High-Dose Propofol Induces Cytotoxicity by Elevating Intracellular Ca²⁺ via GABA_A Receptor and IP₃R in HT22 Cells

Disruption of calcium (Ca²⁺) homeostasis has been implicated as a key pathological mechanism underlying propofol-induced neurodevelopmental and cognitive deficits. However, the mechanisms underlying propofol-induced intracellular Ca²⁺ dysregulation remain incompletely understood. Extending findings of anesthetic-induced metabolic disruptions in non-neuronal models to the central nervous system, this study aimed to elucidate the underlying mechanisms of the Ca²⁺ imbalance in neuronal cells, with implications for the safety of clinical anesthesia in pediatric populations.

Mouse hippocampal neurons (HT22 cells) served as an in vitro model. Cell viability was assessed using the CCK-8 assay. Intracellular Ca²⁺ dynamics were evaluated using the Fluo-4 AM Ca²⁺ fluorescent probe to investigate the mechanisms underlying propofol-induced Ca²⁺ dysregulation.

Propofol exposure at 10 μM and 50 μM across all time points (2, 6, or 24 hours) showed no significant impact on cell viability. Similarly, 100 μM propofol lacked toxicity at 2 or 6 hours, but survival significantly declined after 24 hours exposure (P < 0.0001). Furthermore, 200 μM propofol decreased cell viability after 2 hours of treatment (P < 0.01), with further reduction following prolonged exposure (P < 0.05). A rapid increase in intracellular Ca²⁺ concentration was observed with 200 μM propofol (P < 0.0001), which was entirely abolished by the inhibition of the γ-aminobutyric acid type A (GABA_A) receptor. Conversely, inhibition of the inositol trisphosphate receptor (IP₃R) alone partially mitigated the propofol-induced Ca²⁺ elevation (P < 0.0001). Notably, chelation of elevated intracellular Ca²⁺ using BAPTA-AM fully prevented the propofol-induced decrease in cell viability (P < 0.01).

Propofol induces cytotoxicity in HT22 cells in a concentration- or time-dependent manner. Notably, cytotoxicity at 100 μM propofol was observed only after 24 hours of exposure, whereas 200 μM propofol produced rapid cytotoxicity. This rapid toxicity is mediated by activation of GABA_A receptor and IP₃R, which triggers the endoplasmic reticulum (ER) Ca²⁺ release and elevating intracellular Ca²⁺ concentration.