IR@PKUHSC  > 北京大学药学院
学科主题化学生物学
氨基三乙酸衍生物氧钒配合物的合成及其降糖作用研究
王娜
2015-06-05
学位类型博士
导师杨晓达
专业化学生物学
授予单位北京大学
授予地点北京大学药学院
学位博士
关键词钒,糖尿病,抗氧化剂,药物设计,PPARa,PPARr
其他题名synthesis and anti-diabeticeffects of novel novel nitrilotriacetic acid vanadyl complexes
分类号R914.5
页数164
摘要

钒化合物是具有巨大发展潜力的抗糖尿病药物,然而其潜在的毒性问题限制了进一步的临床应用。如何设计配合物的结构、获得药理作用和毒性作用相平衡的新型钒化合物是当今钒配合物药物设计和合成的重要问题。基于钒化合物毒性的分子机制是诱导细胞活性氧生成、导致氧化应激,我们使用具有抗氧化和抗炎症活性的双功能配体,合成了一系列不同结构的新型氧钒配合物。主要研究结果如下:

6个氨基三乙酸衍生物氧钒配合物的合成及其结构及生物活性的研究:

以氨基三乙酸为碳骨架引入具有羟基苯胺或羟基苯乙胺衍生物结构的抗氧化功能配体,制备了6个新型氧钒配合物(VOL1~VOL6),其结构通式如下:

 

对上述氧钒配合物进行了抗氧化能力和HK-2细胞毒性评价。抗氧化能力评价结果表明:所合成的配体均具有良好的自由基淬灭活性。其中,配合物VOL1,VOL6的抗氧化能力较配体为强,配合物(VOL2~VOL5)的抗氧化能力较配体大幅减弱。其中在上述配合物中VOL1的抗氧化能力最强。对HK-2细胞毒性评价结果表明,配合物的IC50总体和其抗氧化性趋势一致,即抗氧化性越高,细胞毒性越小。其中,VOL1对HK-2细胞的毒性最小,并且其毒性明显低于对照药物BMOV。

利用db/db2型糖尿病小鼠对其降糖效应做了初步测试,结果表明:上述新型氧钒配合物(0.1 mmol/kg/bodyweight/day,P.O.)均表现出一定的降糖活性、改善糖耐量和对肝肾的保护作用。其中VOL1的效果最好。因此,本研究选择VOL1进行了降糖效果、降糖机制和动物毒性的系统研究。

新配合物VOL1对2型糖尿病db/db小鼠的降糖效果、降糖机制和动物毒性研究:

 

VOL1(0.1 mmol/kgbodyweight/day,P.O.)可将db/db 2型糖尿病小鼠血糖降低至正常水平,并增强糖耐受力,还可在未抑制饮食的情况下,显著降低小鼠饮水量。与BMOV(稳定降糖剂量需要0.18 mmol/kg以上)相比,VOL1的降糖剂量小、降糖效果及对血糖代谢功能的改善更稳定。血清生化指标显示,VOL1缓解了高血糖症造成的肝、肾损伤,改善糖尿病小鼠的脂代谢水平。

分析VOL1治疗后小鼠脂肪及肌肉组织中糖尿病病理相关几个重要的生物指标显示:①VOL1可在基因及蛋白水平显著上调脂肪组织及肌肉组织中的PPARα及PPARγ水平;②VOL1显著上调了肌肉组织中AKT和P38-MAPK蛋白水平;③VOL1显著上调了脂肪组织中AKT,P38-MAPK,Adiponectin和p-AMPK蛋白水平;④VOL1在两种组织中均抑制JNK磷酸化活化。说明钒化合物的降糖效应涉及多种机制,而调节PPARγ及PPARα是其中的关键环节。

VOL1小鼠口服给药急性毒性的LD50= 1045 mg/kg,根据世界卫生组织化学物质急性毒性分级标准,VOL1属低毒性物质,其毒性与BMOV(LD50= 902 mg/kg)持平,但比BSOV(LD50= 3200 mg/kg)高。在肾损伤上,western-blotting分析发现钒配合物(VOL1,BMOV)并不升高db/db糖尿病小鼠KIM-1水平,说明短期作用下钒化合物不影响肾功能;但与BMOV相比,VOL1治疗组的KIM-1水平有所降低,预示VOL1长期肾副作用可能比BMOV低。

但是,VOL1并未能改善糖尿病小鼠的血清白蛋白水平。由于钒化合物治疗过程中长期低血清白蛋白水平可能诱发糖尿病小鼠的肾间质水肿(RIE)现象,因此VOL1的长期肾副作用(及其机制)仍有待实验观察。分析钒化合物不提升糖尿病鼠血清白蛋白水平的原因包括:钒化合物通过诱导未折叠蛋白的响应以限制肝脏组织白蛋白合成;钒化合物抑制JNK活化加重糖尿病蛋白尿等。针对这些副作用,理论上可通过膳食补充或设计新的配体活性等方法得到解决。

总结本文实验结果,我们认为合适的配合物热力学和动力学稳定性、良好的抗氧化活性和附加抗炎活性可能调节钒化合物毒性和药效的平衡的关键因素。钒化合物具有多机制的抗糖尿病作用,依然是目前最具发展潜力抗糖尿病金属药物,而本课题组的双功能配体氧钒配合物是未来抗糖尿病钒化合物设计的一个合理策略。

英文摘要

Vanadium compounds are promising insulin-enhancing agent for treatment of both type I & II diabetes, however, the potential metal toxicity, e.g. kidney change, prevented the clinical application. Although the mechanism of the renal toxicity of BMOV/BEOV has not been elucidated, many studies have suggested that vanadium-induced oxidative stress due to interaction with mitochondria could be one major mechanism for vanadium toxicity. In addition, vanadium-induced inflammatory responses could also be involved amongst which induction of cyclooxygenase-1,2 (COX-1,2) has been proposed as the main source of pro-inflammatory factors upon vanadium treatment.

