DPP-Ⅳ抑制剂—异槲皮苷促进2型糖尿病模型小鼠胰岛素分泌及降糖作用的研究
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摘要
糖尿病是一种以高血糖为主要特征的内分泌代谢疾病，主要分为1型糖尿病（胰岛素依赖型糖尿病）和2型糖尿病两种类型（非胰岛素依赖型糖尿病），其中2型糖尿病占发病总数的80%～90%，病因主要是组织对胰岛素抵抗、胰岛素敏感性下降和胰岛β细胞分泌胰岛素相对不足。
胰高血糖素样肽-1(Glucagon-like Polypep Tide1, GLP-1)是食物在肠道内的消化吸收过程中，刺激肠道内分泌细胞产生的，由30个氨基酸残基组成。GLP-1的生理功能主要包括促进胰岛素的分泌、胰岛β细胞增殖并抑制其凋亡、减缓胃的排空、抑制餐后胰高血糖素的分泌、减少肝糖元的合成、提高胰岛素的敏感性以及控制食欲等。临床研究显示，GLP-1及其类似物在2型糖尿病的治疗过程中具有重要的应用价值，其通过增加胰岛素的分泌，抑制胰高血糖素的释放调节患者体内的血糖浓度。因此，GLP-1作为一种安全、有效的促胰岛素分泌物越来越受到关注。但是，GLP-1被体内的二肽基肽酶Ⅳ(Dipeptidyl Peptidase IV, DPP-IV)降解的速度非常快，其半哀期很短，仅1～2min，内源性GLP-1分泌量不足的情况下不能积累达到激动GLP-1受体(GLP-1R)的剂量，故难以应用于临床。因此，研发一种安全、有效的DPP-IV抑制剂是目前2型糖尿病药物开发的一个新热点。
二肽基肽酶IV (Dipeptidyl Peptidase IV, DPP-IV)又称CD26，是由科学家Glenner和Hopsu-Havu在鼠肝脏组织匀浆过程中发现的一种位于细胞表面的丝氨酸肽酶(Serine Peptidase)，广泛表达于哺乳动物组织中，其催化活性中心位于胞外分子的C末端区域，DPP-IV能够特异性地裂解GLP-1的N端二肽残基：Xaa2Pro(Proline)或Xaa2Ala (Alanine)(Xaa为任意氨基酸)，是体内、外主要促使GLP-1降解、失活的关键酶之一。DPP-IV抑制剂的结构与DPP-IV的天然底物相似，含有Xαα-Pro类似结构，可以竞争性地结合DPP-IV的活化部位，但其亲和力却远大于它的天然底物，从而改变DPP-IV的构象，降低其催化活性。
2006年10月由默克公司研制成功的磷酸西格列汀(Sitagliptin)是最早获得FDA审批，并且作为DPP-IV抑制剂上市的治疗2型糖尿病的药物，该药显示出较好的降糖效果。但是磷酸西格列汀是化学合成药物，价格较昂贵，供货量少，且存在着较大的副作用，如可能出现超敏反应、肝酶升高、上呼吸道感染、鼻咽炎、过敏反应、血管性水肿和剥脱性皮肤损害、皮疹、荨麻疹和胰腺炎。因此，筛选出一种有效，经济，安全的DPP-IV抑制剂显得尤为重要。
我们以编码DPP-IV催化结构域(△DPP-IV)的cDNA质粒PMD18为模板，通过PCR反应扩增DPP-IV催化结构域基因，然后将其克隆到pGex-HisC3载体中，并将重组质粒转入大肠杆菌Rosstta DE3plysS中，加入IPTG诱导表达，最后通过亲和层析方法获得了△DPP-IV蛋白。
我们采用发色底物法建立了DPP-IV酶活性分析体系，检测纯化后的△DPP-IV酶的纯度达90%以上，活力为4.2U/mg，酶反应动力学显示在前20分钟内该酶显示了良好的线性关系。从现有的47种中药单体化合物中筛选△DPP-IV酶的抑制剂，在每种单体化合物终浓度为1mmol/L的情况下，抑制效果达到80%以上的单体化合物有两种，其中异槲皮苷对DPP-IV酶的抑制效果最好，达到了98.1%，测定其对△DPP-IV的IC50为96.8μmol/L，同时通过双倒数作图法研究发现异槲皮苷对△DPP-IV的抑制类型为竞争性抑制，抑制常数Ki为236μmol/L。
从细胞水平上检测了异槲皮苷刺激NCI-H716细胞分泌GLP-1的影响。不同浓度的异槲皮苷溶液和磷酸西格列汀溶液(10μmol/L、50μmol/L和100μmol/L)刺激人肠道内分泌细胞NCI-H716细胞系不同时间(6h、24h、48h和72h)，结果显示，随着异槲皮苷和磷酸西格列汀浓度的升高和刺激时间的延长GLP-1的分泌量逐渐增多，但异槲皮苷对GLP-1的刺激效果更明显。
