【作者】 庄秀园；

【导师】 瞿伟菁；

【作者基本信息】 华东师范大学 ， 植物学， 2011， 博士

【摘要】 糖基化终产物(advanced glycation end products, AGEs)是糖尿病慢性并发症的重要风险因子。前期研究发现,多种植物有效成分具有降血糖降血脂等功效,但对AGEs的形成及AGEs诱导的细胞损伤的影响罕见报道。本研究对上述有效成分进行筛选,以寻找有效的AGEs抑制药物,同时结合提取过程中含量发生较大变化的几种糖尿病相关微量元素进行研究,拟在阐明植物有效成分抗AGEs作用机理的同时,明确其与微量元素的关系。采用体外美拉德(Maillard)反应体系,对七种植物有效成分提取物进行筛选。结果发现参试的四种黄酮类提取物均具有抑制AGEs形成的作用,其中以沙棘籽渣黄酮(flavonoids from seed residues of Hippophae rhamnoides L., FSH)效果最佳,对总AGEs和戊糖素(pentosidine)的形成都具有显著的抑制作用。同时,本文也研究了铜、锌、锰三种微量元素对AGEs形成的影响,结果显示,二价锰离子对AGEs的形成具有十分显著的抑制作用,而铜离子促进AGEs形成。在上述AGEs形成干预研究的基础上,本文进一步研究了FSH,锌,锰,以及FSH与这两种微量元素的联合应用对AGEs诱导的血管内皮细胞损伤的影响。将培养的牛主动脉内皮细胞(bovine aortic endothelial cells, BAECs)用AGEs溶液处理30 min后,再以FSH联合微量元素进行治疗。结果发现AGEs的处理能够降低细胞锌元素的水平,抑制内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)活力,减少一氧化氮(nitric oxide, NO)释放；同时,细胞的氧化还原平衡遭到破坏,细胞总抗氧化能力大幅度降低,活性氧簇(reactive oxygen species, ROS)等有害自由基水平上升,胞内主要的抗氧化酶——超氧化物歧化酶(superoxide dismutase, SOD)的活性也受到抑制,炎症相关核转录因子kappa B (nuclear factor kappa B, NF-κB)水平增高、活化增强,AGEs受体蛋白(receptor for AGEs, RAGE)表达增加。在FSH的治疗下,细胞的上述变化大部分得到改善,细胞锌水平、eNOS的mRNA和蛋白表达水平、NO形成和分泌显著提高,细胞氧化还原状态逐渐恢复,并回复正常的形态和分布。微量元素锌能够提高细胞eNOS的表达和酶活,以及NO的生成和分泌,并提高细胞抗氧化能力,抑制NF-κB蛋白的过表达及其活化和核转位。而外源性锰能够有效清除过量的ROS,维持Mn-SOD的基因表达,提高总SOD和Mn-SOD的酶活力。此外,随着二价锰离子的加入,细胞NF-κB的蛋白表达、激活及其核转位得到明显抑制,RAGE的nRNA和蛋白表达水平明显下降。在FSH联合微量元素的治疗中,FSH与锌在多项指标上具有协同作用,而与锰则多表现为拮抗作用。其中锌的补充,可以稳定FSH的细胞保护作用,或加速其生物学效应的发挥,而FSH也能够降低较高浓度微量元素的细胞毒性,有利于微量元素作用的持久发挥。总之,糖尿病相关微量元素锰和FSH能够显著抑制AGEs体外形成,其中二价锰离子对AGEs的抑制作用属于首次报道。适量补给锌、锰、FSH可以改善AGEs引起的内皮细胞功能障碍：锌能够活化eNOS、恢复NO的生物利用度,而锰则是强大抗氧化剂,可以通过提高Mn-SOD酶活等方式清除ROS。FSH既能够显著提高eNOS表达、增加NO分泌,又能提高细胞抗氧化能力。同时,FSH与锌具有较强的协同作用,两者的合理配伍能够提高FSH抵御糖基化损伤的整体药效。更多还原

