CN103285379A - Use of GLP-1 (Glucagon-Like Peptide-1) for preparing medicines for preventing and treating macrovascular complications of type 2 diabetes - Google Patents
Use of GLP-1 (Glucagon-Like Peptide-1) for preparing medicines for preventing and treating macrovascular complications of type 2 diabetes Download PDFInfo
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Abstract
The invention provides use of glucagon-like peptide-1 (GLP-1) for preparing medicines for preventing and treating macrovascular complications of type 2 diabetes. The experiments show that GLP-1 has the anti-apoptosis effect by inhibiting the JNK (Jun N-terminal Kinase) signal paths of aortic endothelial cells of mice, increasing expression of Bc1-2 and inhibiting the activity of caspase-3. The effect is probably completed by promoting ROS (Reactive Oxygen Species) in scavenger-cells to exert anti-oxidative damage effect. GLP-1 in vitro and in vivo can remarkably improve degree of injury of great vessels of mice with type 2 diabetes. Therefore, GLP-1 can be used for preparing medicines for preventing and treating macrovascular complications of type 2 diabetes. The invention further provides a pharmaceutical composition for preparing medicines for preventing and treating macrovascular complications of type 2 diabetes.
Description
Technical field
The invention belongs to the prevention and control field of diabetic complication, relate to the GLP-1(glucagon-like-peptide-1) new purposes, particularly GLP-1 is for the preparation of the purposes of prevention with the medicine for the treatment of type 2 diabetes mellitus trunk complication.
Background technology
Diabetes refer to the absolute or relative deficiency of insulin in the blood, cause blood glucose too high, and then cause a class disease of metabolism disorders such as fat and protein.Control improperly that diabetes have many complication, as infection, heart change, cerebrovascular disease, renal failure, lose the sight of both eyes and lower limb gangrene etc., these complication seriously influence patients ' life quality even life-threatening.Wherein, cardiovascular complication is to cause the diabetics main causes of death.The main pathological basis of diabetes cardiovascular complication is little blood vessel, nerve and trunk damage.The diabetic vascular damage can be accumulated the pathological changes that whole body is organized internal organs more, brings huge burden to patient and society.
The basis of diabetes cardiovascular complication is the vascular cell damage that disease causes, and endothelial cell damage is the pathological phenomenon that occurs the earliest in the diabetic vascular damage process.Under the type 2 diabetes mellitus state, long-term hyperglycemia and hyperinsulinemia stimulate, and cause the endotheliocyte oxidative damage.At first, the plurality of enzymes in the endotheliocyte can produce reactive oxygen free radical (ROS), for example nadph oxidase, xanthine oxidase, (endothelial type nitric oxide synthase) eNOS, adenosine oxygenase, glucoseoxidase and mitochondrion electron transfer enzyme etc.The oxygen-derived free radicals that they produce comprises superoxide anion, hydrogen peroxide, hydroxy radical and nitrito-etc., and these molecules participate in apoptosis, growth and the inflammatory reaction of endotheliocyte as intracellular second message,second messenger.Secondly, glucose mainly promotes endotheliocytes to generate ROS by three approach: i.e. mitochondrion electronics respiratory chain, the autoxidation of oxidasic synthetic and glucose, when in the cell during sugared excessive concentration, glucose produces ROS rapidly by these three approach, the ROS that piles up in the endotheliocyte will promote the increase that advanced glycosylation end product (AGEs) and oxidized low-density lipoprotein (ox-LDL) generate, thereby further causes atherosclerotic generation and development.In addition, long-term hyperinsulinism stimulates, and can cause very low density lipoprotein (VLDL) in the cell (VLDL) and low density lipoprotein, LDL accumulations such as (LDL), and they cause endothelial dysfunction by inducing inflammation, oxidative stress and inhibition eNOS activity etc.As seen, it is significant for the control of diabetes trunk complication to study and filter out the medicine that can stop endothelial cell damage.
The medicine that is used for the treatment of diabetes cardiovascular complication at present has many, but its effect mainly is blood fat reducing, anticoagulant and promote vasodilation etc. and the further deterioration that wards off disease, these medicines can not effectively be cured the diabetes cardiovascular complication.Therefore, develop new medicine, have important social meaning and economic benefit.
