【作者】 安杰；

【导师】 甄彦君；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2003， 硕士

【摘要】 目的：EC损伤是AS病变形成的始动环节，脂质代谢紊乱是导致AS发生的重要因素之一，降低血脂、保护内皮已在AS的治疗中倍受关注。多种中药可从不同环节干预AS始动因素、降低AS早期事件发生率、减轻AS病变程度，且效果明显。为研究木贼是否具有降血脂、保护血管EC及影响AS病变发生的作用，本实验建立了食饵性大鼠高脂血症及早期AS模型，预防性给予木贼煎剂，从不同层面观察大鼠主动脉EC的功能、代谢及形态变化，了解木贼对AS预防作用及可能的作用机理，为木贼临床抗AS应用提供理论依据。方法：（1） 动物随机分为4组：正常组、模型组、木贼高剂量预防组和木贼低剂量预防组，每组12只。（2） 复制大鼠食饵性高脂血症及早期AS模型。造模同时，给药组每日以木贼煎剂2ml/只灌胃，高剂量组合生药12.5g/kg.d，低剂量组合生药 5g/kg.d；正常组及模型组等量生理盐水灌胃。实验共10周。（3） 10周末断尾取血，酶法检测血脂水平；取主动脉分别以肉眼、光镜及电镜观察形态变化；硝酸还原酶法测血清NO含量,化学比色法测血清NOS活性；流式细胞术检测EC凋亡率及相关基因Bax、Bcl-2表达量。结果：（1）实验大鼠一般状况：正常组大鼠毛色光泽，<WP=4>活动自如，活泼，饮食正常。模型组饲喂高脂饲料，体重增长较快，皮下脂肪厚，但随实验进程，毛发色泽暗淡，活动减少，精神状态较差，进食量减少；给药组明显好于模型组。（2）动物血脂的改变（见附表1）：模型组TC、TG、LDL仍较正常对照组明显升高（p<0.01），HDL降低（p<0.01）；木贼预防组总TC、TG、LDL较模型组显著降低（p<0.01），而HDL升高（p<0.01）。高、低剂量组之间无差别。（3）血清NO及NOS变化（见附表2）：模型组NOS活性显著低于正常组（p<0.05），NO浓度显著增高（p<0.01）。木贼低剂量组的NOS活性较模型组增强（p<0.05 29.31±1.97 VS 27.43±2.00），NO显著降低（p<0.01 102.30±18.88 VS 200.92±116.49）。而木贼高剂量组的NOS和NO与模型组相比差异均无显著性。（4）主动脉形态学改变：4.1）肉眼观察：正常组血管弹性好，内膜表面平滑，富有光泽；模型组动脉弹性减低，管壁增厚，内膜面光泽度稍差；木贼预防组与正常组相比变化不明显。4.2）光镜观察（见附图1-8）：正常组动脉内膜光滑，EC排列整齐、无缺失，内膜下间隙较小；模型组血管壁增厚，内膜不光滑，EC呈连续性缺失，底层胶原纤维暴露，大量MC粘附于内皮表面或潜入内皮下，内皮下间隙增宽；木贼预防组内膜较光滑，局部有少量MC粘附，可见EC缺失，但明显好于模型组；高、低剂量组之间未见明显差别。4.3）扫描电镜所见（见附图9-12）：正常组EC界限清楚，排列规整，连接紧密；细胞呈长梭形，核区饱满，膜表面结构清晰，可见典型微绒毛。模型组：EC形态明显异常，<WP=5>部分呈收缩状态，体积缩小，细胞间隙增大或出现深大裂隙；细胞膜表面呈大小不等的虫蚀状、火山口状缺损，洞内嵌有红细胞、白细胞，并可见大片EC崩解，甚至脱落仅余残基，区内有血小板、血细胞、细胞碎屑和纤维素等附着。木贼两预防组EC基本完整，排列较整齐，可见少量缺损，胞膜部分破坏，呈小洞状，损伤明显轻于模型组。4.4）透射电镜观察（见附图13-16）：正常组EC完整，细胞间连接紧密，胞质互相覆盖；模型组EC细胞连接处胞质分离，变直、变宽，细胞间出现较大间隙，并可见MC黏附于EC表面或伸出伪足潜入内皮间隙、内皮下，大部分EC残缺、脱落，表面有血小板黏附。木贼高、低剂量组可见EC较完整，覆盖于内膜表面，细胞连接较好，MC黏附少。（5）流式细胞术检测结果：5.1）EC凋亡率变化（见附表3、附图13-16）：模型组EC凋亡率显著高于正常组（p<0.01），木贼高、低剂量组凋亡率则显著低于模型组（p<0.01 6.12±1.39 VS 7.04±1.12 VS 10.5±1.63）。高、低剂量组之间无差别。5.2）凋亡相关基因Bax、Bcl-2表达的变化（见附表4、附图17-图24）：模型组基因Bax、Bcl-2表达及Bax\Bcl-2值均明显高于正常组（p<0.01）；木贼两组大鼠主动脉EC Bax表达明显低于模型组（p<0.01, 4.89±0.34 VS 4.78±0.27 VS 5.88±0.43），Bcl-2表达量变化虽无统计学意义，但Bax/Bcl-2值较模型组低（p<0.05, 0.95±0.009 VS 1.02±0.005 VS 1.18±0.017）。高、低剂量预防组之间无差别。结论：本实验成功复制SD大鼠食饵性高脂血症及早期AS病变模型，木贼预防性用药后，观察血管EC功能<WP=6>及形态变化。结果表明：（1）木贼可调节食饵性高脂血症大鼠的脂代谢紊乱，降低TC、TG、LDL，升高HDL； （2）木贼可改善EC功能，影响细胞激素分泌，激活NOS，增强NO活性；（3）木贼可下调促凋亡基因Bax表达下降，使Bax与抑制凋亡基因Bcl-2比值下降；（4）木贼可显著降低AS早期大鼠主动脉EC的凋亡率。（5）形态学观察发现（肉眼、光镜及电镜）木贼能保护血管EC，减轻AS早期EC受损的程度更多还原

