Jsh 23

The BV2 mouse microglia cell line (FH0355) and HT22 mouse hippocampal neuron cell line (FH1027) was both purchased from Shanghai FuHeng Biology Co., Ltd. in China. On the first day, BV2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma–Aldrich, MO, USA) and 1% penicillin/streptomycin (Gibco) in humidified 5% CO₂ air environment at 37 °C. On the second day, the BV2 cell culture medium was replaced, and the cells were divided into 4 groups: 1) the Control group (BV2), in which BV2 cells were incubated with medium only for 24 h; 2) the Heme group (BV2 + Heme) in which BV2 cells were treated with 40 μM hemin (H9039, Sigma, USA) for 24 h; 3) the MyD88 inhibition group (BV2 + Heme + T6167923) in which BV2 cells were treated with 40 μM hemin and 2 μM MyD88 inhibitor (T6167923, #HY-19744, MedChemExpress, USA) for 24 h; and 4) the NF-κB inhibition group (BV2 + Heme + JSH-23), in which BV2 cells were treated with 40 μM hemin and 20 μM NF-κB inhibitor (JSH-23, #S7351, Selleck, USA) for 24 h. Then, the BV2 cells were harvested for subsequent experiments.

Recombinant human TNFα (Rhtnf-a, Invivogen/bioconnect,France), Recombinant Human Interferon-γ (11343536, Immunotools, Germany). Cetuximab (5 mg/ml; Merck serono, USA), rituximab (10 mg/ml; Roche, Switzerland), rapamycin (50 nM; Selleckchem, Germany), PD98059 (50 μM; Selleckchem), GW5074 (20 μM; Selleckchem), LY294002(25 μM; Selleckchem),SP600125 (20 μM; Selleckchem). Pamapimod (0,5uM Selleckchem), JSH-23(10uM, Selleckchem), and T-5224(20uM, Apexbt, USA). The concentration of GW5074, SP600125, LY-294,002, PD98059, rapamycin was chosen on basis of previous results [22 (link)]. The concentration of Pamapimod (0,5uM Selleckchem), JSH-23(10uM, Selleckchem), and T-5224(20uM, Apexbt) was according to instruction of the manufacturer.