A1 Peroxynitrite-mediated mitophagy could be a crucial therapeutic target for reducing
cerebral ischemia–reperfusion injury
Jiangang Shen, Jinghan Feng, Hansen Chen
School of Chinese Medicine, Li Ka Shing Faculty of Medicine, The University of Hong
Kong, 10 Sassoon Road, Hong Kong, SAR, China
Correspondence: Jiangang Shen - shenjg@hku.hk
Journal of Chinese Medicine 2018, 13(Supp 2):A1
Basic autophagy/mitophagy is essential for cell survival whereas excessive autophagy/mitophagy
is detrimental during cerebral ischemia–reperfusion (I/R) injury. Peroxynitrite (ONOO−),
a representative of reactive nitrogen species, is a critical neurotoxic factor in
mediating cerebral I/R injury, but its roles in autophagy/mitophagy remain unclear.
Herein, we hypothesized that ONOO− could induce PINK1/Parkin-mediated mitophagy activation
via triggering dynamin-related protein 1 (Drp1) recruitment to damaged mitochondria,
contributing to cerebral I/R injury. The major discoveries revealed that: (1) PINK1/Parkin-mediated
mitophagy activation was predominant among general autophagy, leading to rat brain
injury at the reperfusion phase after cerebral ischemia; (2) increased nitrotyrosine
was found in the plasma of ischemic stroke patients and ischemia–reperfused rat brains,
indicating the generation of ONOO− in ischemic stroke; (3) ONOO− was dramatically
increased in accompanied with mitochondrial recruitment of Drp1, PINK1/Parkin-mediated
mitophagy activation, and progressive infarct size in rat ischemic brains at the reperfusion
phase; (4) FeTMPyP, a peroxynitrite decomposition catalyst, remarkably reversed mitochondrial
recruitment of Drp1, mitophagy activation and brain injury; (5) ONOO− induced tyrosine
nitration of Drp1 peptide and mitochondrial recruitment of Drp1 for mitophagy activation.
Those results suggest that ONOO−-induced mitophagy activation aggravates cerebral
I/R injury via recruiting Drp1 to damaged mitochondria.
Furthermore, we investigated ONOO−-induced mitophagy as a therapeutic target for attenuating
cerebral I/R injury by using a natural antioxidant naringin as an example. Naringin
possessed strong ONOO− scavenging capability and inhibited the production of superoxide
and nitric oxide in SH-SY5Y cells under 10 h oxygen-glucose-deprivation plus 14 h
of reoxygenation or ONOO− donor 3-morpholinosydnonimine conditions. Naringin also
inhibited NADPH oxidases and iNOS in rat brains with 2 h ischemia plus 22 h reperfusion.
Naringin was able to cross the blood–brain barrier, decreased neurological deficit
score and infarct size, and attenuated apoptotic cell death. Naringin reduced 3-nitrotyrosine
formation, decreased the ratio of LC3-II to LC3-I in mitochondrial fraction, and inhibited
the translocation of Parkin to the mitochondria. Taken together, peroxynitrite-mediated
mitophagy activation could be a potential therapeutic target for cerebral I/R injury.
A2 Dual roles of antioxidant enzymes: mechanism and implication
Xin Gen Lei1, Jian-Hong Zhu2
1Department of Animal Science, Cornell University, Ithaca, New York, USA; 2Department
of Preventive Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
Correspondence: Xin Gen Lei - xl20@cornell.edu
Journal of Chinese Medicine 2018, 13(Supp 2):A2
Background: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are
generated from aerobic metabolism, as a result of accidental electron leakage as well
as regulated enzymatic processes. Because ROS/RNS can induce oxidative injury and
act in redox signaling, enzymes metabolizing them will inherently promote either health
or disease, depending on the physiological context. It is thus misleading to consider
conventionally called antioxidant enzymes to be largely, if not exclusively, health
protective. As such a notion that antioxidant enzymes are beneficial is common, we
herein attempted to rationalize why this simplistic view should be avoided [1].
Materials and methods: Physiological phenotypes triggered in mouse models of overexpression
or knockout of major antioxidant enzymes are reviewed and summarized. Mechanisms by
which these phenotypes are mediated are elaborated with regard to chemical, biological,
and metabolic interactions of the antioxidant enzymes with their substrates, downstream
events, and cellular context.
