An endogenous active peptide and use thereof

Abstract

The application discloses an endogenous active peptide and application thereof. The application relates to the field of metabolism and blood glucose regulation, in particular to an endogenous active peptide and application thereof in weight regulation, metabolism and blood glucose regulation. The application provides application of CMGA active peptide or a medicinal salt thereof. The CMGA active peptide contains 11 amino acids, and it is found through animal experiment verification that the peptide has the functions of improving metabolism and regulating blood glucose. Glucose tolerance tests and insulin tolerance tests show that the CMGA active peptide can maintain the homeostasis of glucose in blood, and has important therapeutic significance.

Description

Technical Field

[0001] This invention relates to an endogenous bioactive peptide and its applications in the fields of metabolism and blood glucose regulation, specifically to the use of endogenous bioactive peptides in weight regulation, metabolic thermogenesis, and blood glucose regulation. Background Technology

[0002] Obesity has become a major disease affecting modern people, increasing the risk of a range of illnesses, such as cardiovascular disease, increased cardiac workload, and damage to internal organs due to visceral fat. Regulating weight and helping it return to a normal range is a major concern. Various methods are being used to identify substances that can increase metabolism and accelerate energy expenditure to improve weight gain. Ideally, weight loss should occur naturally and healthily by improving metabolism without changing one's lifestyle.

[0003] Blood glucose homeostasis is a crucial state for maintaining normal physiological functions, and common blood glucose disorders are often associated with metabolic diseases. Insulin is a commonly used bioactive peptide drug for regulating blood glucose. Insulin is composed of 56 amino acids and can be mass-produced through fermentation engineering. However, insulin contains a large number of amino acids, making it easily degraded and resulting in a short duration of action in the body. In daily medication use, hypoglycemia frequently occurs due to incorrect or excessive medication. Therefore, there is an urgent need to develop alternative substances that can avoid these drawbacks, maintain blood glucose homeostasis during medication, and prevent large fluctuations in blood glucose levels.

[0004] Neuropeptides are biologically active peptides secreted by neuroendocrine cells. After intracellular cleavage and modification, they are stored at axonal terminals and released extracellularly upon appropriate stimulation to bind to corresponding receptors and exert their functions. Besides being enriched in the central nervous system, neuropeptides are also widely distributed in systems such as the gastrointestinal tract. These neuropeptides play roles in regulating metabolism, growth, memory, and sleep in various systems. The precursor protein of the active peptide in this study is named chromogranin A (CMGA), a classic precursor protein for expressing neuropeptides. This precursor protein, after cleavage and processing, can form multiple functional neuropeptides, with expression products including chromogranin, trypsin, WE-14, kraftine, GE-25, and serine protease inhibitors. However, currently known mature neuropeptide data represent only a small portion of the neuropeptide segments expressed by these precursor proteins, and the functions of some neuropeptides remain unexplored. Therefore, this study explores the functions of the cleaved peptide segments of CMGA to provide a new approach and broaden the selection of glycemic control drugs. Summary of the Invention

[0005] The problem to be solved by this invention is to find neuropeptides and peptide hormones with potential functions, explore their functions, and study how these neuropeptides can improve animal metabolism, achieve natural and healthy weight loss, and regulate blood sugar.

[0006] From another perspective, the problem that this invention aims to solve is how to quickly reduce the blood glucose concentration when it rises without reducing it too much, thereby maintaining the homeostasis of blood glucose.

[0007] To address the above technical problems, the present invention provides the application of a polypeptide or its pharmaceutical salt, wherein the polypeptide is a CMGA active peptide, and the polypeptide has at least one of the following properties:

[0008] A1. Reduce animal weight.

[0009] A2. Altering metabolic disorders in animals

[0010] A3. Promote increased production capacity

[0011] A4. Improves glucose tolerance in animals.

[0012] A5. Increases the rate at which glucose levels drop in animal blood.

[0013] A6. To maintain the homeostasis of glucose in animal blood.

[0014] A7. Treatment and / or prevention of obesity in animals.

[0015] The polypeptide provided by this invention is the polypeptide whose amino acid sequence is SEQ ID No. 1 in the sequence listing.

[0016] The application of the polypeptides described in this article in the preparation of drugs for regulating blood glucose in animals is also within the scope of protection of this invention.

[0017] The application of the medicinal salt described in this invention in the preparation of drugs for regulating blood glucose in animals is also within the scope of protection of this invention.

[0018] In the above applications, the drug has at least one of the following properties:

[0019] A1. Reduce animal weight.

[0020] A2. Altering metabolic disorders in animals

[0021] A3. Promote increased production capacity

[0022] A4. Improves glucose tolerance in animals.

[0023] A5. Increases the rate at which glucose levels drop in animal blood.

[0024] A6. To maintain the homeostasis of glucose in animal blood.

