本文選題：動(dòng)脈粥樣硬化 + 水蛭酶解提取物��； 參考：《山東大學(xué)》2017年博士論文

【摘要】：研究目的心血管疾病是導(dǎo)致全球中老年人死亡的頭號(hào)病因,嚴(yán)重影響著人們的生活質(zhì)量,每年死亡人數(shù)約有940萬例,低收入和中等收入國(guó)家人群受到的影響尤甚。在2008年有1730萬人死于心血管疾病,占全球死亡總?cè)藬?shù)的30%。估計(jì)到2030年,死于心血管疾病的人數(shù)將增加至2330萬,預(yù)計(jì)心血管疾病將繼續(xù)成為單個(gè)首要死因。動(dòng)脈粥樣硬化是臨床上常見疾病,也被認(rèn)為是各類心血管疾病(如冠心病,腦血管疾病、血栓等)發(fā)病的病理基礎(chǔ)。內(nèi)皮功能紊亂假說認(rèn)為動(dòng)脈粥樣硬化(atherosclerosis,AS)基本發(fā)展過程是由內(nèi)皮功能紊亂(endothelial dysfunction)開始,修飾后的低密度脂蛋白(modified LDL,主要是氧化型ox-LDL)通過內(nèi)皮細(xì)胞的間隙進(jìn)入內(nèi)皮下層,而發(fā)生了功能紊亂的內(nèi)皮細(xì)胞表面表達(dá)的粘附因子增加(如E選擇素,P選擇素,ICAM-1,VCAM-1等),使血液循環(huán)中的單核細(xì)胞易粘附于血管內(nèi)皮表面,進(jìn)而單核細(xì)胞在其釋放的趨化因子的作用下遷移至內(nèi)皮下層,這些單核細(xì)胞在集落刺激因子作用下分化為巨噬細(xì)胞,然后吞噬內(nèi)皮下層的修飾后的低密度脂蛋白,成為泡沫細(xì)胞,此時(shí)即可形成AS早期的脂質(zhì)條紋。當(dāng)巨噬細(xì)胞吞噬了過多的低密度脂蛋白成為泡沫細(xì)胞后,其會(huì)凋亡、裂解致使脂質(zhì)釋放,并進(jìn)一步在內(nèi)皮下層堆積。同時(shí)巨噬細(xì)胞及泡沫細(xì)胞還會(huì)釋放各種趨化因子,招募更多的巨噬細(xì)胞及平滑肌細(xì)胞遷移到內(nèi)皮下層。隨著時(shí)間的推移,上述病理逐漸發(fā)展,動(dòng)脈粥樣斑塊也不斷增大。巨噬細(xì)胞及泡沫細(xì)胞及已經(jīng)凋亡的細(xì)胞及脂質(zhì)顆粒構(gòu)成了粥樣斑塊的脂質(zhì)核心,而血管平滑肌細(xì)胞從血管中膜遷移至內(nèi)皮下,形成覆蓋脂質(zhì)核心的纖維帽結(jié)構(gòu)。復(fù)雜斑塊可依照其中平滑肌細(xì)胞構(gòu)成的纖維帽的厚薄分為穩(wěn)定型斑塊和不穩(wěn)定型斑塊。穩(wěn)定型斑塊纖維帽厚,不容易破裂,在血管內(nèi)膜下逐漸增大而導(dǎo)致管腔狹窄。不穩(wěn)定型斑塊由于纖維帽較薄,容易發(fā)生破裂,釋放出內(nèi)容物,而引起血小板聚集,形成血栓。中藥水蛭作為在臨床上被廣泛使用的一味傳統(tǒng)中藥,具有悠久的歷史。中醫(yī)藥理論認(rèn)為,水蛭具有“破血、逐瘀”的效果,雖然已有很多研究證明了水蛭的抗凝抗血栓作用,而其在臨床中許多非凝血類疾病中的應(yīng)用,表明其的藥理學(xué)機(jī)制尚未被完全闡明。水蛭酶解提取物(leech enzymolysis extract, LEE)是使用生物酶解方法,模擬人體消化過程制備的一種中藥水蛭粗提物,其在動(dòng)脈粥樣硬化中的作用尚未闡明。本研究基于中藥水蛭的藥理學(xué)研究和血管內(nèi)皮功能紊亂假說的研究基礎(chǔ),對(duì)中藥水蛭酶解提取物在抑制動(dòng)脈粥樣硬化進(jìn)展方面的藥理學(xué)機(jī)制從動(dòng)物、細(xì)胞及分子層面進(jìn)行了綜合性研究。