The protective effects of a novel synthetic β-elemene derivative on human umbilical vein endothelial cells against oxidative stress-induced injury: Involvement of antioxidation and PI3k/Akt/eNOS/NO signaling pathways

Article abstract of DOI:10.1016/j.biopha.2018.07.107

Antioxidant therapy is considered as promising strategy for treating oxidative stress-induced cardiovascular disease. Bis (β-elemene-13-yl) glutarate (BEG) is a novel β-elemene derivative. Herein, we examined the antioxidant activity of BEG on human umbil

Full text of DOI:10.1016/j.biopha.2018.07.107

Biomedicine & Pharmacotherapy 106 (2018) 1734–1741
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
The protective effects of a novel synthetic β-elemene derivative on human
umbilical vein endothelial cells against oxidative stress-induced injury:
Involvement of antioxidation and PI3k/Akt/eNOS/NO signaling pathways
a,c
a
b
c
a
b
Khalil Ali Ahmad , Hong Ze , Jichao Chen , Farhan Ullah Khan , Chen Xuezhuo , Jinyi Xu ,
Ding Qilong
a
a,⁎
Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Longmian Avenue, 639, Nanjing, Jiangsu, 211198, China
State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, China
Shanghai Jiao Tong University, School of Pharmacy, 800 Dongchuan Road, Shanghai, 200240, China
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bis (β-elemene-13-yl) glutarate
Antioxidant activity
Vascular endothelial cells
Reactive oxygen species
Endothelial nitric oxide synthase
Nitric oxide
Antioxidant therapy is considered as promising strategy for treating oxidative stress-induced cardiovascular
disease. Bis (β-elemene-13-yl) glutarate (BEG) is a novel β-elemene derivative. Herein, we examined the anti-
oxidant activity of BEG on human umbilical vein endothelial cells (HUVECs) after injury with hydrogen peroxide
(H
2
O
2
) and investigated the mechanism involved. HUVECs were divided into the following groups: control group
untreated cells); treated groups (cells treated with 0.1, 1, 10 μmol/L of BEG); positive control group (cells
alone). Cells were pre-incubated
with or without BEG for 24 h and then incubated for a further 2 h with 0.5 mM H . Our results showed that
BEG significantly reduced H induced loss in endothelial cell viability, reactive oxygen species (ROS) pro-
(
2 2
treated with 0.1 mM Vitamin E); model group (cells treated with 0.5 mM H O
2 2
O
2 2
O
duction, reduced lactate dehydrogenase (LDH) release, and malonyldialdehyde (MDA) level in a concentration-
dependent manner. Also, BEG increased the cellular the superoxide dismutase (SOD) activity. Moreover, we
found that H
production. These effects induced by H
inhibited by a PI3K inhibitor (wortmannin) and eNOS inhibitor (L-NAME). In conclusion, the present study
demonstrated that BEG has antioxidant activity. Furthermore, BEG reduced H -induced endothelial cells in-
jury by the involvement of antioxidation and PI3K/Akt/eNOS/NO signaling pathways.
2
O
2
decreased Akt and eNOS phosphorylation, which perhaps, indirectly reduced nitric oxide (NO)
2 2
O , however, were reduced by pre-treatment with BEG. BEG effects were
2 2
O
1. Introduction
phosphorylation is the phosphatidylinositol 3-kinase (PI3K)-Akt
pathway [7,8]. β-elemene, a sesquiterpene compound isolated from the
essential oil of Curcuma Wenyujin, is a traditional Chinese medicine
(TCM) [9]. β-elemene has been approved by the China Food and Drug
Administration for human cancer treatment [10]. Recently, studies in
endothelial cells damaged by oxidative stress have demonstrated that β-
elemene exerts moderate antioxidant activity against oxidative injury
and in vivo, it inhibits neointimal hyperplasia after vascular injury
Oxidative stress occurs from an imbalance between the oxidant and
the antioxidant defense systems subsequent to physiologic stressors,
disease, or environmental factors [1–3]. Substantial evidence suggests
that increase oxidative stress plays an essential role in the pathogenesis
of cardiovascular diseases including atherosclerosis, and hypertension
[
4]. Nitric oxide (NO) plays an important role in maintaining cardio-
vascular homeostasis [5]. NO wields many vaso-protective effects such
as vasodilatation, anti-inflammatory and anti-aggregation actions to
suppress the lipid oxidation and vascular smooth muscle proliferation
2 2
[11]. Furthermore, it prevents H O -induced monocyte-endothelial
cells interactions in vitro and reduces atherosclerosis development in
vivo [12,13]. Our previous studies showed that β-elemene derivatives
possess antioxidant activities more than β-elemene and the positive
[
6]. Endothelial nitric oxide synthase (eNOS) has been discovered to be
phosphorylated by some kinases such as PKA, Akt, AMPK, PKC, Ca2/
calmodulin-dependent kinase II, etc [7]. Furthermore, studies have
reported that the best-characterized pathway that regulates eNOS
2 2
control vitamin E in HUVECs damaged by H O [14,15].
