Protective Effect Of Protocatechuic Acid On Rotenone-induced Apoptosis In PC12 Cells

Rotenone, as a specific inhibitor of mitochondrial complex I, was used to establish the apoptotic model of Parkinson's disease in vitro in cultured PC12 cells that retain dopaminergic characteristics and have been widely used for neurobiological and neurochemical studies. The results showed that cell viability decreased and the leakage of lactate dehydrogenase (LDH) in PC12 cells increased after the treatment of rotenone for 48 h; by observing the nuclear morphological changes and flow cytometric analysis (FCM), the apoptotic rate was augmented significantly. In addition, expression of Bcl-2 and Bcl-2/Bax ratio declined, while expression of Bax increased. It suggested that rotenone-induced apoptosis was involved in mitochondria-dependent transduction pathway.Combined with our previous fractionation way, protocatechuic acid (PCA) was further isolated from the ethyl acetate extract after the ethanol extract from the fruits of A. oxyphylla was primarily extracted with different solvents according to their different polarities at room temperature. In addition, rotenone-induced apoptosis in PC12 cells was used as a model to investigate the neuroprotective effects of PCA. LDH assay revealed that application of rotenone induced the leakage of LDH and PCA stabilized the cell membrane structure. Pretreatment with PCA for 30 min prior to rotenone significantly suppressed appearance of apoptotic bodies observed by the nuclear morphological changes and declined the number of sub-G1 cells measured using FCM; moreover, PCA markedly reduced the apoptosis/necrosis rate which was precisely quantified using propidium iodide and annexin-V dual staining; the apoptosis in rotenone-induced PC12 cells was associated with loss of mitochondrial membrane potential (MMP), formation of ROS, GSH depletion, suppressions of SOD and CAT, activation of caspase-3 and down-regulation of Bcl-2. In contrast, PC12 cells pretreated with PCA significantly prevented the above-mentioned mitochondria-dependent apoptotic pathway. All these results suggest that PCA may have the capacity to counteract the neurotoxicity of rotenone by attenuating mitochondrial dysfunction leading to apoptosis.