CN107034264A - A kind of method and hypoglycemic medicine for obtaining hypoglycemic medicine - Google Patents
A kind of method and hypoglycemic medicine for obtaining hypoglycemic medicine Download PDFInfo
- Publication number
- CN107034264A CN107034264A CN201710310470.1A CN201710310470A CN107034264A CN 107034264 A CN107034264 A CN 107034264A CN 201710310470 A CN201710310470 A CN 201710310470A CN 107034264 A CN107034264 A CN 107034264A
- Authority
- CN
- China
- Prior art keywords
- medicine
- islet
- insulin
- hypoglycemic
- ability
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/507—Pancreatic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Abstract
The invention discloses a kind of method and hypoglycemic medicine for obtaining hypoglycemic medicine, it is related to medicine and hygiene fieldses, the main insulin secreting ability and insulin resistance ability for making the beta Cell of islet systems of early stage INS 1 and laboratory rodent by suppressing inflammation of the invention improves to realize the reduction of blood glucose, the medicine containing p38 MAPK inhibitor SB203580 compositions is particularly used to realize, the hypoglycemic medicine that the present invention is provided can be prepared for nursing one's health, delay and/or treat the following application in the medicine or health products of diabetes, p38 MAPK inhibitor SB203580 can significantly improve the insulin secreting ability of glycosuria patient's beta Cell of islet and improve insulin resistance, reduce the blood sugar level of glycosuria patient, by suppressing inflammation, delay and improve the development of diabetes, in treating diabetes, there is high application prospect in conditioning.
Description
Technical field
It is used to reduce medicine and its application of blood glucose the present invention relates to a kind of, more particularly to a kind of p38MAPK inhibitor is used
Medicine and its application in reduction blood glucose.
Background technology
Diabetes (diabetes mellitus, DM) are due to clinical caused by h and E factor interaction
Syndrome, is one of high incidence, the principal endocrine disease of high fatal rate of disabling.According to the difference of pathogenesis, diabetes
Mainly include type 1 diabetes and diabetes B.More than the 90% of diabetic population is accounted in China's diabetes B, it is due to pancreas
The clinical syndrome being characterized with long term hyperglycemia caused by the relative shortage of island element and insulin resistance.Chronic hyperglycemia can cause
Various Tissues, particularly eye, kidney, nerve, cardiovascular long-term damage, functional defect and exhaustion.Clinically occur more than three (many
Food, many drinks, diuresis), few (Body weight loss) classical symptom.
Islet beta cell function is damaged and insulin resistance is two important physiological pathology mechanism of diabetes B.It is more and more
Research confirm that islet beta cell function progressive deteriorates or exhaustion is key that prediabetes is changed into diabetes, is glycosuria
The necessary links of disease progression.
P38 (also referred to as CSBP or RK) is a kind of serine/threonine mitogen-activated protein kinase (MAPK), research
Have proven to the effect with regulation Pro-inflammatory mediator.P38 has found in mouse monokaryon cell first, junket in its molecule
Propylhomoserin can be by a kind of kinases of lipopolysaccharides (LPS) phosphorylation.Saklatvala J. et al. (Cell, 781039-1049,1994)
Contacting between the response of p38 and the cell by cell factor is determined first, it was demonstrated that IL-1 activated protein kinases cascades, it is led
Small heat shock protein Hsp27 phosphorylation is caused, this may be by MAPK protein kinase 2 (MAPKAP kinases -2) to realize
's.Han, J. et al., (Science, 265808-811,1994) carries out sequence analysis confirmation by the peptide chain of the kinases to purifying
It is relevant with the p38MAPK of the LPS activation in mouse monokaryon cell.While Rouse, J. et al. (Cell, 781027-1037,
1994) prove p38MAPK in itself response various kinds of cell stress, including contact ultraviolet radiation and heat shock in by upstream kinases
Activated, and determine that MAPKAP kinases -2 is the kinases of Hsp27 phosphorylations.Then, Smith Kline Beecham worker
Lee, J. et al., Nature, 372739-746 prove that p38MAPK is a series of molecular target of Pyridinylimidazoles compounds, lead to
Cross the generation that Pyridinylimidazoles compound suppresses the TNF for the person monocytic cell that LPS is excited.This is a critical discovery, is led
Cause the exploitation of many p38MAPK selective depressants.
P38MAPK activation can stimulate Monocytes/Macrophages to express TNF (Tumor necrosis
Factors, TNF), interleukin (Interleukin, IL) -1, IL-6, IL-8 and cyclo-oxygenase (cyclo-oxygenase,
COX) -2, IL-8 synthesis, oxidative stress and elastin laminin enzyme r e lease in regulation and control neutrophil leucocyte, regulation and control TNF and lipopolysaccharide mediate
Endothelial cell COX-2 expression, prostaglandin synthesis and E-Selectin expression.Generation except promoting inflammatory factor, p38 can
The inflammatory aggregation of mediating neutrophil and chemotactic, are to play a part of promoting inflammation in the startup stage of inflammation, this
What effect was mediated after being activated with p38 downstream molecules MAPK activator proteins 2 (MAPK-activated kinase, MAPKAP2)
HSP27 phosphorylations are relevant.In areas of inflammation the p38 of neutrophil leucocyte can also by nadph oxidase play promote active oxygen and
The effect of complement generation.