研究方法(1)通過動(dòng)物實(shí)驗(yàn)驗(yàn)證LEE是否能夠抑制動(dòng)脈粥樣硬化進(jìn)展。使用ApoE敲除小鼠作為模型動(dòng)物,給予高膽固醇飼料飼養(yǎng)12周,至18周齡后,超聲生物顯微鏡檢查確認(rèn)動(dòng)物已有動(dòng)脈粥樣硬化斑塊形成后,實(shí)驗(yàn)動(dòng)物按照體重排序,隨機(jī)數(shù)表法分為7組:空白對(duì)照組,不給于任何藥物,仍然繼續(xù)給予高膽固醇飼料飼養(yǎng);水蛭酶解提取物組,分別給予0.02g/kg,0.1g/kg和0.5g/kg三個(gè)劑量水平的藥物;淫羊藿苷組,分別給予30mg/kg和60mg/kg兩個(gè)劑量水平的藥物;辛伐他汀組,給予10mg/kg辛伐他汀。分組后持續(xù)給藥飼養(yǎng)12周,至30周齡后,解剖進(jìn)行實(shí)驗(yàn)取材。(2)超聲生物顯微鏡檢查評(píng)價(jià)LEE抗動(dòng)脈粥樣硬化效果ApoE敲除小鼠在飼養(yǎng)至30周齡后,行UBM檢查,測(cè)量主動(dòng)脈弓處斑塊形成情況及主動(dòng)脈弓處血管內(nèi)外徑,以評(píng)價(jià)藥物的抗動(dòng)脈粥樣硬化效果。(3)冰凍組織切片油紅O染色評(píng)價(jià)LEE抗動(dòng)脈粥樣硬化效果取小鼠心臟,制成冰凍組織切片后,油紅O染色方法顯示主動(dòng)脈根部的斑塊大小,以評(píng)價(jià)藥物的抗動(dòng)脈粥樣硬化效果。(4)冰凍組織切片MOMA-2染色評(píng)價(jià)LEE抗動(dòng)脈粥樣硬化效果取小鼠心臟,制成冰凍組織切片后,使用免疫組化染色方法,用MOMA-2抗體顯示主動(dòng)脈中單核巨噬細(xì)胞浸潤(rùn)情況,以評(píng)價(jià)藥物的抗動(dòng)脈粥樣硬化效果,及可能的作用機(jī)制。(5) ELISA檢測(cè)小鼠血清中與單核巨噬細(xì)胞粘附遷移相關(guān)的細(xì)胞因子。(6)構(gòu)建TNF-α誘導(dǎo)的內(nèi)皮細(xì)胞功能紊亂細(xì)胞模型。使用TNF-α刺激誘導(dǎo)的內(nèi)皮細(xì)胞激活,建立內(nèi)皮細(xì)胞功能紊亂細(xì)胞模型,用來研究LEE對(duì)內(nèi)皮細(xì)胞的作用。(7)粘附實(shí)驗(yàn)驗(yàn)證LEE對(duì)內(nèi)皮細(xì)胞異常激活的抑制作用。(8)遷移實(shí)驗(yàn)驗(yàn)證LEE對(duì)內(nèi)皮細(xì)胞異常激活的抑制作用。(9) western blot檢測(cè)與內(nèi)皮細(xì)胞粘附和遷移作用相關(guān)的細(xì)胞因子。(10)檢測(cè)LEE抑制內(nèi)皮細(xì)胞異常激活中NFκB的激活和核轉(zhuǎn)移作用。(11)檢測(cè)LEE抑制MAPK信號(hào)通路中相關(guān)蛋白p38,ERK,JNK的磷酸化情況。(12) qPCR檢測(cè)小鼠主動(dòng)脈血管壁中CX3CR1和CX3CL1表達(dá)水平。(13)western blot檢測(cè)淫羊藿苷組小鼠主動(dòng)脈血管壁中CX3CR1和CX3CL1表達(dá)水平。(14)基因芯片表達(dá)譜分析淫羊藿苷對(duì)LPS刺激的巨噬細(xì)胞RAW264.7中基因表達(dá)差異。(15)qPCR檢測(cè)淫羊藿苷對(duì)LPS刺激的巨噬細(xì)胞RAW264.7中CX3CR1和CX3CL1表達(dá)水平。