Chemicals and reagents
Brucine (purity ≥ 98% by HPLC) (Figure. 1A) was purchased from Nantong Feiyu Medical Biotechnology Corporation (Nantong, China). Brucine was dissolved in dimethyl sulfoxide (DMSO) at 10 mM as stock solution and the final concentration of DMSO in the media was less than 0.1%. 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT, #M8180), Propidium Iodide (PI, #3843838) were from Sigma-Aldrich (St Louis, MO, USA). Fetal bovine serum (FBS, #1763585) and Dulbecco’s modified Eagle’s medium (DMEM, #8121436) were from Gibco BRL (Gaithersburg, MD, USA). Annexin V‑fluorescein isothiocyanate (FITC, #556420) was obtained Becton Dickinson Pharmingen (Franklin Lake, New Jersey, USA). Mouse lactate dehydrogenase (LDH, #RJ17540), tumor necrosis factor alpha (TNF-α, #RJ17928), interleukin 1β (IL-1β, #RJ16944) and interleukin-6 (IL-6, #RJ16958) ELISA kits were from Shanghai Renjie Biotechnology (Shanghai, China). Caspase-3 Activity ELISA kit (#KD16967) was obtained from Guangzhou Kedi Biotechnology (Guangzhou, China). The antibodies against caspase 3 (#ab184787), cleaved caspase-3 (#ab214430), Bax (#ab182733), Bcl-2 (#ab59348), NF-κBp65 (#ab32536), phospho-NF-κBp65 (#ab76302), Peroxisome proliferation activating receptors (PPAR)α (#ab126235), PPARβ/δ (#ab178866), PPARγ (#ab209350) were from Abcam (Cambridge, UK).
Cell culture and drug treatment
The mouse neuroblastoma (Neuro-2a) cells were from American Type Culture Collection (Rockville, MD, USA) and were cultured with DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were grown in a 5% CO2 humidified atmosphere with 5% at 37°C. For brucine treatment, which was then appropriately diluted with serum-free DMEM before application. Neuro-2a cells were treated with different concentrations (1, 4, 16 μM) of brucine or co-treatment with 10 μM Z-DEVE-FMK (caspase 3 inhibitor), 10 μM GW 9662 (PPARγ inhibitor) or 20 μM Gliotoxin (NF-κB inhibitor) for 24 h.
Primary astrocytes were extracted from neonatal rat brains as our previous study (Zheng et al. 2020). The primary astrocytes were fixed with 4% paraformaldehyde, then incubated with anti-GFAP antibody, and visualized by an Alexa Fluor-conjugated secondary antibody. GFAP staining was used to identified the primary astrocytes up to more than 95% for the following experiment. Primary astrocytes were cultured with DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were grown in a 5% CO2 humidified atmosphere with 5% at 37°C. Primary astrocytes were treated with different concentrations (8, 16, 32 μM) of brucine or 10 μM Z-DEVE-FMK (caspase 3 inhibitor) or 10 μM GW 9662 (PPARγ inhibitor) or Gliotoxin (NF-κB inhibitor) 24 h.
Cell viability determination
Neuro-2a cells (1 × 104) or primary astrocytes (1 × 104) were treated as mentioned above. At the end of treatment, 20 μl MTT (5 mg/ml) was added into the medium and cultured for another 4 h. Thereafter, the medium was carefully removed and 150 μl DMSO was added into each well. The optical density value of the cells was measured by a microplate reader at a wavelength of 490 nm. Cell viability was expressed as the percentage relative to the absorbance of the untreated control cells.
Detection of LDH level
The level of LDH of Neuro-2a or primary astrocytes after exposure of brucine was determined using a LDH assay kit. In brief, the Neuro-2a cells or primary astrocytes were treated as mentioned above. Briefly, supernatant was collected by centrifugation (3000 × g, 20 min), and the level LDH release was evaluated using a LDH assay kit according to the protocol’s instructions. Thereafter, absorbance was detected at wavelength of 450 nm..
Cell morphological observation
For morphological observation, Neuro-2a cells or primary astrocytes were seeded in 6-well plates and treated with brucine as mentioned above. Then, the cells were observed by phase contrast microscopy (Olympus, IX53 + DP20, Japan).
Total RNA was extracted from the Neuro-2a cells of control and brucine group using TRIzol reagent according the experimental protocol, then quality of RNA samples quantified were detected based on Nanodrop ND-2000 system (Thermo Scientific, USA) and Agilent Bioanalyzer 4150 system (Agilent Technologies, CA, USA). Finally, qualified RNA transcriptome sequencing was performed with an Illumina Novaseq 6000 /MGISEQ-T7 instrument supported by Shanghai Applied Protein Technology. Principal component analysis (PCA), an unsupervised analysis that reduces the dimension of the data, was carried out to visualize the distribution and grouping of the samples, which was also commonly used to assess differences between groups and the quality of the biological replicates of samples within groups. Differential expression analysis was performed using the DESeq2 (http://bioconductor.org/packages/release/bioc/html/DESeq2.html), differential genes (DGEs) with |log2FC| >1 and adjusted p <0.05 were considered to be significantly different expressed genes. To directly observe the profile of potential DEGs, the up and down regulation of DEGs were visualized by volcano plot. Differential gene clustering is used to judge the changes of differential genes between brucine and control group. KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.kegg.jp/) is one of the databases commonly used in pathway research. We use cluster Profiler R software package for GO function enrichment and KEGG pathway enrichment analysis, and P< 0.05, it is considered that the GO or KEGG function is significantly enrichment.
