Preparation and characterization of ZnO NP suspensions
The composition, shape and primary size of ZnO NPs (50 nm; CAS number:1314-13-2, SigmaAldrich, USA) were determined by XPS (D8 advanced, Bruker, Germany), SEM (Sigma 500, Zeiss, Germany) and TEM (Tecnai G2 F20, FEI, USA), respectively. The hydrodynamic size and zeta potentials of the ZnO NPs were determined with a Malvern Zetasizer (Nano-ZS, Malvern, UK).
To prepare a suspension for the animal experiments, ZnO NPs were dispersed in normal saline (NS) containing 1% hydroxy propyl methyl cellulose (HPMC) at a concentration of 20 mg/mL. HPMC was used as a suspension agent to ensure that the nanoparticles were uniformly dispersed and to increase operability. The suspension was ultrasonically dispersed in an ice bath for 30 min before each use.
To prepare a stock solution for the cell experiments, ZnO NPs were dispersed in phosphate-buffered saline (PBS) at a concentration of 2 mg/mL and autoclaved. The working solution was the stock solution diluted 4 times with basic medium and was ultrasonically dispersed in an ice bath for 15 min before each use.
Animals and treatments
6-week-old female C57BL/6 mice were purchased from the Animal Center of Southern Medical University (Guangdong, China). The mice were housed in a room free of specific pathogens (23 ± 1 °C room temperature, 60 ± 10% relative humidity) and underwent an adaptation period for one week before treatment.
A 6-cm2 area of the dorsal skin was shaved, and 62.5 mg of IMQ (Med-Shine, China) was topically applied daily from the 1st day to the 6th day to establish a psoriasis model. After model establishment as histologically confirmed, the mice were randomly distributed into 2 groups of 8 mice per group. From the 7th to the 9th day, 500 μL of NS (containing 1% HPMC) or a ZnO NP suspension (consisting of NS, 1% HPMC and ZnO NPs) was topically applied twice daily to the negative control group and the ZnO NP-treated group, and the mice were sacrificed on the 10th day.
TEM observation of the epidermis
Skin tissues (1×2 mm2) in area were cut from the central area of the freshly excised dorsal skin using a sharp surgical blade, immersed in 2.5% glutaraldehyde overnight at 4 °C, processed and embedded in resin. The micromorphology of the tissues was observed using TEM (H-7500, Hitachi, Japan).
Inductively coupled plasma mass spectra (ICP-MS) analysis of the dermis
The dorsal skin was cleaned with NS at room temperature, excised and placed in 0.2% dispase II protease (Sigma-Aldrich, USA) overnight at 4 °C. The epidermis was removed, and the dermis was gently rinsed in precooled NS 3 times and dried with clean filter paper. 2 g of each dermis sample was digested with nitric acid and hydrogen peroxide through microwave heating at 160 °C. Then, the samples were heated at 120 °C overnight to fully evaporate the nitric acid. The final solutions were diluted to 2 mL with 1% nitric acid and 0.1% Triton-100 and analysed by ICP-MS (7700s, Agilent, USA).
Freshly excised dorsal skin pieces were immersed in 4% paraformaldehyde for 24 h and embedded in paraffin wax after dehydration using graded ethanol and xylene solutions. The tissues were sliced at a thickness of 4 μm. H&E staining was performed for histological morphological analysis. IHC was used to detect the number of TUNEL (Servicebio, China)-labelled cells and the levels of 8-OHdG (JaICA, Japan), TNF-α (Abcam, USA), IL-1β (Abcam, USA), IL-6 (Abcam, USA) and COX2 (Bimake, USA). Images were captured using a microscope (BX63, Olympus, Tokyo, Japan). Cell counts and grey value analysis were performed using ImageJ software.
qRTPCR analysis of the inflammatory response in vivo
Skin tissues that had been frozen in liquid nitrogen and stored at -80 °C were homogenized in liquid nitrogen and extracted with TRIzol reagent (Gibco, USA) according to the manufacturer’s instructions. The concentration and purity of the total RNA were determined by measuring the absorbance at 260 and 280 nm using a spectrophotometer (Molecular Devices, USA). Complementary DNA (cDNA) was reverse transcribed from mRNA samples using a PrimeScript™ RT Reagent Kit (TaKaRa, Japan). qRTPCR was performed with a LightCycler 480 Sequence Detector System (Roche, Switzerland) using a commercial kit (SYBR Premix Ex Taq II, TaKaRa, Japan). The mouse primer sequences used for qRT-PCR are shown in Table 1.
