【作者】 张斌；

【导师】 高鑫； 金惠铭； 陆志强； 赵耐青；

【作者基本信息】 复旦大学 ， 内科学， 2006， 博士

【摘要】 目的：前期研究发现清活Ⅰ号中药方可以降低糖尿病患者尿微量白蛋白排泄率，并具有与天然抗氧化剂N-乙酰半胱氨酸（NAC）类似的保护血管内皮细胞高糖损伤的作用。本研究目的是探索清活Ⅰ号中药方保护血管内皮高糖损伤的抗氧化应激分子机制。 方法：采用电子顺磁共振波谱仪（EPR）和氧自由基捕获技术检测清活Ⅰ号中药复方及其各组方单味药体外清除氧自由基的能力，观察该中药复方对高糖培养bEnd.3内皮细胞氧自由基生成的影响。应用中药血清药理学研究方法观察该中药复方的药物血清对高糖培养SD大鼠肺微血管内皮细胞增殖的影响。运用Real time RT-PCR检测该中药复方对高糖培养bEnd.3内皮细胞解偶联蛋白（UCP）基因mRNA表达的影响，以及蛋白印迹方法测定细胞UCP蛋白分子的表达情况。 结果：清活Ⅰ号复方可淬灭体外羟自由基模型中82.2％的自由基，其组方单味药除了3号药外，其它组方单药可使模型中羟自由基减少23.1％～46.2％，中药复方和各组方药在一定浓度范围内，其淬灭氧自由基的作用存在量效关系。清活Ⅰ号复方的药物血清和NAC一样可改善高糖培养的SD大鼠肺微血管内皮细胞的增殖和活力。内皮细胞bEnd.3高糖培养时，EPR检测到了细胞生成的超氧阴离子明显增多，清活Ⅰ号中药、超氧岐化酶和NAC均可减少高糖培养内皮细胞氧自由基的生成，各组EPR检测到的氧自由基信号较高糖培养组分别下降了70％、56％和47.3％。Real time RT-PCR检测结果：UCP-1、UCP-3在5.56mM葡葡糖培养组bEnd.3细胞（NG组）和高糖培养的各组bEnd.3细胞中表达量很少或不表达，UCP-2基因mRNA的表达是单纯高糖培养组细胞（HG组）较NG组增高（p=0.09），清活Ⅰ号中药（中药组）和NAC（NAC组）干预后则较HG组进一步明显上调（p分别为0.009、0.036）。Western blot分析则在中药组检测到的较明显的UCP-2蛋白分子的表达，NAC组有少量表达，HG组和NG组未检测到明显的UCP-2分子的表达。 结论：1．清活Ⅰ号中药复方及其各组方药均有较强的体外淬灭氧自由基作用，其中复方淬灭氧自由基的能力要明显强于任何单味组方药；2．清活Ⅰ号复方的药物血清具有类似天然抗氧化剂NAC减轻或是消除高糖致内皮细胞损伤的作用；3．高糖可导致血管内皮细胞超氧阴离子生成增多，清活Ⅰ号中药方具有清除细胞生成的超氧阴离子作用；4．清活Ⅰ号保护血管内皮损伤作用除了与该中药方具有直接淬灭氧自由基作用外，还可能与清活Ⅰ号中药能上调高糖环境中内皮细胞UCP-2的表达，减少线粒体超氧阴离子生成有关。目的：评价冠状动脉粥样硬化性心脏病（冠心病）患者氧化应激状态和血管内皮功能相关血清指标变化，为有抗氧化和保护血管内皮功能作用的清活Ⅰ号中药方用于冠心病患者治疗提供临床循证医学证据。 方法：正常健康对照200人，经冠状动脉造影检查（CAG）诊断的冠心病患者200例（CAG阳性组）和经CAG排除的非冠心病患者135例（CAG阴性组）。评估各组人群的糖、脂代谢等情况，分光比色法测定血清抗超氧阴离子自由基(O₂^-．)活力、脂质过氧化物（MDA）、一氧化氮合酶（总NOS和iNOS）活性和一氧化氮（NO）水平并进行组间比较。 结果：CAG阳性组的血清抗O₂^-．活力、总NOS和iNOS、NO水平均明显低于正常对照组，血清MDA水平则明显高于正常对照组（p均＜0.001）。CAG阳性组血清抗O₂^-．活力与CAG阴性组相近（p=0.634），但MDA水平仍高于CAG阴性组，并且总NOS、iNOS和NO水平低于GAG组（p≤0.001）。CAG阴性组血清抗O₂^-．活力比正常对照组低（p=0.002），MDA水平高于正常对照组（p=0.001），总NOS水平两组见相近（p=0.967），但CAG阴性组的iNOS和NO水平较正常对照组低（p≤0.001）。CAG阳性组中糖调节异常者的比率（55.5％）高于CAG阴性组（37％）（p=0.001）。 结论：冠心病患者存在血管内皮功能障碍，体内氧化应激水平较高，其血清清除超氧阴离子能力下降；冠心病患者常合并糖调节异常；有抗氧化作用和保护内皮细胞高糖损伤作用的中药复方清活Ⅰ号可能有助于糖尿病患者和非糖尿病患者冠心病的防治。更多还原