Results: Paradoxical functions in association with metabolism, health, and diseases
exist in superoxide dismutases, catalase, glutathione peroxidases, thioredoxin reductases,
thioredoxins, glutaredoxins, and peroxiredoxins, along with other selenoproteins and
selenoprotein synthesis-related selenocysteine tRNA. Novel treatments of antioxidant
enzyme-related human diseases are proposed by deliberate targeting of dual roles of
the pertaining enzymes. Paradoxical roles of antioxidant enzymes derive from sophisticated
molecular mechanisms of redox biology and metabolic homeostasis.
Conclusions: Simply viewing antioxidant enzymes as always being beneficial is not
only conceptually misleading but also clinically hazardous if such notions underpin
medical treatment protocols based on modulation of redox pathways.
Reference
Lei XG, Zhu JH, Cheng WH, Bao Y, Ho YS, Reddi AR, Holmgren A, Arner ESJ. Paradoxical
roles of antioxidant enzymes: Basic mechanisms and health implications. Physiol Rev.
2016;96:307–64.
A3 To explore the mechanism of inflammation and degeneration of the central nervous
system: role of brain astrocytes
Hsi-Lung Hsieh1,2
1Department of Nursing, Division of Basic Medical Sciences, Research Center for Chinese
Herbal Medicine, and Graduate Institute of Health Industry Technology, Chang Gung
University of Science and Technology, Tao-Yuan, Taiwan; 2Department of Neurology,
Chang Gung Memorial Hospital, Tao-Yuan, Taiwan
Correspondence: Hsi-Lung Hsieh - hlhsieh@mail.cgust.edu.tw
Journal of Chinese Medicine 2018, 13(Supp 2):A3
Background: Inflammation is a central pathogenic mechanism of various neuropathies
including neurodegenerative diseases. In chronic neurodegenerative diseases such as
Alzheimer’s disease (AD), the pathology is associated with an abnormal inflammatory
response, characterized by the activation of several cell populations in the brain
such as neuroglial cells. The relationships between inflammation and the development
of these neuropathies involve complex molecular networks and processes. One common
feature of various neurodegenerative diseases is activation of large number of astrocytes
and microglia that includes the morphological changes and expression of many inflammatory
mediators. Increasing studies have indicated that the cell–cell interactions between
glial cells and neurons may be important in the regulation of brain inflammation and
neurodegeneration. Thus, these results implicate that activated neuroglial cells,
astrocytes especially, play a critical role in the progression and pathogenesis of
neurodegenerative disorders. Moreover, recent evidence suggests that brain inflammation
may impact on local inflammation in the brain diseases leading to over-production
of several inflammatory mediators, which may in turn influence functions including
apoptosis. Elevated levels of several pro-inflammatory factors including cytokines,
peptides, pathogenic structures, per-oxidants in central nervous system (CNS) have
been detected in the patients with brain disorders like AD. These pro-inflammatory
factors exert as potent stimuli in brain inflammatory responses through up-regulation
of diverse inflammatory mediators.
Results and conclusions: Here, we explored the mechanisms underlying the intracellular
signaling pathways (e.g., protein kinase Cs, reactive oxygen species, or mitogen-activated
protein kinases) involved in the expression of inflammatory mediators induced by pro-inflammatory
factors in brain astrocytes and its effects on neuronal cells. Understanding of the
regulatory mechanisms involved in the relationship of neuroglia and neuronal cells
may provide the helpful therapeutic strategy for brain injury, inflammation, and neurodegenerative
disorders.
Acknowledgements: We thank funding grants from the Ministry of Science and Technology,
Taiwan (NSC102-2320-B-255-005-MY3; MOST106-2320-B-255-005; MOST107-2320-B-255-003)
and Chang Gung Medical Research Foundation (CMRPF1C0193; CMRPF3D0033; CMRPF1F0131;
CMRPF1F0132; CMRPF1H0051).
References
Hsieh HL, et al. Role of redox signaling in neuroinflammation and neurodegenerative
diseases. Biomed Res Int. 2013;2013:484613–30.
Hsieh HL, et al. The role of matrix metalloproteinase-9 in pro-inflammatory factors-induced
brain inflammation and neurodegenerative diseases. Inflamm Cell Signal. 2014;1:88–96.