Measurements of oxidative stress biomarkers
Skin tissues that had been frozen in liquid nitrogen and stored at -80 °C were homogenized in precooled NS using a tissue homogenizer. The homogenates (1:10 w/v) were centrifuged at 3000 rpm for 10 min at 4 °C to obtain supernatants. The protein concentrations of the supernatants were determined with a BSA kit (Thermo Fisher, USA). T-GSH, GSH, and GSSH levels, GPx levels, and MDA levels were quantified with a GSH and GSSH assay kit (Beyotime, China), a GSH peroxidase assay kit (Beyotime, China) and an MDA assay kit (Nanjing Jiancheng Bioengineering Institute, China), respectively, according to the manufacturer’s instructions.
Cell culture and treatments
Normal human immortalized keratinocytes (HaCaT cells) were obtained from Otwo Biotech, Inc. (Shenzhen, China). Cell line authentication by short tandem repeat (STR) profiling indicated that this cell line matched the HaCaT cell line (code: 711) collected by Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). The cells were cultured in Dulbecco’s modified Eagle medium (4.5 g of L-glucose/L) containing 10% foetal bovine serum at 37 °C in a 5% CO2 humidified incubator. The culture medium was replaced every other day, and the cells were passaged when they reached approximately 90% confluence.
HaCaT cells were stimulated with rh-TNF-α (10 ng/mL for 48 h; R&D, USA) to establish an in vitro model of psoriasis. The medium supernatant was collected after rh-TNF-α stimulation and used to dilute the sonicated ZnO NP working solution. rh-TNF-α-stimulated HaCaT cells continued to be cultured in diluted ZnO NP working solution containing 10, 20, 30 or 40 μg/mL ZnO NPs for 1, 3, 6, 12 or 24 h.
Cells were pretreated with inhibitors of the NF-κB and MAPK pathways and oxidative stress for 3 h before ZnO NP treatment for 6 h to determine the mechanism by which ZnO NPs promote inflammation and cell death. QNZ (0.1 μM; TargetMol, USA) is an inhibitor of NF-κB activation and TNF-α production. JSH-23 (10 μM; TargetMol, USA) suppresses the nuclear translocation and transcriptional activity of NF-κB. SCH772984 (10 μM; TargetMol, USA), SB203580 (2 μM; TargetMol, USA) and JNK-IN-8 (1 μM; TargetMol, USA) inhibit the phosphorylation of ERK, p38 and JNK, respectively. N-acetylcysteine (NAC, 1 mM; TargetMol, USA), the N-acetyl derivative of cysteine, inhibits ROS.
To understand the role of cysteine and oxidative stress in the mechanism by which ZnO NPs induce apoptosis, inhibitors of cysteine synthesis and antioxidants were applied. Erastin (200 μM; TargetMol, USA) inhibits the cysteine/glutamate amino acid transporter system xc-, which is the main pathway of cysteine biosynthesis. RSL3 (10 μM; TargetMol, USA) inhibits cysteine synthesis and is a covalent inhibitor of glutathione peroxidase 4. Cells not treated with ZnO NPs were treated with erastin and RSL3 for 6 h. Fer-1 (20 μM; TargetMol, USA) blocks lipid peroxidation. DEP (40 μM; MCE, USA) depletes iron and prevents iron-dependent lipid peroxidation. ZVF (30 μM; Selleck, USA) is an irreversible pan-caspase inhibitor. NAC (1 mM) was also used to inhibit ROS. Cells treated with ZnO NPs were treated with Fer-1, DEP, ZVF and NAC for 6 h.
Cell survival assay
The cells were seeded in 96-well plates at a density of 2000 cells per well and allowed to attach overnight. Then, the cells were treated with rh-TNF-α and ZnO NPs. Cell viability was determined using a CCK-8 assay kit (Dojindo Molecular Technologies, Japan), and the absorbance was measured at a wavelength of 450 nm using a microplate reader.
