Tong 2018
Tong 2018
Tong 2018
PII: S0144-8617(18)30325-4
DOI: https://doi.org/10.1016/j.carbpol.2018.03.061
Reference: CARP 13411
To appear in:
Please cite this article as: Tong, Haibin., Zheng, Xiaoli., Song, Jianxi., Liu,
Jian., Ren, Ting., Zhang, Xu., Huang, Luqi., & Wu, Mingjiang., Radical
scavenging activity of sulfated Bupleurum chinense polysaccharides and their
effects against oxidative stress-induced senescence.Carbohydrate Polymers
https://doi.org/10.1016/j.carbpol.2018.03.061
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Radical scavenging activity of sulfated Bupleurum chinense polysaccharides and
Haibin Tong a, b, c, Xiaoli Zheng b, Jianxi Song c, Jian Liu b, Ting Ren c, Xu Zhang b,
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a
Center for Post-doctoral research , Resource Center for Chinese Materia Medica,
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China Academy of Chinese Medical Sciences, Beijing 100700, China;
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b
College of Life and Environmental Science, Wenzhou University, Wenzhou 325035,
China;
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c
Jilin Provincial Key Laboratory of Molecular Geriatric Medicine, Life Science
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*
To whom correspondence should be addressed:
Highlights
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1
Abstract
Bupleurum chinense was chemically modified to yield two sulfated derivatives, which
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chromatography (HPLC), Fourier transform infrared spectrometry (FT-IR), and gas
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chromatography (GC), and then compared with those of BCPS-1. Furthermore, the
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antioxidant activities of all three polysaccharides were also evaluated using 1,1-
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diphenyl-2-picrylhydrazyl (DPPH) radical assay, superoxide radical assay and
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hydroxyl radical assay, while their effects against H2O2-induced cellular senescence
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were determined using senescence-associated β-galactosidase (SA-β-gal) staining, cell
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results indicated that the sulfate group in the modified B. chinense polysaccharides
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Senescence
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1. Introduction
radical (HO•), and superoxide anion (O2−) are generated from cellular respiration
(Seifried et al., 2007). Accumulated evidence shows that ROS can result in direct or
indirect macromolecular damage, and therefore, ROS has been implicated in the
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pathogenesis of various diseases (Valko et al., 2006; Waris & Ahsan, 2006).
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Excessive production of ROS can overwhelm the cellular antioxidant defense
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system, eventually leading to serious disorders, such as carcinogenesis,
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neurodegeneration, atherosclerosis, diabetes, and accelerated aging (Colavitti &
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Finkel, 2005; Finkel & Holbrook, 2000; Ray, Huang, & Tsuji, 2012). Cellular
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senescence is considered as a form of premature cellular aging characterized by
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certain primary cells with exogenous ROS can trigger the cells to rapidly enter
senescence, whereas lowering the level of free radicals can rescue the cells from
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(Chandrasekaran, Idelchik, & Melendez, 2017; Passos & Von Zglinicki, 2006).
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Therefore, developing natural antioxidants that can protect our bodies from ROS-
induced damage while having little or no toxicity has become a research hotspot.
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Recently, natural polysaccharides from herbs that have been used in traditional
medicine have gained tremendous attention from researchers working with free
radical scavengers and ROS-induced senescence or diseases (Balsano & Alisi, 2009;
3
Ho, So, & Chang, 2009; Li et al., 2014). Examples of these biologically active
mouse kidney, liver, and testicular tissue from H2O2-induced DNA breakage and
oxidative damage (Li, 2007; Luo et al., 2006), and Angelica sinensis polysaccharides
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(ox-LDL) in endothelial progenitor cells (Lai & Liu, 2015). In addition, LBPs can
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protect epithelial cells from human lens against H2O2-induced apoptosis by
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modulating ROS generation, mitochondrial membrane potential, pro-apoptotic
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proteins, antioxidant enzyme activity and attenuating cellular senescence (Qi et al.,
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2014). Another example of these polysaccharides is Guiqi polysaccharide (GQP),
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which is extracted from Angelica and Astragalus roots, and it has been shown to
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These findings suggest that through their antioxidant effects, polysaccharides from
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medicinal herbs can protect cells against oxidative stress-induced senescence and
pharmacological efficacy (Song et al., 2017; Tong et al., 2013, 2014 & 2017).