Natural and synthetic antioxidants were shown to effectively decrease vanadium toxicity. Therefore, incorporation of antioxidant groups in the structure of vanadium complexes could be a feasible strategy in designing novel anti-diabetic vanadium complexes with lower toxicity.  By conjugating nitrilotriacetic acid to provide a coordination group and antioxidative motifs, we synthesized six new vanadium complexes.

Synthesis and characterization of six nine new vanadium complexes

Six new vanadium complexes with formulated as VO(L)2 with aminophenol- derivatized nitrilotriacetic acid have been prepared. Their generic structure is as below:

 

The tests of antioxidant activity and Cytotoxicity on HK2 human kidney cells show that:1)the ligands exhibited significantly antioxidant activity; 2)complexes VOL1 and VOL6 have better antioxidant capacity than ligands, while the antioxidant activity of other complexes (VOL2 ~ VOL5) is weaker than ligand. 3)VOL1 have the dominant antioxidant capacity among the complexes; 4) The toxicity of the complexes were firstly evaluated in vitro on HK-2 kidney cells along with their antioxidant capacity ,That was the higher the antioxidant activity, the smaller the cytotoxicity, and VOL1 had minimal cytotoxicity.

Then new vanadium complexes were chosen to test anti-diabetic therapeutic potential and animal toxicity. Herein, Vanadium complexes (0.1 mmol / kg body weight / day, P.O.) had good hypoglycemic effect, they also improved glucose metabolism ability and protectd liver and kidney, VOL1 treated gtoup exhibited the prominent anti-diabetic effect. Probably because VOL1 vanadium oxygen radicals played a role in anti-diabetic, and ligand-aminophenol had potent antioxidant and anti-inflammatory effects, alleviated symptoms caused by diabetes.

Combining these results, we choosed VOL1 as the agent for the next experiment.The toxicity,hypoglycemic effects and hypoglycemic mechanisms had been investigated in detail.

Investigations on acute toxicity, anti-diabetic mechanism of VOL1

The oral LD50 of VOL1 was calculated to be 1045 mg kg-1.which toxicity is slightly higher than BMOV (LD50= 902.04mg / kg), this situation maybe caused of their excellent water solubility, and elevated bioavailability led to marginally higher toxicity.

Compared with BMOV, VOL1 treated group(0.1 mmol / kg body weight / day, P.O.) effectively lowered blood glucose level, enhanced glucose tolerance, and significantly reducing the daily water intake, showing a more stable blood sugar hypoglycemic effects and improve metabolic function. Serum biochemical parameters indicated, VOL1 alleviated the injuries in liver, heart, and kidney caused by hyperglycemia. But VOL1 did not improve ALB levels in diabetic mice, the reason may be inadequate albumin synthesis in the liver tissue, proteinuria increased, leading to a decline in the level of plasma in Alb.

The QPCR and westernblot analysis revealed that VOL1 exhibited hypoglycemic effect through insulin enhancing mechanism: (i) VOL1 caused significant elevation of Akt, p38-MAPK, Adiponectin, p-AMPK ,indicating activation of insulin signal transduction; (ii) VOL1 could significantly up-regulate PPARα and PPARγ level(primarily in muscle and adipose tissue) both in protein and gene. respectively. PPARα and γ are well recognized targets for insulin enhancers (iii) as the consequence of PPARγ activation,vanadium treatment significantly reduced phosphorylation of JNK (c-Jun NH2-terminal protein Kinase).

The westernblot analysis showed that BMOV and VOL1 treated groups did not cause significant elevation of the kidney injury molecule-1 (KIM-1/TIM-1); while VOL1 even slightly reduced KIM-1/TIM-1 level, suggesting potentially lower long term toxicity of VOL1. However, considering the potential of vanadium-induced inflammatory responses (not investigated herein) and possible consequences of low serum albumin (if last ever), the long term renal toxicity of VOphpada needs to be carefully investigated in future works.

In conclusion, the experimental results suggest VOL1 as a potent anti-diabetic agents. In light of rational drug, the present work the kinetics of complex stabilities and antioxidant property would be important factors determining the toxicity andefficacy of vanadium complexes. Further works using the present strategy of vanadium compound design would be appropriate.

语种中文
相关网址查看原文
文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/149664
专题北京大学药学院
北京大学第三临床医学院_儿科
作者单位北京大学药学院
第一作者单位北京大学药学院
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王娜. 氨基三乙酸衍生物氧钒配合物的合成及其降糖作用研究[D]. 北京大学药学院. 北京大学,2015.
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