CN104399059A - Use of antimicrobial peptide AWRK6 in preparation of drug for treating type 2 diebetes - Google Patents
Use of antimicrobial peptide AWRK6 in preparation of drug for treating type 2 diebetes Download PDFInfo
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- CN104399059A CN104399059A CN201410606268.XA CN201410606268A CN104399059A CN 104399059 A CN104399059 A CN 104399059A CN 201410606268 A CN201410606268 A CN 201410606268A CN 104399059 A CN104399059 A CN 104399059A
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Abstract
The invention relates to a use of an antimicrobial peptide AWRK6 in preparation of a drug for treating type 2 diebetes. The antimicrobial peptide AWRK6 has molecular weight of 2130.5 and a two-stage structure comprising a main structure of alpha-helix, has an isoelectric point of 9.60 and an instability index of -6.74. An experiment utilizing a mouse pancreas islet beta cell line MIN6 and a type 2 diebetes model rat as experiment materials proves that the antimicrobial peptide AWRK6 has obvious insulin secretion promoting activity, reduces blood sugar and observably promotes MIN6 cell proliferation.
Description
Technical field
The invention belongs to medicinal application field, relate generally to the application of a kind of cecropin A WRK6 in preparation treatment type-II diabetes medicine.
Background technology
The chronic hyperglycemia that diabetes are a kind of multi-pathogenesis Metabolic disorder and cause, basic pathology is because of saccharide in body, lipid, protein metabolism disorder and cause insulin secretion and dysfunction, because body is in hyperglycemia environment for a long time during trouble diabetes, can cause other organ chronic disease and dysfunctions, diabetes are killers of the third-largest threat human health after cardiovascular disease, tumor.According to IDF, expect the puzzlement that the whole world will have 3.8 hundred million people can be subject to diabetes in 2025, at present, China's diabetes prevalence occupy second place of the world.Along with people's living standard improves constantly, living-pattern preservation, type Ⅱdiabetes mellitus sickness rate increases day by day.
Research shows, two large mechanism of type Ⅱdiabetes mellitus morbidity are insulin resistant and β cell dysfunction respectively.Increasing research shows, the dysfunction of islets of langerhans particularly beta Cell of islet is the key link of type Ⅱdiabetes mellitus morbidity, this just means, insulin resistant is the factor that starts that type Ⅱdiabetes mellitus produces, but islet beta cell function obstacle is then the key factor that type Ⅱdiabetes mellitus produces, therefore research and development have pancreotropic hormone to discharge active polypeptide drug, by irritation cell excreting insulin, promote the propagation of islet cells and regeneration and regulate function of receptors on its cell membrane to become the main direction for the treatment of type Ⅱdiabetes mellitus.
Summary of the invention
The object of the present invention is to provide a kind of peptide medicament of new generation being used for the treatment of type-II diabetes.By type Ⅱdiabetes mellitus rat model, analyzed by beta Cell of islet system Min6 and rat animal model and detect novel antimicrobial peptide AWRK6 function of blood sugar reduction in vivo and in vitro and mechanism.The present invention is intended to function and the mechanism of clear and definite AWRK6 insulin secretion accelerating, is developed and becomes novel therapeutic type Ⅱdiabetes mellitus medicine.
AWRK6 (SWVGKHGKKFGLKKHKKH) is the novel antimicrobial peptide transformed according to Rana dybowskii cecropin D ybowskin-2CDYa, by 18 Amino acid profiles, molecular weight is 2130.5, it is secondary structure to utilize HNN method to predict, its structure is mainly alpha-helix, isoelectric point, IP is 9.60, and unstability index is-6.74, is the cationic antibacterial peptide with better biological stability.
The present invention, by beta Cell of islet system MIN6 and type Ⅱdiabetes mellitus rat model, detects cecropin A WRK6 insulin secretion accelerating and function of blood sugar reduction in vivo and in vitro.Show, cecropin A WRK6 can insulin secretion accelerating.Its mechanism of action is: cecropin A WRK6 causes islet cells uelralante by transferring intracellular calcium store release calcium ion.AWRK6 urgees the Epac2 protein expression that MIN6 cell mediated by cAMP and causes intracellular free calcium level to increase.
By the present invention, indicating cecropin A WRK6 can prepare the application in treatment type-II diabetes medicine.After injection cecropin A WRK6 30min, model group rat blood sugar starts to decline, and when 180min, blood sugar recovery is to normal level, and hypoglycemic effect is remarkable.The more important thing is, the hypoglycemic effect produced when AWRK6 overcomes insulin injection, experimental result shows that AWRK6 there is no blood sugar reducing function to normal rats.
