S1413

The reagents used in this study are listed as follows: H89 2HCl (10 mM, dissolved in DMSO, S1582, Selleck, Houston, TX), forskolin (FSK) (10 mM, dissolved in DMSO, S2449, Selleck, Houston, TX), PKI (14–22) amide myristoylated (PKI) (0.5 mg, dissolved in DMSO, Sc471154, Santa Cruz, CA), Tetrandrine (10 mM, dissolved in DMSO, CAS:518–34-3, Shanghai Ronghe Medical, Shanghai, China), Caspase inhibitor z-VAD-fmk (10 mM, dissolved in DMSO, S7023, Selleck, Houston, TX), Bafilomycin A1 (dissolved in DMSO, S1413, Selleck, Houston, TX), Chloroquine (CQ) (C6628, Sigma-Aldrich, USA), PD98059 (Beverly, MA, USA), N-acetyl-L-cysteine (NAC) (dissolved in ddH₂O, A0150000, Sigma-Aldrich, USA), 3-Methyladenine (3-MA) (dissolved in ddH₂O, M9281-100MG, Sigma-Aldrich, USA), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Invitrogen Carlsbad, CA), and an FITC Annexin V Apoptosis Detection Kit (556,547, BD Pharmingen). All reagents were formulated as recommended by their suppliers.

Primary LF were isolated from 6-week-old mice as previously described [20 (link)] and cultured with DMEM containing 15% FBS at 37 °C. Passage 2 cells were treated with MitoQ (50 nM, HY-100116, MCE), fenofibrate (10 μM, T1149, Topscience), oleic acid (10 μM, S4707, Selleck), etomoxir (50 μM, S8244, Selleck), bafilomycin (5 nM, S1413, Selleck) and SRT1720 (4 μM, S1129, Selleck) (Additional file 1). The dosages of these compounds were based on published papers. And MTT test for them was performed (Additional file 2).

The 1 h OGD/24 h R model was employed to simulate ischemia/reperfusion injury in vitro. When the neurons were cultured for 7 days, the medium was changed to glucose-free DMEM (11966-025, Gibco, Grand Island, USA) and placed in a 37°C hypoxia incubator (Thermo Scientific, Marietta, OH, USA) under hypoxic conditions (2% O₂/5% CO₂/93% N₂) for 1 h. After that, glucose-free DMEM was replaced by Neurobasal Medium, which contained 2% B27 supplement under normoxic condition 5% CO₂/21% O₂/74% N₂ for 24 h reoxygenation. Recombinant mouse (rm) IL-17A (250 ng/mL, 421-ML-025/CF, R&D System, MN), 3-MA (5 mM, S2767, Selleck Chemicals), Bafilomycin A1 (BafA1) (100 nM, S1413, Selleck Chemicals) and CsA (100 nM, S2286, Selleck Chemicals) dissolved in 4 mM HCL, H₂O or DMSO, respectively, were added into the medium during OGD/R treatment. rmIL-17A dissolved in 40 μM HCL was used as vehicle group.