【作者】 刘志敏；

【导师】 甄艳军；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的:前期实验表明木贼水煎剂可以调节脂代谢、抑制内皮细胞（endothelial cell, EC）凋亡,抗动脉粥样硬化（atherosclerosis, AS）早期事件的发生。本实验对木贼进行初步提取分离,对比观察提取物对大鼠主动脉EC凋亡的影响及其形态学变化,探讨木贼保护血管内皮的有效部位及其可能机制。方法:1木贼有效部位的提取、分离木贼水煎剂经正丁醇萃取、分离为两部分,其中正丁醇萃取液回收至干,蒸馏水定容至1.25g（生药）/ml;剩余液亦浓缩至相同浓度,均冰箱保存备用。2两种木贼提取物的药效动物实验健康雄性SD大鼠50只,随机分为五组:正常组、模型组、吉非罗齐组、木贼正丁醇提取物组、正丁醇提取剩余物组,每组10只。给药组每天饲喂高脂饲料且按10ml/kg标准灌胃给药,分别合吉非罗齐0.16g/（kg.d） ,生药木贼12.5g/（kg.d）,正常组和模型组每天灌等量生理盐水,并随动物体重增加及时调整药量。3指标检测九周末眶后取血,酶法检测:胆固醇（total cholesterol, TC）、甘油三酯（triglyceride, TG）、高密度脂蛋白（high densitylipoprotein, HDL）、低密度脂蛋白（low density lipoprotein, LDL）、极低密度脂蛋白（very low density lipoprotein, VLDL）;流式细胞术检测EC周期、凋亡率及相关基因caspase-3、突变型p53、bcl-2蛋白表达,计算细胞增殖指数（proliferative index, PI）;分别以肉眼、光镜及电镜观察主动脉内膜形态变化。结果:1实验大鼠一般状况正常组大鼠饮食正常,活动自如。模型组体重增长较快;但随实验进程,进食减少、精神较差。吉非罗齐组、两木贼给药组大鼠饮食、活动、精神状态均明显好于模型组。2两种木贼提取物对血脂的影响模型组TC、LDL较正常对照组明显升高（P<0.01）;吉非罗齐组TG、LDL、VLDL较模型组显著降低（P<0.01）,HDL明显升高（P<0.01）;两木贼给药组降低TG、VLDL作用与吉非罗齐相似（P<0.05）,提取剩余物组HDL升高显著（P<0.01）,与正丁醇提取物组相比具有统计学意义（P<0.01）。3两种木贼提取物对主动脉EC凋亡相关基因caspase-3、突变型p53、bcl-2蛋白表达的影响EC凋亡率及突变型p53、caspase-3表达模型组较正常对照组明显升高（P<0.05）,吉非罗齐组较模型组显著降低（P<0.05）;正丁醇提取物下调caspase-3表达作用明显（P<0.05）。Bcl-2蛋白表达在各组中均未见明显变化（P>0.05）。4两种木贼提取物对主动脉EC周期与凋亡的影响模型组停留在G₀/G₁期的EC较正常对照组明显升高（P<0.01）,S和G₂/M期细胞百分比显著减少（P<0.01）,PI降低显著（P<0.01）,凋亡率显著升高（P<0.01）;两种木贼提取物对细胞周期的影响与吉非罗齐相似,较模型组停留在G0/G1期细胞百分比显著降低（P<0.01）,S和G2/M期细胞明显增多（P<0.05）,PI显著升高（P<0.01）,细胞凋亡率显著减少（P<0.01）。5主动脉形态学改变5.1肉眼观察正常组血管弹性好,内膜平滑,富有光泽;模型组动脉弹性减低,管壁稍增厚,光泽度稍差;吉非罗齐组、两木贼给药组与正常组相比变化不明显。5.2光镜观察正常组动脉EC连续、形态规则,表面未见细胞粘附,内皮下间隙较小。模型组血管壁增厚,内皮呈不连续性缺失,底层胶原纤维暴露,内皮下间隙明显增宽。吉非罗齐组内皮较连续,形态规则,明显好于模型组。正丁醇提取物组内膜连续、光滑,EC排列整齐,明显好于模型组。提取剩余物组内膜较光滑,部分EC缺失、单核细胞粘附,但好于模型组。两木贼给药组之间未见明显差别。5.3扫描电镜观察正常对照组大鼠主动脉EC长梭形,核区饱满,连接紧密,可见长短不等的典型微绒毛。模型组EC排列紊乱,表面微绒毛减少甚至消失,呈大小不等的虫蚀状、火山口状缺损,可见细胞碎片、纤维素等附着。吉非罗齐组EC形态完整,轮廓清晰,偶见小洞状破损。木贼正丁醇提取物组EC呈带状排列,核区饱满,可见典型微绒毛,散在的脂肪小颗粒黏附。正丁醇提取剩余物组EC连接较紧密,局部坏死、脱落,可见较大脂肪颗粒黏附。5.4透射电镜观察正常对照组大鼠EC胞浆内吞饮小泡丰富,线粒体、内质网形态正常。模型组EC坏死、脱落严重,残存细胞内吞饮小泡、脂质空泡数量明显增多,线粒体肿胀、融合,粗面内质网扩张、脱颗粒。吉非罗齐组EC吞饮小泡、脂质空泡数量减少,部分线粒体肿胀,粗面内质网轻度扩张。木贼正丁醇提取物组EC吞饮小泡、脂质空泡明显减少,部分线粒体轻度肿胀,粗面内质网扩张减轻、脱颗粒。正丁醇提取剩余物组EC吞饮小泡和脂质空泡较多,线粒体、粗面内质网肿胀,游离核糖体散在于细胞质中。结论:两种木贼提取物对AS早期大鼠降脂和保护内皮的效果与前期水煎剂实验结果一致,且两组间未见显著性差异,表明木贼确有调节脂代谢、从基因水平上抑制内皮凋亡、保护内皮抗AS早期损伤的作用。更多还原

【Abstract】 Objective: Morphological and functional changes of EC are the initial pathogenesis at the early stage of AS, and hyperlipemia induces endothelium injury and AS principally, so cutting down hypolipemia and protecting endothelium are important for preventing the progress of AS. Chinese herb Equisetum have multiple protection on EC, verified by experiments through regulating lipid metabolism, decreasing apoptosis and so on. The present experiment is to investigate the effective part of Equisetum and its possible mechanisms on early atherosclerosis in rats fed with high lipid diet.Methods:1 The Equisetum was extracted by distilled water and n-butano. All extracts were pooled together and concentrated named Equisetum extract by n-butano and the surplusage dividly. Both the concentrates were stored in a freezer during the experiment.2 Fifty