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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2018, Vol. 12 Issue (3) : 307-318    https://doi.org/10.1007/s11684-017-0547-2
RESEARCH ARTICLE |
Neuroprotective effects of Ginkgo biloba extract and Ginkgolide B against oxygen–glucose deprivation/reoxygenation and glucose injury in a new in vitro multicellular network model
Xiaohan Yang1,2,3,4, Tiezheng Zheng1,2,3,4, Hao Hong4, Nan Cai4, Xiaofeng Zhou4, Changkai Sun1,2,3,4(), Liying Wu5, Shuhong Liu5, Yongqi Zhao5, Lingling Zhu5, Ming Fan5(), Xuezhong Zhou6(), Fengxie Jin7
1. Department of Biomedical Engineering, Faculty of Electronic Information and Electrical Engineering, Dalian University of Technology, Dalian 116024, China
2. Research Center for the Control Engineering of Translational Precision Medicine, Dalian University of Technology, Dalian 116024, China
3. State Key Laboratory of Fine Chemicals, Dalian R&D Center for Stem Cell and Tissue Engineering, Dalian University of Technology, Dalian 116024, China
4. Liaoning Provincial Key Laboratory of Cerebral Diseases, Institute for Brain Disorders, Dalian Medical University, Dalian 116044, China
5. Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China
6. School of Computer and Information Technology and Beijing Key Lab of Traffic Data Analysis and Mining, Beijing Jiaotong University, Beijing 100044, China
7. College of Biotechnology, Dalian Polytechnic University, Dalian 116034, China
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Abstract

Acute ischemic stroke (AIS), as the third leading cause of death worldwide, is characterized by its high incidence, mortality rate, high incurred disability rate, and frequent reoccurrence. The neuroprotective effects of Ginkgo biloba extract (GBE) against several cerebral diseases have been reported in previous studies, but the underlying mechanisms of action are still unclear. Using a novel in vitro rat cortical capillary endothelial cell-astrocyte-neuron network model, we investigated the neuroprotective effects of GBE and one of its important constituents, Ginkgolide B (GB), against oxygen–glucose deprivation/reoxygenation and glucose (OGD/R) injury. In this model, rat cortical capillary endothelial cells, astrocytes, and neurons were cocultured so that they could be synchronously observed in the same system. Pretreatment with GBE or GB increased the neuron cell viability, ameliorated cell injury, and inhibited the cell apoptotic rate through Bax and Bcl-2 expression regulation after OGD/R injury. Furthermore, GBE or GB pretreatment enhanced the transendothelial electrical resistance of capillary endothelial monolayers, reduced the endothelial permeability coefficients for sodium fluorescein (Na-F), and increased the expression levels of tight junction proteins, namely, ZO-1 and occludin, in endothelial cells. Results demonstrated the preventive effects of GBE on neuronal cell death and enhancement of the function of brain capillary endothelial monolayers after OGD/R injury in vitro; thus, GBE could be used as an effective neuroprotective agent for AIS/reperfusion, with GB as one of its significant constituents.

Keywords acute ischemic stroke      Ginkgo biloba extract      Ginkgolide B      network model      neuroprotection     
Corresponding Authors: Changkai Sun,Ming Fan,Xuezhong Zhou   
Just Accepted Date: 18 July 2017   Online First Date: 08 November 2017    Issue Date: 04 May 2018
 Cite this article:   
Xiaohan Yang,Tiezheng Zheng,Hao Hong, et al. Neuroprotective effects of Ginkgo biloba extract and Ginkgolide B against oxygen–glucose deprivation/reoxygenation and glucose injury in a new in vitro multicellular network model[J]. Front. Med., 2018, 12(3): 307-318.
 URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0547-2
http://academic.hep.com.cn/fmd/EN/Y2018/V12/I3/307
Fig.1  Chemical structure of GB.
