蚤休醇提物与蚤休皂苷对H_2O_2诱导的ECV304细胞损伤作用的机制研究
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摘要
目的:对中药蚤休进行提取,应用其两种产物研究对H_2O_2诱导的脐静脉内皮细胞ECV304损伤的保护作用及其机制。方法:首先制备蚤休醇提物,并进行蚤休总皂苷的提取; MTT法检测蚤休醇提物和蚤休皂苷对H_2O_2诱导的ECV-304损伤的保护作用及确定两种药物保护作用的实验剂量;体外培养脐静脉内皮细胞ECV304,建立氧化损伤的细胞模型,然后分为八个实验处理组:1正常对照组2氧化损伤组3高浓度蚤休醇提物(EFB)(500pg/ml)组4中浓度蚤休醇提物(50pg/ml)组5低浓度(5pg/ml)组6高浓度蚤休皂苷(Pa)(1ug /ml)组7中浓度蚤休皂苷(100pg/ml)组8低浓度蚤休皂苷(10pg /ml)组;在第二部分实验中,应用流式细胞仪Annexin V/PI染色检测两种药物干预H_2O_2氧化损伤前后的细胞凋亡、PI染色检测细胞周期与凋亡的关系、激光共聚焦显微镜观察Annexin V/PI染色后各组细胞的形态学改变;激光共聚焦显微镜观测药物各组细胞内Ca2+的含量的表达,500pg/ml蚤休醇提物与1ug/ml蚤休皂苷预处理10分钟加入损伤浓度的H_2O_2,观察细胞内Ca2+的浓度的动态变化。RT-PCR检测各实验组caspase-3 mRNA表达的变化;免疫荧光染色检测各实验组caspase-3的定性表达。在第三部分实验中,以RT-PCR检测各实验组血管内皮细胞中细胞间粘附分子(intracelluar adhesion molecule,ICAM-1)、血管细胞粘附分子(vasculer cell adhesion molecule , VCAM-1 )、细胞因子--单核细胞趋化蛋白(monocytechemoattractant protein,MCP-1)mRNA水平的变化的表达;应用流式细胞仪定量检测ICAM-1、VCAM-1的阳性细胞数;免疫荧光染色检测各实验组MCP-1的表达。结果:建立了细胞过氧化损伤的模型;经鉴定正确提取制备了蚤休醇提物和蚤休皂苷;MTT显示加入损伤因素后细胞吸光度OD值低于正常对照组(P<0.01);两种药物预处理后OD值增加,高浓度蚤休醇提物组与高浓度蚤休皂苷组的OD值与正常组相比无显著性差异(P>0.05);流式细胞结果显示:采用流式细胞仪PI染色检测结果对细胞周期进行分析,表明损伤作用于ECV-304细胞后,细胞在G0/G1期的数目增多,在S期的数目减少,同时流式细胞仪检测可见亚二倍体峰,而蚤休醇提物和蚤休皂苷预处理以后,S期细胞数明显增多,各药物干预组G0/G1期细胞数与正常组无差异。流式细胞仪Annexin V/PI检测方法显示,损伤组的早期与晚期凋亡率之和最高,,预处理后细胞的早期与晚期凋亡率之和降低,在蚤休皂苷,其降低凋亡的效应呈剂量依赖性。激光共聚焦显微镜观察Annexin V/PI染色后的各组细胞的形态学表现为,在正常对照组,镜下仅见较少的着色为绿色的细胞膜,未见染成红色的细胞核。在损伤组早期以及晚期凋亡均较明显,表现为密度高的红色的细胞核着色,细胞膜绿色或者细胞核将游移出细胞,即典型的凋亡小体表现。还有较多的细胞仅呈现红色的细胞核,而不见细胞膜,呈现晚期凋亡典型的形态学表现。而蚤休醇提物与蚤休皂苷干预后,均随药物浓度的增加,表现为红色细胞核的细胞量即晚期凋亡量逐渐减少,表现为深红色的胞核显著减少,多表现为细胞膜着色为绿色。各实验组细胞内静态Ca~(2+)的含量测定结果显示,损伤组的Ca~(2+)荧光强度最高,与正常对照组相比差异显著(P<0.01);药物预处理后细胞内Ca~(2+)浓度荧光强度显著下降,此效应在两种药物均呈现剂量依赖性增大。Ca~(2+)的动态含量测定结果显示,H_2O_2作用于细胞后,细胞内Ca~(2+)的浓度呈现升高趋势,荧光强度持续上升达一个高峰,然后缓慢下降至平台值;蚤休醇提物与蚤休皂苷预处理后,再加入损伤因素,则Ca~(2+)的浓度上升峰值下降,峰值与平台期之间的差值减小,与损伤组相比差异显著(P<0.01),细胞内的Ca~(2+)的浓度呈现较为稳定的状态。caspase-3 mRNA水平在各实验组的表达显示;损伤组与内参照相比的灰度值最大,经过预处理之后其值明显下降(P<0.01);免疫荧光染色也证实了这个结果,损伤组的caspase-3荧光强度最高,而预处理后明显下降(P<0.01)。在实验的第三部分中,RT-PCR实验结果显示,两种药物预处理氧化损伤后,ICAM-1、VCAM-1和MCP-1 mRNA的表达水平与损伤组相比明显减弱(P<0.01);损伤组中与内参照的灰度值之比最大,与正常组相比差异显著(P<0.01);免疫荧光染色显示损伤组MCP-1的荧光强度最大,与正常组相比差异显著(P<0.01);而经过预处理之后表达减弱,效应均呈剂量依赖性;且均与损伤组相比有显著性差异(P<0.01);流式细胞仪定量检测ICAM-1、VCAM-1的结果显示,损伤组中表达ICAM-1、VCAM-1的阳性细胞数均最多,与正常组相比差异显著(P<0.01);两种药物预处理氧化损伤后,阳性细胞数明显减少。结论:所提取的两种药物蚤休醇提物以及皂苷可以保护H_2O_2造成的氧化损伤,推测蚤休的保护作用由其有效成分蚤休皂苷发挥。是通过阻抑正常的细胞周期诱导凋亡、抑制凋亡蛋白caspase-3介导的凋亡信号转导通路实现,而这个通路的激活与钙离子激活后介导的德信号转导有关;以及通过抑制内皮细胞粘附分子与细胞因子的表达从而抑制炎症性损伤,达到保护内皮细胞、抗动脉粥样硬化的目的。
