A synthetic steroid 5α-Androst-3β,5,6-Triol blocks hypoxia/reoxygenation - Induced neuronal injuries via protection of mitochondrial function

Article abstract of DOI:10.1016/j.steroids.2013.06.004

Ischemic stroke is a leading cause of death worldwide, yet therapies are limited. During periods of ischemia following reperfusion in ischemic stroke, not only loss of energy supply, but a few other factors including mitochondrial dysfunction and oxidative stress also make vital contribution to neuronal injury. Here we synthesized a steroid compound 5α-androst-3β,5, 6β-triol by 3 steps starting from dehydroepiandrosterone and examined its effect on mitochondrial function and oxidative stress in primary cultured cortical neurons exposed to hypoxia followed by reoxygenation. 5α-Androst-3β,5,6β-triol dose-dependently protected cortical neurons from hypoxia/reoxygenation exposure. Rates of reduction in neuronal viability, loss of mitochondrial membrane potential, disruption of ATP production and oxidative stress were ameliorated in 5α-androst-3β,5, 6β-triol pretreated cultures. In summary, these results suggest that 5α-androst-3β,5,6β-triol is neuroprotective against hypoxia/reoxygenation induced neuronal injuries through mediation of mitochondrial function and oxidative stress.

Full text of DOI:10.1016/j.steroids.2013.06.004

Steroids 78 (2013) 996–1002
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
A synthetic steroid 5a-androst-3b,5,6b-triol blocks hypoxia/reoxygena-
tion-induced neuronal injuries via protection of mitochondrial
function q
Jiesi Chen a,1, Tiandong Leng a,1, Wenli Chen a, Min Yan a, Wei Yin a, Yijun Huang a, Suizhen Lin b,
Dayue Duan c, Jun Lin d, Gongxiong Wu e, Jingxia Zhang a, , Guangmei Yan a,
⇑
⇑
a Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou 510080, PR China
b Guangzhou Cellprotek Pharmaceutical, G Building F/4, 3 Lanyue Road, Science City, Guangzhou 510663, PR China
c Laboratory of Cardiovascular Phenomics, Department of Pharmacology, University of Nevada School of Medicine, CMM 303F, Center for Molecular Medicine, Reno, NV
89557-0318, USA
d Department of Anesthesiology, University Hospital of Brooklyn, Long Island College Hospital, Downstate Medical Center, State University of New York, 339 Hicks Street, Brooklyn,
NY 11201, USA
e Harvard Medical School, One Joslin Place, Boston, MA 02215, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Ischemic stroke is a leading cause of death worldwide, yet therapies are limited. During periods of ische-
mia following reperfusion in ischemic stroke, not only loss of energy supply, but a few other factors
including mitochondrial dysfunction and oxidative stress also make vital contribution to neuronal injury.
Received 21 December 2012
Received in revised form 18 May 2013
Accepted 9 June 2013
Here we synthesized a steroid compound 5
androsterone and examined its effect on mitochondrial function and oxidative stress in primary cultured
cortical neurons exposed to hypoxia followed by reoxygenation. 5 -Androst-3b,5,6b-triol dose-depen-
a-androst-3b,5,6b-triol by 3 steps starting from dehydroepi-
Available online 28 June 2013
a
Keywords:
5a-Androst-3b,5,6b-triol
Hypoxia/reoxygenation
Neuroprotection
Mitochondrial function
Oxidative stress
dently protected cortical neurons from hypoxia/reoxygenation exposure. Rates of reduction in neuronal
viability, loss of mitochondrial membrane potential, disruption of ATP production and oxidative stress
were ameliorated in 5
-androst-3b,5,6b-triol is neuroprotective against hypoxia/reoxygenation induced neuronal injuries
through mediation of mitochondrial function and oxidative stress.
Ó 2013 The Authors. Published by Elsevier Inc. All rights reserved.
a-androst-3b,5,6b-triol pretreated cultures. In summary, these results suggest that
5
a
1. Introduction
Mitochondria comprise the central locus for energetic perturba-
tions during I/R [4]. Besides, concomitant oxidative stress makes
considerable contribution to neuronal injuries [5–7]. Previous evi-
dence pointed to an increase of intracellular reactive oxygen spe-
cies (ROS) accumulation during oxidative stress, with the source
being mitochondria [8,9], logically, mitochondrial function is of
central importance in ischemic/hypoxic and secondary insults.
