Characterization of TiO2NPs
TiO2NPs were purchased from Sigma-Aldrich (particle size < 25 nm, 637254, St. Louis, MO, USA). We quantified the morphology and size of TiO2NPs 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 TiO2NPs was measured by nitrogen absorption methods based on the multipoint BET method (ASAP2020; Micromeritics, Norcross, GA, USA). The hydrodynamic size and zeta potential of TiO2NPs were determined by ELS-8000 (Otsuka Electronic, Tokyo, Japan). The purity of TiO2NPs used in the experiment were determined by energy-dispensive X-ray spectoroscopy (Rayny EDX-700, Shimadzu). The endotoxin levels in TiO2NPs 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 TiO2NPs 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 TiO2NPs, twenty-four healthy female mice were randomly assigned to four experimental groups (n = 6 per group); VC group and three TiO2NPs-treated (5, 10, and 20 mg/kg, respectively) groups. On day 1, 3, and 5, animals of the TiO2NPs treated groups (5, 10, and 20 mg/kg doses in 50 μL of PBS, respectively) received TiO2NPs via intranasal instillation under light anesthesia using Zoletil 50® (Virbac Laboratories, Carros, France). The VC group received 50 μL of PBS via intranasal instillation. TiO2NPs 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 TiO2NPs 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+ TiO2NPs (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 TiO2NPs treatment groups (5, 10, and 20 mg/kg doses in 50 μL of PBS, respectively) received TiO2NPs via intranasal instillation under light anesthesia using Zoletil 50® (Virbac Laboratories). The VC and OVA groups received 50 μL of PBS via intranasal instillation. TiO2NPs 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.
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 . 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).
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% CO2. 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 (4×104 cells/well). After 24 h, the medium was replaced with fresh medium, and various concentrations of TiO2NPs (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 . 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 TiO2NPs or PBS and harvested after 6 h. To investigate the protein expression involved in TXNIP-apoptosis signaling, western blot was performed as mentioned above.
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.