Senegenin alleviates Aß1-42 induced cell damage through triggering mitophagy.

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE Senegenin (SEN), an active compound extracted from the traditional Chinese herb Polygala tenuifolia Willd. (a species in the genus Polygala, family Polygalaceae), could nourish neurons and resist neuronal damage in mouse models of Alzheimer's disease (AD). Amyloid-ß (Aß) depositions in neuronal cells may cause pathological changes such as oxidative stress which one return could cause severe damage to mitochondria in AD patients or animal models. Mitophagy is an important mechanism to selectively remove damaged mitochondria. In neurons, this process is mainly mediated by PTEN-induced putative kinase 1 (PINK1)/Parkin pathway. Previous studies have shown that SEN could reduce mitochondrial damage and inhibit apoptosis in neurons. Therefore, this study speculated that SEN might activate mitophagy to clear damaged mitochondria, thereby mitigating Aß-induced cell damage in neuronal cells. AIM OF THE STUDY This study aimed to determine the effects of SEN on Aß-induced cell damage, and further to explore whether SEN could induce mitophagy. Moreover, the regulatory role of mitophagy in the neuroptrotective effect of SEN would be elucidated. MATERIALS AND METHODS: This study established an in vitro cell damage model using Aß1-42 to treat mouse hippocampal neuron HT22 cells. The effects of SEN on cell damage were determined by MTT assay and lactate dehydrogenase (LDH) release assay. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by Cytation™5 cell imaging microplate detection system. The apoptotic rate was analyzed by flow cytometry. The effects of SEN on mitophagy were detected by transmission electron microscope, immunofluorescence and immunoblotting. RESULTS: Firstly, HT22 cells were treated with 30 µM Aß1-42 for 24 h to establish the damage model. It was found that 30 µM Aß1-42 caused neuronal damages as evidenced by reduced cell viability, increased LDH release and ROS, collapsed MMP and elevated apoptosis. Secondly, Aß1-42-incubated cells were treated with 10, 20, 40 and 60 µM SEN for 24 h. SEN significantly reduced the damage of Aß1-42-incubated cells as shown by recovered cell viability and MMP, reduced apoptosis and ROS. Notably, SEN induced the formation of mitophagosomes and mitolysosomes, and elevated the conversion of LC3 I to LC3 II. Moreover, SEN down-regulated the expression of p62, promoted the accumulation of full-length PINK1 and the translocation of Parkin to mitochondria, decreased the expression of mitochondrial matrix protein HSP60, thus activating the PINK1/Parkin-mediated mitophagy. However, when cells were pretreated with 5 µM CsA (Cyclosporine A, a mitophagy inhibitor) for 2 h and then co-treated with 20 and 40 µM SEN for 24 h, the protective effects of SEN were compromised. CONCLUSIONS: The present study demonstrated that SEN could alleviate Aß1-42-induced cell damage through PINK1/Parkin-mediated mitophagy. Our findings justify the traditional use of P. tenuifolia in China with anti-aging or anti-neurodegenerative effects.