Ethanol extract of Moschus attenuates glutamate-induced cytotoxicity in HT22 cells by regulating the Nrf2 and MAPK pathways

Ethnopharmacological relevance: Moschus is a traditional Chinese materia medica for treating central nervous system disorders. Oxidative stress is a key pathogenic mechanism of Alzheimer's disease (AD) and serves as a critical bridge linking various pathological processes of AD. Previous studies have shown that Moschus can exert neuroprotective effects by inhibiting glutamate-induced neuronal cell damage. However, its underlying mechanisms remain unclear. Aim of the study: This study aimed to evaluate the effects and potential mechanisms of the ethanol extract of Moschus (EEM) on glutamate-induced oxidative damage in HT22 cells. Materials and methods: The components of EEM were identified using GC-MS. An oxidative toxicity cell model was established by exposing HT22 cells to glutamate. Cell viability was assessed through CCK8 and LDH assays, and the modes of cell death were evaluated using FITC-Annexin V staining and TUNEL assays. Intracellular and mitochondrial ROS levels were measured with DCFH-DA and MitoSOX Red probes. Intracellular Ca2+ levels were measured with the Fluo-4 AM fluorescent probe. Mitochondrial function was analyzed using the JC-1 fluorescent probe. Protein expression levels of Bid, Calpain-1, Bax, Bcl-2, AIF, P-ERK, ERK, P-JNK, JNK, P-P38, P38, Nrf2, HO-1, Keap1, and NQO-1 were analyzed through western blotting. The distribution of AIF and Nrf2 in the cytoplasm and nucleus was examined through immunofluorescence staining. Results: Using GC-MS, 18 major components were identified in EEM. EEM significantly inhibited apoptosis, reduced ROS generation, and alleviated Ca2+ overload. EEM restored mitochondrial dysfunction by regulating the expression of mitochondria-related apoptotic proteins, including the downregulation of Calpain-1 and Bax, upregulation of Bid and Bcl-2, and inhibition of AIF nuclear translocation. EEM inhibited MAPK phosphorylation while activating the Nrf2/Keap1 signaling pathway. Conclusions: Our study shows that EEM protects HT22 cells from glutamate-induced damage by regulating the MAPK and Nrf2 pathways, effectively reducing oxidative stress and apoptosis. In summary, this study first demonstrates at the cellular level that EEM exerts neuroprotective effects by modulating the MAPK and Nrf2 pathways. These findings provide new insights into the mechanism of Moschus against AD and establish a foundation for its potential application in AD.