【作者】 徐望龙；

【导师】 郭玉；

【作者基本信息】 南华大学 ， 药理学， 2014， 硕士

【摘要】 目的通过检测山银花黄酮粗提物（CFFLC）体外清除自由基作用、抗细胞氧化损伤能力和对抗急性心肌缺血大鼠氧化损伤作用以及其抗肿瘤作用来研究山银花黄酮粗提物的药理作用，为山银花黄酮类化合物的进一步开发利用提供理论依据。方法用邻苯三酚自氧化法、Fenton反应和DPPH反应测定CFFLC对不同自由基的清除能力；用MTT法检测CFFLC对H2O2诱导的氧化损伤内皮细胞和心肌细胞的保护作用；以灌胃法给予CFFLC，连续给药7天，通过皮下注射异丙肾上腺素的方法建立大鼠急性心肌缺血氧化损伤模型，右颈动脉插管法测定大鼠左心室内压，并通过连接标准肢体Ⅱ导联测心电图变化。左心室内压检测指标包括左心室收缩压、舒张压、平均压；心电图检测指标包括T波辐度、ST段波辐、QT间期及心率的变化。此外，用NBT染色法测心肌缺血面积百分率，硫代巴比妥酸法检测血清中丙二醛(MDA)含量，黄嘌呤氧化酶法检测血清中超氧化物歧化酶(SOD)活性，同时，测定血清中乳酸脱氢酶(LDH)活性、肌酸激酶(CK)活性等氧化损伤蛋白酶。用MTT法检测CFFLC对MCF-7细胞增殖抑制作用的影响；以Hoechst33342染色法观察CFFLC诱导MCF-7细胞的凋亡作用；用流式细胞术检测CFFLC诱导MCF-7细胞凋亡的凋亡率变化。结果1）CFFLC对超氧阴离子自由基（O2-）、羟自由基（OH）、DPPH自由基（DPPH）均有较强的清除作用且呈浓度依赖性，CFFLC对OH的清除作用最强，其次是O2-，然后是DPPH；浓度为250μg/mL时对O2-、 OH、DPPH的清除率分别为42.40%、64.99%和28.90%。2）CFFLC对H2O2诱导氧化损伤的内皮细胞和心肌细胞有较强的抗氧化作用且与浓度相关，500μg/mL的CFFLC使内皮细胞的存活率从49.21%提高到了60.45%；1000μg/mL的CFFLC使心肌细胞的存活率从61.32%提高到了69.98%。3）CFFLC能抵抗心肌缺血大鼠ST段的上抬，抑制T波的升高，缩短QT间期时程，缩小大鼠心肌缺血面积百分率，提高心率。CFFLC明显改善血流动力学和左心室功能，升高左心室收缩压、舒张压和平均压；同时，减少血清中MDA含量，降低CK和LDH活性，升高SOD活性。4）CFFLC对MCF-7细胞增殖有明显的抑制作用，且随浓度的增加而增强，1000μg/mL的CFFLC对MCF-7细胞增殖抑制率达34.60%，与对照组比较呈显著差异性（P＜0.01vs正常组）。经Hoechst33342染色可观察到细胞形态发生明显变，随CFFLC浓度的增加，细胞核被染成亮蓝色的细胞比例明显增加。经流式细胞术检测，0μg/mL、100μg/mL、300μg/mL、600μg/mL的CFFLC诱导的MCF-7细胞凋亡率分别为：3.89%、10.40%、22.02%和27.03%。结论1)CFFLC具有明显的体内和体外抗氧化作用：对O2-、 OH、DPPH有较强的清除能力；对内皮细胞和心肌细胞有明显的抗H2O2氧化损伤作用；有明显抵抗大鼠急性心肌缺血造成氧化损伤的作用，其抗氧化作用呈浓度依赖性。2)CFFLC具有明显的体外抗肿瘤作用：对MCF-7细胞有明显抑制增殖与诱导凋亡作用，且凋亡率与浓度呈正相关。更多还原