Meanwhile, excessive ROS in reverse causes damages to mitochon-
dria and consequently exacerbates mitochondrial dysfunction [10].
Thereupon, that mitochondrial dysfunction acts with oxidative
stress has been widely recognized to imperil neurons during acute
ischemic/hypoxic insult.
Worldwide, stroke is the 2nd leading cause of death [1], yet
therapies are very limited. Ischemic stroke accounts for 83% of all
strokes [2], with the poor outcomes attributed mainly to neuronal
injuries and death. Worse still, almost all identified neuroprotec-
tive compounds from preclinical research have failed to be trans-
lated from bench to bedside [3]. Understanding the process of
neuronal injury resulting from exposure to the distinct environ-
ment during stroke - ischemia/reperfusion (I/R) is of critical
importance in identifying useful targets for the development of
pharmacological treatment of stroke.
Ever since Baulieu and colleagues reported dehydroepiandros-
terone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) as
the first neurosteroids in 1998 [11], there has been a growing
interest in searching neuro-active steroids. Thereafter a trend has
been set in the development of steroid drugs as treatments of both
chronic neurodegenerative disease and acute brain injuries. A large
number of steroids isolated and purified from marine sources were
reported bioactive in the aspects of anti-tumor, anti-inflammation,
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.
⇑
Corresponding authors. Tel.: +86 20 39943029 (J. Zhang), +86 20 84113817
(G. Yan).
E-mail addresses: zhjingx@mail.sysu.edu.cn (J. Zhang), ygm@mail.sysu.edu.cn
(G. Yan).
1
These authors contribute equally to this work.
0039-128X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.06.004

J. Chen et al. / Steroids 78 (2013) 996–1002
997
anti-oxidation and so on, yet the low yields from extraction have
been disconcerting considering the actual pharmacological values,
2.1.2. 5a-3b,6b-Diformyloxyl-androst-5-ol (3)
Compound 2 (7 g, 0.026 mol) was added into 88% formic acid
(140 ml) at room temperature, and heated to 65 °C with agitation
at 400 rpm for 10 min. After cooling down to room temperature,
30% hydrogen peroxide (10 ml) was added with stirring. Reaction
was maintained for 1 h until the solid dissolved completely and
the suspension became pellucid. Next the mixture was heated to
60 °C for 1 h to resolve the rest hydrogen peroxide and poured into
water (600 ml) with stirring. The granular white solids were col-
lected by suction filtration after attaining room temperature. For
dispersed solids, KHCO3 was used for neutralizing the residual acid
followed by vacuum drying. The resulting solids were further puri-
fied by column chromatography to afford compound 3 (7.9 g,
84.9%), and the purity was 94.62%. ½aꢁtD: ꢀ80.2 (dioxane, 10 mg/
which have, consequently, to
preparations of purely synthetic compounds for preclinical studies
and future possible clinical uses. Here we synthesized 5 -androst-
a large degree promoted the
a
3b,5,6b-triol (triol), a previously discovered steroid [12], from
DHEA by 3 steps and obtained an overall yield of 76.0%. The neuro-
protetive effect of triol was afterwards examined on a well-estab-
lished cellular model of hypoxia/reoxygenation (H/R) injury [12]
mimicking the I/R process during stroke focused on mediation of
mitochondrial function and oxidative stress.
2. Experimental procedures
mL). IR (KBr)
m: 3259, 2954, 1375, 1720, 1692, 1058, 1058,
1034 cmꢀ1. UV (nm): 200–210, 225. 1H NMR (DMSO-d6) d: 0.72
2.1. Chemistry
(s, 3H, 18-CH3), 1.00 (m, 1H, 14-H), 1.17 (s, 3H, 18-CH3), 1.40 (d,
1H, 9-H), 1,54 (m, 1H, 8-H), 1.73 (s, 1H, 7a-H), 1.90 (m, 1H, 2b-
DHEA (Wuhan Yuancheng Technology, China) was used as a
starting steroid. All chemicals and solvents were of analytical grade
and purchased from Guangzhou Chemical Reagents Company Chi-
na if not mentioned particularly. All melting points were measured
on an X6 melting point apparatus and were uncorrected. All reac-
tions were monitored by thin-layer chromatography (TLC) on silica
gel G. Optical rotations were measured at 25 °C on Perkin Elmer
Model 341 polarimeter. The IR spectra were recorded as KBr pellets
on an EQUINOX 55 FT spectrophotometer (wave numbers in cmꢀ1).
