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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 Author(s): 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:  
https://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0547-2
https://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.
1 Goyal M, Demchuk AM, Menon BK, Eesa M, Rempel JL, Thornton J, Roy D, Jovin TG, Willinsky RA, Sapkota BL, Dowlatshahi D, Frei DF, Kamal NR, Montanera WJ, Poppe AY, Ryckborst KJ, Silver FL, Shuaib A, Tampieri D, Williams D, Bang OY, Baxter BW, Burns PA, Choe H, Heo JH, Holmstedt CA, Jankowitz B, Kelly M, Linares G, Mandzia JL, Shankar J, Sohn SI, Swartz RH, Barber PA, Coutts SB, Smith EE, Morrish WF, Weill A, Subramaniam S, Mitha AP, Wong JH, Lowerison MW, Sajobi TT, Hill MD; ESCAPE Trial Investigators. Randomized assessment of rapid endovascular treatment of ischemic stroke. N Engl J Med 2015; 372(11): 1019–1030
https://doi.org/10.1056/NEJMoa1414905 pmid: 25671798
2 Zhao L, Liu X, Liang J, Han S, Wang Y, Yin Y, Luo Y, Li J. Phosphorylation of p38 MAPK mediates hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury via mitochondria translocation of Bcl-xL in mice. Brain Res 2013; 1503: 78–88
https://doi.org/10.1016/j.brainres.2013.01.051 pmid: 23399686
3 Heuschmann PU, Wiedmann S, Wellwood I, Rudd A, Di Carlo A, Bejot Y, Ryglewicz D, Rastenyte D, Wolfe CD; European Registers of Stroke.Three-month stroke outcome: the European Registers of Stroke (EROS) investigators. Neurology 2011; 76(2): 159–165
https://doi.org/10.1212/WNL.0b013e318206ca1e pmid: 21148118
4 Lv P, Fang W, Geng X, Yang Q, Li Y, Sha L. Therapeutic neuroprotective effects of ginkgolide B on cortex and basal ganglia in a rat model of transient focal ischemia. Eur J Pharm Sci 2011; 44(3): 235–240
https://doi.org/10.1016/j.ejps.2011.07.014 pmid: 21855632
5 National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med 1995; 333(24): 1581–1587
https://doi.org/10.1056/NEJM199512143332401 pmid: 7477192
6 Emberson J, Lees KR, Lyden P, Blackwell L, Albers G, Bluhmki E, Brott T, Cohen G, Davis S, Donnan G, Grotta J, Howard G, Kaste M, Koga M, von Kummer R, Lansberg M, Lindley RI, Murray G, Olivot JM, Parsons M, Tilley B, Toni D, Toyoda K, Wahlgren N, Wardlaw J, Whiteley W, del Zoppo GJ, Baigent C, Sandercock P, Hacke W; Stroke Thrombolysis Trialists’ Collaborative Group.Effect of treatment delay, age, and stroke severity on the effects of intravenous thrombolysis with alteplase for acute ischaemic stroke: a meta-analysis of individual patient data from randomised trials. Lancet 2014; 384(9958): 1929–1935
https://doi.org/10.1016/S0140-6736(14)60584-5 pmid: 25106063
7 Sun K, Hu Q, Zhou CM, Xu XS, Wang F, Hu BH, Zhao XY, Chang X, Chen CH, Huang P, An LH, Liu YY, Fan JY, Wang CS, Yang L, Han JY. Cerebralcare Granule, a Chinese herb compound preparation, improves cerebral microcirculatory disorder and hippocampal CA1 neuron injury in gerbils after ischemia-reperfusion. J Ethnopharmacol 2010; 130(2): 398–406
https://doi.org/10.1016/j.jep.2010.05.030 pmid: 20580803
8 del Zoppo GJ, Poeck K, Pessin MS, Wolpert SM, Furlan AJ, Ferbert A, Alberts MJ, Zivin JA, Wechsler L, Busse O, Greenlee R, Brass L, Mohr JP, Feldmann E, Hacke W, Carlos SK, Biller J, Gress D, Otis SM. Recombinant tissue plasminogen activator in acute thrombotic and embolic stroke. Ann Neurol 1992; 32(1): 78–86
https://doi.org/10.1002/ana.410320113 pmid: 1642475
9 Bhatia R, Hill MD, Shobha N, Menon B, Bal S, Kochar P, Watson T, Goyal M, Demchuk AM. Low rates of acute recanalization with intravenous recombinant tissue plasminogen activator in ischemic stroke: real-world experience and a call for action. Stroke 2010; 41(10): 2254–2258
