Protective effect of tanshinone IIA on human umbilical vein endothelial cell injured by...

Journal of Ethnopharmacology108(2006)217–222Protective effect of tanshinone IIA on human umbilical vein endothelial cell injured by hydrogen peroxide and its mechanism Rong Lin a,b,∗,Wei-Rong Wang a,Jun-Tian Liu a,Guang-De Yang c,Chun-Jie Han aa Department of Pharmacology,Xi’an Jiaotong University School of Medicine,Xi’an,Shaanxi,PR Chinab Key Laboratory of Environment and Genes Related to Diseases,Xi’an Jiaotong University,Ministry of Education,Xi’an,Shaanxi,PR Chinac Department of Pharmacy,Xi’an Jiaotong University School of Medicine,Xi’an,Shaanxi,PR ChinaReceived26March2006;received in revised form24April2006;accepted8May2006Available online16May2006AbstractTanshinone IIA(Tan IIA)is isolated from Salvia miltiorrhiza,the root of which is widely used as a traditional Chinese medicine to treat atherosclerosis.The aim of the present study was to evaluate the putative protective effect of Tan IIA in a human umbilical vein endothelial cell line(ECV-304)injured by hydrogen peroxide in vitro and the mechanism of its protection.The percentage of cell viability was evaluated by3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.The endothelial cell apoptosis and expression of cluster of differentiation 40(CD40)were detected byflow cytometric analysis.Preincubation with Tan IIA significantly increased the viability of ECV-304cell injured by hydrogen peroxide,which was accompanied with the increased nitric oxide level and superoxide dismutase activity in a dose-dependent manner. Moreover,cell apoptosis and CD40expression were decreased in a dose-dependent manner.In conclusion,our data suggests that Tan IIA protects ECV-304cell damage induced by hydrogen peroxide through its anti-oxidant effect and CD40anti-inflammatory approach.©2006Elsevier Ireland Ltd.All rights reserved.Keywords:Tanshinone IIA;Hydrogen peroxide;Apoptosis;Nitric oxide;CD401.IntroductionAtherosclerosis is one of the most prevalent fatal diseases in current society.For some years,lipid peroxidation of endothelial cell has been accorded crucial roles in the process of atheroscle-rosis.Tanshinone IIA(Tan IIA)is a lipophilic diterpene com-pound found in Salvia miltiorrhiza as a marker component. Many experimental and clinical studies have reported that Tan IIA is an anti-oxidant being effective to atherosclerosis(Li et al.,1991;Lin,1991;Liang et al.,2000).Our previous stud-ies also indicated that Tan IIA has a protective effect against human umbilical vein endothelial cell line(ECV-304)dam-age induced by hydrogen peroxide(H2O2)via anti-oxidative activity(Wang et al.,2006).Increasing evidences support an involvement of inflammation in pathogenesis of atherogenesis (Libby et al.,2002).Studies have identified cluster of differ-Abbreviations:MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-iphenyl tetrazolium bromide;SOD,superoxide dismutase;DMSO,dimethyl sulphoxide∗Corresponding author.Tel.:+862982657691;fax:+862982655188.E-mail address:linrong@(R.Lin).entiation40(CD40)and ligand of cluster of differentiation40 (CD40L)as key regulator of this process and potentially impor-tant atherosclerotic therapeutic target(Schonbeck et al.,2000). Recently our studies have established that lovastatin and fenofi-brate can decrease CD40and CD40L expression induced by C-reactive protein(CRP)in endothelial cell(Lin et al.,2004). The effect of Tan IIA on CD40expression induced by H2O2in ECV-304cell is unknown.Therefore,the aim of the present study was to examine the protective effect of Tan IIA in ECV-304cell injured by H2O2 by measuring cell viability,cell apoptosis,and nitric oxide (NO)level and to assessed role of the anti-oxidative activity or the CD40-CD40L anti-inflammatory pathway in the protection by measuring superoxide dismutase(SOD)activity and CD40 expression.2.Materials and methods2.1.MaterialsRPMI1640medium and fetal bovine serum(FBS)were pur-chased from