冠心合剂主要活性成分对TNF-a损伤血管内皮细胞保护作用的实验研究
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摘要
目的:研究冠心合剂主要活性成分对由TNF-a损伤的血管内皮细胞的保护作用。方法:将人脐静脉内皮细胞进行常规细胞培养、传代,后分为对照组、TNF-α组、药物活性成分组;其中对照组不加任何药物,于培养箱培养24 h后待检;TNF-α组细胞先正常培养18 h,后加入200 ng/m L的TNF-α作用6 h后,PBS洗涤待测;药物活性成分组分别加入80μg/m L、30μg/m L、20μg/m L、20μg/m L浓度的黄芪甲苷、槲皮素、异鼠李素、β-谷甾醇(药物浓度已经前期实验MTS法验证),培养18 h,再加入浓度为200 ng/m L的TNF-α继续培养6 h后,PBS洗涤细胞待测。通过TUNEL(末端脱氧核苷酸转移酶介导的d UTP缺口末端标记测定法)进行定量检测、DNA Ladder(琼脂糖凝胶电泳)进行定性检测。结果:TUNEL实验中,光镜下可看到冠心合剂主要活性成分组细胞的结构完整性优于TNF-α组,且凋亡细胞数明显少于TNF-α组(P﹤0.001)。四种药物相关活性成分中以黄芪甲苷对内皮细胞保护作用明显(P﹤0.001)。结论:冠心合剂相关药物活性成分能明显保护由TNF-α损伤的内皮细胞。四种药物相关活性成分中以黄芪甲苷对内皮细胞的保护作用最明显(P﹤0.001)。本实验琼脂糖凝胶电泳结果中并未出现细胞凋亡条带,考虑与细胞凋亡程度及凋亡时间的非同步性有关。
Objective: To study the protective effect of the main ingredients of Guanxin Decoctionon to the vascular endothelial cells injuried by TNF-α in order to further clarify the material basis and molecular pharmacological mechanism of Guanxin Decoction anti atherosclerosis and at the same time to prove the scientificalness of Guanxin Decoction anti atherosclerosis.Methods: Human umbilical vein endothelial cells according with the Cytological Guide were given the conventional culture and subculture and then were divided into negative control group,positive group and drug group. The negative group was without any drug culture for 24 h. The positive cells were cultured for 18 h,and then joining TNF-α 200 ng / m L for 6 h and PBS washing for test. The drug group added 80 μg / m L,30 μg / m L,20 μg / m L,20 μg / m L of astragaloside,quercetin,isorhamnetin,β-sitosterol( validation of drug concentration was tested by the MTS method before),cultured for 18 h,and then adding 200 ng / m L TNF-α cultured for 6 h after PBS washing the cells to be tested. Through this experiment,two kinds of classical ways to detection of apoptosis: one was TUNEL( Td T-mediated d UTP nick end labeling) for the quantitative detection and the other was DNA Ladder( agarose gel electrophoresis) for qualitative detection.Results: Confirmation of Guanxin Decoction had a protective effect on endothelial cells injured by TNF-α. The results of TUNEL showed the cell structure of drug groups was superior to that ofthe TNF-α group,also the apoptosis rate was far below that of the TNF-α group( P<0.001). It was confirmed that there was difference between them. The astragaloside in all kinds of drugs active ingredients had the significant protective effect on the endothelial cells injured by TNF-α( P<0.001). The result of DNA Ladder was all the groups showed no DNA ladder display,considering it was related to the degree of cell apoptosis and the cell apoptosis not at the same time. Conclusion: Active ingredients of Guanxin Decoction can obviously protect the endothelial cells injuried by TNF-α. Astragaloside in all kinds of drugs active ingredients has thesignificant protective effect on the endothelial cells( P<0.001).The agarose gel electrophoresis results did not appear apoptosis strip,considering it is related to the degree of apoptosis and the time of cell apoptosis not at the same time.