Objectives : To extract traditional Chinese medicine bistortae,investigate the protection of the injury on the umbiliar vein endothelial cell ECV 304 induced by hydrogen peroxide(H_2O_2) of ethanol from bistortae and pariphyllin, and to study the mechanism of anti一atherosclerosis (AS) of these medicines.Methods: Prepration ethanol from bistortae(EFB) ,then extracte pariphyllin(Pa) from this ; examine the protection of the two above medicine to the injury in ECV 304 induced by H_2O_2,decide the protection dose of the two medicine by MTT reduction assay. ECV 304 had been cultured in vitro, a model of endothelial cell oxidative damage induced by H_2O_2 was established.then cells were divided by eight experimental groups:1 normal;2oxidative damage group;3 high density ethanol from bistortae(500pg/ml); 4 medium density ethanol from bistortae(50pg/ml); 5 low density ethanol from bistortae(5pg/ml) ;6 high density pariphyllin(1ug /ml); 7 medium density pariphyllin(100pg/ml); 8 low density pariphyllin(10pg /ml).In second part experiment, Flow cytometry (FCM) methods Annexin V/PI staining and PI staining were used to analysis the effection of two medicine to the apoptosis and cell cycle in all groups, Laser confocal microscopy(LCM) was used to observe the appearance of apoptosis and quiet state and the dynamic change of the density of calcium ion (Ca~(2+)) , Reverse transcription PCR (RT-PCR) and immunofluorescene(IFM) staining were used to detect the protein express in mRNA level of apoptosis associated protein caspase-3 in all experiment groups. In third part experiment, we used RT-PCR to detect the protein expression in mRNA level of intracelluar adhesion molecule (ICAM-1)、vasculer cell adhesion molecule (VCAM-1)、monocyte chemoattractant protein(MCP-1)in all experiment groups,FCM were used to quantitate the ICAM-1 and VCAM-1 and in all group cells,then, and IFM was used to decide the qualitation expression of MCP-1 in all groups Results:The model of oxidative damage was established, ethanol from bistortae and pariphyllin were got out successfully ;MTT assay discovered that cell optical density in oxidation damaged group was lower than nomal control (P<0.01);when pretreated by the two kind of medicine ,the OD value was increased,and was not significant difference in the high density ethanol from bistortae and the high density pariphyllin than normal control(P>0.05).FCM Annexin V/PI staining result showed that early period and advanced stage apoptosis rate was lower when predisposed than that of damaged group,and the effection was dose dependent in pariphyllin groups. FCM PI staining showed that cells of G0/G1 stage were more in cell cycle in oxidation damage group,but less in S stage and hypodiploid peak could observe ,The LCM showed that there appeared apoptosis body in damage group, Furthermore we studied H_2O_2 mediated Ca~(2+) responses in ECV304 and found that the increase of Fluo-3 fluorescence intensity in the presence of external Ca~(2+): H_2O_2 elicited an transient peak of [Ca~(2+)]i and then fell to a subsequent sustained higher plateau.When pretreatment by the ethanol from bistortae(500pg/ml)and