Hsieh HL, et al. Astrocyte as the modulator in brain inflammation and neurodegenerative
disorders. Ann Neurodegener Dis. 2016;1(2):1008.
A4 Evaluation of the Ganoderma effects in pain sensitivity of aging mice with the
homemade tail flick instrument
Chia-Ying Lin1, Ming-Wei Chao2, Wei-Nong Li1, Chao-Ming Tang1, Chia-Yi Tseng1
1Biomedical Engineering, Chung Yuan Christian University, Zhongli District, Taoyuan
City, 320, Taiwan; 2Bioscience Technology, Chung Yuan Christian University, Zhongli
District, Taoyuan City, 320, Taiwan
Correspondence: Chia-Yi Tseng - cytseng@cycu.edu.tw
*Chia-Ying Lin and Ming-Wei Chao contributed equally to this work
Journal of Chinese Medicine 2018, 13(Supp 2):A4
Background: It is become more important on elderly care because the aging of population.
It also leads to the increase of developing health-care products, such as Monacolin,
fish oil, Lactobacillus and plant sterols etc. Ganoderma tsugae have ingredients including
polysaccharide, triterpenes, adenosine and small-molecule proteinase. Ganoderma tsugae
is attributed with therapeutic properties, such as anticancer, regulate blood sugar,
antioxidation, antibiosis, antivirus, liver-protecting and protect stomach damage.
Normal physiological pain is a type of admonition for human to perceive harmful stimulate
and take enough response time to avoid further damage. There are some research show
that increasing oxidative stress in elder’s brain is one of the reason of decreasing
pain sensitivity. Thus elder have lower physiological pain than youth, it may increase
the possibility of suffer injury. So, we suppose that Ganoderma tsugae can reduce
the oxidative stress in elder’s brain and increase their pain sensitivity.
Materials and methods: We gave d-galactose (100 mg/kg B.W./day) by injection to induce
mice aging. And Ganoderma tsugae (200 μg/kg B.W./day) was gave by oral gavage. We
measured the pain sensitivity of galactose-induced aging mice with formalin test,
hot water test and self-made hot-plate pain test instrument to investigate whether
Ganoderma tsugae increase the pain sensitivity or not. We used 2% formalin by inject
into mice paw to observe mice licking response, expecting to see that the mice given
Ganoderma tsugae will licking for a higher frequency than aging mice. We put 2/3 of
mice tail into the hot water (55 °C), then survey whether the tail retract in 15 s,
we also want to see that the mice given Ganoderma tsugae will retract their tail quicker
than aging mice. We let mice stand on the hot-plate instrument for 30 s then record
the latency time, await that the latency time of the mice given Ganoderma tsugae will
shorter than aging mice.
Conclusions: Overall, our expected result is that the Ganoderma tsugae can increase
the pain sensitivity of aging mice.
Keywords: Aging, Ganoderma Tsugae, Pain, d-Galactose
A5 The protective effect of artemisinin against oxidative stress in neuronal/non neuronal
cells and its underlying mechanisms
Xin Xigan, Fang Jiankang, Peng Tangming, Wenhua Zheng
The Faculty of Heath Sciences, University of Macau, Macau, China
Correspondence: Wenhua Zheng - WenhuaZheng@umac.mo
Journal of Chinese Medicine 2018, 13(Supp 2):A5
Artemisinin, also known as Qinghaosu (Chinese: 青蒿素) is an anti-malarial drug that
possess the most rapid action of all the currently available drugs against Plasmodium
falciparum malaria. In past 3 years, we found that artemisinin promoted the survival
of various neuronal/non cell types from oxidative insults like H2O2/SNP and beta amyloid.
