第一部分 “清活Ⅰ号”中药复方拮抗高糖导致血管内皮细胞氧化应激的作用及机制研究 摘要 目的：血管并发症是糖尿病患者首要的致残与致死因素。内皮细胞功能紊乱在糖尿病血管并发症的发生发展中起重要作用。内皮细胞功能紊乱的根本机制在于内皮细胞线粒体活性氧簇(ROS)生成过多。前期研究已证实“清活Ⅰ号”中药复方(QHYH)体内可以降低2型糖尿病患者尿微量白蛋白排泄率，体外可以改善高浓度葡萄糖抑制的微血管内皮细胞的生长。本研究的目的是研究“清活Ⅰ号”拮抗高糖导致的氧化应激、保护内皮细胞的抗氧化机制。最近有研究表明，基因水平抑制解偶联蛋白2 (UCP2)的表达可使内皮细胞ROS生成增加。因此，我们也研究了 UCP2的表达变化是否与高糖导致小鼠脑微血管内皮细胞ROS生成增加有关，及UCP2是否参与QHYH的抗氧化应激作用。 方法：bEnd.3细胞分别以正常浓度葡萄糖(NG，5.6mM)、高浓度葡萄糖(HG，25 mM)、高糖加“清活Ⅰ号”(1:100稀释)培养。分别以H₂DCF-DA和DAF-FM DA荧光探针检测内皮细胞ROS和一氧化氮(NO)的生成。UCP2 mRNA水平以实时荧光定量PCR检测。内皮源性一氧化氮合酶(eNOS)、Akt磷酸化水平及UCP2蛋白表达水平以western blotting检测。以UCP2 siRNA进行RNA干扰抑制UCP2表达。 结果：高糖增加bEnd.3细胞ROS生成，HG组ROS生成较NG组增加(142.27± 11.44% vs. 100.00±1.92% in NG group，P<0.01)，同时使UCP2表达下调：高糖对UCP2 mRNA的抑制呈浓度依赖性，35mM时作用最强(69.90±4.84%，P <0.01 vs. NG group)， 也呈时间依赖性，35 mM葡萄糖作用48小时抑制作用最强(66.93±4.73%，P<0.001 vs. Oh group)。高浓度葡萄糖(35 mM)抑制 eNOS Ser1177和Akt Ser473的磷酸化(45.49±2.51% and 76.71±4.00%，P<0.01 vs. NG group)抑制bEnd.3内皮细胞NO生成(53.83±3.98%，P<0.01 vs.NG group).向高糖培养基中加入“清活Ⅰ号”，可逆转高糖导致的ROS生成增加(53.65±8.11% vs. 142.27±11.44% in HG group，P<0.001)、改善eNOS与Akt磷酸化(72.81 ± 7.02% vs. 45.49±2.51% in HG group，P<0.05 and 120.98±1.20% vs. 76.71±4.00% in HG group，P<0.001)、NO 生成(118.82±2.95% vs. 53.83±3.98% in HG group， P<0.001)、及UCP2蛋白表达(94.09±6.30% vs. 53.83±3.98% in HG group， P<0.001).通过UCP2 siRNA抑制UCP2蛋白表达后，“清活Ⅰ号”拮抗高糖导致的氧化应激的作用减轻，不能改善高糖诱导的ROS生成及NO产生的下调。 结论：“清活Ⅰ号”可通过抑制ROS生成，促进Akt、eNOS磷酸化和NO生成，拮抗高糖诱导的内皮细胞氧化应激。“清活Ⅰ号”保护内皮细胞的作用可能部分与其上调UCP2的表达有关。本研究结果提示“清活Ⅰ号”作为抗氧化剂治疗糖尿病血管并发症具有应用前景。 关键词：“清活Ⅰ号”中药复方；活性氧簇(ROS)；内皮细胞；解偶联蛋白(UCP2)；一氧化氮(NO)；内皮源性一氧化氮合酶(eNOS) 第二部分 “清活Ⅰ号”分离物对内皮细胞ROS生成的影响及机制探讨 摘要 目的：前期研究已证实“清活Ⅰ号”中药复方具有抗氧化应激作用。本实验的目的是对“清活Ⅰ号”中药复方进行分离提纯取，检测其分离物抑制ROS生成的作用，明确“清活Ⅰ号”中药复方中参与氧化应激作用的化合物，并探讨所筛选化合物抗氧化应激的作用机理。解偶联蛋白2 (UCP2)的表达可抑制内皮细胞ROS生成增加。高糖对内皮细胞UCP2的表达具有抑制作用。我们的研究也证实了“清活Ⅰ号”可改善高糖对UCP2的抑制作用，“清活Ⅰ号”的抗氧化应激作用可能与其改善UCP2表达有关。因此检测了 “清活Ⅰ号”分离所得化合物对bEnd.3细胞UCP2 mRNA水平的影响。NADPH氧化酶是内皮细胞中ROS的重要来源。NADPH氧化酶由gp91〓、p22〓、p67〓、p47〓等亚单位组成，因此本实验拟检测有效化合物对bEnd.3细胞NADPH氧化酶gp91〓、p22〓、 p67〓、p47〓基因表达的影响。 方法：以溶剂萃取法对“清活Ⅰ号”水煎剂进行分离，获得3个部分。以大孔树脂法对清活Ⅰ号进行分离，获得5个部分。bEnd.3细胞分别以正常浓度葡萄糖(NG，5.6mM)、高浓度葡萄糖(HG，25mM)、高糖加不同浓度“清活Ⅰ号”分离提取物培养。以H₂DCF-DA荧光探针检测bEnd.3细胞及人脐静脉内皮细胞(HUVEC)ROS的生成。以MTT法检测“清活Ⅰ号”分离物对内皮细胞(bEnd.3细胞与HUVEC细胞)增殖的影响。以MCI硅胶层析、反复柱层析等方法对有效部分进一步分离提取。以实时荧光定量PCR检测“清活Ⅰ号”分离所得有效化合物对 bEnd.3 细胞 UCP2、gp91〓、p22〓、p67〓、p47〓 mRNA 水平的影响。 结果:自“清活Ⅰ号”水煎剂中先后分离出28种粗提部分，通过对抑制高糖刺激ROS生成活性的筛选及进一步分离，共分离出12个化合物单体，其中9个为己知化合物，包括京尼平苷酸、京尼平苷、黄芪甲苷、葛根素、陈皮苷、西红花苷、川芎嗪、阿魏酸、绿原酸，文献均报道过其具有抗氧化活性。另三个化合物：E3-M15-1、E3-M15-2和E1-H1-1，结构暂未知，亦不明确其是否具有抗氧化活性。抑制高糖刺激ROS生成活性检测发现西红花苷、川芎嗪、阿魏酸、绿原酸、E3-M15-1、E3-M15-2和E1-H1-1在两种细胞模型(bEnd.3细胞与HUVEC细胞)上对高糖刺激ROS生成均有抑制作用，且对内皮细胞无明显毒性作用(E3-M15-2高浓度组除外)。 高浓度葡萄糖可抑制bEnd.3细胞UCP2 mRNA水平，“清活Ⅰ号”及其分离物(西红花苷、川芎嗪、阿魏酸、绿原酸、E3-M15-1、E3-M15-2和E1-H1-1)可不同程度使UCP2 mRNA水平恢复。 高浓度葡萄糖可使gp91〓、p22〓、p67〓mRNA表达升高；“清活Ⅰ号”及川芎嗪可抑制高糖诱导的gp91〓、p22〓、p67〓 mRNA升高；西红花苷可抑制高糖上调的gp91〓与p67〓 mRNA水平；E3-M15-1可抑制高糖上调的gp91〓、p22〓 mRNA水平，阿魏酸、绿原酸仅可抑制高糖上调的p22〓 mRNA水平。 结论：“清活Ⅰ号”分离物西红花苷、川芎嗪、阿魏酸、绿原酸、E3-M15-1、 E3-M15-2和E1-H1-1对高糖刺激ROS生成均有抑制作用，为“清活Ⅰ号”抗氧化应激作用的重要组成部分。“清活Ⅰ号”及其分离物(西红花苷、川芎嗪、阿魏酸、绿原酸、E1-H1-1)的抗氧化应激作用可能与其改善高糖对UCP2 mRNA的抑制及抑制高糖上调的NADPH氧化酶mRNA水平有关。 关键词：内皮细胞；“清活Ⅰ号”；ROS；UCP2；NADPH氧化酶