The present invention, further illustrates the blood sugar reducing function of cecropin A WRK6 in vivo in model animal from animal level, is provided solid experiment basis for developing the medicine becoming a new generation's treatment type Ⅱdiabetes mellitus.
Accompanying drawing explanation
Fig. 1: cecropin A WRK6 urgees mouse islets β cell MIN6 excreting insulin result;
In figure, * * represents that difference extremely significantly (p<0.01) compared with blank group.
Fig. 2: CCK-8 method detects AWRK6 and urgees mouse islets β Cells Cell Proliferation result;
In figure, * represents significant difference (p<0.05) compared with matched group, and * * represents that difference extremely significantly (p<0.01) compared with matched group.
Fig. 3: oral glucose tolerance test (OGTT) experimental result picture;
In figure, * represents significant difference (p≤0.05) compared with blood glucose level normal, and * * represents that difference extremely significantly (p≤0.01) compared with blood glucose level normal.
After Fig. 4: Cheng Mo, rat body weight compares;
In figure, * represents significant difference (* P<0.05) compared with matched group, and * * represents that difference extremely significantly (* * P<0.01) compared with matched group
Fig. 5: cecropin A WRK6 to type Ⅱdiabetes mellitus rat model hypoglycemic effect figure;
In figure, * * represents significant difference (P<0.01) compared with model control group, and * * * represents that difference extremely significantly (P<0.001) compared with model control group.
Fig. 6: AWRK6 affects result figure to normal group rat blood sugar.
Fig. 7: calcium ion fluorescent detects AWRK6 to MIN6 intraor extracellular calcium ion concentration effect diagram.
Fig. 8: WesternBlot technology for detection AWRK6 is to MIN6 cell Epac expression of results.
Fig. 9: WesternBlot technology for detection AWRK6 is to the gray scale scanning bar diagram of MIN6 cell Epac expression of results.
Detailed description of the invention
One, AWRK6 is external falls insulin secretion accelerating and short cell-proliferation activity
1.AWRK6 on the impact of MIN6 cell insulin secretion
MIN6 cell is the cell line coming from beta Cell of islet, and In vitro culture has beta Cell of islet feature.The MIN6 cell of exponential phase will be grown to, 24 orifice plates are inoculated into the number of 105/ml after trypsinization, after cell attachment spends the night, cultivate 1-2 days, serum-free DMEM in high glucose culture medium overnight incubation, old culture medium is abandoned in suction, and add high sugared KRB buffer preincubate 30min, experimental group adds the AWRK6 of 10-6M.Collect the cells and supernatant processed by sterile tube, centrifugal 20 minutes of 2000-3000rpm, carefully collects supernatant, and ELISA detects insulin content.
The external insulin secretion accelerating result of AWRK6 is as Fig. 1, and result display AWRK6 significantly can promote the secretion of MIN6 cell insulin.
2.CCK-8 detects AWRK6 to the impact of MIN6 cell proliferation
Method: count MIN6 single cell suspension with blood counting chamber, with 4 × 104, every hole cell density, by above-mentioned cell suspension inoculation in 96 orifice plates, volume is 100 μ L.After night incubation is adherent, discard culture medium, first use PBS fine laundering once, every hole adds 200 μ L serum-free DMEM in high glucose culture medium again, carries out serum starvation 24h.
Buffer in 96 orifice plates is discarded, with the high sugared KRB of 30.6mM at 37 DEG C, 5%CO
2preincubate 30min under condition, then arranges as follows by experimental group:
In 96 orifice plates, AWRK6 group adds the antibacterial peptide 10 μ L of gradient concentration, puts into incubator, 37 DEG C, 5%CO
2hatch 12 hours and 24 hours respectively.Often group arranges three multiple holes.According to 12 hours and 24 hours point, the MIN6 cell after being hatched by variable concentrations antibacterial peptide took out, and 10 μ L CCK-8 reagent are added in every hole, continue to hatch 2 hours.Equilibrium at room temperature 5 minutes after taking out, microplate reader detects its light absorption value at 450nm place, and removal background light absorption value, with blank group for reference, is converted into percentage ratio by other each group.