在动物水平上，我们选用了8周龄、体重约20g的昆明小鼠，采用腹腔注射浓度为35mg/kg/day STZ辅以高脂高糖饲料喂养的方式诱导2型糖尿病小鼠模型的建立，连续注射STZ三天后，小鼠的血糖上升到22.43mmol/L≥11.1mmol/L，即建模成功。小鼠按体重和血糖值随机分为6组：正常组、模型组、异槲皮苷低剂量给药组(20mg/kg)、中剂量给药组(40mg/kg)、高剂量给药组(80mg/kg)和阳性药物磷酸西格列汀组(20mg/kg)，每天观察饮水量及尿量，每周记录体重及血糖浓度的变化。连续给药8周后，结果显示，从外观上来看，正常组小鼠饮食饮水尿量正常，体重增长快，皮毛有光泽，活动灵活；模型组小鼠多饮多食多尿症状明显，体重减轻，皮毛竖立无光泽，精神萎靡，反应迟钝，异槲皮苷给药组小鼠一般情况均好于模型组，其中高剂量给药组(80mg/kg)小鼠整体状况较好，多饮多食多尿症状减轻，磷酸西格列汀给药组小鼠状况良好。体重方面，模型组小鼠体重下降，异槲皮苷给药组小鼠体重与灌胃前体重有明显上升(p<0.001)，磷酸西格列汀给药组小鼠体重与灌胃前相比明显上升(p<0.001)。血糖方面，与模型组相比，给药组小鼠血糖浓度明显下降，其中高剂量组（80mg/kg)小鼠血糖浓度下降明显，下降到14.8mmol/L(p<0.001)；磷酸西格列汀给药组血糖浓度下降到17.4mmol/L(p<0.001)，说明异槲皮苷具有明显的降血糖效果。在葡萄糖耐受实验(OGTT)中，结果显示，与模型组相比，给药组血糖浓度明显降低，即中剂量、高剂量(40mg/kg、80mg/kg)异槲皮苷给药组和磷酸西格列汀给药组能明显拮抗外源葡萄糖引起的血糖升高，低剂量(20mg/kg)异槲皮苷给药组也能降低外源性葡萄糖引起的血糖升高，但并不明显，这说明异槲皮苷能够调节糖尿病小鼠体内的血糖。异槲皮苷对血清中的DPP-IV活性具有明显的抑制作用。而在连续给药8周后，与模型组相比，给药组小鼠体内的甘油三酯和总胆固醇值变化并不明显。从体内GLP-1的分泌情况来看，在未给予葡萄糖灌胃时，给药组小鼠空腹血清GLP-1浓度明显高于模型组，在给予2g/kg体重葡萄糖后的10min时，小鼠体内的GLP-1分泌量明显增多，达到峰值，其中与模型组相比，高剂量组(80mg/kg)和磷酸西格列汀组的GLP-1分泌量增多(p<0.001)。而在给予葡萄糖后的20min时，小鼠体内的胰岛素分泌量达到高峰，其中，模型组小鼠体内的胰岛素分泌偏少，异槲皮苷高剂量组(80mg/kg)和磷酸西格列汀组的小鼠体内胰岛素分泌量明显增多(p<0.001)。小鼠处死后，取胰腺组织，通过HE染色的方法观察小鼠胰岛细胞的形态。结果显示，正常组小鼠胰岛细胞数量多，腺管完整清晰可见，细胞形态饱满，胞质丰富浅染，而模型组小鼠胰岛明显萎缩，胰岛细胞不完整，细胞界限模糊；与模型组相比，异槲皮苷给药组和磷酸西格列汀组小鼠胰岛病理切片腺管部分恢复饱满充盈状态，细胞排列规则，细胞形态完整，尤其是高剂量(80mg/kg)给药组，胰腺细胞形态基本恢复到正常组水平。
Diabetes mellitus is a kind of endocrine and metabolic diseases as the maincharacteristics is high glucose. It mainly divided into type1(insulin dependentdiabetes) diabetes and type2diabetes (non insulin dependent diabetes).Approximately80%to90%of diabetes mellitus belongs to type2diabetes. Thereare many etiologies in type2diabetes, including insulin resistance with organization,decreased insulin sensitivity and insulin secretion insufficient.