Pretreatment of PC12 cells with artemisinin significantly suppressed SNP/H2O2/Aβ-induced
cell death by decreasing the production of intracellular reactive oxygen species (ROS),
preventing the decline of mitochondrial membrane potential, restoring abnormal changes
in nuclear morphology and reducing LDH release and caspase 3/7 activities in PC12
cells and D407 cells. Artemisinin was able to stimulate the phosphorylation/activation
of different signaling protein such as extracellularly regulated protein kinases (ERK)
kinase, AMPK and CREB while it had no effect on the Akt pathway. In addition, inhibitor
or siRNA of ERK pathway and AMPK attenuated the protective effects of artemisinin
whereas the PI3 K inhibitor LY294002 had no effect. Interestingly, intravitreous injection
of artemisinin, concentration-dependently reversed the light exposed damage of rat
retinal physiological function detected by flash electroretinogram. These results,
taken together, suggested that artemisinin is a potential protectant which is able
to suppress cell death induced by oxidative stresses. Our results offer support for
the potential therapeutic application of artemisinin for prevent and treatment of
neuronal degenerative disorders. Supported by NFSC (31771128), FDCT 021/2015/A1, 016/2016/A1
and MYRG2016-00052FSH and MYRG2018-00134-FHS from University of Macau.
A6 Regulation of oxidative acute lung injury and pulmonary infections by anti-inflammatory
reflex
Sitapara RA1,2, Antoine DJ1,2, Patel VS1,2, Ashby CR Jr.1,2, Mantell LL1,2
1St John’s University College of Pharmacy and Health Sciences, NY, USA; 2Feinstein
Institute for Medical Research, Northwell Health System, NY, USA
Correspondence: Mantell LL - mantell@stjohns.edu, lmantell@northwell.edu
Journal of Chinese Medicine 2018, 13(Supp 2):A6
Oxygen therapy with supraphysiological concentrations of oxygen (hyperoxia) is routinely
administered during mechanical ventilation for the management of severe respiratory
distress, such as acute respiratory distress syndrome. However, prolonged exposure
to hyperoxia results in acute lung injury and compromises the host defense to clear
bacteria. Previously, we showed that exposure to hyperoxia can induce the accumulation
of alarmin nuclear protein high mobility group box-1 (HMGB1) in the airways. The elevated
levels of airway HMGB1 is a critical mediator of both inflammatory lung injury and
compromised host defense against bacterial infections in animal models of cystic fibrosis
and ventilator-associated pneumonia (VAP). We investigated the effect of GTS-21, a
selective α7 nicotinic acetylcholine receptor agonist, on hyperoxia-induced acute
lung injury (ALI) and pulmonary infections using mouse models of ALI and VAP. We show
here that GTS-21 dose dependently attenuated hyperoxia-induced lung injury characterized
by a significant decrease in protein leakage into the airways and pronounced leukocyte
infiltration both in the airways as well as lung interstitium. This protective effect
of GTS-21 is associated with a significant decrease in hyperoxia-induced accumulation
of HMGB1 in the airways. Intriguingly, although inflammation was dampened by the treatment
with GTS-21, bacterial clearance in the airways and the lungs was markedly improved.
Moreover, hyperoxia-compromised macrophage function in phagocytosis was enhanced by
GTS. Our results indicate that GTS-21 is effective in improving bacterial clearance
and reducing acute lung injury by enhancing macrophage function via inhibiting the
release of nuclear HMGB1 through the reduction of hyperacetylation and oxidation.
Therefore, the α7 nicotinic acetylcholine receptor represents a possible pharmacological
target to improve the clinical outcome of patients on ventilators by augmenting host
defense against bacterial infections.
A7 Redox imbalance and oxidative stress in diabetes
Liang-Jun Yan
Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University
of North Texas Health Science Center, Fort Worth, TX, 76107
Correspondence: Liang-Jun Yan - liang-jun.yan@unthsc.edu
Chinese Medicine 2018, 13(Supp 2):A7
Cellular redox imbalance refers to the perturbation of the balance between NAD and
NADH that are involved in metabolism, cell signaling and stress management. While
this NADH/NAD redox imbalance has been implicated in the pathogenesis of diabetes
and its implications, the detailed underlying mechanisms are yet to be elucidated.
We hypothesize that NADH/NAD redox imbalance with excessive NADH generated by persistent
hyperglycemia can overload mitochondrial electron transport chain, in particular complex
I (NADH/ubiquinone oxidoreductase), leading to subsequent induction of elevated mitochondrial
production of reactive oxygen species (ROS) that then results in increased cell death
and accentuation of diabetes and its complication. Using both type 1 and type 2 diabetic
animal models, we have proven the correctness of this hypothesis in several organs
in that complex I is hyperactive in response to NADH overload and both oxidative damage
and cell death are increased. Our study points to the possibility of restoring NADH/NAD
redox balance as potential approaches to fighting diabetes and its complications.
