Caffeoyl Triterpenoid Esters as Potential Anti-ischemic Stroke Agents from Celastrus orbiculatus

Article abstract of DOI:10.1021/acs.jnatprod.6b00314

Three new triterpenoids, celastrusins A-C (1-3), together with 3-O-caffeoyl-α-amyrin (4) were isolated from the root bark of Celastrus orbiculatus. Their structures were identified by spectroscopic analysis, X-ray crystallography using Cu Kα radiation, and the comparison of both observed and reported spectroscopic data. An in vitro bioassay revealed that the caffeoyl triterpenoid esters 1, 3, and 4 possess neuroprotective effects against oxygen-glucose deprivation (OGD) induced SH-SY5Y cell damage. Further animal studies indicated that compound 1 significantly reduced brain infarction after transient middle cerebral artery occlusion (MCAO) in rats using a 10 mg/kg (i.v.) dose.

Full text of DOI:10.1021/acs.jnatprod.6b00314

Article
pubs.acs.org/jnp
Caffeoyl Triterpenoid Esters as Potential Anti-ischemic Stroke Agents
from Celastrus orbiculatus
Jin-Long Li,†,§ Lei Wu,‡,§ Jian Wu,†,§ Hong-Xuan Feng,‡ Hong-Min Wang,† Yan Fu,‡ Ru-Jun Zhang,†
Hai-Yan Zhang,*,‡ and Wei-Min Zhao*,†
‡
†State Key Laboratory of Drug Research and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China
S
* Supporting Information
ABSTRACT: Three new triterpenoids, celastrusins A−C (1−3), together with 3-O-caffeoyl-α-amyrin (4) were isolated from the
root bark of Celastrus orbiculatus. Their structures were identified by spectroscopic analysis, X-ray crystallography using Cu Kα
radiation, and the comparison of both observed and reported spectroscopic data. An in vitro bioassay revealed that the caffeoyl
triterpenoid esters 1, 3, and 4 possess neuroprotective effects against oxygen-glucose deprivation (OGD) induced SH-SY5Y cell
damage. Further animal studies indicated that compound 1 significantly reduced brain infarction after transient middle cerebral
artery occlusion (MCAO) in rats using a 10 mg/kg (i.v.) dose.
neurasthenia, palpitation, and amnesia.8 Previous investigations
troke ranks as the second highest leading killer in the
1
Sworld. There are two broad categories of stroke: ischemic,
showed β-dihydroagarofuran-type sesquiterpenoids as the
due to lack of blood flow, and hemorrhagic, due to bleeding in
the brain. According to a recent study, ischemic strokes account
for approximately 87% of all strokes.2 Effective therapeutic
strategies remain a challenge, despite advances made in
understanding the pathophysiology of cerebral ischemia. Most
marketed drugs for stroke prevention act as antiplatelet agents
(aspirin, among others), anticoagulants (warfarin), HMG-CoA
reductase inhibitors (statins), ACE inhibitors (ramipril), or
angiotensin II blockers (sartans). Currently, the only FDA-
approved drug for the treatment of ischemic stroke,
recombinant tissue-plasminogen activator (rt-PA), is still
restricted by its short time window, narrow eligible range,
and risk of hemorrhage.3−5 Therefore, discovering new lead
compounds that are both safe and effective for minimizing
postischemic damage is a priority in the field of antistroke drug
development.
Natural products have been shown to be reliable resources in
drug discovery and development processes. In the area of
stroke research, natural products have played a significant role
in providing potentially valuable compounds for drug discovery,
such as butylphthalide from Apium graveolens6 and ginkgolide B
from Ginkgo biloba,7 among others. Celastrus orbiculatus Thunb.
(Celastraceae) is widely distributed in China. The roots and
fruits have been used to treat snakebite, arthritis, insomnia,
major secondary metabolites of the seeds and the presence of
triterpenoids mainly in the roots. Bioassays of these terpenoids
were mainly focused on their antitumor9,10 and anti-
inflammatory11,12 aspects. Inspired by the traditional use of
C. orbiculatus and C. paniculatus seeds in China and India for
memory-enhancing effects, neuroprotective assays of compo-
nents isolated from the genus Celastrus were undertaken.