https://doi.org/10.1161/STROKEAHA.110.592535 pmid: 20829513
10 Kalogeris TJ, Kevil CG, Laroux FS, Coe LL, Phifer TJ, Alexander JS. Differential monocyte adhesion and adhesion molecule expression in venous and arterial endothelial cells. Am J Physiol 1999; 276(1 Pt 1): L9–L19
pmid: 9887050
11 Durukan A, Tatlisumak T. Acute ischemic stroke: overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia. Pharmacol Biochem Behav 2007; 87(1): 179–197
https://doi.org/10.1016/j.pbb.2007.04.015 pmid: 17521716
12 DeFeudis FV, Drieu K. Ginkgo biloba extract (EGb 761) and CNS functions: basic studies and clinical applications. Curr Drug Targets 2000; 1(1): 25–58
https://doi.org/10.2174/1389450003349380 pmid: 11475535
13 Müller WE, Heiser J, Leuner K. Effects of the standardized Ginkgo biloba extract EGb 761® on neuroplasticity. Int Psychogeriatr 2012; 24(S1 Suppl 1): S21–S24
https://doi.org/10.1017/S1041610212000592 pmid: 22784424
14 Jahanshahi M, Nikmahzar E, Yadollahi N, Ramazani K. Protective effects of Ginkgo biloba extract (EGB 761) on astrocytes of rat hippocampus after exposure with scopolamine. Anat Cell Biol 2012; 45(2): 92–96
https://doi.org/10.5115/acb.2012.45.2.92 pmid: 22822463
15 Zhang C, Ren C, Chen H, Geng R, Fan H, Zhao H, Guo K, Geng D. The analog of Ginkgo biloba extract 761 is a protective factor of cognitive impairment induced by chronic fluorosis. Biol Trace Elem Res 2013; 153(1-3): 229–236
https://doi.org/10.1007/s12011-013-9645-4 pmid: 23605048
16 Xia SH, Fang DC. Pharmacological action and mechanisms of ginkgolide B. Chin Med J (Engl) 2007; 120(10): 922–928
pmid: 17543184
17 Maclennan KM, Darlington CL, Smith PF. The CNS effects of Ginkgo biloba extracts and ginkgolide B. Prog Neurobiol 2002; 67(3): 235–257
https://doi.org/10.1016/S0301-0082(02)00015-1 pmid: 12169298
18 Erecińska M, Silver IA. Tissue oxygen tension and brain sensitivity to hypoxia. Respir Physiol 2001; 128(3): 263–276
https://doi.org/10.1016/S0034-5687(01)00306-1 pmid: 11718758
19 Martins AH, Hu J, Xu Z, Mu C, Alvarez P, Ford BD, El Sayed K, Eterovic VA, Ferchmin PA, Hao J. Neuroprotective activity of (1S,2E,4R,6R,-7E,11E)-2,7,11-cembratriene-4,6-diol (4R) in vitro and in vivo in rodent models of brain ischemia. Neuroscience 2015; 291: 250–259
https://doi.org/10.1016/j.neuroscience.2015.02.001 pmid: 25677097
20 Huang J, Kodithuwakku ND, He W, Zhou Y, Fan W, Fang W, He G, Wu Q, Chu S, Li Y. The neuroprotective effect of a novel agent N2 on rat cerebral ischemia associated with the activation of PI3K/Akt signaling pathway. Neuropharmacology 2015; 95: 12–21
https://doi.org/10.1016/j.neuropharm.2015.02.022 pmid: 25725335
21 Kis B, Deli MA, Kobayashi H, Abrahám CS, Yanagita T, Kaiya H, Isse T, Nishi R, Gotoh S, Kangawa K, Wada A, Greenwood J, Niwa M, Yamashita H, Ueta Y. Adrenomedullin regulates blood-brain barrier functions in vitro. Neuroreport 2001; 12(18): 4139–4142
https://doi.org/10.1097/00001756-200112210-00055 pmid: 11742253
22 Nakagawa S, Deli MA, Kawaguchi H, Shimizudani T, Shimono T, Kittel A, Tanaka K, Niwa M. A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int 2009; 54(3-4): 253–263
https://doi.org/10.1016/j.neuint.2008.12.002 pmid: 19111869
23 Nakagawa S, Deli MA, Nakao S, Honda M, Hayashi K, Nakaoke R, Kataoka Y, Niwa M. Pericytes from brain microvessels strengthen the barrier integrity in primary cultures of rat brain endothelial cells. Cell Mol Neurobiol 2007; 27(6): 687–694
https://doi.org/10.1007/s10571-007-9195-4 pmid: 17823866
24 Willis CL. Imaging in vivo astrocyte/endothelial cell interactions at the blood-brain barrier. Methods Mol Biol 2012; 814: 515–529
https://doi.org/10.1007/978-1-61779-452-0_34 pmid: 22144329