1H NMR and 13C NMR spectra were recorded on a nuclear magnetic
resonance spectrometer Bruker (Avancell, 400 MHz). Mass spectra
were recorded on GC–MS (Agilent, USA) to confirm the structure of
the compounds. All purity data were recorded on 7890A
GC(Agilent, USA) with the GC column DB-5MS (Agilent, USA).
The octanol–water partition coefficient (logP) was obtained as
previously described [13,14] and concentrations of triol in the
aqueous phase and organic phase were measured from 7890A GC
system.
H), 4.82 (s, 1H, 6-H), 5.29 (m, 1H, 3-H), 7.98 (s, 1H, 20-H), 8.00
(s, 1H, 21-H). 13C NMR (DMSO-d6) d: 16.39 (CH3), 17.57 (CH3),
20.45 (CH2), 21.04 (CH2), 25.36 (CH2), 26.57 (CH2), 30.97 (CH),
31.78 (CH2, CH2), 36.73 (CH2), 38.55 (C), 38.68 (CH2), 40.25 (CH2),
40.95 (C), 45.37 (CH), 53.78 (CH), 70.64 (CH), 74.83 (C), 75.95
(CH), 160.28 (C), 160.74 (C). EI-MS m/z: 364 (M+), 336 (M-CO),
318 (M-CO–H2O), 300 (M-CO–2H2O), 272 (M-2CO–2H2O), 254
(M-2CO–3H2O). Analysis calculated for C21H32O5 C 69.20, H 8.85;
found C 69.39, H 8.85.
2.1.3. 5a-Androst-3b,5,6b-triol (4)
25% Sodium hydroxide (20 ml) was added to a solution of com-
pound 3 (7 g, 0.019 mol) in methanol (140 ml). The mixture solu-
tion was heated to 60 °C for 30 min and cooled down to room
temperature. Then the mixture was diluted in water (650 ml)
and acidified. The precipitates were filtered and washed with
water, then thoroughly by vacuum drying to obtain a white crude
solid 4 (5.5 g, 93.5%). The precipitates were filtered and washed
with water, then thoroughly by vacuum drying to obtain a white
crude solid 4 (5.5 g, 93.5%), the purity of which was 96.74%. The
resulting solid was further purified by recrystallization with ace-
tone/water (2:1) to afford compound 4 (3.6 g, 65.5%) with high
2.1.1. 5-En-androst-3b-ol (2)
Diethylene glycol (100 ml) was added into a four-necked flask
with each neck used either as a stirrer, temperature controller,
condenser pipe or inlet port. When reaching a temperature of
130 °C, DHEA 1 (10 g, 0.034 mol) and hydrazine hydrate (50 ml)
were added into the flask. After heating and refluxing for 2 h,
KOH (10 g, 0.178 mol) was added carefully into the mixture. The
residual liquid and hydrazine hydrate were distilled until the tem-
perature reached 210 °C, the mixture was then refluxed for 4 h and
cooled down to room temperature [15]. 98% Sulfuric acid (10 ml)
was added carefully to 1.5 L of water with a glass rod stirring syn-
chronously, after well-distributed, the mixture from last reaction
was poured into this acidic solution to counteract the residual al-
kali. Afterwards, the mixture was suction filtrated and the solid
was dispersed into the water. Suction filtration was repeated twice
following the vacuum drying to yield a white solid 2 (9.1 g, 95.8%),
and the purity was 98.95%. ½aꢁtD: ꢀ67.18 (anhydrous ethanol,
purity (99.51%).
221 °C. IR (KBr)
½
aꢁtD: ꢀ27.7 (dioxane, 10 mg/mL). Mp. 216–
m
: 3421, 2925, 2854, 1459, 1376, 719, 1043 cmꢀ1
.
1H NMR (DMSO-d6) d: 0.67(s, 3H, 18-CH3), 0.88 (m, 1H, 14-H),
1.034 (s, 3H, 19-CH3), 1.86 (dd, J = 12.0, 1H, 4b-H), 3.30 (s, 1H,
6a-H), 3.65 (s, 1H, 5a-OH), 3.80 (m, 1H, 3a-H), 4.16 (d, J = 5.7,
1H, 3b-OH), 4.40 (d, j = 4.0, 1H, 6b-OH). 13C NMR (DMSO-d6) d:
16.30 (CH3), 17.40 (CH3), 20.14 (CH2), 20.75 (CH2), 25.20 (CH2),
30.36 (CH), 31.12 (CH2), 32.09 (CH2), 34.85 (CH2), 37.92 (CH2),
38.68 (C), 40.05 (CH2), 40.55 (CH2), 40.91 (C), 44.88 (CH), 53.87
(CH), 65.75 (CH), 74.16 (CH), 74.30 (C). EI-MS m/z: 308 (M+), 290
(M-H2O) , 272 (M-2H2O), 257 (M-2H2O–CH3). Analysis calculated
for C19H32O3 C 74.03, H 8.51; found C 74.09, H 10.54.
