【作者】 毛张凡；

【导师】 孙宗全；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 多酚类化合物是中药和药食两用植物的重要有效成分，广泛存在于众多植物中，在日常蔬菜、水果及饮料中含量丰富。多酚类化合物如白藜芦醇、槲皮素等的药理作用非常广泛，如降血压、抗氧化、抗脂质过氧化、抗缺血再灌注损伤、抗肿瘤等，因此受到医药界的高度关注。但天然的多酚类化合物由于含有多个羟基而容易被氧化，不利于提取、制备和保存。另外其生物利用度低，半衰期短、效价低等缺点也防碍了对其的开发和利用。多甲氧基黄酮是黄酮的甲基衍生物，具有明显的药代动力学特点和药效动力学优势。但天然多甲氧基黄酮的提取工艺至今尚无重大突破，得量有限，价格昂贵，使得深入研发难以进行。我们对槲皮素进行了甲基化改造，获得高纯度低价位的五甲基槲皮素（Pentamethylquercetin，PMQ），克服了药源的瓶颈，使我们得以深入研究PMQ的药理学特性。本实验组之前研究发现PMQ能对抗乳鼠心肌细胞的氧化损伤；能内皮依赖性和内皮非依赖性的舒张离体血管；能对抗离体和在体的心肌缺血再灌注损伤；能抑制乳鼠心脏成纤维细胞的增值。这些发现提示PMQ对心血管系统的作用值得进一步研究。考虑到临床上行冠状动脉旁路移植术（CABG）的冠心病患者仍面临着心肌重构和静脉移植桥管新生内膜再狭窄的问题，目前仍没有有效的治疗手段，本实验尝试探索PMQ对这类疾病是否能有所益处。故本实验的目的是研究PMQ对AngⅡ诱导的心肌肥大、间质纤维化的作用及对血管平滑肌细胞增值和自体移植静脉内膜增生的影响，并探讨其可能的机制。另外，在实验中改进了成年大鼠心肌细胞分离和培养的方法及大鼠颈部自体静脉移植的连续缝合方法。第一部分五甲基槲皮素减轻AngⅡ所致大鼠心肌肥厚和细胞凋亡目的：研究五甲基槲皮素（PMQ）对血管紧张素Ⅱ（AngⅡ）致大鼠心肌肥厚和凋亡的作用。方法：30只SD大鼠随机分为5组，试验持续21天。（1）空白组：每晨生理盐水灌胃；（2）PMQ组：每晨PMQ50mg／kg灌胃；（3）AngⅡ组：于第15天开始皮下注射AngⅡ（288μg／kg·d）；（4）：PMQ+AngⅡ组：PMQ和AngⅡ处理同前；（5）溶剂+AngⅡ组：每晨溶剂灌胃，AngⅡ处理同前。于第22天处死大鼠，测量心脏指数、左心指数，real time-pcr检测BNP mRNA的表达，TUNNEL法测心肌凋亡，免疫组化测Bax、Bcl-2表达。结果：AngⅡ能明显引起高血压、心肌肥厚（提高心脏指数、左心指数及BNPmRNA的表达），诱导心肌细胞凋亡及上调凋亡相关蛋白Bax表达、提高Bax／Bcl-2。PMQ能明显抑制AngⅡ引起的上述改变。结论：PMQ能对抗AngⅡ引起的高血压、心肌肥厚及凋亡。第二部分五甲基槲皮素减轻AngⅡ所致大鼠心肌间质纤维化目的：研究PMQ对AngⅡ所诱导的大鼠心肌间质纤维化的作用。方法：SD大鼠30只随机分为5组，试验持续21天。（1）空白组：每晨生理盐水灌胃；（2）PMQ组：每晨PMQ（50mg／kg）灌胃；（3）AngⅡ组：于第15天开始皮下注射AngⅡ（288μg／kg·d）；（4）：PMQ+AngⅡ组：PMQ和AngⅡ处理同前；（5）溶剂+AngⅡ组：每晨溶剂灌胃，AngⅡ处理同前。第22天处死大鼠，测量心肌羟脯氨酸含量，real time-pcr检测collagenⅠ及collagenⅢmRNA的表达，免疫组化测collagenⅠ、collagenⅢ容积分数（CVF）及其比值CVFⅠ／CVFⅢ。结果：AngⅡ能明显提高大鼠心肌羟脯氨酸含量，上调collagenⅠ、collagenⅢ的mRNA表达，增加collagenⅠ、Ⅲ容积分数及CVFⅠ／CVFⅢ比值。PMQ能显著抑制AngⅡ引起的上述改变。结论：PMQ能对抗AngⅡ引起的心肌间质纤维化。第三部分五甲基槲皮素抑制AngⅡ诱导的大鼠心肌NADPH氧化酶mRNA表达上调目的：研究PMQ对AngⅡ所诱导的大鼠心肌重构的作用机制。方法：SD大鼠30只随机分为5组，试验持续21天。（1）空白组：每晨生理盐水灌胃；（2）PMQ组：每晨PMQ（50mg／kg）灌胃；（3）AngⅡ组：于第15天开始皮下注射AngⅡ（288μg／kg·d）；（4）：PMQ+AngⅡ组：PMQ和AngⅡ处理同前；（5）溶剂+AngⅡ组：每晨溶剂灌胃，AngⅡ处理同前。第22天处死大鼠，测量心肌SOD活力、MDA含量，real time-pcr检测NADPH oxidase亚单位Nox2和p47^phox mRNA的表达。结果：AngⅡ能明显提高大鼠心肌NADPH oxidase亚单位Nox2和p47^phox的mRNA表达，并降低心肌SOD活力、增加MDA含量。PMQ能显著抑制AngⅡ引起的上述改变。结论：PMQ能对抗AngⅡ引起的心肌重构可能与其抗氧化及下调NADPH oxidase mRNA表达有关。第四部分五甲基槲皮素抑制AngⅡ诱导的血管平滑肌增殖和NADPH氧化酶mRNA表达上调目的：研究PMQ对AngⅡ诱导血管平滑肌（VSMC）增殖的作用及其机制。方法：AngⅡ（0.1μmol／L，24h）刺激血管平滑肌细胞增殖，同时分别给予0.1、0.3、1、3、10、30μmol／L的PMQ干预，用MTT法测细胞活力，用DCFH-DA测ROS，用real-time PCR测NADPH oxidase亚单位Nox1，p47^phox，p22^phox的mRNA表达。结果：PMQ 0.3μmol／L开始可以显著抑制VSMC的活力并减少ROS的产生，3μmol／L作用最强，10μmol／L、30μmol／L作用减弱。PMQ同时显著抑制Nox1，p47^phox，p22^phox表达。结论：PMQ能显著抑制AngⅡ诱导的VSMC增殖，此作用可能与其抗氧化及抑制NADPH氧化酶有关。第五部分五甲基槲皮素抗大鼠颈部自体静脉移植物内膜增生目的：研究PMQ对大鼠自体移植静脉内膜增生的作用。方法：建立大鼠颈部自体静脉移植模型，随机分为模型对照组和给药组。对照组每晨溶剂灌胃，给药组每晨按PMQ 12.5mg／kg、25mg／kg、50mg／kg三个剂量分别灌胃。于28天后取材测新生内膜和中膜的厚度及面积比。结果：与对照相比，三个剂量组的PMQ均能显著减小内膜和中膜的厚度及面积比，其中50mg／kg组的效果较其他两个剂量弱。