Characterization of silica nanoparticle (SiNPs)
Silicon dioxide nanoparticles used in this study were obtained from Sigma-Aldrich ((particle size: 10–20 nm, 99.5% purity) St Louis, MO, USA) and was dispersed in distilled water as stock, and sonicated before use (160 W, 20 kHz, 5 min; JY 92-IIN; Scientz, Ningbo, China). The morphology of SiNPs was examined by transmission electron microscopy (Tecnai G2 Spirit 120Kv, FEI, Czech). The hydrodynamic diameter and zeta potential of SiNPs in distilled water and RPMI-1640 culture media with 10% fetal bovine serum (FBS) were measured using dynamic light scattering (Nano-S90, Malvern Instruments, UK) and Zetasizer Nano Series (Malvern Instruments, UK), respectively. To confirm the endotoxin content of SiNPs, Bioendo EC Endotoxin Test Kit (EC325454S, Bioendo, Xiamen, China) was used and 0.0748 ± 0.0111 EU/mL was detected in SiNPs at 100 μg/mL.
The surface area of SiNPs was measured by the BET method from N2 sorption isotherm, which was recorded by a 24-h vacuum outgassing process at 120 °C on automatic specific surface and aperture analysis instrument (AUTOSORB-IQ2-MP). The weight changes in dehydration and dihydroxylation of SiNPs were determined on a Q50 thermogravimeter (TA instruments, USA), in which 3 mg of SiNPs was gradually heated to 800 °C in air with a heating rate of 10 °C/min. Fourier transform infrared (FTIR) spectrum was recorded on a Bruker Vector 22 spectrometer with KBr pellet. The ability of SiNPs to generate free radicals was examined using a Bruker A300 electron paramagnetic (EPR) instrument. The X-band (9.8 GHz) spectrum was recorded at room temperature using 5,5-dimethyl-pirroline-N-oxide (DMPO) as the spin-trapping molecule. SiNPs (5% in w:v) were dispersed in the mixture of equal volume of DMPO aqueous solution (12.5 mg/mL) and H2O2 (30%).
Hemolysis assay
Mouse blood samples were obtained from three healthy mice and stabilized with EDTA. After the serum was removed by centrifugation and suction, red blood cells (RBCs) were washed several times with sterile isotonic PBS until the supernatant was transparent. Following the last wash, RBCs were diluted to 1/10 of the original volume with sterile isotonic PBS. 300 μL of RBC suspension was mixed with 1200 μL of isotonic PBS as a negative control, 1200 μL of nanopure water as a positive control, or 1200 μL of SiNPs or calcined-SiNPs at 600 °C with concentrations from 25 to 100 μg/mL. The mixtures were vortexed and left to rest for 3 h at room temperature. The samples were centrifuged at 10000 x g at 4 °C for 3 min and the absorbance of the supernatants at 541 nm was measured using a microplate reader (Tecan Infinite M200, Switzerland). Hemolysis was calculated by the difference in the absorption between SiNPs-treatment sample and negative control, as percentage of the difference in the absorption between positive and negative controls.
Animals and exposure
Eight-week-old C57BL/6 WT and Trpm2−/− male mice were used in this study. The WT mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China), and the Trpm2−/− mice were bred in Zhejiang University after introduced from University of Leeds, where the transgenic mice were generated. Mouse embryonic clones carrying the mutated allele lacking exons 17 and 18 of the trpm2 gene and a neomycin resistance and thymidine kinase selection cassette were injected into C57BL/6-derived blastocysts. Homozygous Trpm2−/− mice were obtained by several rounds of cross-breeding and validated by PCR of genomic DNA [74]. All mice in this study were group-housed in cages with regular rodent chow and mineral water provided ad libitum under standard breeding conditions with a 12-h reversed light/dark cycle and ~ 22 °C. All animal experiments were performed strictly in accordance with the ethical guidelines by the Ethics Committee of Laboratory Animal Care and Welfare, Zhejiang University School of Medicine. The WT mice were randomized into four groups (n = 8 for each group): control group, SiNPs-treatment group, PJ34-treatment group, and SiNPs/PJ34-treatment group. The Trpm2−/− mice were divided into two groups (n = 8 for each group): control group and SiNPs-treatment group. All mice in the SiNPs-treatment groups were anesthetized using isoflurane and SiNPs suspended in normal saline (6 mg/mL) were intratracheally instilled slowly with a dosage of 10 mg/kg body weight (40 μL). PJ34 (HY-13688A, MCE, USA) was dissolved in normal saline and administered to mice intraperitoneally 1 h with a dosage of 10 mg/kg body weight before i.t. instillation with SiNPs and were daily injected for another 7 days without SiNPs exposure. The control groups received normal saline.