CCK-8 detects Cell proliferation results as shown in Figure 2.As seen from Figure 2, add after cecropin A WRK6 compared with matched group, under 450nm, OD increases, and this illustrates that AWRK6 has proliferation to MIN6 cell, after wherein hatching 12 hours, and 10
-7m concentration AWRK6 effect is extremely remarkable.
Above two experimental results show, it is active that AWRK6 has obvious insulin secretion accelerating, and can significantly promote MIN6 cell proliferation, imply that this peptide is expected to be developed to the Cenozoic antibacterial peptide medicament of type-II diabetes very much.
Two, AWRK6 is to the hypoglycemic activity of type-II diabetes rat model
By Wistar rat animal housing 25 DEG C, adaptability is raised one week, and divide cage by rat at random, every four is a cage, and weighs up body weight, is divided into normal group (4) and type Ⅱdiabetes mellitus model group (model group 20).According to the method for generally acknowledging, streptozotocin (STZ) is adopted to build type-II diabetes rat model, and oral glucose tolerance test (OGTT) is carried out to rat model, blood glucose value blood glucose after oral glucose of two groups of rats all raises, wherein normal group is raised to peak value from 15min, and reach maximum when 30min, from 60min, blood glucose declines gradually, and is returned to normal level at 120min.And diabetic model group rat blood sugar is raised to from 15min until 120min does not decline yet, illustrate that it declines greatly to sugared tolerance, glucose metabolism in vivo does not cause the release of insulin, shows modeling success (see Fig. 3).
Observe from morphology in addition, type Ⅱdiabetes mellitus model group rats lethargy, hair color is dark yellow and occur " three-many-one-little " phenomenon that polyphagia polydipsia body weight obviously declines.Within every two days, measure a rat body weight, Measuring Time continues 16 days.As shown in Figure 4, model group rats is compared with rats in normal control group, and body weight significantly declines for the result that after Cheng Mo, rat body weight compares.
1. cecropin A WRK6 analyzes type Ⅱdiabetes mellitus rat model hypoglycemic effect
Choose the successful Wistar rat of modeling, every 6 is one group, chooses two groups and carries out the experiment of medicine blood sugar lowering.Overnight fast 12 hours before experiment, and detect its fasting blood sugar, be recorded as 0min blood glucose value.
Setup Experiments is three groups, is respectively:
AWRK6 group: be dissolved in 0.9% normal saline with 100nmol/kg.bodyweight by AWRK6, lumbar injection volume is 5ml/kg.bodyweight.
Positive controls: Exenatide injection, lumbar injection dosage is 1 μ g/kg.weight, and injection volume is 5mL/kg.weight.
Blank group: only lumbar injection 0.9% normal saline.
Respectively at 15min, 30min, 45min, 60min, 90min, 120min, 150min, 180min after injection, tail vein gets blood, detects its blood glucose value.
As shown in Figure 5, antibacterial peptide group blood glucose starts to decline at 30min hypoglycemic effect, and reaches normal level at 150min; Positive controls blood glucose declines at 30min and reaches normal level.Blank group continues to detect 180min blood glucose and there is no significance reduction.
The detection that 2.AWRK6 affects normal group rat blood sugar
The antidiabetic drugs of current use has hypoglycemic effect can be made diabetics take to occur hypoglycemia phenomenon, as insulin injection, whether sulfonylurea drugs, thus to normal group rats by intraperitoneal injection 100nmol/kg.bodyweight AWRK6, have impact to detect AWRK6 to euglycemia.
The testing result that AWRK6 affects normal group rat blood sugar is as Fig. 6, after the AWRK6 of visible normal rats lumbar injection 100nmol/kg.bodyweight, blood glucose value in 180min, compares with 0min and there is no significant difference, illustrates that AWRK6 there is no blood sugar reducing function to normal rats.
Indicate the hypoglycemic effect of cecropin A WRK6 in animal level by above-mentioned experiment, animal level, confirm further the blood sugar reducing function of cecropin A WRK6 in vivo in model animal.Showing that AWRK6 still has insulin secretion accelerating and hypoglycemic activity in vivo by experiment, being provided solid experiment basis for developing the medicine becoming a new generation's treatment type Ⅱdiabetes mellitus.
