Available via license: CC BY-NC-ND 4.0
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Protective effect of Cordyceps sinensis
extract on rat brain microvascular
endothelial cells injured by oxygeneglucose
deprivation
Xue Bai
a,1
, Yibo Tang
b,1
, Yan Lin
b
, Yuqing Zhao
b
,
Tianyang Tan
b
, Shuyan Wang
b
, Meiqi Liu
a
, Zhenghui Chang
a
,
Ying Liu
b
, Zhenquan Liu
b,
*
a
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China
b
School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
Received 20 July 2017; received in revised form 13 November 2017; accepted 14 December 2017
Available online 2 January 2018
KEYWORDS
Cordyceps sinensis
extract;
Brain microvascular
endothelial cells;
Oxygeneglucose
deprivation;
Anti-oxidation;
Anti-inflammation
Abstract Objective: To investigate the protective effect of Cordyceps sinensis extract (CSE)
on injury of primary cultured rat brain microvascular endothelial cells (rBMECs) induced by ox-
ygeneglucose deprivation (OGD).
Methods: We isolated and cultured primary rBMECs in order to establish an in vitro OGD model.
Cellular activity was detected using a cell counting kit to determine the appropriate dosage. The
rBMECs were divided into control, model, low-, mid-, and high-dose (5, 10, 20 mg$mL
1
) CSE
groups under OGD for 6 hours. CSE was dissolved in cell culture medium to the appropriate con-
centration, passed through a 0.22 mm sterile filter, and administered for 12 hours before and dur-
ing OGD. Cellular morphology was observed under a microscope. Lactate dehydrogenase level in
cultural supernatant, superoxide dismutase activity, and the content of nitric oxide and malon-
dialdehyde in cells were tested by colorimetric methods. Levels of tumor necrosis factor-aand
interleukin-1 beta in cells were determined by enzyme-linked immunosorbent assay.
Results: After 12-hour administration of CSE at the concentration of 5, 10, 20 mg$mL
1
before
and during OGD, compared with the model group, the CSE groups obviously alleviated the dam-
age of rBMECs induced by OGD, inhibited the apoptosis and the necrosis of the cells, and
improved cellular morphology of rBMECs. Additionally, compared with the model group, CSE also
restrained lactate dehydrogenase leakage in hypoxic cells (P<.01), significantly increased su-
peroxide dismutase activity (P<.05), and reduced the levels of nitric oxide, malondialdehyde,
tumor necrosis factor-a, and interleukin-1 beta (P<.05).
* Corresponding author.
E-mail address: lzqbzy@sina.com (Z. Liu).
Peer review under responsibility of Beijing University of Chinese Medicine.
1
These authors equally contributed to this article.
https://doi.org/10.1016/j.jtcms.2017.12.002
2095-7548/ª2018 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: http://www.elsevier.com/locate/jtcms
Journal of Traditional Chinese Medical Sciences (2018) 5,64e71
Conclusion: C. sinensis extract plays a significant role in protecting injured primary cultured
rBMECs induced by OGD. The mechanism may be related with the increase of cellular anti-
oxidative capacity and anti-inflammatory effect.
ª2018 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Introduction
Cerebral ischemia is a devastating disease of the brain and
is one of the major causes of death and disability.
1
Insuf-
ficient blood supply to the brain causes cerebral ischemia
and cell death in certain areas of the brain, such as the
striatum, neocortex, and hippocampus, leading to demen-
tia.
2,3
Endothelial cells, basement membrane, astrocyte
end-feet, and pericytes constitute the bloodebrain barrier
(BBB).
4e7
Under physiological conditions, the BBB prevents
entry of toxic components into the cerebral cortex and
maintains a healthy balance of the brain, which is a
specialized barrier.
4
Cerebral ischemia results in disruption
of the BBB, and then peripheral immune cytotoxic mole-
cules penetrate into the cerebral cortex, which lead to
ischemic neuronal death.
8,9
Besides cerebral ischemia,
multiple sclerosis and Alzheimer’s disease may both destroy
the BBB.