Objective: To study the protective effect of the main ingredients of Guanxin Decoctionon to the vascular endothelial cells injuried by TNF-α in order to further clarify the material basis and molecular pharmacological mechanism of Guanxin Decoction anti atherosclerosis and at the same time to prove the scientificalness of Guanxin Decoction anti atherosclerosis.Methods: Human umbilical vein endothelial cells according with the Cytological Guide were given the conventional culture and subculture and then were divided into negative control group,positive group and drug group. The negative group was without any drug culture for 24 h. The positive cells were cultured for 18 h,and then joining TNF-α 200 ng / m L for 6 h and PBS washing for test. The drug group added 80 μg / m L,30 μg / m L,20 μg / m L,20 μg / m L of astragaloside,quercetin,isorhamnetin,β-sitosterol( validation of drug concentration was tested by the MTS method before),cultured for 18 h,and then adding 200 ng / m L TNF-α cultured for 6 h after PBS washing the cells to be tested. Through this experiment,two kinds of classical ways to detection of apoptosis: one was TUNEL( Td T-mediated d UTP nick end labeling) for the quantitative detection and the other was DNA Ladder( agarose gel electrophoresis) for qualitative detection.Results: Confirmation of Guanxin Decoction had a protective effect on endothelial cells injured by TNF-α. The results of TUNEL showed the cell structure of drug groups was superior to that ofthe TNF-α group,also the apoptosis rate was far below that of the TNF-α group( P<0.001). It was confirmed that there was difference between them. The astragaloside in all kinds of drugs active ingredients had the significant protective effect on the endothelial cells injured by TNF-α( P<0.001). The result of DNA Ladder was all the groups showed no DNA ladder display,considering it was related to the degree of cell apoptosis and the cell apoptosis not at the same time. Conclusion: Active ingredients of Guanxin Decoction can obviously protect the endothelial cells injuried by TNF-α. Astragaloside in all kinds of drugs active ingredients has thesignificant protective effect on the endothelial cells( P<0.001).The agarose gel electrophoresis results did not appear apoptosis strip,considering it is related to the degree of apoptosis and the time of cell apoptosis not at the same time.
引文
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[3]刘宏飞,祝光礼,盛志超.冠心合剂对冠心病患者PCI术后血清NO、ET-1水平的影响[J].中西医结合心脑血管病杂志,2012,10(10):1179-1180.
[4]农凌波,肖正伦.肿瘤坏死因子a、白细胞介素1对牛肺动脉内皮细胞凋亡的影响及意义[J].中华结核和呼吸杂志,2003,26(11):668-670.
[5]张翠丽,林琳,邹昕.内皮细胞凋亡与动脉粥样硬化[J].心血管病学进展,2007,28(2):235-237.
[6]张欣胜,鹿育萨.血管紧张素Ⅱ对动脉粥样硬化斑块发生和发展过程中炎性因子的影响作用[J].医学综述,2006,12(12):1414-1415.
[7]董红梅,喻伦银,贾宗智,等.TNF-α对培养的脐静脉内皮细胞的损伤作用[J].临床与实验病理学杂志,2002,18(4):401-403
[8]常江平,朱旭东,刘子娟.TNF-α增加血管内皮通透性与其激活P38MAPK和音符因子重新分布有关[J].西安交通大学学报,2004,25(6):538-541.
[9]Hubbard,A.K.C.R.Timblin,A,shukla,M.Rincon B.T.Mossman,Activation of NF-KB-dependent gene expression by silica in lungs of luciferase reporter mice[J].Am J Physiol Lung cell Mol physiol,2002,282(5):968-975.
[10]吴军,李闪,朱建华,等.TNF-α对人脐静脉内皮细胞中NO和e NOS的影响[J].浙江医学,2006,28(8):636-638.
[11]Z annad F,Dousset B,Al la F.Treatment of con gestive heart failure:interfering the ald osterone-cardiac extracellul armatrixrelatonship[J].Hypertension,2001,38(5):1221-1232.
