpreted the results of experiments. J.D.H. and J.C.K. conceived and supervised the study. The manuscript was written by J.O.L. and I.S.S., and revised critically by J.C.K. All authors read and approved the final manuscript. Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIT) (NRF-2020R1A4A1019395). Availability of data and materials The data sets supporting the conclusions of this article are included within the article and its Additional files 1. Ethics approval and consent to participate All experimental procedures were carried out in accordance with the National Institute Health Guidelines for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of Chonnam National University approved experimental protocols involving animals (CNU IACUC-YB-2020-19). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. 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Over-expression of thioredoxin-interacting protein promotes apoptosis of MIN6 cells via activating p38MAPK pathway. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2017;33(10):1323–7. Izquierdo JL, Almonacid C, González Y, Del Rio-Bermudez C, Ancochea J, Cárdenas R, et al. The impact of COVID-19 on patients with asthma. Eur Respir J. 2021;57(3):2003142. https://doi.org/10.1183/13993003.03142-2020 . Abdulnasser Harfoush S, Hannig M, Le DD, Heck S, Leitner M, Omlor AJ, et al. High-dose intranasal application of titanium dioxide nanoparticles induces the systemic uptakes and allergic airway inflammation in asthmatic mice. Respir Res. 2020;21(1):168. https://doi.org/10.1186/s12931-020-01386-0 . Ma J, Chan CC, Huang WC, Kuo ML. Berberine inhibits pro-inflammatory cytokine-induced IL-6 and CCL11 production via modulation of STAT6 pathway in human bronchial epithelial cells. Int J Med Sci. 2020;17(10):1464–73. https://doi.org/10.7150/ijms.45400 . Shi H, Magaye R, Castranova V, Zhao J. 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Malone CF, Emerson C, Ingraham R, Barbosa W, Guerra S, Yoon H, et al. mTOR and HDAC inhibitors converge on the TXNIP/thioredoxin pathway to cause catastrophic oxidative stress and regression of RAS-driven tumors. Cancer Discov. 2017;7(12):50–1463. https://doi.org/10.1158/2159-8290.CD-17-0177 . Shin IS, Shin NR, Park JW, Jeon CM, Hong JM, Kwon OK, et al. Melatonin attenuates neutrophil inflammation and mucus secretion in cigarette smoke-induced chronic obstructive pulmonary diseases via the suppression of Erk‐Sp1 signaling. J Pineal Res. 2015;58(1):50–60. https://doi.org/10.1111/jpi.12192 . Lim JO, Shin NR, Seo YS, Nam HH, Ko JW, Jung TY, et al. Silibinin attenuates silica dioxide nanoparticles-induced inflammation by suppressing TXNIP/MAPKs/AP-1 signaling. Cells. 2020;9(3):678. https://doi.org/10.3390/cells9030678 . Supplementary Files SupplMaterial.doc Additional file 1: Fig. S1 Effects of TiO2NPs exposure on inflammatory cell counts in BALF. Fig. S2 Effects of TiO2NPs exposure on cytokine levels in BALF. Fig. S3 Effects of TiO2NPs exposure on inflammatory cell infiltration and mucus production in the lungs. Fig. S4 Effects of TiO2NPs exposure on the expression of TXNIP, p-ASK1, t-ASK1, Bax, Bcl2, and cleaved-Cas3 in the lungs. Table S1. Primer sequences for qRT-PCR. Cite Share Download PDF Status: Published Journal Publication published 14 Sep, 2021 Read the published version in International Journal of Molecular Sciences → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-754255","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":42565275,"identity":"41620398-821f-430b-8eca-e972f46b08eb","order_by":0,"name":"Je-Oh Lim","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Je-Oh","middleName":"","lastName":"Lim","suffix":""},{"id":42565276,"identity":"efe545ab-7c8d-4b51-bb1a-d7b40b51a54f","order_by":1,"name":"Se-Jin Lee","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Se-Jin","middleName":"","lastName":"Lee","suffix":""},{"id":42565277,"identity":"2d2c8df1-caa1-48ab-90d3-0cc5f49c1d2e","order_by":2,"name":"Woong-Il Kim","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Woong-Il","middleName":"","lastName":"Kim","suffix":""},{"id":42565278,"identity":"97e64260-9ad7-4208-bb03-950c066b0b29","order_by":3,"name":"So-Won Pak","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"So-Won","middleName":"","lastName":"Pak","suffix":""},{"id":42565279,"identity":"87f03724-ec63-48f9-8079-1ecd14f42b0f","order_by":4,"name":"Changjong Moon","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Changjong","middleName":"","lastName":"Moon","suffix":""},{"id":42565280,"identity":"75e9a03d-42ab-41ec-8c96-48f18926f558","order_by":5,"name":"In-Sik Shin","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"In-Sik","middleName":"","lastName":"Shin","suffix":""},{"id":42565281,"identity":"b394d999-d26f-43a6-a2e3-2b039a99b1d8","order_by":6,"name":"Jeong-Doo Heo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIie3OsQrCMBCA4QsdutwDVCj4Cg2FuBR8lUihU9wrOFwWu+QBHMTHcG6XTPUhxFUERxexgoLbdRTMPwQS8nEHEAr9YILMcNYaPw/ZSNJr/BieAAxEbDSMJ1Gz7M6rfZXOY5dFdQ35jLjF3LWUx4NBRK9F34NKW45sjZrYQ42YlK2wGygSbrGBzO529yILEvYxjihhybynECieuEs+IV8h9l535JOcJbIx8kbrch43Tp5oXcgtS+jr0gKwMwCm/JdQKBT6+551pjhDCMsaeAAAAABJRU5ErkJggg==","orcid":"","institution":"Korea Institute of Toxicology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jeong-Doo","middleName":"","lastName":"Heo","suffix":""},{"id":42565282,"identity":"afa9273a-fb77-46f0-8e01-186633b7d0e2","order_by":7,"name":"Jong-Choon Kim","email":"","orcid":"https://orcid.org/0000-0002-8265-9911","institution":"Chonnam National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jong-Choon","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2021-07-27 10:33:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-754255/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-754255/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.3390/ijms22189924","type":"published","date":"2021-09-14T17:20:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":12083311,"identity":"3d29d002-b7b3-44f8-9013-b106c5b3a548","added_by":"auto","created_at":"2021-08-03 20:47:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":466936,"visible":true,"origin":"","legend":"Morphology and physicochemical properties of TiO2NPs. a Morphology of TiO2NPs measured using transmission electron microscopy. b Morphology of TiO2NPs measured using scanning electron microscopy. c Zeta potential of TiO2NPs measured using ELS-8000 (-31 mV). d Purity of TiO2NPs measured using energy-dispersive X-ray spectroscopy (Ti: 21.35%, O: 78.65%). e Hydrodynamic size of TiO2NPs in PBS solution measured using ELS-8000.","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/b6afbc5c8dff2019f8cb0a39.png"},{"id":12083369,"identity":"3ae3aecb-a06c-4dcb-91ed-da930d2d691e","added_by":"auto","created_at":"2021-08-03 