结论：PMQ有抑制自体静脉移植物新生内膜增生的作用。附录1多用途成年大鼠心室肌细胞分离方法目的：建立稳定的可同时用于细胞培养和膜片钳实验的成年大鼠心室肌细胞分离方法。方法：应用Langendorff灌流，生物酶（胶原酶Ⅱ加BSA）消化法分离耐钙心肌细胞。分别用左右心室肌做细胞培养和全细胞膜片钳研究。结呆：左室心肌细胞数3.7±0.6×10⁶，存活率96.0±2.1％，杆状细胞得率84.8±2.7％，横纹清晰。膜片钳实验时容易封接破膜，记录到典型的I_Ca．L。结论：本方法简单、节约、重复性好，改善了杆状细胞得率、细胞质量，左右心室肌细胞分别能很好的用于培养和膜片钳实验。附录2大鼠颈部自体静脉移植模型的两种吻合方法目的：比较间断吻合和连续吻合法建立静脉桥狭窄动物模型的优劣。方法：SD大鼠20只，分间断吻合和连续吻合组，取颈外静脉与颈总动脉行端端吻合。术后4周取下静脉桥，观察桥管通畅性，分析新生内膜与中膜的厚度、面积比。结果：连续组与间断组相比手术时间更短，出血更少，但桥管通畅率低，两组内膜增生程度没有显著差异。结论：连续吻合用时短，出血少，对术者要求更高，更易形成吻合口狭窄。两者造模效果一样。更多还原

【Abstract】 Polyphenol compounds are very important active ingredient in plants for Chinesemedicine and medical food; they are plenty in many plants, vegetables, fruits and drinks.Polyphenolic compounds, like resveratrol （RES） and quercetin （QUE）, have extensivepharmacological effects, such as blood pressure lowing, anti-oxidation, anti-lipidperoxidation, anti-repeffusion injury and anti-tumor and so on; which are highly concernedin the medicine kingdom. However, natural polyphenol compounds could be oxidatedeasily because of their many hydroxyls, thus impact the extraction, manufacture and storing.Their low bioavailability, short half-life and low effect-price rate obstacle the exploitationand utilization as well. Polymethoxylflavonoids are methylation derivatives frompolyphenolflavonoid, which have obvious pharmacokinetics characteristics andpharmacodynamics advantage. Because of no big progress on the extraction process,polymethoxylflavonoids are limited sourced, expensive, and hard to be researched.Therefore, we methylized quercetin and obtained high-purified and inexpensivePentamethylquercetin （PMQ）, which overcame the bottleneck of PMQ source and made thein-depth pharmacological study of PMQ a reality.In the earlier studies, we found that PMQ reduced the oxidation injury of H₂O₂ onisolated neonatal rat myocardial cells; extenuated ischemia reperfusion injury in vivo and invitro heart; suppressed proliferation of cultured cardiac fibroblast induced by aldosterone;relaxed isolated vascular rings by endothelium-dependent and endothelium-independentmechanisms. All these suggest that the advantages of PMQ on cardiovascular systemdeserve to be investigated. The patients with coronary heart disease after accepted coronaryartery bypass grafting operation faced two fundamental problems: chronic ventricalremodeling and vein graft restenosis caused by neointima. We wondered if PMQ had anyeffect on this kind of disease. In this study, we investigated the effects of PMQ on cardiachypertrophy and fibrosis induced by AngⅡ; the influences of PMQ on proliferation ofvascular smooth muscle cell induced by AngⅡ; the impacts on intimal hyperplasia model of autologous vein graft