 Protective effects of a prescribed traditional Chinese medicine preparation on oxidative stress induced by high glucose in bEnd.3 cells. Aims: vascular diseases are the principal causes of morbidity and mortality in patients with diabetes mellitus. It has been suggested that endothelial dysfunction may be a critical and initiating factor in the pathogenesis of diabetic vascular complications. An underlying mechanism for endothelial dysfunction is the enhanced generation of endothelial mitochondrial reactive oxygen species (ROS). We previously observed 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. This study was designed to gain the insight into the antioxidant mechanism of QHYH against oxidative stress induced by high glucose in endothelial cells. We studied the effect of QHYH on ROS production, NO generation and the phosphorylation of Akt and eNOS. Since a recent study indicates that genetic ablation of UCP2 produces more ROS in endothelial cells, we therefore investigated the effect of QHYH on UCP2 expression. Finally, using siRNA technique, we studied whether UCP2 is involved in the increase of ROS in mice brain microvascular cells (bEnd.3 cells) induced by high glucose, and whether it is accounted for the antioxidant mechanism of QHYH in endothelial cells. Materials and Methods: bEnd.3 cells were cultured in normal glucose (NG group, 5.6mM), high glucose (HG group), high glucose in the presence of QHYH (1:100 dilution). 35 mM glucose was used as high glucose working concentration except 25 mM in ROS detection experiments. ROS and nitric oxide (NO) production was measured by the fluorescent marker H2DCF-DA and DAF-FM DA, respectively. Endothelial nitric oxide synthase (eNOS), and uncoupling protein 2 (UCP2) mRNA were detected by quantitative real-time reverse transcription- polymerase chain reaction. Phosphorylation of eNOS, protein kinase B (Akt) and UCP2 protein expression were measured by western blotting. UCP2 siRNA was used to evaluate the involvement of UCP2 in the antioxidant effect of QHYH. Results: High glucose (25 mM) increased ROS production (142.27 ± 11.44% vs. 100.00 ± 1.92% in NG group, P＜0.01) in bEnd.3 cells. UCP2 mRNA and protein expression were decreased at the same time, glucose reduction of UCP2 mRNA in a dose-dependent manner, with the maximum effect at 35 mM glucose (69.90 ± 4.84%, P ＜0.01 vs. NG group), and was time-dependent, with maximal effect occurring after 48 hours of incubation (66.93 ± 4.73%, P ＜0.001 vs. Oh group). High glucose (35 mM) downregulated phosphorylation of eNOS at Seri 177 and Akt at Ser473 (45.49 ± 2.51% and 76.71 ± 4.00%, respectively, P＜0.01 vs. NG group) and decreased NO generation (53.83 ± 3.98%, P＜0.01 vs. NG group) in bEnd.3 cells compared to NG group. Addition of QHYH reversed high glucose's effects on ROS production (53.65± 8.11% vs. 142.27 ± 11.44% in HG group, P＜0.001),eNOS and Akt phosphorylation (72.81 ± 7.02% vs. 45.49 ± 2.51% in HG group, P＜0.05 and 76.71 ± 4.00% vs. 120.98± 1.20% in HG group, P＜0.001, respectively), NO generation (118.82 ± 2.95% vs. 53.83 ± 3.98% in HG group, P＜0.001), and UCP2 protein expression (94.09 ± 6.30% vs. 53.83 ± 3.98% in HG group, P＜0.001). When UCP2 expression was blocked by UCP2 siRNA, the anti-oxidant effects of QHYH against high glucose were attenuated; QHYH could not reverse the high glucose's effects on ROS production and NO generation Conclusions: QHYH could protect endothelial cells from high glucose induced ROS production, downregulation of Akt and eNOS phosphorylation and reduction of NO generation. The protective effects of QHYH were partially related to UCP2 protein expression, since deletion of UCP2 by siRNA lead to the loss of the effects. These results suggest the therapeutic potential of QHYH in treatment of diabetic vascular complications. Key words: traditional Chinese medicine compound recipe Qinghuoyihao; reactive oxygen species (ROS); uncoupling protein 2 (UCP2) ; endothelial cell; nitric oxide (NO); eNOS (endothelial NO synthase) Protective effects of QHYH extraction on ROS production in endothelial cells Background: We have confirmed that QHYH has antioxidative effects. The purpose of this experiment is to investigate the chemical constituents of QHYH, determine their antioxidative effects on high glucose induced ROS production, and study the underlying mechanism of their antioxidative effects. Genetic ablation of UCP2 produces more ROS in endothelial cells, and our study has confirmed that QHYH could improve glucose induced inhibition of UCP2 mRNA, deletion of UCP2 by siRNA lead to the loss of protective effects of QHYH, QHYH's protective effects were partially related to UCP2 protein expression, so we detected effects of the chemical compounds extracted from QHYH on UCP2 mRNA level in bEnd.3 cells. NADPH oxidase is one of the most important sources of ROS. It is composed of gp91〓, p22〓, p67〓 and p47〓, so we also detected the effects of chemical compounds extracted from QHYH on NADPH submits mRNA levels. Materials and methods: We got 3 fractions from QHYH by solvent extraction