According to recent investigations, dimeric trinorditerpenoids
from the root barks of C. orbiculatus were found to exhibit
significant neuroprotective effect against H2O2-induced injury
of PC12 cells.13 Furthermore, β-dihydroagarofuran-type
sesquiterpenoids from the seeds of C. flagellaris and C. angulatus
were shown to rescue Aβ25−35-induced SH-SY5Y cells from
viability reduction, some of which were also observed to
possess significant cognition-enhancing effects in rats with
doses of 20 mg/kg (p.o.).14 Herein the structure elucidation of
caffeoyl triterpenoid esters isolated from C. orbiculatus and their
pharmacological evaluation against postischemic brain damage
are reported.
Received: April 8, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00314
J. Nat. Prod. XXXX, XXX, XXX−XXX

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1
RESULTS AND DISCUSSION
Table 1. H NMR (500 MHz) and 13C NMR (125 MHz)
■
Data of Compounds 1−3 (CDCl3)
Celastrusin A (1) was obtained as a white, amorphous powder,
and its molecular formula was determined as C39H56O5 based
on the HREIMS molecular ion at m/z 604.4131 and 13C NMR
1 and 2
3
δH, multiplicity (J in
δH, multiplicity (J in
1
data. The H and 13C NMR data of celastrusin A (1) showed
position
Hz)
δC
Hz)
δC
characteristic signals for one caffeoyl group and one
triterpenoid moiety [an aliphatic carbonyl (δC 214.3), six sp3
quaternary carbons, four methines, 12 methylenes, including an
oxymethylene at δC 75.4/δH 3.87 and 3.88, and seven methyl
groups, including six tertiary methyls and one secondary
methyl] (Table 1). In the HMBC spectrum of 1, cross-peaks
were observed between H2-2 (δH 2.39, 2.30), H-4 (δH 2.25),
H3-23 (δH 0.88) and C-3 (δC 214.3); between H3-23 (δH 0.88)
and C-5 (δC 42.4); between H3-24 (δH 0.73) and C-4 (δC
58.4), C-6 (δC 41.7); between H3-25 (δH 0.86) and C-10 (δC
59.5), C-8 (δC 53.4), C-11 (δC 35.7); between H3-26 (δH 1.03)
and C-8 (δC 53.4), C-15 (δC 32.7); between H3-27 (δH 1.04)
and C-18 (δC 42.0), C-12 (δC 30.7); between H3-28 (δH 1.22)
and C-18 (δC 42.0), C-22 (δC 39.4), C-16 (δC 35.9); and
between H3-30 (δH 1.10) and C-29 (δC 75.4), C-19 (δC 30.2),
C-21 (δC 28.3) (Figure 2), which enabled confirmation of the
presence of 29-hydroxyfriedelin as its triterpenoid moiety.15
NOE correlations between H3-25 (δH 0.86) and H3-24 (δH
0.73) and H3-27 (δH 1.04); between H-18 (δH 1.65) and H3-27
and H3-28 (δH 1.22); and between H-8 (δH 1.39) and H3-26
(δH 1.03) and H-10 (δH 1.53) indicated a trans A/B/C/D ring
fusion and a cis D/E ring fusion. The NOESY cross-peaks of
H3-30/H3-28 and H-4/H-10 revealed their axial orientations
(Figure 2). Its absolute configuration was defined by X-ray
diffraction analysis of its acetylated derivative 1a, crystallized
from acetonitrile (Figure 3) as (4R,5S,8S,9S,10S,13S,-
14R,17S,18R,20R)-3-oxo-29-(6′,7′-di-O-acetyl)-
caffeoyloxyfriedelin.
