Bc0300

Glucose (Solarbio, BC2500), lactic acid (LA; Solarbio, BC2230), ATP (Solarbio, BC0300) and NADPH (Solarbio, BC1100) detection kits were used to detect intracellular glucose levels, LA levels, ATP levels, and the NADPH/NADP+ ratio, respectively. ECAR was detected using the Seahorse XF Glycolysis Stress Test Kit (Seahorse, 103,020–100).

Each well was seeded with 1×10⁵ Cal-27 cells. After 24 h of incubation, different concentrations of ICD were added (0, 0.15, 0.30, 0.60, and 1.20 mM) for 24 h, 48 h, and 72 h, respectively. After the cells were harvested, the levels and activities of the MMP (M8650, Solarbio, Beijing, China), ATP (BC0300, Solarbio, Beijing, China), ROS (CA1410, Solarbio, Beijing, China), and mitochondrial complex enzymes I–IV (BC0630; BC0950; BC3245; BC0945, Solarbio, Beijing, China) were detected according to the manufacturer’s instructions. The changes in the MMP in each group were detected with a 490 nm excitation wave and a 530 nm irradiation wave. The ATP content of each group was detected at a 340 nm wavelength. ROS levels were measured at a 488 nm excitation wavelength and a 525 nm emission wavelength. In addition, the cellular ROS content was calculated using flow cytometry (CytoFlex, Indianapolis, IN, USA) after 24 h, 48 h, and 72 h of treatment with 0.60 mM ICD, respectively. The detection wavelength of complex enzyme I was 600 nm, while that of complex enzyme II was 600 nm, that of complex enzyme III was 550 nm, and that of complex enzyme IV was 550 nm. The activity of the respiratory electron transport chain complex enzymes I–IV was detected in each group.

Mitochondria were isolated from freshly acquired brain samples using a Mitochondria Isolation Kit (C3606, Beyotime). ATP content (BC0300, Solarbio) and mitochondrial ETC (I‐IV) complex (BC0515, BC3230, BC3240, BC0945; Solarbio) activities were assayed using the appropriate commercially available kits according to the manufacturer's instructions.