Uncaria rhynchophylla alkaloid extract exerts neuroprotective activity against Parkinson's disease via activating mitophagy with the involvement of UCHL1

Ethnopharmacological relevance: Uncaria rhynchophylla (Miq.) Miq. ex Havil. (UR), a traditional Chinese medicinal plant, plays an active role in neuroprotection. Clinical medication and modern pharmacological studies have proved the efficacy of UR against Parkinson's disease (PD), with alkaloids being recognized as the main bioactive components. But the therapeutic effect and mechanism of U. rhynchophylla alkaloid extract (URA) against PD need further exploration. Aim of study: The study aimed to investigate the therapeutic effect and potential mechanism of URA on PD. Materials and methods: LC-MS methodology was used to evaluate the chemical constituents of URA. The anti-PD activity of URA in vivo was measured on the mouse and rat models of PD. Neuroprotective effect of URA on PC12 cells was measured by MTT assay. Dopamine (DA) and its metabolites were detected by LC-MS for probing the protection ability on dopaminergic neurons. The differentially expressed proteins between model group and URA administrated group were analyzed by proteomics, suggesting oxidative phosphorylation as possible pathway of URA. Considering the critical role of mitochondria in oxidative phosphorylation, JC-1 staining, MitoSOX staining, transmission electron microscopy (TEM) observation and adenosine triphosphate (ATP) levels detection were used to analyze the effects of URA on mitochondrial morphology and function. Biolayer interferometry (BLI) was used to search for the possible UCHL1-bonding compounds in URA. Results: URA significantly mitigated the behavioural defects by improving coordination, shortening the time to climb down the whole pole (T-LA) and increasing the forelimbs' muscle strength of MPTP-induced PD mice and 6-OHDA-induced PD rats. In addition, URA improved tyrosine hydroxylase expression in dopaminergic neurons by immunohistochemistry (IHC) staining, and thus increased the neurotransmitter levels of DA and relevant metabolites. Furthermore, URA promoted mitophagy as reflected by a significant decrease in reactive oxygen species (ROS) generation, an increase in ATP levels and clearance of damaged mitochondria. Subsequently, Ubiquitin C-terminal hydrolase 1 (UCHL1), which is associated with the mitochondrial dysfunction in PD, is suggested to be a promising target based on the proteomics result, and proved by the blocked protective effects of URA by UCHL1 inhibitor. Furthermore, hirsuteine (HTE) was identified as a potential active compound of URA binding to UCHL1 by BLI, and the binding capacity and site were verified by surface plasmon resonance (SPR) and molecular docking. Conclusion: This work demonstrates that URA exerts effective neuroprotective activity against PD via activation of mitophagy with the involvement of UCHL1, and HTE may be a potential active compound of URA.