1
1.96, m; 1.69, m
2.39, m; 2.30, m
22.5
41.4
214.3
58.4
42.4
41.7
18.4
53.4
37.5
59.5
35.7
30.7
40.0
38.4
32.7
35.9
30.5
42.0
30.2
32.0
28.3
39.4
7.0
1.59, m; 1.05, m
1.56, m
38.2
23.7
81.1
37.9
55.2
18.2
34.2
41.2
49.3
36.8
40.0
21.2
38.7
42.9
26.4
28.4
35.4
47.8
41.4
75.1
37.2
38.2
27.8
16.5
15.8
16.0
14.8
18.2
17.1
20.8
2
3
4.45, dd (9.9, 6.0)
4
2.25, m
5
0.70, m
6
1.74, m; 1.27, m
1.49, m; 1.39, m
1.39, m
1.40, m; 1.27, m
1.27, m
7
8
9
1.16, m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1.53, m
1.61, m; 1.37, m
1.45, m; 1.23, m
1.19, m; 1.10, m
1.34, m; 1.18, m
1.05, m
1.47, m; 1.22, m
1.47, m; 1.26, m
1.61, m; 0.85, m
1.88, m; 1.03, m
1.65, m
0.92, m
1.43, m
1.32, m; 1.24, m
1.46, m; 0.90, m
1.44, m; 1.05, m
0.88, d (6.6)
0.73, s
1.35, m; 1.05, m
1.70, m; 0.90, m
0.75, s
14.8
18.0
20.7
18.6
32.2
75.4
26.6
0.79, s
0.86, s
0.76, s
1.03, s
0.91, s
1.04, s
0.82, s
1.22, s
0.77, s
Compound 2 gave an [M]+ peak at m/z 588.4176 in its
HREIMS, 16 Da less than that of 1 and consistent with a
3.87, brs
0.92, d (6.6)
0.94, s
1.10, s
molecular formula of C39H56O4. Comparison of the 1H and 13
C
Caffeoyl moiety of 1
Caffeoyl moiety of 3
1′
2′
3′
4′
5′
6′
7′
8′
9′
168.2
115.9
144.9
127.7
114.4
144.0
146.5
115.6
122.6
168.0
115.9
145.0
126.6
113.9
144.9
147.4
115.2
121.8
NMR data of 2 and 1 revealed the absence of the 6′-OH group
on the aromatic nucleus in 2, as evidenced by the appearance of
an A2B2 spin system in 2 (δH 7.43 and 6.88, each 2H, d, J = 8.2
Hz) instead of the ABX spin system in 1 (Table 1). In addition,
hydrolysis of 2 with 10% KOH yielded 29-hydroxyfriedelin and
p-hydroxycinnamic acid, as identified by comparison with
authentic samples. Thus, the structure of 2 was identified as 29-
O-p-hydroxycinnamoylfriedelin as shown in Figure 1 and
named celastrusin B.
6.30, d (15.9)
7.59, d (15.9)
6.09, d (15.9)
7.39, d (15.9)
7.13, d (1.8)
6.91, d (2.1)
6.89, d (8.2)
6.68, d (8.2)
7.02, dd (8.2, 1.8)
6.81, dd (8.2, 2.1)
p-Hydroxycinnamoyl moiety
of 2
The molecular formula of 3 was deduced as C39H58O5 on the
basis of HREIMS (m/z 606.4292) and 13C NMR data.