25 Guo S, Kim WJ, Lok J, Lee SR, Besancon E, Luo BH, Stins MF, Wang X, Dedhar S, Lo EH. Neuroprotection via matrix-trophic coupling between cerebral endothelial cells and neurons. Proc Natl Acad Sci USA 2008; 105(21): 7582–7587
https://doi.org/10.1073/pnas.0801105105 pmid: 18495934
26 Iadecola C. Neurovascular regulation in the normal brain and in Alzheimer’s disease. Nat Rev Neurosci 2004; 5(5): 347–360
https://doi.org/10.1038/nrn1387 pmid: 15100718
27 Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis 2010; 37(1): 13–25
https://doi.org/10.1016/j.nbd.2009.07.030 pmid: 19664713
28 Abbott NJ. Blood-brain barrier structure and function and the challenges for CNS drug delivery. J Inherit Metab Dis 2013; 36(3): 437–449
https://doi.org/10.1007/s10545-013-9608-0 pmid: 23609350
29 Wang CP, Li JL, Zhang LZ, Zhang XC, Yu S, Liang XM, Ding F, Wang ZW. Isoquercetin protects cortical neurons from oxygen-glucose deprivation-reperfusion induced injury via suppression of TLR4-NF-кB signal pathway. Neurochem Int 2013; 63(8): 741–749
https://doi.org/10.1016/j.neuint.2013.09.018 pmid: 24099731
30 Wakita H, Tomimoto H, Akiguchi I, Kimura J. Protective effect of cyclosporin A on white matter changes in the rat brain after chronic cerebral hypoperfusion. Stroke 1995; 26(8): 1415–1422
https://doi.org/10.1161/01.STR.26.8.1415 pmid: 7631347
31 Siao CJ, Tsirka SE. Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II. J Neurosci 2002; 22(9): 3352–3358
pmid: 11978811
32 Rubin LL, Staddon JM. The cell biology of the blood-brain barrier. Annu Rev Neurosci 1999; 22(1): 11–28
https://doi.org/10.1146/annurev.neuro.22.1.11 pmid: 10202530
33 Zehendner CM, Librizzi L, Hedrich J, Bauer NM, Angamo EA, de Curtis M, Luhmann HJ. Moderate hypoxia followed by reoxygenation results in blood-brain barrier breakdown via oxidative stress-dependent tight-junction protein disruption. PLoS One 2013; 8(12): e82823
https://doi.org/10.1371/journal.pone.0082823 pmid: 24324834
34 Zhou T, He Q, Tong Y, Zhan R, Xu F, Fan D, Guo X, Han H, Qin S, Chui D. Phospholipid transfer protein (PLTP) deficiency impaired blood-brain barrier integrity by increasing cerebrovascular oxidative stress. Biochem Biophys Res Commun 2014; 445(2): 352–356
https://doi.org/10.1016/j.bbrc.2014.01.194 pmid: 24513285
35 Kim DH, Lee HE, Kwon KJ, Park SJ, Heo H, Lee Y, Choi JW, Shin CY, Ryu JH. Early immature neuronal death initiates cerebral ischemia-induced neurogenesis in the dentate gyrus. Neuroscience 2015; 284: 42–54
https://doi.org/10.1016/j.neuroscience.2014.09.074 pmid: 25301746
36 Chen Y, Wu X, Yu S, Fauzee NJ, Wu J, Li L, Zhao J, Zhao Y. Neuroprotective capabilities of Tanshinone IIA against cerebral ischemia/reperfusion injury via anti-apoptotic pathway in rats. Biol Pharm Bull 2012; 35(2): 164–170
https://doi.org/10.1248/bpb.35.164 pmid: 22293345
37 Cheng F, Zhong X, Lu Y, Wang X, Song W, Guo S, Wang X, Liu D, Wang Q. Refined Qingkailing protects MCAO mice from endoplasmic reticulum stress-induced apoptosis with a broad time window. Evid Based Complement Alternat Med 2012; 2012: 567872
https://doi.org/10.1155/2012/567872 pmid: 22536287
38 Chen L, Wei X, Hou Y, Liu X, Li S, Sun B, Liu X, Liu H. Tetramethylpyrazine analogue CXC195 protects against cerebral ischemia/reperfusion-induced apoptosis through PI3K/Akt/GSK3b pathway in rats. Neurochem Int 2014; 66: 27–32
https://doi.org/10.1016/j.neuint.2014.01.006 pmid: 24462584
39 Eskes R, Desagher S, Antonsson B, Martinou JC. Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol 2000; 20(3): 929–935
https://doi.org/10.1128/MCB.20.3.929-935.2000 pmid: 10629050
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