Polysaccharide extracts of Cirsium japonicum protect rat H 9c2 m yocardial cells from oxidative stress induced by hydrogen peroxide

Zheng-Bo Tao,Li-Yan Xiong,Li-Hui Wang,Chuan Zhang,*

1 Administrative Office for Undergraduates,Second Military Medical University,Shanghai,China.2 Department of Identification of Traditional Chinese Medicine,School of Pharmacy,Second Military Medical University,Shanghai,China.3 Business Unit of Traditional Chinese Medicine Resources,Shanghai Traditional Chinese Medicine Co.,Ltd,ShanghaiPharma,Shanghai,China.

Background

Ischemic heart disease is one of the leading causes of mortality globally[1].It is vital to preventmyocardial ischemia and reperfusion injury and to preserve myocardial cell function in coronary heartdisease surgery[2,3].A large amount of reactive oxygen species(ROS)is produced by myocardial ischemia and reperfusion injury in cardiomyocytes.ROS formation might be triggered by ATP depletion and calcium cation overload[4,5].During reperfusion,ROS formation can occur when oxygen interrupts the mitochondrial respiratory chain[6].It has been proven that ROS affect intercellular communications and cell skeletons,as well as modify the active sites of intracellular targets involved in processes such as cell detachment,morphology alteration,or even cell death[7].The blockage of excessive ROS was supposed to create a promising therapeutic approach against cardiovascular diseases[8].Mitogen-activated protein kinase(MAPK)is one of the important cell signaling pathways that is involved in the development of heart disease[9].

Daji(Cirsium japonicum),a spiny deciduous shrub of the family asteraceae,has recently gained worldwide attention due to its nutritional and medicinal potential[10].This plant is naturally distributed widely acrossAsia and Europe and is currently domesticated in various parts of the world[11].Various parts of Daji(Cirsium.japonicum),especially berries,have been traditionally utilized by the Tibetan and Mongolian people since the Tang Dynasty of China(618 A.D.-907A.D.).The earliest medical use of the plant was found in the Tibetan medicinal system [12]for the treatment of gastric disorders,cardiovascular problems,liver injury,and skin diseases[13,14].Current research has reported a wide spectrum of pharmacological properties of Daji(Cirsium japonicum)including anti-oxidative,immunomodulatory,hepatoprotective,and anti-inflammatory effects[11].Daji(Cirsium.japonicum)berries are rich in nutrients and compounds such as vitamins,carotene,flavonoids,essential oils,carbohydrates,organic acids,amino acids,and minerals.However,the specific components in Daji(Cirsium japonicum)that are responsible for these effects have not yetbeen elucidated.Hence,in the present study,we aimed to investigate the effect of Daji(Cirsium japonicum) polysaccharide extracts (CJP) against oxidative stress in the rat H9c2 myocardial cell line.Results showed that CJP had a protective effect against hydrogen peroxide(H2O2)injury in rat H9c2 myocardial cells.These results laid the preliminary foundation for the therapeutic application of Daji(Cirsium japonicum).

Methods

Materials and reagents

Daji(Cirsium japonicum)was collected from Matoushan in Liangcheng,Inner Mongolia,China,and authenticated by Professor Xuejun Jiang.A voucher specimen of Daji(Cirsiumjaponicum) (herbarium voucher number Shaji-2012-1215)was deposited at the Department of Pharmaceutical Engineering.Sephadex G-150 was purchased from Pharmacia Chemicals (Uppsala,Sweden).

Preparation of polysaccharide extracts

Polysaccharide extracts from the berries of Daji(Cirsium japonicum)were prepared as previously described with small modifications[15].Briefly,the powdered Daji(Cirsium japonicum)dry berries were defatted with light petroleum(boiling point 30-60°C)for 16 h.Then the residue was sonicated for extraction four times with distilled water(solid/liquid ratio 1:25 g/m L)at 80°C for 40 min.The extracts were combined,concentrated,and incubated with papain(0.4 g/100 m L)at 65°C for 6 h.The Sevage method [16]was further adopted to deproteinate concentrated solution, which was precipitated with four volumes of 95%ethanol at 4°C for 48 h and was followed by centrifugation at 1500×g for 10 min at room temperature(25-30°C).And the pellet was washed by ethanol followed by acetone,and then dried.The polysaccharide solution(50 g/L,pH=8.0)was dialyzed extensively against doubly distilled water with an 8 kDa cut-off dialysis bag for 48 h.And to yield the CJP fraction,the dialyzed solution was fractionally precipitated with 80%concentration of ethanol,followed by chromatograph analysis on a Sephadex G-150 column(type:1.5×50 cm),and elution with distilled water(0.5 m L/min).