10
Brain microvascular endothelial cell, as an essential
component of the BBB, plays an important role in main-
taining cerebral dynamic balance and decreasing perme-
ability of the brain.
11
A variety of factors can damage brain
microvascular endothelial cells, such as hypoxia and
glucose deficiency, which lead to inflammation and oxida-
tive stress.
12,13
Once brain microvascular endothelial cells
destructed, it will increase the permeability of cell mem-
brane by damaging its structure and function,
14
and further
destroys the BBB. Dysfunction of the BBB can result in
irreversible cerebral and neurological damage.
15,16
Many
researchers endeavor to search for neuroprotectants asso-
ciated with the mechanisms including inflammation,
oxidative stress, apoptosis, and BBB destruction.
17
In
experimental models of cerebral ischemia, many neuro-
protective agents have been used, but the effect is poor.
18
Therefore, to develop the effective drugs for the preven-
tion and treatment of cerebral ischemia is necessary.
Cordyceps (Cordyceps sinensis (B
ERK
.) S
ACC
., abbr. CS),
fungal parasitic larva moth, is an important traditional Chi-
nese herbal medicine, which has been used for over one
thousand years. Since the 17th century, Chinese doctors
have used CS for invigorating the lungs and kidneys,
19,20
which is indicated for chronic cough, cough caused by
consumptive disease, hemoptysis, and impotence and sem-
inal emission, etc.
21
C. sinensis is mainly distributed in
Sichuan and Qinghai Provinces in China, Kathmandu Valley
and a few high-altitude locations of Nepal, etc.
22
Recent
research has found that CS contains amino acids, cyclic
peptides, nucleosides, sterols, organic acids, poly-
saccharides, and a variety of inorganic elements.
23e25
C.
sinensis has been paid great attention because it has the
properties of decreasing blood glucose, regulating immunity,
and resisting oxidation, bacterial and fungal infection, and
tumor.
26
In our previous study, C. sinensis extract (CSE) showed a
protective effect on cerebral ischemia induced by middle
cerebral artery occlusion in rats.
27
We found that CSE
reduced the behavioral scores in rats, improved neurolog-
ical symptoms of the model rats, and significantly increased
levels of glutathione reductase, glutathione S transferase,
glutathione peroxidase, Na
þ
-K
þ
-ATPase, and catalase.
Another study indicated that CSE significantly decreased
the synthesis and release of inducible nitric oxide synthase,
cyclooxygenase-2, intercellular cell adhesion molecule-1,
tumor necrosis factor-a(TNF-a), and interleukin-1b(IL-
1b), and inhibited the expression of nuclear factor kappa-
light-chain-enhancer of activated B cells.
28
Ethanol
extract of CS has a neuroprotective effect in the focal ce-
rebral ischemia/reperfusion (IR) model in rats. Ethanol
extracts of CS significantly suppressed IR-induced brain
production of inducible nitric oxide synthase, intercellular
cell adhesion molecule-1, cyclooxygenase-2, TNF-a, and IL-
1b, and inhibited infiltration of polymorphonuclear cells.
29
Additionally, ethanol extracts of CS significantly reduced
the infarct size of the middle cerebral artery occlusion.
Cordymin, one of the active components of CS, has a neu-
roprotective effect in the focal cerebral IR model. Cordy-
min strengthens the defense of cerebral ischemia, which is
related to an increase of antioxidant activity. Antioxidant
homeostasis after cerebral IR may be helpful for recovery
of cerebral ischemia injury. Besides, cordymin inhibits
polymorphonuclear cell infiltration and IR-induced C3 pro-
tein, TNF-a, and IL-1blevels, and markedly improves neu-
robehavioral function after cerebral IR in rats.
30
Although the protective effects of CS on cerebral
ischemia have been well reported from the perspective of
animal model, cellular and molecular biological experi-
ments are seldom mentioned. Therefore, in this study, we
try to examine the protective effect of CSE on rat brain
microvascular endothelial cells (rBMECs) by establishing an
ischemic injury model of rBMECs induced by oxygen-glucose
deprivation (OGD) at the cellular and molecular levels.