P38MAPK inhibitor SB203580 (can also write SB 203580 or SB-203580) synthesis is earliest, chemically
Matter is relatively stablized, and commercialization is more ripe, it is easy to obtain, so being most widely used in scientific research.SB203580 English
Entitled (4- (4-Fluorophenyl) -2- (4-methyl sulfinylphenyl) -5- (4-pyridyl) -1H-
Imidazole), Chinese is 4- (4- fluorophenyls) -2- (4- methylsulfinylphenyls) -5- (4- pyridine radicals) -1H- miaows
Azoles, its molecular structural formula is as follows：
SB203580 molecular weight is 377.43, and molecular formula is C21H16N3FOS, CAS Number：152121-47-6.This production
Product purity is more than 99%.
SB203580 energy selective depression p38MAPK, IC50 are 600nM；For JNK/SAPK and p44/42MAPK (i.e.
Erk1/2) without significant inhibitory action, IC50 is only 100 μM.
SB203580 can play the effect for suppressing p38MAPK in the cell through cell membrane, suppress follow-up MAPKAP
The effect of Kinase-2 and MAPKAP Kinase-3 activation, so as to effectively suppress some inflammatory factors (such as IL-1 β,
TNF-α) induction part signal transduction.Important regulation due to p38MAPK to proinflammatory inflammation factor (IL-1 β, TNF α and IL-6)
Effect, p38MAPK is in the chronic inflammatory diseases such as rheumatoid arthritis, Crohn disease, ankylosing spondylitis and chronic asthma
Effect turns into the new focus studied recently.P38 inhibitor treats chronic inflammatory diseases by blocking p38 paths to suppress inflammatory development
Possibility be also widely studied and report.
The treatment of diabetes mainly has self-management, blood sugar monitoring, dietetic treatment, kinesiatrics and drug therapy, medicine
Treatment includes insulin and the like and oral hypoglycemic drug.In general, if OHA can not make blood glucose control
System is up to standard or has serious acute or chronic complication, then will be treated with insulin or the like.Insulin type is
Treat the only effective treatment method of type 1 diabetes.The medicine for the treatment diabetes B delivered in the market has：(1) pancreas
Island element promotees secretion medicine, such as sulfonylurea drugs, meglitinide；(2) biguanides, such as melbine；(3) alpha-glucosidase inhibitor,
Such as acarbose；(4) Studies of The Insulin Sensitizer Thiazolidinediones, such as Pioglitazone；(5) it is new：Such as GLP-1 receptor stimulating agents and
DPP4 inhibitor, SGLT2 receptor blocking pharmacons.Wherein sulfonylurea drugs are one of more common medicines for the treatment of diabetes, and it leads to
The acceptor promotion insulin releasing for acting on beta Cell of islet film surface is crossed, its hypoglycemic effect, which is depended on, to be remained a great deal of
(more than 30%) functional beta Cell of islet.Do not find that any drug therapy diabetes B patient can be from pathogenesis at present
Upper basic improvement and protection islet beta cell function and increase target tissue delay disease to develop the sensitiveness of insulin.
One (comes from the insulinoma of Induced by Radioactive Ray, with higher insulin point to mouse RINm5F cell lines
Secrete rate) culture studies find that IL-1 β and IFN-γ joint can promote β Apoptosis, and confirm this apoptosis-promoting effect
At least partly it is to be mediated through inducible nitric oxide synthase (iNOS).Xu Jing et al. research shows p38MAPK inhibitor
SB203580 can mitigate mice pancreatic tissue damage by suppressing inflammatory reaction.Kim et al. research is then shown in C2C12 bones
In bone myocyte system, p38MAPK inhibitor SB203580 can significantly inhibit the intake of glucose, point out p38MAPK take part in outer
All insulin sensitivity regulations.
Whether SB203580 can improve the blood sugar level of diabetic individual, delay to have no relevant report in terms of diabetes de-velopment
Road.
The content of the invention
The purpose of the present invention be directed to existing Course of Diabetes Treatment in medication exist technological deficiency there is provided a kind of glycosuria
The novel targets of disease treatment, by suppressing inflammation, delay and improve the development of diabetes.Present invention observation p38MAPK inhibitor
SB203580 beta Cell of islet is survived and apoptosis influence, and SB203580 to the improvement of diabetes animal model blood glucose with
And mechanism is studied, so as to provide a kind of hypoglycemic medicine using p38MAPK inhibitor SB203580 as main component, Ke Yiyong
In delaying and/or treat diabetes.
To realize the technical purpose of the present invention, first aspect present invention provides a kind of method for reducing blood glucose, and it passes through suppression
Inflammation processed makes the insulin secreting ability of beta Cell of islet and the improvement of insulin resistance ability realize the reduction of blood glucose.
Particularly, it is described to improve beta Cell of islet insulin secreting ability and body insulin resistance energy by suppressing inflammation
Power is realized using the medicine containing p38MAPK inhibitor SB203580 compositions.
Especially, the method for the reduction blood glucose can be used for nursing one's health, delay and/or treating diabetes.
Wherein, the diabetes are type 1 diabetes, diabetes B, secondary diabetes.Especially diabetes B.
To realize the technical purpose of the present invention, second aspect of the present invention provides a kind of method for obtaining hypoglycemic medicine, including：
Search the inflammation with suppressing inflammation-related and suppress medicine；
From the inflammation of all inflammation-relateds suppress medicine in filter out with improve beta Cell of islet insulin secreting ability or/
The drug target relevant with insulin resistance ability；
According to the drug test result carried out to the drug target, the reduction blood glucose is selected from the drug target
Medicine.
Particularly, it is p38MAPK inhibitor SB203580 that the reduction hypoglycemic medicament is selected from the drug target.
