Paeoniflorin, a potent natural compound, protects PC12 cells from MPP+ and acidic damage via autophagic pathway

Paeoniflorin (PF) is the principal bioactive component of Radix Paeoniae alba, which is widely used in Traditional Chinese Medicine for the treatment of neurodegenerative disorders such as Parkinson's disease (PD). To evaluate the neuroprotective effects of PF on MPP+- or acid- (pH 5.0) induced injury in cultured PC12 cells and to investigate the activity of autophagy-lysosome pathway (ALP). Amiloride (Ami), a non-selective blocker of acid-sensing ion channels (ASICs), as a positive control drug, since it is neuroprotective in rodent models of PD. The cell viability was analyzed with MTT assay. The cell injury was assessed by lactate dehydrogenase (LDH) assay. Flow cytometry and Western blot analysis were used to study the apoptotic, calcium influx and autophagic mechanisms. Ami (100 μM) and PF (50 μM) both protected PC12 cells against MPP+- or acid-induced injury as assessed by MTT assay, lactate dehydrogenase release, and apoptosis rate. The concentrations of cytosolic free Ca2+ were raised after exposure to MPP+ or acidosis, while Ami and PF both reduced the influx of Ca2+. More importantly, we found that the mechanisms of neuroprotective effects of Ami and PF were closely associated with the upregulation of LC3-II protein, which is specifically associated with autophagic vacuole membranes. Furthermore, application of MPP+ or acid induced the overexpression of LAMP2a, which is directly correlated with the activity of the chaperone-mediated autophagy pathway. However, Ami and PF inhibited the overexpression of LAMP2a. Our data provide the first experimental evidence that PF modulates autophagy in models of neuron injury, as well as providing the first indication of a relationship between ASICs and ALP.