Materials and methods
Ethical approval
The experiments were performed according to the guid-
ance of the National Institutes of Health for the care and
use of laboratory animals, and approved by the Ethics
Committee of Beijing University of Chinese Medicine
(BUCM-4-2016082601-3005).
Protective effect of Cordyceps sinensis extract on rat brain 65
Primary culture of rat brain microvascular
endothelial cells
SpragueeDawley rats (SPF/VAF) were supplied from Beijing
Vital River Laboratory Animal Technology (Beijing, China).
Eighteen male SD rats weighed 20 g were sacrificed on ice.
The rats were then soaked in 75% cold ethanol for
3e5 minutes. Under sterile conditions, the skull was cut
along the rat brain midline, the dura was removed, and the
hemispheres were then rinsed in cold D-Hank’s solution
(NaCl 8 g, KCl 0.4 g, KH
2
PO
4
0.06 g, NaHCO
3
0.35 g, Na
2
H-
PO
4
$12H
2
O 0.134 g) twice. The leptomeningeal, vessels and
cerebral medulla were then carefully removed. The cere-
bral cortex was collected and cut into pieces at the size of
about 1 mm
3
which were then homogenized in endothelial
cell flushing fluid containing fetal bovine serum. The liquid
was then filtered via a 200 mm and a 76 mm strainer suc-
cessively. Capillary sections from the 76 mm strainer were
collected and centrifuged for 5 minutes at 4C (201 g).
The precipitate was added to 0.2% type II collagenase so-
lution for digestion 20 minutes at 37C, and then centri-
fuged for 5 minutes at 4C (201 g). The digested
production was rinsed with endothelial cell flushing solution
and centrifuged twice. After that, the production was
suspended in endothelial cell culture medium, inoculated
into gelatin-covered 25-cm
2
culture flasks, and then
cultured in a 37C, 5% CO
2
incubator. After 7e8 days, the
cells were grown into a dense monolayer and the third
generation was used in the experiment.
31
Preparation of the oxygen-glucose deprivation
model
Third generation rBMECs were in secondary culture for
24 hours. The cells were incubated with sugar-free Kreb’s
solution (NaCl 6.954 g, KCl 0.3504 g, KH
2
PO
4
0.1633 g,
NaHCO
3
2.l g, CaCl
2
0.277 g, MgCl
2
0.203 g) and were then
placed in an anoxic incubator (37C, continuous access of
95% N
2
and 5% CO
2
) for 6 hours.
32e34
After a successful
modeling, the cells were used for the following experi-
ments according to the test indicators.
Preparation of Cordyceps sinensis extract
To prepare the extract, 10 g CS powder purchased from
Beijing Tongrentang Pharmacy (Beijing, China) was added
to 100 mL ultra-pure water and ultrasonically extracted at
40C for 90 minutes. The extract was then filtered and the
supernatant was collected and lyophilized. Before use, the
lyophilized powder was dissolved in cell culture medium to
the appropriate concentration, passed through 0.22 mm
sterile filter, and stored at 4C.
27,28,35,36
Grouping and administration
The third generation rBMECs were adjusted to a cell density
of 1 10
5
mL
1
and seeded into 96-well plates. Cells were
cultured in an incubator to 70%e80% confluence after
24 hours. The cells were divided into nine groups: normal
control group (NCG) without intervention, model group
without medication, and CSE groups (5, 10, 20, 50, 100,
200, and 400 mg$mL
1
), with six wells for each group.
Except for the normal group, the other eight groups
involved the OGD model as described above. The last seven
groups were administered CSE for 12 hours before and
during OGD. After OGD, the survival rate of the cells was
calculated by absorbance using a cell counting kit-8 (CCK-8;
Dojindo, Kumamoto, Japan) as follows:
Cell survival rate (%) Z(TG absorbance - BH absor-
bance)/(NCG absorbance eBH absorbance).
Note: TG: test group; BH: blank hole. Blank hole: no cell
in the hole, only add CCK-8 test solution.
According to the CCK-8 results, the optimum concen-
trations of CSE were 5, 10, and 20 mg$mL
1
and then used in
the following steps.