Particularly, it is to include p38MAPK inhibitor that the reduction hypoglycemic medicament is selected from the drug target
The medicine of SB203580 compositions.
Wherein, according to the result of the drug test carried out to the drug target, selected from the drug target described
Reduction hypoglycemic medicament includes：
Drug target is subjected to cell and zoopery respectively, so that it is thin to judge whether drug target can improve pancreas islet β
The insulin secreting ability of born of the same parents system, if improve the blood sugar level of diabetes animal model and whether by improving β cell work(
What energy and insulin resistance were realized.
Moved if experimental result display target medicine can improve the insulin secreting ability of beta Cell of islet system, diabetes
Blood sugar level, β cell functions and the insulin resistance of thing model, then judge the medicament can as reduction blood glucose medicine.
To realize the technical purpose of the present invention, third aspect present invention provides a kind of hypoglycemic medicine, with p38MAPK inhibitor
SB203580 is main component, including pharmaceutically acceptable carrier and/or excipient.
Pharmaceutically acceptable carrier generally by sanitarian approve for this purpose and as medicament it is nonactive into
Point.Can be about the compilation of pharmaceutically acceptable carrier《Handbook of pharmaceutical excipients》(Handbook of
Pharmaceutical excipients, second edition is edited by A.Wade and P.J.Weller；American
Pharmaceutical Association are published, Washington and The Pharmaceutical Press,
London, 1994) etc. find in reference book.
Described carrier includes excipient, such as starch, water；Lubricant, such as magnesium stearate；Disintegrant, such as crystallite are fine
Tie up element etc.；Filler, such as lactose；Binding agent, such as pregelatinized starch, dextrin；Sweetener；Antioxidant；Preservative, flavoring
Agent, spices etc.；
The hypoglycemic medicine be by through gastrointestinal administration and it is non-through gastrointestinal administration approach be administered.Described is non-through stomach and intestine
Canal drug administration strategy and suggestion drug administration by injection, respiratory tract administration, percutaneous drug delivery, mucosa delivery or cavity/canal drug administration.
The hypoglycemic medicine exists in forms such as tablet, capsule, patches.
It is non-to select injection, spray, aerosol, patch etc. through gastrointestinal administration medicament；Selected through gastrointestinal administration preparation
Select tablet, capsule, powder, granule, pill, solution or syrup etc..
Medicine of the present invention consisting of the compound p38MAPK inhibitor SB203580 containing treatment effective dose,
The compound can be single use or be used in combination with other drugs in medicine.Other preventions or/and treatment
The medicine of diabetes especially diabetes B includes but is not limited to following medicine：Such as melbine, insulin.
Wherein, the medicine type of hypoglycemic medicine of the invention can be prepared by disclosed method in the prior art.
To realize the purpose of the present invention, fourth aspect present invention provide it is a kind of by hypoglycemic medicine preparing conditioning, delay and/
Or the application for the treatment of diabetes medicament.
Fifth aspect present invention provides a kind of hypoglycemic medicine and is preparing improvement diabetic's beta Cell of islet insulin secretion
Application in ability medicine.
Sixth aspect present invention provides a kind of hypoglycemic medicine in improvement diabetic's insulin resistance ability is prepared
Using.
Compared with prior art, the present invention has following obvious advantage：
1st, the present invention makes beta Cell of islet insulin secreting ability and insulin resistance ability improve come real based on inflammation is suppressed
The thought of existing blood glucose reduction, discovery is a kind of can to reduce the medicine of blood glucose, and the medicine is with commercially available compound p38MAPK suppression
Agent SB203580 is main component, and R＆D costs are low, pervasive to answer height.
2nd, the present invention has excavated new medical value to known compound p38MAPK inhibitor SB203580, uses it for
Delay and/or treat diabetes, improve beta Cell of islet insulin secreting ability and improve diabetic's insulin resistance, drop
Low blood sugar in diabetic patients, and the medicine or health food for delaying and/or treating diabetes can be prepared into, so as to be known chemical combination
A new field has been opened up in thing p38MAPK inhibitor SB203580 application.
3rd, campaign research of the invention have shown that p38MAPK inhibitor SB203580 have significant treatment diabetes,
The effect of blood glucose is reduced, with improvement beta Cell of islet insulin secreting ability.
4th, p38MAPK inhibitor SB203580 of the invention can be prepared as oral formulations, injection and use, its pharmacological action
By force, notable for preventing, nursing one's health and treating effect of diabetes, instant effect, toxic side effect are small, security is good, can take for a long time
With, and its hypoglycemic mechanism of action can be analyzed with modern pharmacology, with good prospect in medicine.