Glucagon-like polypep tide1(GLP-1) is a member of vasoactive intestinalpeptide/secretin/Pancreas liters of Chitosan, which consists of30amino acidresidues. GLP-1is an incretin hormone, which is released from gut endocrine L cellsin response to food ingestion. It has many physiological functions, includingpromoting insulin secretion, inhibiting of apoptosis, slowing the emptying of thestomach, inhibiting postprandial glucagon secretion, reducing the synthesis ofhepatic glycogen, improving insulin sensitivity and controlling appetite. GLP-1isefficient in improving β cell function according to animal experiments.
According to clinical researchs, GLP-1and its analogues have a great of valuein treating type2diabetes. Therefore, GLP-1aroused more and more attention forthe safety and efficiency in promoting insulin secretion. GLP-1is degraded streamby dipeptidyl peptidase IV (DPP-IV) in the blood, the mourning period is veryshort,only1~2min. Its mourning period is too short to accumulating sufficientGLP-1to activate GLP-1R(the receptor of GLP-1). Therefore, it is difficult to applyclinic.It is a new hot spot for researching type2diabetes to find a safe, effectiveinhibitor of DPP-IV.
Dipeptide based peptidase IV (DPP-IV) called the CD26, is a kind of serinepeptidase on the cell surface, which is found in the process of homogenate byGlenner and Hopsu–Havu. DPP-IV is widely expressed in mammalian tissues,such as kidney, connective tissue, the gastrointestinal tract and lymph nodes. DPP-Ⅳ gene locats on chromosome22q24.3. Its catalytic activity center is the end of Carea located in extracellular molecules. If the second bit of the N-terminal of the peptide is Pro and Ala peptide, it is main substrate of DPP-IV. DPP-IV is a keydegraded enzymes to GLP-1in vivo or vitro by specifically cleavaging of GLP-1N-terminal dipeptide residue: Xaa2Pro or Xaa2Ala. The inhibitor is far more naturalsubstrate in binding to the enzyme. Hence, it reduces its catalytic activity bychanging the conformation of DPP-IV.Owing to the fact that inhibitor is competitivewith the natural substrate at the same structure of DPP-IV containing Xαα-Pro.
Sitagliptin is a inhibitor of DPP-IV.It has an obvious hypoglycemic effect in thetreatment of type2diabetes, which is researched by Merck at October,2006.However, Sitagliptin is expensive as a chemical synthetic drug, and there are manykinds of side effects, such as allergic reactions, elevating liver enzymes, infectingrespiratory tract, nasopharyngitis, anaphylaxis, angioedema and stripping of skindamage, skin rashes, hibes and pancreatitis. Therefore, it is important to find aeffective, economic and srcurity DPP-IV inhibitor.
The cDNAs is PMD18template, which encoding the catalytic domain ofDPP-IV (△D PP-IV), were synthesized from the polyadenylated mRNA, and therecombinant plasmid was transformed into E.coli Rosstta DE3plysS, induced byIPTG. The Catalytic Domain of DPP-IV was amplified by PCR and then thetruncated enzyme was purified by GST Resin column.