2.2. Biological assays
10 mg/ml). Mp. 132–136 °C. IR (KBr)
1058, 2933, 1058 cmꢀ1 1H NMR (DMSO-d6) d: 0.71 (s, 3H, 19-
CH3), 0.88 (m, 1H, 14-H), 0.95 (m, 1H, 9-H), 1.00 (s, 3H, 18-CH3),
1.38 (m,1H,8-H), 1.58 (m,1H, 7b-H), 1.62 (m, 1H, 2 -H), 1.84 (m,
1H, 2b-H), 2.0 (m, 1H, 7
-H), 2.25 (m, 2H, 4-CH2), 13C NMR
m: 3242, 1453, 1058, 2933,
.
2.2.1. Primary cultures
New born Sprague–Dawley rats were provided by Experimental
Animal Center of Sun Yat-sen University. Neurobasal A medium,
fetal bovine serum (FBS), B27 Supplements, GlutaMAX, Trypsin
solution, Penicillin–Streptomycin (P/S) was purchased from
GibcoÒ, Life Technologies. Other chemicals, if not mentioned
particularly, were all purchased from Sigma–Aldrich. Cortical
neurons (CNs) were prepared from new born Sprague–Dawley rats
as described previously [16] with little modification. Briefly, the
cortex was dissected and placed in ice-cold aseptic dissection solu-
tion (DS). After mincing, place the tissue in DS containing 0.25%
a
a
(DMSO-d6) d: 17.22 (CH3), 19.39 (CH3), 20.47 (CH2), 21.11 (CH2),
25.59 (CH2), 31.63 (CH), 32.17 (CH2), 32.21 (CH2), 36.61 (C),
37.32 (CH2), 38.69 (CH2), 40.25 (CH2), 40.54 (C), 42.28 (CH2),
50.43 (CH), 54.83 (CH), 71.68 (CH), 121.62 (CH), 140.76 (C); 3.5
(m,1H,3a
-H), 5.34 (t, 1H,6-H). EI-MS m/z: 274(M+), 259 (M-CH3),
256 (M-H2O), 241 (M-CH3–H2O). Analysis calculated for C19H30
O
C 83.50, H 11.02; found C 83.43, H 10.99.

998
J. Chen et al. / Steroids 78 (2013) 996–1002
trypsin for 15 min at 37 °C. Digestion was terminated by DS con-
taining 10% FBS followed by centrifuging for 5 min at 1500 rpm
and the supernatant was discarded. The cell pellet was resus-
pended in DS containing DNase l (8 mg/ml), Mg2+ and FBS, and
homogenized by pipetting up and down for about 20 times. After
sitting for 15 min, the supernatant was centrifuged for 5 min at
1500 rpm. The cell pellet was then collected and resuspended in
Neurobasal A medium supplemented with 10% FBS, 2% B27,
2 mM GlutaMAX and 2 mM P/S. Cells were then seeded with den-
sity of 1 ꢂ 106 cells/ml into poly-l-lysine (0.5 mg/ml) coated
dishes. Cells were incubated in a CO2 chamber. Medium was re-
placed with non-serum formula 4 h after seeding, afterwards half
medium was changed in every 3–4 days.
2.2.6. ROS formation
Intracellular levels of reactive oxygen species (ROS) was de-
tected by a fluorescent marker 5-(and-6)-carboxy-20,70-dichlorodi-
hydrofluorescein
Probes™, Invitrogen). Cells were cultured on 96-well plates with
black-wall flat bottom for fluorescent quantitation. After H/R treat-
diacetate
(carboxy-H2DCFDA,
Molecular
ment, cells were incubated with 25 lM of carboxy-H2DCFDA for
30 min at 37 °C in darkness. The microplate was gently washed
for three times in warm HBSS/Ca2+/Mg2+ and mounted with equiv-
alent warm buffer. Fluorescence with excitation 495 nm and emis-
sion 529 nm was then read on a microplate fluorescence reader
(TECAN). Results were expressed as percentage of normoxic
controls.