3.AWRK6 urgees MIN6 cells secrete insulin mechanism
3.1 calcium ion fluorescents detect AWRK6 to be affected MIN6 intraor extracellular calcium ion concentration
Fluo-3 AM be a kind of can the fluorescent dye of permeates cell membranes.The fluorescence of Fluo-3 AM is very weak, and its fluorescence intensity can not raise with calcium ion concentration and strengthen.Fluo-3 AM is just sheared by intracellular esterase after entering cell and forms Fluo-3, thus is trapped in cell.Fluo-3 can and calcium binding, can produce stronger fluorescence in conjunction with after calcium ion, detection excitation wavelength is 488nm, and emission wavelength is 525-530nm.
As shown in Figure 7, visible cecropin A WRK6 concentration is 5 × 10 to result
-6during M, comparing with high glucose concentration (30.6mM) with normal glucose concentration (25mM) and obviously can observe intracellular calcium ion fluorescence signal and strengthen, illustrating that AWRK6 realizes short MIN6 excreting insulin function by increasing intraor extracellular calcium ion concentration.
The impact that 3.2 WesternBlot technology for detection AWRK6 express MIN6 cell Epac
There is GLP-1R on islet cells film surface, GIPR and PACAPR tri-kinds of receptors, after receptor activation, is all activated Epac and is expressed by second message,second messenger cAMP and bring out intracellular calcium and increase, realize promoting the release function of insulin.We adopt general WesternBlot technology, have detected the impact that AWRK6 expresses MIN6 cell Epac, and result shows, and after AWRK6 acts on 1 hour, the Epac expression of MIN6 cell obviously raises (Fig. 8 and Fig. 9).
The present invention with mouse islets hybridoma cell line MIN6 for experiment material, by ELISA and CCK-8 method, prove that cecropin A WRK6 has short Islet Cells Insulin secretion and short cell proliferation is active, and tentatively illustrate AWRK6 by bringing out Epac expression rising, calcium ion concentration in elevate cellular, realizes the molecular mechanism of pancreotropic hormone release.In addition by type Ⅱdiabetes mellitus rat model, prove that cecropin A WRK6 has function of blood sugar reduction in vivo by zoopery, and on normal rat blood sugar without impact.
Claims (5)
1. the application of cecropin A WRK6 in preparation treatment type-II diabetes medicine.
2. apply as claimed in claim 1, it is characterized in that: cecropin A WRK6 promotes the application in insulin secretion medicine in preparation.
3. apply as claimed in claim 2, it is characterized in that: cecropin A WRK6 promotes the application in beta Cell of islet hyperproliferation agent in preparation.
4. apply as claimed in claim 3, it is characterized in that: described beta Cell of islet is MIN6 cell.
5. the application as described in as arbitrary in claim 1-4, is characterized in that: the sequence of described cecropin A WRK6 is SWVGKHGKKFGLKKHKKH.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109030804A (en) * | 2018-08-16 | 2018-12-18 | 青岛大学 | The insulin secretion rating model of glucose stimulation |
| CN114045258A (en) * | 2021-10-21 | 2022-02-15 | 辽宁盛京干细胞科技有限公司 | Serum-free medium for mesenchymal stem cell culture and application thereof |
| CN116549602A (en) * | 2023-05-24 | 2023-08-08 | 广州雄韬生物医药科技有限公司 | Application of AWRK6 polypeptide in preparation of psoriasis treatment drugs |
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| CN103772507A (en) * | 2013-09-11 | 2014-05-07 | 辽宁大学 | Fusion antibacterial peptide, as well as preparation method and application thereof |
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| CN103772507A (en) * | 2013-09-11 | 2014-05-07 | 辽宁大学 | Fusion antibacterial peptide, as well as preparation method and application thereof |
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| JIN L ET AL: "expression of antimicrobial peptide dybowskin-2CAMa in pichia pastoris and characterization of its antibacterial activity", 《ADVANCE JOURNAL OF FOOD SCIENCE AND TECHNOLOGY》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109030804A (en) * | 2018-08-16 | 2018-12-18 | 青岛大学 | The insulin secretion rating model of glucose stimulation |
| CN114045258A (en) * | 2021-10-21 | 2022-02-15 | 辽宁盛京干细胞科技有限公司 | Serum-free medium for mesenchymal stem cell culture and application thereof |
| CN114045258B (en) * | 2021-10-21 | 2024-04-16 | 辽宁盛京干细胞科技有限公司 | Serum-free culture medium for mesenchymal stem cell culture and application |
| CN116549602A (en) * | 2023-05-24 | 2023-08-08 | 广州雄韬生物医药科技有限公司 | Application of AWRK6 polypeptide in preparation of psoriasis treatment drugs |
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