水蛭酶解提取物和淫羊藿苷抑制动脉粥样硬化进展的药理学机制研究

发布时间：2018-05-13 09:57

本文选题：动脉粥样硬化 + 水蛭酶解提取物　；参考：《山东大学》2017年博士论文

【摘要】：研究目的心血管疾病是导致全球中老年人死亡的头号病因,严重影响着人们的生活质量,每年死亡人数约有940万例,低收入和中等收入国家人群受到的影响尤甚。在2008年有1730万人死于心血管疾病,占全球死亡总人数的30%。估计到2030年,死于心血管疾病的人数将增加至2330万,预计心血管疾病将继续成为单个首要死因。动脉粥样硬化是临床上常见疾病,也被认为是各类心血管疾病(如冠心病,脑血管疾病、血栓等)发病的病理基础。内皮功能紊乱假说认为动脉粥样硬化(atherosclerosis,AS)基本发展过程是由内皮功能紊乱(endothelial dysfunction)开始,修饰后的低密度脂蛋白(modified LDL,主要是氧化型ox-LDL)通过内皮细胞的间隙进入内皮下层,而发生了功能紊乱的内皮细胞表面表达的粘附因子增加(如E选择素,P选择素,ICAM-1,VCAM-1等),使血液循环中的单核细胞易粘附于血管内皮表面,进而单核细胞在其释放的趋化因子的作用下迁移至内皮下层,这些单核细胞在集落刺激因子作用下分化为巨噬细胞,然后吞噬内皮下层的修饰后的低密度脂蛋白,成为泡沫细胞,此时即可形成AS早期的脂质条纹。当巨噬细胞吞噬了过多的低密度脂蛋白成为泡沫细胞后,其会凋亡、裂解致使脂质释放,并进一步在内皮下层堆积。同时巨噬细胞及泡沫细胞还会释放各种趋化因子,招募更多的巨噬细胞及平滑肌细胞迁移到内皮下层。随着时间的推移,上述病理逐渐发展,动脉粥样斑块也不断增大。巨噬细胞及泡沫细胞及已经凋亡的细胞及脂质颗粒构成了粥样斑块的脂质核心,而血管平滑肌细胞从血管中膜迁移至内皮下,形成覆盖脂质核心的纤维帽结构。复杂斑块可依照其中平滑肌细胞构成的纤维帽的厚薄分为稳定型斑块和不稳定型斑块。稳定型斑块纤维帽厚,不容易破裂,在血管内膜下逐渐增大而导致管腔狭窄。不稳定型斑块由于纤维帽较薄,容易发生破裂,释放出内容物,而引起血小板聚集,形成血栓。中药水蛭作为在临床上被广泛使用的一味传统中药,具有悠久的历史。中医药理论认为,水蛭具有“破血、逐瘀”的效果,虽然已有很多研究证明了水蛭的抗凝抗血栓作用,而其在临床中许多非凝血类疾病中的应用,表明其的药理学机制尚未被完全阐明。水蛭酶解提取物(leech enzymolysis extract, LEE)是使用生物酶解方法,模拟人体消化过程制备的一种中药水蛭粗提物,其在动脉粥样硬化中的作用尚未阐明。本研究基于中药水蛭的药理学研究和血管内皮功能紊乱假说的研究基础,对中药水蛭酶解提取物在抑制动脉粥样硬化进展方面的药理学机制从动物、细胞及分子层面进行了综合性研究。研究方法(1)通过动物实验验证LEE是否能够抑制动脉粥样硬化进展。使用ApoE敲除小鼠作为模型动物,给予高胆固醇饲料饲养12周,至18周龄后,超声生物显微镜检查确认动物已有动脉粥样硬化斑块形成后,实验动物按照体重排序,随机数表法分为7组:空白对照组,不给于任何药物,仍然继续给予高胆固醇饲料饲养;水蛭酶解提取物组,分别给予0.02g/kg,0.1g/kg和0.5g/kg三个剂量水平的药物;淫羊藿苷组,分别给予30mg/kg和60mg/kg两个剂量水平的药物;辛伐他汀组,给予10mg/kg辛伐他汀。分组后持续给药饲养12周,至30周龄后,解剖进行实验取材。(2)超声生物显微镜检查评价LEE抗动脉粥样硬化效果ApoE敲除小鼠在饲养至30周龄后,行UBM检查,测量主动脉弓处斑块形成情况及主动脉弓处血管内外径,以评价药物的抗动脉粥样硬化效果。(3)冰冻组织切片油红O染色评价LEE抗动脉粥样硬化效果取小鼠心脏,制成冰冻组织切片后,油红O染色方法显示主动脉根部的斑块大小,以评价药物的抗动脉粥样硬化效果。(4)冰冻组织切片MOMA-2染色评价LEE抗动脉粥样硬化效果取小鼠心脏,制成冰冻组织切片后,使用免疫组化染色方法,用MOMA-2抗体显示主动脉中单核巨噬细胞浸润情况,以评价药物的抗动脉粥样硬化效果,及可能的作用机制。(5) ELISA检测小鼠血清中与单核巨噬细胞粘附迁移相关的细胞因子。(6)构建TNF-α诱导的内皮细胞功能紊乱细胞模型。使用TNF-α刺激诱导的内皮细胞激活,建立内皮细胞功能紊乱细胞模型,用来研究LEE对内皮细胞的作用。(7)粘附实验验证LEE对内皮细胞异常激活的抑制作用。(8)迁移实验验证LEE对内皮细胞异常激活的抑制作用。(9) western blot检测与内皮细胞粘附和迁移作用相关的细胞因子。(10)检测LEE抑制内皮细胞异常激活中NFκB的激活和核转移作用。(11)检测LEE抑制MAPK信号通路中相关蛋白p38,ERK,JNK的磷酸化情况。(12) qPCR检测小鼠主动脉血管壁中CX3CR1和CX3CL1表达水平。(13)western blot检测淫羊藿苷组小鼠主动脉血管壁中CX3CR1和CX3CL1表达水平。(14)基因芯片表达谱分析淫羊藿苷对LPS刺激的巨噬细胞RAW264.7中基因表达差异。(15)qPCR检测淫羊藿苷对LPS刺激的巨噬细胞RAW264.7中CX3CR1和CX3CL1表达水平。(16) western blot检测淫羊藿苷对LPS刺激的巨噬细胞RAW264.7中CX3CR1和CX3CL1表达水平。(17)迁移实验验证淫羊藿苷抑制巨噬细胞RAW264.7的迁移效果。实验结果(1)通过UBM观察和测量主动脉弓处血管内外径比值(ID/OD),LEE组中剂量水平(0.1g/kg)和高剂量水平(0.5g/kg)、淫羊藿苷组低剂量水平(30mg/kg)和高剂量水平(60mg/kg)的ID/OD值与对照组相比明显较大。LEE高剂量水平(0.5g/kg)、淫羊藿苷高剂量水平(60mg/kg)和辛伐他汀组(10mg/kg)的ID/OD值之间没有明显的统计学差异。表明LEE及淫羊藿苷可以有效抑制AS的进展,并且在高剂量水平时,其效果与辛伐他汀效果相当。(2)通过油红O染色和计算斑块面积占主动脉横截面积的比例,LEE组高剂量水平(0.5g/kg)、淫羊藿苷组低剂量水平(30mg/kg)和高剂量水平(60mg/kg)斑块面积占主动脉横截面积与对照组相比明显较小。LEE高剂量水平(0.5g/kg)和淫羊藿苷低剂量水平(30mg/kg)之间没有明显的统计学差异。淫羊藿苷高剂量水平(60mg/kg)和辛伐他汀组(10mg/kg)之间没有明显的统计学差异。表明LEE及淫羊藿苷可以有效抑制AS的进展,且淫羊藿昔在高剂量水平时,其效果与辛伐他汀效果相当。(3)通过MOMA-2染色评价单核巨噬细胞浸润,LEE组中剂量水平(0.1g/kg)和高剂量水平(0.5g/kg)、淫羊藿苷高剂量水平(60mg/kg)的MOMA-2染色与对照组相比明显较低。表明LEE及淫羊藿苷可以有效抑制AS中的单核巨噬细胞浸润情况。