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Moderate structural modification, including carboxymethylation, acetylation,
phosphorylation and sulfation could yield variants of the polysaccharides with better
pharmacological effect against ROS and oxidative damage (Tang & Huang, 2016; Tu
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antioxidant activity of the modified polysaccharides and the extent of their protective
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effect against H2O2-induced premature senescence in mouse lung endothelial cells
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(MLECs) were evaluated and discussed.
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2. Materials and Methods
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2.1. Materials and chemicals
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(Jilin Province, China) in September 2016, and authenticated by Dr. Yu Hou, School
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of Life Science, Beihua University. The specimen was deposited in School of Life
described (Sun et al., 2010). Rabbit polyclonal antibodies against p53 (FL-393) and β-
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actin (H-196) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-
United Kingdom). Dulbecco's modified eagle medium (DMEM) Low Glucose and
Ham’s F12 Nutrient Mixture were purchased from Gibco Invitrogen Co (Grand
Island, NY). Endothelial cell growth supplement (ECGS) was purchased from BD
5
Biosciences (San Jose, CA). Fetal bovine serum (FBS) was purchased from Hangzhou
obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemical
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Modification of BCPS-1 by sulfation was carried out using the chlorosulfonic
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acid–pyridine (CSA-Pyr) method (Tu et al., 2016). Briefly, an esterification agent was
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first prepared from a mixture of CSA and Pyr using either a CSA:Pyr ratio of 1:4 or
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1:8 ratio. Next, BCPS-1 (200 mg) was dissolved in anhydrous formamide (FA) with
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constant stirring, and then mixed with the esterification reagent followed by
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incubation at 80 ◦C. After that, the mixture was neutralized with NaOH, and dialyzed
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against water, and the sulfated polysaccharides were then precipitated with 95%
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ethanol, and freeze-dried. The two sulfated derivatives were designated as S-BCP1-4
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and S-BCP1-8, with the numbers in subscript refer to the ratio of CSA to Pyr in the
esterification agent: a ratio of 1:4 for S-BCP1-4 and 1:8 for S-BCP1-8.
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substitution (DS) in the sulfated derivatives was calculated according to the following
equation:
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The content of total carbohydrate was determined using the phenol-sulfuric acid
6
colorimetric method. The relative molecular weights of the polysaccharides were
described (Sun et al., 2010). Sample of the polysaccharide was applied to an Agilent
1200 HPLC system equipped with a TSK-GEL G3000 PWXL column. The column
was eluted with 0.1 mol/L Na2SO4 solution and the eluent was detected by a RID-10A
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refractive index detector. The column was calibrated with T-series dextran standards
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with different molecular weights (T-200, T-70, T-40, T-20, and T-10). The relative
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molecular weight of each polysaccharide was then estimated by reference to the
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standard curve. Gas chromatography (GC) was used to identify and quantify the
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monosaccharide composition of the polysaccharides. Briefly, the polysaccharide
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sample was hydrolyzed with 2 M TFA, and the hydrolyzed product was converted
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into the alditol acetates as described by Lehrfeld (1985), and analyzed by GC using a
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according to a previously published method (Xia et al., 2011). The superoxide radical
method of NBT (Tong et al., 2013). Hydroxyl radical scavenging activity was
7
determined using the method of Fenton reaction (Tong et al., 2013). Half maximal
effective concentration (EC50) value was calculated from the plot of scavenging
Mouse lung endothelial cells (MLECs) isolated from mouse lungs and
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immortalized by retroviral delivery of a SV40 large T antigen, were generously
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provided by Prof. Martin Schwartz (Yale University at New Haven, CT). The cells
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were maintained in a medium consisting of a 1:1 mixture of Dulbecco's Modified
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Eagle Medium (DMEM) Low Glucose and Ham’s F12 Nutrient Mixture. The medium
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was supplemented with 0.05 mg/ml endothelial cell growth supplement (ECGS), 10%
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heat inactivated fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml
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platelet endothelial cell adhesion molecule-1 (PECAM-1) on the cell surface was
routinely checked by flow cytometry to ensure that the cells could still retain their
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detection kit (Beyotime, Haimen, China), which was based on the method described
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by Lai and Liu (2015). First, MLECs (70% confluent) in a 6-well plate were
pretreated with 400 μg/ml polysaccharide, and then exposed to H2O2 for 12 h. Next,
the cells were washed twice with PBS, fixed in fixative solution for 10 min, and then
8
washed with PBS. After that, the cells were incubated with fresh staining solution for
counting the number of stained cells (blue) within five random fields under a bright-
field microscope.