Analysis of polysaccharide extracts

The total carbohydrate contents in the CJP fraction were determined by the phenol-sulfuric acid method as previously described[17],with d-glucose as a standard.The absorbance was measured at 490 nm.The content of uronic acid in the CJP was determined using the m-hydroxydiphenyl colorimetric method with glucuronic acid as the standard[18].The protein content was determined by the bicinchoninic acid assay using bovine serum albumin(BSA)as the standard.High-performance gel permeation chromatography was adopted to primarily determine the molecules of the polysaccharides and the monosaccharide composition of CJPwasanalyzed by gas chromatography.Briefly,2mg CJP was hydrolyzed with 2 mol/L CF3COOH(1.0 m L)at 120°C for 3 h.The monosaccharide solution of CJPwas derivatized with 0.5 mol/L 1-phenyl-3-methyl-5-pyrazolone and 0.3 mol/L NaOH.After neutralization with 0.3 mol/L HCl and extraction with chloroform,the supernatant was filtered(0.22 µm),analyzed by high performance liquid chromatography equipped with a ZORBAX Eclipse C18 column(250 × 4.6 mm,5 μm),and monitored with UV absorbance at 245 nm.For infrared spectral analysis,10 mg CJPwasmixed with 100mg potassium bromide and compressed to a tablet,followed by conduction on a fourier transform infrared spectrophotometer(Thermo Election Corporation,USA)in the range of 4000-400 cm-1.

Cell cultureand cellviability assay

H9c2 ratcardiomyocyte cells obtained from the American Type Culture Collection(Cedarlane Laboratories,Ontario,Canada)were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10%heat-inactivated fetal bovine serum,100 U/m L penicillin,and 100 g/m L streptomycin at 37°C with 5% CO2.When cell confluency reached 70-80%,H9c2 cells were used for experiments.

Cell viability was measured quantitatively using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Briefly,the ratH9c2 cells(5×104cells/m L)were seeded in a 96-well plate followed by 24 h H2O2incubation of 25,50,100mol/L or CJP incubation of 50,100,200,400,and 600µg/m L.Then,10µLMTT solution(5 mg/m L)was added to each well,and incubated at 37°C for 4 h.After removal of the supernatant,200µL dimethyl sulfoxide was added to each well to dissolve the formazan.The absorbance was measured at 570 nm by an ELISA reader(Tecan,Männedorf,Switzerland).After subtraction of the blank,the cell viability was calculated and presented as percentagesof the control.

Effect of CJPon hydrogen peroxide injury model

The model of oxidative stress injury was established by adding a free radical donor,H2O2,to the cultured myocardial cells.To determine the protective effect of CJP against H2O2-induced cell loss in H9c2 cells,cells were incubated with increasing concentrationsof CJP(50,100,200,400,and 600µg/m L)for 24 h and then treated with 100mol/L H2O2for another 24 h.Cell viability was determined colorimetrically using MTT assay.

Fluorescence-activated cell sorting(FACS)analysis of cell apoptosis

Cell apoptosis was detected using fluorescein isothiocyanate-conjugated Annexin-V and propidium iodide double staining as described previously[19].Briefly,the rat H9c2 cells(1×106cell/m L),subjected to be shock with H2O2and treat with or without CJP of 100µg/m L,were seeded into 6-well plates.Staining was performed using an Annexin-V and propidium iodide double staining kit(Biosea Biotechnology Co.,Beijing,China), in accordance with the manufacturer’s instructions.Ten thousand cells were counted,and the data acquisition and analysis were performed using a flow cytometer FACSCaliburTM(BD Biosciences,San Diego,CA,USA)and CellQuest software(BD Biosciences,San Diego,CA,USA).The alteration of nuclearmorphology was observed under a fluorescence microscope(Olympus,IX53,Tokyo,Japan)by Hoechst33258 staining.