(4)ELISA法检测ApoE-/-小鼠血清中的VCAM-1,1CA1-11,CX3C1和CX3CL1的水平,LEE中剂量水平(0.1g/kg)和高剂量水平(0.5g/kg)可以明显降低小鼠血清中VCA1M-1和ICAM-1的水平,而对CX3CR1和CX3CL1无明显影响。淫羊藿苷高剂量水平(60mg/kg)可以明显降低小鼠血清中CX3CR1和CX3CL1的水平,而对VCAM-1和ICAM-1无明显影响。(5)使用TNF-α刺激内皮细胞建立内皮细胞功能紊乱模型,以eNOS转录情况为指标,PCR检测不同TNF-α浓度诱导下eNOS的转录水平,筛选了 TNF-α的诱导浓度为10ng/ml,而LEE的处理剂量为200μig/ml。(6)粘附实验中与对照组相比,TNF-α可以明显增加THP-1向内皮细胞的粘附,而LEE可以降低粘附率,抑制THP-1向内皮细胞的粘附。(7)Transwell实验中与对照组相比,TNF-α可以明显引起THP-1向内皮细胞的迁移,而LEE可以抑制THP-1向内皮细胞的迁移。(8)使用western blot方法,检测内皮细胞所表达的与粘附和迁移相关的粘附分子ICAM-1和趋化因子MCP-1。实验结果表明,TNF-α可以显著诱导内皮细胞表达ICAM-1和MCP-1,而LEE可以降低ICAM-1和MCP-1的表达。(9)使用荧光染色及Western blot检测NF-κB激活和p65亚基的核转移,实验结果显示TNF-α可以强烈诱导p65向核内转移,而在给予LEE处理之后再使用TNF-α诱导的LEE组,p65向细胞核内转移的情况受到了显著的抑制。这表明LEE能够抑制NF-κB的激活。(10)Western blot检测LEE对MAPK信号通路上相关蛋白p38、ERK、JNK的磷酸化的影响,实验结果显示LEE可以抑制ERK、JNK的磷酸化。(11)实时定量PCR及western blot检测小鼠主动脉壁中CX3CR1和C3CL1的水平,结果表明淫羊藿苷能够降低ApoE敲除小鼠主动脉壁中CX3CR1和CX3CL1的水平(12)基因芯片表达谱分析结果表明,淫羊藿苷的处理能够使LPS诱导的巨噬细胞RAW264.7中CX3CR1出现明显的表达差异。(13)实时定量PCR及western blot对RAW264.7细胞CX3CR1表达水平分析,结果显示淫羊藿苷处理可以明显抑制LPS诱导的CX3CR1。(14) Transwell迁移实验结果表明,淫羊藿苷可以剂量依赖性的抑制CX3CL1所诱导的巨噬细胞迁移。实验结论(1) LEE及淫羊藿苷可以明显抑制AS的进展;(2) LEE及淫羊藿苷可以明显抑制AS斑块中单核巨噬细胞浸润情况;(3) LEE和淫羊藿苷可以影响小鼠血清中粘附分子和趋化因子的水平(4) LEE可以抑制TNF-α诱导的内皮细胞功能紊乱(5) LEE可以抑制TNF-α诱导的内皮细胞中的NFκB的激活和p65亚基的核转移;(6) LEE可以抑制MAPK信号通路中ERK、JNK的磷酸化。(7)淫羊藿苷可以降低ApoE敲除小鼠主动脉壁中CX3CR1和CX3CL1的水平;(8)淫羊藿苷可以降低LPS诱导的巨噬细胞中CX3CR1的表达水平;(9)淫羊藿苷可以抑制巨噬细胞在CX3CL1诱导下的迁移。LEE通过抑制内皮细胞中MAPK信号通路中ERK、JNK的磷酸化而阻断NFκB的激活,使相应的粘附分子(ICAM-1)和趋化因子(MCP-1)表达减少,而减少了单核细胞向内皮细胞的粘附和迁移,从而使AS斑块中单核巨噬细胞的浸润,而发挥其抗动脉粥样硬化作用。淫羊藿苷通过抑制巨噬细胞中CX3CR1的表达,而减少巨噬细胞在趋化因子CX3CL1作用下的迁移,从而使AS斑块中单核巨噬细胞的浸润,而发挥其抗动脉粥样硬化作用。
[Abstract]:Research objective cardiovascular disease is the leading cause of death in the middle and old people around the world, which seriously affects the quality of life. There are about 9 million 400 thousand deaths per year, especially in low and middle income countries. In 2008, 17 million 300 thousand people died of cardiovascular disease, accounting for 30%. of the total number of deaths worldwide to 2030, The number of people dying of cardiovascular disease will increase to 23 million 300 thousand, and it is expected that cardiovascular disease will continue to become a single leading cause of death. Atherosclerosis is a common clinical disease, and is also considered as a pathological basis for various cardiovascular diseases (such as coronary heart disease, cerebrovascular disease, thrombus, etc.). The endothelial dysfunction hypothesis believes that atherosclerosis (athe Rosclerosis, AS) the basic development process is initiated by the endothelial dysfunction (endothelial dysfunction). The modified low density lipoprotein (modified LDL, mainly oxidized ox-LDL) enters the subendothelium through the space of endothelial cells, and the adhesion factors of the endothelial cells that are disordered are increased (such as E selectin, P selection. The mononuclear cells in the blood circulation are easily adhered to the surface of the vascular endothelium, and then the monocytes migrate to the subendothelium under the action