pariphyllin(1ug/ml)for 10 min and which significantly prevented the transient increase and sustained increase of [Ca~(2+)]i,RT-PCR showed that the expression of caspase-3 weakened when predisposed by the two kind of medicine than without this disposal(P<0.01);IFM staining result showed that same effection;RT-PCR result showed that the expression of ICAM-1、VCAM-1 and MCP-1 mRNA was significantly descended(P<0.01) in medicine treatment groups than in H_2O_2 damage group, IFM staining showed that MCP-1 had the greatest fluorescence intensity in H_2O_2 damage group,but in all groups pretreatment with medicine it obviously weakened(P<0.01); FCM result discovered that the numbers of masculine cells which express ICAM-1 or VCAM-1 was obviously more than other groups(P<0.01),but pretreated groups the was lower.
Conclusion: Ethanol from bistortae and Pariphyllin has the protective action on oxidative damage of ECV304 induced by H_2O_2, the mechanism of protective action may relate to decrease the cell signal transduction mediated of apoptosis,which may be activated by calcium ion mediated signal transduction ; and related the stabilize the cell wall, inhibit the inflammatory reaction induced by ICAM-1、VCAM-1 and MCP-1, and then protect endothelial cell,prevent AS come true.
Objectives : To extract traditional Chinese medicine bistortae,investigate the protection of the injury on the umbiliar vein endothelial cell ECV 304 induced by hydrogen peroxide(H_2O_2) of ethanol from bistortae and pariphyllin, and to study the mechanism of anti一atherosclerosis (AS) of these medicines.Methods: Prepration ethanol from bistortae(EFB) ,then extracte pariphyllin(Pa) from this ; examine the protection of the two above medicine to the injury in ECV 304 induced by H_2O_2,decide the protection dose of the two medicine by MTT reduction assay. ECV 304 had been cultured in vitro, a model of endothelial cell oxidative damage induced by H_2O_2 was established.then cells were divided by eight experimental groups:1 normal;2oxidative damage group;3 high density ethanol from bistortae(500pg/ml); 4 medium density ethanol from bistortae(50pg/ml); 5 low density ethanol from bistortae(5pg/ml) ;6 high density pariphyllin(1ug /ml); 7 medium density pariphyllin(100pg/ml); 8 low density pariphyllin(10pg /ml).In second part experiment, Flow cytometry (FCM) methods Annexin V/PI staining and PI staining were used to analysis the effection of two medicine to the apoptosis and cell cycle in all groups, Laser confocal microscopy(LCM) was used to observe the appearance of apoptosis and quiet state and the dynamic change of the density of calcium ion (Ca~(2+)) , Reverse transcription PCR (RT-PCR) and immunofluorescene(IFM) staining were used to detect the protein express in mRNA level