Liu Zi-shan¹, Zhang Yan-xue¹, Wu Xiao-lin¹, Gu Ning¹, Xu Xin-ying^{1, 2,}, Yu Zhu-qin^{1, 3,}

Objective: This study aims to delineate whether autophagy plays an critical role in the neuroprotective effects of picroside II in vitro and further explore potential molecular mechanism. Methods: The oxygen glucose deprivation/reoxygenation (OGD/R) cell model were established in SH-SY5Y cells. The cell viability, cells morphologic change and apoptosis were observed by cell counting kit-8 (CCK-8), inverted microscope and flow cytometry respectively. Autophagy-related proteins Beclin 1, microtubule associated protein 1 light chain 3 (LC3), and p62 levels of brain tissues and cells were detected by Western Blot; furthermore, LC3 expression and autophagy flux of cells were further detected by immunofluorescence labeling and GFP-mRFP-LC3 adenovirus transfection. Reactive oxygen species (ROS) levels and phospho-AMPK, phospho-mTOR, phospho-ULK 1 levels were were detected using ROS assay Kit, Western blot and immunocytochemistry, respectively. Results: Picroside II down-regulated of autophagy-related Beclin-1 and LC3 levels, and up-regulated of p62 protein; meanwhile ameliorated the abnormal morphological structures of nerve cells and apoptosis; and increased cell viability. Furthermore, accompanied by decreasing autophagy flux, picroside II prevented the generation of ROS, down-regulated of AMPK and the up-regulated of mTOR and Unc-51-like kinase 1 (ULK1) levels. Conclusion: Picroside II exerts neuroprotective effect against OGD/R injury by inhibiting ROS-mediated AMPK-mTOR-ULK1 autophagy signaling pathway

autophagy, OGD/R, AMPK-mTOR-ULK 1, signaling pathway