20:50:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95042,"visible":true,"origin":"","legend":"Morphology and physicochemical properties of TiO2NPs. a Morphology of TiO2NPs measured using transmission electron microscopy. b Morphology of TiO2NPs measured using scanning electron microscopy. c Zeta potential of TiO2NPs measured using ELS-8000 (-31 mV). d Purity of TiO2NPs measured using energy-dispersive X-ray spectroscopy (Ti: 21.35%, O: 78.65%). e Hydrodynamic size of TiO2NPs in PBS solution measured using ELS-8000.","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/b38ef5350d42f794d0ea2e70.png"},{"id":12083215,"identity":"4becdc44-3c41-456e-af6f-9dd18689d517","added_by":"auto","created_at":"2021-08-03 20:44:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107736,"visible":true,"origin":"","legend":"Effects of TiO2NPs exposure on cytokines levels in BALF and OVA-specific IgE levels in serum. a IL-1β level in BALF. b IL-6 level in BALF. c TNF-α level in BALF. d IL-5 level in BALF. e IL-13 level in BALF. f OVA-specific IgE level in serum. VC, PBS intranasal instillation; OVA, OVA challenge + PBS intranasal instillation; TiO2NPs 5, 10, and 20, OVA challenge + 5, 10, and 20 mg/kg of TiO2NPs intranasal instillation, respectively. Data are represented as means ± SD, n = 6. # p \u003c 0.05, ## p \u003c 0.01, significantly different from the VC group; * p \u003c 0.05, ** p \u003c 0.01, significantly different from the OVA group.","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/1aaf61f13af8dbc4f96a6eb2.png"},{"id":12083370,"identity":"352b3930-7852-4222-97ea-9448656acb74","added_by":"auto","created_at":"2021-08-03 20:50:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1458155,"visible":true,"origin":"","legend":"Effects of TiO2NPs exposure on inflammatory cell infiltration and mucus production in the lungs. a Lung tissue stained with hematoxylin and eosin (× 200). b Lung tissue stained with periodic acid-Schiff stain (× 200). VC, PBS intranasal instillation; OVA, OVA challenge + PBS intranasal instillation; TiO2NPs 5, 10, and 20, OVA challenge + 5, 10, and 20 mg/kg of TiO2NPs intranasal instillation, respectively. Data are represented as means ± SD, n = 6. ## p \u003c 0.01, significantly different from the VC group; * p \u003c 0.05, ** p \u003c 0.01, significantly different from the OVA group. Bar = 50 μm.","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/87fd8f611d766850537597eb.png"},{"id":12083221,"identity":"7c03ad1a-428d-4c54-b685-8e1050c396ff","added_by":"auto","created_at":"2021-08-03 20:44:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1247000,"visible":true,"origin":"","legend":"Effects of TiO2NPs exposure on the expression of TXNIP and cleaved-Cas3 in the lungs. a Expression of TXNIP (× 400, alveolar). b Expression of cleaved-Cas3 (× 400, alveolar). VC, PBS intranasal instillation; OVA, OVA challenge + PBS intranasal instillation; TiO2NPs 5, 10, and 20, OVA challenge + 5, 10, and 20 mg/kg of TiO2NPs intranasal instillation, respectively. Data are represented as means ± SD, n = 6. ## p \u003c 0.01, significantly different from the VC group; ** p \u003c 0.01, significantly different from the OVA group. Bar = 50 μm.","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/64382a1ac30ee6dba80f0244.png"},{"id":12083219,"identity":"ed9fd5b2-396f-44c8-93ef-9c6a96f1de4d","added_by":"auto","created_at":"2021-08-03 20:44:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":119031,"visible":true,"origin":"","legend":"Effects of TiO2NPs exposure on the expression of TXNIP, p-ASK1, t-ASK1, Bax, Bcl2, and cleaved-Cas3 in the lungs. a Protein expression was determined using western blotting. b Relative densitometric values of protein expression. VC, PBS intranasal instillation; OVA, OVA challenge + PBS intranasal instillation; TiO2NPs 5, 10, and 20, OVA challenge + 5, 10, and 20 mg/kg of TiO2NPs intranasal instillation, respectively. Data are represented as means ± SD, n = 6. # p \u003c 0.05, ## p \u003c 0.01, significantly different from the VC group; ** p \u003c 0.01, significantly different from the OVA group.","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/cf692f890634644fcb523774.png"},{"id":12083213,"identity":"5c0c64ef-2946-4052-bf5e-c2d8515116eb","added_by":"auto","created_at":"2021-08-03 20:44:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":79570,"visible":true,"origin":"","legend":"Effects of TiO2NPs treatment on cell viability and inflammatory cytokines in NCI–H292 cells. a Cell viability. b IL-1β level. c IL-6 level. d TNF-α level. Control, PBS treatment; 1.5625, 3.125, 6.25, 12.5, and 25 μg/mL of TiO2NPs treatment; respectively. Data are represented as means ± SD, n = 3. ** p \u003c 0.01, significantly different from the control group.","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/f1de9813c71a562469ef8f1b.png"},{"id":12083216,"identity":"524fc229-a9bc-4de4-b627-b1ea0761ed4a","added_by":"auto","created_at":"2021-08-03 20:44:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":70762,"visible":true,"origin":"","legend":"Effects of TiO2NPs treatment on mRNA expression of inflammatory cytokines measured by qRT-PCR in NCI–H292 cells. a TNF-α mRNA expression. b IL-6 mRNA expression. c IL-1β mRNA expression. d IL-8 mRNA expression. Control, PBS treatment; 3.125, 6.25, 12.5, and 25 μg/mL of TiO2NPs treatment; respectively. Data are represented as means ± SD, n = 3. ** p \u003c 0.01, significantly different from the control group.","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/4666645e35bec1fb7bf92f4f.png"},{"id":12083313,"identity":"1c3e492d-f4e1-4c13-a2bb-6392910e90bf","added_by":"auto","created_at":"2021-08-03 20:47:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":102242,"visible":true,"origin":"","legend":"Effects of TiO2NPs treatment on the expression of TXNIP, p-ASK1, t-ASK1, Bax, Bcl2, and cleaved-Cas3 in NCI–H292 cells. a Protein expression was determined using western blotting. b Relative densitometric values of protein expression. Control, PBS treatment; 3.125, 6.25, 12.5, and 25 μg/mL of TiO2NPs treatment; respectively. Data are represented as means ± SD, n = 3. ** p \u003c 0.01, significantly different from the control group.","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/095311e18503f846b4332228.png"},{"id":12083218,"identity":"ca2a7df7-7361-409f-b036-1ba26b05c14f","added_by":"auto","created_at":"2021-08-03 20:44:27","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":84602,"visible":true,"origin":"","legend":"Effects of knockdown of TXNIP on TiO2NPs-induced apoptosis in NCI–H292 cells. a Proteins expression by western blotting. b