【Abstract】 Objective: Morphological and functional changes of VEC are the initial pathogenesis at the early stage of atherosclerosis. Chinese herbs have multiple protection on VEC, including prevention of endothelium injury, antioxidation, influence upon the production and release of some vascular active substance, cytokines and related genes expressions.The present experiment is to investigate the effect of Equisetum on the experimental hypercholesterolemia and early atherosclerosis in rats fed with high lipid diet and its possible mechanisms.Methods: Forty-eight Sprague-Daw ley rats were divided at random into: normal group, model group, group treated with Equisetum of 12.5g/kg·d(high dosage group) and group treated with Equisetum of 5g/kg?d(low dosage group) . Equisetum is administered to rats via gavage for ten 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 a high fat diet. The levels of plasma total cholesterol, triglyceride, high<WP=8>density lipoprotein and low density lipoprotein were examined after ten weeks. Morphological changes of aorta were observed under gross, light and electron microscopes. Nitric oxide and endothelial nitric oxide synthase in the serum were measured. The rate of apoptosis and the expressions of Bax and Bcl-2 in aorta endothelium were detected by flow cytometry.Result:（1）(table.1) The levels of plasma total cholesterol, triglyceride，low density lipoprotein were higher and that of high density lipoprotein was lower in the model group compared with the normal group (p<0.01). Equisetum decreased the levels of plasma total cholesterol, triglyceride、low density lipoprotein and increased high density lipoprotein (p<0.01). （2）(table. 2)The bioactivity Nitric oxide synthase was lower and the content of nitric oxide was higher in model group compared with the normal group (p<0.01). The low dosage Equisetum increased nitric oxide synthase bioactivity (p<0.01, 29.31±1.97 VS 27.43±2.00 ), regulated the release of NO (p<0.01, 102.30±18.88 VS 200.92±116.49).（3）(table.3 fig.13-fig.24) The rate of apoptosis of endothelium in model group was higher than that in normal group (p<0.01). The expressions of Bax were enhanced (p<0.01). The rate of apoptosis, the expression of Bax and the amount of Bax/Bcl-2 in Equisetum groups were decreased compared with those in model group (p<0.01,6.12±1.39 VS 7.04±1.12 VS 10.5±1.63; 4.89±0.34 VS 4.78±0.27 VS 5.88±0.43; 0.95±0.009 VS<WP=9>1.02±0.005 VS 1.18±0.017). There was no significant difference between the high and low dosage groups.（4）(fig.1- fig.16) We 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 animals were less compared with model group.Conclusion：（1）Equisetum decreased the levels of plasma cholesterol, triglyceride, low density lipoprotein and increased that of high density lipoprotein.（2）Equisetum increased nitric oxide synthase bioactivity, regulated the release of No and promoted arterial vasodilatation. （3）Equisetum appeared action on regulating the apoptosis of aorta endothelium and promoting cell survival by regulating the expression of Bax . （4）Morphological changes of aorta were showed a significant effect of protecting aorta endothelial cell in rats fed with Equisetum.As a result, Equisetum protected aorta endothelial cell and prevented the progression of atherosclerosis更多还原