The GLP-1(glucagon-like-peptide-1) is a kind of intestinal secretion hormone, because stimulating insulin secretion, reduce the volume of appetite and increase islet cells and become the focus and emphasis of diabetes study in recent years, its analog has been used to the treatment of clinical diabetes at present.Except islet cells is had the effect, the multiple histoorgan that studies show that the body of GLP-1 all tool has certain effect, and can delay gastric emptying as GLP-1, increases feeling of repletion and reduces and ingests; GLP-1 can activate the Akt signal path, regulates glycogen synthase kinase 3 β (GSK3 β), and heme oxygenase (HO-1) etc. has the expression of Cardioprotective functional molecular, thereby improves the function of diabetic animal cardiac index and endotheliocyte; Find that in addition the analog Li Lalu peptide of GLP-1 has the effect of antiinflammatory and anti-oxidation stress, it is by PKC-α and NF-κ B signal path in the inhibition endotheliocyte, and the activity of reduction nadph oxidase reduces inflammatory factor to cells injury.As seen GLP-1 has very huge potential using value for the control of diabetes cardiovascular complication.
Summary of the invention
The object of the present invention is to provide the GLP-1(glucagon-like-peptide-1) for the preparation of the purposes of prevention with the medicine for the treatment of type 2 diabetes mellitus trunk complication, thus provide new approach for prevention and treatment type 2 diabetes mellitus trunk complication.
The present invention shows by experiment, GLP-1 brings into play anti-apoptotic effect by the activity that suppresses JNK signal path in the rat aorta endotheliocyte, increases the expression of Bcl-2 and suppress caspase-3, and above-mentioned effect is likely to be finished by the mechanism of action that promotes ROS in the scavenger cell to bring into play anti-oxidative damage; GLP-1 all can significantly improve the degree of injury of type 2 diabetes mellitus rat trunk in vivo and in vitro.Therefore, GLP-1 can be used for preparing prevention and the medicine for the treatment of type 2 diabetes mellitus trunk complication.
It is a kind of for the pharmaceutical composition of prevention with treatment type 2 diabetes mellitus trunk complication that the present invention provides simultaneously, comprises the GLP-1 of effective dose, and acceptable accessories.Preferably, described pharmaceutical composition is made intravenous drip or hypodermic pharmaceutical dosage form.
At present, GLP-1 can be applied to clinical by lasting intravenous drip or lasting subcutaneous injection, for example is used for the treatment of acute illness.Therefore, the pharmaceutical composition that comprises the GLP-1 of effective dose provided by the present invention also is expected to be applied to clinically prevention and treatment type 2 diabetes mellitus trunk complication.
For the short restricted problem of plasma half-life that solves natural GLP-1, people have further developed the GLP-1 analog, have both possessed the effect of GLP-1, can resist degraded again, for example, GLP-1 are carried out structural modification, make it be difficult for being degraded by DPP-4.At present, existing multiple GLP-1 analog is used for clinical practice.
Based on similar structure, the GLP-1 analog also can be used for preparing prevention and the medicine for the treatment of type 2 diabetes mellitus trunk complication.
Therefore, the present invention also provides the GLP-1 analog for the preparation of the purposes of prevention with the medicine for the treatment of type 2 diabetes mellitus trunk complication.
It is a kind of for prevention and treatment type 2 diabetes mellitus trunk complication pharmaceutical composition that the present invention also provides, and comprises the GLP-1 analog of effective dose, and acceptable accessories.Preferably, described pharmaceutical composition is made intravenous drip or hypodermic pharmaceutical dosage form.
The specific embodiment
Below the present invention is done specifying, should be noted that, following examples are used for explanation of the present invention and also unrestricted the present invention.Although with preferred embodiment the present invention is had been described in detail, those of ordinary skill in the art should be appreciated that and can make amendment, be out of shape the present invention under not departing from the scope of the present invention or be equal to replacement, all belongs to protection scope of the present invention.
Embodiment one: the structure of type 2 diabetes mellitus endothelial cell damage model
The high sugar of present embodiment use in conjunction and hyperinsulinism stimulate vascular endothelial cell, and high sugar and hyperinsulinism environment under the simulation type 2 diabetes mellitus state make up the endothelial cell damage model.
Laboratory animal and cell:
Healthy adult is male/SD female (Sprague-Dawley) rat is available from Nanfang Medical Univ's Experimental Animal Center, the rat aorta endotheliocyte from healthy adult male/thoracic aorta of female sd inbred rats separates.