Compound 3 possessed a caffeoyl moiety according to the
UV absorption bands (λmax 330, 244, and 217 nm) and NMR
data. The remaining 13C NMR signals of 3 (five sp3 quaternary
carbons, six sp3 tertiary carbons, 11 sp3 secondary carbons, and
eight methyl groups) suggested a pentacyclic triterpenoid
1′
2′
3′
4′
5′/9′
6′/8′
7′
168.1
6.32, d (15.7)
7.64, d (15.7)
115.2
144.6
126.7
130.0
115.9
158.4
7.43, d (8.3)
6.88, d (8.3)
2
skeleton different from those of both 1 and 2. J correlations
between the eight methyl signals and their attached carbons, as
3
well as J couplings in the HMBC spectrum (Figure 4), led to
the deduction of an α-amyrin-type triterpenoid moiety in the
structure of 3.16,17 The downfield shift of H-3/C-3 (δH 4.45/δC
81.1) and the HMBC correlation between H-3 (δH 4.45) and
C-1′ (δC 168.0) localized the caffeoyl group at C-3. The
coupling constants of H-3 (δH 4.45, dd, J = 9.9, 6.0 Hz)
suggested its α-orientation. In an NOE experiment (Figure S31,
Supporting Information), selective irradiation of the signal at δH
0.94 (H3-30) enhanced the signals at δH 1.57 (H3-27) and δH
1.43 (H-19), revealing the β-orientation of the 20-OH function.
Therefore, compound 3, celastrusin C, was characterized as 3β-
caffeoyloxy-20β-hydroxyursane (Figure 1).
3β-Caffeoyloxy-Δ12-ursene (4) was also isolated and
identified through comparing its spectroscopic data to those
reported (Figure 1).18
B
DOI: 10.1021/acs.jnatprod.6b00314
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Figure 1. Structures of compounds 1−4.
Figure 2. Key HMBC and NOESY correlations of 1.
Figure 5. Effect of compounds 1−4 on SH-SY5Y cells subjected to
OGD. The cell viability of the control was taken as 100%, and the
average value of cell viability under OGD exposure was 56.18 3.34%.
The data are expressed as the mean SEM (n = 3), ###p < 0.001 vs
control, **p < 0.01 vs OGD group.
Dose-Dependent Effects of 1 in Protecting SH-SY5Y
Cells from OGD Damage. SH-SY5Y cells were subjected to
OGD for 1 h and reoxygenation for 24 h, which resulted in
dendrite fragmentation and simultaneous decreases in cell
Figure 3. ORTEP drawing of 1a.
number (Figure 6A) and cell viability (55.05
2.03%, p <
0.001 vs control) (Figure 6B). However, post-treatment with
compound 1 effectively reduced the OGD-induced morpho-
logical damage in SH-SY5Y cells (Figure 6A). In accordance
with the result from morphological observations, compound 1
treatment notably attenuated OGD-induced injury in a dose-
dependent (1 to 10 μM) manner with maximal protection at 10
μM (115.41 7.16%, p < 0.001 vs OGD group) (Figure 6B).
Additionally, compound 1 showed no significant effects on
normal SH-SY5Y cells at those experimental concentrations
(Figure S1, Supporting Information).
Figure 4. Key HMBC correlations of 3.
Compound 1 Attenuated Brain Injury after Transient
Middle Cerebral Artery Occlusion (MCAO) in Rats. With
the best neuroprotective activity found in the bioassay,
compound 1 was further evaluated for its in vivo neuro-
protective activity using a classic MCAO model for ischemic
stroke study. As shown in Figure 7, the MCAO insult produced
a large infarct (29.45 3.65%), as shown in the white region of
rat brain sections. Compound 1 was administered intravenously
immediately 2 h after the onset of ischemia. As a result, post-
treatment of 1 at the dose of 10 mg/kg reduced infarct volume
by 14.20 4.54% (p < 0.05 vs MCAO group). Edaravone was
used as a positive control. Consistent with its protective effect
on infarct areas, 1 also produced significant attenuation in
neurological scores (p < 0.05 vs MCAO group).
Compounds 1−4 were evaluated for their neuroprotective
effects against oxygen-glucose deprivation (OGD)-induced
damage of SH-SY5Y cells. Exposing SH-SY5Y cells to OGD
for 1 h and reoxygenating in normoxic and normoglycemic
conditions for 24 h induced a significant decrease in cell
viability (56.18 3.34%, p < 0.001 vs control). Compounds 1−
4 were added at the beginning of reoxygenation to obtain a final
concentration of 10 μM for evaluating efficacy. Compared with
the inactive compound 2, compounds 1, 3, and 4, which have a
caffeoyl moiety in their structures, exhibited more potent
activity (Figure 5). Among the three bioactive compounds, 1
most significantly protected SH-SY5Y cells from OGD damage.
