Germacrone protects against oxygen-glucose deprivation/reperfusion injury by inhibiting autophagy processes in PC12 cells

Jianxing Zhang,Jingjing Li,Sujie Wang,Guojuan Chen,Huiqin Bi,Jiang Liu,Lili Chen,Li Yuan

doi:10.1186/s12906-020-2865-1

Abstract

BackgroundGermacrone is an anti-inflammatory ingredient in the Chinese medicine zedoary turmeric. The purpose of this study was to explore the protective mechanism of germacrone against PC12 cells injury caused by oxygen-glucose deprivation/reperfusion (OGD/R).MethodsOGD/R injury model of PC12 cells was established by using OGD/R (2 h/24 h). The cell viability was assessed by MTT assay and LDH release. The ultrastructure of cells was observed by transmission electron microscopy (TEM). The expression of autophagy related proteins in cells was determined by Western Blot.ResultsThe results of ultrastructural observation showed that PC12 cells damaged by OGD/R showed typical autophagy characteristics. In addition, OGD/R observably up-regulated the expression of autophagy related proteins: the class III type phosphoinositide 3-kinase (PI3K III), light chain 3(LC3), and Beclin-1 in PC12 cells, and inhibited the expression of the class I type phosphoinositide 3-kinase (PI3K I), Protein kinase B (Akt), the mammalian target of rapamycin (mTOR), and B-cell lymphoma 2(Bcl-2) proteins. Furthermore, germacrone increased the cell viability of OGD/R-damaged PC12 cells by down-regulating the expression of LC3 protein in cells in a concentration-dependent manner. More importantly, germacrone significantly inhibited the expression of PI3K III, LC3, and Beclin-1 in OGD/R-injured PC12 cells, and up-regulated the expressionof PI3K I, Akt, mTOR, and Bcl-2 proteins in cells, and this inhibited or up-regulated effect was reversed by PI3K I inhibitor (ZSTK474).ConclusionThe above results indicated that germacrone could inhibit the autophagy effect in OGD/R injury model of PC12 cells, the mechanism of inhibition was regulated by PI3K III/Beclin-1/Bcl-2 and PI3K I/Akt/mTOR pathways, thereby improving the cell viability of PC12 cells and playing a neuroprotective role, which provided a new drug for the treatment of OGD/R.

Highlights

Germacrone is an anti-inflammatory ingredient in the Chinese medicine zedoary turmeric
The results found that the internal structure of cells in the control group was normal, while cells in the oxygen-glucose deprivation/reperfusion (OGD/R) 0-h group began to show vesicles with auto-phagosome characteristics, indicating that OGD/R had caused the activation of autophagy effect in PC12 cells (Fig. 2C)
The number of vesicles was the highest in the OGD/R 24 h group, and the typical characteristics of apoptosis and necrosis were observed in cells (Fig. 2C), suggesting that the autophagy effect of PC12 cells induced by OGD/R 24 h was the most significant

Summary

Introduction

Germacrone is an anti-inflammatory ingredient in the Chinese medicine zedoary turmeric. According to the regulation of autophagy related genes, autophagy involves the encapsulation of damaged organelles and proteins and degradation of its contents by lysosomes, so as to form auto-phagosome with doublelayer membrane structure and complete the turnover and metabolism of intracellular substances [1,2,3]. Studies have found that PI3K I/Akt/ mTOR and PI3K III/Beclin-1/Bcl-2 are important signaling pathways involved in cell autophagy [10, 11]. In PI3K III/Beclin-1/Bcl-2 signaling pathway, Beclin-1 is a specific autophagy gene, and Beclin-1 protein can form a complex with PI3K III to induce autophagy related proteins to locate in the autophagosome membrane, promoting the formation of auto-phagosome [14]

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