Though β-elemene (Fig. 1A) has limited bioavailability due to poor
water solubility, rapid clearance and short half-life, the introduction of
⁎
Corresponding author.
E-mail address: dqlcpu@163.com (D. Qilong).
https://doi.org/10.1016/j.biopha.2018.07.107
Received 8 May 2018; Received in revised form 17 July 2018; Accepted 18 July 2018
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.

K.A. Ahmad et al.
Biomedicine & Pharmacotherapy 106 (2018) 1734–1741
Fig. 1. Chemical structure of β-elemene (A) and Bis (β-elemene-
1
3-yl) glutarate (B).
a hydrophilic group such as oxygen or nitrogen-containing groups into
the skeleton of b-elemene could favorably impact its water solubility
and activity [14]. Bis (β-elemene-13-yl) glutarate (Fig. 1B), a novel β-
elemene derivative, was designed and synthesized to increase the an-
tioxidant activity of the parent compound and to improve its pharma-
cological properties. In this study, we examine its antioxidant activity,
and the potential antioxidant mechanisms involved in oxidative stress-
induced HUVECs damage.
J = 3.7 Hz, 2H), 4.81 (s, 2H), 4.76 (s, 2H), 4.59 (s, 4H), 4.52 (s, 2H),
3.39 (s, 2H), 2.00 – 1.90 (m, 4H), 1.64 (s, 6H), 1.60 – 1.50 (m, 3H), 1.46
– 1.32 (m, 6H), 0.93 (s, 6H). HRMS (ESI) calculated for C33
+H] 509.3625, found 509.3612.
H
49
O
4
[M
+
2.3. Cell culture and treatment
Human umbilical vein endothelial cells (HUVECs) were obtained
from the center for new drug safety evaluation and research, school of
pharmacy, China pharmaceutical university and maintained in
Dulbecco’s modified Eagle medium supplemented with 10% fetal bo-
vine serum (Gibco), 100 U/ml penicillin, 100 U/ml streptomycin. Cells
2. Materials and methods
2.1. Materials
2
were incubated at 37 C with 95% humidity and 5% CO . The medium
Hydrogen peroxide (H
2
O
2
), 3-(4,5-dimethyl– thiazol-2-yl)- 2,5-di-
was changed every 2–3 days until the cells reached (∼90) confluence
and cells between 4 and 8 passages were used for the experiments.
HUVECs were divided into the following experimental groups: 1) con-
trol group, untreated cells; 2) treated group, cells pretreated with dif-
ferent concentrations of BEG (0.1, 1 and 10 μmol/L) for 24 h; 3) positive
control group, cells incubated with Vitamin E 0.1 mM for 24 h; and 4)
phenyl tetrazolium bromide (MTT), dimethylsulfoxide (DMSO) were
purchased from (Nanjing KeyGen Biotech. Co.Ltd, Nanjing, China).