[0025] A7. Treatment and / or prevention of obesity in animals.

[0026] The pharmaceutical salts of the aforementioned polypeptides also fall within the scope of protection of this invention.

[0027] The pharmaceutical-grade polypeptide salts of the present invention include acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, and methyl bromomethane. omide, bromide, methyl nitrate, calcium edetate, methyl sulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium salts Onion salt, dihydrochloride, oleate, ethylenediaminetetraacetate, oxalate, ethylenedisulfonate, emmonate (dihydroxynaphthyl salt), estolate, palmitate, esylate, pantothenate, fumarate, phosphate / diphosphate ), gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycolyllarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine, succinateHydrobromide, tannate, hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, triethyliodide, lactate, valerate, etc. Depending on their intended use, pharmaceutical salts can be formed from cations such as sodium, potassium, aluminum, calcium, lithium, manganese, zinc, and bismuth, or from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts can be prepared using standard methods, such as by reacting free acids with organic or inorganic bases. In the presence of a basic group, such as an amino group, acidic salts such as hydrochlorides, hydrobroms, acetates, and pamoates can be used as dosage forms; in the presence of an acidic group (such as -COOH) or an alcohol group, pharmaceutically acceptable esters such as acetates, maleates, and pivaloyloxymethyl acetate, as well as esters known in the literature for improving solubility and hydrolysis, can be used as sustained-release and prodrug formulations.

[0028] This invention provides a composition for regulating blood glucose in animals.

[0029] The above composition contains the aforementioned polypeptide or pharmaceutical salt. The active ingredient of the above composition may be the polypeptide or its pharmaceutical salt, and the active ingredient of the above composition may also contain other biological or non-biological components. Other active ingredients of the above composition can be determined by those skilled in the art based on the composition's effect on regulating blood sugar.

[0030] The above composition may be a drug.

[0031] In practical applications, the polypeptides or their pharmaceutical salts of the present invention can be administered directly to patients as drugs, or mixed with suitable carriers or excipients before administration. The carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Various dosage forms can be formulated using these materials, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and lyophilized powder injections. Suppositories can be vaginal suppositories, vaginal rings, or ointments, creams, or gels suitable for vaginal application. They can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose. Various carriers known in the art can be widely used to formulate unit dosage forms into suppositories. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.To formulate unit-dose dosage forms for injection, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Additionally, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injection formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Moreover, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation.

[0032] The above dosage forms can be used for administration via injection, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracerebrospinal injection or infusion, etc.; cavity administration, such as rectal, vaginal and sublingual; respiratory administration, such as nasal administration; and mucosal administration.

[0033] The dosage of the polypeptides and their derivatives of the present invention, or their pharmaceutical salts or compositions thereof, depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific active ingredient used, the route of administration and the frequency of administration, etc. The above dosage can be administered in a single dose or in several doses, such as two, three or four doses.

[0034] For any given patient, the specific effective therapeutic dose level must be determined based on a number of factors, including the disorder being treated and its severity; the activity of the specific active ingredient used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific active ingredient; the duration of treatment; medications used in combination with or concurrently with the specific active ingredient; and similar factors known in the medical field. For example, it is practiced in the art to start the dose of the active ingredient below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0035] In this document, the composition has at least one of the following properties:

[0036] A1. Reduce animal weight.

[0037] A2. Altering metabolic disorders in animals

[0038] A3. Promote increased production capacity

[0039] A4. Improves glucose tolerance in animals.

[0040] A5. Increases the rate at which glucose levels drop in animal blood.

[0041] A6. To maintain the homeostasis of glucose in animal blood.

[0042] A7. Treatment and / or prevention of obesity in animals.

[0043] In this document, the animal may be a vertebrate. The vertebrate may be a mammal. The mammal may be a human or a mouse, etc.

[0044] In this article, the changes described can be improvements.

[0045] This invention relates to a bioactive endogenous peptide discovered and identified in the mouse intestine. It is a naturally occurring bioactive peptide released after cleavage modification following the expression of the CMGA precursor protein. This bioactive peptide consists of 11 amino acids, is readily soluble in water, and can be dissolved in high concentrations in water or physiological saline. It is stable when stored at -80°C. Mouse animal experiments have demonstrated that this peptide enhances metabolic thermogenesis and regulates blood glucose levels. It can rapidly reduce blood glucose concentration when elevated without significantly decreasing it, thus playing an important therapeutic role in maintaining blood glucose homeostasis. In vivo studies reveal the activity of the CMGA bioactive peptide and its application in metabolic thermogenesis and blood glucose regulation. Mouse animal models confirm the role of the CMGA bioactive peptide in metabolic and blood glucose regulation, providing direction for the development of metabolic drugs and the treatment of clinical diseases. Attached Figure Description

[0046] Figure 1 This is a principal component analysis diagram of peptides.