Relative densitometric values of protein expression. Si-scr, scrambled siRNA 20 nM treatment; si-scr+TiO2NPs, scrambled siRNA 20 nM + TiO2NPs 25 μg/mL treatment; si-TXNIP+TiO2NPs, TXNIP siRNA 20 nM + TiO2NPs 25 μg/mL treatment. Data are represented as means ± SD, n = 3. ## p \u003c 0.01, significantly different from the si-scr group; ** p \u003c 0.01, significantly different from the si-scr+TiO2NPs group.","description":"","filename":"fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/a66a1d0902c93b86397c0798.png"},{"id":60607262,"identity":"1090b17a-02af-4d18-8325-5b53ede31489","added_by":"auto","created_at":"2024-07-18 17:20:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4249095,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/37d08f5a-4488-4023-9f24-abdc2f6855fe.pdf"},{"id":12083318,"identity":"aac80faa-ba54-4fe6-8437-f9baacf8704f","added_by":"auto","created_at":"2021-08-03 20:47:43","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5030912,"visible":true,"origin":"","legend":"Additional file 1: Fig. S1 Effects of TiO2NPs exposure on inflammatory cell counts in BALF. Fig. S2 Effects of TiO2NPs exposure on cytokine levels in BALF. Fig. S3 Effects of TiO2NPs exposure on inflammatory cell infiltration and mucus production in the lungs. Fig. S4 Effects of TiO2NPs exposure on the expression of TXNIP, p-ASK1, t-ASK1, Bax, Bcl2, and cleaved-Cas3 in the lungs. Table S1. Primer sequences for qRT-PCR.","description":"","filename":"SupplMaterial.doc","url":"https://assets-eu.researchsquare.com/files/rs-754255/v1/ebbf823baba999bdba9a4b3b.doc"}],"financialInterests":"","formattedTitle":"Titanium dioxide nanoparticles exacerbate allergic airway inflammation via TXNIP upregulation in a mouse model of asthma","fulltext":[{"header":"Background","content":"\u003cp\u003eAir pollutants, such as yellow dust and fine dust, have become a critical social issue and are life-threatening to patients with respiratory diseases. In particular, titanium dioxide nanoparticles (TiO\u003csub\u003e2\u003c/sub\u003eNPs), a component of Asian dust and air pollutants, have been reported to exacerbate respiratory distress in several studies [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, TiO\u003csub\u003e2\u003c/sub\u003eNPs induce an intensive inflammatory response by triggering inflammatory cell migration and pro-inflammatory cytokine secretion, and consequently, contributing to the development and exacerbation of respiratory diseases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the mechanisms of action associated with the toxic effects of titanium on the respiratory system and its diseases have not been clearly identified.\u003c/p\u003e \u003cp\u003eOf the known respiratory disease, asthma is an inflammatory disease of the respiratory airways that affects proximately 300\u0026nbsp;million people globally [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It is characterized by excessive inflammation of the bronchi and obstruction of the airflow due to increased immune responses, resulting in varying respiratory symptoms, mainly difficulty breathing, wheezing, coughing and tightness in the chest [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Proinflammatory cytokines are a key player in the development and progression of asthma, and they induce elevated immune responses, resulting in characteristic responses in asthma, such as eosinophilia, airway hyperresponsiveness and mucus production [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previous studies have shown that fine dust and air pollutants contribute to asthma exacerbation; however, the mechanism underlying the toxicity is not well established.\u003c/p\u003e \u003cp\u003eThioredoxin-interacting protein (TXNIP), a critical regulator of pathological responses, is induced by various of stressors, including inflammation, metabolic dysfunction, apoptosis, and lung dysfunction [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. TXNIP is expressed in the lungs of experimental animals exposed to lipopolysaccharide and ovalbumin (OVA), which induce increased inflammatory responses via the activation of inflammasomes [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In contrast, TXNIP is involved in the apoptotic response of lung tissues. Elevation of TXNIP expression by various stimuli results in the activation of apoptotic signaling molecules, such as mitochondrial apoptosis signal-regulating kinase 1 (ASK1), B-cell lymphoma 2 associated X (Bax), p38 mitogen-activated protein kinase and cleaved-caspase 3 (Cas3), which eventually trigger the apoptosis of lung tissues [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The association between TiO\u003csub\u003e2\u003c/sub\u003eNPs and apoptosis under asthmatic conditions has not been well established. Therefore, an in-depth study is needed to understand the apoptosis mechanisms triggered via TXNIP in OVA-induced mice and how TiO\u003csub\u003e2\u003c/sub\u003eNPs pathologically exacerbate the development of asthma.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the exacerbation of asthma in response to TiO\u003csub\u003e2\u003c/sub\u003eNPs exposure in OVA-induced asthmatic mice and explored the underlying mechanisms involving TXNIP and apoptosis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhysicochemical characterization of TiO\u003csub\u003e2\u003c/sub\u003eNPs\u003c/h2\u003e \u003cp\u003eThe morphology, primary size, and hydrodynamic size of TiO\u003csub\u003e2\u003c/sub\u003eNPs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The primary size and hydrodynamic size in phosphate-buffered saline (PBS) were 48.89\u0026thinsp;\u0026plusmn;\u0026thinsp;15.49 and 238.94\u0026thinsp;\u0026plusmn;\u0026thinsp;57.94 nm, respectively. The specific surface area of TiO\u003csub\u003e2\u003c/sub\u003eNPs was 40.45 and 39.38 m\u003csup\u003e2\u003c/sup\u003e/g as determined using Brunauer\u0026ndash;Emmett\u0026ndash;Teller (BET) and single point methods, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The zeta potential of the TiO\u003csub\u003e2\u003c/sub\u003eNPs was \u0026minus;\u0026thinsp;31.01 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Purity of TiO\u003csub\u003e2\u003c/sub\u003eNPs was measured as 21.35% of Ti and 78.65% of O using energy-dispersive X-ray spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). TiO\u003csub\u003e2\u003c/sub\u003eNPs suspensions did not show detectable endotoxin levels (data not shown). TiO\u003csub\u003e2\u003c/sub\u003eNPs concentrations in the lung tissues were determined using inductively coupled plasma mass spectrometry (ICP-MS) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); the amount of TiO\u003csub\u003e2\u003c/sub\u003eNPs in TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated groups was markedly increased in a dose-dependent manner compared with that in the vehicle control (VC) group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMeasurement of surface area and ICP-MS of TiO\u003csub\u003e2\u003c/sub\u003eNPs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBET Surface area (m\u003csup\u003e2\u003c/sup\u003e/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eICP-MS (mg/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e40.