The apoptosis of Neuro-2a cells was detected using the TUNEL assay by the One Step TUNEL Apoptosis Assay Kit according to the manufacturer’s instructions. Briefly, the Neuro-2a cells were treated as described above, then the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and then incubated with 0.3% Triton X-100 in PBS for 10 min. Thereafter, the Neuro-2a cells were incubated in TUNEL test solution at 37°C for 1 h in the dark; while, the cell nuclei were labeled using DAPI staining. And TUNEL-positive cells were observed using a fluorescence microscope (Olympus IX73, Olympus, Tokyo, Japan).
Detection of apoptosis using flow cytometry
The apoptosis of treated cells was examined using an Annexin V‑FITC/PI apoptosis reagents according to the manufacturer's protocol. Briefly, the Neuro-2a cells were treated as mentioned above. The cells were then labeled with 500 µl binding buffer containing 2 µl Annexin V and 5 µl PI in the dark for 15 min after washed with PSB. Subsequently, apoptosis was detected using a flow cytometer (Navios, Beckman coulter, USA), and the Annexin V and PI values were set as the horizontal and vertical axes, respectively. Mechanically damaged, late apoptotic, dual negative/normal, and early apoptotic cells were located in the upper left, upper right, lower left and lower right quadrants of the flow cytometric dot plot, respectively.
Measurement of caspase 3 activity
The Neuro-2a cells or primary astrocytes were treated as described above. Cells supernatant was collected by centrifugation (3000 × g, 20 min, 4°C), and the caspase 3 assay was evaluated using caspase 3 assay kit according to the manufacturer’s recommended protocol. Thereafter, absorbance was detected at wavelength of 450 nm, and all values of % caspase 3 activity were normalized to the untreated control group.
PPARγ activity assay
The primary astrocytes were treated as described above. The cells were dissolved in RIPI lysis buffer containing protease inhibitor and PMSF and kept on ice for 30 min. Then cells supernatant was collected by centrifugation (1000 g, 15 min, 4°C), protein concentration was determined by the BCA assay. The DNA-binding activity of PPARγ was evaluated by ELISA kit according to the manufacturer’s recommended protocol. Thereafter, absorbance was detected at wavelength of 450 nm.
NF-κB DNA binding activity assay
Th primary astrocytes were treated with or without brucine as described above. Afterward, the NF-κB binding activity was following determined using the NF-κBp65 Transcription Factor Assay Kit according the manufacturer’s instructions. Thereafter, absorbance was detected at wavelength of 450 nm.
Evaluation of inflammatory factors
The levels of inflammatory factors were detected using ELISA assay. Briefly, the Neuro-2a cells or primary astrocytes were treated as mentioned above. Then the supernatants were collected with centrifugation (3000 × g for 10 min at 4°C). Thereafter, the levels of IL-1β, IL-6 and TNF-α were detected according to the manufacturer’s instructions.
Western blot analysis
RIPA buffer containing a protease and phosphatase inhibitor cocktail were added to the cells and homogenized at 4°C for 30 min. Then protein concentration was evaluated by BCA assay, and lysates were normalized to equal amounts of protein. Further, 10 μg proteins were separated by 12% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Subsequently, the membranes were blocked with 5% fat-free dry milk at room temperature for 2 h. The blots were incubated with anti-caspase 3 (1:1000), anti-cleaved caspase 3 (1:1000), anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-NF-κBp65 (1:1000), anti-phospho-NF-κBp65 (1:1000), anti-PPARα (1:1000), anti- PPAR-β/δ (1:1000), anti-PPARγ (1:1000), anti-β-actin (1:5000) and GAPDH (1:5000) overnight at 4 °C. The membranes were washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (1:5000) at room temperature for 30 min. The proteins were finally examined using an enhanced chemiluminescence system and Image J microsoft was used to analyze the bands.
Molecular docking analysis between brucine and caspase 3 were performed using Autodock 4.2 and Autodock Tools (ADT). In brief, the caspase 3 (Protein Data Bank ID:3KJF) protein were obtained from the Protein Data Bank database. A three-dimensional structure of brucine was established using the ChemBio3D Ultra 14.0 (PerkinElmer Informatics, USA), which was further proceeded using ADT. Thereafter, the molecular docking of the brucine and caspase 3 proteins was performed using Autodock 4.2.
All data were obtained from at least three independent experiments and expressed as mean ± SD. Comparisons of the different groups were performed with Student’s t-test. P< 0.05 was considered the minimal level of significance.