Gibco(Gibco Industries Inc.).3-(4,5-Dimethyl-0378-8741/$–see front matter©2006Elsevier Ireland Ltd.All rights reserved. doi:10.1016/j.jep.2006.05.004218R.Lin et al./Journal of Ethnopharmacology108(2006)217–222thiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT),trypsin, penicillin,streptomycin,dimethyl sulphoxide(DMSO),and hydrogen peroxide were obtained from Sigma(St.Louis,MO, USA).Mouse anti-human CD40antibody and goat anti-mouse FITC-conjugated IgG were purchased from Santa Cruz Biotech-nology(Santa Cruz,CA,USA).SOD and NO assay kits were produced by the Institute of Nanjing Jiancheng Biology Engineering(Nanjing,PR China).AnnexinV-fluorescin isoth-iocyanate(Annexin V-FITC)apoptosis detection kit was from Beckman Coulter Company(France).All other chemicals used were of the commercially available grade.2.2.Preparation of Tan IIA extractThe dried root of Salvia miltiorrhiza Bunge was purchased from the western region of Shaanxi Province,PR China and authenticated by Professor Guangde Yang(Department of Phar-macy,Xi’an Jiaotong University,Xi’an,PR China).Tan IIA for reference was supplied by the Central Control Institute of Pharmaceutical and Biological Products(Beijing,PR China). Air-dried and powdered Salvia miltiorrhiza Bunge root(250g) was soaked in1500mL of95%ethanol for60min,and then refluxed twice in a water bath for about2h.The combined extracts were thenfiltered through a0.45␮m Type JH mem-branefilter(Ø47mm,Millipore,Billerica,USA).Thefiltrate concentrated in vacuo was evaporated at40◦C under the reduced pressure to obtain crude Tan IIA.The conditions of determination of Tan IIA by high perfor-mance liquid chromatography(HPLC)were applied according to the method for assay of Tan IIA in Chinese Pharmacopoeia. The experimental conditions of the reverse phase-high perfor-mance liquid chromatography(RP-HPLC)method were as fol-lows:planetsil C18column(150mm×4.6mm,5␮m),mobile phase of methanol–water(75:25),flow rate at1.0mL/min,detec-tion wavelength at270nm and room temperature.2.3.Cell cultureThe human umbilical vein endothelial cell line ECV-304was obtained from Shanghai Institutes for Biological Sciences.The cell was maintained in an atmosphere of5%CO2at37◦C in RPMI1640medium supplemented with10%fetal bovine serum, 100U/mL penicillin and100mg/mL streptomycin.2.4.Oxidative damage induced by H2O2ECV-304cell was cultured in24-well plates at a density of 2×105cells/mL and allowed to grow to the desired confluence. Tan IIA(15,30and60␮g/mL)dissolved in DMSO(no more than0.1%in v/v)was added into the wells for24h incuba-tion,and then the medium was changed and cell was exposed to 0.25mM H2O2for another4h except normal control.2.5.Measurement of cell viabilityThe viability of ECV-304cell was measured by a colori-metric MTT assay.Briefly,after the different concentrations of Tan IIA in RPMI1640were added to the wells to incubate for 24h,0.25mM H2O2was added for further4h incubation.Fol-lowing addition of MTT solution(5mg/mL)to each well,the plates were incubated for4h at37◦C.After the medium had been removed,the dye crystal was dissolved in150␮L DMSO. Finally,the optical density(OD)of each well was immediately measured on ELISA micro-plate reader(Metertech)at570nm to represent cellular viability.The OD of formazan formed in control cell was taken as100%viability.2.6.Determination of SOD activity and NO releaseAfter exposed to the different concentrations of Tan IIA for24h,ECV-304cell was incubated with0.25mM H2O2for another4h.The medium sample was collected to measure SOD activity and NO release.Detection of SOD activity was immedi-ately performed on an ultraviolet/visible scanning spectropho-tometer at550nm.The level of NO in the conditioned medium was immediately measured as previously described(Feng et al., 2001).2.7.Determination of apoptotic cellsAfter exposure to the various concentrations of Tan IIA for24h,ECV-304cell was incubated with0.25mM H2O2 for another4h.The medium sample was collected immedi-ately