Brief description of the drawings
Fig. 1 is tetra- kinds of subtype protein expression of results figures of p38MAPK in the embodiment of the present invention 1 under different condition of culture, wherein
1A is control group, and condition of culture is that glucose 11.1mmol/L, 1B are high sugared group, and condition of culture is glucose 25mmol/L, 1C
For high fat group, condition of culture is palmitic acid 0.2mmol/L, and 1D is high glucose and high fat group, and condition of culture is glucose 25mmol/L, palm fibre
Palmitic acid acid 0.2mmol/L, 1E are cell factor stimulation group, and condition of culture is IL-1 β 5ug/ml, IFN-γ 5ug/ml, glucose
11.1mmol/L, 12H, 24H, 48H are different incubation times；
Fig. 2 is level of insulin secretion result figure in the embodiment of the present invention 2 under different incubation times；
Fig. 3 is the different week old fasting blood-glucose curves of each group mouse in the embodiment of the present invention 3；Wherein, FPG represents fasting blood-glucose
Concentration；Con groups represent normal control C57 mouse；Dmi groups represent db/db mouse SB203580 gavage groups；Dmo groups represent db/db
Mouse positive controls,
Fig. 4 is the blood glucose curve figure of the IPGTT experiments of each group mouse in the embodiment of the present invention 3, and wherein Dmi groups are db/db
Mouse SB203580 gavage groups；Dmo groups are db/db mouse distilled water positive controls.4A is 7 week old (before pharmaceutical intervention) mouse
IPGTT experiment blood glucose curve figure；The blood glucose curve figure that figure B tests for the IPGTT of 12 week old (pharmaceutical intervention 5 weeks) mouse；
The blood glucose curve figure that 4C tests for the IPGTT of 14 week old (pharmaceutical intervention 7 weeks) mouse；4D is that 16 week old (pharmaceutical intervention 9 weeks) are small
The blood glucose curve figure of the IPGTT experiments of mouse；
Fig. 5 is the Area under the curve of blood glucose figure of different week old mouse IPGTT experiments, wherein：AUC is represented below blood glucose curve
Product, Dmi represents db/db mouse SB203580 gavage groups, and Dmo represents db/db mouse distilled water control groups；
Fig. 6 compares figure for the HOMA- β of different week old control groups and experimental mice, wherein, Dmi groups are db/db mouse
SB203580 gavage groups；Dmo groups are db/db mouse distilled water control groups.P between #Dmi and Dmo groups<0.05；
Fig. 7 compares figure for the HOMA-IR of different week old control groups and experimental mice.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.But these embodiments be only limitted to explanation the present invention without
For limiting the scope of the present invention.The experimental method of unreceipted specific experiment condition in the following example, generally according to conventional strip
Part, or according to the condition proposed by manufacturer.
The beneficial effect of medicine of the present invention is expanded on further below by way of test example, these test examples include this
The pharmacodynamics test of invention medicine.
P38MAPK inhibitor SB203580 used in the present invention is purchased from LC Laboratories companies of the U.S..
The method that embodiment 1 obtains hypoglycemic medicine
1st, search the inflammation with suppressing inflammation-related and suppress medicine
Lookup method is that, by retrieving various medical WEBs, the approach such as bibliographic data base obtains inflammation and suppresses medicine, and leads to
Cross commercially available mode and obtain inflammation suppression medicine.
2nd, screen
From the inflammation of all inflammation-relateds suppress medicine in filter out improvement beta Cell of islet insulin secreting ability or/and
The relevant drug target of insulin resistance ability, is specifically that rat Langerhans islet INS-1 β cell lines are being contained into different inflammation depressants
Cultivated under the condition of culture of thing, observe existence and the apoptosis of beta Cell of islet, and different inflammation are suppressed into medicine applied to sugar
The sick animal model of urine, observes blood sugar level, so as to judge that different inflammation suppress whether medicine causes β cell functions progressive to deteriorate
Or exhaustion (specific process of the test can be found in following test example 1-3).
3rd, according to the drug test result carried out to the drug target, the reduction blood is selected from the drug target
Sugared medicine, finally found that p38MAPK inhibitor SB203580 can effectively improve blood sugar level.
Realized in order to which the insulin secreting ability and insulin resistance ability that make beta Cell of islet by suppressing inflammation improve
The reduction of blood glucose, inventor carry out lot of experiments, draw p38MAPK inhibitor SB203580 can as hypoglycemic medicine knot
Really, tests below example is experimental study content and result of the inventor to p38MAPK inhibitor SB203580.
Test example 1p38MAPK inhibitor SB203580 to INS-1 beta Cell of islet system, tetra- kinds of subtype expressions of p38MAPK
Influence
1st, experiment material
1.1 experimental cell
Rat Langerhans islet INS-1 β cell lines：Because INS-1 cells expression oxygen radical removing enzyme is few, therefore to oxidative stress
Damage is extremely sensitive, similar to primary pancreas islet.This feature becomes dysfunction and the cell toxicant that research oxygen radical is advocated
Property model, INS-1 be widely used in research β cell growths and survival factor research.Being purchased from Shanghai, to visit power biotechnology limited
Company.This is to come from radiation-induced pancreas islet J3 cytomas, is by adding 2 mercapto ethanol (2- in tumor cell culture base
ME) set up.Most cells insulin stained positive is found by immunofluorescence dyeing, glucagon, growth is not detected by
The secretion of chalone or pancreatic polypeptide.
1.2 experimental pharmacy
Cell culture related reagent：
1640 culture mediums (22400-089) and hyclone (16000-044) are purchased from Life Teconology companies；
0.5% trypsase (containing EDTA without phenol red) and green grass or young crops-streptomysin mixed liquor are purchased from Life Teconology companies；Pipette tips, 6
Hole, 12 holes, 96 well culture plates, centrifuge tube, the consumptive material such as glass scales suction pipe and disposable filter are purchased from Corning companies of the U.S..
IL-1 β and IFN-γ are purchased from sigma companies.
