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Tong 2018

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Accepted Manuscript

Title: Radical scavenging activity of sulfated Bupleurum


chinense polysaccharides and their effects against oxidative
stress-induced senescence

Authors: Haibin Tong, Xiaoli Zheng, Jianxi Song, Jian Liu,


Ting Ren, Xu Zhang, Luqi Huang, Mingjiang Wu

PII: S0144-8617(18)30325-4
DOI: https://doi.org/10.1016/j.carbpol.2018.03.061
Reference: CARP 13411

To appear in:

Received date: 10-10-2017


Revised date: 13-3-2018
Accepted date: 18-3-2018

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

This is a PDF file of an unedited manuscript that has been accepted for publication.
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apply to the journal pertain.
Radical scavenging activity of sulfated Bupleurum chinense polysaccharides and

their effects against oxidative stress-induced senescence

Haibin Tong a, b, c, Xiaoli Zheng b, Jianxi Song c, Jian Liu b, Ting Ren c, Xu Zhang b,

Luqi Huang a, *, Mingjiang Wu 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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Research Center, Beihua University, Jilin 132013, China.


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*
To whom correspondence should be addressed:

Tel. /fax: + 86-577-86689078


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E-mail: wmj@wzu.edu.cn (M. Wu); huangluqi01@126.com (L. Huang)


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Highlights
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★Sulfated derivatives of BCPS-1 exhibit distinct physicochemical properties.

★Sulfated derivatives possess stronger radical scavenging activities.

★Sulfated derivatives protect MLECs against H2O2-induced senescence.

★Sulfate group in modified BCPS-1 might play essential roles in bioactivities.

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Abstract

In this study, BCPS-1, a polysaccharide previously isolated and characterized from

Bupleurum chinense was chemically modified to yield two sulfated derivatives, which

we designated as S-BCP1-4 and S-BCP1-8. The physicochemical properties of these

sulfated derivatives were then determined by high performance liquid

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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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cycle assay and immunoblotting in H2O2-induced mouse lung endothelial cells


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(MLECs). Compared to BCPS-1, S-BCP1-4 and S-BCP1-8 exhibited remarkable


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antioxidant effect, and in a concentration-dependent manner. They also provided

stronger protection against H2O2-induced cellular senescence in MLECs. These


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results indicated that the sulfate group in the modified B. chinense polysaccharides
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might play an important role in radical scavenging and resistance to H2O2-induced


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senescence. These sulfated polysaccharides could be considered as novel

pharmaceutical products with potential antioxidant and anti-senescence effects.


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Keywords: Bupleurum chinense; Polysaccharide; Sulfated modification; Antioxidant;

Senescence

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1. Introduction

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl

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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irreversible proliferative arrest, and is accompanied by characteristic changes in gene


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expression, metabolism and cell morphology (Childs et al., 2015). Treatment of


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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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ROS-induced senescence and reduce the progression of aging-related disorders


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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;

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Ho, So, & Chang, 2009; Li et al., 2014). Examples of these biologically active

polysaccharides include Lycium barbarum polysaccharides (LBPs), which can protect

mouse kidney, liver, and testicular tissue from H2O2-induced DNA breakage and

oxidative damage (Li, 2007; Luo et al., 2006), and Angelica sinensis polysaccharides

(ASP), which can attenuate senescence induced by oxidized low-density lipoprotein

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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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attenuate senescence-related pathological features in WI-38 cells (Pu et al., 2015).


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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

prevent senescence-related diseases.


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Bupleurum chinense is a well-known and widely used traditional Chinese


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medicine (Sun et al., 2010). Accumulated evidence from pharmacological research


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and clinical practice indicate that B. chinense polysaccharides possess notable

pharmacological efficacy (Song et al., 2017; Tong et al., 2013, 2014 & 2017).
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Polysaccharides are bio-macromolecular polymers and their activity is greatly

affected by their structural and physicochemical properties, such as monosaccharide

composition, types of glycosidic bond, molecular weight, and spatial conformation.

