Mu-Xiang-You-Fang protects PC12 cells against OGD/R-induced autophagy via the AMPK/mTOR signaling pathway

Ethnopharmacological relevance: Mu-Xiang-You-Fang (MXYF) is a classic prescription of Hui medicine. It is composed of five herbs and has been used to treat ischemic stroke for many years. However, the potential pharmacological mechanisms of MXYF remain unclear. The present research is aimed to investigate the protective effect and possible mechanisms of MXYF treatment in an in vitro model of cerebral ischemia-reperfusion injury.

Materials and methods: An oxygen-glucose deprivation and reperfusion (OGD/R) model of PC12 cells was established. The effect of MXYF on the cell viability after OGD/R injury was determined using a cell counting kit (CCK-8) assay. The colorimetric method was used to determine the Lactate Dehydrogenase (LDH) leakage rate. The calcium concentration was determined by the chemical fluorescence method, and mitochondrial membrane potential was determined using flow cytometry. Monodansylcadaverine (MDC) staining and electron microscopic analysis were then conducted to detect Autophagy after oxygen-glucose deprivation and reperfusion in PC12 cells. Immunofluorescence and western blot analyses were used to detect the expression of proteins associated with Autophagy.

Results: It was found that MXYF (1, 2, 4 μg/mL) could significantly increase cell viability and mitochondrial membrane potential and decrease the calcium concentration and LDH release rate in PC12 cells. After OGD/R injury in PC12 cells, the number of autophagosomes and autophagolysosome significantly increased. MXYF (4 μg/mL) inhibited the Autophagy induced by OGD/R and inhibited the expression of LC3, Beclin1, p-AMPK, and ULK1. In contrast, the expression of p-mTOR, p-p70s6k, and p62 was significantly enhanced.

Conclusions: These findings suggest that MXYF inhibits Autophagy after OGD/R-induced PC12 cell injury through the AMPK-mTOR pathway. Thus, MXYF might have therapeutic potential in treating ischemic stroke.