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Cell cycle analysis was performed as described by Zhang et al. (2016). MLECs
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were cultured in 6-well plates and then pretreated with BCPS-1 or its sulfated
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derivatives. Hydrogen peroxide was added to the cells to a final concentration of 200
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mM followed by 12-h incubation. The cells were then harvested by centrifugation,
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and fixed in 70% ethanol followed by staining with propidium iodide containing 10
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μg/mL RNase A. The cells were analyzed with a BD FACSAria flow cytometer.
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2.8. Immunoblotting
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lysed in ice-cold RIPA lysis buffer, and the cell debris was removed from the lysate
with a bicinchoninic acid (BCA) assay kit (Beyotime, Haimen, China) according to
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the manufacturer’s instruction. The extract was then subjected to SDS-PAGE, and the
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proteins in the gel were then transferred to a nitrocellulose (NC) membrane. The blot
secondary antibody. Positive bands in the blot were detected using the enhanced
chemiluminescence (ECL) reagents and quantitated using the NIH ImageJ software.
9
Expression of the p53 and p16 genes was determined by qRT-PCR, performed with
an ABI Prism 7900HT Real-time PCR system (Applied Biosystems) and a real-time
PCR kit (SYBR® Premix Ex Taq™, Takara, Japan). First, total RNA was isolated
using TRIzol reagent (Invitrogen). The concentration of total RNA concentration was
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electrophoresis. Purified RNA (1 g) was reversely transcribed to cDNA using gene
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specific primers [p53 (Forward, 5’-TGCAGTTGTGGGTCAGCG -3’; Reverse, 5’-
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AGGATAGGTCGGCGGTTC-3’); p16INK4a (Forward, 5’-
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TGGTGGTGCTGCACGGGTC-3’; Rev, 5’-GCACGATGTCTTGATGTCCC-3’); β-
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actin (Forward, 5’-TGAACCCTAAGGCCAACC-3’; Reverse, 5’-
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TGATGTCACGCACGATTT-3’)]. All the primers were synthesized by Shanghai
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Sangon Company. The reaction mixture which contained SYBR Green Mix, cDNA,
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primer pair and H2O was incubated for 30 min at 48°C, followed by initial
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denaturation at 95◦C and 30 s of annealing and extension at 60°C. At the end of the 40
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cycles, a melting curve analysis was performed to confirm the presence of a single
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amplified product of the expected size. The fold difference in mRNA transcripts was
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Data were presented as mean ± standard deviation. All data were analyzed by one
way ANOVA performed using SPSS version 13.0. Statistical significance was
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3. Results and discussion
The properties of BCPS-1 have been determined in our previous study (Sun et al.,
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ratio of 2.1:2.5:1, and the carbohydrate content of BCPS-1 is 97.5% as determined by
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the phenol-sulfuric acid colorimetric method. No protein or nucleic acid was found in
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the purified BCPS-1 preparation. Furthermore, BCPS-1 has a backbone of (1→5)-
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linked Ara, (1→4)-linked Gal and (1→3)-linked Gal residues which occasionally
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branches at O-6. The branches are composed of (1→4)-linked Glc, and terminated
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Gal residues.