of their chemokine release. These monocytes are differentiated into macrophages under the action of colony stimulating factor and then phagocytic low density lipoprotein, which is the underlayer of the endothelium. Foam cells, at this time, form a lipid stripe in the early AS. When macrophages engulf too much low density lipoprotein to become a foam cell, it is apoptotic, cracking and causing lipid release and accumulates further in the underlayer of the endothelium. Meanwhile, macrophages and foam cells will release various chemokines and recruit more macrophages and flat. The smooth muscle cells migrate to the underlayer of the endothelium. As the time goes on, the pathology develops gradually and the atherosclerotic plaques are increasing. The macrophage and foam cells and the already apoptotic cells and lipid particles constitute the lipid core of the atherosclerotic plaque, and the vascular smooth muscle cells migrate from the membrane of the blood tube to the endothelium and form the lipid core. The fibrous cap structure of the heart. Complex plaques can be divided into stable and unstable plaques according to the thickness of the fibrous cap made up of the smooth muscle cells. The fibrous cap of the stable plaque is thick and is not easy to break up. The stenosis of the lumen is caused by the gradual enlargement of the vascular intima. The unstable plaque is thinner in the fibrous cap, easy to break and release. Chinese medicine Hirudo is a traditional traditional Chinese medicine which is widely used in clinical practice. It has a long history. The theory of Chinese medicine holds that Hirudo has the effect of "breaking blood and removing blood stasis", although many studies have proved the anticoagulant and anti thrombus effect of leech, and it has many non coagulation in clinical. The pharmacological mechanism of the disease shows that its pharmacological mechanism has not been fully elucidated. Leech enzymolysis extract (LEE) is a kind of crude leech crude extract prepared by using biological enzyme solution to simulate the digestive process of human body, and its role in atherosclerosis has not been clarified. The basis of the study and the hypothesis of vascular endothelial dysfunction, a comprehensive study of pharmacological mechanisms of leech extracts in inhibiting the progression of atherosclerosis from animal, cell and molecular levels. The study method (1) verified whether LEE could inhibit the progression of atherosclerosis by animal experiments. ApoE knockout was used. The mice were fed with high cholesterol feed for 12 weeks and after 18 weeks of age. After ultrasound biomicroscopy confirmed the formation of atherosclerotic plaques in animals, the experimental animals were divided into 7 groups according to the weight sequencing and random number table method: the blank control group was not given any drugs, and still continued to feed high cholesterol feed; water The leech enzymolysis extract group was