Nitric oxide (NO), lactate dehydrogenase (LDH), malondialdehyde
(
MDA) and superoxide dismutase (SOD) commercial kits were obtained
from Jiancheng Bioengineering Institute (Nanjing, China). 2,7-di-
chlorofluorescein diacetate (DCFH-DA), BCA protein assay kit, phe-
nylmethylsulfonyl fluoride (PMSF) and Ripa lysis buffer were pur-
chased from Beyotime (Jiangsu, China). Quantitative RT-PCR kit and
SYBR Green Premix Ex Taq were obtained from Takara Biomedical Inc
Japan). NG-nitro-L-arginine methyl ester (L-NAME), and PI3K inhibitor
wortmannin were from Sigma (MO, USA). eNOS, phospho-eNOS
Ser1177), Akt, p-Akt (serine 473) and β-actin antibodies and horse-
radish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody were
purchased from Santa Cruz Biotechnology Inc (USA). Dulbecco’s mod-
ified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were ob-
tained from Gibco (MD, USA).
model group, cells incubated with 0.5 mM H
treated and positive control groups were further induced by incubation
with 0.5 mM H for 2 h, while the control group was kept untreated.
In western blot experiments, HUVECs were pre-incubated with eNOS
for 1 h and PI3k/Akt inhibitors. H (0.5 mM) and Vitamin E (0.1 mM)
2 2
O alone for 2 h. The
2 2
O
(
2 2
O
concentrations were determined from the pilot study by our research
group and based on similar methods described previously [12,14 and
16].
(
2.4. MTT assay of HUVECs
MTT cell assay was performed to study the cytotoxic effect of BEG in
2.2. Chemical synthesis and physicochemical properties of BEG
HUVECs. Cells were seeded in 96-well plates at a density of
3
5
× 10 cells/well for 24 h. After 24 h incubation, cells were treated
The chlorinated β-elemene 2 was prepared by chlorination of β-
with different concentrations of BEG (0.1, 1, 10, 50, 100, 500 and
1000 μmol/L) and the control group was treated only with DMEM for
3
elemene 1 with NaClO, followed by treatment with CH COONa to give
the acylated compound 3. Then 3 was undergone alkaline hydrolysis to
produce 13-β-elemol 4, which was further reacted with glutaric anhy-
dride in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
2
24 h at 37 °C in 5% CO incubator. MTT solution (50 μL) was added to
each group and cells were incubated at 37 °C for a further 4 h. After 4 h
incubation, DMEM containing MTT solution was discarded. Cells were
then dissolved by adding (200 μL) DMSO to each well and the solutions
were mixed thoroughly for 5 min. Finally, the absorbance was de-
midehydrochloride (EDCI)/4-N,N-dimethylaminopyridine(DMAP) to
1
yield the target compound 5 (Fig. 2). H NMR (300 MHz, CDCl
3
) δ 5.74
(
dd, J = 17.7, 10.5 Hz, 2H), 5.00 (s, 2H), 4.95 (s, 2H), 4.86 (d,
termined at 570 nm with a microplate spectrophotometer (BD
Fig. 2. Chemical synthesis of Bis (β-elemene-13-yl) glutarate.
1735

K.A. Ahmad et al.
Biomedicine & Pharmacotherapy 106 (2018) 1734–1741
Bioscience, San Jose, CA, USA). The absorbance of untreated cells was
regarded as 100% of cell survival. Cell Viability = (treated viable cells)
and the protein concentration of each sample was determined using a
BCA Protein Assay kit (Pierce, US) according to the manufacturer in-
structions. The lysates were denatured by boiling them in the SDS
sample buffer. Then equal amounts of protein (50 g) from each group
were loaded onto 10% SDS-PAGE and transferred to PVDF membranes
/
(non-treated control viable cells) × 100%.
2 2
2.5. Cell viability assay of HUVECs damage by H O
(
Millipore, Billerica, MA, USA). The membranes were blocked for 1 in
To study the protective effects of BEG in HUVECs damaged by
5% phosphate buffered saline (PBS) containing 0.1% (v/v) tween 20
and 5% non-fat dry milk and then incubated overnight at 4 °C with
primary antibody against eNOS or phospho-eNOS or Akt, or phospho-
Akt Ser 473, and β-actin. The dilution factor for the primary antibodies
against 1:500 eNOS, 1:500 phospho-eNOS,1: 1000 Akt, 1: 1000 p-Akt
and 1: 1000 β-actin. After washing, the membranes were incubated
with the anti-IgG secondary antibody (1:2000) conjugated to horse-
radish peroxide for 1 h at room temperature. Then the membranes were
washed in TBST for 30 min and exposed to enhanced chemilumines-
cence reagents. Densitometric analysis was performed to quantify the
signal intensity. β-actin was used as a standard reference. The relative
density of each protein band was normalized to β-actin.