Experimental technique:
With healthy adult male/female sd inbred rats cervical vertebra dislocation puts to death, separate rat chest aorta, peel off clean blood vessel fatty tissue on every side, inner membrance turns up, and, digests 45 minutes under 37 ℃ of conditions with I Collagen Type VI enzyme (2g/L) the ligation of blood vessel two ends with ligature, with the fritter of vascular scissors into about 3mm * 3mm, inner membrance is affixed in the Tissue Culture Dish of spreading Mus tail collagen down, adds the normal sugared DMEM culture medium that contains 20%FBS in culture dish, and Tissue Culture Dish is placed 5%CO
2, cultivate in 37 ℃ the cell culture incubator.Cultivated the 3rd day, and namely had endotheliocyte from piece of tissue, to dissociate out.Treat the cytosis that dissociates out from piece of tissue, remove piece of tissue, cell is cultivated normally.Cultivate based on 37 ℃ 5%CO with the normal sugared DMEM that contains 10%FBS
2Continue cultured cell in the incubator, treat that cell enters the logarithmic growth after date, had digestive transfer culture, the endotheliocyte in 3～7 generations is used for follow-up experimentation.
Rat aorta endotheliocyte bed board treats that cell grows at the bottom of the ware 85%, spends the night with the hungry cell of sugar-free serum-free medium, makes the metabolism of cell be in synchronized state.Cell is divided into 3 groups: normal glucose concentration processed group (being called for short normal sugared processed group) is about to cell culture in the DMEM culture medium that contains the 5.5mmol/L concentration of glucose; High concentration glucose processed group (being called for short high sugared processed group) is about to cell culture in the DMEM culture medium that contains the 25mmol/L concentration of glucose; High concentration glucose and high concentration insulin processed group (being called for short high sugared hyperinsulinism processed group) are about to cell culture in the DMEM culture medium that contains 25mmol/L glucose and 100nmol/L insulin concentration.All contain 2% FBS in above-mentioned 3 groups of cell culture mediums.Cell stimulates 72h or 96h respectively under these conditions, carries out follow-up endothelial cell apoptosis experiment then, and Caspase-3 is active to be detected and the oxidative stress detection.
Apoptosis detects (DAPI dyeing)
The rat aorta endotheliocyte is laid in 96 orifice plates, stimulate 72h under these conditions after, 4% paraformaldehyde fixed cell 10min of pre-cooling, penetrating 10min under the 0.1%Triton X-100 room temperature, DAPI dyeing 3min then, the glycerol mounting, microscopically is observed and is taken pictures.Apoptosis rate=(cell number that apoptosis changes/total cell number) * 100%
Caspase-3 is active to be detected
Colorimetry detects the activity of Caspase-3 in the rat aorta endotheliocyte.The rat aorta endotheliocyte is laid on six orifice plates, stimulate 72h under these conditions, collecting cell, extract total protein of cell, the Bradford method is measured protein concentration, and the adjustment protein concentration is 2mg/ml, get 10 μ l protein samples in 96 orifice plates, every hole adds the substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) of 10 μ l caspase-3 and 80 μ l detect buffer, hatches 4h under 37 ℃, uses microplate reader to detect the light absorption value in each hole at 405nm wavelength place.
ROS detects
The rat aorta endotheliocyte is laid on six orifice plates, stimulate 72h under these conditions, collecting cell, adding concentration is the DCFH-DA probe of 10 μ M, under 37 ℃, hatch 30min, PBS(pH=7.4) washed cell is 3 times, and flow cytometer detects and respectively organizes fluorescence intensity in the cell, and fluorescence intensity is namely reacted and respectively organized ROS level in the cell.
Statistical analysis
Experimental data adopts SPSS13.0 software to carry out statistical analysis, the method that relatively adopts the independent sample t check of data between two groups; The comparison of three groups or three groups above data adopts One-way ANOVA to analyze relatively.
