Calcium Overload and Apoptosis in Mitochondrial Pathways under Ischemia, Hypoxia, and Epilepsy-Like Conditions in an In Vitro Study

Objective Ischemia and hypoxia are frequently associated with epileptic characteristics, and irregular calcium metabolism can exacerbate neuronal damage. To investigate calcium homeostasis and its effect on neuronal injury, hippocampal HT22 cells were subjected to oxygen-glucose deprivation (OGD), followed by reoxygenation and culturing in Mg ²⁺ -free medium, to mimic cerebral ischemic and hypoxic brain injury combined with epilepsy. Methods An oxygen-glucose deprivation combined with epileptiform discharge (OGD&ED) model was established to simulate the pathological state of cerebral ischemia-hypoxia combined with epilepsy. The model was evaluated based on cell viability and Ca²⁺ overload, and the optimal induction conditions were determined as 3-hour OGD, 3-hour culture without Mg²⁺, and 8-hour reoxygenation. A calcium-sensing receptor (CaSR) agonist (R568) and inhibitor (NPS-2143), combined with a Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) inhibitor (KN-93), were used to study the regulatory effect of Ca²⁺ overload on the mitochondrial apoptotic pathway. The apoptosis rate, mitochondrial damage, intracellular Ca²⁺ concentration, and changes in the expression of calcium-related proteins were detected. Results Compared with the single OGD group or the ED group, the OGD&ED group showed a significant increase in Ca²⁺ influx, more severe cell and mitochondrial damage, and a higher apoptosis rate. The CaSR agonist R568 aggravated Ca²⁺ overload, while the inhibitor NPS-2143 and the CaMKII inhibitor KN-93 effectively inhibited Ca²⁺ influx. Ca²⁺ overload activated the mitochondrial apoptotic pathway, resulting in abnormal expression of apoptosis-related proteins and further aggravating neuronal injury. Conclusion In the OGD&ED model with an intervention time of OGD3h, Mg ²⁺ -free, and reoxygenation8h, Ca ²⁺ overload exacerbates the damage to cells and mitochondria and promotes apoptosis through the mitochondrial pathway.