in rat and the mechanisms. In addition, we improved the methodsof isolation of adult rat cardiomyocytes for culture and experiment, of patch clamp andanastomosis to institute vein graft model.PartⅠ3,3’,4’,5,7-Pentamethylquercetin reduces cardiachypertrophy and apoptosis in AngiotensinⅡ-infused ratsObjective The hypothesis that pentamethylquercetin （PMQ） reduces cardiac hypertrophyand myocyte apoptosis was tested in AngiotensinⅡ（AngⅡ）-infused rats. Methods: Thirtyrats were randomly assigned to the 5 groups with 6 rats in each group: （1） control group:Saline gavage was performed daily for 21 days; （2） PMQ group: PMQ （50mg/kg） gavagewas performed daily for 21 days; （3） AngⅡgroup: AngⅡ（288μg/kg·d） was daily injectedsubcutaneously from the 15th day; （4） PMQ+ AngⅡgroup: PMQ gavage and AngⅡinjection were performed as the same as above; and （5） solvent+ AngⅡgroup: Vehiclegavage was performed daily for 21 days, AngⅡinjection was performed as the same asabove. Blood pressure was monitored daily utilizing tail-cuff. After the rats wereeuthanized at 22st day, the heart weight index （HW/BW） and the left ventricular weightindex （LVW/BW） were calculated, and the expression of BNP mRNA was determined byreal time-PCR. Myocyte apoptosis was measured by TUNEL assay and the expression ofBax and Bcl-2 were determined by immunohistochemistry. Results: PMQ reduces bloodpressure and cardiac hypertrophy induced by AngⅡby decreasing the heart weight index,the left ventricular weight index and the expression of BNP mRNA; inhibits myocyteapoptosis by reducing the expression of Bax, Bax/Bcl-2. Conclusion: PMQ reducescardiac hypertrophy and myocyte apoptosis in AngⅡinduced hypertension rats. Theresults suggest that PMQ may represent an attractive therapeutic approach to treat CHF. PartⅡ3,3’,4’,5,7-Pentamethylquercetin reduces cardiacfibrosis in AngiotensinⅡ-infused ratsObjective: To investigate the effect of PMQ on AngⅡinduced cardiac fibrosis. Methods:Thirty rats were randomly assigned to the 5 groups, 6 each: （1） control group: Saline wasadministrated daily via gavage for 21 days; （2） PMQ group: PMQ （50mg/kg） wasadministrated daily via gavage for 21 days; （3） AngⅡgroup: AngⅡ（288μg/kg·d） wasinjected subcutaneously daily from the 15th day; （4） PMQ+ AngⅡgroup: PMQ and AngⅡwere administrated as above; and （5） solvent+ AngⅡgroup: Solvent and AngⅡwereadministrated as above. After the rats were euthanized on the 22nd day, the myocardialhydroxyproline content was measured, and the expression of collagenⅠand collagenⅢmRNA were determined by real time-PCR. Collagen volume fraction （CVF）ⅠandⅢwere detected by immunohistochemistry, and CVFⅠ/CVFⅢwas calculated. Results:PMQ reduced cardiac