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eVC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003eTiO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eNPs 5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003eTiO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eNPs 10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003eTiO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eNPs 20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e2.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e5.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.597\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e6.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.613\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSingle point Surface area (m\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eVC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003eOVA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003eOVA+\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eTiO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eNPs 5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u003cb\u003eOVA+\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eTiO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eNPs 10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eOVA+\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eTiO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eNPs 20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e39.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.056\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e2.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.397\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e6.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.673\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of TiO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eNPs on hyperresponsiveness (AHR) and inflammatory cell counts\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn pulmonary toxicity study, exposure to TiO\u003csub\u003e2\u003c/sub\u003eNPs significantly increased inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) of mice compared with the BALF of VC mice. In particular, a marked increase in the number of neutrophils and macrophages was observed (Fig. S1). BALF of the OVA group showed increased inflammatory cell counts, especially that of eosinophils, which was significantly increased compared with the VC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). OVA-induced mice exposed to TiO\u003csub\u003e2\u003c/sub\u003eNPs exhibited elevated counts of eosinophils, macrophages, and neutrophils compared with the OVA group, and this increase occurred in a dose-dependent manner. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, the mean Penh value was augmented in the OVA group compared with the VC group. In addition, Penh values increased in OVA\u0026thinsp;+\u0026thinsp;TiO\u003csub\u003e2\u003c/sub\u003eNPs mice compared with OVA mice, and this increase was dose-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of TiO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eNPs on cytokine production and OVA-specific IgE levels in serum\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether TiO\u003csub\u003e2\u003c/sub\u003eNPs affect the production of inflammatory cytokines in addition to increasing the number of inflammatory cells, the cytokine levels in BALF samples were measured. In pulmonary toxicity study, the levels of proinflammatory cytokines, namely tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, were significantly increased in TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated groups compared with the VC group (Fig. S2). The levels of TNF-α, IL-6, and IL-1β in BALF were significantly increased in OVA group compared with the VC group. Exposure to TiO\u003csub\u003e2\u003c/sub\u003eNPs increased the levels of TNF-α, IL-6, and IL-1β compared with the OVA group, and this increase was dose-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). Similar to the levels of TNF-α, IL-6, and IL-1β, the levels of IL-5 and IL-13 in BALF were increased in the OVA group. Moreover, the levels of IL-5 and IL-13 were increased dose-dependently in TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated groups compared with the OVA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and e). The OVA-specific IgE level in serum was elevated in the TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated groups compared with the OVA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of TiO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eNPs on airway inflammation and mucous secretion in OVA-induced mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMice exposed to TiO\u003csub\u003e2\u003c/sub\u003eNPs showed an accumulation of inflammatory cells around the alveoli and bronchi as well as increased mucus production (Fig. S3). OVA-induced mice showed a significantly higher degree of airway inflammation then VC mice. When the OVA-induced mice were treated with TiO\u003csub\u003e2\u003c/sub\u003eNPs, airway inflammation worsened in a dose-dependent manner compared to that in OVA mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Changes in the mucus production index exhibited a pattern similar to changes in airway inflammation wherein mucus production in TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated groups was markedly increased in a dose-dependent manner compared with that in the OVA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eEffects Of Tionps On Txnip And Apoptotic Protein Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry (IHC) was used to estimate the expression levels of TXNIP and cleaved- Cas3 in lung tissues in response to OVA and TiO\u003csub\u003e2\u003c/sub\u003eNPs treatment. The lungs of normal mice exposed to TiO\u003csub\u003e2\u003c/sub\u003eNPs showed a dose-dependent increase in the expression of TXNIP and cleaved-Cas3 compared with those of VC mice. Similarly, exposure to TiO\u003csub\u003e2\u003c/sub\u003eNPs led to increases in the expression of TXNIP, phospho-ASK1 (p-ASK1), Bax, and cleaved-Cas3 and a decrease in the expression of B-cell lymphoma 2 (Bcl2) compared with that in VC mice (Fig. S4). TXNIP expression in the OVA group was increased compared with that in the VC group. TXNIP expression increased in a dose-dependent manner in TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated groups compared with the OVA group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Likewise, cleaved-Cas3 expression in the OVA group increased compared with that in the VC group and in the TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated groups compared with that in the OVA group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). The effects of TiO\u003csub\u003e2\u003c/sub\u003eNPs on TXNIP activation were determined using immunoblotting. