Stigmasterol isolated from Azadirachta indica flowers attenuated glutamate-induced neurotoxicity via downregulation of the Cdk5/p35/p25 signaling pathway in the HT-22 cells

Glutamate, an excitatory neurotransmitter, was elevated in the brain of neurodegenerative disease (ND) patients. The excessive glutamate induces Ca2+ influx and reactive oxygen species (ROS) production which exacerbates mitochondrial function, leading to mitophagy aberration, and hyperactivates Cdk5/p35/p25 signaling leading to neurotoxicity in ND. Stigmasterol, a phytosterol, has been reported for its neuroprotective effects; however, the underlying mechanism of stigmasterol on restoring glutamate-induced neurotoxicity is not fully investigated.We investigated the effect of stigmasterol, a compound isolated from Azadirachta indica (AI) flowers, on ameliorating glutamate-induced neuronal apoptosis in the HT-22 cells.To further understand the underlying molecular mechanisms of stigmasterol, we investigated the effect of stigmasterol on Cdk5 expression, which was aberrantly expressed in glutamate-treated cells. Cell viability, Western blot analysis, and immunofluorescence are employed.Stigmasterol significantly inhibited glutamate-induced neuronal cell death via attenuating ROS production, recovering mitochondrial membrane depolarization, and ameliorating mitophagy aberration by decreasing mitochondria/lysosome fusion and the ratio of LC3-II/LC3-I. In addition, stigmasterol treatment downregulated glutamate-induced Cdk5, p35, and p25 expression via enhancement of Cdk5 degradation and Akt phosphorylation. Although stigmasterol demonstrated neuroprotective effects on inhibiting glutamate-induced neurotoxicity, the efficiency of stigmasterol is limited due to its poor water solubility. We conjugated stigmasterol to soluble soybean polysaccharides with chitosan nanoparticles to overcome the limitations. We found that the encapsulated stigmasterol increased water solubility and enhanced the protective effect on attenuating the Cdk5/p35/p25 signaling pathway compared with free stigmasterol.Our findings illustrate the neuroprotective effect and the improved utility of stigmasterol in inhibiting glutamate-induced neurotoxicity.