We established the detection methods for the enzyme. It is chromogenicsubstrate method. The activity of DPP-IV was4.2U/mg, and the enzyme showed alinear relationship during the first20min. There are two kinds of monomercompound detected. They had obvious inhibitory to DPP-IV, in which the inhibitoryeffect of Isoquercitrin reached98.1%. We found that Isquercitrin was a competitiveinhibitor of DPP-IV, with the half maximal inhibitory concentration (IC50) andapparent inhibition constant (Ki) were96.8and236μM.
NCI-H716cells were treated with different concentration of Isquercitrin(10μmol/L、50μmol/L and100μmol/L)or Sitagliptin at10μmol/L、50μmol/L and100μmol/L in different times (6h,24h,48h or72h).The results show that, aobvious dose-dependent increase in the secretion of GLP-1was found by Elisa Kitmethods.
20g mice were acclimated for1week and injected with35mg/kg/day STZ inorder to induce type2diabetes model. When the blood glucose was up to22.43mmol/L≥11.1mmol/L, the mice were randomly divided into six groupsaccording the body weight and blood glucose:(1)normal group,(2)model group,(3)soquercitrinlow-dose group (20mg/kg),(4) medium-dose group (40mg/kg),(5)highdose group (80mg/kg),(6) positive drug sitagliptin phosphate (20mg/kg).After8weeks, the normal group looks like very well, and the modeling mice havepolyphagia polyuria symptoms and they are unresponsive. However, theIsoquercitrin group were better than the model group. The Isoquercitrin treatmentinhibited plasma DPP-IV activity in a dose-dependent manner. The weight of micein Isoquercitrin group and Sitagliptin group were higher than those in model group(p<0.001), and the blood glucose in Isoquercitrin group and Sitagliptin group werelower than that in model group (p<0.001). The oral glucose tolerance test showedthat the Isoquercitrin significantly inhibited postprandial blood glucose excursions ina dose-dependent manner. The levels of serum GLP-1and insulin of mice inIsoquercitrin group and Sitagliptin group were higher than those in model group(p<0.001). However, the level of Triglycerides and cholesterol had not significantdifferences compared with model group. Observation of mouse islet cells by HEstaining morphology showed that, there were many full islet cells in normal, andglandular tube was very clear. In contrast those in model group mice was notcomplete and atrophied. And islet cells in Isoquercitrin groups glandular tube fulledwith those of model group, especially in high dose group, which the islet cellsrecovered to the level of normal group.