2.2.2. Hypoxia/reoxygenation exposure and drug treatment
2.3. Statistic analysis
Triol was dissolved in 20% hydroxypropyl-beta-cyclodextrin
(HPBCD) solution (Xi’an Deli Biological Chemistry, China) in differ-
ent concentrations as stock. At 8 day in vitro (DIV), cultures were
pretreated with different concentrations of triol (0.1, 0.3, 1.0,
ANOVA followed by Holm–Sidak post hoc tests were used for
MTT reduction, MMP, ATP levels and ROS formation analysis. Dif-
ferences at P < 0.05 were considered statistically significant.
3.0 lM), or vehicle with a concentration equaling that in the high-
est triol dose group half an hour before hypoxia/reoxygenation
exposure. For comparative study on the effects, cultures were pre-
3. Results
treated with DHEA or triol at the concentrations of 0.1 and 3.0 lM
3.1. Chemistry
or vehicle half an hour before H/R exposure. Cultures were then
placed in a hypoxic chamber (CoyLab, USA) perfused with 1% O2,
5% CO2 and 94% N2 for at 37 °C for 24 h then removed to the regular
CO2 chamber for 3 h for reoxygenation. Control cultures were incu-
bated under normoxic condition in the regular incubator men-
tioned above for the corresponding duration.
The synthetic route from DHEA (1) to the target compound and-
rost-3b,5a,6b-triol (4) is shown in Scheme 1. The first reaction
known as Wolf–Kishner–Huang Reaction, was optimized to
achieve better yields as high as 95.8%. The double bond of com-
pound 2 was oxidized to form 5a,6b-dihydroxy by H2O2/formic
acid, meanwhile 3b-hydroxy and 6b-hydroxy were esterified to
form 3b,6b-diformate which were eliminated after by alkaline
2.2.3. Neuronal viability
Neuronal viability was measured by (3-(4,5-dimethylthiazolyll-
2)-2,5-diphenyltetrazolium bromide) (MTT) assay. MTT reduction
was measured in each group after H/R exposure and data was pre-
sented as percentage of normoxic control group. Fluorescein diac-
etate (FDA) and propidium iodide (PI) were used as indicators of
living and dead cells, respectively. Images were acquired from a
fluorescent microscope.
reaction. The 5a-hydroxy was not esterified for steric hindrance.
Ultimately the target compound 4 with high purity (99.51%) was
obtained by recrystallization. The characteristic features of DHEA
include the appearance of the 13C NMR peak at 220.08 (17-C),
71.76 (3-CH), 121.95 (6-CH), 140.93 (5-C). The disappearance of
13C NMR peak of compound 2 at 220.08 (17-C), together with the
appearance of 13C NMR peak at 40.54 (17-CH2) implied that 17-
carbonyl was reduced to methylene. The disappearance of the
13C NMR peak at 121.95 (6-CH), 140.93 (5-C) and the appearance
of 13C NMR peak at 74.83 (5-C), 75.95 (6-CH) illustrated that the
2.2.4. Mitochondrial membrane potential
5,50,6,60-Tetrachloro-1,10,3,30-tetraethylbenzimidazol carbocya-
nine iodide (JC-1, Sigma–Aldrich) was loaded into cultured neurons
as a MMP fluorescent probe. Briefly, primary cortical neurons were
seeded onto 35 mm dishes for imaging and black-walled 96-well
plates with flat bottom (Nunc) for fluorescent quantitation. After
5,6-double bonds formed 5a
,6b-dihydroxy; the appearance of 13C
NMR peak at 160.28 (20-C), 160.74 (21-C) indicated the formation
of 3,6-diformate which was not detected in compound 4 and the
appearance of 65.75 (3-CH), 74.16 (6-CH), 74.30 (5-C) and 308
(M+) from MS analysis proved the formation of the target com-
H/R treatment, cultures were incubated with 5 lM of JC-1 for
pound 5
a-androst-3b,5,6b-triol (4). The Partition Coefficient (logP)
20 min at 37 °C then washed twice with phenol free Neurobasal
A medium. Images were then acquired from a fluorescent micro-
scope (Olympus) using a ‘‘dual-bandpass’’ filter. Fluorescent values
were read on a microplate fluorescent reader (Molecular Devices,
USA) at excitation 488 nm and emission 600 nm for red fluores-
cence or 530 nm for green fluorescence. Results were expressed
as red/green ratio and the ratio was normalized by comparison
with the normoxic control group.
of triol is 3.31, indicating that triol is lipophilic.