given three doses of 0.02g/kg, 0.1g/kg and 0.5g/kg, respectively. The icariin group was given two doses of 30mg/kg and 60mg/kg, respectively, and simvastatin group was given 10mg/kg simvastatin. After grouping, the drug was kept for 12 weeks and after 30 weeks of age. (2) ultrasound biological microscopy. Evaluation of LEE anti atherosclerotic effect in ApoE knockout mice after feeding to 30 weeks old, UBM examination was performed to measure the plaque formation in the aortic arch and the internal and external diameter of the aortic arch to evaluate the anti atherosclerotic effect of the drug. (3) frozen tissue section oil red O staining was used to evaluate the anti atherosclerotic effect of LEE to take the heart of mice and take the heart of mice to take the heart of mice After making the frozen tissue slices, the oil red O staining method showed the size of the plaque in the root of the aorta to evaluate the anti atherosclerotic effect of the drug. (4) the frozen tissue section MOMA-2 staining was used to evaluate the anti atherosclerotic effect of LEE to take the heart of the mice. After the frozen tissue section was made, the immunohistochemical staining method was used to display the MOMA-2 antibody. The infiltration of mononuclear macrophages in the aorta in order to evaluate the anti atherosclerotic effect of the drug and the possible mechanism of action. (5) ELISA detection of cell factors associated with the adhesion and migration of mononuclear macrophages in the serum of mice. (6) construction of TNF- alpha induced endothelial cell dysfunction cell model with TNF- alpha induced endothelial cells Activation, the establishment of endothelial cell dysfunction cell model, used to study the role of LEE on endothelial cells. (7) adhesion experiments verify the inhibitory effect of LEE on the abnormal activation of endothelial cells. (8) migration tests verify the inhibitory effect of LEE on abnormal activation of endothelial cells. (9) Western blot detection of endothelial cell adhesion and migration related cells Factor. (10) detection of activation and nuclear transfer of NF kappa B in the inhibition of abnormal activation of endothelial cells by LEE. (11) detection of the phosphorylation of related protein p38, ERK, JNK in the LEE inhibition of MAPK signaling pathway. (12) qPCR detection of CX3CR1 and CX3CL1 expression in the aorta wall of the mice. (13) detection of the aorta wall of icariin group in mice by western The expression level of CX3CR1 and CX3CL1. (14) gene chip expression profile analysis of icariin stimulated LPS stimulated macrophage RAW264.7 expression difference. (15) qPCR detection of Icariin on LPS stimulated macrophage RAW264.7 CX3CR1 and CX3CL1 expression level. (16) Western blot detection of icariin against LPS stimulated macrophages CR1 and CX3CL1 expression level. (17) migration experiment verified the effect of icariin inhibiting the migration of macrophage RAW264.7. Experimental results (1) observe