Bronchoalveolar lavage fluids (BALFs) and cell counts
All mice were sacrificed on the 7th day after exposure to SiNPs. The trachea was clearly visualized after separating the tissues and skin, and a 20-gauge cannula was inserted into the trachea. The lungs were lavaged with 0.8 mL of ice-cold PBS for twice and 1.2 mL of BALFs collected from each mouse. Then BALFs were centrifuged at 500x g at 4 °C for 15 min. The supernatant was transferred to a new tube and frozen for subsequent analysis. The cell pellet was suspended in 500 μL of PBS and the total cell counts were counted using hemocytometer. Counting different cells (macrophages, neutrophils and lymphocytes) was evaluated on a cytospin slide stained with Wright-Giemsa dyes (BA-4017, Baso, Zhuhai, China) and 300 cells per mouse were examined under a light microscope.
Analysis of BALFs
The concentration of total proteins in the BALFs was measured using Enhanced BCA Protein Assay Kit (P0009, Beyotime, Shanghai, China). The levels of IL-1β, IL-6, TNF-α in the BALFs were determined using ELISA Kit (ELM-IL1β-1/ELM-IL-6-1/ELM-TNFα-1, Raybiotech, GA, USA), and the amount of LDH released in the BALFs was assessed using a LDH Cytotoxicity Assay Kit (C0017, Beyotime, Shanghai, China), according to the manufacturers’ instructions.
Histological examination
Mice were euthanized under ether anesthesia on the 7th day after SiNPs exposure. All mice were placed on an iced table. The right lung was stored in liquid nitrogen, and the left lung was fixed in 4% paraformaldehyde for 48 h at 4 °C, embedded in paraffin, and serially cut into 5-μm sections. After dewaxing, the sections selected from each mouse were stained with hematoxylin and eosin (H&E) and evaluated the histology of the lung tissues under a light microscope (Olympus BX53, Tokyo, Japan).
Cell culture
The non-tumorigenic human bronchial epithelial cells (Ad12-SV40 immortalized) BEAS-2B were kindly provided by Prof. Xiangwei Gao (Institute of Environmental Medicine, Zhejiang University School of Medicine, China) and cultured in Roswell Park Memorial Institute medium (RPMI-1640, 31,800, Gibco, USA) with 10% FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin in a 5% CO2 humidified atmosphere at 37 °C. Cells were seeded at a density of 5 × 103, 1.5 × 104, 3 × 105 cells/well in 96-well, 24-well and 6-well plates, respectively, to conduct subsequent different experiments. Treatment with SiNPs was performed as described previously. Briefly, BEAS-2B cells were seeded overnight at a 60–70% confluence and treated with SiNPs or with an equal volume of PBS. The immortalized bone marrow derived macrophages (iBMDMs) derived from C57BL/6 mice were kindly provided by Prof. Feng Shao (National Institute of Biological Sciences, China) [75, 76]. iBMDM cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, 12800, Gibco, USA) with 10% FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin in a 5% CO2 humidified atmosphere at 37 °C. Both of two types of cells were exposed to SiNPs following pretreatment with various inhibitors and chelators for 30 min.
Cell viability assay
The viability of BEAS-2B cells was determined using Cell Counting Kit-8 (C0043, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates at a density of 5 × 103 cells/well and treated with SiNPs (12.5, 25, 50 and 100 μg/mL) with or without PJ34 (10 μM), NAC (5 mM, A7250, Sigma, USA), compound A1 (10 μM), TPEN (5 μM, P4413, Sigma, USA) and BAPTA-AM (1 μM, A1076, Sigma, USA) for 24 or 48 h. Cells were washed twice with PBS and CCK-8 was added to each well. After further incubated for 1.5 h, the absorbance at 450 nm was evaluated using a microplate reader (Tecan Infinite M200, Switzerland).
Detection of intracellular ROS
ROS was detected using DCFH-DA staining and fluorescence imaging. BEAS-2B cells were grown on glass-bottom dishes (Cellvis, CA, USA) to 70% confluence, and treated with SiNPs (100 μg/mL) for 12 h in the presence or absence of NAC (5 mM), and SiNPs-calcined (100 μg/mL) at 600 °C. Thirty minutes prior to imaging, cells were fed with fetal bovine serum free RPMI-1640 loaded with DCFH-DA (10 μM, S0033, Beyotime, Shanghai, China) in dark and kept in a CO2 incubator at 37 °C. Cells were washed twice with HBSS (#14025092, Gibco, USA) and visualized under an Olympus FV1000 confocal microscope. Data were analyzed using ImageJ software. DCFH-DA intensity was analyzed by integrated intensity across the whole image divided by total cell number in the same mage using ImageJ.