3.2. Neuroprotective effect of 5a-androst-3b,5,6b-triol (triol)
3.2.1. Triol was neuroprotective against H/R exposure
Twenty-four hours of hypoxia followed by 3 h of reoxygenation
resulted in visible morphological changes and cell death in primary
cortical cultures (Fig. 1). In the normoxic control group, cortical
neurons were showing well-rounded cell bodies with intact neu-
rites forming a complete network, after H/R exposure, fractured
neurites were seen. Live/dead staining by FDA/PI showed a de-
crease in intact living cells (green) and an increase in dead cells
(red). Pretreatment with triol improved neuronal survival (in-
creased FDA stained cell number and decreased PI stained cell
number), and protected the morphology of cultured neurons from
H/R exposure, with the maximum effect at a concentration of
2.2.5. ATP levels
Intracellular ATP levels were detected with an ATP Determina-
tion Kit (Molecular Probes™, Invitrogen) according to the manufac-
turer’s instruction. Briefly, after H/R treatment, cultures in each
group were treated with 100 lL cell lysis buffer (Thermo). Cell ly-
sates were then moved to a white-walled 96-well plate (Nunc)
with flat bottom, and incubated with luciferin and luciferase for
15 min in darkness. Luminescence was then read from a micro-
plate luminometer (TECAN) with an emission 560 nm. Results
were expressed as reduction percentage of normoxic controls.
3
lM. Consistently 24 h of hypoxia followed by 3 h of reoxygena-
tion resulted in a decline in neuronal viability by about 40.0%

J. Chen et al. / Steroids 78 (2013) 996–1002
999
Scheme 1. (a) KOH, diethylene glycol, hydrazine hydrate, 210 °C. (b) H2O2, HCOOH, RT. (c) NaOH, MeOH, 60 °C.
Fig. 1. Protective effects of triol on hypoxia/reoxygenation (H/R)-induced neuronal injuries in primary cultured cortical neurons. (a) Representative phase contrast and FDA/PI
stained images. 24 h Hypoxia followed by 3 h reoxygenation (H/R) induced morphological alterations including mild shrinkage of cell bodies and fracture of neurites. FDA/PI
staining showed a decrease in live cells (green) and increase in dead cells (red). Treatment with triol alleviated morphological damages. FDA/PI staining revealed a raised
neuronal survival rate in triol treated groups. (b) The statistic graph of neuronal viability by MTT assay. H/R significantly reduced neuronal viability and treatment with triol
enhanced neuronal viability in a dose-dependent manner. Data was presented as mean S.D. from seven independent experiments. ⁄⁄⁄P < 0.001 vs. control group; #P < 0.05,
##P < 0.01 ###P < 0.001 triol pretreated groups vs. H/R group. Bar = 20
lm. (For interpretation of color in this Figure, the reader is referred to the web version of this article).
compared with normoxic control. In contrast, neurons pretreated
with triol were showing better cell viability in a dose-dependent
manner with the maximum effect reaching 90.0% of normal
control.
3.2.3. Triol ameliorated mitochondrial dysfunction
3.2.3.1. Triol alleviated loss of MMP after H/R. MMP is an early surro-
gate biomarker and a commonly used indicator for mitochondrial
function. JC-1 used in this study is a voltage sensitive probe to
monitor MMP, when MMP polarizes, JC-1 accumulates in the mito-
chondrial matrix as a polymer (red), while if MMP depolarizes, JC-1
becomes a monomer and locates in the cytoplasm (green). As
shown in Fig. 3a, in normoxic control group, red fluorescence
markedly dominated green fluorescence, cells were showing a
bright orange color. After H/R exposure, there was a significant
increase in green fluorescence (orange turned partly green),
suggesting loss of MMP, while cultures pretreated with triol were
showing an enhancement of orange fluorescence consistent with
the increase of triol doses, indicating an alleviation of MMP loss
in these cultures. Similar effect was not seen in the cultures
pretreated with DHEA.