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea and b, TXNIP expression in the lungs was increased in the OVA group compared with the VC group. The expression of p-ASK1, Bax, and cleaved-Cas3 in the OVA group was also increased compared with that in the VC group. However, Bcl2 expression decreased in OVA group compared with the VC group. Compared with the OVA group, exposure to TiO\u003csub\u003e2\u003c/sub\u003eNPs induced a marked increase in the expression of TXNIP, p-ASK1, Bax, and cleaved-Cas3 in a dose-dependent manner. The Bcl2 level in TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated groups was lower than that in the OVA group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of TiO\u003c/strong\u003e \u003csub\u003e\u0026nbsp;\u003cstrong\u003e2\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e \u003cstrong\u003eNPs on the production of proinflammatory mediators in NCI-H292 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the \u003cem\u003ein vitro\u003c/em\u003e experiments, concentrations of TiO\u003csub\u003e2\u003c/sub\u003eNPs for treatment groups were decided based on the results of the cell viability assay (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea). TiO\u003csub\u003e2\u003c/sub\u003eNPs treatment significantly elevated the levels of IL-1\u0026beta;, IL-6, and TNF-\u0026alpha; in NCI-H292 cells in a dose-dependent manner compared to untreated cells (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb-d).\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea-d, real-time reverse-transcription polymerase chain reaction (qRT-PCR) results revealed that the expression of \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-8\u003c/em\u003e, and \u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e was markedly increased in a dose-dependent manner in NCI-H292 cells treated with TiO\u003csub\u003e2\u003c/sub\u003eNPs compared with those in the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of TiO\u003c/strong\u003e \u003csub\u003e\u0026nbsp;\u003cstrong\u003e2\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e \u003cstrong\u003eNPs on TXNIP and apoptosis protein expression in NCI-H292 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunoblotting revealed that TXNIP expression increased in TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated cells compared to untreated cells, and this increase occurred in a dose-dependent manner. TiO\u003csub\u003e2\u003c/sub\u003eNPs treatment also led to an increase in the levels of p-ASK1, Bax, and cleaved-Cas3 compared to untreated cell, this also occurred in a dose-dependent manner (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003ea and b). To determine the role of TXNIP in mediating the effects of TiO\u003csub\u003e2\u003c/sub\u003eNPs, we transfected NCI-H292 cells with \u003cem\u003eTXNIP\u003c/em\u003e-specific small interfering RNA (siRNA). The control siRNA did not have any effect on the increased expression of p-ASK1, Bax and cleaved-Cas3 and decreased Bcl2 expression seen with TiO\u003csub\u003e2\u003c/sub\u003eNPs treatment; however, treatment with \u003cem\u003eTXNIP\u003c/em\u003e-specific siRNA decreased the expression of Bax and cleaved-Cas3 and increased the expression of Bcl2 in TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated cells, restoring them to levels similar to those of the control group (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003ea and b).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere has been an increase in the number of patients with underlying respiratory diseases, and they are a vulnerable subpopulation that should be considered when evaluating the potential respiratory toxicity of various substances [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The aim of this study was to examine the effect of TiO\u003csub\u003e2\u003c/sub\u003eNPs on asthma exacerbation and elucidate the mechanism that underlies this aggravation. We showed that exposure to TiO\u003csub\u003e2\u003c/sub\u003eNPs aggravated asthma, increased TXNIP expression, and activated apoptosis in the lungs of OVA-induced mice. In addition, TiO\u003csub\u003e2\u003c/sub\u003eNPs treatment of NCI-H292 cells led to an upregulation of apoptotic machinery via upregulation in TXNIP.\u003c/p\u003e \u003cp\u003eIn this study, exposure to TiO\u003csub\u003e2\u003c/sub\u003eNPs was found to increase the inflammatory response in the respiratory tract and worsen the major symptoms of asthma, namely, airway inflammation, mucus overproduction, and AHR, in mice with OVA-induced asthma. Eosinophilic inflammatory response, which is characteristic of asthma, is known to be induced by IL-4, IL-5, and IL-13 produced by CD4\u003csup\u003e+\u003c/sup\u003e T helper type 2 (Th2) cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition, proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, function as growth factors for B cells and play an important role in the differentiation of CD4\u003csup\u003e+\u003c/sup\u003e Th2 cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These cytokines have been reported to increase the secretion of mucus by stimulating the goblet cells of bronchi, resulting in AHR [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Asthmatic mice exposed to TiO\u003csub\u003e2\u003c/sub\u003eNPs showed a characteristic increase in cytokines, and the aforementioned major symptoms of asthma further worsened. In NCI-H292 cells treated with TiO\u003csub\u003e2\u003c/sub\u003eNPs, cytokine production was significantly increased, similar to our \u003cem\u003ein vivo\u003c/em\u003e results. Thus, we demonstrated that TiO\u003csub\u003e2\u003c/sub\u003eNPs cause respiratory toxicity and exacerbate asthma; this is in conformity with previous reports [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTXNIP can directly bind to thioredoxin (TRX) and inhibit TRX