Experimental result
1, high sugared hyperinsulinism promotes the rat aorta endothelial cell apoptosis
The result shows, high sugar and high sugared hyperinsulinism irritation cell 72h, be the star moon shape and the nucleus of fragment shape increase, the endotheliocyte generation apoptosis of the sugared and high sugared hyperinsulinism processed group of height be described.Statistics is respectively organized the apoptosis rate of cell, and the apoptosis rate of normal sugared processed group cell is (12.46 ± 2.05) %, and the apoptosis rate of high sugar and high sugared hyperinsulinism processed group cell is respectively (36.90 ± 5.90) % and (52.00 ± 8.14) %.Namely the apoptosis rate of high sugar and high sugared hyperinsulinism processed group cell is respectively 3.0 times and 4.2 times that normal sugar is organized, difference all have statistical significance (P=0.000, P=0.000); In addition, compare with the sugared processed group of height, high sugared hyperinsulinism processed group apoptosis rate is 1.4 times of high sugared processed group, significant difference (P=0.006), and visible high sugared hyperinsulinism inducing endothelial cell effect of apoptosis is more obvious.
2, high sugared hyperinsulinism improves the activity of caspase-3 in the rat aorta endotheliocyte
The result shows, the activity of high sugar and high sugared hyperinsulinism group caspase-3 is respectively 1.5 times and 1.8 times that normal sugar is organized, and difference have statistical significance (P=0.000, P=0.000); High sugared group is compared with the sugared hyperinsulinism group of height, and the activity of high sugared hyperinsulinism group caspase-3 is 1.2 times of high sugar group, i.e. the activity of high sugared hyperinsulinism group caspase-3 rising is (P=0.013) more obviously.In sum, high sugared hyperinsulinism is by strengthening the activity inducement apoptosis of caspase-3 in the rat aorta endotheliocyte, and the success of prompting vascular endothelial cell damage model construction is for follow-up experimentation provides method and foundation.
3, ROS level in the high sugared rising rat aorta endotheliocyte
The result shows that the level of ROS is 267.98 ± 5.01 in the normal sugared processed group endotheliocyte, and the level that high sugar is organized ROS is 401.89 ± 62.11, and namely high sugar group ROS level is 1.5 times that normal sugar is organized, and the two has statistical significance (P=0.018).As seen, the high sugar ROS level in the rat aorta endotheliocyte that significantly raise points out that oxidative stress has played the effect that can not be ignored in the high sugared inducing endothelial cell apoptotic process, for screening and the research of follow-up medicine provides theoretical foundation.
In sum, under the long-time stimulus of high sugar and hyperinsulinism, the propagation of rat aorta endotheliocyte has been subjected to inhibition, and the active of caspase-3 significantly strengthens in the cell, and the endothelial cell apoptosis rate improves; The analysis showed that further ROS content obviously raises in the endotheliocyte that high sugar is induced, disclose these variations and result from intracellular oxidative damage.
Mechanism for the damage of research diabetes trunk, be the object of observation with the rat aorta endotheliocyte directly in this experiment, the result finds that also high sugared hyperinsulinism processing can increase the ROS level of rat aorta endotheliocyte, the trigger cell apoptosis, these results have proved that directly the endotheliocyte oxidative stress that high sugar and hyperinsulinism cause is apoptotic important mechanism unusually.
Present embodiment has successfully prepared the rat aorta endotheliocyte, the trunk endothelial cell damage environment that adopted high sugared hyperinsulinism treatment of simulated, and by morphology, molecular biology and cell biology method clear and definite high sugared hyperinsulinism cause the mechanism of rat aorta endothelial cell damage, for the preventive and therapeutic effect of later observation GLP-1 provides foundation and basis.
Embodiment two: the influence of the rat aorta endothelial cell apoptosis that the sugared hyperinsulinism of the height of GLP-1 is induced
Present embodiment is on the constructed diabetic vascular cell injury model based of embodiment one; systematically study the protective effect of the vascular endothelial cell diabetic damage of GLP-1; and disclose its mechanism, for the method for preventing and treating of diabetes trunk complication provides foundation.
Laboratory animal and cell:
Healthy adult is male/female sd inbred rats is available from Nanfang Medical Univ's Experimental Animal Center, the rat aorta endotheliocyte from healthy adult male/thoracic aorta of female sd inbred rats separates.
Experimental technique:
The GLP-1 that this experiment is adopted is GLP-1(7-36), available from Shanghai Chu peptide bio tech ltd.
