摘要
目的:对以红景天苷为先导化合物合成的一系列化合物进行抗缺血缺氧活性筛选,期望从大量的化合物中筛选出具有抗缺血缺氧活性的物质并为新药的开发提供科学的实验依据。
方法:(1)不同浓度药物预处理细胞后制备原代心肌细胞缺氧—复氧模型和谷氨酸诱导的SH-SY5Y细胞损伤模型,全自动生化分析仪检测乳酸脱氢酶(LDH)释放抑制率的变化。(2)选出其中活性较好、合成方法简单的S01为代表化合物,初步研究其药效及抗缺血缺氧作用机制。以不同浓度S01药物(2,10 and 50μg·ml~(-1))预处理心肌细胞或者神经细胞后,建立心肌细胞的缺氧—复氧损伤或者谷氨酸诱导的SH-SY5Y细胞损伤模型,检测心肌细胞上清液中的乳酸脱氢酶(LDH)、天冬氨酸转氨酶(AST)、肌酸激酶(CK)的含量和细胞凋亡程度及SH-SY5Y细胞上清中乳酸脱氢酶(LDH)含量和细胞存活率,同时检测细胞内Ca~(2+)浓度、活性氧(ROS)水平、ATP水平,及细胞上清中NO水平、超氧化物歧化酶(SOD)活性和丙二醛含量及S01对正常小鼠游泳耐疲劳能力的影响和常压耐缺氧能力的影响。
结果(1)利用细胞模型的筛选,发现有4种化合物的体外抗缺血缺氧活性优于先导化合物。(2)预先给予S01能够降低损伤细胞中乳酸脱氢酶、天冬氨酸转氨酶(AST)、肌酸激酶(CK)的含量和细胞凋亡程度及SH-SY5Y细胞上清中乳酸脱氢酶(LDH)含量,升高细胞存活率,改善细胞功能。另外结果表明,S01能够升高SOD活性,且可减小胞内ATP水平降低程度,且能降低损伤细胞中ROS升高水平、MDA含量、NO水平、细胞内钙超载和细胞凋亡,从而保护细胞,同时S01能够显著延长小鼠的存活时间,增强小鼠耐常压缺氧的能力。
结论:这些化合物通过多种机制来对缺血缺氧的细胞进行保护作用,可能对脑缺血或者心肌缺血具有治疗作用。
AIM(1)To find compounds possessing anti-ischemia and anti-anoxia activity from compounds derived from Salidroside and provides the scientific experimental data for newdrug -research.
METHODS(1)SH-SY5Y cells and cultured neonatal rat myocardial cells were pretreated with each compound(40,200 and 1000 ug·ml~(-1),respectively)and then induced damage by glutamate or hypoxia/reoxygenation,the inhibition rate of Lactate dehydrogenase(LDH) release was detected by colorimetry.(2)S01 which has better acticity and simpler synthesis method was selected as the representative to research the drug action and anti-ischemia/anti-anoxia mechanism.SH-SY5Y cells and cultured neonatal rat myocardial cells were pretreated with S01(2,10 and 50 ug·ml~(-1),respectively)and then induced damage by glutamate or hypoxia/reoxygenation,then the content of LDH,AST,CK enzymes and the cell apoptosis degree in cultured neonatal rat myocardia cells,the content of LDH enzyme and cell viability in SH-SY5Y cells,cell the free Ca~(2+)concentration([Ca~(2+)]_i),intracellular reactive oxygen species(ROS)level,adenosine triphosphate(ATP)level,nitric oxide(NO)level, oxide dismutase(SOD)activity and malondialdehyde(MDA)level were detected.The anti-fatigue and anti-hypoxia ability of mice were also detected.
RESULTS(1)4 compounds were found to have better anti-ischemia and anti-anoxia activity than lead compounds.(2)Compared with model groups,in cultured neonatal rat myocardial cells,S01(2,10 and 50 ug·ml~(-1))can reduce LDH,AST,CK release induced by hypoxia/reoxygenation,in SH-SY5Y cells,S01 can reduce LDH release and levated the cell survival rate induced by glutamate.Moreover,results indicate that S01 can increase SOD activity and ATP level,and inhibited the increase of MDA,ROS,Ca~(2+)overload,apoptosis and NO levels induced by glutamate or hypoxia/reoxygenation.Furthermore,S01 can increase the anti-fatigue and anti-hypoxia ability of mice.