胰高血糖素样肽-1(Glucagon-like Polypep Tide1, GLP-1)是食物在肠道内的消化吸收过程中，刺激肠道内分泌细胞产生的，由30个氨基酸残基组成。GLP-1的生理功能主要包括促进胰岛素的分泌、胰岛β细胞增殖并抑制其凋亡、减缓胃的排空、抑制餐后胰高血糖素的分泌、减少肝糖元的合成、提高胰岛素的敏感性以及控制食欲等。临床研究显示，GLP-1及其类似物在2型糖尿病的治疗过程中具有重要的应用价值，其通过增加胰岛素的分泌，抑制胰高血糖素的释放调节患者体内的血糖浓度。因此，GLP-1作为一种安全、有效的促胰岛素分泌物越来越受到关注。但是，GLP-1被体内的二肽基肽酶Ⅳ(Dipeptidyl Peptidase IV, DPP-IV)降解的速度非常快，其半哀期很短，仅1～2min，内源性GLP-1分泌量不足的情况下不能积累达到激动GLP-1受体(GLP-1R)的剂量，故难以应用于临床。因此，研发一种安全、有效的DPP-IV抑制剂是目前2型糖尿病药物开发的一个新热点。
二肽基肽酶IV (Dipeptidyl Peptidase IV, DPP-IV)又称CD26，是由科学家Glenner和Hopsu-Havu在鼠肝脏组织匀浆过程中发现的一种位于细胞表面的丝氨酸肽酶(Serine Peptidase)，广泛表达于哺乳动物组织中，其催化活性中心位于胞外分子的C末端区域，DPP-IV能够特异性地裂解GLP-1的N端二肽残基：Xaa2Pro(Proline)或Xaa2Ala (Alanine)(Xaa为任意氨基酸)，是体内、外主要促使GLP-1降解、失活的关键酶之一。DPP-IV抑制剂的结构与DPP-IV的天然底物相似，含有Xαα-Pro类似结构，可以竞争性地结合DPP-IV的活化部位，但其亲和力却远大于它的天然底物，从而改变DPP-IV的构象，降低其催化活性。
2006年10月由默克公司研制成功的磷酸西格列汀(Sitagliptin)是最早获得FDA审批，并且作为DPP-IV抑制剂上市的治疗2型糖尿病的药物，该药显示出较好的降糖效果。但是磷酸西格列汀是化学合成药物，价格较昂贵，供货量少，且存在着较大的副作用，如可能出现超敏反应、肝酶升高、上呼吸道感染、鼻咽炎、过敏反应、血管性水肿和剥脱性皮肤损害、皮疹、荨麻疹和胰腺炎。因此，筛选出一种有效，经济，安全的DPP-IV抑制剂显得尤为重要。
我们以编码DPP-IV催化结构域(△DPP-IV)的cDNA质粒PMD18为模板，通过PCR反应扩增DPP-IV催化结构域基因，然后将其克隆到pGex-HisC3载体中，并将重组质粒转入大肠杆菌Rosstta DE3plysS中，加入IPTG诱导表达，最后通过亲和层析方法获得了△DPP-IV蛋白。
我们采用发色底物法建立了DPP-IV酶活性分析体系，检测纯化后的△DPP-IV酶的纯度达90%以上，活力为4.2U/mg，酶反应动力学显示在前20分钟内该酶显示了良好的线性关系。从现有的47种中药单体化合物中筛选△DPP-IV酶的抑制剂，在每种单体化合物终浓度为1mmol/L的情况下，抑制效果达到80%以上的单体化合物有两种，其中异槲皮苷对DPP-IV酶的抑制效果最好，达到了98.1%，测定其对△DPP-IV的IC50为96.8μmol/L，同时通过双倒数作图法研究发现异槲皮苷对△DPP-IV的抑制类型为竞争性抑制，抑制常数Ki为236μmol/L。
从细胞水平上检测了异槲皮苷刺激NCI-H716细胞分泌GLP-1的影响。不同浓度的异槲皮苷溶液和磷酸西格列汀溶液(10μmol/L、50μmol/L和100μmol/L)刺激人肠道内分泌细胞NCI-H716细胞系不同时间(6h、24h、48h和72h)，结果显示，随着异槲皮苷和磷酸西格列汀浓度的升高和刺激时间的延长GLP-1的分泌量逐渐增多，但异槲皮苷对GLP-1的刺激效果更明显。
在动物水平上，我们选用了8周龄、体重约20g的昆明小鼠，采用腹腔注射浓度为35mg/kg/day STZ辅以高脂高糖饲料喂养的方式诱导2型糖尿病小鼠模型的建立，连续注射STZ三天后，小鼠的血糖上升到22.43mmol/L≥11.1mmol/L，即建模成功。小鼠按体重和血糖值随机分为6组：正常组、模型组、异槲皮苷低剂量给药组(20mg/kg)、中剂量给药组(40mg/kg)、高剂量给药组(80mg/kg)和阳性药物磷酸西格列汀组(20mg/kg)，每天观察饮水量及尿量，每周记录体重及血糖浓度的变化。连续给药8周后，结果显示，从外观上来看，正常组小鼠饮食饮水尿量正常，体重增长快，皮毛有光泽，活动灵活；模型组小鼠多饮多食多尿症状明显，体重减轻，皮毛竖立无光泽，精神萎靡，反应迟钝，异槲皮苷给药组小鼠一般情况均好于模型组，其中高剂量给药组(80mg/kg)小鼠整体状况较好，多饮多食多尿症状减轻，磷酸西格列汀给药组小鼠状况良好。体重方面，模型组小鼠体重下降，异槲皮苷给药组小鼠体重与灌胃前体重有明显上升(p<0.001)，磷酸西格列汀给药组小鼠体重与灌胃前相比明显上升(p<0.001)。血糖方面，与模型组相比，给药组小鼠血糖浓度明显下降，其中高剂量组（80mg/kg)小鼠血糖浓度下降明显，下降到14.8mmol/L(p<0.001)；磷酸西格列汀给药组血糖浓度下降到17.4mmol/L(p<0.001)，说明异槲皮苷具有明显的降血糖效果。