Western blot analysis

H9c2 cells growing on 6-well plateswere pretreated with or withoutCJP(100µg/m L)for 24 h prior to exposing to 400 μM H2O2,as indicated.Then cells were harvested and lysed using Radio-Immunoprecipitation Assay lysis buffer containing 50 mM Tris-HCl(pH 7.4),150 mM NaCl,1%sodium deoxycholate,1%Nonidet P-40,1mM phenylmethylsulfonyl fluoride,1 mMethylene diamine tetra acetic acid,and protease inhibitors.The protein concentration of each sample was determined using a bicinchoninic acid protein assay kit(Pierce Chemical,Rockford,IL).A sample of 40 μg of the total protein was electrophoresed on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes.After being washed three times with phosphate-buffered saline solution, the polyvinylidene difluoridemembranes were blocked in 5%BSA for 2 h and incubated overnight at 4°C with the primary antibodies against caspase-3, caspase-8,caspase-9,p38,c-Jun N-terminal kinase(JNK)or β-actin.Subsequently, the membranes were washed in Tris-buffered saline-Tween 20 containing 1%BSA,and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature,and developed using the ECL chemiluminescence detection system(Amersham).

Statistical analysis

The data are expressed as the mean±SEM.Statistical analysis was performed using one-way analysis of variance.P<0.05 was considered to be statistically significant.

Results

Phytochem icalanalysisof CJP

CJP,a dark-brown powder,is soluble in water.CJP is comprised of carbohydrate(77.21%)and uronic acid(36.69%).Furthermore,Coomassie brilliant blue staining showing that the protein content in CJP was 6.17%.High-performance gel permeation chromatography analysis showed that CJP contained one major polysaccharide with severalminor ones(Figure 1A).The monosaccharide composition of CJP by gas chromatographic analysis found that CJP is mainly composed of glucose,galactose,arabinose,and rhamnose in the ratio of 3.40:2.14:1.17:1.00,together with trace amountsofmannose and xylose.

The fourier transform infrared spectrophotometer spectrum analysis of CJP is shown in Figure 1B.The strong stretching peak at 3398.1 cm-1represents a hydroxyl group(-OH).Peaks at 1740.7 cm-1and 1617.3 cm-1could be attributed to the aldehyde group(-CHO)stretching vibration and double bond(C=C)stretching vibration,respectively.The peak at 1383.9 cm-1might represent themethyl(-CH3)saturated C-H plane bending vibration.The peak at 1101.1 cm-1represents C-O-C glycosidic rings asymmetric vibration,indicating the existence of pyranose in CJP.The peak at 1020.6 cm-1might representC-O stretching vibration.

CJPattenuated H9c2 cell injury induced by H 2O2

H9c2 cell viability(a cell line of rat cardiomyocytes),assessed by the MTT assay,decreased dramatically after H2O2treatment for 24 h in a dose-dependent manner(Figure 2A).CJPpretreatmentalone did not significantlyalter the cell viability level of H9c2 cells.Notably,CJP(100, 200, 400, and 600 µg/m L) pretreatment significantly protected the H9c2 rat cardiomyocytes from H2O2injury dose-dependently(Figure 2B).Therefore,CJP pretreatment exhibited protective effects in free radical-induced myocardial cell injury.

Figure1 Phytochem ical analysisof CJP

CJPattenuated H2O2-induced H 9c2 cell apoptosis

The nucleus in the normal group was showed as a typical round or oval shape;those in the H2O2-injured group were typical of karyopyknotic stain,with chromatin concentrated fragmentation(Figure 2C).Quantitative analysis using FACS analysis confirmed that the number of cells in the CJP treated group was significantly less than in the H2O2-injured group(Figure 2D).Furthermore,as shown in Figure 2E,H2O2induced caspase-3,-8,and-9 cleavages in H9c2 cells;however,the effects were inhibited by CJP,further demonstrating that CJP suppressed the induced apoptosis.

Protection of CJP against H2O2 injury m ight involve the MAPK pathway

Apoptosis was finally induced by H2O2,which could be partially attributed to the activated MAPK signaling pathway by H2O2.To further investigate the mechanism underlying the effect of CJP on MAPK,the intracellular signal transduction cascade wasexamined(Figure 3).The activation of p38 MAPK and JNK was detected by western blotanalysis.The phosphorylation of p38 MAPK and JNK were significantly enhanced in the H2O2group,demonstrating that pretreatment with CJP 100µg/m L could ameliorate the phosphorylation levels.