CONCLUSION The compounds can protect cells from the damage induced by glutamate and oxygen-glucose deprivation through some mechanism,which indicates that they may have the potential to treat cerebral or myocardium ischemia.
引文
[1]王浩春,刘蔚,史寅奎,等.细胞保护作用的抗心肌缺血药物研究进展[J].解放军药学学报,2004,20(6):460-461.
[2]Hu R.,Siu C.W.,Lau E.O.,et al.Impaired nitrate-mediated dilatation could reflect nitrate tolerance in patients with coronary artery disease[J].International Journal of Cardiology,2007,120,351-356.
[3]Xi Y.,Sun N.L..Progress in tolerance of nitrate[J].Chinese journal of clinical pharmacology,2007,23(3):213-216.
[4]Hollenberg N.K.,The role of β-blockers as a cornerstone of cardiovascular therapy[J].The American Journal of Hypertension,2005,18,165-168.
[5]Jenkins N.P.,Keevil B G.,Hutchinson I.V..Beta-blockers are associated with lower C-Reactive protein concentrations in patients with coronary artery disease[J].The American Journal of Medicine,2002,112,269-274.
[6]Li H.,Xu X.X..Adverse reactions and rational utilization of β-receptor antagonists[J].Chinese Journal of Pharmacovigilance,2006,3(6):365-366.
[7]Hiroshi A.,Tetsuo M.,Shoji S.,et al.A calcium channel blocker amlodipine increases coronary blood flow via both adenosine- and NO-dependent mechanisms in ischemic hearts[J].Journal of Molecular and Cellular Cardiology,2005,39,605-614.
[8]杨彦玲,刘运德.抗心肌缺血再灌注损伤药物的研究进展[J].心血管病学进展,2003,24(2):121-124.
[9]史旭波,胡大一.血栓形成与凝血机制及调节[J].临床荟萃,2007,22(14):989-991.
[10]刘波.急性心肌梗死溶栓药物进展[J].中国急救复苏与灾害医学杂志,2007,2(2):117-120.
[11]Gotto A.M..Statins,cardiovascular disease,and drug safety[J].The American Journal of Cardiology,2006,97(8):3-5.
[12]Xu Y.Y.,Jabbour S.,Goldberg R.,et al.Usefulness of statin drugs in protecting against atrial fibrillation in patients with coronary artery disease[J].The American Journal of Cardiology,2003,92,1379-1383.
[13]李昱柳,苏巧娟,李灵芝.红景天苷药理活性研究进展[J].武警医学院学报,2007,16(1):98-100.
[14]肖好,李绍平,王一涛.红景天苷药理研究进展[J].实用医院临床杂志,2006,3(3):98-99.
[15]李莉,孔乐凯,陈融.红景天苷对脑缺血再灌注脑组织NO代谢的影响[J].山东医药,2001,41(19):17-18.
[16]张文生,朱陵群,牛福玲.红景天苷对缺氧/缺糖损伤神经细胞的保护作用[J].中国中药杂志,2004,29(5):459-462.
[17]何晶.天麻素的药理作用及临床应用[J].天津药学,2006,18(5),62-63.
[18]林秀峰,俞惠新,谭成,等.红景天苷的碘标记及其在小鼠体内的分布[J].核技术,2006,29(12):913-916.
[19]程刚,郝秀华,刘国良,等.天麻素在大鼠体内的药动学研究[J].中国药学杂志,2003,38(2):127-129.
[20]游金辉,谭天秩,匡安仁,等.~3H-天麻甙元和~3H-天麻素在小鼠体内的分布和代谢[J].华西医大学报,1994,25(3):325-328.
[21]Mikkelsen L.M.,Hernaiz M.J.,Martin-pastor M.,et al.Conformation of Glycomimetics in the free and protein-bound state:Structural and binding featuresof the C-glycosyl analogue of the core trisaccharide α-D-Man-(1→3)-[α-D-Man-(1→6)]-D-Man[J].Journal of the American Chemical Society 2002,124,14940-14951.