【Abstract】 Aims: Our previous study has demonstrated that a prescribed traditional Chinese medicine preparation — Qinghuoyihao（QHYH） can decrease urinary microalbumin excretion in type 2 diabetic patients and improve microvascular endothelial cells growth in high glucose medium with similar effects to a natural antioxidant N-acetylcysteine（NAC）. This study was to explore the antioxidative molecular mechanism of QHYH against the noxious effects of high glucose level on endothelial cells.Methods: Electron paramagnetic resonance（EPR） together with spin trap technique was applied to determine the antioxidative capacity of QHYH and its each constitutional material in vitro. And ffects of QHYH on cellular superoxide anion production was observed when bEnd.3 cells were cultured in a high glucose medium. The effects of QHYH on SD rat pulmonary microvascular endothelial cells cultured in high glucose media was investigated with serum pharmacological approaches. Expression of uncoupling proteins（UCPs） mRNA in bEnd.3 cells cultured in media with 5.56mM glucose（NG group）, 35mM glucose（HG group）, 35mM glucose and 1:100 QHYH （QHYH group）, and 35mM glucose plus 10mM NAC（NAC group） were analyzed by real time quantitive RT-PCR, and expression of UCP molecule were studied by Western blot analysis.Results: EPR determination revealed that QHYH could extinguish 82.2% of the radicals generated in a hydroxyl free radical model system and the scavenging capacity of single component medicine ranged from 23.1% to 46.2%. Like NAC, serum from SD rats which were administered with QHYH could improve SD rat pulmonary microvascular endothelial cells’ vitality in high glucose milieu. Cellularsuperoxide anion production increased when bEnd.3 cells were cultured in media with 35mM glucose and QHYH could potently scavenge the superoxide anions with a similar effect to superoxide dismutase. No obvious expression of UCP-1 or UCP-3 mRNA was observed in bEnd.3 cells cultured in media with 5.56mM or 35mM glucose while distinct expression of UCP-2 mRNA was detected in bEnd.3 cells of NG, HG, QHYH, and NAC groups. Expression of UCP-2 mRNA in HG group was higher than that in NG group（p=0.009） and UCP-2 mRNA was further upregulated in cells of QHYH and NAC groups（p=0.009, 0.036 respectively）. In Western blot analysis, expression of UCP-2 molecule were detected in cells of QHYH group and a blear band of UCP-2 molecule appeared in NAC group while no apparent UCP-2 molecule band occurred in NG or HG groups.Conclusions: The results suggest that the prescribed traditional Chinese medicine preparation — Qinghuoyihao and its each constitutional material can scavenge oxygen free radicals in vitro with Qinghuoyihao compound recipe being more potent than any of its single component remedium. Serum materia medica of Qinghuoyihao recipe, like NAC, can protect endothelial cells against the noxious effects of high level glucose in culture media. Cellular superoxide anion production increases when endothelial cells are exposed to high level glucose and the Qinghuoyihao preparation is effective in extinguishing superoxide anions produced by the endothelial cells. In addition to its direct effects of scavenging oxygen free radicals, the traditional Chinese medicine compound recipe can upregulate expression of UCP-2 molecule in cells exposed to high glucose milieu resulting in decrease of mitochondrial superoxide anion production and this may also play an important role in its antioxidative and protective effects on endothelial cells in high glucose surroundings.Aims: To determine the oxidative stress status and endothelial function in patients with coronary atherosclerotic heart disease （CAD）.Methods: The serous scavenging activities for superoxide anion radicals（O₂^-·）, malondialdehyde （MDA） level, nitric oxide synthase（NOS） and its subtype inducible nitric oxide synthase（iNOS） activity, and nitric oxide（NO） level were measured by spectrophotometry in 200 healthy individuals（healthy group）, 200 patients with CAD diagnosed by coronary angiography（CAD group） and 135 patients with normal coronary angiographic manifestation（CAG negative group）. Glucose metabolism status and plasm lipid profile were also analyzed in all the individuals. Results: The average serous scavenging activities for O₂^-·, NOS and iNOS activity, and NO level in CAD group were much lower than that in healthy group（p<0.001） while MDA level was higher than that in healthy group（p<0.001）. No obvious difference in serous scavenging activities for O₂^-· was observed between CAD group and CAG negative group（p=0.634） while MDA level in CAD group was higher than that in CAG negative group（p<0.001） and NOS and iNOS activities were lower than that in CAG negative group. Compared with healthy group, CAG negative group had lower average serous scavenging activities for O2^-· （p=0.002）, a higher MDA level（p=0.001）, similar NOS activities（p=0.967）, decreased iNOS activities（p=0.001）, and a lower NO level（p<0.001）. The prevalence of impaired glucose regulation（IGR） in patients with CAD was 55.5%, which was much higher than that in patients in CAG negative group（p=0.001）.Conclusions: Endothelial dysfunction, increased oxidative stress, decreased serous scavenging activities for O2^-· and high prevalence of IGR can be observed in patients with CAD. Antioxidative therapy may benefit patients with CAD.更多还原