oxidative stress, MTT cell assay was used as described above with some
modification. Cells were seeded in 96-well plates at a density of 5 × 103
cells/well. After 24 h incubation, HUVECs were pre-treated with dif-
ferent concentrations of BEG (0.1, 1, 10 μM) and 100 μM Vitamin E
(
positive control) for 24 h at 37 °C in 5% CO
cells were treated with 0.5 mM H for further 2 h. The model group
alone for 2 h while the control
2
incubator. Then, these
2 2
O
cells were incubated with 0.5 mM H
2 2
O
group was kept untreated.
2
.6. Measurement of LDH, NO, SOD and MDA
The contents of LDH, NO, SOD and MDA were determined by fol-
lowing the manufacturer’s instructions (Nanjing Jiancheng
Bioengineering Institute Nanjing, China). All procedures entirely de-
scribed previously [11,17]. The levels of LDH, NO, SOD and MDA were
expressed as unit’s micromoles per liter supernatant, units per liter
supernatant, per milligram protein and nanomoles per milligram pro-
tein, respectively. The absorbance of LDH, NO, SOD and MDA were
measured at wavelengths of 440, 550, 550 and 530 nm respectively.
Each experiment was performed in triplicate.
2.10. Statistical analysis
Statistical analysis was performed using GraphPad Prism Version
7.00 (GraphPad Software, Inc.). Data were expressed as means ± S.D
from three independent experiments. The ANOVA test was used to
determine the statistical significance. The P value (p < 0.05) was
considered significant among all the analysis.
3. Results
2.7. Reactive oxygen species (ROS) assay
3.1. Cytotoxic effect of BEG in HUVECs
Intracellular ROS generation was investigated using ROS detection
kit following the manufacturer instructions. Briefly, HUVECs were in-
cubated with different concentrations of BEG for 24 h and then stimu-
lated with 0.5 mM H O for further 2 h, the control group was left
2 2
untreated. After that, HUVECs were labeled with 10 μM DCFH-DA and
incubated at 37 °C for 30 min. After three times washes with PBS, the
DCFH fluorescence of the cells from individual cells was measured at
excitation and emission wavelengths, 485 nm and 530 nm respectively,
Cytotoxicity of BEG in HUVECs was investigated using MTT assay.
Our results indicated that incremental doses of BEG treatment for 24 h
did not produce any obvious cytotoxic effect (Fig. 3A). Therefore, doses
of 0.1 μmol/L, 1 μmol/L, and 10 μmol/L of BEG were used in the sub-
sequent experiments for examining its possible protective role against
H
2
O
2
induced toxicity.
using
a
fluorescence microplate reader (Tecan, Männedorf,
3.2. Effect of BEG on H O induced HUVECs damage
2 2
Switzerland). ROS fluorescent intensity index was expressed as the
percentage of the control group fluorescence intensity. Each experiment
was performed in triplicate.
In order to evaluate whether BEG protects HUVECs against oxida-
tive stress, cell viability was measured by MTT assay. As shown in
(
Fig. 3B), the control group did not show any significant difference
while the model group incubated with H alone for 2 h decreased the
cell viability to 63%. In the treated group, treatment of BEG reduced the
cell growth inhibition caused by H and increased the cell viability in
2
.8. Quantitative real-time PCR analysis of mRNA expression
2 2
O
Quantitative real-time PCR was used to detect eNOS mRNA ex-
2 2
O
pression in each group. HUVECs were treated with BEG (0.1, 1, 10 μM)
a dose-dependent manner. The cell viability was restored up to
97.6667% by pretreatment with 10 μmol/L of BEG, which was almost
superior to the positive control group (100 μM Vit-E).
for 24 h and then stimulated with (0.5 Mm) H for further 2 h. Total
2 2
O
RNA was extracted by trizol reagent according to the manufacturer
instructions (Invitrogen). cDNAs were synthesized with Prime Script RT
Master Mix kit (Takara, Japan) following the manufacturer instructions.
cDNAs were used for PCR amplification using SYBR Premix Ex TaqTM
2 2
3.3. Effect of BEG on H O induced LDH leakage in HUVECs
(
Takara, Japan) according to the manufacturer instructions on ABI
Increased LDH release from HUVECs into the culture medium also
Prism 7900 Sequence Detection System (Applied Biosystems, CA, USA).
