Chu Zhili, Chen Yubing, Xie Danni, Song Caiyou, Yang Lin, Qin Tao, Zhai Zhenwei, Cao Zhixing, Xu Ying, Sun Tao
State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, China.
Traditional Chinese Medicine Factory Co. Ltd, Taiji Group Chongqing, Chongqing, 402284, China.
J Ethnopharmacol. 2025 May 28;348:119879. doi: 10.1016/j.jep.2025.119879. Epub 2025 Apr 25.
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.
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.
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 Ca 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.
Using GC-MS, 18 major components were identified in EEM. EEM significantly inhibited apoptosis, reduced ROS generation, and alleviated Ca 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.
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.
麝香是一种用于治疗中枢神经系统疾病的传统中药。氧化应激是阿尔茨海默病（AD）的关键致病机制，也是连接AD各种病理过程的关键桥梁。先前的研究表明，麝香可通过抑制谷氨酸诱导的神经元细胞损伤发挥神经保护作用。然而，其潜在机制仍不清楚。
本研究旨在评估麝香乙醇提取物（EEM）对谷氨酸诱导的HT22细胞氧化损伤的影响及潜在机制。
采用气相色谱-质谱联用（GC-MS）法鉴定EEM的成分。通过将HT22细胞暴露于谷氨酸建立氧化毒性细胞模型。通过CCK8和LDH测定评估细胞活力，并使用FITC-Annexin V染色和TUNEL测定评估细胞死亡模式。用DCFH-DA和MitoSOX Red探针测量细胞内和线粒体活性氧水平。用Fluo-4 AM荧光探针测量细胞内钙水平。使用JC-1荧光探针分析线粒体功能。通过蛋白质印迹分析Bid、钙蛋白酶-1、Bax、Bcl-2、AIF、P-ERK、ERK、P-JNK、JNK、P-P38、P38、Nrf2、HO-1、Keap1和NQO-1的蛋白质表达水平。通过免疫荧光染色检测AIF和Nrf2在细胞质和细胞核中的分布。
采用GC-MS法在EEM中鉴定出18种主要成分。EEM显著抑制细胞凋亡，减少活性氧生成，并减轻钙超载。EEM通过调节线粒体相关凋亡蛋白的表达恢复线粒体功能障碍，包括下调钙蛋白酶-1和Bax，上调Bid和Bcl-2，并抑制AIF核转位。EEM抑制丝裂原活化蛋白激酶（MAPK）磷酸化，同时激活Nrf2/Keap1信号通路。
我们的研究表明，EEM通过调节MAPK和Nrf2途径保护HT22细胞免受谷氨酸诱导的损伤，有效降低氧化应激和细胞凋亡。总之，本研究首次在细胞水平上证明EEM通过调节MAPK和Nrf2途径发挥神经保护作用。这些发现为麝香抗AD的机制提供了新的见解，并为其在AD中的潜在应用奠定了基础。