3.2.2. Triol exhbited stronger neuroprotection compared to DHEA
against H/R-induced injuries
DHEA is a well known neurosteroid with various neurobiologi-
cal activities [17]. We next compared the neuroprotective effects of
triol and DHEA in the above-mentioned H/R injury model. We ob-
served better cell morphology and higher survival rate in the cul-
tures pretreated with triol compared with cultures pretreated
with DHEA at the same concentration level (Fig. 2a). Cell viability
obtained by MTT assay revealed that DHEA was neurprotective
against H/R exposure at the concentration of 3.0
lM, however this
effect was much weaker compared to triol (Fig. 2b).

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J. Chen et al. / Steroids 78 (2013) 996–1002
Fig. 2. Comparison of neuroprotective effects between triol and DHEA against H/R exposure (a) Representative phase contrast and FDA/PI stained images. Notably, cultures
treated with triol are showing better morphology (phase contrast and FDA staining) and fewer dead cells (PI staining) compared to cultures treated with the same
concentration of DHEA (3.0
high doses of triol, but only high dose of DHEA with weaker effect. Data was presented as mean S.D. from four independent experiments. ⁄⁄⁄P < 0.001 vs. control group;
#P < 0.05, ##P < 0.01, ###P < 0.001 triol/DHEA pretreated groups vs. H/R group. Bar = 50
m.
lM). (b) The statistic graph of neuronal viability by MTT assay. H/R significantly reduced neuronal viability, which was improved by both low and
l
Fig. 3. Amelioration of mitochondrial dysfunction in triol and DHEA pretreated neurons after H/R. (a) Representative images of JC-1 stained neurons. Mitochondrial
membrane potential (MMP) was detected by a fluorescent probe JC-1 and images were acquired from a fluorescent microscope with a ‘‘dual-bandpass’’ filter. In the control
group, cells were showing a bright orange color and H/R exposure resulted in a turn from orange partly to green, indicating loss of MMP, while in triol pretreated neurons, red
fluorescence was enhanced, indicating an improvement of MMP loss. Similar effect was not seen in DHEA pretreated cultures. (b) Statistic graph of MMP. Fluorescence values
of JC-1 were obtained from fluorescent microplate reader, then calculated as red/green fluorescence ratio and normalized as percentage of the control group. Data was
presented as mean S.D. from three independent experiments. (c) Intracellular ATP levels. ATP levels were detected by a luciferin and luciferase assay. Data was presented as
mean S.D. from four independent experiments. ⁄⁄⁄P < 0.001, control vs. H/R group; #P < 0.05, ##P < 0.01, ###P < 0.001 triol/DHEA pretreated groups vs. H/R group.
Bar = 20 lm. (For interpretation of color in this Figure, the reader is referred to the web version of this article).

J. Chen et al. / Steroids 78 (2013) 996–1002
1001
Quantification performed in the fluorescent microplate reader
presented consistent results (Fig. 3b). H/R exposure reduced red/
green fluorescence ratio by almost 40.0%, while treatment with
triol improved loss of MMP in a dose-dependent manner. In the
DHEA pretreated groups, no statistical significance was shown.
neuroprotective at the concentration of 3.0
l
M however, the effect
was much weaker compared to that of triol. Meanwhile, there was
a consistent improvement in triol pretreated groups in H/R-in-
duced MMP loss and disruption of ATP production, indicating that
regulation of mitochondrial function may be involved in the mech-
anism of action. Intracellular ROS formation, a commonly used
indicator of oxidative stress, was also reduced in triol pretreated
neurons, suggesting that triol also acts by reducing oxidative stress
during H/R exposure. Also we found that both in terms of protec-
tion of mitochondrial function and inhibition of oxidative stress,
triol exhibited notable advantages over DHEA.
3.2.3.2. Triol preserved intracellular ATP levels. Mitochondria are the
main source of ATP production, which reflects directly mitochon-
drial respiratory function. Therefore we next examined the intra-
cellular ATP levels. In the H/R challenged cortical neurons,
intracellular ATP levels were reduced by almost 40% (Fig. 3c) com-
pared to normoxic controls. Intracellular ATP levels in the cultures
were increased significantly during H/R when both treated with
It has been well demonstrated that H/R leads to massive distur-
bance in energy metabolism involving a series of cellular events
and causes oxidative stress which is also very complicated process.