β-actin was used as an internal control. Primer sequences were as fol-
lows: 5′-AAG ATC TCC GCC TCG CTC A-3′(sense) and 5′-GCT GTT GAA
GCG GAT CTT A-3′(antisense) for eNOS; 5′-CGC AAA GAC CTG TAC
GCC AAC-3′ (sense) and 5′-CAC GGA GTA CTT GCG CTC AGG-3′(an-
tisense) for β-actin.
reflects cells damage. Therefore, LDH leakage was investigated to fur-
ther confirm the protective effect of BEG on H O induced HUVECs
2 2
damage. For this purpose, cells were pretreated with different con-
centrations of BEG (0.1, 1, 10 μmol/L) and Vit E 100 μM for 24 h fol-
lowed by further treatment with 0.5 mM H
(Fig. 4A), cells treated with 0.5 mM H O
2 2
2
O
2
for 2 h. As shown in
alone for 2 h significantly
increased cells LDH leakage into the medium, while pretreatment of
2.9. Western blot analysis
HUVECs with various concentrations of BEG inhibited H
LDH leakage in a dose-dependent manner.
2 2
O -induced
Cells were treated as described above. HUVECs were washed twice
with ice-cold PBS and then lysed by using RIPA lysis buffer containing
3.4. Antioxidant effect of BEG against H
2 2
O induced HUVECs damage
1
1
% PMSF at 4 °C. The lysates were centrifuged at 12,000g at 4 °C for
5 min to remove the insoluble materials. Supernatants were collected
SOD activity and MDA content were evaluated to reflect the level of
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K.A. Ahmad et al.
Biomedicine & Pharmacotherapy 106 (2018) 1734–1741
Fig. 3. Effect of BEG on cell viability. (A) Concentration-dependent effect of BEG on cell viability of HUVECs incubated for 24 h. (B) The protective effect of BEG in
HUVECs after H
induced cells damage. MTT assay was used to evaluate the cell viability. Values are presented as means ± SD, n = 3. ##Greater significant
2 2
O
difference from the control group at P < 0.01. *Significant difference from the model group at P < 0.05. **Greater significant difference from the model and VE
groups at P < 0.01.
oxidative damage in HUVECs and to study the antioxidant effect of
BEG. As shown in (Fig. 4B and C) pre-incubation with H alone for
h significantly increased intracellular MDA level and decreased the
2 2
manner. Cells treated with 0.5 mM H O alone for 2 h increased the
intracellular ROS concentration compared with the control group
(P < 0.01). However, pre-incubation with different concentrations of
2
O
2
2
intracellular SOD activity compared to the control group. While treat-
ment with the indicated concentrations of BEG and Vit-E results in-
BEG significantly reduced H
production in cells treated with H
2
O
2
-induced ROS overproduction. ROS
(model group) reduced from
2 2
O
dicated that H
2
O
2
-induced oxidative stress injury was significantly
260.9 ± 19.44 to 191.7 ± 2.64, 173.6 ± 2.476, and 133.1 ± 6.10
in the cells treated with BEG (0.1, 1, 10 μmol/L) respectively. These
results suggest that BEG significantly reduced ROS overproduction in
attenuated by pretreatment with BEG in dose-dependent manner.
2 2
HUVECs after H O induced oxidative stress in a dose-dependent
2 2
3.5. Effect of BEG on ROS production induced by H O
manner.
The intracellular ROS generation was evaluated by the dichloro-
fluorescein (DCFH) assay. Effect of BEG on ROS level in H -treated
cells is shown in Fig. 5A & B. Results indicated that BEG reduced the
ROS generation in HUVECs damage by H in a dose-dependent
2
O
2
3.6. Effect of BEG on NO release in H
2 2
O -treated HUVECs
2 2
O
NO is a molecule that has obtained identification as an important
Fig. 4. Antioxidant Effect of BEG against H
activity in endothelial cells after H
induced cells damage. Values are presented as means ± SD, n = 3. ##Greater significant difference from the control group at
P < 0.01. *Significant difference from the model group at P < 0.05. **Greater significant difference from the model group at P < 0.01.
