Neuroprotective Effects of BYHW Decoction

Primary hippocampal culture was prepared [46 (link)] as previously described. Primary hippocampal neurons cultured in 6-well plates were underwent OGD or not (naïve group) after 5–6 days in culture [19 (link)]. To obtain the extract, the BYHW decoction was suspended in HBSS at a concentration of 35 mg/mL, mixed thoroughly with a vortex for 5 minutes, and centrifuged at 150 g for 30 minutes to collect the supernatant. Mitochondria damage, apoptosis, ROS, and ATP content were detected using MitoTracker kit, Annexin V-FITC/PI kit, MitoSOX red kit, and ATP content kit according to the instruction of the manufactures, respectively. Damage to the mitochondria and neuronal apoptosis was measured in a BD FACSAriaTM llu flow cytometer and analyzed using the DIVA software from BD Biosciences (San Jose, CA, USA). ROS in cells were evaluated by fluorescent microplate reader (PerkinElmer EnSpire™, USA). ATP content was measured (PerkinElmer EnSpire™, USA) at 340 nm immediately and after 3-minute incubation at 37°C. All experiments were performed at least three times.