of apoptosis associated protein caspase-3 in all experiment groups. In third part experiment, we used RT-PCR to detect the protein expression in mRNA level of intracelluar adhesion molecule (ICAM-1)、vasculer cell adhesion molecule (VCAM-1)、monocyte chemoattractant protein(MCP-1)in all experiment groups,FCM were used to quantitate the ICAM-1 and VCAM-1 and in all group cells,then, and IFM was used to decide the qualitation expression of MCP-1 in all groups Results:The model of oxidative damage was established, ethanol from bistortae and pariphyllin were got out successfully ;MTT assay discovered that cell optical density in oxidation damaged group was lower than nomal control (P<0.01);when pretreated by the two kind of medicine ,the OD value was increased,and was not significant difference in the high density ethanol from bistortae and the high density pariphyllin than normal control(P>0.05).FCM Annexin V/PI staining result showed that early period and advanced stage apoptosis rate was lower when predisposed than that of damaged group,and the effection was dose dependent in pariphyllin groups. FCM PI staining showed that cells of G0/G1 stage were more in cell cycle in oxidation damage group,but less in S stage and hypodiploid peak could observe ,The LCM showed that there appeared apoptosis body in damage group, Furthermore we studied H_2O_2 mediated Ca~(2+) responses in ECV304 and found that the increase of Fluo-3 fluorescence intensity in the presence of external Ca~(2+): H_2O_2 elicited an transient peak of [Ca~(2+)]i and then fell to a subsequent sustained higher plateau.When pretreatment by the ethanol from bistortae(500pg/ml)and pariphyllin(1ug/ml)for 10 min and which significantly prevented the transient increase and sustained increase of [Ca~(2+)]i,RT-PCR showed that the expression of caspase-3 weakened when predisposed by the two kind of medicine than without this disposal(P<0.01);IFM staining result showed that same effection;RT-PCR result showed that the expression of ICAM-1、VCAM-1 and MCP-1 mRNA was significantly descended(P<0.01) in medicine treatment groups than in H_2O_2 damage group, IFM staining showed that MCP-1 had the greatest fluorescence intensity in H_2O_2 damage group,but in all groups pretreatment with medicine it obviously weakened(P<0.01); FCM result discovered that the numbers of masculine cells which express ICAM-1 or VCAM-1 was obviously more than other groups(P<0.01),but pretreated groups the was lower.
Conclusion: Ethanol from bistortae and Pariphyllin has the protective action on oxidative damage of ECV304 induced by H_2O_2, the mechanism of protective action may relate to decrease the cell signal transduction mediated of apoptosis,which may be activated by calcium ion mediated signal transduction ; and related the stabilize the cell wall, inhibit the inflammatory reaction induced by ICAM-1、VCAM-1 and MCP-1, and then protect endothelial cell,prevent AS come true.