to measure apoptotic cells by AnnexinV-FITC detection kit according to manufacturer’s protocol.ECV-304cell was analyzed in Becton Dickinson FACScanflow cytometer using CellQuest software(Becton Dickinson).The cell was identified as follows:normal ECV-304cell locating in the lower left quad-rant of theflow cytometric result,necrotic ECV-304cell in the upper right quadrant,apoptotic ECV-304cell in the lower right quadrant.2.8.Determination of CD40expressionAfter ECV-304cell grew for24h in free FBS,the differ-ent concentrations of Tan IIA in RPMI1640were added to the wells to incubate for further24h and then cell was incubated with0.25mM H2O2for another4h.Expression of CD40was assessed byflow cytometry under the above condition.The cells (1×106/mL)harvested by trypsinization were incubated with 10␮L(200␮g/mL)of the mouse anti-human CD40monoclone antibody for1h at4◦C.Subsequently,the cells were washed twice with PBS and centrifuged at1000rpm for3min before incubated with30␮L(1.4␮g/mL)of the goat anti-mouse FITC-conjugated Ig G for1h at4◦C.Finally,the washed ECV-304 cells were immediatelyfixed in4%paraformaldehyde PBS and analyzed in theflow cytometer.At least5000viable ECV-304 cells per condition were analyzed.2.9.Statistical analysisData were analyzed using a one-way ANOV A.The data repre-sented means±S.E.M.and values of P<0.05were statistically significant.R.Lin et al./Journal of Ethnopharmacology 108(2006)217–2222193.Results3.1.Effect of Tan IIA on H 2O 2injured cell viability in ECV-304cellTo evaluate whether Tan IIA protected the cell from oxidative damage induced by H 2O 2,ECV-304cell was incubated with Tan IIA (7.5,15,30and 60␮g/mL)for 24h,then H 2O 2was used as a damage agent for another 4h.The cell viability was measured by MTT assay.H 2O 2decreased the viability of cell markedly,while Tan IIA relived the cell damage induced by H 2O 2in a dose-dependent manner (Fig.1).Tan IIA significantly increased the cell viability with increase of the used concentration.The result suggests that Tan IIA protects ECV-304cell from oxidative dam-age.3.2.Effects of Tan IIA on SOD activity and NO release in H 2O 2injured ECV-304cellThe activity of SOD in the conditioned medium,as an indica-tor of cell anti-oxidation,was measured spectrophotometrically using a commercially available assay kit.The level of NO in the conditioned medium was determined indirectly as the content of nitrite and nitrate.Incubation of the cell with 0.25mM H 2O 2for 4h caused a significant decrease in SOD activity and NO release in the medium.While preincubation of the cell with Tan IIA (15,30and 60␮g/mL)significantly attenuated the decreased NO level and SOD activity in a dose-dependent manner (Table 1).3.3.Effect of Tan IIA on apoptosis in H 2O 2injured ECV-304cellTo evaluate the effect of different concentrations of Tan IIA on cell apoptosis,AnnexinV-FITC detection kit and flowcytom-Fig.1.Effect of Tan IIA on cell viability in H 2O 2injured ECV-304cell.The cell was incubated with different concentrations of Tan IIA (7.5,15,30and 60␮g/mL)for 24h and followed by incubation with 0.25mM H 2O 2.Cell viability was measured by MTT assay (n =8).Results were presented as mean ±S.E.M.values of the data obtained from three independent experiments performed in duplicate.*P <0.05,compared with H 2O 2group;**P <0.01,com-pared with H 2O 2group.Table 1Effects of Tan IIA on SOD activity and NO release in H 2O 2injured ECV-304cell a Group Dose (␮g/mL)NO (nmoL/mL)SOD (U/mL)Control 80.87±3.60**47.71±1.61**H 2O 264.16±3.2935.74±1.96Tan IIA 1568.58±2.3438.73±1.01*Tan IIA 3074.15±3.07*43.80±1.76**Tan IIA6078.52±1.81**45.99±1.57**a All the data were shown as the mean ±S.E.M.,n =8.*P <0.05,compared with H 2O 2group.