College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, 610032, China; Translational Chinese Medicine Key Laboratory of Sichuan Province, Sichuan Academy of Chinese Medicine Science, Chengdu, 610000, China.
Sichuan Industrial Institute of Antibiotics, School of Pharmacy, Chengdu University, Chengdu, 610000, China.
J Ethnopharmacol. 2024 Jan 30;319(Pt 2):117192. doi: 10.1016/j.jep.2023.117192. Epub 2023 Sep 19.
Musk, a traditional Chinese medicine, is broadly used in inducing resuscitation and refreshing the mind, activating blood and alleviating pain. It is commonly used for the treatment of ischemic stroke, and muscone is its core medicinal component.
The aim of this study was to explore whether muscone ameliorates neuronal damage through cholinergic signaling of muscarinic receptors.
The effects of muscone were tested in a rat model of middle cerebral artery occlusion (MCAO) as well as injured neurons induced by oxygen-glucose deprivation (OGD) in PC12 cells. Cell counting kit 8 (CCK8) assay was used to measure the cell viability, and the production of lactate dehydrogenase (LDH) and adenosine-triphosphate (ATP) were examined by kit. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA), tetramethylrhodamine ethyl ester (TMRE) and Fluo-4 acetoxymethyl ester (Fluo-4 AM) staining were used to demonstrate effect of muscone on the reactive oxygen species (ROS) level, mitochondria membrane potential (MMP) and intracellular Ca measurement in cells respectively, in which all of those staining was visualized by laser confocal microscope. For in vivo experiments, rats' cerebral blood flow was measured using laser Doppler blood flowmetry to evaluate the MCAO model, and a modified neurological severity score (mNSS) was used to assess the recovery of neurological function. Calculate infarct rate was measured by 2,3,5-Triphenyl Tetrazolium Chloride (TTC) staining. Except DCFH-DA and Fluo-4 AM staining, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolylcarbocyanine iodide (JC-1) staining was used to observe intracellular Ca measurement in brain cells. Protein levels in cells and tissues were detected by Western blot.
Pretreatment with muscone significantly improved the cell viability, lactic acid production, mitochondrial membrane potential collapse and function, Ca overload, ROS generation, and cell apoptosis in OGD PC12 cells. Muscone also regulated PI3K, ERK and AKT signal pathways by activating cholinergic signaling of muscarinic receptors in PC12 cells induced with OGD. More importantly, the blocking of cholinergic signaling of muscarinic receptors by atropine significantly reduces the neuroprotective effects of muscone, including the cell viability, Ca efflux, and mitochondrial repair. Furthermore, muscone was found to effectively alleviate mitochondrial dysfunction and elevated levels of ROS induced by the MCAO in the brain tissue. Notably, this beneficial effect of muscone was attenuated by atropine but not by (+)-Sparteine.
Our study indicates that muscone exerts its neuroprotective effects by activating muscarinic receptors of cholinergic signaling, thus providing a promising therapeutic target for the treatment of OGD-induced nerve injury in stroke. The findings suggest that these treatments may hold potential benefits for stroke patients.
ETHNOPHARMACOLOGICAL 相关性：麝香，一种传统的中药，广泛用于诱导复苏和提神醒脑，活血止痛。它通常用于治疗缺血性中风，而麝香酮是其核心药用成分。
本研究旨在探讨麝香酮是否通过毒蕈碱型乙酰胆碱受体的胆碱能信号改善神经元损伤。
在大脑中动脉闭塞（MCAO）大鼠模型以及氧葡萄糖剥夺（OGD）诱导的 PC12 细胞损伤中测试了麝香酮的作用。使用细胞计数试剂盒 8（CCK8）测定细胞活力，试剂盒测定乳酸脱氢酶（LDH）和三磷酸腺苷（ATP）的产生。2'，7'-二氯二氢荧光素二乙酸酯（DCFH-DA）、四甲基罗丹明乙酯（TMRE）和 Fluo-4 乙酰氧基甲酯（Fluo-4 AM）染色分别用于示踪麝香酮对细胞内活性氧（ROS）水平、线粒体膜电位（MMP）和细胞内 Ca 测量的影响，所有这些染色均通过激光共聚焦显微镜观察。在体内实验中，使用激光多普勒血流仪测量大鼠脑血流以评估 MCAO 模型，并使用改良神经严重程度评分（mNSS）评估神经功能恢复情况。通过 2,3,5-三苯基四唑氯（TTC）染色计算梗死率。除 DCFH-DA 和 Fluo-4 AM 染色外，使用 5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基碳环化碘（JC-1）染色观察脑细胞内 Ca 测量。Western blot 检测细胞和组织中的蛋白水平。
麝香酮预处理可显著提高 OGD PC12 细胞的细胞活力、乳酸生成、线粒体膜电位崩溃和功能、Ca 超载、ROS 生成和细胞凋亡。麝香酮还通过激活 OGD 诱导的 PC12 细胞毒蕈碱型乙酰胆碱受体调节 PI3K、ERK 和 AKT 信号通路。更重要的是，毒蕈碱型乙酰胆碱受体的阻断剂阿托品可显著降低麝香酮的神经保护作用，包括细胞活力、Ca 外流和线粒体修复。此外，麝香酮可有效减轻大脑组织中 MCAO 引起的线粒体功能障碍和 ROS 水平升高。值得注意的是，这种麝香酮的有益作用被阿托品减弱，但不是 (+)-sparteine 减弱。
本研究表明，麝香酮通过激活胆碱能信号毒蕈碱型乙酰胆碱受体发挥神经保护作用，为治疗中风引起的 OGD 诱导的神经损伤提供了有希望的治疗靶点。研究结果表明，这些治疗方法可能对中风患者有益。