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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

et al., 2016). By focusing on sulfation, we produced two variants of the B. chinense

polysaccharide (BSCP-1) that we previously isolated and characterized. The

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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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Bupleurum chinense roots were purchased from a local pharmaceutical market


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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

Science, Beihua University, with voucher specimen number 2016-00315. The


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polysaccharide BCPS-1 was purified from the roots of B. chinense as previously


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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-

p16/INK4a rabbit mAb (EP435Y-129R) was obtained from Abcam (Cambridge,


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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

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Biosciences (San Jose, CA). Fetal bovine serum (FBS) was purchased from Hangzhou

Sijiqing Corp (Hangzhou, China). 1,1-diphenyl-2-picrylhydrazyl (DPPH) was

obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemical

reagents used were analytical grade.

2.2. Sulfated modification

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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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The content of sulphur (S%) in the sulfated derivatives was determined by a


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calibration curve constructed with sodium sulfate as a standard. The degree of


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substitution (DS) in the sulfated derivatives was calculated according to the following

equation:
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DS = (1.62 × S%)/(32 - 1.02 × S%)

2.3. Physicochemical properties of the sulfated derivatives

The content of total carbohydrate was determined using the phenol-sulfuric acid

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colorimetric method. The relative molecular weights of the polysaccharides were

determined by high performance liquid chromatography (HPLC) as previously

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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Varian 3400 instrument (Hewlett–Packard Component, USA) equipped with a DM-


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2330 capillary column and flame-ionization detector (FID). Ultraviolet-visible spectra

were recorded on a Varian Cary 100 spectrophotometer. Fourier transform infrared


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spectrometry (FT-IR) spectra were recorded on a Nicolet 5700 FT-IR spectrometer


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instrument in the frequency range of 500-4000 cm-1.


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2.4. Antioxidant activity assay

The DPPH radical scavenging activity of the polysaccharides was determined


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according to a previously published method (Xia et al., 2011). The superoxide radical

scavenging activity of the polysaccharides was determined using the photoreduction

method of NBT (Tong et al., 2013). Hydroxyl radical scavenging activity was

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determined using the method of Fenton reaction (Tong et al., 2013). Half maximal

effective concentration (EC50) value was calculated from the plot of scavenging

effect (%) versus drug concentration (mg/ml).

2.5. Cell culture

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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streptomycin. The cultures were incubated at 37°C in a 5% CO2 humidified


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atmosphere, and the expression of intercellular adhesion molecule-2 (ICAM-2) and


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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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normal endothelial cell (EC) characteristics (Lim & Luscinskas, 2006).


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2.6. Senescence-associated β-galactosidase (SA-β-Gal) staining


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Cellular senescence was determined by a senescence-associated β-galactosidase

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

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washed with PBS. After that, the cells were incubated with fresh staining solution for

overnight at 37°C. The percentage of SA-β-gal positive cells was determined by

counting the number of stained cells (blue) within five random fields under a bright-

field microscope.

2.7. Cell cycle analysis

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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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Immunoblotting was performed as described by Tong et al. (2017). Cells were


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lysed in ice-cold RIPA lysis buffer, and the cell debris was removed from the lysate

by centrifugation. The concentration of protein in the supernatant was determined


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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

was probed with anti-p53 or anti-p16 antibody followed by HRP-conjugated


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secondary antibody. Positive bands in the blot were detected using the enhanced

chemiluminescence (ECL) reagents and quantitated using the NIH ImageJ software.

2.9. Quantitative real-time PCR (qRT-PCR) analysis

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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

determined by spectrophotometry, whereas the quality of the RNA was evaluated by

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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 for 30s, and 40 amplification cycles, each consisted of 5 s of

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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calculated according to formula 2−ΔΔct.

2.10. Statistical analysis


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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

considered at the p<0.05 and p<0.01 levels.

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3. Results and discussion

3.1. The physicochemical properties of sulfated derivatives

The properties of BCPS-1 have been determined in our previous study (Sun et al.,

2010). BCPS-1 has a molecular weight of 29 kDa as determined by HPLC analysis. It

is a heteropolysaccharide composed of arabinose, galactose, and glucose with a molar

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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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conformation of the molecule, resulting in enhanced bioactivity (Jiang et al., 2015).


