| Background and Objectivediabetes mellitus, DM, is a syndrome from various pathopoiesises including hereditary factor, immunity dysfunction, bacterial infection combined with its toxin and free radical toxin that result in the functional recession of islet and the resistance of islet, which lead to disorders of glucose, protein, fat, water and electrolyte. It’s typical in hyperglycemia with diuresis, polydipsia, polyphagia and weigh losing.Recently, the provenance of diabetes in China has been rising with the developing economic condition, enhancing living standard, changed diet structure and decreased labor intensity, which aggravates the health gradually. In the "international diabetes forum" of2009that was hold by Ministry of Health and Word Diabetes Foundation, it was reported that the number of diabetic patients in China was the second largest, followed by the number in India, with an astonishing increasing.Type Ⅱ diabetes is a chronic disease of complex and polygene hyperglycemia results in the lack secretion of β cells accompany insulin resistance, the interaction between gene and environmental factors. It’s estimated that the heritability rate of Type Ⅱ diabetes reaches70%-80%, the essential reason of pathogenesis. Furthermore, the morbidity varies in the population, from5%in the white to50%in the Asian.The occurrence of type Ⅱ diabetes is mostly after the age from35to40, accounting for more than90%. Insulin can be secreted by the oral administration. The short-term target is to control the level of blood sugar and the long-term is to prevent from the occurrence and the development of its complication. The basic therapy consists mainly of exercise and diet when it’s crucial for the pharmacotherapy and the monitoring of blood sugar.The conventional medicine of type Ⅱ diabetes divide into sulfonylurea, biguanide, thiazolidinedione (euglycemic agent) and α-glucosidase inhibitor.Novel anti-diabetic of different mechanism from all kinds of pathogenesis have come out to the market recently as the further study of molecular biology and the diabetic pathogenesis, which give the various therapy to the diabetes, and DPP Ⅳ inhibitor is of this kind.Dipeptidyl peptidase Ⅳ (DPP Ⅳ or DPP-4; EC3.4.14.5) is a prolyl peptidase which preferentially cleaves proteins and peptides after a proline amino acid residue. DPP Ⅳ is commonly characterised by an ability to cleave Xaa-Pro or Xaa-Ala dipeptides preferentially from the N-terminus of polypeptides (where Xaa is any amino acid except Pro).Since the definition of incretin has been clear and further study in incretin has focus on, the protection and proliferation of β cells, the increased secretion of insulin and the secretive inhibition of glucagons have found favor in the researchers. However, the main elements of incretin, glucagons-like peptide-1(GLP-1) and glucose-dependent insulinotropic peptide (GIP) are vulnerable to degradation by the DPP Ⅳ so that the half-life period of incretin is short which is hard to be active biologically. Hence, the study of DPP Ⅳ inhibitor has become a hit point in the diabetic therapy. So far, the study has reached progress, and various kinds of macromolecule of DPP Ⅳ inhibitor has been emerging, such as sitagliptin phosphate (Januvia), making it a promising strategy in type Ⅱ diabetes treatment.At present, the study of DPP Ⅳ is deeper abroad. Dominantly, DPP Ⅳ is extracted from Caco-2cells and the animal’s liver and kidney as constructed selected model of DPP Ⅳ inhibitor by the reaction with specific substrate, Gly-Pro-pNA. By contrast, the study of this selected model is limited. In the paper, we aim to establish a convenient, fast and effective selection model of DPP Ⅳ inhibitor to select potential inhibitor.The selected model in the paper is the optimal from the present model has established in the laboratory, of the concentration of cell crush, of substrate, pH and the time of incubation. Orthogonal experiment was used to evaluate the three conditions and enzyme specific activity as index. A more economic and effectively model was applied from the orthogonal result to the selection of a pile of compounds whether they have inhibitory activity from DPP Ⅳ. The Z’factor and the signal to noise ratio (S/N) to evaluated the model were calculated by the experiment sitagliptin as positive control and distill water as negative control instead of sitagliptin. Different temperature were set including-20℃,4℃,25℃,37℃and in liquid nitrogen and deposited in different period, respectively to estimate the activity of DPP Ⅳ in cell crush. Gradient concentration of DMSO, alcohol and ethyl acetate from1%to30%dissolved the cell crush before testing the activity of DPP Ⅳ. The inhibitory type of compounds, JD-1and JD-1was judged by the comparison with the kinetic parameters of enzyme, Km and v max; a set of compound calculated by the model containing activity to DPP Ⅳ was selected.Methods1. the extraction of DPP Ⅳ from Caco-2cells, the determination of protein concentrationCaco-2cell was cultured in DMEM medium with10%fatal calf serum,1%non-essential amino-acid,1%glutamine. The cells were crushed by multigelation; supernatant was obtained after centrifugation with-70centigrade deposition. Bradford assay was conducted for the examination of protein concentration of the cell crush. A total of10tubes was tested, from which one is standard protein of1.00mg·mL-1bovine serum albumin (BSA) and nine is the combination of different volumes with distill water, reagent of coomassie brilliant blue and cell crush. Standard curve was drawn by the standard protein concentration (mg·mL-1) as abscissa, the value of OD595as vertical coordinate from which the protein concentration of cell crush was obtained.2. Orthogonal experimentThe orthogonal experiment was taken with four factors which contained three levels to measure the optimal conditions. The factors were divided into the cell crush100%,70%and50%, the substrate (GLy-Pro-pNA) concentration were100μM,200μM,300μM, pH were8.3,8.0,9.0and the incumbent time were15min,30min and60min. The value of OD in405nm was tested when the ration time reached and the enzyme specific activity (μmoL·min-1·g-1) was the index estimated the result of orthogonal experiment3. The factor Z’and the single to noise ratio (S/N)The experiment of testing the factor Z’and S/N was arranged by sitagliptin with100μM as positive control and distill water as negative control replaced sitagliptin. A total of twenty times repeated determination with the value of OD in405nm after fiftern minutes incubanation in37centigrade. The value of factor Z’ and S/N were calculated by the equation.4. The affect of different concentration of DMSO, ethanol and ethyl acetate to the activity of DPP ⅣThe affect in the selected model was evaluated by a set of gradient concentration added in the model,1%,5%,10%,15%,20%,25%and30%. Distil water as negative control and the inhibitory rate was measured by regression analysis as data were showed in Mean±SD, Microsoft Office Excel2003statistic software after fifteen minutes incubation in37centigrade5. The affects of reserved period at37℃,25℃,4℃,-20℃,-70℃and in the liquid nitrogen to the activity of DPP Ⅳ respectivelyThe cell crush was divided into three groups, of which37℃,25℃as a group one,4℃a group two and the left including liquid nitrogen,-70℃and-20℃a group three. The OD value of group one was tested every two hours and at the twentieth hour, six times totally. The OD value of group two was tested on the second day, every two days and the thirtieth day, nine times totally. The OD value of group three was tested very other week, six times totally.6. The measurement of DPP Ⅳ kinetic parameters (Km, v max)The reciprocals of initial concentration of substrate from different substrate concentrations and the initial velocities,1/[S] as horizontal ordinate,1/v as vertical coordinate were drawn regression curve, and calculate the values of Km, v max by the regression equation.7. The inhibitory judgment of JD-1, JD-2A total of six concentrations of JD-1, JD-2was prepared and the initial velocities were calculated by adding different concentrations of substrate. The initial velocities and the initial concentrations of substrate from different substrate concentrations at the same concentrations of JD-1, JD-2respectively were dawn a regression curve by1/[S] as horizontal ordinate,1/v as vertical coordinate. The values of Km’ and v max’ were calculated from the regression equation; the inhibitory property of JD-1and JD-2was judged by comparing Km’ and v max’ with Km and v max and the value of Ki was calculated by the equation.8. The estimation of inhibitory rate on the testing compoundsThe testing compounds and the positive control, sitagliptin were dissolved in3%ethanol with3%ethanol as negative control and the three-duplicate test. The inhibitory rate was calculated by the value of OD405nm after one hour ice-bath and fifteen-minute incubation. Results1. The result of orthogonal experiment showed that the optimal conditions were50%cell crush,300μM substrate concentration, pH8.3and fifteen-minute incubation. Of these conditions, the most effective was the substrate concentration; the least was pH with a sequence of B>D>A>C. There was significant difference (P<0.05) in each factor and level.2. The values of Z’and S/N evaluated the selected model were0.775and16.411, respectively. the value of Z’is over than0.5, S/N over than5.3. the effects of DMSO, ethanol and ethyl acetate to the activity of DPP IV in the selected model. The result showed that when the concentration of the three organic solvents up to30%, the activity of DPP IV reduced to77.06%,74.87%and75.58%. According the statistic analysis, there were significant difference between1%and the concentrations up to15%.4. the effect of different centigrade in which deposited different periods to the stability of DPP IV. There was significant difference in the reducing of the activity between the0-hour group and the hour groups after six hours in25℃癈and37℃and between the0-day group and the groups after three days in4℃. In the deposited six-week, the activity of DPP IV decreased to67.5%,60.0%and62.0%in the groups of liquid nitrogen,-70℃and-20℃respectively and there was significant difference between liquid nitrogen and-70℃,-20℃.5. The estimation of inhibitory property to JD-1and JD-2was held. The values of Km and Vmax were100.7μM and2.105μM·min-1. The gradients (Km/Vmax and1/Vmax) of JD-1, JD-2and sitagliptin from Lineweaver-Burk plot were rising with the increasing concentrations which showed that they were competitive inhibitors.6. The inhibitory rates of the testing compounds indicated that the rates of WL-01and W1-04with the1X106nM concentration were63.51%±0.98%and 73.18%±1.53%, over than50%.ConclusionsIt was show that the model was accurate, repeatable and stable which is suitable for high throughput when the values of Z’factor and the S/N were over than0.5and5, respectively by using the conditions from the result of orthogonal experiment. It is optimal using the concentration lower than15%of DMSO as solvent from the experiment result. It’s more stable putting at37℃than25℃according to the statistic analysis since it is less effectiveatn37℃; the reducing effect to DPP IV after thirty days deposited at4℃presents that it’s suitable not in a long period at this centigrade; the three centigrades of liquid nitrogen,-70℃and-20℃leads to the reducing effect to DPP IV and it’s the least effective at-70℃. The value of Km is100.7μM from the experiment which is approximate to the value reported. From the Lineweaver-Burk plot, JD-1, JD-1and sitagliptin are competitive inhibitors, the same result of sitagliptin reported. There are two compounds, WL-01and WI-04which are over50%inhibitory rate in the concentration of1×106nM from the selected experiment. |
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