Therefore, compound 1 was selected for further investigations.
The above experiments suggest that further exploration of
both the pharmacological mechanism of action of 1 and the
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DOI: 10.1021/acs.jnatprod.6b00314
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Figure 6. Compound 1 attenuated OGD-induced SH-SY5Y cell injury.
(A) Morphology of SH-SY5Y cells after different treatments. (B) Cell
viability was measured by MTT assay after treatment with different
concentrations of 1 and OGD exposure. The data are presented as the
percentage of surviving cells relative to control cells from three
independent experiments and as the mean SEM. ###p < 0.001 vs
control, *p < 0.05, ***p < 0.001 vs OGD group.
structure−activity relationship of caffeoyl triterpenoid esters as
potential lead compounds against ischemic stroke is warranted.
Figure 7. Effect of compound 1 on infarct area and neurological
function after 2 h of middle cerebral artery occlusion (MCAO) and 24
h of reperfusion. (A) Triphenyltetrazolium chloride-stained coronal
sections from representative rats given either vehicle or 1. (B)
Quantification of the infarct volume. (C) Effect of 1 on neurological
score outcomes. *p < 0.05 vs vehicle-treated MCAO group, n = 11−
14.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
measured on an SGW X-4B melting instrument and are uncorrected.
Optical rotations were measured with a PerkinElmer 341 polarimeter
in MeOH. MS data were measured with a Finnigan LCQ/DECA or
Finnigan LTQ mass spectrometer. UV spectra were measured using a
Varian Cary 50 UV/vis spectrophotometer. IR spectra were measured
with a PerkinElmer 577 instrument. NMR spectra were recorded on a
Bruker AVANCE III 500 instrument with tetramethylsilane as the
reference. Semipreparative HPLC separations were conducted using a
Unimicro 2010 instrument and YMC-Pack ODS-A column (250 × 20
mm, 5 μm; YMC Co., Ltd., Kyoto, Japan). Low-pressure column
chromatography (CC) was conducted using silica gel (200−400 mesh;
Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Sephadex
LH-20 (GE Healthcare Biosciences AB, Uppsala, Sweden) as packing
materials.
Extraction and Isolation. The EtOAc fraction of the 95% EtOH
extract of the root barks of C. orbiculatus (20 kg) was separated over a
silica gel column eluted with a mixture of petroleum ether/EtOAc
(10:1 to 1:1, v/v) to give Fr. 1−4.13 Fr. 2 was further separated using
RP-18 CC and eluted with MeOH/H2O (3:2 to 1:0, v/v) to obtain
four subfractions (Fr. 2a−2d). Fr. 2d was purified by semipreparative
HPLC (MeCN/H2O, 9:1) to give compounds 2 (0.028 g) and 4
(0.035 g). Fr. 3 was subjected to CC over MCI and eluted with a
gradient of MeOH/H2O (1:4 to 10:0, v/v) to yield fractions 3a−3c.
Fr. 3c was separated by Sephadex LH-20 with 95% EtOH as eluent to
yield 1 (4.0 g) and 3 (0.03 g).
Plant Material. The root barks of Celastrus orbiculatus were
collected in the suburb of Huaihua, Hunan Province. A voucher
specimen (No. SIMM0906-2) has been identified by Prof. Jin-Gui
Shen of SIMM, CAS.
D
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Celastrusin A (1): canary yellow, amorphous powder; [α]2D2 −23 (c
0.1, acetone); UV (MeOH) λmax 329, 244, and 218 nm; IR (KBr) νmax
3369, 2935, 2872, 1701, 1678, 1630, 1599, 1518, 1261, and 754 cm−1;
1D NMR data, see Table 1; HREIMS m/z 604.4131 [M]+ (calcd for
C39H56O5, 604.4128).