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Growing evidence indicates that polysaccharide sulfation can change the chain
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activities (Román et al., 2016; Xu et al., 2016). Obvious changes were found in the
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two sulfated derivatives of BCPS-1. The DS of S-BCP1:4 and S-BCP1:8 were 0.38 ±
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0.05 and 0.61 ± 0.08, respectively, indicating that the sulfation process was
successful. The average molecular weights of S-BCP1-4 and S-BCP1-8 were 37.6 and
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51.7 kDa, respectively, which were higher than that of BCPS-1 (29 kDa). The
attributed to the different ratios of Pyr:CSA in the esterification used in each sulfated
11
modification reaction. The FT-IR spectra of S-BCP1-4 and S-BCP1-8 revealed two new
characteristic absorption bands not found in BCPS-1 (Fig. 1). The band at 1234 cm−1
was assigned to the S=O asymmetry stretching vibration, while the band at around
830 cm-1 was assigned to C-O-S symmetry stretching vibration associated with a C-O-
SO3 group. This further confirmed that both S-BCP1-4 and S-BCP1-8 were products of
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successful sulfated modification.
R IP
BCPS-1
SC
100
95
1744.818482
U
90
N
Transmittance [%]
A
627.136918
85
2922.182071
1403.003246
1609.177515
1006.931624
M
1099.167481
80
3410.489550
75
ED
1733.967204
CC 95 90
Transmittance [%]
2960.161541
1630.880070
A
567.454892
85
827.885548
80
1034.059817
3448.469021 1234.808448
75
100
95
90
Transmittance [%]
1744.818482
85
2965.587180
578.306170
80
1620.028792 833.311187
T
1039.485456
75
3448.469021
IP
1234.808448
70
R
3500 3000 2500 2000 1500 1000 500
Wavenumber cm-1
SC
Sample Name hongjt16 Sample Scans 16
Path of File D:\宋见喜\20161227 Resolution 4
(Xia et al., 2011). BCPS-1, S-BCP1-4 and S-BCP1-8 all exhibited radical scavenging
PT
activity as shown by the conversion of DPPH to its corresponding non radical form,
and this activity was dependent of the concentration of the polysaccharides (Fig. 2A).
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BCP1-4 and S-BCP1-8 were significantly (p<0.05) higher than that of BCPS-1. S-BCP1-
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1.6 mg/ml. At 1.6 mg/ml, the DPPH radical scavenging rate of BCPS-1, S-BCP1-4 and
S-BCP1-8 reached 66.3 ± 2.6%, 85.5 ± 4.4% and 90.6 ± 2.1%, respectively. The EC50
13
values of S-BCP1-4 and S-BCP1-8 were 0.26 and 0.19 mg/ml, respectively, while that
of BCPS-1 was 0.41 mg/ml. Thus both sulfated derivatives of BCPS-1 appeared to
concentrations.
Superoxide anions, which are formed from the mitochondrial electron transport
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systems, can activate and induce the production of other free radicals, such as
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hydroxyl radical and lipid peroxidation (Balaban, Nemoto, & Finkel, 2005). The
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superoxide scavenging activity of BCPS-1 and its two sulfated derivatives increased
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with increasing polysaccharide concentrations (Fig. 2B). At 1.6 mg/ml, the
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superoxide scavenging rates of BCPS-1, S-BCP1-4 and S-BCP1-8 were 56.7 ± 3.6%,
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65.0 ± 1.9% and 72.8 ± 3.6%, respectively. The EC50 values of BCPS-1, S-BCP1-4
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and S-BCP1-8 were 0.77, 0.42 and 0.28 mg/ml, respectively. These findings showed
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that modification of BCPS-1 by sulfation could turn it into a stronger inhibitor against
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Hydroxyl radicals are highly reactive oxygen radicals that can damage almost all
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(Valko et al., 2007). The scavenging ability of BCPS-1, S-BCP1-4 and S-BCP1-8
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2C). BCPS-1, S-BCP1-4 and S-BCP1-8 exhibited similar scavenging activities at 0.05
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to 0.4 mg/ml, but at 0.8 to 1.6 mg/ml, S-BCP1-4 and S-BCP1-8 were better scavengers
than BCPS-1 (p<0.05). At 1.6 mg/ml, the scavenging rates of BCPS-1, S-BCP1-4 and
S-BCP1-8 were 57.9 ± 2.9%, 80.3 ± 4.2% and 84.5 ± 1.9%, respectively. The EC50
14
values of BCPS-1, S-BCP1-4 and S-BCP1-8 were 0.60, 0.29 and 0.22 mg/ml,
scavenger that was used as positive control) at 1.6 mg/ml was 88.1 ± 2.5% and its
EC50 values was about 0.12 mg/ml. The hydroxyl radical-scavenging capacity of the
sulfated derivatives was therefore almost the same as that of BHT. These findings
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demonstrated that the addition of sulfate group to B. chinense polysaccharide could
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potentially boost its scavenging activity against hydroxyl radicals.