Western blot related reagents：
The anti-p38MAPK monoclonal antibodies (#9212) of the anti-rats of I, the anti-p38MAPK phosphorylations monoclonal antibody (# of the anti-rats of I
4511), the anti-p 38 alpha MAPK monoclonal antibodies (#9218) of the anti-rats of I, the anti-p38 β MAPK monoclonal antibodies (#2339) of the anti-rats of I, I
The anti-anti- p38 γ MAPK monoclonal antibodies (#2307) of rat, the anti-p38 δ MAPK monoclonal antibodies (#9214) of the anti-rats of I, the anti-rats of I
Anti-bax monoclonal antibody (#2772), the anti-Bcl-2 monoclonal antibodies (#2870) of the anti-rats of I are purchased from Cell Signaling
Technology companies.The anti-actin monoclonal antibodies of the anti-rats of I are purchased from green skies Bioisystech Co., Ltd；II anti-peroxidations
The goat anti-mouse IgG (SC-2005) that thing enzyme is combined, peroxidase-conjugated goat anti-rabbit igg (SC-2004) is purchased from
Santa Cruz Biotechnology companies；The enhanced luminescent solutions of Western blot ECL (#34075) are purchased from the U.S.
Thermo companies；Nitrocellulose filter is purchased from MilliPore companies.
1.3 laboratory apparatus
High-precision electronic assay balance (Sartorius, Switzerland), desk-top cryogenic freezing supercentrifuge (eppendorf,
Germany), electronic balance (Adventurer, the U.S.), ultraviolet specrophotometer (Shimadzu UV3000, Japan), carbon dioxide cell
It is incubator (PRECISION, the U.S.), superclean bench (Baker, the U.S.), inverted phase contrast microscope (Olympus, Japan), thin
Born of the same parents' blake bottle (Thermo, the U.S.), Tissue Culture Plate (Coning, the U.S.), Nano Drop 2000 (Thermo, the U.S.), PCR
Instrument (Gene, Hong-Kong), chemiluminescence detection system (Kodak Image Station 4000MM PRO, the U.S.), enzyme mark
Instrument (Bio-Rad, the U.S.), single track adjustable pipette (Eppendorf, Germany), electrophoresis tank, transferring film groove, the power supply (instrument of Beijing 61
Device factory, China).
2nd, experimental method
By rat Langerhans islet INS-1 β cell lines in 1640 containing 10%FBS and the basis culture state of 11.1mmol/L glucose
After middle culture 48 hours, it is divided into five groups, is respectively：The control group of 11.1mmol/L glucose is continuously added, 25mmol/L is added
The sugared group of height of glucose, adds the high fat group of 0.2mmol/L palmitic acids, adds 25mmol/L glucose and 0.2mmol/L palms
The high glucose and high fat group of acid, and add the positive control of 5ug/ml IL-1 β, 5ug/ml IFN-γs and 11.1mmol/L glucose
Group (i.e. cell factor stimulation group), and carry out INS-1 β cell line cultures, training to the SB203580 of addition 20uM concentration in every group
The cell of culture is collected in 12 hours, 24 hours, 48 hours three timing nodes respectively during supporting, and albumen is carried out to it and is carried
Take, and Western blot technology for detection p38MAPK tetra- kinds of subtype proteins of α, β, γ, δ expression change, made with Actin
For protein expression internal reference.Obtain expression of results as shown in Figure 1.
Expression of results according to Fig. 1 can be seen that p38MAPK α and β hypotype under different glycolipid condition of culture,
Expression quantity has no significant change (Figure 1A, B, C, D).γ from δ hypotypes under different condition of culture, its variation tendency phase not to the utmost
Together：After high sugar is cultivated 48 hours (Figure 1B), it is seen that the expression quantity of SB203580 intervention group γ hypotypes increases；In high fat culture bar
Under part, the γ subtype expression quantities of SB203580 intervention groups since after culture in 12 hours increase, and be continued until 48 hours (figures
1C)；In control group and high sugared group, δ hypotypes have no significant change, but under high fat condition of culture, it is seen that SB203850 intervention groups
δ subtype expressions slightly have rise (Fig. 1 C), and the still visible same trend (Fig. 1 D) in high glucose and high fat co-cultivation；In positive control
Group (Fig. 1 E), can significantly be observed after SB203580 intervenes from after adding stimulation 12 hours, γ subtype expression quantities are gradually increasing, and 48 is small
When expression significantly rise, while visible δ hypotypes are with the extension of incubation time, its expression quantity is also in rising trend.
It can be seen that, SB203580 use can improve p38MAPK γ and the δ subtype expression quantities of INS-1 beta Cell of islet system,
INS-1 Apoptosis is reduced, and improves the insulin secreting ability of early stage INS-1 beta Cell of islet system, with culture in 24 hours most
To be notable.
Evaluations of the p38MAPK inhibitor SB203580 of test example 2 to INS-1 beta Cell of islet system insulin secretion function
The cell that step 2 in test example 1 is collected into culture carries out supernatant culture respectively, is pierced by international insulin
Swash glucose production (GSIS) and evaluate insulin secretion function, specific experiment method is:
1) cell culture medium is sucked, physiology salt washing cell 3 times adds 3.3mmol/L glucose KRHB solution and carried out in advance
Processing 30 minutes；2) supernatant is sucked, 1.0mmol/L glucose KRHB solution is added, sucted after 1 hour clearly to 1.5ml EP pipes ,-
20 DEG C freeze insulin to be measured.3) while adding 16.7mmol/L glucose KRHB solution stimulates cell, sucted after 1 hour clearly extremely
1.5ml EP are managed, and -20 DEG C freeze insulin to be measured.4) after glucose stimulation terminates, by cell extraction RNA, RNA concentration is determined,
As insulin correction.Wherein total insulin content is determined by putting method of exempting from after collection cells and supernatant centrifugation, is obtained such as Fig. 2
Shown result of the test.