2 2
O induced HUVECs damage. (A) Concentration-dependent effect of BEG on LDH leakage. (B) MDA level and (C) SOD
2 2
O
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K.A. Ahmad et al.
Biomedicine & Pharmacotherapy 106 (2018) 1734–1741
Fig. 5. Effect of BEG on ROS production NO and eNOS mRNA in endothelial cells after H
2
O
2
induced cell damage. (A) Intracellular ROS generation in HUVECs was
determined by measuring DCFH fluorescence. (B) ROS fluorescent intensity index was presented as the percentage of the control group. (C) Effect of BEG on HUVECs
NO release (D) RT-PCR analysis. Effect of BEG on eNOS mRNA expression. Values are presented as means ± SD, n = 3. ##Greater significant difference from the
control group at P < 0.01. *Significant difference from the model group at P < 0.05. **Greater significant difference from the model group at P < 0.01.
modulator of cardiovascular diseases, and one of the hallmarks of en-
dothelial dysfunction is a reduction in the bioavailability of NO, which
is catalyzed by eNOS [18]. In this study, NO release was assessed. As
shown in Fig. 5C, NO production was significantly decreased in the
model group compared to the control group (P > 0.01). Meanwhile,
pre-incubation with indicated concentrations of BEG and Vit-E for 24 h
3.8. BEG exerts protective effects on endothelial cells through Akt/eNOS
pathways
We further examined the underlying mechanisms by which in-
creased eNOS modulates specific cellular functions. Previous studies
found that ROS regulates eNOS activity and expression via PI3K/Akt
signaling pathway. Akt stimulation leads to eNOS activation and in-
creased the phosphorylation of eNOS (p-eNOS) that in turn increase NO
generation. In this study, we found that, in comparison to other ex-
perimental groups, BEG treated group up-regulated the Akt phosphor-
ylation and the total Akt level (P < 0.05 vs groups 2 and 5) and im-
proved phosphorylation of eNOS (P < 0.05 vs groups 2 and 4) and
total eNOS levels (P < 0.05 vs groups 2 and 3). Furthermore, these
effects were inhibited by PI3K inhibitor wortmannin and eNOS in-
and further incubation for 2 h with H
2 2
O significantly improved NO
production in a dose-dependent manner.
3.7. Effect of BEG on decreased Akt/eNOS expression induced by H
2 2
O in
endothelial cells
To determine the preliminary molecular mechanisms accounting for
the protective effects of BEG on HUVECs against H , we investigated
2
O
2
2 2
hibitor L-NAME (P < 0.01vs BEG + H O group). Therefore, these
the gene expression of eNOS mRNA (Fig. 5D) and the protein expres-
sions of Akt and eNOS (Fig. 6). As shown in (Fig. 5D), treatment with
results further demonstrated that the Akt/eNOS signaling pathway was
involved in the BEG-induced protective effects on HUVECs as shown in
Fig. 7.
2 2
0.5 mM H O for 24 h decreased eNOS mRNA expression compared
with the control group. While eNOS mRNA expression was effectively
improved by pre-treatment with different concentrations of BEG (0.1, 1,
4. Discussion
1
2 2
0 μmol/L) for 24 h and further incubation with 0.5 mM H O for 2 h in
a dose-dependent manner. We also observed that the expression of
eNOS and Akt proteins were enhanced significantly by treatment with
BEG in a dose-dependent manner compared with model group alone.
Oxidative stress influences various biological functions of vascular
endothelial cells and plays a critical role in the pathophysiology of
cardiovascular diseases [19]. High concentrations of oxidative stress,
such as H O is associated with increased vascular endothelial cell in-
2 2
These results showed that BEG protects HUVECs from H O induced
2
2
damage and improved the expression of eNOS mRNA (Fig. 5D), Akt,
and eNOS proteins (Fig. 6).
jury [20]. Therefore, there are increasing interests to use antioxidant
agents against ROS stimulus in cardiovascular disease, which has been
demonstrated in studies of vitamin C, sphingosine 1-phosphate and
propofol [21–23]. An abnormality in ROS production and the sub-
sequent decrease in vascular NO bioavailability have been long
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K.A. Ahmad et al.