**P <0.01,compared with H 2O 2group.etry analysis were used according to manufacturer’s protocol.As shown in Fig.2,Tan IIA reduced the apoptotic cells in a dose-dependent manner.Normal control cells showed minimal apoptosis rate 5.67±0.30%(Fig.2A),the H 2O 2injured cells for 4h showed apoptosis rate 55.43±1.71%(Fig.2B),the per-centages of apoptotic cells pretreated with 15,30and 60␮g/mL Tan IIA were 43.90±1.73%,28.85±2.29%and 12.4±0.87%,respectively (Fig.2C–E).3.4.Effect of Tan IIA on CD40expression in H 2O 2injured ECV-304cellTo examine whether Tan IIA influenced the expression of CD40in H 2O 2injured ECV-304cell involved in the pathogene-sis of atherosclerosis,we explored the expression of CD40with flow cytometry.As shown in Fig.3,the percentage of CD40positive cells in H 2O 2injured cells significantly increased and incubation of the cell with Tan IIA (15,30and 60␮g/mL)for 24h prior to H 2O 2prevented the increase.This suggests that Tan IIA reduces the expression of CD40in a dose-dependent manner.4.DiscussionSalvia miltiorrhiza Bunge is a famous Chinese herbal plant,and its root,known as Danshen in Chinese,is an ancient Chinese drug.The chief bioactive ingredients of Danshen are the diterpenoid pigments,particularly the phenanthrofurane quinone derivatives generally known as tanshinones (Tang and Eisenbrand,1992).Tan IIA is the most effective member of this family and has been used as a reference for some medicine.Tan IIA functions as an anti-oxidant (Ng et al.,2000).The in vitro experiments indicate that Tan IIA can decrease the expression of intercellular adhesion molecular-1(ICAM-1)in human umbil-ical vein endothelial cell (HUVEC)induced by tumor necro-sis factor-␣(TNF-␣)and inhibit the oxidation of low-density lipoprotein (Jiang et al.,1998;Niu et al.,2000).Moreover,our studies show that Tan IIA has a protective effect against apopto-sis of endothelial cell injured by H 2O 2via decreasing the content of malondialdehyde (Wang et al.,2006).There are considerable evidences that oxidative damage contributes to the develop-ment of atherosclerosis through the formation of reactive oxygen species (Shi et al.,2000)and lipid peroxidation,and so Tan IIA can be used for atherosclerosis in traditional Chinese medicine.220R.Lin et al./Journal of Ethnopharmacology 108(2006)217–222Fig.2.Effect of Tan IIA on apoptosis in H 2O 2injured ECV-304cell After the preincubation of cell with Tan IIA (15,30and 60␮g/mL)for 24h and then using 0.25mM H 2O 2as a injury agent for another 4h,the apoptosis of ECV-304cell was measured by the flow cytometry (n =6).(A):control;(B):H 2O 2group;(C):cell preincubation with 15␮g/mL Tan IIA;(D):cell preincubation with 30␮g/mL Tan IIA;(E):cell preincubation with 60␮g/mL Tan IIA.Our present studies were to assess the possible H 2O 2-induced oxidative damage in endothelial cells involved in atherosclerosis and the effect of Tan IIA on activity of SOD being an important endogenous anti-oxidant enzyme.The result showed that Tan IIA (15,30and 60␮g/mL)led to a significant increase of SOD activity in H 2O 2damaged cell in a dose-dependent manner,indi-cating that Tan IIA has an ability to protect ECV-304cell from H 2O 2induced injury through anti-oxidative approach.Inflammation is now recognized as a major force driving atherosclerosis (Ross,1999).Recent studies have demonstrated that CD40–CD40L inflammatory signaling pathway is a potent activator of endothelial cell and promoter of atherosclerosis (Phipps,2000).The expression of CD40on endothelial cell is lit-tle in sections of normal arterial tissue (Mach et al.,1997).How-ever,our study shows that lovastatin and fenofibrate decrease CD40expression on endothelial cell induced by CRP (Lin et al.,2004).Tan IIA possesses an anti-inflammatory effect.Stud-ies indicate that Tan IIA inhibits migration of human leukocyte and decrease the expression of ICAM-1in HUVEC induced by TNF-␣in vitro (Zhou et al.,1997;Jiang et al.,1998).Another research also indicates that Tan IIA decreases the expression of ICAM-1and alleviated chronic inflammatory reaction of heart in rats (Zhan et al.,2005).Our present studies were to assess the possible expression of CD40in H 2O 2injured endothelialR.Lin et al./Journal of Ethnopharmacology108(2006)217–222221Fig.3.Effect of Tan IIA on CD40expression in H2O2injured ECV-304cell.The cell was incubated with different concentrations of Tan IIA(15,30,60␮g/mL) for24h and then,exposed to H2O2for4h,expression of CD40was explored by flow cytometry.The columns indicated the mean value of4times of experiment. The bars were the S.E.M.