Determination of lactate dehydrogenase level
As mentioned above, after 24-hour incubation, the cells
were divided into five groups: NCG, model group, and CSE
groups (5, 10, and 20 mg$mL
1
), with six wells in each group.
Except for NCG, the other four groups involved the OGD
model described above. After OGD, the culture medium was
removed and the cells were washed with phosphate buff-
ered solution (PBS). The absorbance was measured by
490 nm in accordance with the instructions of the lactate
dehydrogenase (LDH) kit from Beyotime Institute of
Biotechnology (Shanghai, China). By correction, absorbance
in the blank hole was subtracted in these tested groups.
LDH level (%) Z(TG absorbance NCG absorbance)/
(MEAC absorbance NCG absorbance).
Note: MEAC: maximum enzyme activity of cells.
Determination of superoxide dismutase,
malondialdehyde, and nitric oxide levels
For this part, the third generation rBMECs were adjusted to
a cell density of 1 10
5
mL
1
and seeded into 6-well plates.
Cells were cultured in the incubator to 70%e80% confluence
after 24 hours, and then divided into NCG, model group,
and CSE groups (5, 10, and 20 mg$mL
1
), with six wells in
each group. Except for NCG, the other four groups involved
the OGD model described above. After OGD, the culture
medium was removed and the cells were digested with
0.125% trypsin after washing twice with PBS and being
transferred to a centrifuge tube. The cells were ultrasoni-
cally crushed, centrifuged for 5 minutes at 4C (1600 g),
and the supernatant was collected by 1.5 mL precooled
microcentrifuge tube. The levels of superoxide dismutase
(SOD), malondialdehyde (MDA), and nitric oxide (NO) were
determined in accordance with the instructions of the
corresponding kit from Beyotime Institute of Biotechnology.
Determination of tumor necrosis factor-aand
interleukin-1blevels
Grouping and modeling were performed as described
above. The medium was removed and the cells were
washed twice with PBS. Cell lysis buffer was added to blow
and split the cells, and the lysate was collected. The levels
of TNF-aand IL-1bwere determined according to the in-
structions of the enzyme-linked immunosorbent assay kit
from Beyotime Institute of Biotechnology.
66 X. Bai et al.
Statistical analysis
SPSS 22.0 (IBM, Armonk, NY) and Graphpad Prism 6.0
(GraphPad Software, CA) were used for statistical analysis
and graphing. The measured data are expressed as the
mean (SD). If data were normally distributed with variance
homogeneity, single factor analysis of variance between
groups was used. P<.05 was considered to be statistically
significant.
Results
Morphological observation
In the NCG group, rBMECs were spindle-shaped or polyg-
onal, with a clear outline, full shape, and good uniform
growth. The volume and count of cells in the model group
were both reduced, also presented with rupture, apoptosis,
and necrosis. Compared with the model group, the CSE
groups increased the number of cells, shortened the space
between cells, enlarged the volume of cells, and alleviated
the damage of cells. Additionally, the damage of cell
morphology induced by OGD was effectively alleviated in a
dose-concentration relation (Fig. 1).
Cell survival rate
The cell survival rate was significantly decreased in the
model group and CSE groups compared with the normal
control group (P<.01, Table 1). This finding indicated that
OGD caused cell damage and decreased the activity of
rBMECs. Compared with the model group, CSE at concen-
trations of 5e400 mg$mL
1
significantly improved the
Figure 1 Morphological changes of rBMECs among five groups.
Note: Images were taken at original magnification 100. (A) rBMECs were provided endothelial cell culture medium without OGD.
(B) rBMECs were not supplied with CSE for 12 hours before and during OGD. (C) rBMECs were administered 5 mg$mL
1
CSE for
12 hours before and during OGD. (D) rBMECs were administered 10 mg$mL
1
CSE for 12 hours before and during OGD. (E) rBMECs
were administered 20 mg$mL
1
CSE for 12 hours before and during OGD. rBMECs: rat brain microvascular endothelial cells;
CSE: Cordyceps sinensis extract; OGD: oxygeneglucose deprivation.