TEM observation of cells
rh-TNF-α and ZnO NP (40 μg/mL for 6 h)-treated cells were washed with 37 °C PBS and fixed with 2.5% glutaraldehyde. The fixed cells were collected using a cell scraper and centrifuged at 1000 g for 5 min at 4 °C. The samples were then embedded, and ultrathin slices were prepared. Intracellular structural changes were observed using a Hitachi H-7500 TEM instrument (Hitachi, Japan).
Cellular ROS assay
Cellular ROS production was determined by using 2',7'-dichlorofluorescin diacetate (DCFH-DA) probes (Beyotime, China). The treated cells were washed, collected with EDTA-free trypsin and incubated with the probes (10 µM) for 30 min at 37 °C, washed twice with PBS and resuspended in 500 μL of PBS. The fluorescence intensity of the cells was analysed using a flow cytometer (FACSAria III, BD, USA) at excitation/emission wavelengths of 485/535 for DCFH-DA.
Measurements of cellular GSH and cysteine levels
Cells were lysed and collected. T-GSH and GSH levels and cysteine levels were quantified with a GSH and GSSH assay kit (Beyotime, China) and a cysteine assay kit (Solarbio, China), respectively, according to the manufacturer s’ instructions.
ELISA and q-RT-PCR analysis of the inflammatory response in vitro
The medium supernatant was collected and centrifuged at 5000 rpm for 10 min at 4 °C. The levels of TNF-α, IL-1 and IL-6 in the medium supernatant were quantified using ELISA kits (BioLegend, USA) according to the manufacturer’s instructions. The cells were lysed with TRIzol reagent (Gibco, USA) according to the manufacturer’s instructions. mRNA extraction, cDNA synthesis and qRT-PCR were carried out as described for the animal experiments. The human primer sequences used for qRT-PCR are shown in Table 2.
Annexin V/PI analysis
Cells were washed, digested with EDTA-free trypsin and stained with FITC-conjugated Annexin V and PI (Beyotime, China) according to the manufacturer’s instructions. The final cell suspensions in PBS were analysed using flow cytometry (FACSAria III, BD, USA).
The cells were seeded on slides, treated with rh-TNF-α and ZnO NPs, fixed with 4% paraformaldehyde overnight and permeabilized with 0.5% Triton X-100 for 30 min. After being blocked with 5% FBS for 1 h, the cells were incubated with an anti-p-NF-κB p65 antibody (CST, USA) at 4 °C overnight and incubated with a FITC-conjugated secondary antibody (Proteintech, China) at room temperature for 1 h. Fluorescence images were captured using a fluorescence microscope (BX63, Olympus, Tokyo, Japan).
Western blot analysis
Cells were collected and lysed using reagent from a nuclear protein and cytoplasmic protein extraction kit (Beyotime, China) containing 1 mM protease and phosphatase inhibitor (Beyotime, China). The cell lysates were heated in SDS-PAGE sample buffer (Genstar, China) at 99 °C for 5 min. The proteins were separated by SDS-PAGE and transferred to polyvinylidene diﬂuoride membranes (Merck Millipore, Germany) that were then blocked with 5% skim milk for 1 h. The membranes were incubated overnight at 4 °C with primary antibodies, including anti-NF-κB p65, phospho-NF-κB p65, phospho-44/42 MAPK, phospho-p38, phospho-JNK, Bcl-2, cleaved-caspase 3 (CST, USA), ERK, p38, JNK, BAX, CD98, CBS, CTH, TBP, GAPDH (Proteintech, China), and xCT (Bimake, USA) antibodies. The membranes were then washed with TBST and incubated at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (CST, USA) for 1 h. Proteins were detected using an ECL kit (WBLKS0500, Merck Millipore, USA) and an automatic chemiluminescence image analysis system (Tanon, China). Quantitative analysis was performed using ImageJ software.
All the data are presented as the mean ± standard deviation (SD) and were analysed with SPSS 22.0 software. Comparisons among each group were assessed using one-way ANOVA when the variance in the data was homogenous or using a non-parametric test when the variance in the data was not homogenous. Differences for which P < 0.05 were considered to be statistically significant.