引文
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[57] Li H, Cybulsky MI, Gimbrone MA Jr, et al. Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma[J].Am J Pathol,1993,43:1551-1559
[58]温海涛,李莉,陈融,任冬梅,李瑞峰,阎晓梅,胡维诚.蚤休总皂甙对大鼠动脉内皮细胞保护作用的实验研究.中国中西医结合杂志2002,26(6):7-9
[59]杜艳芝,阎晓梅,胡维诚等.蚤休总皂甙对大鼠动脉内皮细胞保护作用的实验研究.中国病理生理杂志1999,15(12):1134-1137
[1] Hong H , Aksenov S , Guan X, Follon JT, Waters D , Chen C. Remodeling of small intramyocardial cornory arteries distal to a severe epicardial cornory arterystenosis. Arterioscler Thromb Vasc Biol , 2002 , 22 : 2 0592065
[2] Yanagisawa M,Kurihara H,Kimura S,Tomobe Y,Kobayashi M, Mitsui Y, et al.A novel potent vasoconstrictor produced by vascular endothelial cells. Nature,1988, 332 (6163) : 411-415
[3] Jones LG,Gause KC,Meier KE. Effects of endothelin on mitogen-activated protein kinase activity and protein synthesis in isolated adult feline cardiac myo-cytes. Lifes Sciences,1996,58 (7) : 617-630
[4] Zamora MA,Dempsey EC, Walchak SJ , Stelzner TJ . BQ123 ,an ETA receptor antagonist,inhibits endothelin212mediated proliferation of human pulmo- nary artery smooth muscle cells. Am J Respir Cell Mol Biol,1993,9 (4) : 329-433
[5]张继峰,田青,汤健,苏静怡,唐朝枢,杨连春,等.从抗蛇毒药探寻内皮素拮抗剂的初步报告.南京医学院学报,1993,13(4): 365-368
[6]王峰,杨连春,刘敏,吉小莉,吕敏,程言亮,等.抗蛇毒中草药拮抗内皮素1和S6b作用的初步研究.中国中药杂志,1997,22(10):620-622
[7]胡政力,吴尧忠,陈彦,邢海燕,贾晓斌.“必通汤”降低原发性高血压患者血浆内皮素的初步报告.江苏中医,2000,21(2): 10-11
[8]李瑞峰,温海涛,李莉,陈融,任冬梅,郭成浩,等.半边莲不同组分对内皮细胞内皮素及内皮源一氧化氮合酶代谢的影响.中国动脉硬化杂志,2002,10 (1) : 19-22
[9]张慧渊,余胜珠,张愍,正脂丸对人血管内皮细胞产生一氧化氮、内皮素的影响.安徽中医学院学报2003,22(2):38-40
[10]王华亭,蔡生业,姚成芳,朱宗涛,王丽,张维东,复方花刺参粘多糖对家兔血管成形术后内皮功能及超微结构的影响.中国动脉硬化杂志2004,12(5): 497-499
[11]李宝华,郑广娟,张文高.软脉降脂胶囊对实验性高脂血症大鼠血脂及MDA、SOD、NO的影响.中西医结合心脑血管病杂志,2004,2(4):220-222.
[12]吴智春,吴凯,王浩,王晶.金匮泻心汤抗动脉粥样硬化的实验研究.中国老年学杂志,2003,23(7): 461-462
[13]张子新,曾定尹,王绽菲,通心络对动脉粥样硬化家兔血管成形术后血管内皮功能的保护作用.中国动脉硬化杂志2003,11(6):569-571
[14]吉瑞瑞,李付英,张雪静,段重高,周亚伟,淫羊藿苷对缺氧诱导血管内皮细胞损伤的保护作用中国中西医结合杂志2005,25(6):525-529
[15]李越华,刘宇,顾仁樾.三七有效组分Rx对兔动脉粥样硬化的实验研究.中西医结合心脑血管病杂志2003,1(10):582-587
[16]陈冰,宋剑南,牛晓红,金红,李玉红,血管细胞信号分子钙离子和蛋白激酶C表达的影响.中国动脉硬化杂志2004,12(2): 143-146
[17]顾耘,软脉煎对氧化低密度脂蛋白造成血管内皮细胞损伤的影响.中医药学刊2005,23(1): 107-108
[18]高维明,张会临,山葡萄多酚对血管内皮细胞损伤的保护作用.中国公共卫生2006,22(6): 715-716
[19]王维蓉,林蓉,彭宁,韩纯洁,丹参酮Ⅱa对过氧化氢损伤人血管内皮细胞的保护作用.中药材2006,29(1): 49-51
[20]那娜,徐沛龙,钟进义,葡多酚对血管内皮细胞自由基损伤的影响.营养学报2005,27(1): 58-61
[21]刘锦,鲁映青,金雀异黄素对低密度脂蛋白氧化修饰及氧化性低密度脂蛋白诱导的血管内皮细胞c-myc mRNA表达的抑制作用.中国动脉硬化杂志2002,10(6):509-512
[22]何书英、钱之玉,西红花苷对氧化性低密度脂蛋白致内皮细胞损伤的保护作用.中国新药杂志2005,14(2): 169-171
[23]张园园,张运,张梅,高月花,李秀昌,李贵华,等,舒降之、普罗布考、开搏通和祛淤消斑胶囊对食饵性兔动脉粥样硬化血管内皮舒缩功能的影响.中国动脉硬化杂志2003,11(2): 118-122
[24]金明华,吴伟康,秦鉴,陈树清,四逆汤抗血管内皮脂质过氧化损伤作用.中国临床康复,2003,7(15):2164-2165.
