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EGb761 Ameliorates Neuronal Apoptosis and Promotes Angiogenesis in Experimental Intracerebral Hemorrhage via RSK1/GSK3β Pathway

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Abstract

Neuronal apoptosis after intracerebral hemorrhage (ICH) plays an essential role in neurological deterioration. Preclinical studies have shown that EGb761, an extract of Ginkgo biloba, is neuroprotective in some other neurological diseases with apoptosis. This study was conducted to investigate the potential neuroprotective effect of EGb761 on neuronal apoptosis in experimental ICH. A model of ICH was induced in C57BL/6 mice by injecting collagenase. EGb761 was administered for 21 days and neurologic behaviors were assessed at 1, 3, 7, 14, and 21 days after ICH. RNAi-mediated knockdown of p90 ribosomal S6 kinase 1 (RSK1) was used to further investigate the role of RSK1 in EGb761-induced neuroprotective effects. Neuronal death was determined by TUNEL staining. The image datasets of neurovascular networks were acquired via micro-optical sectioning tomography (MOST). The glycogen synthase kinase-3β (GSK3β) activity was assayed using commercial kit. Primary cultured cortical neurons were exposed to ferrous iron and treated with EGb761. Apoptotic neurons were counted by flow cytometry. RSK1, GSK3β, phosphorylated-GSK3β (pGSK3β), Bcl2, Bax, cleaved-caspase3 (CC3), and VEGF were measured by Western blot. The pGSK3β was also detected by immunofluorescence staining. We found that mice in EGb761 group performed better on rotarod test. Reduced TUNEL-positive neurons and richer microvascular networks were observed in mice treated with EGb761. EGb761 attenuates neuronal apoptosis induced by ferrous iron counted by flow cytometry in vitro. Decreased GSK3β activity was observed in EGb761-treated mice compared with mice with ICH. EGb761 increased the expression of pGSK3β (Ser9), RSK1 and the Bcl2/Bax ratio, and VEGF and decreased CC3 expression. In conclusion, EGb761 reduces neuronal apoptosis and promotes angiogenesis in experimental intracerebral hemorrhage via RSK1/GSK3β pathway.

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Acknowledgements

This investigation was supported by Prof. Zhouping Tang’s National Natural Science Foundation of China (81171089, 81471211). We would like to express our gratitude to Dr. Teng Zhang for his assistance in this experiment.

We are very grateful to Wuhan Oebio Biology Co., Ltd. (http://www.oebio.com/) for kindly assisting us with the MOST technique used in the reconstruction of 3D neurovascular networks. Ginkgo biloba (EGb761) was kindly provided by Dr. Willmar Schwabe Pharmaceuticals, Germany.

Authors’ Contributions

Chao Pan performed most of the experiments and drafted the manuscript; Na Liu and Ping Zhang conducted the neuronal culture studies; Qian Wu performed the flow cytometry; Hong Deng established the animal models; Feng Xu bred the mice and conducted the behavioral tests; Lifei Lian and Qiming Liang performed the Western blot; Yang Hu conducted the statistical analysis; and Prof. Zhouping Tang and Suiqiang Zhu designed and guided the experiments.

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Authors and Affiliations

  1. Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China

    Chao Pan, Na Liu, Ping Zhang, Qian Wu, Hong Deng, Feng Xu, Lifei Lian, Qiming Liang, Yang Hu, Suiqiang Zhu & Zhouping Tang

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  1. Chao Pan
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Correspondence to Suiqiang Zhu or Zhouping Tang.

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Glossary

AD

Alzheimer’s disease

CC3

Cleaved-caspase3

GSK3β

Glycogen synthase kinase-3β

ICH

Intracerebral hemorrhage

mNSS

Modified neurological severity score

MOST

Micro-optical sectioning tomography

PD

Parkinson’s disease

pGSK3β

Phosphorylated-GSK3β

PI

Propidium iodide

RNAi

RNA interference

RSK1

p90 ribosomal S6 kinase 1

SAH

Subarachnoid hemorrhage

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Pan, C., Liu, N., Zhang, P. et al. EGb761 Ameliorates Neuronal Apoptosis and Promotes Angiogenesis in Experimental Intracerebral Hemorrhage via RSK1/GSK3β Pathway. Mol Neurobiol 55, 1556–1567 (2018). https://doi.org/10.1007/s12035-016-0363-8

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  • DOI: https://doi.org/10.1007/s12035-016-0363-8

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