fibrosis in AngⅡinduced hypertension rats by decreasing themyocardial hydroxyproline content, downregulating the expression of collagen I andcollagenⅢmRNA, and decreasing CVFⅠ, CVFⅠ/CVFⅢ. Conclusion: PMQ couldreduce cardiac fibrosis.PartⅢ3,3’,4’,5,7-Pentamethylquercetin downregulatesexpression of NADPH oxidase mRNA inAngiotensinⅡ-infused ratsObjective: To investigate the mechanism of anti-ventricular remodelingof PMQ on AngⅡ-infused rats. Methods: Thirty rats were randomly assigned to the 5 groups, 6 each: （1） control group: Saline was administrated daily via gavage for 21 days; （2） PMQ group:PMQ （50mg/kg） was administrated daily via gavage for 21 days; （3） AngⅡgroup: AngⅡ（288μg／kg·d） was injected subcutaneously daily from the 15th day; （4） PMQ+ AngⅡgroup: PMQ and AngⅡwere administrated as above; and （5） solvent+ AngⅡgroup:Solvent and AngⅡwere administrated as above. After the rats were euthanized on the22nd day, the myocardial SOD activity and MDA content were measured, and theexpression of NADPH oxidase subunits Nox2 and p47^phox mRNA were determined by realtime-PCR. Results: PMQ exerted antioxidant function by increasing SOD activity anddecreasing MDA content and reducing the mRNA expression of NADPH oxidase subunitsNox2 and p47^phox. Conclusion: PMQ could reduce cardiac remodeling, which may resultfrom antioxidant functionPartⅣ3,3’,4’,5,7-Pentamethylquercetin suppresses theproliferation of VSMC and downregulates the mRNA expressionof NADPH oxidase induced by AngⅡObjective: To investigate the effect of PMQ on AngⅡinduced proliferation of vascularsmooth muscle cells and its mechanism. Methods: The proliferation of vascular smoothmuscle cells were induced by AngⅡ（0.1μmol/L, 24h） while PMQ was administrated atdifferent dosages（0.1, 0.3, 1, 3, 10 and 30μmol/L）. Cell viability was detected by MTT;ROS was measured by DCFH-DA; and the expressions of NADPH oxidase subunits Noxl,p47^phox, and p22^phox mRNA were measured by real-time PCR. Results: PMQ suppressedthe cell viability and ROS of vascular smooth muscle cells induced by AngⅡ. PMQ startedto demonstrated therapeutic effects from 0.3μmol/L, got the peak at 3μmol/L, and theeffects weakened from 30μmol/L to 10μmol/L. PMQ also downregulated the mRNA expressions of NADPH oxidase subunit Nox1, p47^phox and p22^phox induced by AngⅡ.Conclusion: PMQ suppressed the proliferation of vascular smooth muscle cells induced byAngⅡ, which maybe result from the anti-oxidation activity and mRNA expressiondownregulation of NADPH oxidase.PartⅤ3,3’,4’,5,7-Pentamethylquercetin suppresses intimalhyperplasia of the vein graftObjective: To investigate the effect of PMQ on intimal hyperplasia of the vein grafts inrats. Methods: SD rats were randomly assigned to control group and PMQ group. Solventwas administrated daily in rats of control group via gavage, PMQ （12.5mg/kg, 25mg/kg,50mg/kg） was administrated daily in rats of PMQ group via gavage. The reversed jugularvein was implanted into the carotid artery. 