在葡萄糖耐受实验(OGTT)中，结果显示，与模型组相比，给药组血糖浓度明显降低，即中剂量、高剂量(40mg/kg、80mg/kg)异槲皮苷给药组和磷酸西格列汀给药组能明显拮抗外源葡萄糖引起的血糖升高，低剂量(20mg/kg)异槲皮苷给药组也能降低外源性葡萄糖引起的血糖升高，但并不明显，这说明异槲皮苷能够调节糖尿病小鼠体内的血糖。异槲皮苷对血清中的DPP-IV活性具有明显的抑制作用。而在连续给药8周后，与模型组相比，给药组小鼠体内的甘油三酯和总胆固醇值变化并不明显。从体内GLP-1的分泌情况来看，在未给予葡萄糖灌胃时，给药组小鼠空腹血清GLP-1浓度明显高于模型组，在给予2g/kg体重葡萄糖后的10min时，小鼠体内的GLP-1分泌量明显增多，达到峰值，其中与模型组相比，高剂量组(80mg/kg)和磷酸西格列汀组的GLP-1分泌量增多(p<0.001)。而在给予葡萄糖后的20min时，小鼠体内的胰岛素分泌量达到高峰，其中，模型组小鼠体内的胰岛素分泌偏少，异槲皮苷高剂量组(80mg/kg)和磷酸西格列汀组的小鼠体内胰岛素分泌量明显增多(p<0.001)。小鼠处死后，取胰腺组织，通过HE染色的方法观察小鼠胰岛细胞的形态。结果显示，正常组小鼠胰岛细胞数量多，腺管完整清晰可见，细胞形态饱满，胞质丰富浅染，而模型组小鼠胰岛明显萎缩，胰岛细胞不完整，细胞界限模糊；与模型组相比，异槲皮苷给药组和磷酸西格列汀组小鼠胰岛病理切片腺管部分恢复饱满充盈状态，细胞排列规则，细胞形态完整，尤其是高剂量(80mg/kg)给药组，胰腺细胞形态基本恢复到正常组水平。
Diabetes mellitus is a kind of endocrine and metabolic diseases as the maincharacteristics is high glucose. It mainly divided into type1(insulin dependentdiabetes) diabetes and type2diabetes (non insulin dependent diabetes).Approximately80%to90%of diabetes mellitus belongs to type2diabetes. Thereare many etiologies in type2diabetes, including insulin resistance with organization,decreased insulin sensitivity and insulin secretion insufficient.
Glucagon-like polypep tide1(GLP-1) is a member of vasoactive intestinalpeptide/secretin/Pancreas liters of Chitosan, which consists of30amino acidresidues. GLP-1is an incretin hormone, which is released from gut endocrine L cellsin response to food ingestion. It has many physiological functions, includingpromoting insulin secretion, inhibiting of apoptosis, slowing the emptying of thestomach, inhibiting postprandial glucagon secretion, reducing the synthesis ofhepatic glycogen, improving insulin sensitivity and controlling appetite. GLP-1isefficient in improving β cell function according to animal experiments.
According to clinical researchs, GLP-1and its analogues have a great of valuein treating type2diabetes. Therefore, GLP-1aroused more and more attention forthe safety and efficiency in promoting insulin secretion. GLP-1is degraded streamby dipeptidyl peptidase IV (DPP-IV) in the blood, the mourning period is veryshort,only1~2min. Its mourning period is too short to accumulating sufficientGLP-1to activate GLP-1R(the receptor of GLP-1). Therefore, it is difficult to applyclinic.It is a new hot spot for researching type2diabetes to find a safe, effectiveinhibitor of DPP-IV.