Discussion

In the present study,we demonstrated that the polysaccharide extracts of Daji(Cirsium japonicum)exerted a protective effect against oxidative stress in rat H9c2 cells treated with H2O2.It was found that CJP attenuated H2O2-induced cell apoptosis via inhibiting the MAPK pathway.These results imply that CJP could regulate the apoptosis-related pathway in response to oxidative stress.

Figure 2 CJPsignificantly m itigated the H2O2-induced injury through inhibition of apoptosis

Figure 3 CJPsignificantly inhibited the phosphorylation of p38 and JNK MAPK pathway in H9c2 cardiomyocytes treated by H2O2

Polysaccharidesare reported to have pleiotropic effects,including hypolipidemic [20], anti-radiation [21],pro-immune[22],anti-oxidative[23],and other effects.As a long applied herbal medicine,Daji(Cirsium japonicum)has been extensively studied for its possible anti-inflammatory [24], anti-diabetic [25], and hepatoprotective effects[11].Additionally,the cytotoxic effect of the planthas been investigated[26].One reason that could not be neglected for the pleiotropic effects is the complex content of active components present in the plant.Therefore,itwas vital to examine the effect of an individual component rather than the whole plant,or even the medical part of the plant.Our study showed that the polysaccharide extracts inhibited cell apoptosis in H2O2-treated rat H9c2 cells,which was at least partially involved in theMAPK pathway.

Oxidative stress-induced cardiomyocyte damage plays an important role in the pathophysiology of cardiovascular diseases[27].It is a state when the free radical production exceeds the defense ability of the antioxidative system.ROS is one main class of free radical,among which H2O2is a metabolite generated by various enzyme-catalyzed redox reactions.Cardiac oxidative stress is reported to be associated with increased cardiac fibrosis and hypertrophy,and reduced cardiac performance and contractility,which might lead to severe cardiac dysfunction and potentially fatal cardiac events[28].ROS could be produced in response to alcohol, nonsteroidal anti-inflammatory drugs,ischemia-reperfusion injury,chronic infections,and inflammatory disorders[29].Agents that reduce ROS production and inhibit oxidative stress that produce little adverse effects could have great therapeutic potential in the prevention or treatment of cardiovascular diseases.The present study provides CJP as a promising therapy for oxidative stress-related cardiovascular diseases.The detailed mechanism underlying the protection of CJP against oxidative stress warrant further research.It was reported by Nickeletal.[30]that rather than the cytoplasmic ROS,the mitochondrial ROS play a more important role in the pathogenesis of cardiovascular diseases.

MAPK is a family of serine/threonine kinases that mediate intracellular signals in response to various stimuli.The activation of the MAPK pathway,linked to oxidative stress,has been demonstrated to be involved in the process of cell apoptosis[31].Mammals typically express at least three distinct groups of MAPKs,including p38MAPK,ERK 1/2 and JNK.Itwas reported thatantagonism of the expression of JNK and p38 or their phosphorylation might inhibitmyocardial infarction and apoptosis,restoring cardiac function subsequent to myocardial ischemia[32,33].Similar to these previous studies,our study showed that H2O2significantly enhanced the phosphorylation levels of p38 MAPK and JNK,indicating activation of theMAPK pathway.

Conclusions

In the presentstudy,we investigated the protective effects of Daji(Cirsium japonicum)polysaccharide extracts against H2O2-induced oxidative stress.The results confirmed that,in H2O2-treated rat H9c2 cardiomyocytes,cell apoptosis was attenuated via the inhibition of the MAPK pathway.The present study provides a foundation for further exploration of agents against cardiovascular disease-related oxidative stress.

Acknow ledgement

The current study was designed by Dr Zhang Chuan and Mr Wang Li-Hui.The experiments were conducted by Mr Tao Zheng-Bo with the direction of Dr Xiong Li-Yan.The data was collected and analyzed by Mr Tao and Dr Xiong.The manuscript was drafted by Mr.Tao and Dr Xiong,and edited by MrWang and Dr Zhang.

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