3rd, experimental result
12 hours groups of results showed that culture as shown in Figure 2, with stimulation incubation time under no SB203580 interventions
Extension, the Basal insulin secretion ability of INS-1 cells is gradually reduced, and high sugared and/or high fat is to INS-1 cell base pancreas islet
Plain secretion capacity does not have obvious effect；After 24 hours of incubation, the basal insulin of the INS-1 cells of SB203580 intervention groups point
Secrete ability not being better than not plus SB203580 culture groups, and there is significant difference (p ＜ 0.05) in control group and high sugared culture group；
After culture 48 hours, the Basal insulin secretion ability of INS-1 cells is decreased obviously, but addition SB203580 intervenes and can be substantially apt to
Under the Basal insulin secretion ability (p ＜ 0.05) of INS-1 cells under cell factor stimulation, but high sugared and/or high fat stimulation
The Basal insulin secretion ability of INS-1 cells, which has no, to be obviously improved, and is significantly reduced under the stimulation of high fat；It is thin in positive control
Intracellular cytokine stimulation group, it is not dry that the Basal insulin secretion abilities of the INS-1 cells of SB203580 intervention groups is better than SB203580
Pre- group (Fig. 2A).Under different condition of culture incubation times, the insulin secreting ability of the glucose stimulation of observation INS-1 cells
Change.1. after culture 24 hours, the glucose stimulated insulin secretion capacity of the INS-1 cells of SB203580 intervention groups is strong
In the non-intervention groups of SB203580；2. after culture 48 hours, under the insulin secreting ability for the INS-1 cells that glucose is stimulated is obvious
Drop, but add the insulin secreting ability that SB203580 intervenes the INS-1 cells that can obviously improve under cell factor is stimulated；3.
Positive control cell factor stimulation group, the glucose stimulated insulin secretion capacity of the INS-1 cells of SB203580 intervention groups exists
It is significantly stronger than within 24 hours the non-intervention groups of SB203580, but the small rear and non-intervention group indifference (Fig. 2 B) of culture 48.
It can be seen that：
1) in positive control cell factor stimulation group, the Basal insulin secretion of the INS-1 cells of SB203580 intervention groups
Ability is better than the non-intervention groups of SB203580 (p ＜ 0.05)；
2) in positive control cell factor stimulation group, the glucose of the INS-1 cells of SB203580 intervention groups stimulates pancreas islet
Plain secretion capacity was significantly stronger than the non-intervention groups of SB203580 at 24 hours.
The p38MAPK inhibitor SB203580 of test example 3 improves to blood glucose
1st, experiment material
1.1 experimental animal
C57 mouse：The week old of SPF levels 6 female C57BL mouse 24.Db/db mouse：6 week old female db/db mouse 42.
Standard mice pellet, free water, feed, the round the clock illumination of (12h/12h) light, room temperature 20 DEG C~26
DEG C, humidity 40%~70%.
1.2 experimental pharmacy
As shown in table 1：
The test medicine of table 1
1.3 laboratory apparatus
The test apparatus that this implementation is used is as shown in table 2：
The laboratory apparatus of table 2
2nd, experimental method
2.1 animal packet
Adaptability is raised one week after mouse purchase.After one week, 6 C57 mouse and 6 db/db mouse are put to death at random.
Remaining mouse is divided into 3 groups：Blank control group (Con groups) is C57 mouse, 18；Experimental group (Dmi groups) and the positive
Control group (Dmo groups) is db/db mouse, each group 18.First group is given distilled water gavage；2nd group is given 15mg/ (kg*d)
SB203580 gavages；3rd group is given equivalent distilled water gavage.
Body weight, the food-intake of mouse are monitored weekly, and fasting plasma glucose and insulin level are surveyed in blood sampling.Treat Dmo groups and
Dmi group mouse fasting blood-glucoses occur starting to be calculated as 0 week (9 week old) during significant difference, do abdominal cavity within the 2nd week, the 4th week, the 6th week
Inject carbohydrate tolerance test (Intraperitoneal glucose tolerance test, IPGTT).
Wherein, body weight determination：Measured after fasting 16h using animal balance.
Food-intake：In units of cage, claim to feed feed weekly and remaining difference is obtained.
Blood sugar detection：The a small amount of blood blood glucose meter of tail end is determined.
Plasma insulin：Enzyme-linked immunosorbent assay.
3rd, experimental result
3.1 blood glucose
Mouse fasting 16h overnight is defined as fasted conditions.Since being measured fasting blood-glucose 7 week old, db/db mouse are to show
Write and be higher than C57 mouse.And start before pharmaceutical intervention and intervene one week after, experimental group (Dmi groups) and positive controls (Dmo groups) are empty
Abdomen blood glucose is not significantly different.2nd week (9 week old) after SB203580 gavages, the fasting blood-glucose between Dmi groups and Dmo groups starts
The fasting blood-glucose difference of appearance significant difference, the 3rd week experimental group and control group disappears, since after intervening the 4th week, and two groups empty
Abdomen blood glucose significant difference continued to exist up to 5 weeks, and experimental group fasting blood-glucose is substantially less than positive controls, and blood sugar detection result is such as
Shown in Fig. 3.