A8 Loganin prevents peripheral nerve injury-induced neuropathic pain via the modulation
of TNF-a/NF-kB signaling pathway
Bin-Nan Wu1, Su-Ling Hsieh2, Yu-Chin Chang1
1Department of Pharmacology, Graduate Institute of Medicine, College of Medicine,
Kaohsiung Medical University, Kaohsiung, 807, Taiwan; 2Department of Pharmacy, Kaohsiung
Medical University Hospital, Kaohsiung, 807, Taiwan
Correspondence: Bin-Nan Wu - binnan@kmu.edu.tw
Journal of Chinese Medicine 2018, 13(Supp 2):A8
Background: Neuropathic pain, largely resulting from primary lesions in the peripheral
nerve or from malfunctions in the central nervous system, has an extremely negative
impact on the quality of life of patients affected by this condition. The chronic
constriction injury (CCI) model of peripheral nerve injury has provided a deeper understanding
of nociception and the events contributing to the pathogenesis of chronic pain conditions.
Loganin is isolated from Corni fructus, a well-known herb with glucose-lowering action
and neuroprotective activity. This study aimed to investigate the molecular mechanisms
of loganin in a rat model of CCI-induced neuropathic pain.
Methods: Sprague–Dawley rats were randomly divided into four groups: sham, sham + loganin,
CCI and CCI + loganin. Loganin (5 mg/kg/day) was injected intraperitoneally starting
at day 1 after surgery. Mechanical and thermal responses were assessed before surgery
and at day 3, 7, 14 after CCI. Proximal and distal sciatic nerves (SNs) were isolated
for western blots, confocal microscopy and enzyme-linked immunosorbent assay to analyze
protein expression, immunoreactivity and proinflammatory cytokines.
Results: Behavior data show that thermal hyperalgesia and mechanical allodynia were
reduced in loganin treated group as compared to CCI group. The neurobehavioral changes
was correlated with the demyelination of Schwann cells, particularly in the distal
stump of injured SN. Inflammatory proteins (p-NF-kB, p-IkB, iNOS) and proinflammatory
cytokines (TNF-a, IL-1b) induced by CCI were attenuated in the loganin treated group
at day 7 after CCI. Loganin also blocked IkB phosphorylation (p-IkB). Double immunofluorescent
staining further demonstrated that p-NF-kB protein was reduced by loganin in peripheral
glial cells at day 7 after CCI.
Conclusion: Based on these findings, we concluded that loganin has antiinflammatory
and antihyperalgesia properties in CCI-induced neuropathic pain via decreases in TNF-a/NF-kB
activation.
Acknowledgements: This study was supported by grant from the Ministry of Science and
Technology (MOST 106-2320-B-037-009-MY3), Taiwan.
A9 Taiwan database of extracts and compounds (TDEC)
Juan-Cheng Yang3, Guan-Yu Chen3, Tsung-Yu4, Chia-Lin Lee Yao2, Yang-Chang Wu1
1Graduate Institute of Natural Products/Research Center for Natural Products and Drug
Development, Kaohsiung Medical University, Kaohsiung, Taiwan; 2Department of Cosmeceutics,
China Medical University, Taichung, Taiwan; 3Chinese Medicine Research and Development
Center, Taichung, Taiwan; Kaohsiung Medical University, Kaohsiung, Taiwan
Correspondence: Chia-Lin Lee Yao - yachwu@kmu.edu.tw; Yang-Chang Wu - chlilee@mail.cmu.edu.tw
Journal of Chinese Medicine 2018, 13(Supp 2):A9
Taiwan database of extracts and compounds (TDEC) is an academic and scientific website
which offers a platform for investigators in different fields to share their own research
information. Furthermore, the most significant aim of TDEC is to preserve the Taiwan’s
important resources, including crude extracts, pure natural isolates, and chemically
synthesized derivatives from Chinese Herbal Medicines, marine organisms, and microbes
etc. To integrate aforementioned substances from the academic research institutions,
industrial units, and botanic conservation centers to make more drugs and products
developments efficiently.
