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The chemical regents and antibodies were purchased as follows: potassium antimonyl tartrate trihydrate (Sigma, 60063); Hoechst (Sigma, 94403); TAK1 inhibitor (TargetMol, T4264); BAY11-7082 (MCE, HY-13453); anti-β-actin (Santa Cruz Biotechnology, sc-47778); anti-p65 (Abcam, ab16502); anti-p-p65 (Abcam, ab86299); anti-GFAP (CST, 12389S); anti-iNOS (GeneTex, GTX130246); anti-Lamin A/C (Proteintech, 10298-1-AP); anti-Tubulin (Proteintech, 10068-1-AP); anti-TAK1 (Abcam, ab109526); anti-p-TAK1 (Abcam, ab109404); and anti-Cyclin D1 (Cell Signaling Technology, 2978).
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We performed mouse experiments in accordance with the ethical guidelines from the Ethics Committee of Laboratory Animal Care and Welfare (Nantong University School of Medicine). Thirty ICR male mice (weight ≥ 27 g, 6 weeks of age) were provided by the Experimental Animal Center of Nantong University. After acclimatization to a 12/12 h dark/light cycle for one week, mice were randomly divided into three groups. In each group, 100 μL antimony (0, 10, and 20 mg/kg) dissolved in 0.9% saline solution was injected intraperitoneally. This dose was used to explore antimony-associated neurotoxicological mechanism because it had a significant toxic effect on neurobehavioral change in mice under present conditions[3]. Moreover, during treatment of schistosomiasis, the patients were exposed to antimony potassium tartrate at a higher dose (2.5 g)[19]. More importantly, in the human brain, the antimony concentration is ≥ 2.5 × 10−6–170.7 × 10−6 g/g[2]. Antimony treatment was performed as follows. Antimony was injected intraperitoneally on the first, third, and fifth days of every week for eight weeks, and the mice were sacrificed on the first day of the ninth week. All mice were anesthetized by intraperitoneal injection of 7% chloral hydrate (5 mL/kg). Mice were perfused intracardially with saline and 4% paraformaldehyde for paraffin embedding. Moreover, cerebral cortices were collected and frozen at −80 °C for immunoblot analysis.
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C6 cells were cultured aseptically according to conventional culture methods. F12K (Gibco) medium was supplemented with 2.5% fetal bovine serum (FBS, Gibco) and 15% horse serum (HyClone, SH30074.02) and cells were grown at 37 °C and with 5% CO2. Antimony was dissolved in 0.9% normal saline, and C6 cells were seeded for 24 h and then exposed to an Antimony concentration gradient (0, 0.625, 1.25, 2.5, 5 μmol/L) for another 24 h. Moreover, cells were exposed to 2.5 μmol/L antimony for the time gradient assay (0, 3, 6, 12, 24 h). C6 cells were pretreated with TAK1 and p65 inhibitors for 1 h and exposed to 2.5 μmol/L for 24 h, then protein or RNA were collected and stored at −80 °C.
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C6 cells were seeded in 96-well plates (≥ 10,000 cells/well) and exposed to varying antimony concentrations for 24 h. After exposure, 10 μL of the reaction solution were added to each well according to the instructions of the CCK8 kit (DOJINDO, Japan, CK04) as described by Zhao et al.[20]. The optical density (OD) value at 450 nm was detected with a microplate reader 2 h later (Thermo Scientific, Varips Kan Flash).
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Total protein was extracted from mouse brain tissue and C6 cells, and the protein concentrations were measured using the BCA kit (Beyotime, P0009). An equal amount of protein per sample was subjected to electrophoresis (10% SDS-PAGE) to separate the proteins, which were then transferred to a PVDF membrane. The membrane was blocked in a TBST (Tris-buffered saline) buffer containing 3% BSA (bovine serum albumin) for at least 2 h at room temperature, and then incubated with primary antibody at 4 °C overnight. The corresponding secondary antibodies were then incubated with the membrane for 1 h, and finally, protein bands were detected using an enhanced chemiluminescence system (ECL; Millipore Corporation, Billerica, P90720).
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C6 cells exposed to antimony were collected, their cytoplasmic proteins were extracted with cytoplasmic protein extractants A and B according to the instructions for the Nuclear Separation kit (Beyotime, P0027)[21], and the nuclear protein was extracted with the nuclear protein extraction reagents. Finally, the expression of p65 protein was detected by immunoblotting.