Dipeptide based peptidase IV (DPP-IV) called the CD26, is a kind of serinepeptidase on the cell surface, which is found in the process of homogenate byGlenner and Hopsu–Havu. DPP-IV is widely expressed in mammalian tissues,such as kidney, connective tissue, the gastrointestinal tract and lymph nodes. DPP-Ⅳ gene locats on chromosome22q24.3. Its catalytic activity center is the end of Carea located in extracellular molecules. If the second bit of the N-terminal of the peptide is Pro and Ala peptide, it is main substrate of DPP-IV. DPP-IV is a keydegraded enzymes to GLP-1in vivo or vitro by specifically cleavaging of GLP-1N-terminal dipeptide residue: Xaa2Pro or Xaa2Ala. The inhibitor is far more naturalsubstrate in binding to the enzyme. Hence, it reduces its catalytic activity bychanging the conformation of DPP-IV.Owing to the fact that inhibitor is competitivewith the natural substrate at the same structure of DPP-IV containing Xαα-Pro.
Sitagliptin is a inhibitor of DPP-IV.It has an obvious hypoglycemic effect in thetreatment of type2diabetes, which is researched by Merck at October,2006.However, Sitagliptin is expensive as a chemical synthetic drug, and there are manykinds of side effects, such as allergic reactions, elevating liver enzymes, infectingrespiratory tract, nasopharyngitis, anaphylaxis, angioedema and stripping of skindamage, skin rashes, hibes and pancreatitis. Therefore, it is important to find aeffective, economic and srcurity DPP-IV inhibitor.
The cDNAs is PMD18template, which encoding the catalytic domain ofDPP-IV (△D PP-IV), were synthesized from the polyadenylated mRNA, and therecombinant plasmid was transformed into E.coli Rosstta DE3plysS, induced byIPTG. The Catalytic Domain of DPP-IV was amplified by PCR and then thetruncated enzyme was purified by GST Resin column.
We established the detection methods for the enzyme. It is chromogenicsubstrate method. The activity of DPP-IV was4.2U/mg, and the enzyme showed alinear relationship during the first20min. There are two kinds of monomercompound detected. They had obvious inhibitory to DPP-IV, in which the inhibitoryeffect of Isoquercitrin reached98.1%. We found that Isquercitrin was a competitiveinhibitor of DPP-IV, with the half maximal inhibitory concentration (IC50) andapparent inhibition constant (Ki) were96.8and236μM.
NCI-H716cells were treated with different concentration of Isquercitrin(10μmol/L、50μmol/L and100μmol/L)or Sitagliptin at10μmol/L、50μmol/L and100μmol/L in different times (6h,24h,48h or72h).The results show that, aobvious dose-dependent increase in the secretion of GLP-1was found by Elisa Kitmethods.
20g mice were acclimated for1week and injected with35mg/kg/day STZ inorder to induce type2diabetes model. When the blood glucose was up to22.43mmol/L≥11.1mmol/L, the mice were randomly divided into six groupsaccording the body weight and blood glucose:(1)normal group,(2)model group,(3)soquercitrinlow-dose group (20mg/kg),(4) medium-dose group (40mg/kg),(5)highdose group (80mg/kg),(6) positive drug sitagliptin phosphate (20mg/kg).After8weeks, the normal group looks like very well, and the modeling mice havepolyphagia polyuria symptoms and they are unresponsive. However, theIsoquercitrin group were better than the model group. The Isoquercitrin treatmentinhibited plasma DPP-IV activity in a dose-dependent manner. The weight of micein Isoquercitrin group and Sitagliptin group were higher than those in model group(p<0.001), and the blood glucose in Isoquercitrin group and Sitagliptin group werelower than that in model group (p<0.001). The oral glucose tolerance test showedthat the Isoquercitrin significantly inhibited postprandial blood glucose excursions ina dose-dependent manner. The levels of serum GLP-1and insulin of mice inIsoquercitrin group and Sitagliptin group were higher than those in model group(p<0.001). However, the level of Triglycerides and cholesterol had not significantdifferences compared with model group. Observation of mouse islet cells by HEstaining morphology showed that, there were many full islet cells in normal, andglandular tube was very clear. In contrast those in model group mice was notcomplete and atrophied. And islet cells in Isoquercitrin groups glandular tube fulledwith those of model group, especially in high dose group, which the islet cellsrecovered to the level of normal group.
引文
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