MCAO. Saline, edaravone (10 mg/kg), and compound 1 (10 mg/kg)
were administered intravenously immediately after reperfusion. The
animal’s body temperature was maintained at 37.0 0.5 °C during the
whole surgical procedure.
Neurological Deficit Evaluation. After 24 h of reperfusion, the
neurological deficits were tested according to the modified Neuro-
logical Severity Score (mNSS) evaluations.21 The mNSS was scored
on an 18-point scale including motor, visual, and tactile responses.22 A
higher score represents worse neurological performance.
Brain Injury Measurement. After mNSS evaluation, rat brain
coronal sections (2 mm thick) were prepared and stained with 1%
triphenyltetrazolium chloride (Sinophar Chemical Reagent Co.,
Shanghai, China) for 15 min followed by fixation in paraformaldehyde
solution (4%) for 24 h. The red staining represented normal and
healthy brain tissue, and the white represented infarct tissue. After
scanning with a digital camera, different brain areas were analyzed and
calculated using an image analysis system (Image-ProPlus). The infarct
volumes were calculated as follows: (area of right hemisphere − area of
noninfarct left area)/right hemisphere × 100%.23
(4R,5S,8S,9S,10S,13S,14R,17S,18R,20R)-3-Oxo-29-(6′,7′-di-O-
acetyl)caffeoyloxyfriedelin (1a): colorless block crystal; mp 218−219
°C; 1H NMR (500 MHz, CDCl3) δH 7.62 (d, J = 16.0 Hz, H-3′), 7.41
(dd, J = 8.4, 2.1 Hz, H-9′), 7.38 (d, J = 2.1 Hz, H-5′), 7.22 (d, J = 8.4
Hz, H-8′), 6.41 (d, J = 16.0 Hz, H-2′), 3.88 (brs, H-29), 2.31 and 2.30
(s, CH3CO−), 1.22 (s, H-28), 1.10 (s, H-30), 1.04 (s, H-27), 1.03 (s,
H-26), 0.88 (d, J = 6.6 Hz, H-23), 0.87 (s, H-25), 0.72 (s, H-24); 13C
NMR (125 MHz, CDCl3) δC 213.3 (C-3), 168.2 (C-1′), 168.1 and
167.1 (each CH3CO−), 143.6 (C-7′), 142.6 (C-6′), 142.6 (C-3′),
133.5 (C-4′), 126.6 (C-8′), 124.1 (C-5′), 122.8 (C-9′), 119.7 (C-2′),
75.4 (C-29), 59.6 (C-10), 58.3 (C-4), 53.4 (C-8), 42.3 (C-5), 42.0 (C-
18), 41.7 (C-6), 41.4 (C-2), 40.0 (C-13), 39.3 (C-22), 38.4 (C-14),
37.6 (C-9), 35.9 (C-16), 35.7 (C-11), 32.69 (C-15), 32.23 (C-28),
32.0 (C-20), 30.7 (C-12), 30.5 (C-17), 30.2 (C-19), 28.3 (C-21), 26.6
(C-30), 22.4 (C-1), 20.8 (2 × CH3CO−), 20.7 (C-26), 18.6 (C-27),
18.4 (C-7), 18.0 (C-25), 14.8 (C-24), 7.0 (C-23); HREIMS m/z
688.4336 [M]+ (calcd for C43H60O7, 688.4339). The crystallographic
data of 1a have been deposited in the Cambridge Crystallographic
Data Center (CCDC) with the entry number 1454973.
Statistical Analysis. All data were reported only if at least three
independent experiments showed consistent results. The data were
expressed as the mean
SEM and analyzed by one-way ANOVA
followed by Tukey’s test for multiple comparisons or by Student’s t-
test for single comparison. Statistical significance was established using
a p value of <0.05.
Celastrusin B (2): canary yellow, amorphous powder; [α]2D2 −8 (c
0.1, CHCl3); UV (MeOH) λmax 313, 227 nm; IR (KBr) νmax 3365,
2933, 2872, 1705, 1635, 1608, 1587, 1514, 1456, 1388, 1275, 1169,
982, and 829 cm−1; 1D NMR data, see Table 1; HREIMS m/z
588.4176 [M]+ (calcd for C39H56O4, 588.4179).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00314.