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C6 cells on coverslips were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min at 4 °C, washed another three times with PBS and permeabilized with 0.2% Triton X-100 for 15 min at 4 °C. Next, the cells were blocked with goat serum for 2 h at room temperature. Then, the slides were incubated with primary anti-p65 antibody (diluted 1:200 in PBS) at 4 °C overnight. The slides were then washed three times with PBS and incubated with the Alexa Fluor 488-conjugated goat anti-rabbit antibody (Invitrogen, R37116) for 1 h followed by Hoechst treatment for 15 min. Cells were observed using a fluorescence microscope (Leica, Microsystems, GmbH, Germany). For fluorescence excitation, an Ar/UV laser was used for Hoechst, and an Ar/Blue laser was used for p65.
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Paraffin-embedded brain tissue slices (5 μm thickness) were cut in the coronal plane and subjected to immunohistochemistry to detect the expression of GFAP as described by Piccolini et al.[22]. Embedded sections were deparaffinized in xylene and rehydrated through a series of graded alcohol treatments. Then, slices were boiled in 10 mmol/L citrate buffer for 30 min for antigen retrieval. Endogenous peroxidation was blocked with 3% H2O2 for 30 min, then slices were incubated with 5% BSA for 2 h at room temperature in order to block non-specific antigen binding sites. Then, the slices were incubated with GFAP antibody (diluted 1:200 in PBS) at 4 °C overnight, and then incubated with the HRP-conjugated secondary antibody. Sections were washed in PBS three times and finally stained with DAB and hematoxylin. Sections were dehydrated in ethanol, cleared in xylene, and mounted with neutral balsam mounting medium. Slices were imaged using a micro Oscilloscope (Leica, Microsystems, GmbH, Germany) for visualization.
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Following the kit instructions, total RNA was extracted from C6 cells using Trizol reagent (TakaRa, 9109) and reversed-transcribed to generate cDNA. For RT-PCR, Taq DNA polymerase and specific primers were used. The PCR products of each sample were analyzed by electrophoresis in a 2% agarose gel. The intensity of resulting bands was measured by densitometry and then normalized to the corresponding GAPDH bands. For qRT-PCR, PCR analysis was performed in a 10 μL reaction using SYBR GREEN PCR Master Mix (Applied Biosystems). We performed qRT-PCR to measure gene expression, and GAPDH was chosen as a reference and was calibrated on untreated cells. The delta-delta Ct (
ΔΔCT) method was applied for relative quantification. The corresponding primers are shown in Table 1. Gene name Primer name Sequence (5’–3’) RAT-GAPDH GAPDH-F TGATGACATCAAGAAGGTGGTGAAG GAPDH-R TCCTTGGAGGCCATGTGGGCCAT RAT-iNOS iNOS-F GCTTTGTGCGGAGTGTCAGT iNOS-R CCTCCTTTGAGCCCTCTGTG RAT-GFAP GFAP-F GGCGCTCAATGCTGGCTTCA GFAP-R TCTGCCTCCAGCCTCAGGTT RAT-IL-6 IL-6-F GAAAGTCAACTCCATCTGCC IL-6-R CATAGCACACTAGGTTTGCC RAT- IL-1β IL-1β-F TCAGGAAGGCAGTGTCACTCATTG IL-1β-R ACACACTAGCAGGTCGTCATCATC RAT-TNF-α TNF-α-F GCCGATTTGCCATTTCAT TNF-α-R CAGTCGCCTCACAGAGCAA Table 1. Oligonucleotide sequences used in PCR
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Quantitative results are presented as the mean ± standard deviation (SD) from at least three independent experiments. Then, analysis of variance (ANOVA) or Student’s t-test were used for statistical analysis. A P value ≤ 0.05 was considered significant.
Nuclear Factor-κB Signaling Mediates Antimony-induced Astrocyte Activation
doi: 10.3967/bes2021.005
- Received Date: 2020-03-19
- Accepted Date: 2020-07-14
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Key words:
- Antimony /
- Astrocyte activation /
- Neurotoxicity /
- NF-κB /
- TAK1
Abstract:
Citation: | ZHANG Tao, ZHENG Yu Dan, JIAO Man, ZHI Ye, XU Shen Ya, ZHU Piao Yu, ZHAO Xin Yuan, WU Qi Yun. Nuclear Factor-κB Signaling Mediates Antimony-induced Astrocyte Activation[J]. Biomedical and Environmental Sciences, 2021, 34(1): 29-39. doi: 10.3967/bes2021.005 |