[Frontiers in Bioscience, Landmark, 25, 798-816, Jan 1, 2020]

Amentoflavone induces cell cycle arrest, apoptosis, and autophagy in BV-2 cells

Zheng Liu1, 2, Feng Wang1, 2, Hui Ma1, Hechun Xia1, Jihui Tian1, Tao Sun1, 2

1Department of Neurosurgery, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia, China, 2Ningxia Key Laboratory of Cerebrocranial Diseases, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, Ningxia, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
    3.1. Chemicals and reagents
    3.2. Cell line and cell culture
    3.3. Proteomic, pathway and network analysis
    3.4. MTT assay
    3.5. Cell cycle distribution analysis
    3.6. Quantification of cellular apoptosis
    3.7. Quantification of cellular autophagy
    3.8. Confocal fluorescence microscopy
    3.9. Western blot analysis
    3.10. Statistical analysis
4. Results
    4.1. AF reduces viability of BV-2 cells
    4.2. Proteomic response to AF treatment in BV-2 cells
    4.3. AF induces apoptosis in BV-2 cells
    4.4. AF induces cell cycle arrest at G2/M phase in BV-2 cells
    4.5. AF induces autophagy in BV-2 cells
5. Discussion
6. Acknowledgments
7. References

1. ABSTRACT

Previous studies have shown that amentoflavone (AF) elicits anti-inflammatory and neuroprotective effects. To further investigate the effects of AF on the microglia cell line BV-2, proteomic analysis was performed to screen potential key regulators. The top 5 canonical pathways associated with AF treatment were EIF2 signaling, regulation of eIF4 and p70s6k signaling, mTOR signaling, protein ubiquitination pathway and phagosome maturation. The top up-regulated genes were DOCK2, SEC23A, ME1, UGGT1 and STOM, while the most down-regulated molecules were IGF2R, ATP5O, DDX47, WBP11 and IKBIP. AF significantly decreased BV-2 cell proliferation. It induced cell cycle arrest at G2/M, increased CDK2, p27Kip1 and p53/p-p53, and decreased CDK1/CDC2 and cyclin B1. Cell apoptosis was induced, with increased levels of BAX, c-caspase-3 and c-caspase-9, and decreased levels of BCL-XL. Increased level of autophagosome induced by AF was observed, and increased Beclin-1 and decreased phosphorylation of PI3K and Erk1 were found as well. In conclusion, AF induces cell cycle arrest at G2/M, promotes apoptosis and autophagy in BV-2 cells, which may account for the anti-inflammatory effect of AF in epilepsy.

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Abbreviations: CNS: central nervous system; AEDs: antiepileptic drugs; AF: Amentoflavone; DMSO: Dimethyl sulfoxide; FBS: fetal bovine serum; RNase A: ribonuclease; PI: propidium iodide; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-ditetrazoliumbromide; PBS: phosphate-buffered saline; EDTA: ethylene diaminetetraacetic acid; BCA: bicinchoninic acid; RIPA: radioimmunoprecipitation assay; DMEM: Dulbecco's modified Eagle's medium; SDS: sodium dodecyl sulfate; 7-AAD: 7-aminoactinomycin D; PVDF: polyvinylidene difluoride; SD: standard deviation; ANOVA: analysis of variance; CDKs: cyclin-dependent kinases

Key Words: Epilepsy, Amentoflavone, BV-2 cells, Apoptosis, Cell cycle arrest, Autophagy

Send correspondence to: Tao Sun, Department of Neurosurgery, General Hospital of Ningxia Medical University, No. 804 South Shengli Street, Yinchuan 750004, Ningxia, China, Tel: 86-951-6980002, Fax: 86-951-6980002, E-mail: ssldeng@sina.com