In our study, particular attention was given to mitochondria in vir-
tue of its irreplaceable role in energy metabolism. It is very hard to
directly and quantitatively measure mitochondrial function, for
this reason, served as a surrogate marker, MMP has been com-
monly used to indicate mitochondrial functional alterations. Loss
of MMP during H/R was evident suggesting mitochondrial dysfunc-
tion, whereas in the triol pretreated groups MMP was retrieved,
that is, mitochondrial dysfunction was alleviated. Meanwhile it is
documented that hypoxic insult depolarizes mitochondria and
consequently impacts ATP production by inhibition of oxidative
phosphorylation [18]. In our study there was an enhancement of
intracellular ATP levels in triol pretreated cultured neurons, show-
ing that energy supply was preserved. Noteworthily, intracellular
ATP levels reflect a combination of production and consumption.
It needs further investigation to determine whether triol acts di-
rectly by promoting mitochondrial respiration. Furthermore, mito-
chondrial dysfunction is linked to oxidative stress, together they
act as determinants of cell death or survival in stroke [19]. Oxida-
tive stress can be defined as an imbalance between ROS production
and/or impaired ROS metabolism that leads to an excessive intra-
cellular ROS accumulation [20]. Consistent with previous reports,
the intracellular ROS level was remarkably increased after H/R
exposure whereas a significant reduction was seen in triol pre-
treated groups, which means that triol also reduces oxidative
stress during H/R. Ulteriorly, it will be interesting to determine
the specific role of triol in attenuating oxidative stress during H/R.
There is another concern that although it has been well estab-
lished that inhibition of mitochondrial respiration causes electron
leak to generate ROS in response to hypoxia [8,21,22], during dif-
ferent phases of H/R, ROS generation or oxidative stress is of con-
siderable complexity [5,23–26], thus a more specific look into
this issue seems necessary in further ascertaining that triol pro-
tects neurons against H/R by targeting specific mitochondrial sites.
Notwithstanding, maintenance of MMP, ATP production and ROS
generation are closely linked events with mitochondria being
headquarters, and our experimental data overall supported that
triol protected cultured neurons against H/R injury by blocking
or ameliorating the occurrence of detrimental mitochondrial
events, yet the exact molecular mechanisms especially the direct
target of triol need further investigation.
low (0.1
lM) and high (3.0 lM) of triol. At the concentration of
3.0 M, DHEA also statistically increased the intracellular ATP le-
l
vel, however this effect was much weaker compared to that of triol.
3.2.4. Triol inhibited accumulation of ROS
Intracellular ROS level is a commonly used indicator of oxida-
tive stress. Here we incubated cortical neurons with carboxy-H2-
DCFDA and fluorescent intensities were read and normalized as
percentage of control. As shown in Fig. 4, oxidative stress in H/R
treated neurons was evident, ROS level enhanced to almost 1.7-
fold of control group, and treatment with triol reduced ROS accu-
mulation in a dose-dependent manner. At the concentration of
3.0
trol group in the triol pretreated group. Notably, treatment with
3.0 M of DHEA also significantly reduced the intracellular ROS
lM, intracellular ROS level reduced to almost 1.2-fold of con-
l
accumulation during H/R, however, with a much weaker effect.
4. Discussion
Development of novel neuroprotectants has been indispensable
in the treatment of ischemic stroke for the limitations of current
therapies. In the present study DHEA was used as a starting drug
to obtain the target compound 5a-androst-3b,5,6b-triol (triol)
from 3 steps of reaction with an overall yield as high as 76.0%.
We herein for the first time report the bioactivity of triol in a well
established H/R injury model which has been used an in vitro
ischemic stroke model, and triol exhibited robust neuroprotective
effect in primary cultured cortical neurons against H/R exposure.
As a starting material, DHEA itself is known to exert neuroprotec-
tive effects although its effect on H/R-induced neuronal injury is
controversial [17]. In our model, we found that DHEA was
Finally, logP is a crucial parameter in the mathematical predic-
tion of membrane structure penetration of chemicals, and the logP
value of triol is 3.31, which indicates that triol is lipophilic in nat-
ure and is expected to across the blood–brain barrier and other
membrane structures. Taken together, triol is conclusively of great
potential as a pharmaceutical candidate in the treatment of acute
ischemic stroke.
Fig. 4. Reduction of intracellular ROS formation in triol and DHEA pretreated
neurons after H/R exposure. ROS levels were detected by a fluorescent probe
H2DCF-DA (DCF). Data were from three independent experiments as mean S.D.
⁄⁄⁄P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 triol/DHEA pre-
treated groups vs. H/R group.
Acknowledgements
This work was supported by South China Comprehensive Plat-
form for New Medicine R&D (Project No. 2009ZX09301-015), and

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J. Chen et al. / Steroids 78 (2013) 996–1002
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