Rat aorta endotheliocyte bed board treats that cell grows at the bottom of the ware 85%, spends the night with the hungry cell of sugar-free serum-free medium, makes cell be in synchronized state.Cell is divided into 5 groups: normal glucose concentration processed group (being called for short normal sugared processed group) is about to cell culture in the DMEM culture medium that contains the 5.5mmol/L concentration of glucose; High concentration glucose processed group (being called for short high sugared processed group) is about to cell culture in the DMEM culture medium that contains the 25mmol/L concentration of glucose; High sugar+GLP-1 processed group is about to cell culture in the DMEM culture medium that contains 25mmol/L glucose and 10nmol/L GLP-1 concentration; High concentration glucose and high concentration insulin processed group (being called for short high sugared hyperinsulinism processed group) are about to cell culture in the DMEM culture medium that contains 25mmol/L glucose and 100nmol/L insulin concentration; High sugared hyperinsulinism+GLP-1 processed group is about to cell culture in containing the 25mmol/L glucose, in the DMEM culture medium of 100nmol/L insulin and 10nmol/LGLP-1 concentration.All contain 2% FBS in above-mentioned 5 groups of cell culture mediums.Cell stimulates 72h respectively under these conditions, carries out follow-up endothelial cell apoptosis experiment then, and Caspase-3 is active to be detected and the oxidative stress detection, and method is with embodiment one.
Experimental result
1, GLP-1 suppresses the rat aorta endothelial cell apoptosis that high sugar and hyperinsulinism are induced
The result shows, in high sugar and high sugared hyperinsulinism processed group, has many cells to demonstrate karyopyknosis, and chromatic agglutination, nucleus are star moon shape or fragment shape, i.e. typical apoptosis form.And in the GLP-1 processed group, the nucleus minimizing that apoptosis changes takes place.Calculate the apoptosis rate (seeing Table 1) respectively organize cell, the apoptosis rate of high sugar and high sugared hyperinsulinism processed group cell is respectively 3.0 times and 4.2 times of normal sugared processed group, all have statistical significance (P=0.011, P=0.008); And the GLP-1 processed group has descended 44% and 66% respectively than high sugar and high sugared hyperinsulinism processed group apoptosis rate, have statistical significance (P=0.035, P=0.017).
Table 1 different disposal condition is to the influence of rat aorta endothelial cell apoptosis
2, GLP-1 suppresses caspase-3 activity in the rat aorta endotheliocyte that high sugar and hyperinsulinism induce
The result shows (table 2), and the normal sugared processed group of specific activity of caspase-3 has raise 50% and 80% respectively in high sugar and the high sugared hyperinsulinism processed group cell, difference all have statistical significance (P=0.000, P=0.000); And the high sugar of the specific activity of cell caspase-3, hyperinsulinism processed group have descended 27% and 28% respectively in the GLP-1 processed group, and significant difference (P=0.000, P=0.000).In sum, GLP-1 can significantly reduce caspase-3 in the rat aorta endotheliocyte that height is sugared and hyperinsulinism is induced, and then suppresses apoptosis.
Table 2: the different disposal condition is to the influence of caspase-3 activity in the rat aorta endotheliocyte
3, GLP-1 promotes the expression of anti-apoptotic proteins Bcl-2 in the rat aorta endotheliocyte
Bcl-2 is most important adjusting molecule in the apoptosis signal pathway, and it has the inhibition effect of apoptosis.Therefore, the influence that Bcl-2 expresses in the high sugar of research, hyperinsulinism and the rat aorta endotheliocyte of GLP-1 is the important step that discloses the anti-apoptosis molecular mechanism of GLP-1.
Adopt western blot method to detect the influence that anti-apoptosis molecule Bcl-2 expresses in the endotheliocyte of GLP-1 at protein level.The result shows, the expression of Bcl-2 is respectively 1.54 times and 1.38 times of normal sugared processed group in high sugar+GLP-1 and the high sugared hyperinsulinism+GLP-1 processed group, difference all have statistical significance (P=0.001, P=0.003); The Bcl-2 expression of high sugar+GLP-1 processed group is 1.25 times of high sugared processed group, significant difference (P=0.017); The expression of high sugared hyperinsulinism+GLP-1 processed group Bcl-2 is 1.38 times of high sugared hyperinsulinism processed group, and difference has statistical significance (P=0.003).These observations show that GLP-1 can improve the expression of Bcl-2 in the rat aorta endotheliocyte that height is sugared and hyperinsulinism is handled, for the signal path that discloses the anti-apoptosis of GLP-1 provides foundation.