Before SB203580 intervenes, each time point blood sugar level and no difference of science of statistics are (such as Fig. 4 A institutes between Dmi and Dmo groups
Show).Pharmaceutical intervention 5 weeks (week old of mouse 12) surveys IPGTT (intraperitoneal injection carbohydrate tolerance test) discoveries, Dmi groups and Dmo group phases afterwards
Than sugar tolerance makes moderate progress, and each time point blood sugar level of experimental group and Area under the curve of blood glucose are below positive controls, difference
With statistical significance (as shown in Figure 4 B).The blood glucose curve during week old of mouse 14 (pharmaceutical intervention 7 weeks) and during 12 week old of mouse
Similar, the IPGTT of Dmi groups each time point blood sugar level and Area under the curve of blood glucose are below Dmo groups, and difference has statistics meaning
Adopted (as shown in Figure 4 C).Gap reduces between two groups when 16 week old of mouse, but each time point blood sugar level Dmi groups are below Dmo
There is significant difference (as shown in Figure 4 D) between group, two groups of 90min and 120min blood glucose.
Calculate IPGTT TG-AUCs and find that before intervention, there was no significant difference for Dmi and Dmo groups TG-AUC；12nd
Week, there is within the 14th week significant difference, Dmi groups are less than Dmo groups, but significant difference disappearance (as shown in Figure 5) in the 16th week, Dmi
Compare p ＜ 0.05 with Dmo mouse.
3.2nd, β cell functions and insulin resistance are assessed
Dmo groups are assessed using HOMA steady-state models (Homeostasis model assessment) formula and Dmi groups are small
Mouse islet beta cell function (i.e. HOMA- β=20 × Fasting insulin level (mIU/L)/(fasting blood glucose level (mmol/L)-
3.5) (%)；HOMA IR=fasting blood glucose levels (mmol/L) × Fasting insulin level (mIU/L)/22.5.).
From pharmaceutical intervention 4 weeks (week old of mouse 11), the β cell work(of the HOMA steady-state models of experimental group (Dmi groups) mouse
Positive controls (Dmo groups) can be persistently better than, at the 12nd week (pharmaceutical intervention the 5th week) difference reach statistical significance, such as table 3,
Shown in Fig. 4.Wherein, Dmi groups are db/db mouse SB203580 gavage groups in Fig. 4；Dmo groups compare for db/db mouse distilled water
Group.P between #Dmi and Dmo groups<0.05.
The insulin resistance index (i.e. HOMA-IR) of Dmi group mouse has downward trend, and at the 12nd week, constantly difference had statistics
Meaning is learned, as shown in table 4 and Fig. 5.From pharmaceutical intervention the 4th week, even if after correction HOMA-IR, HOMA- β between Dmi and Dmo groups
Difference still has conspicuousness.
HOMA- β, HOMA-IR are calculated according to equation below：
HOMA- β=20 × Fasting insulin level (mIU/L)/(fasting blood glucose level (mmol/L) -3.5) (%)；
HOMA-IR=fasting blood glucose levels (mmol/L) × Fasting insulin level (mIU/L)/22.5]
The each group mouse of table 3 difference week old HOMA- β (%)
Note：Data are represented in means standard deviation form.Examined when comparing between two groups with Mann-Whitney U.
paIt is worth for the p value after correction insulin resistance.
The each group mouse of table 4 difference week old HOMA IR (%)
Note：Data are represented in means standard deviation form.Examined when comparing between two groups with Mann-Whitney U.
From above-mentioned experimental result：P38MAPK inhibitor SB203580 has the effect for improving islet beta cell function,
And play the role of to improve body insulin resistance, so as to reduce blood glucose.P38MAPK inhibitor can turn into treating diabetes
One class antidiabetic drug, inflammation and improving islet beta cell function and insulin resistance.
Claims (10)
1. a kind of method for obtaining hypoglycemic medicine, it is characterised in that including：
Search the inflammation with suppressing inflammation-related and suppress medicine；
Improvement beta Cell of islet insulin secreting ability or/and pancreas islet are filtered out from the inflammation suppression medicine of all inflammation-relateds
The relevant drug target of plain resistivity；
According to the drug test result carried out to the drug target, the reduction blood glucose medicine is selected from the drug target
Thing.
2. the method as described in claim 1, it is characterised in that the reduction hypoglycemic medicament is selected from the drug target is
P38MAPK inhibitor SB203580.
3. method as claimed in claim 2, it is characterised in that the reduction hypoglycemic medicament is selected from the drug target is
Include the medicine of p38MAPK inhibitor SB203580 compositions.
4. a kind of hypoglycemic medicine, including pharmaceutically acceptable carrier and/or excipient, it is characterised in that it is by suppressing scorching
Disease makes the insulin secreting ability of beta Cell of islet and the improvement of insulin resistance ability realize the reduction of blood glucose, its mainly into
It is p38MAPK inhibitor SB203580 to divide.
5. hypoglycemic medicine as claimed in claim 4 is in the application for preparing conditioning, delaying and/or treat diabetes medicament.
6. the hypoglycemic medicine described in claim 4 is preparing improvement diabetic's beta Cell of islet insulin secreting ability medicine
In application.
7. the hypoglycemic medicine described in claim 4 is preparing the application in improving diabetic's body insulin resistance ability.
8. a kind of method for reducing blood glucose, it is characterised in that it makes the insulin secreting ability of beta Cell of islet by suppressing inflammation
And insulin resistance ability improves to realize the reduction of blood glucose.
9. method as claimed in claim 8, it is characterised in that described to enable the insulin secretion of beta Cell of islet by suppressing inflammation
Power and insulin resistance ability, which improve, to be realized using the medicine containing p38MAPK inhibitor SB203580 compositions.
