Haikun Shenxi Capsule alleviates Alzheimer's disease by targeting mitophagy to clear turbidity toxin.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: "Turbidity toxin damages brain" has been identified as a critical etiopathogenetic mechanism in Alzheimer's disease (AD). Haikun Shenxi Capsule (HKSX), a traditional Chinese medicine (TCM) formula with efficacy in dissolving turbidity and eliminating toxicant, is clinically used for chronic renal failure (CRF). AIM OF THE STUDY: Based on the TCM theory of "treating different diseases with the same therapy", this study aimed to investigated the neuroprotective effects and molecular mechanisms of HKSX on AD. MATERIALS AND METHODS: SAMP8 mice and N2aApp695 cells were used as AD models, behavioral, pathological assessments, as well as metabolomics, transmission electron microscopy (TEM), immunofluorescence (IF), immunohistochemistry (IHC) and Western blot analyses, were conducted to validate HKSX's therapeutic effects and underlying mechanisms on AD. RESULTS: Behavioral testing results showed that HKSX significantly improved learning and memory impairments of SAMP8 mice in novel object recognition (NOR) test and Morris Water Maze (MWM) test, along with reduced levels of Aβ and p-Tau at Thr231 in hippocampus. In metabolomics profiling, HKSX was demonstrated to modulate 73 metabolites and key metabolic pathways, including unsaturated fatty acid biosynthesis, tricarboxylic acid (TCA) cycle, and D-glutamine/D-glutamate metabolism, which are closely related with mitochondria. TEM showed that HKSX improved mitochondrial swelling and cristae rupture in SAMP8 mice, accompanied by increased autolysosomes. HKSX also enhanced mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) levels, reduced reactive oxygen species (ROS) content, suggesting that it significantly improved mitochondrial structure and function. IHC showed that HKSX administration significantly increased PINK1-positive staining. IF double-labeling results indicated that HKSX promoted the co-localization of Parkin-Tom20, LC-3-LAMP, and mitochondria-lysosomes, providing direct evidence of mitophagy activation. However, inhibition of mitophagy with Mdivi-1 abrogated HKSX-induced activation of PINK1-Parkin signaling, blocked clearance of turbidity toxins, such as Aβ, p-Tau and ROS, and suppressed autophagosome formation in N2a/APP695 cells, confirming that HKSX-mediated neuroprotection is mitophagy-dependent. CONCLUSIONS: This study established that HKSX alleviated AD-like pathological features and cognitive deficits by activating PINK1-mediated mitophagy pathway. These results suggest that mitophagy may be involved in the cellular process of "eliminating turbidity toxins and detoxification," which provide a novel therapeutic angle for exploring TCM remedies that resolve turbidity in the treatment of AD.