Donepezil

The human brain microvascular endothelial cells (HBMECs) used in this study were supplied by the cell bank of Shanghai Biology Institute (Shanghai, China). These cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin solution in a humidified atmosphere with 5% CO₂ at 37°C. For the establishment of I/R conditions in vitro, HBMECs were cultured in a glucose- and serum-free DMEM placed in a hypoxic cell culture system for 2 h at 37°C, 5% CO₂ and 95% N₂. Then, cells were maintained in a glucose-containing DMEM with 10% FBS in an incubator with 95% air and 5% CO₂ for 6 h. Those cultured in a complete medium only at 37°C, 5% CO₂ were served as a control [23 (link)].
To investigate the effect of donepezil on the cells, we treated the cells with donepezil (Eisai, Suzhou, China) at concentrations of 20, 50, and 100 μM for 2 h.

Donepezil was provided by Eisai China Inc. (Suzhou, China). Aβ_25-35, dimethyl sulfoxide (DMSO), a mouse monoclonal anti-β-actin antibody and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were provided by Sigma-Aldrich (St. Louis, MO, USA). The protein kinase C (PKC) inhibitor GF109203X was provided by Calbiochem (San Diego, CA, USA). A rabbit polyclonal anti-phospho-PKC (P-PKC) antibody and a rabbit polyclonal anti-phospho-myristoylated alanine-rich C kinase (MARCKS) antibody were provided by Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti-PKCα and PKCε antibodies were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). A Dulbecco’s modified Eagle medium (DMEM), foetal bovine serum (FBS), penicillin, streptomycin and pancreatin-EDTA (ethylene diamine tetraacetic acid) mixture was provided by Gibco (Carlsbad, CA, USA). RIPA cell lysis buffer was provided by Shenneng Bocai (Shanghai, China). A BCA protein assay kit was provided by Pierce (Pierce Biotechnology, Rockford, IL, USA). An ECL Western Blotting Detection kit was provided by Amersham-Pharmacia Biotech (GE Healthcare, Little Chalfont, UK).

Scoplamine hydrobromide injection was purchased from Guangzhou Pharmaceuticals Corporation (Guangzhou, China). Donepezil (Eisai China Inc) and edaravone (Boda Pharmaceutical) were dissolved in 0.9% physiological saline. The gavage doses of BSYZ-F were 5, 10 and 20 times the clinical dosage (17.5 g dosage, 60 kg human), respectively. Since 0.88 g of BSYZ-E was determined to contain 100 g of BSYZ-F. The oral dose of BSYZ-E is 1.46 mg/kg, 2.92 mg/kg and 5.84 mg/kg. Mice were randomly sorted into the following groups, with 12 mice in each group: the vehicle control group (CON, 0.9% Saline), scoplamine group (SCOP 3 mg/kg), low dose BSYZ-E group (BSYZ-E L, SCOP 3 mg/kg + BSYZ-E 1.46 mg/kg), medium dose BSYZ-E group (BSYZ-E M, SCOP 3 mg/kg + BSYZ-E 2.92 mg/kg), high dose BSYZ-E group (BSYZ-E H, SCOP 3 mg/kg + BSYZ-E 5.84 mg/kg), Donepezil group (DON, SCOP 3 mg/kg + DON 3 mg/kg) and Edaravone group (EDA, SCOP 3 mg/kg + Edaravone 33.2 mg/kg). Animals were treated by oral gavage with Saline, BSYE-E, Donepezil or Edaravone continuously once per day. Mice underwent the behavioural tests from the 8th to the 17th day. Except CON group received saline intra-peritoneally (ip) 30 min after treatment with Saline, all mice were injected with SCOP 30 min after oral administration of BSYE-E, DON or EDA. They were taken the behavioural tests 30 min after SCOP injection.