背景:马斯克,中药,广泛用于诱导复苏和提神,活血缓解疼痛.它通常用于治疗缺血性中风,麝香酮是其核心药用成分。
目的:本研究的目的是探讨麝香酮是否通过毒蕈碱受体的胆碱能信号改善神经元损伤。
方法:在大鼠大脑中动脉阻塞(MCAO)模型以及PC12细胞中氧糖剥夺(OGD)诱导的神经元损伤中,测试了麝香酮的作用。细胞计数试剂盒8(CCK8)测定用于测量细胞活力,用试剂盒检测乳酸脱氢酶(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-4AM)染色用于证明麝香酮对活性氧(ROS)水平的影响,线粒体膜电位(MMP)和细胞内Ca2+分别测定,其中所有这些染色都通过激光共聚焦显微镜可视化。对于体内实验,使用激光多普勒血流仪测量大鼠脑血流量以评估MCAO模型,并使用改良的神经系统严重程度评分(mNSS)来评估神经功能的恢复。通过2,3,5-三苯基氯化四唑(TTC)染色测量计算梗死率。除DCFH-DA和Fluo-4AM染色外,5,5\',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基碳花青碘化物(JC-1)染色用于观察脑细胞中细胞内Ca2测量。通过蛋白质印迹检测细胞和组织中的蛋白质水平。
结果:用麝香酮预处理可显著提高细胞活力,乳酸生产,线粒体膜电位崩溃和功能,Ca2+过载,ROS生成,OGDPC12细胞凋亡。Muscone还调节PI3K,ERK和AKT信号通路通过激活OGD诱导的PC12细胞中毒蕈碱受体的胆碱能信号传导。更重要的是,阿托品阻断毒蕈碱受体的胆碱能信号显著降低麝香酮的神经保护作用,包括细胞活力,Ca2+流出,和线粒体修复。此外,发现麝香酮可有效缓解线粒体功能障碍和MCAO诱导的脑组织中ROS水平升高。值得注意的是,阿托品减弱了麝香酮的这种有益作用,但()-Sparteine没有减弱。
结论:我们的研究表明,麝香酮通过激活胆碱能信号的毒蕈碱受体发挥其神经保护作用,从而为OGD诱导的卒中神经损伤的治疗提供了有希望的治疗靶点。研究结果表明,这些治疗可能对中风患者具有潜在的益处。
BACKGROUND: 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.
OBJECTIVE: The aim of this study was to explore whether muscone ameliorates neuronal damage through cholinergic signaling of muscarinic receptors.
METHODS: 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 Ca2+ 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 Ca2+ measurement in brain cells. Protein levels in cells and tissues were detected by Western blot.
RESULTS: Pretreatment with muscone significantly improved the cell viability, lactic acid production, mitochondrial membrane potential collapse and function, Ca2+ 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, Ca2+ 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.
CONCLUSIONS: 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.