Fig.2  Schematic illustration of the construction of in vitro basal EAN models. The primary SD rat cortical astrocytes, capillary endothelial cells, and neurons were isolated and cultured alone at days 1, 2, and 3, respectively; 5–7 days primary endothelial cells were seeded on the interior side of collagen-coated polyester membrane of the inserts and maintained in E medium II for approximately 4 days. On the 5th day, the insert containing endothelial cells was inverted and the astrocytes suspension was seeded on the exterior side for 3 h. After 3 h, the E–A cocultured insert was maintained in E medium II for approximately 2 days and then replaced in the well containing 10 days primary neurons.
Fig.3  Characterization of cells in the basal EAN model. (A) Immunofluorescence characterization of endothelial cells, astrocytes, and neurons after 2 days of coculturing in the basal EAN model. Bar= 50 mm. (B) TEER values of endothelial cells from the first day when endothelial cells were seeded on the interior side of the Transwell inserts to the sixth day. (C) Astrocyte processes directly reach the surface of endothelial cells through the pore of the insert, which was observed using an electronic microscope.
Fig.4  TEER and Na-F permeability of endothelial cells in different groups after OGD/R injury. (A) Except for the control group, the other groups were subjected to OGD/R injury. The TEER values are presented as mean±SEM (n = 4). **P<0.01 compared with the OGD/R group, ## P<0.01 compared with the GBE group. (B) Except for the control group, the other groups were subjected to OGD/R injury. Pe for Na-F is presented as the mean±SEM (n = 4). **P<0.01 compared with OGD/R group, ## P<0.01 compared with GBE group.
Fig.5  The ZO-1 and occludin protein expression levels of endothelial cells in different groups were analyzed by Western blot analysis. (A) Except for the control group, the other groups were subjected to the OGD/R injury. The ZO-1 and occludin protein expression of neurons in different groups was analyzed by Western blot analysis. (B) ZO-1 protein expression is presented as the mean±SEM (n = 4). **P<0.01 compared with OGD/R group, ## P<0.01 compared with GBE group. (C) Quantification of occludin protein expression is presented as the mean±SEM (n = 4). **P<0.01 compared with the OGD/R group, ## P<0.01 compared with the GBE group.
Fig.6  Immunofluorescence staining for ZO-1 in endothelial cells in different groups. (A) In the control group, ZO-1 was expressed in endothelial cells, (B) in the OGD/R group, ZO-1 was expressed in endothelial cells, (C) in the GBE group, ZO-1 was expressed in endothelial cells, (D) in the GB group, ZO-1 was expressed in endothelial cells. Bar= 50 mm.
Fig.7  The neuron cell viability in different groups was analyzed by MTT assay. Except for the control group, the other groups were subjected to the OGD/R injury. The cell viability is presented as mean±SEM (n = 4). **P<0.01 compared with OGD/R group, # P<0.05 compared with GBE group.
Fig.8  LDH leakage of neurons in different groups was analyzed by LDH assay. Except for the control group, the other groups were subjected to the OGD/R injury. The LDH leakage of neurons is presented as mean±SEM (n = 4). **P<0.01 compared with OGD/R group, # P<0.05 compared with GBE group.
Fig.9  The apoptosis percent of neurons in different groups was analyzed by the TUNEL assay. (A) Except for the control group, the other groups were subjected to OGD/R injury. The TUNEL assay of apoptosis was conducted in different groups. Green represents apoptotic cells, whereas blue is DAPI. (B) The neuronal apoptosis percent is presented as mean±SEM (n = 4). **P<0.01 compared with OGD/R group, ## P<0.01 compared with GBE group.
Fig.10  The Bcl-2 and Bax protein expression of neurons in different groups was analyzed by Western blot analysis. (A) Except for the control group, the other groups were subjected to OGD/R injury and Bcl-2 protein expression of neurons in different groups was analyzed by Western blot analysis. (B) Bcl-2 protein expression is presented as mean±SEM (n = 4). **P<0.01 compared with OGD/R group, ## P<0.01 compared with GBE group. (C) The Bax protein expression in neurons in different groups was analyzed by Western blot analysis. (D) Bax protein expression was quantified and presented as mean±SEM (n = 4). **P<0.01 compared with the OGD/R group, ## P<0.01.
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