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Compared to BCSP-1, the sulfated derivatives exhibited stronger biological activities,

such as anti-oxidant, immunomodulating, anti-coagulant, antitumor and antiviral


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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

differences in physicochemical properties between S-BCP1-4 and S-BCP1-8 were

attributed to the different ratios of Pyr:CSA in the esterification used in each sulfated

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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
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90

N
Transmittance [%]

A
627.136918
85

2922.182071
1403.003246
1609.177515

1006.931624
M

1099.167481
80

3410.489550
75

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3500 3000 2500 2000 1500 1000 500


Wavenumber cm-1
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Sample Name hongjt Sample Scans 16


S-BCP1-4 Path of File D:\宋见喜\20161227
Operator Name Wang Yujia
Resolution 4
Instrument Type Tensor 27
Sample Form 粉末 北华大学分析测试中心红外光谱室
Page 1 of 1
100

1733.967204
CC 95 90
Transmittance [%]

2960.161541

1630.880070
A

567.454892
85

827.885548
80

1034.059817
3448.469021 1234.808448
75

3500 3000 2500 2000 1500 1000 500


Wavenumber cm-1
Sample Name hongjt14 Sample Scans 16
Path of File D:\宋见喜\20161227 Resolution 4
Operator Name Wang Yujia Instrument Type Tensor 27
Sample Form 粉末 12
北华大学分析测试中心红外光谱室
Page 1 of 1
S-BCP1-8

100
95
90
Transmittance [%]

1744.818482
85

2965.587180

578.306170
80

1620.028792 833.311187

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1039.485456
75

3448.469021

IP
1234.808448
70

R
3500 3000 2500 2000 1500 1000 500
Wavenumber cm-1

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Sample Name hongjt16 Sample Scans 16
Path of File D:\宋见喜\20161227 Resolution 4

Figure 1. FT-IR spectra of BCPS-1, S-BCP1-4 and S-BCP1-8.


Operator Name Wang Yujia
Sample Form 粉末
Instrument Type Tensor 27
北华大学分析测试中心红外光谱室
Page 1 of 1

3.2. Radical scavenging activity of BCPS-1, S-BCP1-4 and S-BCP1-8


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As a stable nitrogen-centered and lipophilic free radical, DPPH has been widely
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used to evaluate the free radical-scavenging activity of antioxidants. DPPH radical has
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a characteristic absorption peak at 517 nm (purple), and in the presence of antioxidant,

DPPH radical is reduced to a non-radical form (DPPH-H), which appears as yellow


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(Xia et al., 2011). BCPS-1, S-BCP1-4 and S-BCP1-8 all exhibited radical scavenging
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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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At a polysaccharide concentration of 0.8 and 1.6 mg/ml, the scavenging effects of S-

BCP1-4 and S-BCP1-8 were significantly (p<0.05) higher than that of BCPS-1. S-BCP1-
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8 exhibited a significantly (p<0.05) higher scavenging capacity at 0.8 mg/ml compared

to S-BCP1-4, but no significant difference between S-BCP1-4 and S-BCP1-8 occurred at

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

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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

possess excellent DPPH free radicals scavenging capacity, especially at high

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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the formation of superoxide radical.

Hydroxyl radicals are highly reactive oxygen radicals that can damage almost all
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kinds of macromolecules, and they cannot be eliminated by enzymatic reaction


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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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increased significantly with increasing concentrations of the polysaccharides (Fig.

2C). BCPS-1, S-BCP1-4 and S-BCP1-8 exhibited similar scavenging activities at 0.05
A

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

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values of BCPS-1, S-BCP1-4 and S-BCP1-8 were 0.60, 0.29 and 0.22 mg/ml,

respectively. In addition, the hydroxyl radical scavenging rate of BHT (a strong

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

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N
50
A s c o r b ic a c id
A
B C P S -1
S - B C P 1 -4
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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)
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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 ( % )

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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

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S - B C P 1 -4
S - B C P 1 -8

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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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a “double-edged sword”, as accumulated evidence indicates that excessive ROS might

be linked to a number of chronic diseases, such as Alzheimer's disease, diabetes and


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cardiovascular disease (Li et al., 2017). Therefore, there has been increasing interest
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in research that aims to develop exogenous antioxidants from natural sources. It is


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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.

medicinal herbs, mushroom, seaweed) are considered as dietary nutritional

supplements or medicines for the prevention of oxidative stress-related diseases

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because their remarkable health benefits (Deniaud-Bouët et al., 2017; Jiao et al.,

2016).