Protective effect of Cordyceps sinensis extract on rat brain 67
survival rate against cell damage induced by OGD
(P<.01). The 200 mg$mL
1
and 400 mg$mL
1
CSE groups had
lower cell survival rates compared with the 5e100 mg$mL
1
CSE groups. The cell survival rates in the 20e100 mg$mL
1
CSE groups did not significantly increase, which indicated
that no definite dose-concentration relation was existent in
this range. Additionally, the ED
50
of the survival rate after
medication was 9.804. Therefore, low, medium, and high
concentrations of CSE were determined as 5, 10, and
20 mg$mL
1
.
Lactate dehydrogenase leakage
In the model group, LDH leakage was rather more obvious
compared with that in the normal group (P<.01, Fig. 2). This
finding indicated that the cell membrane was greatly
damaged by OGD with an increase of LDH leakage. Based on
our observation, CSE at 5, 10, and 20 mg$mL
1
could signifi-
cantly inhibit the LDH leakage in cell supernatant (P<.01).
In these three CSE groups, we found inhibition of LDH leakage
was displayed a dose-concentration relationship.
Levels of superoxide dismutase, malondialdehyde,
and nitric oxide
The activity of SOD was significantly lower (P<.01), and
the levels of MDA and NO (P<.01) were significantly higher
in the model group compared with the normal group.
Groups of CSE at 5, 10, and 20 mg$mL
1
resulted in higher
intracellular SOD activity, and lower MDA and NO levels
compared with the model group, which were displayed
statistical differences (P<.05 or P<.01; Table 2).
Levels of tumor necrosis factor-aand interleukin-
1b
The levels of TNF-aand IL-1bwere significantly lower in the
model group compared with the normal group (P<.01).
Table 1 Effect of CSE on the cell survival rate of rBMECs injured by OGD (n Z6)
a
.
Index NC Model CSE (mg$mL
L1
)
5 10 20 50 100 200 400
Absorbance 1.13 (0.02)** 0.39 (0.02) 0.58 (0.04)** 0.67 (0.09)** 0.73 (0.04)** 0.76 (0.05)** 0.77 (0.03)** 0.70 (0.05)** 0.65 (0.03)**
Cell survival rate (%) 100.00 (1.34)** 26.38 (2.15) 44.69 (4.18)** 53.86 (8.94)** 59.82 (4.36)** 63.11 (4.59)** 63.23 (3.02)** 57.48 (5.24)** 52.05 (3.44)**
Note: CSE: Cordyceps sinensis extract; rBMECs: rat brain microvascular endothelial cells; OGD: oxygeneglucose deprivation. NC: normal control (without oxygeneglucose deprivation);
Model: without medication.
Compared with model group, **P<.01.
a
Values were represented as mean (SD).
Figure 2 CSE inhibits LDH leakage in rBMECs injured by OGD.
Note: CSE: Cordyceps sinensis extract; LDH: Lactate dehydro-
genase; rBMECs: rat brain microvascular endothelial cells;
OGD: oxygeneglucose deprivation. Compared with model
group,
**
P<.01.
68 X. Bai et al.
Groups of CSE at 5, 10, and 20 mg$mL
1
resulted in lower
intracellular TNF-aand IL-1blevels compared with the
model group (P<.05 or P<.01) except of IL-1blevel at
5mg$mL
1
CSE (Table 3).
Discussion
Cerebral ischemia is one of the major causes of death and
disability, and thus effective therapeutic agents are urgent
in current research. C. sinensis is a rare and valued Chinese
medicine, also known as Chinese caterpillar fungi, and is
used for treating human diseases and health care.
37
Ac-
cording to the Pharmacopoeia of the People’s Republic of
China, CS is mainly beneficial for the kidneys and the lungs,
and is indicated for chronic cough, hemoptysis, and impo-
tence and seminal emission, etc.
21
Cerebral ischemic stroke initiates a cascade of signal
molecules, leading to breakdown of the BBB.
5
Damage to
the BBB is the main pathological feature of cerebral
ischemia, and the BBB is thought to be a target in the
treatment of cerebral ischemia.
