Cell Culture and Treatment
Full details on cell lines are provided in the Supplementary Table 1. BV-2 microglia cells were cultured in 1,640 medium, NSC34 and HT-22 cells were cultured in DMEM medium, with 10% fetal bovine serum (Gibco, Shanghai, China). The BV-2 microglia cells were randomly divided into six groups including Control group, PK11195 (0.5 µM, Sigma, Aldrich, USA) group, LPS (1 µg/mL, Solarbio, Beijing, China) group, PK11195 + LPS group, Midazolam (15 µM, NHWA, Jiangsu, China) group, Midazolam + LPS group. In both PK11195 + LPS group and Midazolam + LPS group, cells were pretreated with PK11195(0.5 µM) or Midazolam (15 µM) for 1 h before treated with LPS for 6 h, and all the cells were harvested after treatment.
BV-2 Microglia and NSC34 Neuronal Cells Co-culture System
NSC34 neuronal cells were co-cultured with conditioned medium (CM) from BV-2 microglia cells or were co-cultured with BV-2 microglia cells in Transwell (24 mm Transwell with 0.4 µm pore size insert; Corning, New York, USA) system. For CM co-culture system (Supplementary Fig. 1a), the cells were randomly divided into four groups: Control, LPS, PK11195 + LPS and Midazolam + LPS. In LPS group, BV-2 microglia were activated by 10 ng / mL LPS. The last two groups, BV-2 microglia cells were pretreated with PK11195(0.5 µM) or Midazolam (15 µM) for 1 h followed by LPS treatment for 6 h, then, the BV-2 microglia cells were replaced with serum-free culture medium for another 12 h. The microglia culture supernatant continued culture NSC34 neuronal cells for 12 h. For Transwell co-culture system (Supplementary Fig. 1b), the upper insert chamber was planted with BV-2 microglia cells and the lower six-well plate was used to implant NSC34 neuronal cells. In LPS group, BV-2 microglia cells treated with LPS at a concentration of 10 ng/mL for 6 h. The last two groups, BV-2 microglia cells pretreated with PK11195(0.5 µM) or Midazolam (15 µM) for 1 h followed by LPS treatment for 6 h. Then, two kinds of cells were replaced with serum-free culture medium at the same time. The insert chamber was placed in combination with the six-well plate co-cultivation for 12 h.
BV-2 Microglia and HT-22 Neuronal Cells Co-culture System
As mentioned above, the steps of the BV-2-HT-22 co-culture system (Supplementary Fig. 2) were consistent with those of BV-2-NSC34. The concentration of LPS was 1 ug/mL, the co-culture time was 24 in BV-2-HT-22 co-culture system.
Cell Viability Assay
Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. Briefly, cells were seeded in 96-well culture plates and received the various treatments. After that, cells were incubated with 10 µL CCK-8 reagent and finally the absorbance at 450 nm was measured using a microplate reader (BIO-RAD iMark).
Western blot Analysis
Each sample contained with 20 µg of protein was separated by 10% or 12% SDS-PAGE gels and transferred to PVDF membrane. The PVDF membranes were incubated with the primary antibodies at 4℃ respectively overnight. Full details on primary antibodies used are provided in Supplementary Table 2. The horseradish peroxidase (HRP)-conjugated secondary antibodies (Goat anti-Rabbit, 1:7000 or Goat anti-Mouse, 1:7000; Proteintech, Wuhan, China) were used for 2 h at room temperature and protein expressions were evaluated by using the enhanced chemiluminescence plus detection system (Tanon 4600, Shanghai, China).
RNA Isolation and Quantitative Real-Time PCR Assays
Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, Shanghai, China) following the manufacturer's instructions. Isolated RNA was then reverse-transcribed into cDNA using the HiScript® Ⅲ RT SuperMix (Vazyme, Nanjing, China) following the standard protocol. For real-time quantitative PCR analysis, the resultant cDNA products were amplified using a 2× ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) in triplicate. The mRNA level was normalized to β-actin and expressed as fold-increase. The forward and reverse primer sequences are shown in Supplementary Table 3.
After treatment, the cells were treated with 4% paraformaldehyde for 30 min, then blocked in 5% goat blocking serum (Solarbio, Beijing, China) for 30 min at room temperature. Primary antibodies were incubated at 4 ℃ overnight. Full details on primary antibodies used are provided in Supplementary Table 4. Then, the cells were incubated with goat anti-rabbit IgG (1:500; Multi Sciences, Hangzhou, China) secondary antibody for 2 h in the dark at 37 ℃. Finally, fluorescence images were captured with a fluorescence microscope (Olympus, Tokyo, Japan), and the analysis of the fluorescence images was performed by Image J.
Transmission electron microscopy (TEM)
The cell precipitation was fixed in ice cold 3% glutaraldehyde in phosphate buffer, pH 7.4 for 1 h, then post fixed in 1% OsO4. Following dehydration and embedding in Epon resin, ultrathin sections were cut and stained with uranyl acetate and lead citrate. Finally, the sample were imaged under the transmission electrons microscope (HT 7700-SS, HITACHI, Japan).
Detection of Mitochondrial-Derived DCFH-ROS Levels
After treatment, the BV-2 microglia cells incubated with 10 µM DCFH-DA (Beyotime, Shanghai, China) in the dark at 37 ℃ for 40 min. After washing the cells for three times with serum-free culture medium, images were captured using a fluorescence microscope (Olympus, Tokyo, Japan) and analyzed with Image J.
Enzyme-Linked Immunosorbent Assay (ELISA)
IL-1β (Multi Sciences, Hangzhou, Zhejiang, China) and IL-18 (Multi Sciences, Hangzhou, Zhejiang, China) that the cells had secreted into the culture supernatant in lower chamber in Transwell co-culture system were measured by ELISA according to the manufacturer’s instructions.
The apoptosis of neuronal cells was detected by CoraLite®594 TUNEL Assay Apoptosis Detection kit (Proteintech Group, Inc) according to the manufacturer's directions. The apoptotic cells were TUNEL positive which were labeled with red fluorescence using a fluorescence microscopy. The ratio of TUNEL-positive cells to total neuronal cells indicates the apoptosis index.
Data are presented as means ± SEM of three independent experiments. The data were analyzed with one-way ANOVA followed by Tukey’s post hoc test for significance via SPSS 25.0. Asterisks indicate statistically significant differences between the compared groups: P < 0.05.