TDEC provides functional services such as drug management, drug search, investigator
matching, and data statistics systems etc. Drug information consisted of compounds’
structures, physical and chemical properties, and biological activities etc. could
be shared for every investigators in the world. Especially for the matching function,
it could offer a point-to-point link between drug providers and investigators to efficiently
help them to cooperate with each other and promote the powerful researches.
TDEC is a new conceptual data sharing platform for drug researches and developments.
So far, this platform is developing under system construction. On the timeline, α
version will be online at 2019. (https://tdec.kmu.edu.tw).
Keywords: Taiwan database of extracts and compounds, Extracts, Compounds
Acknowledgements: TDEC was supported by the grants from Ministry of Science and Technology
(MOST 106-2321-B-037-004-)(MOST 107-2321-B-037-004-), Taiwan awarded to Y.C. Wu; We
are grateful to the Office of Library and Information Services (OLIS), Kaohsiung Medical
University, Taiwan provides technical support.
A10 Novel Cordyceps vinegar functions as free radical scavenger
Lu Liu1, Humin Fu2,3, Yong Hu1, Shenghan Lai4, Chao Wang1, Jun Wang2,3,4
1Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory
of Industrial Microbiology, Hubei Provincial Cooperative Innovation Center of Industrial
Fermentation, Hubei University of Technology, Wuhan, 430068, China; 2Department of
Pharmacology, School of Food and Biological Engineering, Hubei University of Technology,
Wuhan, Hubei, 430068, China; 3National 111 Center for Cellular Regulation and Molecular
Pharmaceutics, Wuhan, Hubei, 430068, China; 4Department of Pathology, Johns Hopkins
University School of Medicine, Baltimore, MD, 21287, USA
Correspondence: Jun Wang - jun_wang@hbut.edu.cn
Journal of Chinese Medicine 2018, 13(Supp 2):A10
Background: Free radical serves as a “double-blade” sword for human immune system
[1]. Imbalance of free radical causes severe diseases including cardiovascular diseases,
cancer, diabetes, and so on [2]. Antioxidants are known to defend cells against damages
caused by free radicals [3]. Preventative medicine has obtained more and more attention,
including functional food that can provide antioxidants [4]. Rice vinegar is one of
the most widely used condiments on earth, especially in the traditional Chinese diet
[5]. However, antioxidants have been rarely reported in rice vinegar [6, 7].
Results: In this study, we developed one new method to ferment vinegar with treatment
of Cordyceps militaris (C. militaris) solid medium. This cultivated medium is usually
discarded, resulting in a large waste of rice resources. In fact, the medium contains
the mycelium of C. militaris and roots of fruiting bodies in addition to rice, which
has extremely high nutritional value. A new process has been applied to brew liquid
vinegar by reusing this waste medium. Compared with traditional rice vinegar, the
concentration of total flavones (0.810 mg RE/mL), total phenolics (0.451 mg GAE/mL)
and adenosine (614.4 μg/mL) in this new Cordyceps vinegar (2.86% acetic acid) was
significantly increased. The antioxidant capacity of Cordyceps vinegar was identified
by monitoring DPPH free radical clearance and FRAP iron ion reduction. On the other
hand, this vinegar was found to contain high concentration of cordycepin (19.64 μg/mL),
which hasn’t been reported in other rice vinegars and might contribute to its health
benefits as well.
Conclusion: Therefore, this Cordyceps vinegar may help remove extra harmful free radicals
in human body, which deserves further investigation. In addition, it may benefit human
health with its rich nutritional ingredients.
References
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in chronic diseases? Curr Med Chem. 2013;20(27):3397–415.
Dreher D, Junod AF. Role of oxygen free radicals in cancer development. Eur J Cancer.
1996;32(1):30–8.
Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods:
impact on human health. Pharmacogn Rev. 2010;4(8):118–26.
Viuda-Martos M, Ruiz-Navajas Y, Fernández-López J, Pérez-Álvarez JA. Spices as functional
foods. Crit Rev Food Sci Nutr. 2011;51(1):13–28.
Liu D, Zhu Y, Beefink R, Ooijkaas L, Rinzema A, Chen J, Tramper J. Chinese vinegar
and its solid-state fermentation process. Food Rev Int. 2004;20(4):407–24.