38
One proposal is that
microvascular lesions are the result of decreased micro-
circulation, leading to cerebral ischemia.
39
As an important
component of the BBB, brain microvascular endothelial
cells (BMECs) are monolayer cells that are located between
the subcutaneous tissue and blood circulation. Brain
microvascular endothelial cells have extensive physiolog-
ical functions, such as anticoagulation, antithrombosis, and
fibrinolysis. Brain microvascular endothelial cells are more
vulnerable than the other BBB-associated cells and pe-
ripheral endothelial cells when suffering from ischemia/
hypoxia.
40
Therefore, the study of BMECs induced by OGD is
important to understand the treatment of cerebral
ischemia.
Metabolism of the brain is high. Cerebral hypoxia/
ischemia reduces production of ATP, leading to energy
failure, anaerobic depolarization, functional damage of ion
pumps, and receptor activation of the N-methyl-D-aspartic
acid receptor. This triggers Ca
2þ
influx leading to brain cell
damage or even death. Destruction of the BBB activates
enzymatic reactions including some destructive lipases,
proteases, and nucleases, which result in membrane lipid
peroxidation and membrane damage. Once membrane is
broken, large amounts of intracellular LDH are released,
and brain cells are further damaged. Meanwhile, oxygen
free radicals are produced, and apoptosis ensues. In our
study, the CCK-8 assay showed that CSE increased cell ac-
tivity and alleviated cell injury, indicating that CSE could
protect rBMECs from ischemia. Besides, the effect of CSE in
low concentration (5, 10, 50, 100 and 20 mg$mL
1
) was
better than that of the higher one (200 and 400 mg$mL
1
),
which can be introduced to clinical application. Detection
of LDH in the cell supernatant showed that CSE significantly
inhibited LDH leakage and markedly reduced cytotoxicity.
This prevented cell damage and protected the integrity of
the cell membrane.
Brain tissue is rich in lipids, which easily react with free
radicals to produce lipid peroxides. In the mechanism of
cerebral ischemic injury, the free radical chain reaction is
an important part that causes brain damage.
41
MDA is the
major lipid peroxidation product and causes significant
damage to cell structure and function. The amount of MDA
reflects the extent of cell damage. In our body, there are a
series of free radical scavenging systems that involve free
radicals, and SOD plays an important role in removal of
these free radicals. Superoxide dismutase activity is usually
the main indicator of scavenging oxygen free radicals.
42e44
Therefore, the level of MDA and activity of SOD in cells
Table 2 Effects of CSE on SOD activity, MDA level, and NO content of rBMECs injured by OGD (n Z6)
a
.
Index NC Model CSE (mg$mL
L1
)
51020
SOD (U$mg
1
) 19.94 (1.33)** 7.04 (0.13) 12.93 (1.44)* 15.65 (1.98)** 18.19 (2.51)**
MDA (mmol$mg
1
) 3.74 (0.55)** 12.62 (1.97) 9.31 (0.56)* 8.61 (0.97)* 8.23 (0.63)**
NO (mmol$mg
1
) 5.71 (0.46)** 14.62 (4.58) 11.41 (1.81)* 9.13 (1.29)** 5.33 (0.91)**
Note: CSE: Cordyceps sinensis extract; SOD: superoxide dismutase; MDA: malondialdehyde; NO: nitric oxide; rBMECs: rat brain micro-
vascular endothelial cells; OGD: oxygeneglucose deprivation. NC: normal control; (without oxygeneglucose deprivation); Model group:
without medication.
Compared with model group, *P<.05, **P<.01.
a
Values were represented as mean (SD).
Table 3 Effects of CSE on TNF-aand IL-1blevels of rBMECs injured by OGD (n Z6).