【Abstract】 The accumulation of advanced glycation end products (AGEs) is considered one of the hallmarks in the pathogenesis of diabetes and various macro- or micro-vascular complications. Our previous research had confirmed that several ingredients extracted from medicinal plants, such as flavonoids, saponins, and polysaccharides are effective in controlling serum glucose and lipid levels, whereas their effects on AGEs formation and AGEs-mediated endothelial cell dysfunction are poorly known. The present study investigated these effects in order to find effective AGEs inhibitors. We also studied the effects of trace elements copper, zinc, and manganese on AGEs formation, whose content varied significantly after extraction when compared with their original plant, to clarify the functional relationship between extracted ingredients and trace elements.Fluorescence detection was used to monitor the Maillard reaction in vitro. Flavonoids exhibited excellent inhibitory effects on AGEs formation, especially the flavonoids from seed residues of Hippophae rhamnoides L. (FSH). Incubation with FSH significantly reduced levels of total AGEs and the specific pentosidines. MnCl2 also inhibited the formation of AGEs, while ZnCl2 had the opposite effect.Then we studied the effects of FSH, Zn, Mn, and the combination of FSH with Zn or Mn on AGEs induced endothelial cell dysfunction. Primary cultured bovine aortic endothelial cells (BAECs) were exposed to AGEs for 30 min. followed by FSH and trace element treatments. Cell viability of BAECs was significantly inhibited by AGEs exposure, while the addition of FSH, ZnCl2, and MnCl2 protected the cells from the injuries with cell viability greatly increased. Furthermore, AGEs exposure declined intracellular zinc levels, nitric oxide (NO) release, and endothelial NO synthase (eNOS) levels (including mRNA levels, protein expression levels, and the enzymatic activity). Simultaneously, cellular redox equilibrium was destroyed due to AGEs exposure, total anti-oxidation competence was greatly reduced, intracellular reactive oxygen species (ROS) production was obviously increased, antioxidant enzymatic ability of superoxide dismutase (SOD) was inhibited, and inflammation relevant nuclear transcription factor kappa B (NF-κB) was activated with excessive mRNA levels and protein expression. The addition of FSH improved these poor situations described above. FSH significantly increased the declined intracellular zinc levels, NO release, and eNOS expression. FSH could also restore the anti-oxidant capability, the SOD enzymatic ability, and even the abnormal cell configuration and distribution. ZnCl2 supplementations enhanced the AGEs-decreased intracellular zinc levels, NO release, eNOS levels and their enzymatic capability. ZnCl2 could also elevate the anti-oxidant capability and SOD enzymatic ability of BAECs, inhibit the activation, nuclear translocation, and overexpression of NF-κB. The MnCl2 supplementation significantly enhanced intracellular zinc levels, cleared the excessive formation of intracellular ROS, and impaired the sharpened activation and nuclear translocation of NF-κB in BAECs, as well as restored the inhibited mRNA expression and enzymatic capability of Mn-SOD.Synergistic effects of the combined treatments of FSH and ZnCl2 appeared in NO release detection, eNOS mRNA and protein expression, and eNOS enzymatic activity. Though there were no synergistic effects on cell viability and intracellular zinc uptake, the addition of FSH overcame the negative, cytotoxic effects of ZnCl2, cell viability remained high even 20μmol/L ZnCl2 solutions were supplemented. In the same manner, the presence of ZnCl2 accelerated and maintained the benefit effects of FSH on AGEs-induced endothelial cell dysfunction. With FSH supplementations intracellular zinc levels reached 150% that of the control, regardless of the increasing concentration of exogenous ZnCl2 supplementations. The combination of FSH and MnCl2 was more complicated. FSH overcame the inhibitory effects of the high leveled-MnCl2 on cell viability. With the presence of FSH, viability ratio of the AGEs-pretreated BAECs did not suppressed even when MnCl2 was supplemented at 20μmol/L, a high and toxic concentration of manganese for cells. The MnCl2 supplementations accelerated and maintained the effects of FSH just as ZnCl2 did. However, unlike the effect on increasing zinc levels, FSH hardly had any influences on intracellular manganese levels. Furthermore, the combined therapy of FSH and MnCl2 resulted in antagonistic effects on cellular zinc levels:zinc levels rapidly declined with 50μg/mL FSH and increasing amount of MnCl2. Antagonistic effects also appeared on intracellular ROS formation and NO release.In conclusion, FSH, zinc, and manganese resisted the AGEs-induced injuries through different manners. FSH and MnCl2 significantly inhibited the formation of AGEs in vitro; moderate FSH, ZnCl2, and MnCl2 supplementations improved AGEs-mediated endothelial cell dysfunction. ZnCl2 benefited the injured BAECs probably through an NO and NF-κB related mechanism, whereas MnCl2 was a potentially powerful antioxidant by refreshing Mn-SOD activity. FSH showed significant effects on eNOS activity and NO release elevation, cellular anti-oxidation ability restoration, and intracellular ROS reduction. FSH also had synergistic effect with zinc, the combination of FSH and zinc improved the general effect of FSH on AGEs inhibition, and might have advantage in further therapy for diabetic complications.更多还原