| 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. |
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