[0047] Figure 2 The expression intensity of CMGA active peptide in the sample.

[0048] Figure 3 Comparison of secondary spectra of endogenous peptides (top) and synthetic peptides (bottom).

[0049] Figure 4 This is a graph showing the changes in body weight of mice after continuous drug interference.

[0050] Figure 5 This is a graph showing the changes in heat production in mice.

[0051] Figure 6 This is a graph showing the total heat production of mice during the day and night.

[0052] Figure 7 This is a graph showing changes in blood glucose levels during glucose tolerance testing.

[0053] Figure 8 This is a graph showing changes in blood glucose levels due to insulin resistance.

[0054] Figure 9 This study compares and analyzes the conservation of CMGA active peptides at the amino acid sequence and base level. Detailed Description of the Embodiments

[0055] The present invention is further described in detail below with reference to specific embodiments. The given examples are only for the purpose of illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation on the present invention.

[0056] Unless otherwise specified, all experimental methods in the following examples are conventional methods, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents and the like used in the following examples are all commercially available unless otherwise specified.

[0057] Example 1 Discovery and Preparation of CMGA Active Peptide

[0058] 1. Modeling method of fasting model

[0059] Neuropeptides are polypeptides widely present in both the nervous system and the gastrointestinal system. The secretion of polypeptides in the intestinal tract is affected by food intake and the environment. It has been reported that fasting can promote the secretion of neuropeptides by intestinal cells or stimulate their expression.

[0060] 7-week-old male C57BL / 6 mice weighing 23-25 g (purchased from SPF (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0010, wherein the maintenance feed for rats and mice was purchased from SPF (Beijing) Biotechnology Co., Ltd. and meets the GB14924.3-2010 standard) were randomly divided into three groups, with 5-6 mice in each group. The three groups of mice were treated with normal feeding (con), 1-day fasting (1D), and 2-day fasting (2D) respectively. The feed for the adapted and normally fed mice was the maintenance feed for rats and mice. After the modeling was completed, the entire colon of the mice was sampled.

[0061] 2. Extraction of neuropeptides in the colon

[0062] The colon, which is relatively enriched in neuropeptides, was selected as the research target for the changes of neuropeptides in the intestinal tract. The colons of the three groups of mice after successful modeling were ground and extracted with 90% acidified methanol, and the extraction steps are as follows:

[0063] The tissues were respectively placed into glass grinders with a volume of 1000 μL, 500 μL of acidified methanol was added for grinding, until the tissues and the grinding liquid became a homogenate state without obvious tissue blocks and precipitates. The homogenate was transferred into EP tube 1 of 1.5 mL, and the grinder was washed repeatedly 2 times with 200 μL of acidified methanol, and the washing liquids were combined into EP tube 1. The collected homogenate was subjected to water bath ultrasonication, and then placed in a low-temperature centrifuge (4°C) for centrifugation at 14000 g for 15 min.

[0064] Collect the supernatant into a new EP tube 2. Resuspend the precipitate in 90% acidified methanol, centrifuge at low temperature for 14000g for 15 minutes, and collect the supernatant, combining it with EP tube 2. Evaporate the supernatant in EP tube 2 to dryness at 45℃ in a centrifugal dryer for later use.

[0065] Sample desalting: Resuspend the peptides in 0.1% formic acid (phase A), vortex for 30 seconds until the peptides are completely dissolved in phase A, and then use a C18 reverse-phase chromatography column to remove salts from the sample. The desalting steps are as follows:

[0066] Activation: The desalting column was rapidly activated with a high organic phase of 100% acetonitrile, repeated 3 times.

[0067] Equilibrium: Column equilibration was performed using 0.1% formic acid, repeated 3 times.

[0068] Sample adsorption: The peptides were adsorbed onto the membrane slowly using a suction method, repeated 10 times.

[0069] Desalination: Wash away salt with 0.1% formic acid, repeat 3 times.

[0070] Elution: Elute the peptides with 50% acetonitrile by rapidly blowing up and down 40 times.

[0071] The eluted peptides were then evaporated again at 45°C using a centrifuge until no moisture was visible and no obvious precipitation was observed. These peptides were then used for subsequent mass spectrometry analysis.

[0072] 3. Mass spectrometry data analysis

[0073] Raw mass spectrometry data were used for non-standard quantification after database searching in Peaks Studio. Principal component analysis was performed on all peptides from the three groups of mice. The results are as follows: Figure 1 As shown, the total peptide clusters of the three groups of mice were significantly separated, and the clusters of the fasting group became increasingly separated from the con group as the fasting time increased, indicating that the neuropeptide composition of mice changed more significantly with increasing fasting time. Based on this, screening revealed that the expression abundance of CMGA active peptides increased with increasing fasting time, such as... Figure 2 As shown, the expression abundance of CMGA active peptides gradually increased, and the significance gradually increased with the increase of fasting time. One-way ANOVA was used for analysis.