and 5 fractions by using of macroporous resin. bEnd.3 cells were cultured in normal glucose (NG group, 5.6 mM), high glucose (HG group), high glucose in the presence of QHYH (1:100 dilution) or the chemical compounds extracted from QHYH. 35 mM glucose was used as high glucose working concentration except 25 mM in ROS detection experiments. ROS production was measured by the fluorescent marker H2DCF-DA. Column chromatogram was used for the furhther separation and extraction. The effects of chemical compounds on endothelial cell viatality were determined by MTT chromatometry. The mRNA levels of UCP2, p91〓, p22〓, p67〓, p47〓 were determined by real-time quantitive PCR. Results: 28 fractions were isolated from QHYH, and by detecting their effects on high glucose induced ROS production in bEnd.3 cells and further separation and extraction, 12 chemical compounds were extracted from QHYH, 9 of which are known, including geniposidic acid, geniposide, astragaloside, puerarin, hesperidin, crocin, tetramethylpyrazine (TMP), ferulic acid (FA) and chlorogenic acid (CHA) , and all of them have been reported to have antioxidative effects. The structure of the other 3 compounds: E3-M15-1, E3-M15-2 and E1-H1-1 have not been determined yet. crocin, TMP, FA, CHA, E3-M15-1, E3-M15-2 and El-Hl-1 could inhibit high glucose induced ROS production in both bEnd.3 and HUYEC cell, and have no significant cytotoxicity effects on both cell lines (except E3-M15-2 in high concentration). High glucose could decrease UCP2 mRNA level in bEnd.3 cells. Addition of QHYH and extractions (crocin, TMP, FA, CHA, E3-M15-1, E3-M15-2 and E1-H1-1) could inhibit the effect of high glucose. High glucose increased the mRNA level of gp91〓 and p67〓. QHYH and TMP could inhibit high glucose induced increase of gp91〓, p22〓and p67〓mRNA. E3-M15-1 could inhibit high glucose induced increase of gp91〓and p22〓 mRNA. Crocin could inhibit high glucose induced increase of gp91〓 and p67〓 mRNA level. FA, CHA could only inhibit high glucose induced increase of p22〓 mRNA. Conclusions: The chemical compounds extracted from QHYH including crocin, TMP, FA, CHA, E3-M15-1, E3-M15-2 and E1-H1-1 could inhibit high glucose induced ROS production in bEnd.3 and HUVEC cells, they are among the important constituents of QHYH for its antioxidative effects. The protective effects of QHYH and its extraction (crocin, TMP, FA, CHA, E3-M15-1) were partially related to their improvement of UCP2 mRNA suppressed by high glucose, and their inhibition of NADPH oxidase mRNA expression induced by high glucose. Key words: endothelial cell; Traditional Chinese medicine compound recipe Qinghuoyihao; reactive oxygen species (ROS); uncoupling protein 2(UCP2); NADPH oxidase