*P<0.05,compared with H2O2group;**P<0.01, compared with H2O2group.cell involved in atherosclerosis and the effect of Tan IIA on the expression.The results indicated that Tan IIA prevented the increase of CD40expression in H2O2injured ECV-304cell in a dose-dependent manner,suggesting that Tan IIA has a protec-tive effect against ECV-304cell damage induced by H2O2via CD40–CD40L inflammatory signaling pathway.Hydrogen peroxide also can decrease the viability of cell and induce cell apoptosis(Stridh et al.,1998).Apoptosis is a form of programmed cell death that occurs naturally in cells (Zimmermann et al.,2001).Either insufficient or excessive apoptosis may lead to diseases(Evan and Littlewood,1998).For example,too little apoptosis may lead to tumorigenesis(Debatin et al.,2002),whereas an excess of apoptosis has been implicated in pathogenesis of many diseases including atherosclerosis.Our results also showed that Tan IIA inhibited apoptosis of H2O2 injured ECV-304cell and increased viability of cell injured by H2O2in a dose-dependent manner.Moreover,Tan IIA also increased the release of NO in a dose-dependent manner.NO is known to be an important effector molecule that has a broad spectrum of physiological and pathophysiologic effects,NO has been reported to have protective effects against apoptosis in a variety of cell types including endothelial cell(Dimmeler et al., 1999;Rossig et al.,2000).NO plays an important role in reg-ulating the functional integrity of endothelium,which acts as a barrier between the circulating blood and the underlying tis-sue(Harrison,1994;Dimmeler and Zeiher,1999).Thus,NO regulates the vascular tone,provides anti-thrombotic and anti-inflammatory activity,and inhibits endothelial cell apoptosis (Tsao and Cooke,1998;Murohara et al.,1999;Grub et al.,2000).In conclusion,we demonstrate that Tan IIA has a protec-tive effect against ECV-304cell damage induced by H2O2 via anti-oxidative and anti-inflammatory pathways.The anti-inflammatory pathway is mediated by the decreased CD40 expression induced by H2O2in ECV-304cell.Further,Tan IIA inhibits the cell apoptosis induced by H2O2through promotion of NO release.It is noteworthy that thefinding of the present study may shed light on the pharmacological basis for the clin-ical application of traditional Chinese medicine in treatment of atherosclerosis relevant to endothelial cell damage. AcknowledgmentThis work is supported by grant from the National Natural Science Foundation of China,No.:30371759and Key Science Research Project of Shaanxi Province of China(2005K10-G1). ReferencesDebatin,K.M.,Poncet,D.,Kroemer,G.,2002.Chemotherapy:targeting the mitochondrial cell death pathway.Oncogene21,8786–8803. Dimmeler,S.,Fleming,I.,Fisslthaler,B.,Hermann,C.,Busse,R.,Zeiher,A.M.,1999.Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.Nature399,601–605.Dimmeler,S.,Zeiher,A.M.,1999.Nitric oxide–an endothelial cell survival factor.Cell Death Differentiation6,964–968.Evan,G.,Littlewood,T.,1998.A matter of life and cell death.Science281, 1317–1322.Feng,Q.,Lu,X.,Jones,D.L.,Shen,J.,Arnold,J.M.,2001.Increased inducible nitric oxide synthase expression contributes to myocardial dysfunction and higher mortality after myocardial infarction in mice.Circulation104, 700–704.Grub,S.,Persohn, E.,Trommer,W.E.,Wolf, A.,2000.Mechanisms of cyclosporine A-induced apoptosis in rat hepatocyte primary cultures.Toxi-cology and Applied Pharmacology163,209–220.Harrison, D.G.,1994.Endothelial dysfunction in atherosclerosis.Basic Research in Cardiology1,87–102.Jiang,K.Y.,Ruan,C,G.,Gu,Z,L.,Zhou,W.Y.,Guo,C.Y.,1998.Effects of tanshinone II-A