and measure the ratio of internal and external diameter of aorta at the aortic arch (ID/OD), the dose level (0.1g/kg) and high dose level (0.5g/kg) in group LEE, low dose level (30mg/kg) and high dose of icariin group in group LEE. The ID/ OD value of (60mg/kg) was significantly higher than that of the control group (0.5g/kg), and there was no significant difference between the high dose level of icariin (60mg/kg) and the ID/ oat (10mg/kg) in the simvastatin group (10mg/kg), indicating that LEE and icariin could effectively inhibit the progress of AS, and the effect was with simvastatin at a high dose level. (2) high dose level (0.5g/kg) in group LEE, low dose level (30mg/kg) and high dose level (60mg/kg) patch area of icariin group were significantly lower than that of control group (0.5g/kg) and icariin low dose (0.5g/kg) and icariin low dose (.LEE) by oil red O staining and calculation of the proportion of plaque area to aorta cross section area. There was no significant statistical difference between the level of 30mg/kg and the high dose of icariin (60mg/kg) and simvastatin group (10mg/kg), which showed that LEE and icariin could effectively inhibit the progress of AS, and the effect of epimedium in high doses of water was equal to that of simvastatin. (3) through MOMA-2 Staining was used to evaluate mononuclear macrophage infiltration, medium dose level (0.1g/kg) and high dose level (0.5g/kg) in group LEE, MOMA-2 staining of icariin high dose level (60mg/kg) was significantly lower than that of control group. It showed that LEE and icariin could effectively inhibit the infiltration of mononuclear macrophage in AS. (4) ELISA method was used to detect the serum of ApoE-/- mice. The level of VCAM-1,1CA1-11, CX3C1 and CX3CL1, the dose level (0.1g/kg) and the high dose level (0.5g/kg) in LEE could obviously reduce the level of VCA1M-1 and ICAM-1 in the serum of mice, but had no obvious effect on CX3CR1 and CX3CL1. There was no obvious effect on ICAM-1. (5) the endothelial cell dysfunction model was stimulated by TNF- alpha, and the transcription of eNOS was detected by eNOS transcription. PCR was used to detect the transcriptional level of eNOS under the concentration of TNF- a, and the induced concentration of TNF- alpha was 10ng/ml, and the dosage of LEE was 200 mu ig/ml. (6) in adhesion experiment compared with the control group, TNF- Alpha can obviously increase the adhesion of THP-1 to endothelial cells, and LEE can reduce the adhesion rate and inhibit the adhesion of THP-1 to endothelial cells. (7) compared with the control group, TNF- alpha can obviously cause the migration of THP-1 to endothelial cells in the Transwell experiment, and LEE can inhibit the migration of THP-1 to endothelial cells. (8) Western blot method is used to detect endothelial cells. The results of cell adhesion and migration related adhesion molecules ICAM-1 and chemokine MCP-1. showed that TNF- alpha could significantly induce the expression