Cholinergic signaling of muscarinic receptors directly involves in the neuroprotection of muscone by inducing Ca2+ antagonism and maintaining mitochondrial function

Ethnopharmacological relevance: 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.

Aim of the study: The aim of this study was to explore whether muscone ameliorates neuronal damage through cholinergic signaling of muscarinic receptors.

Materials and 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.

Atropine; Cerebral ischemia; Europrotective effects; Muscarinic receptors; Muscone.