Ascorbic acid 6-palmitate modulates microglia M1/M2 polarization in lipopolysaccharide-stimulated BV-2 cells via PERK/elF2α mediated endoplasmic reticulum stress

Cell Culture and Treatment

Immortalized murine microglia cell line BV-2 cells (Procell Life Science & Technology Co., Ltd, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin at 37°C in humidified 5% CO2 and 95% air. To establish the BV-2 cells M1 polarization model, BV-2 cells were incubated with LPS (1 µg/mL; Sigma-Aldrich, USA) for 24 h. To confirm the role of ER stress on BV-2 cells’ M1 polarization, cells were incubated with tunicamycin (TM; Sigma-Aldrich, USA) 6 nM or 4-phenyl butyric acid (4-PBA; Sigma-Aldrich, USA) 30 µM for 24 h.

Cytotoxicity assay

50 mg L-AP powder was dissolved in 1 mL of dimethylsulfoxide (DMSO) to prepare a 125 mM L-AP storage solution. Cells were plated into 96-well plates at the density of 1×103 / well and incubated with different concentrations of ascorbic acid 6-palmitate (0, 2, 4, 8, 16, 32, 62.5, 125, 250, 500 µM) for 24 h. Then, 10 µL of cell counting kit-8 (CCK-8) reagent (Dojindo, Japan) was added to each well and incubated at 37°C for 4 h. After incubation, OD values were measured at 450 nm using an Epoch microplate spectrophotometer (Bio-Tek, USA).

Enzyme-linked immunosorbent assay (ELISA)

Cells were plated into 6-well plates at the density of 1 × 106 / well. After treatment, the concentrations of interleukin-6 (IL-6) and TNF-α in cell supernatant were measured using a microplate reader (Bio-tek Epoch) at 450 nm with ELISA kits (Multisciences Biotech, Co., Ltd, Hangzhou, China) according to the manufacturer’s instructions. The absorbance of the reaction products was calibrated at 570 nm.

Real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted with Trizol reagent and reverse-transcribed using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). Real-time PCR was performed using qTOWER3G (Analytik Jena, Germany). PCR amplification conditions involved the initial denaturation for 2 min at 95°C and 40 cycles of 15 s at 95°C and 60 s at 60°C. PCR primer sequences were as follows (forward primer and reverse primer, respectively): gapdh (122 bp): 5′-AGGTCGGTGTGAACGGATTTG-3′, 5′-TGTAGACCATGTAGTTGAGGTCA-3′; inos (118 bp): 5′-ACGAGACGGATAGGCAGAGA-3′, 5′-CACATGCAAGGAAGGGAACT-3′; il-10 (100 bp): 5′-CTTACTGACTGGCATGAGGATCA-3′, 5′-GCAGCTCTAGGAGCATGTGG-3′; arg-1 (118 bp): 5′-GACCTGGCCTTTGTTGATGT-3′, 5′-CCATTCTTCTGGACCTCTGC-3′; chop (382 bp): 5′-TGTTGAAGATGAGCGGGTGG-3′, 5′-GATTCTTCCTCTTCGTTTCCTGG-3′; grp78 (117 bp): 5′-TTCTCAGCATCAAGCAAGGA-3′, 5′-CATGGTAGAGCGGAACAGGT-3′. The data were analyzed with LightCycler®96 software (Roche Diagnostics, Germany).

Protein preparation and Western blotting analysis

Total proteins were extracted using RIPA Lysis Buffer (Beyotime Institute of Biotechnology, China) and the protein concentrations were measured with a BCA protein assay kit (Beyotime Institute of Biotechnology, China). Equal amounts of total protein (40 µg/10 µL) were loaded onto 8% SDS PAGE gels at 120 v for 60 min, electrotransferred to PVDF membranes, cropped the PVDF membranes according to the position of the marker, blocked in 5% no-fat milk at room temperature for 2 h, and incubated with the diluted primary antibodies (Table 1) at 4 °C overnight in turn. After washing three times with TBST, the membranes were probed with horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. β-Actin was the loading control. The protein was detected by chemiluminescence reagent (GE Healthcare) and the protein band density was quantified using Image J software.

Molecular docking

Molecular docking studies were performed using Accelrys Discovery Studio 4.0 (DS, BIOVIA Software, Inc., USA) to gain insights into the plausible binding modes of L-AP and PERK. The crystal structure of PERK (PDB bound to GSK6924) was obtained from the Protein Data Bank (http://www.rcsb.org/). The molecular structure of L-AP was pre-treated by DS and defined as the ligand. Also, the protein molecule of PERK was pre-treated by DS to remove water molecules and original ligands and defined as the receptor. The binding sites of L-AP and PERK were specified by the center of the co-crystallized ligand GSK6924. Then, the pre-processed receptor and ligand were batch-processed using MGLTooLs-1.5.6 to generate files in pdbqt format. Finally, molecular docking of the ligand to the receptor was performed with AutoDock Vina. Other parameters were set as default. Various binding modes were evaluated based on the docking energy, and the score was obtained. Reasonable docking results were selected according to the lowest score. In general, if the binding energy is less than − 1.2 kcal/mol, the docking result is feasible.

Statistical analysis

All values were expressed as mean ± SEM and statistically analyzed by one-way analysis of variance (ANOVA) with Bonferroni correction (GraphPad Prism version 5). P < 0.05 was considered as a statistically significant difference.

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