【Abstract】 AIM Through investigation the effects of crude flavonoids extracts from FlosLonicerae Confusae(CFFLC) on clearing the diverse free radicals in vitro andactivities to protect oxidative cells as well as protection effects on the rats with acutemyocardial ischemia disease even containing the abilities to fight against tumor cells,it is the aim to provide the material foundation and scientific basis for furtherexploration and utilization.METHODS The abilities to clear diverse free radicals of CFFLC were evaluated bythe assays of pyrogallic acid autoxidation reaction, fenton reaction and DPPH reaction.The protective effect of CFFLC against the oxidative damage induced by H2O2onendothelial cell and myocardial cell were tested by the method of MTT. Afterreceiving the extracts by successive oral administration for seven days, an acutemyocardial ischemia(AMI) model rat was constructed by subcutaneous injected withisoproterenol hydrochloride. Then the intraventricular pressure and hemodynamicscontained systolic blood pressure(SBP), diastolic blood pressure(DBP), mean bloodpressure(MBP) were detected and the electrocardiogram contained T-wave, ST-segment, QT interval was recorded. Area of myocardial ischemia was determined bynitroblue tetrazol(NBT) staining. The content of malondialdehyde(MDA) in bloodserum was detected by a method of penthiobarbital acid, the bioactivity of superoxidedismutase(SOD) in blood serum was detected by xanthine oxidase. Meanwhile, thebioactivities lactate dehydrogenase(LDH) and creatine kinase(CK) in blood serumwere also measured. The inhibition effects on MCF-7cells proliferation were testedby MTT. The morphological changes of MCF-7apoptosis induced with CFFLC wereobserved by Hoechst33342staining. Flow cytometry was involved to measure therate of apoptosis of MCF-7cells. RESULTS CFFLC possessed with a great activities of scavenging on superoxideanion radical (O2-), hydroxyl radical(OH) and DPPH free radical(DPPH). By takingsolution with the concentration of250μg/mL of CFFLC, the scavenging rates on O2-, OH and DPPH were42.40%,64.99%and28.90%, respectively. So, CFFLC showedthe best effect on scavenging OH, followed with O2-, and then DPPH. CFFLCpossessed with a significant antioxidant effects on oxidative damaged endothelial cellwhich were induced by H2O2.The survival rate of endothelial cell treated with500μg/mL CFFLC increased from49.21%to60.45%and the survival rate ofmyocardial cell treated with1000μg/mL CFFLC increased from61.32%to69.98%,respectively. But CFFLC manifested a great inhibition effect depending on theconcentration. CFFLC could depress ST-segment and T-wave of electrocardiogramupper and shorten the time of QT interval in an AMI rat. And the area of myocardialischemia was cut down and the heart rate was raised. The hemodynamics and leftventricular function were improved clearly. SBP, DBP and MBP were recoveredsignificantly compared with the model group. Furthermore, the content of MDAreduced, the bioactivities of CK and LDH decreased, while the bioactivities of SODincreased. Great inhibition effect of CFFLC on MCF-7cells proliferation wasmanifested and it was to be dose dependent. When it was1000μg/mL, the inhibitionratio on the MCF-7cells reached34.60%. The significant morphological changes ofMCF-7were observed after Hoechst33342staining. The ratio of nucleus stained intosapphirine cells increased along with the concentration. Flow cytometry detectedresults displayed that the apoptosis rates of MCF-7induced by a concentration of100μg/mL,300μmg/mL and600μg/mL were10.40%,22.02%and27.03%,respectively.CONCLUSION1) CFFLC possessed with great antioxidant bioactivities in vivo and in vitro. It hasshowed that CFFLC has good potential abilities to scavenge O2-, OH, DPPH. Therewere obvious protective effects on against the oxidative damage endothelial cell andmyocardial cell induced by H2O2. There were evident protective effects of CFFLC onAMI induced with subcutaneous injected isoproterenol hydrochloride. 2) Inhibition effects on MCF-7cells proliferation and the phenomenon of MCF-7apoptosis could be occurred after being treated with CFFLC and apoptosis rate was tobe positive correlation with its concentration.更多还原