4, GLP-1 suppresses that ROS generates in the rat aorta endotheliocyte that high sugar induces
Influence by ROS level in the high sugar of Flow cytometry and the rat aorta endotheliocyte of GLP-1.The result shows that the ROS level is respectively 267.98 ± 2.51,401.89 ± 31.06 and 140.48 ± 26.64 in normal sugar, high sugar and the high sugar+GLP-1 processed group.The content that is ROS in the high sugared processed group cell has increased by 50% than normal sugared processed group, and difference has statistical significance (P=0.002); The content of ROS has descended 65% than high sugared processed group in high sugar+GLP-1 processed group, significant difference (P=0.000).As seen, GLP-1 can significantly reduce ROS content in the endotheliocyte that high sugar induces.
5, the transcriptional level of SOD2 and catalase mRNA in the GLP-1 raising rat aorta endotheliocyte
Influence by SOD2 and catalatic mRNA transcriptional level in the endotheliocyte of Q-PCR detection GLP-1.SOD2 and catalatic main effect are the ROS in the scavenger cell, and the protection cell is avoided oxidative damage.The result shows that GLP-1 all can remarkable increased SOD 2 and catalatic mRNA transcriptional level (table 3 and table 4).Compare with normal sugared processed group, SOD2 and catalatic mRNA transcriptional level have risen 33% and 77% respectively in high sugar+GLP-1 processed group cell, difference have statistical significance (P=0.001, P=0.011).High sugared hyperinsulinism+GLP-1 processed group is compared with normal sugared processed group, and SOD2 and catalatic mRNA transcribe and raise 27% and 58% respectively, and significant difference (P=0.003, P=0.031).In addition, high sugared processed group is compared with high sugar+GLP-1 processed group, SOD2 and catalatic mRNA transcriptional level have risen 25% and 70% respectively in the GLP-1 processed group cell, difference has statistical significance (P=0.002, P=0.018), high sugared hyperinsulinism group is compared with high sugared hyperinsulinism+GLP-1 processed group, and SOD2 and catalatic mRNA transcriptional level have risen 18% and 55% respectively in the GLP-1 processed group cell, significant difference (P=0.022, P=0.020).
Table 3: the different stimulated condition is to the influence of the mRNA transcriptional level of SOD2 in the rat aorta endotheliocyte
Table 4: the different stimulated condition is to the influence of catalatic mRNA transcriptional level in the rat aorta endotheliocyte
6, the activity of SOD2 in the GLP-1 rising rat aorta endotheliocyte
In order to verify that further GLP-1 reduces the mechanism of endotheliocyte ROS content, detects the influence of the SOD2 activity of GLP-1 from protein level.Found that the activity of normal sugared processed group SOD2 is respectively high sugar and high sugared hyperinsulinism processed group 1.23 times and 1.3 times, and significant difference (P=0.001, P=0.031); High sugar is compared with high sugar+GLP-1 processed group, and the activity of high sugar+GLP-1 processed group SOD2 is 1.25 times of high sugared processed group, and difference has statistical significance (P=0.040); Equally, the activity of SOD2 is 1.24 times of high sugared hyperinsulinism group in high sugared hyperinsulinism+GLP-1 processed group, significant difference (P=0.033).Above description of test GLP-1 is by promoting SOD2 and catalatic mRNA transcriptional level, improve the activity of SOD2 and reduce the generation of ROS, be that GLP-1 suppresses to be accompanied by the minimizing that ROS generates in the cell in the process of the rat aorta endothelial cell apoptosis that high sugared hyperinsulinism induces, for the molecular mechanism of the anti-apoptosis anti-oxidation stress of follow-up study GLP-1 provides theoretical foundation.
7, GLP-1 suppresses the expression of p-JNK in the rat aorta endotheliocyte
The JNK signal transduction pathway is an important branch of MAPK path, is the bridge that connects apoptosis and oxidative stress path.Above experiment prompting GLP-1 anti-apoptotic effect may to reduce in the cell ROS level relevant with GLP-1, research knows that the effect of JNK signal path in this process is significant for the relation that discloses the anti-apoptosis of GLP-1 and anti-oxidation stress.Therefore, by the expression that western blot detects phosphorylation JNK in the rat aorta endotheliocyte, study the adjusting whether the JNK signal path participates in high sugar, high sugared hyperinsulinism and GLP-1.