(16) western blot檢測(cè)淫羊藿苷對(duì)LPS刺激的巨噬細(xì)胞RAW264.7中CX3CR1和CX3CL1表達(dá)水平。(17)遷移實(shí)驗(yàn)驗(yàn)證淫羊藿苷抑制巨噬細(xì)胞RAW264.7的遷移效果。實(shí)驗(yàn)結(jié)果(1)通過UBM觀察和測(cè)量主動(dòng)脈弓處血管內(nèi)外徑比值(ID/OD),LEE組中劑量水平(0.1g/kg)和高劑量水平(0.5g/kg)、淫羊藿苷組低劑量水平(30mg/kg)和高劑量水平(60mg/kg)的ID/OD值與對(duì)照組相比明顯較大。LEE高劑量水平(0.5g/kg)、淫羊藿苷高劑量水平(60mg/kg)和辛伐他汀組(10mg/kg)的ID/OD值之間沒有明顯的統(tǒng)計(jì)學(xué)差異。表明LEE及淫羊藿苷可以有效抑制AS的進(jìn)展,并且在高劑量水平時(shí),其效果與辛伐他汀效果相當(dāng)。(2)通過油紅O染色和計(jì)算斑塊面積占主動(dòng)脈橫截面積的比例,LEE組高劑量水平(0.5g/kg)、淫羊藿苷組低劑量水平(30mg/kg)和高劑量水平(60mg/kg)斑塊面積占主動(dòng)脈橫截面積與對(duì)照組相比明顯較小。LEE高劑量水平(0.5g/kg)和淫羊藿苷低劑量水平(30mg/kg)之間沒有明顯的統(tǒng)計(jì)學(xué)差異。淫羊藿苷高劑量水平(60mg/kg)和辛伐他汀組(10mg/kg)之間沒有明顯的統(tǒng)計(jì)學(xué)差異。表明LEE及淫羊藿苷可以有效抑制AS的進(jìn)展,且淫羊藿昔在高劑量水平時(shí),其效果與辛伐他汀效果相當(dāng)。(3)通過MOMA-2染色評(píng)價(jià)單核巨噬細(xì)胞浸潤(rùn),LEE組中劑量水平(0.1g/kg)和高劑量水平(0.5g/kg)、淫羊藿苷高劑量水平(60mg/kg)的MOMA-2染色與對(duì)照組相比明顯較低。表明LEE及淫羊藿苷可以有效抑制AS中的單核巨噬細(xì)胞浸潤(rùn)情況。(4)ELISA法檢測(cè)ApoE-/-小鼠血清中的VCAM-1,1CA1-11,CX3C1和CX3CL1的水平,LEE中劑量水平(0.1g/kg)和高劑量水平(0.5g/kg)可以明顯降低小鼠血清中VCA1M-1和ICAM-1的水平,而對(duì)CX3CR1和CX3CL1無明顯影響。淫羊藿苷高劑量水平(60mg/kg)可以明顯降低小鼠血清中CX3CR1和CX3CL1的水平,而對(duì)VCAM-1和ICAM-1無明顯影響。(5)使用TNF-α刺激內(nèi)皮細(xì)胞建立內(nèi)皮細(xì)胞功能紊亂模型,以eNOS轉(zhuǎn)錄情況為指標(biāo),PCR檢測(cè)不同TNF-α濃度誘導(dǎo)下eNOS的轉(zhuǎn)錄水平,篩選了 TNF-α的誘導(dǎo)濃度為10ng/ml,而LEE的處理劑量為200μig/ml。(6)粘附實(shí)驗(yàn)中與對(duì)照組相比,TNF-α可以明顯增加THP-1向內(nèi)皮細(xì)胞的粘附,而LEE可以降低粘附率,抑制THP-1向內(nèi)皮細(xì)胞的粘附。(7)Transwell實(shí)驗(yàn)中與對(duì)照組相比,TNF-α可以明顯引起THP-1向內(nèi)皮細(xì)胞的遷移,而LEE可以抑制THP-1向內(nèi)皮細(xì)胞的遷移。(8)使用western blot方法,檢測(cè)內(nèi)皮細(xì)胞所表達(dá)的與粘附和遷移相關(guān)的粘附分子ICAM-1和趨化因子MCP-1。