[12]尚正录,尚云.黄芪保心汤预防实验性大鼠急性心肌缺血的研究[J].上海中医药杂志,2004,38(11):47-50.
[13]贾贞超,陈锦瑶,张立实.黄芪甲苷药理作用与毒理学评价研究[A].药物与健康,2011(8):116-119.
[14]张彩英.中药蒲黄防治动脉粥样硬化机制的研究[J].衡阳医学院学报,1991,19(3):75-78.
[15]唐绪刚,黄文权,姜利鲲.五灵脂抗动脉粥样硬化炎症作用研究[J].中药药理与临床,2009,25(1):35-38.
[16]李家富,何涛,杨烨,等.槲皮素、异鼠李素对体外低密度脂蛋白氧化修饰的影响[J].心脏杂志,2003,15(5):414-416.
[17]祝光礼,魏丽萍,钱宝庆.黄芪失笑散对血管内皮功能的影响[J].浙江临床医学,2008,8(10):1016-1018.
[2]许英淑,崔惠润,李成芳.TNF-α的生物学功能及与ACS的关系[J].北华大学学报,2005,10:428.
[3]刘宏飞,祝光礼,盛志超.冠心合剂对冠心病患者PCI术后血清NO、ET-1水平的影响[J].中西医结合心脑血管病杂志,2012,10(10):1179-1180.
[4]农凌波,肖正伦.肿瘤坏死因子a、白细胞介素1对牛肺动脉内皮细胞凋亡的影响及意义[J].中华结核和呼吸杂志,2003,26(11):668-670.
[5]张翠丽,林琳,邹昕.内皮细胞凋亡与动脉粥样硬化[J].心血管病学进展,2007,28(2):235-237.
[6]张欣胜,鹿育萨.血管紧张素Ⅱ对动脉粥样硬化斑块发生和发展过程中炎性因子的影响作用[J].医学综述,2006,12(12):1414-1415.
[7]董红梅,喻伦银,贾宗智,等.TNF-α对培养的脐静脉内皮细胞的损伤作用[J].临床与实验病理学杂志,2002,18(4):401-403
[8]常江平,朱旭东,刘子娟.TNF-α增加血管内皮通透性与其激活P38MAPK和音符因子重新分布有关[J].西安交通大学学报,2004,25(6):538-541.
[9]Hubbard,A.K.C.R.Timblin,A,shukla,M.Rincon B.T.Mossman,Activation of NF-KB-dependent gene expression by silica in lungs of luciferase reporter mice[J].Am J Physiol Lung cell Mol physiol,2002,282(5):968-975.
[10]吴军,李闪,朱建华,等.TNF-α对人脐静脉内皮细胞中NO和e NOS的影响[J].浙江医学,2006,28(8):636-638.
[11]Z annad F,Dousset B,Al la F.Treatment of con gestive heart failure:interfering the ald osterone-cardiac extracellul armatrixrelatonship[J].Hypertension,2001,38(5):1221-1232.
[12]尚正录,尚云.黄芪保心汤预防实验性大鼠急性心肌缺血的研究[J].上海中医药杂志,2004,38(11):47-50.
[13]贾贞超,陈锦瑶,张立实.黄芪甲苷药理作用与毒理学评价研究[A].药物与健康,2011(8):116-119.
[14]张彩英.中药蒲黄防治动脉粥样硬化机制的研究[J].衡阳医学院学报,1991,19(3):75-78.
[15]唐绪刚,黄文权,姜利鲲.五灵脂抗动脉粥样硬化炎症作用研究[J].中药药理与临床,2009,25(1):35-38.
[16]李家富,何涛,杨烨,等.槲皮素、异鼠李素对体外低密度脂蛋白氧化修饰的影响[J].心脏杂志,2003,15(5):414-416.
[17]祝光礼,魏丽萍,钱宝庆.黄芪失笑散对血管内皮功能的影响[J].浙江临床医学,2008,8(10):1016-1018.