function, leading to the activation of apoptotic signaling pathway. Under normal conditions, TRX inhibits the activation of ASK1 via formation of a complex with ASK1. However, activated TXNIP induces the dissociation of this TRX-ASK1 complex, resulting in the activation of ASK1 and, consequently, apoptosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The association between nanoparticles and TXNIP has been reported in several studies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For example, exposure to silica dioxide nanoparticles has been shown to exacerbate asthma and increase pulmonary toxicity via upregulation of TXNIP. In this study, exposure to TiO\u003csub\u003e2\u003c/sub\u003eNPs increased TXNIP expression and activated TXNIP downstream signaling in the lungs of normal and asthmatic mice. Furthermore, we found that the TiO\u003csub\u003e2\u003c/sub\u003eNPs-activated apoptosis was suppressed by down-regulation of the TXNIP gene in human airway epithelial cells. In contrast, upregulation of TXNIP promotes apoptosis by increasing the Bax/Bcl2 ratio and cleaved-caspase 3 expression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Taken together, exposure of mice to TiO\u003csub\u003e2\u003c/sub\u003eNPs increases the expression of TXNIP in the lungs, demonstrating that TXNIP may be involved in the molecular pathogenesis of asthma. This suggests that TXNIP may be responsible for the aggravating effect of TiO\u003csub\u003e2\u003c/sub\u003eNPs-induced apoptosis in asthmatic lungs.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn light of the corona virus disease of 2019 pandemic, interest in respiratory-related diseases is increasing, and the prevention and management of underlying respiratory diseases has become more important. In this study, we showed that TiO\u003csub\u003e2\u003c/sub\u003eNPs induced respiratory toxicity and exacerbated asthma via TXNIP upregulation. Underlying diseases of the respiratory system can be further exacerbated by various environmental allergens such as viruses, house dust mites, and air pollutants. Thus, not only patients, but also normal people may suffer health disorders. As mentioned above, TiO\u003csub\u003e2\u003c/sub\u003eNPs may contribute to the development of respiratory diseases and is life-threatening as it reduces resistance to environmental allergens by exacerbating any underlying respiratory disease. Therefore, this study presents toxicological information about TiO\u003csub\u003e2\u003c/sub\u003eNPs, which has not been previously reported, and provides evidence for the toxicological mechanism underlying TiO\u003csub\u003e2\u003c/sub\u003eNPs-mediated respiratory toxicity and diseases.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCharacterization of TiO\u003csub\u003e2\u003c/sub\u003eNPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003eNPs were purchased from Sigma-Aldrich (particle size \u0026lt; 25 nm, 637254, St. Louis, MO, USA). We quantified the morphology and size of TiO\u003csub\u003e2\u003c/sub\u003eNPs using transmission electron microscopy (JEM-1210, JEOL, Tokyo, Japan) at an accelerating voltage of 200 kV and scanning electron microscopy (Zeiss EVO-MA10; Carl Zeiss Meditec AG, Jena, Germany) at an accelerating voltage of 15 kV. The specific surface area of TiO\u003csub\u003e2\u003c/sub\u003eNPs was measured by nitrogen absorption methods based on the multipoint BET method (ASAP2020; Micromeritics, Norcross, GA, USA). The hydrodynamic size and zeta potential of TiO\u003csub\u003e2\u003c/sub\u003eNPs were determined by ELS-8000 (Otsuka Electronic, Tokyo, Japan). The purity of TiO\u003csub\u003e2\u003c/sub\u003eNPs used in the experiment were determined by energy-dispensive X-ray spectoroscopy (Rayny EDX-700, Shimadzu). The endotoxin levels in TiO\u003csub\u003e2\u003c/sub\u003eNPs suspension were determined using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, USA). After completion of treatment procedures, lung tissue was harvested, weighed, and digested overnight with concentrated nitric acid, and the resultant samples were analyzed for elemental TiO\u003csub\u003e2\u003c/sub\u003eNPs using ICP-MS (Perkin Elmer, Waltham, MA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental procedure for allergic asthma induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecific pathogen-free female BALB/c mice (6 weeks old) were purchased from Samtako Co. (Osan, Republic of Korea) quarantined and acclimatized for seven days. The animals were maintained at 22 \u0026plusmn; 2 \u0026deg;C in a room with a relative humidity of 50 \u0026plusmn; 5%, artificial lighting from 08:00\u0026ndash;20:00, and 13\u0026ndash;18 air changes per hour. Animals were provided with \u003cem\u003ead libitum\u003c/em\u003e access to a standard laboratory diet and water. All experimental procedures were carried out in accordance with the National Institute Health Guidelines for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of Chonnam National University approved experimental protocols involving animals (CNU IACUC-YB-2020-19).\u003c/p\u003e\n\u003cp\u003eTo investigate pulmonary toxicity of TiO\u003csub\u003e2\u003c/sub\u003eNPs, twenty-four healthy female mice were randomly assigned to four experimental groups (n = 6 per group); VC group and three TiO\u003csub\u003e2\u003c/sub\u003eNPs-treated (5, 10, and 20 mg/kg, respectively) groups. On day 1, 3, and 5, animals of the TiO\u003csub\u003e2\u003c/sub\u003eNPs treated groups (5, 10, and 20 mg/kg doses in 50 \u0026mu;L of PBS, respectively) received TiO\u003csub\u003e2\u003c/sub\u003eNPs via intranasal instillation under light anesthesia using Zoletil 50\u003csup\u003e\u0026reg;\u003c/sup\u003e (Virbac Laboratories, Carros, France). The VC group received 50 \u0026mu;L of PBS via intranasal instillation. TiO\u003csub\u003e2\u003c/sub\u003eNPs were prepared in PBS and sonicated in an ultrasonicator (VCX-130, Sonics and Materials, Newtown, CT) for 3 min (130 W, 20 kHz, pulse 59/1) before intranasal instillation.