[22]yang G.L.,Franck R.W.,Bittman R.,et al.Synthesis and growth inhibitory properties of Glucosamine-derived Glycerolipids[J].Organic Letters,2001,3(2):197-200.
[23]Yang G.L.,Schmieg J.,Tsuji M.,et al.The C-glycoside analogue of the immunostimulant α-Galactosyiceramide(KRN7000):Synthesis and striking enhancement of activity[J].Angewandte Chemie (International ed.in English),2004,43,3818-3822.
[24]杨晓宁,田宗文,黄焕斌,等.新生大鼠心肌细胞的体外培养[J].解剖学杂志,2005,28(3):361-363.
[25]冯波,王蓉,盛树力.神经退行性疾病研究中拟神经细胞模型:人神经母细胞瘤株SH-SY5Y的来源特性及应用[J].中国临床康复,2006,10(6):121-123.
[26]Dongdong Li,Zuohui Shao,Terry L.Vanden Hoek,et al.Reperfusion accelerates acute neuronal death induced by simulated ischemia[J].Experimental Neurology,2007,206(2):280-287.
[27]何其华,周慧芳,薛冰,等.雷公藤单体T10对谷氨酸所致PC12细胞损伤的保护作用及机制研究[J].北京大学学报(医学版)),2003,35(3):252-255.
[28]Kyung Ah Koo,Seung Hyun Kim,Mi Kyeong Lee,et al.15-Methoxypinusolidic acid from Biota orientalis attenuates glutamate-induced neurotoxicity in primary cultured rat cortical cells[J].Toxicology in Vitro,2006,20(6):936-941.
[29]宋月英,韩慧文,郝素云,等.红景天研究进展[J].武警医学院学报,2004,13(1):66-68.
[30]Christian Kupatta,Rabea Hinkela,Jan Horstkottea,et al.Selective retroinfusion of GSH and cariporide attenuates myocardial ischemia-reperfusion injury in a preclinical pig model[J].Cardiovascular research,2004,61(3):530-537.
[31]Hiroaki Sasaki,Partha S.Ray,Li Zhu,et al.Oxidative stress due to hypoxia/reoxygenation induces angiogenic factor VEGF in adult rat myocardium:possible role of NFκB[J].Toxicology,2000,155(1-3):27-35.
[32]范礼理,滕健,张润东,赵德育.羟苯氨酮保护大鼠心脏对抗心肌缺血-再灌注损伤.[J].药学学报,2005,40(6):507-512.
[33]邓晓红,许予明,张苏明,许康 生物发光法测定PC12细胞微量ATP含量[J].华中医学杂志),2004,28(1):3-5.
[34]Perchenet L,et al.Journal of cardiovascular pharmacology,1995,26:831.
[35]Vixtor H.Guaiquil,David W.Golde,Daniel L.Beckles et al.Vitamin c inhibits hypoxia-induced damage and apoptotic signaling pathways in cardiomyocytes and ischemic hearts[J].Free radical biology & medicine,2004,37(9):1419-1429.
[36]Hongjiang Chen,Dayuan Li,Gregory J.Roberts,Tom Saldeen,Jawahar L.Mehta.Eicosapentanoic acid inhibits hypoxia-reoxygenation-induced injury by attenuating upregulation of MMP-1in adult rat mvocvtes[J].Cardiovascular research.2003.59(1),7-13.
[37]徐琪,祝世功,周家文,等.红景天保护缺血再灌注损伤鼠脑细胞的作用及机理研究[J].中风与神经疾病杂志,1999,16(3),144-146.
[38]宋斌,黄山杉,刘庆国,等.红景天苷对大鼠心肌缺血再灌注损伤的保护作用[J].中医杂志,2005,32(3),256-258.
[39]Yetik-Anacak G.,Catravas J.D..Nitric oxide and the endothelium:History and impact on cardiovascular disease[J].Vascular Pharmacology,2006,45,268-276.
[40]Razavi H.,M.,Hamilton J.A.,Feng Q..Modulation of apoptosis by nitric oxide:implications in myocardial ischemia and heart failure[J].Pharmacology & therapeutics,2005,105(2):147-162.
[41]陈敏榕,陈燕惠.缺氧缺血性脑损伤与凋亡的进展[J].中国病理生理杂志,2004,20(7)1318-1321.