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SC
A
D P P H r a d ic a l s c a v e n g in g a b ilit y ( % )
100
U
N
50
A s c o r b ic a c id
A
B C P S -1
S - B C P 1 -4
M
S - B C P 1 -8
0
0 .0 0 .4 0 .8 1 .2 1 .6
C o n c e n t r a t io n ( m g /m l)
ED
B
S u p e r o x id e r a d ic a l s c a v e n g in g a b ilit y ( % )
PT
100
E
CC
50
A s c o r b ic a c id
B C P S -1
A
S - B C P 1 -4
S - B C P 1 -8
0
0 .0 0 .4 0 .8 1 .2 1 .6
C o n c e n t r a t io n ( m g /m l)
15
C
H y d r o x y l r a d ic a l s c a v e n g in g a b ilit y ( % )
100
50
BHT
B C P S -1
T
S - B C P 1 -4
S - B C P 1 -8
IP
0
0 .0 0 .4 0 .8 1 .2 1 .6
C o n c e n t r a t io n ( m g /m l)
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Figure 2. Scavenging rate of BCPS-1, S-BCP1-4 and S-BCP1-8 against DPPH radical
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(A), superoxide radical (B) and hydroxyl radical (C). Data are the means ± SDs (n=6).
ROS are chemically reactive species that include peroxides, superoxide, hydroxyl
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N
radical and singlet oxygen (Waris & Ahsan, 2006). They are natural byproducts of
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cellular metabolisms that use oxygen, and are extremely important and indispensable
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to life because of their roles in cell signaling and homeostasis. However, ROS is also
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cardiovascular disease (Li et al., 2017). Therefore, there has been increasing interest
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worth mentioning that, among the exogenous antioxidants, polysaccharides are the
prominent species responsible for the strong resistance against oxidative stress-
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induced damage (Wang et al., 2017). Polysaccharides from natural sources (e.g.
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because their remarkable health benefits (Deniaud-Bouët et al., 2017; Jiao et al.,
2016).
that chemical modifications can transform their chemical structures and improve their
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bioactivities, such as radical scavenging activity (Mei, Yi, & Huang, 2017). Sulfated
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modification can often result in the emergence of stronger bioactivities for the
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modified polysaccharides. For example, regioselective sulfation of Artemisia
SC
sphaerocephala polysaccharide (SRSASP) can enhance its antioxidant activity as
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demonstrated by radical scavenging assay (Wang et al., 2016). Another example is the
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sulfated polysaccharide from Annona squamosa, which possesses stronger DPPH
A
radical and hydroxyl radical scavenging activities than its unmodified counterpart (Tu
M
et al., 2016). The underlying mechanism might have to do with the fact that sulfation
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can improve the water solubility and relieve the chain conformation of the
polysaccharides. In addition, sulfated group can activate the hydrogen atoms of the
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oxidative damage in human tissue and cells can lead to cellular senescence
(Chandrasekaran, Idelchik, & Melendez, 2017). Senescent cells are usually large and
flattened, and display increased SA-β-gal activity. These cells are often in the stage of
17
cell cycle arrest. SA-β-gal is the first molecular marker used for the specific