Quantitative RT-PCR
Total RNA was isolated using a RNAiso Plus kit (9109, Takara, Shiga, Japan) and was reverse-transcribed to cDNA using a PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A, Takara, Shiga, Japan), according to the manufacturer’s instructions. The cDNA were stored at − 80 °C until used. Quantitative PCR was performed using a SYBR Premix Ex Taq™ II Kit (RR820A, Takara, Shiga, Japan) and a 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, USA). Relative mRNA expression was calculated using the 2−ΔΔCT method, and normalized to GAPDH. The primers for IL-1β, IL-6, CXCL-1, CXCL-8 and GAPDH used are listed as follows:
IL-1β-human.
Forward: 5′-AGCTGATGGCCCTAAACAGA-3′.
Reverse: 5′-TGGTGGTCGGAGATTCGTAG-3′.
IL-1β-mouse.
Forward: 5′-TGGTGGTCGGAGATTCGTAG-3′.
Reverse: 5′-CTAATGGGAACGTCACACACCA-3′.
IL-6-human.
Forward: 5′-CCACTCACCTCTTCAGAACG-3′.
Reverse: 5′-CATCTTTGGAAGGTTCAGGTTG-3′.
IL-6-mouse.
Forward: 5′-CATCTTTGGAAGGTTCAGGTTG-3′.
Reverse: 5′-TTGTATCTCTGGAAGTTTCAGATTGTT-3′.
TNF-α-human.
Forward: 5′-TAGCCCATGTTGTAGCAAACC-3′.
Reverse: 5′-ATGAGGTACAGGCCCTCTGAT-3′.
TNF-α-mouse.
Forward: 5′-GCCACCACGCTCTTCTGTCTAC-3′.
Reverse: 5′-GCCACCACGCTCTTCTGTCTAC-3′.
CXCL-1-human.
Forward: 5′- CCAAACCGAAGTCATAGCCAC-3′.
Reverse: 5′- TGCTCCCCTTGTTCAGTATCT-3′.
CXCL-8-human.
Forward: 5′- ACTGAGAGTGATTGAGAGTGGAC-3′.
Reverse: 5′- AACCCTCTGCACCCAGTTTTC-3′.
GAPDH-human.
Forward: 5′-ACAGTCCATGCCATCACTG-3′.
Reverse: 5′-AGTAGAGGCAGGGATGATG-3′.
GAPDH-mouse.
Forward: 5′-AGTAGAGGCAGGGATGATG-3′.
Reverse: 5′-AGTAGAGGCAGGGATGATG-3′.
Elisa
The levels of IL-1β, IL-6, CXCL-1 and CXCL-8 in culture medium were assessed using ELISA kits (ELH-IL1β-1/ELH-IL-6-1/ELH-GROα-1/ELH-IL8–1, Raybiotech, GA, USA) according to the manufacturer’s recommendations. In the experiments testing the effects of inhibitors, cells were treated with PJ34 (10 μM), compound A1 (10 μM) or MCC950 (100 nM) 30 min before and during subsequent exposure to SiNPs (100 μg/ml) for 24 h.
Western blotting
BEAS-2B cell were lysed in RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) containing PMSF (1 mM). After centrifugation at 20000 x g for 10 min, the supernatant was collected and its concentration was determined using an Enhanced BCA Protein Assay Kit (P0009, Beyotime, Shanghai, China). Forty micrograms of proteins were separated by SDS-PAGE using 8% gels for detecting TRPM2 and LAMP1, or 12% gels for SQSTM1, LC3B and Cathepsin-D. β-actin was used as the protein loading control. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P, Millipore, MA, USA), and blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (v/v) (TBST) for 2 h at room temperature. The membranes were incubated with primary antibodies for TRPM2 (ab96785, 1:1000 dilution, Abcam, USA), LAMP1 (#9091, 1:1000 dilution, CST, USA), SQSTM1 (PM045, 1:1000 dilution, MBL, Japan), LC3B (#12741, 1:1000 dilution, CST, USA), Cathepsin-D (ab75852, 1:1000 dilution, Abcam, USA), and β-actin (A5316, 1:5000 dilution, Sigma, USA) overnight at 4 °C. After subjected to three 5-min washes in TBST, the membranes were incubated with secondary antibodies conjugated with various fluorophores (926–68,020, anti-mouse; 926–32,211, anti-rabbit; 1:5000 dilution, LI-COR, USA), and proteins were visualized and analyzed using a LI-COR Odyssey Infrared Fluorescent System.