The result shows, the expression of high sugar and high sugared hyperinsulinism processed group phosphorylation JNK is respectively 5.1 times and 3.3 times of normal sugared processed group, difference all have statistical significance (P=0.001, P=0.027); High sugared processed group is compared with high sugar+GLP-1 processed group, and the expression of high sugared processed group phosphorylation JNK is 1.7 times of high sugar+GLP-1 processed group, significant difference (P=0.035).But high sugared processed group is compared with the sugared hyperinsulinism processed group of height, and the expression of phosphorylation JNK does not have significant difference, and the prompting hyperinsulinism may pass through other signal path inducing endothelial cell apoptosis.In sum, high sugar induces in the rat aorta endotheliocyte ROS to generate by activation JNK signal path to increase and apoptosis, and GLP-1 can suppress the activation of JNK, thereby reduces the generation of ROS, suppresses apoptosis.
Comprehensive above-mentioned experimental result, GLP-1 has the effect of significant protection endothelial cell damage.Therefore, the present invention is for GLP-1 is laid a good foundation for the preparation of prevention and medicine and the clinical practice for the treatment of diabetes trunk complication.
At present, GLP-1 analog Exenatide (Lilly Co., Eli.) and Li Lalu peptide (Denmark Novo Nordisk Co.,Ltd) all successively enter clinical, are used for treating diabetes.The Li Lalu peptide with compare an aminoacid difference (changing the 20th lysine into glutamic acid) with natural GLP-1 molecular structure, and increased by 16 carbon palmityl fatty acid side chains, with natural human GLP-1 95% homology is arranged.Because the existence of fatty acid side chain, its molecule are difficult for being degraded by DPP-IV, and can thereby higher metabolic stability be arranged with albumin bound, the half-life reaches 12-14 hour.Exenatide is a kind of natural GLP-1 analog, is 53% homology with the aminoacid sequence of GLP-1.Because structural homology, and the similarity of the mechanism of action, these GLP-1 analog also can have the effect of significant protection endothelial cell damage, thereby also can be used for preparing prevention and the medicine for the treatment of diabetes trunk complication.
Embodiment three: pharmaceutical preparation
Natural GLP-1 or GLP-1 analog as active component, are allocated in pharmaceutically acceptable excipient, carrier or diluent, be adjusted to fill concentration, injection is made in filtration sterilization, depyrogenation, fill, further can be made into lyophilized injectable powder.Relevant technology can be with reference to the pharmacy common process of this area.
These pharmaceutical preparatioies can be used for intravenous drip or subcutaneous injection clinically.
Claims (6)
1. glucagon-like-peptide-1 is for the preparation of the purposes of prevention with the medicine for the treatment of type 2 diabetes mellitus trunk complication.
2. pharmaceutical composition comprises the glucagon-like-peptide-1 of effective dose and acceptable accessories.
3. pharmaceutical composition according to claim 2, it is characterized in that: described pharmaceutical composition is made intravenous drip or hypodermic pharmaceutical dosage form.
4. the glucagon-like-peptide-1 analog is for the preparation of the purposes of prevention with the medicine for the treatment of type 2 diabetes mellitus trunk complication.
5. pharmaceutical composition comprises the glucagon-like-peptide-1 analog of effective dose and acceptable accessories.
6. pharmaceutical composition according to claim 5, it is characterized in that: described pharmaceutical composition is made intravenous drip or hypodermic pharmaceutical dosage form.
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| CN106574233A (en) * | 2014-08-21 | 2017-04-19 | 达福企业有限公司 | Peptide for treatment of type 2 diabetes mellitus and its complications |
| CN110433282A (en) * | 2018-05-04 | 2019-11-12 | 上海交通大学医学院附属瑞金医院 | Application of the glucagon-like-peptide-1 in terms of calcific aortic disease medicament is treated in preparation |
| CN110433282B (en) * | 2018-05-04 | 2023-03-14 | 上海交通大学医学院附属瑞金医院 | Application of glucagon-like peptide-1 in preparation of medicine for treating calcified aortic valve diseases |
| CN111789939A (en) * | 2019-04-09 | 2020-10-20 | 南京大学 | Application of liraglutide in preparation of tumor immunotherapy medicine |
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