10. the method described in claim 1 is applied to conditioning, delays and/or treat the application of diabetes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710310470.1A CN107034264A (en) | 2017-05-05 | 2017-05-05 | A kind of method and hypoglycemic medicine for obtaining hypoglycemic medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710310470.1A CN107034264A (en) | 2017-05-05 | 2017-05-05 | A kind of method and hypoglycemic medicine for obtaining hypoglycemic medicine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107034264A true CN107034264A (en) | 2017-08-11 |
Family
ID=59538325
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710310470.1A Pending CN107034264A (en) | 2017-05-05 | 2017-05-05 | A kind of method and hypoglycemic medicine for obtaining hypoglycemic medicine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107034264A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110898225A (en) * | 2019-12-23 | 2020-03-24 | 黑龙江中医药大学 | Application of mTOR/S6K1 and MAPK signal pathway inhibition in improvement of insulin resistance |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090074676A1 (en) * | 2005-05-23 | 2009-03-19 | Smithkline Beecham Corporation | Inhibition of p38 MAPK For Treatment Of Obesity |
| US20110318751A1 (en) * | 2010-06-24 | 2011-12-29 | Yoko Mullen | PUMA, A PRO-APOPTOTIC GENE, AS A NOVEL MOLECULAR BIOMARKER FOR TNFalpha-INDUCED HUMAN ISLET DAMAGE |
| CN103930165A (en) * | 2011-09-02 | 2014-07-16 | 纽约市哥伦比亚大学理事会 | CaMKII, IP3R, calcineurin, p38, and MK2/3 inhibitors to treat metabolic disturbances of obesity |
-
2017
- 2017-05-05 CN CN201710310470.1A patent/CN107034264A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090074676A1 (en) * | 2005-05-23 | 2009-03-19 | Smithkline Beecham Corporation | Inhibition of p38 MAPK For Treatment Of Obesity |
| US20110318751A1 (en) * | 2010-06-24 | 2011-12-29 | Yoko Mullen | PUMA, A PRO-APOPTOTIC GENE, AS A NOVEL MOLECULAR BIOMARKER FOR TNFalpha-INDUCED HUMAN ISLET DAMAGE |
| CN103930165A (en) * | 2011-09-02 | 2014-07-16 | 纽约市哥伦比亚大学理事会 | CaMKII, IP3R, calcineurin, p38, and MK2/3 inhibitors to treat metabolic disturbances of obesity |
Non-Patent Citations (3)
| Title |
|---|
| LINGLI ZHOU等: "P38 Plays an Important Role in Glucolipotoxicity-Induced Apoptosis in INS-1 Cells", 《JOURNAL OF DIABETES RESEARCH》 * |
| 许曼音: "《糖尿病学》", 31 May 2010, 上海科学技术出版社 * |
| 谭惠文等: "糖脂毒性对肥胖大鼠β细胞功能和凋亡的影响研究", 《四川大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110898225A (en) * | 2019-12-23 | 2020-03-24 | 黑龙江中医药大学 | Application of mTOR/S6K1 and MAPK signal pathway inhibition in improvement of insulin resistance |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10624938B2 (en) | Total flavone extract of flower of abelmoschus manihot L. medic and preparation method thereof | |
| CN106822599B (en) | Dendrobium loddigesii Rolfe extract and anti-diabetic application thereof | |
| CN108403686A (en) | Application and pharmaceutical composition of the artemisinin derivative in preparing prevention and treatment diabetes B and its complication medicine | |
| CN103599117B (en) | Use of szechuan melandium root pentacyclic triterpenoid saponin compound in preparation of drug for reducing blood sugar | |
| CN107034264A (en) | A kind of method and hypoglycemic medicine for obtaining hypoglycemic medicine | |
| CN106420697B (en) | A kind of polymethoxyflavone, composition and its preparation are used to prevent or treat the purposes of diabetes | |
| CN107115338A (en) | Application of the bicyclic alcohols in diabetes medicament is prepared | |
| WO2022033200A1 (en) | Use of sorafenib in treatment of type 1 diabetes | |
| CN101411781A (en) | Use of pu'er tea in preparing medicament for treating or preventing diabetes | |
| CN113425734A (en) | Application of lobetyolin in preparation of medicine for treating diabetes | |
| CN106727469A (en) | Application of the kukoamine B in treatment type II diabetes medicine is prepared | |
| CN106562985A (en) | Medicinal health care applications of linarin | |
| WO2020168741A1 (en) | Use of canagliflozin in preparation of anti-tumor medication | |
| CN106943408B (en) | Application of tetramethyluric acid in preventing and treating diabetes | |
| CN105801663A (en) | Preparation method and medical application of ursolic acid and berberine conjugate | |
| CN110314160A (en) | Berbamine prevents and treats the application in medicine for treating diabetic nephropathy in preparation | |
| CN101433534B (en) | Use of resveratrol dimer for preparing medicament for reducing blood sugar | |
| WO2004032924A1 (en) | The use of berberine as insulin sensitizer | |
| CN115671105B (en) | Application of LY2922470 in preparation of medicines for preventing or treating kidney diseases | |
| CN108143756A (en) | A kind of plant hypoglycemic agent and preparation method thereof | |
| CN105663152A (en) | Application of triacetyl-3-hydroxyphenyl adenosine in preparing medicines for improving insulin resistance and related diseases | |
| CN110613755A (en) | Preparation method of rhizoma coptidis total alkaloids and application of rhizoma coptidis total alkaloids in treatment of diabetes | |
| CN109010335A (en) | Purposes of the 2,4- thiazolidine dione compounds K145 in preparation treatment diabetes medicament | |
| CN104582730A (en) | Method for improving metabolic function of mitochondria and use thereof | |
| CN111195247B (en) | Alpha-glucosidase inhibitor and application thereof in hypoglycemic drugs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170811 |