Sprague-Dawley rats were randomly divided into five groups: normal group, model group, control group received Gemifibrozil, two Equisetum extract groups treated with Equisetum extract by n-butano and the surplusage dividly. The two Equisetum extracts were administered to rats by gastric perfusion for nine weeks. Normal saline was given to rats of normal group and model group at the same volume and same way. The normal group was fed with standard chow, other groups were fed with standard chow and a high fat diet.3 The levels of plasma total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein and very low density lipoprotein were examined after nine weeks. Morphological changes of aorta were observed under gross, light and electron microscopes. The rate of apoptosis and bcl-2, p53, caspase-3 protein related in aorta endothelium was detected by flow cytometry.Results: 1 The total cholesterol, triglyceride, low density lipoprotein, very low density lipoprotein and high density lipoprotein contents in blood serumThe levels of plasma total cholesterol, low density lipoprotein were higher in the model group compared with the normal group (p<0.05). Both Equisetum extracts decreased the levels of plasma triglyceride and very low-density lipoprotein (p<0.05). The surplusage can increase the level of high density lipoprotein (p<0.05).2 The effects of Equisetum extracts on expression of mutant-p53, caspase-3, bcl-2 in endotheliumThe expression of mutant-p53 and caspase-3 in model group was higher than that in normal group (p<0.05). There was no significant difference between the control group and both Equisetum extracts groups: they decreased them evidently, especially the Equisetum extract by n-butano. The expression of bcl-2 changed litter during groups.3 The effects of Equisetum extracts on the the rate of apoptosis and cell cycle in endotheliumThe rate of apoptosis in model group was higher than that in normal group (p < 0.01) and there was no significant difference during the treatment groups: it was decreased evidently (p<0.01). Most cells retained in G0/G1-phase and little cells retained in S and G2/M phase in model group (p<0.01). The ratio of S and G2/M phase of cells in all the treatment groups were higher than that in the model group (p<0.01), especially Equisetum extract by n-butano.4 The effects of Equisetum extracts on morphosis of aortaWe found the changes of the early stage of atherosclerosis in model group: there were focal or transient areas of endothelium loss, monocyte adhered to the subendothelial space or moved into the vascular wall, et al. Those changes of aorta endothelium in fed Equisetum extracts or Gemifibrozil animals were less compared with model group.Conclusion: Both Equisetum extracts can protect aorta endothelial cell and prevent the progress of atherosclerosis in different ways, which are similar with Equisetum. This study verified that Equisetum has certain effect on early atherosclerosis through protecting endothelial cell, but the active component of Equisetum is not clear and it needs a profound study.更多还原