Chou CH, Liu CW, Yang DJ, Wu YH, Chen YC. Amino acid, mineral, and polyphenolic profiles
of black vinegar, and its lipid lowering and antioxidant effects in vivo. Food Chem.
2015;168:63–9.
Wei K, Cao X, Li X, Wang C, Hou L. Genome shuffling to improve fermentation properties
of acetic acid bacterium by the improvement of ethanol tolerance. Int J Food Sci Technol.
2012;47(10):2184–89.
A11 Aβ1-42 oligomers disrupted bEnd.3 brain endothelial cell junction integrity: a
potential in vitro model to study TCM protective compounds for blood–brain barrier
in Alzheimer’s pathology
Erin Qian Yue, Xinhua Zhou, Linmin Chen, Maggie Pui Man Hoi
State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese
Medical Sciences, University of Macau, Macau, China
Correspondence: Maggie Pui Man Hoi - maghoi@umac.mo
Journal of Chinese Medicine 2018, 13(Supp 2):A11
Background: Blood–brain barrier (BBB) is an important gatekeeper for homeostasis and
exchange of substances in and out of the brain. Brain endothelial cells (BECs) together
with astrocytes and pericytes function as the neurovascular unit (NVU) and is critical
for BBB physiology. The permeability of BBB is regulated by two types of endothelial
cell–cell junctions, tight junctions (TJs) and adherens junctions (AJs). Disruption
of BBB integrity is implicated in many neurodegenerative diseases such as Alzheimer’s
disease (AD). It is suggested that healthy function of the BBB is important for the
clearance of amyloid-beta protein (Aβ). In addition, insults including reactive oxygen
species (ROS) and Aβ might induce damages in BBB resulting in deleterious cycle. In
recent years, soluble Aβ oligomers rather than the insoluble aggregates are suggested
to be the toxic species that cause neuronal cell death. The present study aimed to
establish a cellular model of BBB and to evaluate the effects of Aβ on the endothelial
cell–cell junctions.
Method: We employed immortalized mouse brain endothelial cell (bEnd.3) and mouse astrocyte
(C8-D1A) to construct BBB monolayer and co-culture system in vitro. Soluble Aβ1-42
oligomer was prepared by dissolving in DMEM supplemented with 0.5% FBS and 1% penicillin–streptomycin
and was stored at − 4 °C for 24 h. Cells were incubated with Aβ1-42 oligomers (5–40 μM)
for 24 h. The integrity of the BBB model was evaluated by Trans Endothelial Electrical
Resistance (TEER) and permeability of FITC-conjugated dextran. Proteins of TJs (ZO-1,
Claudin-5), AJs (PECAM-1), and cytoskeleton (F-actin) were assessed by immunofluorescence
staining.
Result and conclusion: Aβ1-42 oligomers exerted significant cytotoxicity on bEnd.3
cells (Fig. 1a) and disrupted BBB functions in both monolayer and co-culture system
(Fig. 1b, c). Aβ1-42 oligomers also disrupted the expression and arrangement of cell
junction proteins and cytoskeleton (Fig. 1d).
Fig. 1
Cell viability of bEnd.3 cell under Aβ1-42 oligomers treatment (a). Permeability and
TEER in bEnd.3 monoculture and co-culture with C8-D1A cells (b, c). Immunofluorescent
staining of tight junction proteins (ZO-1, Claudin-5), adherens junction protein (PECAM-1),
and cytoskeleton F-actin (d). Data represent mean ± SEM of three independent experiments
each performed in triplicate (n = 3). *P < 0.05 vs bEnd.3 group; #P < 0.05 vs co-culture
group. Cells were imaged by IN Cell Analyzer 2000 system (general electric) and analyzed
by ImageJ software
Keywords: Brain microvascular cell (BMEC), blood–brain barrier (BBB), amyloid-beta1-42
(Aβ1-42), tight junctions (TJs), adherens junctions (AJs), drug screening.
Acknowledgements: This work was supported by Research Committee of University of Macau
(MYRG2015-00161-ICMS-QRCM, MYRG2017-00150-ICMS), Macau Science and Technology Development
Fund (FDCT/127/2014/A3), and National Natural Science Foundation of China (NSFC81403139).