Celastrusin C (3): canary yellow, amorphous powder; [α]2D2 +31 (c
0.1, CHCl3/MeOH, 1:1); UV (MeOH) λmax 330, 244, and 217 nm; IR
(KBr) νmax 3535, 3431, 2935, 1685, 1633, 1603, 1529, 1446, 1386,
1275, 1192, 978, and 758 cm−1; 1D NMR data, see Table 1; HREIMS
m/z 606.4292 [M]+ (calcd for C39H58O5, 606.4284).
Spectra of new compounds (PDF)
Crystallographic data file of diacetylated celastrusin A
(1a) (CIF)
Cell Culture. Human neuroblastoma SH-SY5Y cells were cultured
in a 1:1 mixture of Eagle’s minimum essential medium and F12
medium (Gibco, USA), supplemented with 10% fetal bovine serum
(FBS; Gibco, USA), 100 U/mL penicillin, and 100 μg/mL
streptomycin, in a humidified atmosphere of 5% CO2 at 37 °C. SH-
SY5Y cells (2 × 104 cells/100 μL per well) were seeded into 96-well
plates.
AUTHOR INFORMATION
Corresponding Authors
*Tel/fax: 86-21-50806710. E-mail: hzhang@simm.ac.cn (H.
Zhang).
■
OGD Exposure. Neuronal ischemia/reperfusion was induced by
OGD and reoxygenation.19 The culture medium of the OGD group
was replaced with glucose-free EBSS buffer (mmol/L: 116 NaCl, 5.4
KCl, 0.8 MgSO4·7H2O, 1.0 NaH2PO4·2H2O, 26.2 NaHCO3, 0.01
glycine, 8.0 CaCl2·2H2O, pH 7.4) after washing twice and transferred
to a hypoxia chamber (H35 Hypoxystation, Don Whitley Scientific) in
an atmosphere of 85% N2, 10% H2, and 5% CO2 at 37 °C. OGD was
terminated after 1 h by returning to normal cultured conditions and
reoxygenated for another 24 h. The control group was treated without
OGD insult. Compounds were added at the beginning of
reoxygenation.
Cell Viability Assay. Cell viability was measured by using a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma,
USA) assay. After 24 h of reoxygenation, 10 μL of MTT (5 mg/mL)
was added to each well and incubated at 37 °C in the dark for 3 h. All
culture medium was replaced with 100 μL of DMSO to dissolve the
resulting formazan. The absorbance of each well was assessed using a
DTX 800 multimode detector (Beckman Coulter, Fullerton, CA,
USA) at 490 nm.
*Tel/fax: 86-21-50806052. E-mail: wmzhao@simm.ac.cn (W.
Zhao).
Author Contributions
§J.-L. Li, L. Wu, and J. Wu contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Science
and Technology Major Projects for Major New Drugs
Innovation and Development (No. 2013ZX09507002), Na-
tional Natural Science Foundation of China (Nos. 81473113
and 81522045), the State Key Laboratory of Drug Research
(No. SIMM1601KF-03), and Institutes for Drug Discovery and
Development, Chinese Academy of Sciences (No. CA-
SIMM0120162019).
MCAO Model in Rats. Focal cerebral ischemia was induced in
male Sprague−Dawley rats (SD rats, Shanghai Laboratory Animal
Center, Chinese Academy of Sciences, China) using the MCAO
model described previously with minor modification.20 Rats weighing
230−280 g were subjected to MCAO. In brief, rats were anesthetized
with chloral hydrate (400 mg/kg, i.p.). MCAO was induced using a 4−
0 monofilament nylon suture (Beijing Sunbio Biotech Co., China),
inserted into the left internal carotid artery and positioned to occlude
the MCA origin through the proximal external carotid artery. The
suture was withdrawn to restore blood flow (reperfusion) after 2 h of
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