Biomedicine & Pharmacotherapy 106 (2018) 1734–1741
2 2
Fig. 6. Effect of BEG on decreased Akt/eNOS expression induced by H O in endothelial cells.
(A) Concentration-dependent effect of BEG on Akt and eNOS protein expressions in endothelial cells after H
2
O
2
induced damage as determined by Western blot
analysis. (B) Relative levels of eNOS protein expression. (C) Relative levels of Akt protein expression. Optical densities were achieved after normalization to β-actin.
Values are presented as means ± SD, n = 3. #Significant difference from the control group at P < 0.05. Greater significant difference from the control group at
P < 0.01. *Significant difference from the model group at P < 0.05. **Greater significant difference from the model group at P < 0.01.
##
suggested to be the common pathogenic mechanism of endothelial
dysfunction that leads to various cardiovascular risk factors such as
hypertension, atherosclerosis, diabetes mellitus [25]. The present study
aimed to examine the antioxidant activity of BEG on vascular en-
dothelial cells damaged by oxidative stress and to investigate the in-
volved mechanisms. Results presented here indicated that BEG in-
creased the survival rate of endothelial cells and possesses significant
viability was diminished to approximately 60% when exposed to
0.5 mM H , while it was enhanced significantly by pre-treatment
with BEG (Fig. 3B). These findings indicated that BEG can effectively
2 2
O
prevent oxidative damage induced by H
rate of endothelial cells.
2 2
O and increase the survival
Lipid peroxidation is one of the fundamental consequences of free
radical-induced cell damage [24]. MDA is a byproduct of lipid perox-
idation mediated by ROS overproduction and is commonly used as an
protective effects against
2 2
H O -induced damage. Moreover, cell
2 2
Fig. 7. Involvement of Akt/eNOS pathways in the protective effect of BEG on endothelial cells after H O induced damage. (A) Western blot analysis for phospho-
eNOS (p-eNOS), eNOS, phospho-Akt (p-Akt), and Akt (B, C). Densitometric data are presented as means ± SD, n = 3. #Significant difference from the control group
ф
at P < 0.05. **Greater significant difference from the model group at P < 0.01. Greater significant difference from the BEG and Control groups at P < 0.01.
1739

K.A. Ahmad et al.
Biomedicine & Pharmacotherapy 106 (2018) 1734–1741
oxidative stress biomarker [26]. In the present study, MDA level sig-
phosphorylated Akt leads to activation of its downstream eNOS sub-
strate and therefore improved NO production, which is an essential
molecule for maintaining the biological functions of endothelial cells
nificantly increased in the H
groups, MDA level reduced significantly in a dose-dependent manner
Fig. 4B). From this, we speculate that BEG can attenuate the cell da-
mage caused by H . As we know that cells are often armed with
2 2
O damage group while in BEG pre-treated
(
2 2
[4]. Here in this study, our results showed that H O decreased ex-
2
O
2
pression of eNOS mRNA, eNOS, Phospho-Akt and NO release in en-
dothelial cells indicating that sequences of beneficial physiological ac-
tivities are mediated by ROS/Akt/eNOS signaling pathways. However,
several antioxidant agents for the protection against free-radicals. SOD,
among others enzymatic and non-enzymatic antioxidants, perform a
critical role in preventing cellular injury caused by oxidative stress
2 2
BEG pretreatment successfully reversed H O induced cell damage and
[
27]. Therefore, the intracellular ROS can be effectively eliminated by
activates the Akt/eNOS pathways (Fig. 6). To further investigate the
association between the Akt/eNOS pathway and the antioxidant effect
of BEG, we assessed the effect of PI3k/Akt inhibitor (wortmannin) and
eNOS inhibitor (L-NAME) on the antioxidant action of BEG. In agree-
ment with previous studies showing that co-treatment of Wortmannin
and L-NAME significantly blunted the phosphorylation of Akt and eNOS
respectively in endothelial cells [36,37]. Our current study showed that
co-treatment of BEG and L-NAME and Wortmannin reduced eNOS and
Akt phosphorylation significantly in HUVEs respectively, under oxida-
tive stress conditions (Fig. 7). Though L-NAME did not reduce the effect
of BEG on Akt phosphorylation level, it significantly eradicated the