of ICAM-1 and MCP-1 in endothelial cells, and LEE could reduce the expression of ICAM-1 and MCP-1. (9) fluorescence staining and Western blot were used to detect NF- kappa B activation and nuclear metastases. Experimental results showed that - alpha can strongly induce the transfer of p65 into the nucleus, and the transfer of p65 into the nucleus after LEE treatment is significantly inhibited by the use of TNF- alpha induced LEE. This indicates that LEE can inhibit the activation of NF- kappa B. (10) Western blot detection LEE on MAPK signal transduction, phosphorylation of phosphorylation, experimental junction The results showed that LEE could inhibit the phosphorylation of ERK and JNK. (11) real-time quantitative PCR and Western blot were used to detect the level of CX3CR1 and C3CL1 in the aorta wall of mice. The results showed that icariin could reduce the level of CX3CR1 and CX3CL1 in the aorta wall of the ApoE knockout mice (12) gene chip expression analysis. Significant difference in expression of CX3CR1 in RAW264.7 induced macrophages. (13) real-time quantitative PCR and Western blot were used to analyze the CX3CR1 expression level of RAW264.7 cells. The results showed that icariin treatment could inhibit LPS induced CX3CR1. (14) Transwell migration experiment, and icariin could inhibit CX3CL1 in a dose-dependent manner. Induction of macrophage migration. Experimental conclusions (1) LEE and icariin can significantly inhibit the progress of AS; (2) LEE and icariin can significantly inhibit the infiltration of mononuclear macrophages in AS plaques; (3) LEE and icariin can affect the level of adhesion molecules and chemokines in the serum of mice (4) LEE can inhibit the endothelial cells induced by TNF- alpha. Cell dysfunction (5) LEE can inhibit the activation of NF kappa B in TNF- alpha induced endothelial cells and the nuclear transfer of p65 subunits; (6) LEE can inhibit the phosphorylation of ERK and JNK in MAPK signaling. (7) icariin can reduce CX3CR1 and CX3CL1 levels in the aortic wall of ApoE knockout mice; (8) icariin can reduce the size of macrophages. The expression level of CX3CR1 in the cell; (9) icariin inhibited the migration of macrophages under CX3CL1 induced.LEE by inhibiting the activation of NF kappa B by inhibiting the phosphorylation of ERK and JNK in the MAPK signaling pathway in the endothelial cells, reducing the expression of the corresponding adhesion molecules (ICAM-1) and chemokine (MCP-1), and reducing the adhesion of mononuclear cells to endothelial cells. The migration of mononuclear macrophages in AS plaques, thus exerts its anti atherosclerotic effect. Icariin reduces the migration of macrophages under chemokine CX3CL1 by inhibiting the expression of CX3CR1 in macrophages, thus making the monocytic macrophage infiltration in the AS plaque and exerting its anti atherosclerosis. Effect.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

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