Saikosaponin d causes apoptotic death of cultured neocortical neurons by increasing membrane permeability and elevating intracellular Ca ²⁺ concentration

Saikosaponins (SSs) are a class of naturally occurring oleanane-type triterpenoid saponins found in Radix bupleuri that has been widely used in traditional Chinese medicine. As the main active principals of Radix bupleuri, SSs have been shown to suppress mouse motor activity, impair learning and memory, and decrease hippocampal neurogenesis. In the present study, we investigated the effect of five SSs (SSa, SSb1, SSb2, SSc, and SSd) on neuronal viability and the underlying mechanisms in cultured murine neocortical neurons. We demonstrate that SSa, SSb1 and SSd produce concentration-dependent apoptotic neuronal death and induce robust increase in intracellular Ca ²⁺ concentration ([Ca ²⁺ ] _i ) at low micromolar concentrations with a rank order of SSd > SSa > SSb1, whereas SSb2 and SSc have no detectable effect on both neuronal survival and [Ca ²⁺ ] _i . Mechanistically, SSd-induced elevation in [Ca ²⁺ ] _i is the primary result of enhanced extracellular Ca ²⁺ influx, which likely triggers Ca ²⁺ -induced Ca ²⁺ release through ryanodine receptor activation, but not SERCA inhibition. SSd-induced Ca ²⁺ entry occurs through a non-selective mechanism since blockers of major neuronal Ca ²⁺ entry pathways, including L-type Ca ²⁺ channel, NMDA receptor, AMPA receptor, Na ⁺ -Ca ²⁺ exchanger, and TRPV1, all failed to attenuate the Ca ²⁺ response to SSd. Further studies demonstrate that SSd increases calcein efflux and induces an inward current in neocortical neurons. Together, these data demonstrate that SSd elevates [Ca ²⁺ ] _i due to its ability to increase membrane permeability, likely by forming pores in the surface of membrane, which leads to massive Ca ²⁺ influx and apoptotic neuronal death in neocortical neurons.