4 weeks after operation, vein grafts wereharvested, and intimal hyperplasia of the vein grafts was assessed. Results: Compared withcontrol group, PMQ decreased intima/media area index and intima/media thickness index atthree dosages after implantation. The effects of 50mg/kg PMQ were weaker than PMQ at12.5mg/kg and 25mg/kg. Conclusion: PMQ could inhibit neointima hyperplasia of veingraft in rats.SupplementⅠIsolation of adult rat cardiomyocytesfor culture and experiment of patch clampObjective: To improve current enzymatic methods to isolate a high yield of high-quality adult rat cardiomyocytes for both culture and experiment of patch clamp. Methods:Calcium tolerant cardiomyocytes were isolated with collagenaseⅡby langendorff perfusion,Left and right ventricle myocytes were used respectively for culture with or without FBSand patch clamp. The currents of L-type calcium channels were recorded by patch clamp inthe entire cell mode. Results: With this method, we routinely obtained a highyield（3.7±0.6×10⁶/left ventricle） and high percentage（84.8±2.7%） of rod-shaped myocytes,most of which were clearly defined sarcomeric striations and quiescent state. A typicalcurrent of L-type calcium channel was recorded in the cardiomyocytes from right ventricle.Conclusion: this is a simple and reliable myocyte isolation method that greatly improvesthe yield, cell quality, and reproducibility of cardiomyocytes isolation and is suit to bothculture and patch clamp.SupplementⅡTwo types of anastomosisto institute vein graft modelObjective: To compare two types of anastomosis in an animal model of intimal hyperplasiaof autologous vein graft in rats. Methods: SD rats were divided into two groups randomly.The external jugular veins were implanted into the external carotid of the same side withinterrupted suture and twice continuous suture respectively. Samples of tissues wereharvested at 4 weeks after operation. Situation of vein grafts were observed and tissuesections were analyzed by HE staining. Results: Compared with interrupted suture group,continuous suture had less time, less bleeding; but less graft patency. The intimalhyperplasia of two anastomosis methods had no obviously difference. Conclusion:Continuous suture has the advantages of costing less time and less bleeding, but it requiresmore skillful. It is no difference in the degree of intimal hyperplasia of autologous vein graft in rat.更多还原