實(shí)驗(yàn)結(jié)果表明,TNF-α可以顯著誘導(dǎo)內(nèi)皮細(xì)胞表達(dá)ICAM-1和MCP-1,而LEE可以降低ICAM-1和MCP-1的表達(dá)。(9)使用熒光染色及Western blot檢測(cè)NF-κB激活和p65亞基的核轉(zhuǎn)移,實(shí)驗(yàn)結(jié)果顯示TNF-α可以強(qiáng)烈誘導(dǎo)p65向核內(nèi)轉(zhuǎn)移,而在給予LEE處理之后再使用TNF-α誘導(dǎo)的LEE組,p65向細(xì)胞核內(nèi)轉(zhuǎn)移的情況受到了顯著的抑制。這表明LEE能夠抑制NF-κB的激活。(10)Western blot檢測(cè)LEE對(duì)MAPK信號(hào)通路上相關(guān)蛋白p38、ERK、JNK的磷酸化的影響,實(shí)驗(yàn)結(jié)果顯示LEE可以抑制ERK、JNK的磷酸化。(11)實(shí)時(shí)定量PCR及western blot檢測(cè)小鼠主動(dòng)脈壁中CX3CR1和C3CL1的水平,結(jié)果表明淫羊藿苷能夠降低ApoE敲除小鼠主動(dòng)脈壁中CX3CR1和CX3CL1的水平(12)基因芯片表達(dá)譜分析結(jié)果表明,淫羊藿苷的處理能夠使LPS誘導(dǎo)的巨噬細(xì)胞RAW264.7中CX3CR1出現(xiàn)明顯的表達(dá)差異。(13)實(shí)時(shí)定量PCR及western blot對(duì)RAW264.7細(xì)胞CX3CR1表達(dá)水平分析,結(jié)果顯示淫羊藿苷處理可以明顯抑制LPS誘導(dǎo)的CX3CR1。(14) Transwell遷移實(shí)驗(yàn)結(jié)果表明,淫羊藿苷可以劑量依賴性的抑制CX3CL1所誘導(dǎo)的巨噬細(xì)胞遷移。實(shí)驗(yàn)結(jié)論(1) LEE及淫羊藿苷可以明顯抑制AS的進(jìn)展;(2) LEE及淫羊藿苷可以明顯抑制AS斑塊中單核巨噬細(xì)胞浸潤(rùn)情況;(3) LEE和淫羊藿苷可以影響小鼠血清中粘附分子和趨化因子的水平(4) LEE可以抑制TNF-α誘導(dǎo)的內(nèi)皮細(xì)胞功能紊亂(5) LEE可以抑制TNF-α誘導(dǎo)的內(nèi)皮細(xì)胞中的NFκB的激活和p65亞基的核轉(zhuǎn)移;(6) LEE可以抑制MAPK信號(hào)通路中ERK、JNK的磷酸化。(7)淫羊藿苷可以降低ApoE敲除小鼠主動(dòng)脈壁中CX3CR1和CX3CL1的水平;(8)淫羊藿苷可以降低LPS誘導(dǎo)的巨噬細(xì)胞中CX3CR1的表達(dá)水平;(9)淫羊藿苷可以抑制巨噬細(xì)胞在CX3CL1誘導(dǎo)下的遷移。LEE通過抑制內(nèi)皮細(xì)胞中MAPK信號(hào)通路中ERK、JNK的磷酸化而阻斷NFκB的激活,使相應(yīng)的粘附分子(ICAM-1)和趨化因子(MCP-1)表達(dá)減少,而減少了單核細(xì)胞向內(nèi)皮細(xì)胞的粘附和遷移,從而使AS斑塊中單核巨噬細(xì)胞的浸潤(rùn),而發(fā)揮其抗動(dòng)脈粥樣硬化作用。淫羊藿苷通過抑制巨噬細(xì)胞中CX3CR1的表達(dá),而減少巨噬細(xì)胞在趨化因子CX3CL1作用下的遷移,從而使AS斑塊中單核巨噬細(xì)胞的浸潤(rùn),而發(fā)揮其抗動(dòng)脈粥樣硬化作用。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5

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