histochemical staining (Dimri et al., 1995). SA-β-gal is probably derived from the
increased lysosomal biogenesis that commonly occurs in senescent cells (Lee et al.,
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2006). To investigate whether BCPS-1 and its sulfated derivatives could inhibit H2O2-
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induced senescence in MLECs, the activity of SA-β-gal in these cells was first
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investigated. Treatment of MLECs with H2O2 caused a significant increase in the
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number of SA-β-gal-positive cells (Fig. 3). S-BCP1-4 and S-BCP1-8 significantly
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alleviated H2O2-induced damage in MLECs. S-BCP1-4- and S-BCP1-8 -treated MECLs
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both exhibited reduced percentage of SA-β-gal-positive cells, 26.7 ± 2.6% in the case
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of S-BCP1-4-treated cells, and 24.7 ± 2.9% for S-BCP1-8-treated cells. BCPS-1 also
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the effect was weaker than that exerted by S-BCP1-4 and S-BCP1-8, indicating that the
presence of sulfate group in BCPS-1 could increase its protective effect against ROS-
PT
18
P e r c e n t o f S A - β - G a l p o s it iv e c e lls ( % )
PBS H2O2
60
40
*
** **
20
T
IP
0
C
R
-B
-B
o
C
o
n
C
tr
P
l
o
-1
1
l
-4
-8
SC
Figure 3. Percentage of cell senescence in H2O2-induced MLECs as detected by SA-
β-gal staining. Data are the means ± SDs from five random fields. ‘*’ and ‘**’
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N
indicate significantly different from the control at the p<0.05 and p<0.01 levels,
A
respectively.
M
One of the hallmarks of cellular senescence is cell cycle arrest (Campisi & d'Adda
ED
di Fagagna, 2007). Once growth is arrested, the cells fail to initiate DNA replication
because of the expression of dominant cell cycle inhibitors. The effect of BCSP-1 and
PT
its sulfated derivatives on cell cycle was determined by pretreating MLECS with the
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polysaccharides, and then exposed the cells to H2O2 and measured their distribution in
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the different phases of the cell cycle using flow cytometry. The result revealed 84.4%
of the H2O2-treated MLECs in the G0/G1 phase and 10.8% in the S phase, compared
A
to 56.7% in the G0/G1 phase and 32.9% in the S phase in the case of untreated cells,
suggesting that the cell cycle of H2O2-treated MLECs was arrested at the G0/G1 phase
(Fig. 4). Pretreatment with S-BCP1-4 or S-BCP1-8 significantly reduced the proportion
19
of cells in the in G0/G1 phase (63.6% and 60.3%, respectively) and a concomitant
increase in S phase fraction (28.8% and 30.5, respectively) when these cells were
exposed to H2O2, indicating that the cell cycle arrest in G0/G1 phase was significantly
attenuated by S-BCP1-4 and S-BCP1-8. On the other hand, the protective effect of
BCPS-1 against H2O2-induced cell cycle arrest in MLECs was weaker than that of its
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sulfated derivatives. These observations suggested that S-BCP1-4 and S-BCP1-8 could
IP
inhibit the oxidative stress-induced cell cycle arrest, allowing the cells to enter the S
R
phase, thereby avoiding senescence.
SC
120 C e ll C y c le P h a s e D is t r ib u t io n
100
U G 2 /M
S
% C e ll p o p u la t io n
N
G 0 /G 1
80
A
60
M
40
20
ED
0
C
S
o
-B
C
o
-B
n
C
tr
C
el
P
o
P
-1
l
1
PT
1
-4
-8
Figure 4. Effect of BCPS-1, S-BCP1-4 and S-BCP1-8 on H2O2-induced cell cycle arrest
E
CC
in MLECs.