\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of TiO\u003csub\u003e2\u003c/sub\u003eNPs on the development of asthma, 30 animals were randomly assigned to five experimental groups (each group, n = 6); VC group, OVA group, and three OVA+ TiO\u003csub\u003e2\u003c/sub\u003eNPs (5, 10, and 20 mg/kg) groups. On day 1 and 15, mice were sensitized with an intraperitoneal injection of 20 \u0026mu;g of OVA (Sigma-Aldrich) emulsified with 2 mg of aluminum hydroxide (Thermo Scientific) in 200 \u0026mu;L of PBS (pH 7.4). On day 22, 24, and 26, the mice received a 1 h airway challenge with 1% (w/v) OVA solution aerosolized using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan). On day 21, 23, and 25, animals of the TiO\u003csub\u003e2\u003c/sub\u003eNPs treatment groups (5, 10, and 20 mg/kg doses in 50 \u0026mu;L of PBS, respectively) received TiO\u003csub\u003e2\u003c/sub\u003eNPs via intranasal instillation under light anesthesia using Zoletil 50\u003csup\u003e\u0026reg;\u003c/sup\u003e (Virbac Laboratories). The VC and OVA groups received 50 \u0026mu;L of PBS via intranasal instillation. TiO\u003csub\u003e2\u003c/sub\u003eNPs were prepared in PBS and sonicated in an ultrasonicator for 3 min before intranasal instillation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of airway AHR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePenh values were indirectly assessed at 24 h after the final intranasal instillation via single-chamber whole body plethysmography (Allmedicus, Seoul, Republic of Korea). Briefly, mice were anesthetized with an intraperitoneal injection of a mixture of Zoletil and Xylazine (40 mg/kg and 10 mg/kg, respectively), placed in a chamber, and nebulized with aerosolized PBS or methacholine in increasing concentrations (10, 20, and 40 mg/mL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection of BALF and cell counting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were sacrificed at 24 h after measurement of AHR via an intraperitoneal injection of Zoletil 50\u003csup\u003e\u0026reg;\u003c/sup\u003e (Virbac Laboratories), and a tracheostomy was performed. To obtain BALF, ice-cold PBS (0.7 mL) was infused into the lungs twice and was withdrawn each time using a tracheal cannula (a total volume of 1.4 mL). The BALF samples were centrifuged, and its supernatant was collected for biochemical analysis. Collected cells were resuspended in ice-cold PBS (0.5 mL), and 200 \u0026mu;L of the resuspended solution was centrifuged (200 g, 4 \u0026deg;C, 10 min) onto slides using a Cytospin (Hanil Science Industrial Co., Ltd., Seoul, Republic of Korea). The slides were dried, and the cells were fixed and stained. Differential cell counts were performed using the Diff-Quik\u0026reg; staining reagent (Sysmex Corporation, Kobe, Japan) according to the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokines assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe levels of several cytokines, namely TNF-\u0026alpha;, IL-6, IL-1\u0026beta;, IL-5, and IL-13, were measured in BALF using commercial enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, San Jose, CA, USA) according to the manufacturer\u0026rsquo;s protocol. The serum level of OVA-specific IgE was measured using an ELISA kit (BioLegend, San Diego, CA, USA). Absorbance was measured at 450 nm using an ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology and IHC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter BALF samples were collected, the lung tissue was fixed with 4% (v/v) paraformaldehyde for 48 h. The tissues were paraffin-embedded, sectioned at a thickness of 4 \u0026mu;m and stained using hematoxylin and eosin (Sigma-Aldrich) or periodic acid-Schiff solution (IMEB Inc., San Marcos, CA, USA) to evaluate airway inflammation and mucus production, respectively. In addition, the sectioned tissues were processed for IHC, as previously described (Lim et al., 2020b). Primary antibodies used for detection of protein expression were anti-TXNIP (NBP1-54578; 1:200 dilution; Novus Biologicals, Littleton, CO, USA) and anti-cleaved-Cas3 (#9661; 1:200 dilution; Cell signaling, Danvers, MA, USA). Each slide was examined manually by investigators blind to the treatment groups using a light microscope (Leica, Wetzlar, Germany) with 10 and 20x objective lenses and a 100x oil immersion lens. Ten randomly selected nonoverlapping areas per slide were captured with a digital camera (IMTcamCCD5; IMT Inc., Daejeon, Republic of Korea), and quantitative analyses of airway inflammation, mucus production, and protein expression were performed using an image analyzer (IMT i-Solution software, Vancouver, BC, Canada).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify protein expression, we performed immunoblotting as previously described [27]. Primary antibodies used are as follows: anti-TXNIP (NBP1-54578; Novus Biologicals), anti-p-ASK1 (SAB4504337; Sigma-Aldrich), anti-total-ASK1 (t-ASK1, ab45178; Abcam, Cambridge, UK), anti-Bcl2 (#2876; Cell signaling), anti-Bax (#2772; Cell signaling), anti-cleaved-Cas3 (#9661; Cell signaling), and anti-\u0026beta;-actin (\u0026beta;-act, #4967; Cell signaling). Densitometric analysis of expression was performed using Chemi-Doc (Bio-Rad Laboratories).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human airway epithelial cell line NCI-H292 was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in RPMI 1640 medium (WELGENE, South Korea) with 10% fetal bovine serum, streptomycin (100 \u0026mu;g/mL), and penicillin (100 U/mL) and incubated in a humidified chamber maintained at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were serum-starved for 1 h before use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was performed using an EZ-Cytox cell viability assay kit (DAELIL lab, Seoul, Republic of Korea). Briefly, NCI-H292 cells were seeded in 96 well-plate (4\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well). After 24 h, the medium was replaced with fresh medium, and various concentrations of TiO\u003csub\u003e2\u003c/sub\u003eNPs (1.56, 3.13, 6.25, 12.5, 25 \u0026micro;g/mL) were added. The culture plate was incubated for another 24 h. Subsequently, the viable cells were determined by adding 10 \u0026micro;L of the kit solution to each well and incubating for 4 h. Absorbance was measured at 450 nm using an ELISA reader (Bio-Rad Laboratories).