It is widely accepted that the biological activity of polysaccharides is dependent on

the structural characteristics of the polysaccharides, and growing evidence indicates

that chemical modifications can transform their chemical structures and improve their

T
bioactivities, such as radical scavenging activity (Mei, Yi, & Huang, 2017). Sulfated

IP
modification can often result in the emergence of stronger bioactivities for the

R
modified polysaccharides. For example, regioselective sulfation of Artemisia

SC
sphaerocephala polysaccharide (SRSASP) can enhance its antioxidant activity as

U
demonstrated by radical scavenging assay (Wang et al., 2016). Another example is the
N
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
ED

can improve the water solubility and relieve the chain conformation of the

polysaccharides. In addition, sulfated group can activate the hydrogen atoms of the
PT

anomeric carbon to provide the polysaccharides with a strong hydrogen-donating


E

capacity, resulting in the alteration of their biological activities.


CC

3.3. Sulfated derivatives inhibit H2O2-induced premature senescence in MLECs

ROS is continuously produced in the process of metabolism and ROS-induced


A

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

identification of senescent cells, and in most senescent cells, it is detectable by

histochemical staining (Dimri et al., 1995). SA-β-gal is probably derived from the

lysosomal β-galactosidase, and the high expression of SA-β-gal might reflect

increased lysosomal biogenesis that commonly occurs in senescent cells (Lee et al.,

T
2006). To investigate whether BCPS-1 and its sulfated derivatives could inhibit H2O2-

IP
induced senescence in MLECs, the activity of SA-β-gal in these cells was first

R
investigated. Treatment of MLECs with H2O2 caused a significant increase in the

SC
number of SA-β-gal-positive cells (Fig. 3). S-BCP1-4 and S-BCP1-8 significantly

U
alleviated H2O2-induced damage in MLECs. S-BCP1-4- and S-BCP1-8 -treated MECLs
N
both exhibited reduced percentage of SA-β-gal-positive cells, 26.7 ± 2.6% in the case
A

of S-BCP1-4-treated cells, and 24.7 ± 2.9% for S-BCP1-8-treated cells. BCPS-1 also
M

exhibited inhibitory effect against H2O2-induced premature senescence in MLECs, but


ED

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

induced cellular senescence.


E
CC
A

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 ‘**’
U
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
E

polysaccharides, and then exposed the cells to H2O2 and measured their distribution in
CC

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

T
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

underlying mechanism of the protective effect of sulfated polysaccharides against

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

transduction pathways in these cells, consistent with H2O2-induced premature

senescence. Most importantly, treatment with S-BCP1-4 and S-BCP1-8 significantly

T
inhibited the expression of p53 (Figure 5B) and p16 (Figure 5C). The effects of these

IP
polysaccharides on the expression of two H2O2-induced senescence regulators, p53

R
and p16INK4a, were evaluated by qRT-PCR. Consistent with the data from

SC
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.
A

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
ED

expression levels of tumor suppressor protein p53 and cyclin-dependent kinase

inhibitor protein p16, which can bind to and inhibit some CDK-cyclin complexes,
PT

eventually leading to G0/G1 arrest in cell cycle and oxidative stress-induced


E

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

and p16-pRb pathways in oxidative stress-induced cellular senescence.


A

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.

T
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

Modification of BCPS-1 by the addition of sulfated group resulted in significant

changes in the physicochemical properties of the sulfated polysaccharides S-BCP1-4

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

Natural Science Foundation of China (31401203 and 31470430), the China

Postdoctoral Science Foundation (2017M610155), Natural Science Foundation of


A

Zhejiang Province (LY18C020006).

24
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