LysoTracker green staining
BEAS-2B cells were seeded on glass-bottom dishes. After exposure to SiNPs (100 μg/mL) for 24 h, the cells were washed twice with HBSS and incubated for 30 min with 1 mL of prewarmed HBSS containing LysoTracker Green DND-26 dye (200 nM, L7526, Invitrogen, USA) [27]. After washing with HBSS, cells were viewed and imaged using a confocal fluorescence microscope (Olympus FV1000, Tokyo, Japan). The number and size of lysosomes in each cell was analyzed using ImageJ.
Plasmids and transient transfection
The mApple-LAMP1-pHluorin-N-8 construct was a gift from Dr. Michael Davidson (#54918, Addgene, USA). BEAS-2B cells at 60% confluence were transiently transfected with mApple-LAMP1-pHluorin or RFP-LC3-GFP plasmids using Lipofectamine 2000 (11,668,019, Invitrogen, USA) according to the manufacturer’s instructions. Transfected cells were cultured in glass-bottom dishes. For lysosome assay, BEAS-2B cells expressing mApple-LAMP1-pHluorin were treated with PJ34 or compound A1 (both at 10 μM), 30 min prior to and during exposure to SiNPs (100 μg/mL) for 24 h, and imaged under a confocal fluorescence microscope. For autophagy assay, BEAS-2B cells expressing RFP-LC3-GFP were treated with SiNPs (100 μg/mL) for 24 h in the presence or absence of PJ34 (10 μM). Cells treated with 50 μM CQ (C6628, Sigma, USA) for 3 h were used as a positive control. Images from at least three different fields per dish were captured using a confocal microscope. Cells were examined using ImageJ for lysosome function and autophagic flux. Normal lysosomes show red puncta, and impaired lysosomes show yellow puncta, and the ratio of red to yellow puncta in each cell was derived to indicate lysosome function. Autolysosomes show red puncta, and autophagosomes show yellow puncta. Autophagic flux was assessed by the ratio of autophagosomes to autolysosomes in each cell.
Acridine orange staining
Acridine orange (AO), a lysosomotropic weak base that accumulates in intracellular acidic compartments due to proton trapping, was used to measure the degradation function of lysosomes [77]. It is a metachromatic lysosomotropic dye that fluoresces red when accumulated at high concentrations in the lysosomes and green at low concentrations in the cytosol and the nucleus. Cells seeded on glass-bottom dishes were exposed to SiNPs (100 μg/mL) in the absence or presence of compound A1 (10 μM), TPEN (5 μM), or BAPTA-AM (1 μM) for 24 h, and stained with 5 μg/mL AO (A6014, Sigma, USA) at 37 °C for 30 min. After washing twice with PBS, cells were imaged under a confocal fluorescence microscope (Olympus FV1000, Tokyo, Japan) with excitation at 488 nm (green fluorescence) or 546 nm (red fluorescence).
Analysis of lysosomal degradation capacity
DQ-BSA, a BSA derivative with green fluorescence quenched after cleavage by proteolytic enzyme, was utilized to detect lysosomal degradation capacity. Cells seeded on glass-bottom dishes were incubated with 10 μg/mL DQ-BSA-Green (D12050, Invitrogn, USA) for 12 h (37 °C, 5% CO2) and washed with PBS before exposed to SiNPs (100 μg/mL) in the absence or presence of PJ34 or compound A1 (both at 10 μM). Images were captured using a confocal microscope with excitation at 488 nm. The degradation capacity was estimated by measuring the green fluorescence intensity using imageJ.
Intracellular calcium measurement
The effect of SiNPs on intracellular free Ca2+ concentration was determined by using Fluo-3/AM (F1242, Invitrogen, USA) following the manufacturer’s instructions. In brief, BEAS-2B cells were seeded in 96-well plates (3603, Costar, USA) and incubated with Fluo-3/AM (3.5 μM) at 37 °C for 1 h. The cells were washed with HBSS and incubated with PJ34 and compound A1 (both at 10 μM) 30 min prior to addition of SiNPs (100 μg/mL). Fluorescence was measured at 0.5, 1, 2, 3 h in a SynergyMx M5 microplate reader (Molecular Devices, USA) with excitation at 488 nm and emission at 525 nm. The intensity of samples was normalized to that of the control. Each group had 3 replicate wells and all procedures were performed in the dark.
Statistical analysis
Data were expressed as mean ± standard deviation (SD) from at least three independent experiments. All the data shown in this study showed a normal distribution and therefore unpaired Student’s t-test was used to compare two groups and one-way ANOVA with post hoc Bonferroni test to compare more than two groups, using SPSS 22.0. A p-value of less than 0.05 was considered statistically significant. All graphics were prepared using Prism 6 (GraphPad Software, La Jolla, CA, USA).