sulfonate on adhesion molecule expression of endothelial cells and platelets in vitro.Zhongguo Yao Li Xue Bao19,47–50.Li,Z.T.,Yang,B.J.,Ma,G.E.,1991.Chemical studies of Salvia miltiorrhiza f.alba.Yao Xue Xue Bao26,209–213.Liang,Y.,Yang,Y.M.,Yuan,S.L.,2000.Studies on pharmic mechanism and clinic application of Tanshinone.Traditional Herbal Drugs31,304–306. Libby,P.,Ridker,P.M.,Maseri,A.,2002.Inflammation and atherosclerosis.Circulation105,1135–1143.Lin,R.,Liu,J.,Gan,W.,Yang,G.,2004.C-reactive protein-induced expres-sion of CD40-CD40L and the effect of lovastatin and fenofibrate on it in human vascular endothelial cells.Biological and Pharmaceutical Bulletin 27,1537–1543.Lin,T.J.,1991.Antioxidation mechanism of schizandrin and tanshinonatic acidA and their effects on the protection of cardiotoxic action of adriamycin.Sheng Li Ke Xue Jin Zhan22,342–345.Mach,F.,Schonbeck,U.,Sukhova,G.K.,Bourcier,T.,Bonnefoy,J.Y.,Pober, J.S.,Libby,P.,1997.Functional CD40ligand is expressed on human vascular endothelial cells,smooth muscle cells,and macrophages:implications for CD40–CD40ligand signaling in atherosclerosis.Proceedings of the National Academy of Sciences of the United States of America94,1931–1936. Murohara,T.,Witzenbichler,B.,Spyridopoulos,I.,Asahara,T.,Ding,B.,Sul-livan,A.,Losordo,D.W.,Isner,J.M.,1999.Role of endothelial nitric oxide synthase in endothelial cell migration.Arteriosclerosis Thrombosis and Vas-cular Biology19,1156–1161.Ng,T.B.,Liu,F.,Wang,Z.T.,2000.Antioxidative activity of natural products from plants.Life Science66,709–723.Niu,X.L.,Ichimori,K.,Yang,X.,Hirota,Y.,Hoshiai,K.,Li,M.,Nakazawa,H.,2000.Tanshinone IIA inhibits low density lipoprotein oxidation in vitro.Free Radical Research33,305–312.Phipps,R.P.,2000.Atherosclerosis:the emerging role of inflammation and the CD40-CD40ligand system.Proceedings of the National Academy of Sci-ences of the United States of America97,6930–6932.Ross,R.,1999.Atherosclerosis:an inflammatory disease.New England Journal of Medicine340,115–126.222R.Lin et al./Journal of Ethnopharmacology108(2006)217–222Rossig,L.,Haendeler,J.,Hermann,C.,Malchow,P.,Urbich,C.,Zeiher,A.M., Dimmeler,S.,2000.Nitric oxide down-regulates MKP-3mRNA levels: involvement in endothelial cell protection from apoptosis.The Journal of Biological Chemistry275,25502–25507.Schonbeck,U.,Sukhova,G.K.,Shimizu,K.,Mach,F.,Libby,P.,2000.Inhibition of CD40signaling limits evolution of established atherosclerosis in mice.Proceedings of the National Academy of Sciences of the United States of America97,7458–7463.Shi,W.B.,Haberland,M.E.,Jien,M.L.,Shih,D.M.,Lusis,A.J.,2000.Endothe-lial responses to oxidized lipoproteins determine genetic susceptibility to atherosclerosis in mice.Circulation102,75–81.Stridh,H.,Kimland,M.,Jones,D.P.,Orrenius,S.,Hampton,M.B.,1998.Cytochrome C release and caspase activation in hydrogen peroxide and tributyltin-induced apoptosis.FEBS Letters429,351–355.Tang,W.,Eisenbrand,G.,1992.Chdrugs of Plant Origin:Chemistry,Pharmacol-ogy and Use in Traditional and Modern Medicine.Springer-Verlag,Berlin, pp.891–902.Tsao,P.S.,Cooke,J.P.,1998.Endothelial alterations in hypercholesterolemia: more than simply vasodilator dysfunction.Journal of Cardiovascular Phar-macology32,S48–S53.Wang,W.R.,Lin,R.,Peng,N.,Han,C.J.,2006.The protective effects of tanshi-none.II A on vascular endothelial cells injury induced by hydrogen peroxide.Journal of Chinese Medicinal Materials29,53–55.Zhan,C.,Tao,X.,Tian,C.,Xiong,W.,Zheng,Z.,2005.Expression of intercellu-lar adhesion molecule-1in the development of hypertensive left hypertrophy and inhibitory effect of tanshinone II A.Chinese Journal of Clinical Reha-bilitation9,254–256.Zhou,Z.,Zheng,J.,Xu,W.,1997.Study on the effect of ofloxacin and tanshi-none.II.A on human leukocyte chemotactic migration in vitro.Zhongguo Yi Xue Ke Xue Yuan Xue Bao19,232–235.Zimmermann,K.C.,Bonzon,C.,Green,D.R.,2001.The machinery of pro-grammed cell death.Pharmacology and Therapeutics92,57–70.。