Index NC Model CSE (mg$mL
L1
)
51020
TNF-a(pg$mg
1
) 57.18 (8.03)** 120.53 (9.32) 99.78 (4.78)* 83.65 (4.39)** 65.25 (7.52)**
IL-1b(pg$mg
1
) 4.75 (0.95)** 9.37 (0.94) 8.7 (0.56) 6.7 (1.05)* 5.86 (0.47)**
Note: CSE: Cordyceps sinensis extract; TNF-a: tumor necrosis factor-a; IL-1b: interleukin-1b; rBMECs: rat brain microvascular endo-
thelial cells; OGD: oxygeneglucose deprivation. NC: normal control (without oxygeneglucose deprivation); Model: without medication.
Compared with model group, *P<.05, **P<.01.
a
Values were represented as mean (SD).
Protective effect of Cordyceps sinensis extract on rat brain 69
reflect the extent of damage to rBMECs. Our study showed
that CSE could decrease MDA level in ischemic injury and
increase the activity of SOD. This situation improves energy
metabolism, relieves oxidative stress, and combats free
radicals in rBMECs in OGD.
Accumulation of Ca
2þ
in cells activates nitric oxide
synthase, and a large amount of NO generate. The combi-
nation of NO and peroxide produces toxic peroxynitrite.
Nitric oxide also promotes further release of glutamate,
which leads to a vicious circle. In the study, we found that
CSE could obviously decrease NO content in cells, thus
protect cells from further damage.
Tumor necrosis factor-ais an immunomodulatory and
proinflammatory cytokine that influences growth, differ-
entiation, proliferation, and survival of cells. It also regu-
lates immune inflammatory reactions and modulates the
function of immune system-related cells. Interleukin-1bis a
pleiotropic cytokine and an important mediator that trig-
gers the inflammatory response in cerebral ischemia. The
interleukin-1bis mainly expressed by vascular endothelial
cells, glial cells, and neurons.
45
The levels of TNF-aand IL-
1bare low in normal brain tissue. Previous studies have
shown that brain tissue produces TNF-a, IL-1b, and other
inflammatory factors during cerebral ischemia.
46,47
Up-
regulation of IL-1bexpression after cerebral ischemia
stimulates production of cell adhesion molecules, causes
neutrophil aggregation, and induces expression of a variety
of cytokines.
48
Tumor necrosis factor-ainduces cells to
produce matrix metalloproteinases, which destroy the basal
lamina and tight junction proteins of endothelial cells,
causes migration of glial cells, and destroys the integrity of
neurovascular unit. This process increases permeability of
the BBB. Opening of the BBB allows inflammatory cells to
enter the central nervous system, exposing nerve cells to
peripheral immune inflammatory cells and leads to brain
damage.
49
It was testified that CSE could obviously reduce
TNF-aand IL-1blevels in rBMECs in OGD. To be specific,
compared with the model group, TNF-alevels in 5, 10, and
20 mg$mL
1
CSE groups were displayed statistical differ-
ences (P<.05). With regard to IL-1blevel, no great dif-
ference between the model group and 5 mg$mL
1
CSE
group, but compared with the other two groups, there were
statistical differences (P<.05). Therefore, based on our
study, the concentration of CSE at 10 mg$mL
1
and
20 mg$mL
1
are the optimal choice for rBMECs in OGD.
Conclusion
C. sinensis extract plays a remarkable protective role in
rBMECs induced by OGD. The mechanism of CSE may be
related with reduction in oxygen free radicals, enhancement
of scavenging ability of cells in response to oxygen free
radicals, inhibition of the inflammatory reaction, or main-
tenance of endothelial function. These mechanisms still
remain to be examined in subsequent experimental studies.
Funding
This study was supported by the National Natural Science
Foundation of China (81403319 and 81603453) and the
Beijing Excellent Talent Project (2014000020124G114).
Conflicts of interest
None declared.
Author contributions
Xue Bai conducted the experiment, analyzed the data, and
finished the manuscript. Zhenquan Liu designed the ex-
periments and revised the manuscript. Yibo Tang provided
effective guidance for the experimental operation,
checked the data and revised the manuscript. Yuqing Zhao
carried out the experiments. Shuyan Wang assisted on
experimental performance. Tianyang Tan, Yan Lin, Meiqi
Liu, Zhenghui Chang, and Ying Liu did literature collecting
and assistance in the experiment.
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