Accumulated evidence shows that the p53-p21 and p16-pRb pathways are closely
A
associated with cellular senescence (Childs et al., 2015). In order to investigate the
H2O2-induced cellular senescence in MLECs, the expression levels of p53 and p16,
which are the major regulators of cellular senescence in endothelial cells, were
20
determined. H2O2 treatment dramatically up-regulated the expression levels of the
potent senescence inducers, p53 and p16, in H2O2-treated MLECs compared to the
control group (Fig. 5), indicating the activation of the p53-p21 and p16-pRb signal
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inhibited the expression of p53 (Figure 5B) and p16 (Figure 5C). The effects of these
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polysaccharides on the expression of two H2O2-induced senescence regulators, p53
R
and p16INK4a, were evaluated by qRT-PCR. Consistent with the data from
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immunoblotting, H2O2–treated MLECS exhibited significant enhancement in p53
U
(Fig. 6A) p16INK4a (Fig. 6B) mRNA levels, but co-treatment with H2O2 and either S-
N
BCP1-4 or S-BCP1-8 significantly suppressed H2O2-induced expression of p53 (Fig.
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6A) and p16INK4a (Fig. 6B). Exogenous H2O2 or other types of reactive oxygen species
M
could induce a number of biochemical changes in the cells. Most notably, a rise in the
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inhibitor protein p16, which can bind to and inhibit some CDK-cyclin complexes,
PT
senescence (Colavitti & Finkel, 2005; Finkel & Holbrook, 2000). Our data obviously
CC
suggested that S-BCP1-4 and S-BCP1-8 could significantly down-regulate the p53-p21
21
T
IP
B
R
1 .2 PBS
R a t io o f p 5 3 /β - a c t in
SC
1 .0 H 2O 2
0 .8 *
0 .6
0 .4 ** U
N
**
0 .2
A
0 .0
M
C
S
-B
-B
o
C
o
n
C
tr
P
l
o
-1
1
l
-4
-8
ED
C 0 .4
PBS
PT
R a t io o f p 1 6 /β - a c t in
H 2O 2
0 .3
E
0 .2
*
CC
**
0 .1
A
0 .0
C
S
-B
-B
o
C
o
n
C
tr
P
l
o
-1
1
l
-4
-8
Figure 5. Effect of BCPS-1, S-BCP1-4 and S-BCP1-8 on the protein expression levels
22
of p53 and p16 in H2O2-treated MLECs by immunoblotting. Representative images
(A) from three independent experiments are shown. Quantitative analysis p53 (B) and
p16 INK4a (C) was performed using ImageJ software. Data are the means ± SDs. ‘*’
and ‘**’ indicate significantly different from the control at the p<0.05 and p<0.01
levels, respectively.
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IP
A
)
8
- c t
PBS
H 2O 2
R e la t iv e m R N A le v e l (2
R
6
SC
*
p53
4
**
**
2
U
N
0
A
C
S
-B
-B
o
C
o
n
C
tr
P
l
o
M -1
1
l
-4
-8
ED
B
)
6
- c t
PBS
H 2O 2
R e la t iv e m R N A le v e l (2
PT
4 **
IN K 4 a
**
p16
2
CC
0
C
S
A
-B
-B
o
C
o
n
C
tr
P
l
o
-1
1
l
-4
-8
Figure 6. Effect of BCPS-1, S-BCP1-4 and S-BCP1-8 on the expression of p53 and
p16INK4a in H2O2-treated MLECs. Relative mRNA levels of p53 (A) and p16 INK4a (B)
23
were determined by qRT-PCR. Data are the means ± SDs. ‘*’ and ‘**’ indicate
significantly different from the control at the p<0.05 and p<0.01 levels, respectively.
4. Conclusion
T
and S-BCP1-8. It also increased the antioxidant activity of these polysaccharides,
IP
conferring better protective effect against H2O2-induced oxidative stress and cellular
R
senescence. Furthermore, sulfation of BCPS-1 also led to stronger induction of SA-β-
SC
gal activity, abrogation of cell-cycle arrest, and inhibition of p53-p21 and p16-pRb
U
pathways in H2O2-treated MLECs. The health benefit of sulfated polysaccharides,
N
such as antioxidant and anti-senescent effects, would be attractive to the development
A
of novel dietary nutritional supplements or drugs for oxidative stress and aging-related
M
diseases.
ED
Acknowledgements
PT
We thank Dr Alan K Chang (Wenzhou University) for helpful discussion and for
E
revising the language of the manuscript. This work was supported by the National
CC
24
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