[25]张荣怀,李源,王晓明,郭海涛,银杏黄酮甙对人巨细胞病毒促进AS发生发展的保护作用.中国临床康复2004,8(9): 1714-1717
[26]鞠镐,程文立,董晞,丹参粉针剂对氧化低密度脂蛋白诱导的内皮细胞表达MMP9及ICAM-1的影响.中日友好医院学报2005,19(4):230-233
[27]吕虎,华萍,余继红,蒋显猷,冷和平,茶黄素对ox-LDL诱导单核-内皮细胞粘附作用影响的研究.中药材2005,28(4): 304-306
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[1] Hong H , Aksenov S , Guan X, Follon JT, Waters D , Chen C. Remodeling of small intramyocardial cornory arteries distal to a severe epicardial cornory arterystenosis. Arterioscler Thromb Vasc Biol , 2002 , 22 : 2 0592065
[2] Yanagisawa M,Kurihara H,Kimura S,Tomobe Y,Kobayashi M, Mitsui Y, et al.A novel potent vasoconstrictor produced by vascular endothelial cells. Nature,1988, 332 (6163) : 411-415
[3] Jones LG,Gause KC,Meier KE. Effects of endothelin on mitogen-activated protein kinase activity and protein synthesis in isolated adult feline cardiac myo-cytes. Lifes Sciences,1996,58 (7) : 617-630
[4] Zamora MA,Dempsey EC, Walchak SJ , Stelzner TJ . BQ123 ,an ETA receptor antagonist,inhibits endothelin212mediated proliferation of human pulmo- nary artery smooth muscle cells. Am J Respir Cell Mol Biol,1993,9 (4) : 329-433
[5]张继峰,田青,汤健,苏静怡,唐朝枢,杨连春,等.从抗蛇毒药探寻内皮素拮抗剂的初步报告.南京医学院学报,1993,13(4): 365-368
[6]王峰,杨连春,刘敏,吉小莉,吕敏,程言亮,等.抗蛇毒中草药拮抗内皮素1和S6b作用的初步研究.中国中药杂志,1997,22(10):620-622
[7]胡政力,吴尧忠,陈彦,邢海燕,贾晓斌.“必通汤”降低原发性高血压患者血浆内皮素的初步报告.江苏中医,2000,21(2): 10-11
[8]李瑞峰,温海涛,李莉,陈融,任冬梅,郭成浩,等.半边莲不同组分对内皮细胞内皮素及内皮源一氧化氮合酶代谢的影响.中国动脉硬化杂志,2002,10 (1) : 19-22
[9]张慧渊,余胜珠,张愍,正脂丸对人血管内皮细胞产生一氧化氮、内皮素的影响.安徽中医学院学报2003,22(2):38-40
[10]王华亭,蔡生业,姚成芳,朱宗涛,王丽,张维东,复方花刺参粘多糖对家兔血管成形术后内皮功能及超微结构的影响.中国动脉硬化杂志2004,12(5): 497-499
[11]李宝华,郑广娟,张文高.软脉降脂胶囊对实验性高脂血症大鼠血脂及MDA、SOD、NO的影响.中西医结合心脑血管病杂志,2004,2(4):220-222.
[12]吴智春,吴凯,王浩,王晶.金匮泻心汤抗动脉粥样硬化的实验研究.中国老年学杂志,2003,23(7): 461-462
[13]张子新,曾定尹,王绽菲,通心络对动脉粥样硬化家兔血管成形术后血管内皮功能的保护作用.中国动脉硬化杂志2003,11(6):569-571
[14]吉瑞瑞,李付英,张雪静,段重高,周亚伟,淫羊藿苷对缺氧诱导血管内皮细胞损伤的保护作用中国中西医结合杂志2005,25(6):525-529
[15]李越华,刘宇,顾仁樾.三七有效组分Rx对兔动脉粥样硬化的实验研究.中西医结合心脑血管病杂志2003,1(10):582-587
[16]陈冰,宋剑南,牛晓红,金红,李玉红,血管细胞信号分子钙离子和蛋白激酶C表达的影响.中国动脉硬化杂志2004,12(2): 143-146
[17]顾耘,软脉煎对氧化低密度脂蛋白造成血管内皮细胞损伤的影响.中医药学刊2005,23(1): 107-108
[18]高维明,张会临,山葡萄多酚对血管内皮细胞损伤的保护作用.中国公共卫生2006,22(6): 715-716
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