the combined action of SOD, and other endogenous antioxidants, which
provide a repairing mechanism against increased oxidative stress. Our
results showed that the activity of SOD in the H O damage group was
2 2
significantly decreased compared with the untreated group (Fig. 4C). It
indicates that the antioxidant ability of endothelial cells was decreased
by H O treatment. However, BEG treated group significantly improved
2 2
SOD activity, suggesting that BEG strengthens the ability of SOD against
free radicals to protect the endothelial cells from oxidative damage
induced by H
positive control group vitamin E (100 μmol/L) in preventing oxidative
damage in endothelial cells induced by H . Previously, we demon-
2 2
O . In addition, the effect of BEG was superior to the
2
O
2
2 2
antioxidant effect of BEG on H O treated endothelial cells. Conversely,
strated that some of the β-elemene derivatives have cytoprotective ef-
fects and antioxidant activity against oxidative stress-induced vascular
endothelial cells damage [14]. Consistent with previous reports, our
our study found that BEG-induced eNOS phosphorylation was blunted
by the PI3K/Akt inhibitor wortmannin in endothelial cells co-treated
with H O . We found that BEG induced protection of HUVECs and
2 2
results indicated that BEG can successfully inhibit H
dothelial cells injury and possess considerable antioxidant activity.
Accumulative evidence has shown that H can cause endothelial
cell injury by inducing ROS generation through different pathways
28–30]. To confirm the ability of BEG to scavenge the intracellular
ROS generation induced by H , endothelial cells were pre-treated
with different concentrations of BEG and further incubation for 2 h with
.5 mM Non-toxic fluorescence probe dichlorofluorescein
DCFH) was used to confirm ROS production considering its sensitivity
for reliable measurement [31]. Incubation of endothelial cells with
increased the intracellular ROS production that is significantly
2
O
2
-induced en-
multiple biological functions of endothelial cells were significantly re-
duced, suggesting that these mechanisms are implicated in the protec-
tive role of BEG against endothelial cells injury. In light of these find-
ings, we speculate that BEG has the protective effects on human
umbilical vein endothelial cells against oxidative stress-induced injury
in a dose-dependent manner and it works through PI3k/Akt/eNOS/NO
signaling pathways.
2 2
O
[
2 2
O
0
(
2 2
H O .
5. Conclusion
H
2
O
2
This study demonstrated that BEG has antioxidant activity and su-
inhibited by pre-incubation with BEG in a concentration-dependent
perior to the positive control (vitamin E). BEG offers protection against
manner. These findings, convincingly suggest that a significant inhibi-
2 2
oxidative stress induced by H O by inhibiting ROS production, which
tion of H
2
O
2
-induced ROS generation by BEG may participate in re-
in turn inactivates downstream signaling events through PI3K/Akt/
eNOS/NO pathways. In this regards, BEG might be considered as a
future candidate for developing a novel therapeutic agent in the pre-
vention of oxidative stress-induced cardiovascular diseases.
storing the viability of endothelial cells. Moreover, nitric oxide (NO)
plays a fundamental role in maintaining the vasculature homeostasis,
inhibiting the aggregations of blood platelets and preventing con-
glutination between endothelial cells and between blood cells and en-
dothelial cells [32]. Furthermore, abnormal production of NO is asso-
ciated with increased oxidative stress and consequently impaired
endothelium-dependent vasodilation. There have been several studies
demonstrated that down-regulation of NO production is implicated in
the clinical course and pathophysiology of all known cardiovascular
diseases [33]. As shown in Fig. 5C, our results indicated that NO pro-
duction was significantly decreased in the model group compared to the
control group. On the other hand, pre-incubation with indicated con-
centrations of BEG and Vit-E significantly improved NO production in a
dose-dependent manner. eNOS expression is the main factor for NO
Acknowledgment
Authors gratefully acknowledge the support of the Experimental
Basic and Teaching Pharmacy Center especially teacher Li.
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