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of mRNA expression of proinflammatory cytokines in NCI\u0026ndash;H292 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify proinflammatory cytokines mRNA expression, we measured using qRT-PCR as described previously [28]. qRT-PCR experiments were performed using specific forward and reverse primers (Table S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSmall interfering RNA transfection of NCI-H292 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTXNIP\u003c/em\u003e-specific siRNA (4392420) and scrambled siRNA (4390843) were purchased from Ambion (Waltham, MA, USA). Each siRNA (20 nM) was transfected into NCI-H292 cells using LipofectamineTM RNAiMAX reagent (Invitrogen, Waltham, MA, USA) following the forward transfection method, as prescribed by the manufacturer. After suppression of endogenous TXNIP expression, the cells were treated with 25 \u0026micro;g/mL TiO\u003csub\u003e2\u003c/sub\u003eNPs or PBS and harvested after 6 h. To investigate the protein expression involved in TXNIP-apoptosis signaling, western blot was performed as mentioned above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are expressed as means \u0026plusmn; standard deviation (SD). Statistical significance was determined using analysis of variance followed by Dunnett\u0026rsquo;s test for multiple comparisons. \u003cem\u003eP\u003c/em\u003e values less than 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003eNPs: Titanium dioxide nanoparticles; TXNIP: Thioredoxin-interacting protein; OVA: Ovalbumin; ASK1: Apoptosis signal-regulating kinase 1; Bax: B-cell lymphoma 2 associated X; Cas3: Caspase 3; PBS: Phosphate-buffered saline; BET: Brunauer\u0026ndash;Emmett\u0026ndash;Teller; ICP-MS: Inductively coupled plasma mass spectrometry; VC: Vehicle control; AHR: Hyperresponsiveness; BALF: Bronchoalveolar lavage fluid; TNF-\u0026alpha;: Tumor necrosis factor-\u0026alpha;; IL: Interleukin; IHC: Immunohistochemistry; p-ASK1: Phospho-ASK1; t-ASK1: Total-ASK1; Bcl2: B-cell lymphoma 2; qRT-PCR: Real-time reverse-transcription polymerase chain reaction; siRNA: Small interfering RNA; Th2: T helper type 2; TRX: Thioredoxin; ELISA: Enzyme-linked immunosorbent assay; \u0026beta;-act: \u0026beta;-actin; SD: standard deviation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.O.L, S.J.L., W.I.K., S.W.P., and C.M. designed and performed the experiments, analyzed the data, and interpreted the results of experiments. J.D.H. and J.C.K. conceived and supervised the study. The manuscript was written by J.O.L. and I.S.S., and revised critically by J.C.K. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIT) (NRF-2020R1A4A1019395).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets supporting the conclusions of this article are included within the article and its Additional files 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were carried out in accordance with the National Institute Health Guidelines for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of Chonnam National University approved experimental protocols involving animals (CNU IACUC-YB-2020-19).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eCollege of Veterinary Medicine and BK21 FOUR Program, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 61186, Republic of Korea\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eBioenvironmental Science \u0026amp; Technology Division, Korea Institute of Toxicology, Jinju 52834, Republic of Korea\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJonasson S, Gustafsson A, Koch B, Bucht A. 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Cells. 2020;9(3):678. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cells9030678\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Titanium dioxide nanoparticle, Asthma, Airway inflammation, Thioredoxin-interacting protein, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-754255/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-754255/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTitanium dioxide nanoparticles (TiO\u003csub\u003e2\u003c/sub\u003eNPs) are widely used in the fields of industry and medicine and in various consumer products. With the increasing use of TiO\u003csub\u003e2\u003c/sub\u003eNPs, there has been an increase in the number of toxicity studies; however, studies investigating the mechanism underlying its toxicity are very rare. In this study, we evaluated the potential toxic effects of TiO\u003csub\u003e2\u003c/sub\u003eNPs exposure on the lungs as well as the development of asthma in ovalbumin (OVA)-induced mouse model of asthma. We also investigated the related toxic mechanism.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003eNPs caused pulmonary toxicity by exacerbating the inflammatory response, indicated by an increase in the number of inflammatory cells and levels of inflammatory mediators. Exposure of mice with OVA-induced asthma to TiO\u003csub\u003e2\u003c/sub\u003eNPs led to significant increases in inflammatory mediators, cytokines, and airway hyperresponsiveness compared with non-exposed mice with asthma. This was also accompanied by an increase in inflammatory cell infiltration and mucus production in the lung tissues. TiO\u003csub\u003e2\u003c/sub\u003eNPs also decreased the expression of B-cell lymphoma 2 (Bcl2) and increased the expression of thioredoxin-interacting protein (TXNIP), phospho-apoptosis signal-regulating kinase 1, Bcl2-associated X, and cleaved-caspase 3 in the lungs of asthmatic mice compared with those of non-exposed asthmatic mice. These responses were consistent with \u003cem\u003ein vitro\u003c/em\u003e results obtained using human airway epithelial cells. TiO\u003csub\u003e2\u003c/sub\u003eNPs treated cells exhibited an increase in the mRNA and protein expression of IL-1β, IL-6, and TNF-α with an elevation of TXNIP signaling compared to non-treated cells. Moreover, pathophysiological changes induced by TiO\u003csub\u003e2\u003c/sub\u003eNPs treatment were significantly decreased by TXNIP knockdown in the airway epithelial cells.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eTaken together, TiO\u003csub\u003e2\u003c/sub\u003eNPs exposure induced toxicological changes in the respiratory tract and exacerbated the development of asthma via activation of the TXNIP-apoptosis pathway. These results provide insights into the mechanism underlying TiO\u003csub\u003e2\u003c/sub\u003eNPs-mediated respiratory toxicity.\u003c/p\u003e","manuscriptTitle":"Titanium dioxide nanoparticles exacerbate allergic airway inflammation via TXNIP upregulation in a mouse model of asthma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-03 20:44:25","doi":"10.21203/rs.3.rs-754255/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f2de04c9-cf30-40f2-9f74-ec7ea94a6c99","owner":[],"postedDate":"August 3rd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":6173351,"name":"Health Policy"},{"id":6173352,"name":"Toxicology"}],"tags":[],"updatedAt":"2024-07-18T17:20:49+00:00","versionOfRecord":{"articleIdentity":"rs-754255","link":"https://doi.org/10.3390/ijms22189924","journal":{"identity":"international-journal-of-molecular-sciences","isVorOnly":true,"title":"International Journal of Molecular Sciences"},"publishedOn":"2021-09-14 17:20:49","publishedOnDateReadable":"September 14th, 2021"},"versionCreatedAt":"2021-08-03 20:44:25","video":"","vorDoi":"10.3390/ijms22189924","vorDoiUrl":"https://doi.org/10.3390/ijms22189924","workflowStages":[]},"version":"v1","identity":"rs-754255","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-754255","identity":"rs-754255","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}