[0074] Compare the original spectra of the peptide in the Peaks Studio analysis software as follows: Figure 3 As shown in the figure above, its secondary spectrum is complete and the data is reliable, suggesting that it may have a certain regulatory effect in fasting mice.

[0075] The precursor protein of this peptide, chromogranin A, is one of the expression precursors of neuropeptides. After aligning this peptide segment with the precursor protein, it is found that the amino acid sequences before and after it conform to the classical cleavage model of neuropeptides, indicating that it is a potential neuropeptide or peptide hormone. This peptide contains 11 amino acids, with molecular weights of MH 1248.6140; MH-H2O 1230.6035; MH-NH3 1231.5875. Its precursor protein is CMGA, and it is tentatively defined as CMGA active peptide (CMGA peptide).

[0076] The expression level of this peptide in the colon increases after fasting, and the expression level is up-regulated as the fasting time prolongs. Based on this, we conducted verification on the function of this peptide. After the peptide segment was artificially synthesized, the synthesized peptide segment matched the original endogenous secondary spectrogram consistently. The analyzed spectrogram is as shown in Figure 3 below.

[0077] The CMGA active peptide is a polypeptide whose amino acid sequence is Sequence 1 in the Sequence Listing. The CMGA active peptide used in the following tests was synthesized by Anhui Guoping Pharmaceutical Co., Ltd., with a purity greater than 98% and stored in a lyophilized state.

[0078] Example 2. Application of CMGA active peptide in body weight regulation of model animal mice

[0079] 1. Laboratory animals and treatment

[0080] Intervention method of CMGA active peptide: 7-week-old male C57BL / 6 mice weighing 23-25 g (purchased from SPF Biotechnology Co., Ltd., License No.: SCXK(Jing)2019-0010) were used. The maintenance feed for mice and rats was purchased from SPF Biotechnology Co., Ltd., which meets the GB14924.3-2010 standard. The mice were randomly divided into two groups, with 6-7 mice in each group. Normal feeding was maintained during the period, and the feed for the acclimatized and normally fed mice was the maintenance feed for mice and rats. The two groups of mice were divided into two groups: one group was injected with 100 μL of normal saline and designated as the normal saline group, and the other group was injected with 100 μL of CMGA active peptide and designated as the active peptide group. The CMGA active peptide was dissolved in normal saline to a final concentration of 0.1 g / μL. Mice in each group were intraperitoneally injected with the corresponding drug daily, and their body weights were weighed.

[0081] After the mice were administered for one week, the variation range of their body weights is as shown in Figure 4 below. The body weight of the mice in the active peptide group increased at a slower rate than that of the mice in the normal saline group, indicating that the CMGA active peptide can slow down the body weight growth rate of mice.

[0082] Example 3. Application of CMGA active peptide in model animal mice

[0083] 1. Laboratory animals and treatment

[0084] Male C57BL / 6 mice aged 7 weeks and weighing 23-25 g (purchased from SPF (Beijing) Biotechnology Co., Ltd., License No.: SCXK (Beijing) 2019-0010) were adaptively reared in an SPF mouse room at 22-25°C with free access to drinking water and food. After 7 days of adaptation, the mice for metabolic detection were reared adaptively for 3 days in metabolic cages at one mouse per cage, and fed with maintenance feed. The maintenance feed for rats and mice was purchased from SPF (Beijing) Biotechnology Co., Ltd. and meets the GB14924.3-2010 standard. During the adaptation period, the mice had free access to food and unlimited drinking water. Healthy male mice aged 8 weeks, weighing 23-25 g and with blood glucose levels in the normal range were randomly divided into 2 major groups, which were used for metabolic detection, glucose tolerance test and insulin resistance test respectively.

[0085] 1) Metabolic thermogenesis detection

[0086] There are 3-4 mice in each group: 3 mice in the fasting + 0.9% normal saline group (normal saline group) and 4 mice in the fasting + 40 μg CMGA active peptide group (CMGA active peptide group). For the mice in the fasting + normal saline group, the feed was removed after administration, and each mouse was intraperitoneally injected with 0.1 mL of 0.9% normal saline; for the mice in the fasting + CMGA active peptide group, the feed was removed after administration, and each mouse was intraperitoneally injected with 0.1 mL of CMGA active peptide solution (the solute is CMGA active peptide, the solvent is 0.9% normal saline, and the final concentration is 400 μg / mL), that is, the administration dose is 40 μg CMGA active peptide per mouse, and the thermogenesis change of the mice was detected.

[0087] 2) Glucose tolerance test and insulin resistance test

[0088] There are 5 mice in each group: free feeding + 0.9% normal saline group (normal saline group), free feeding + 10 μg CMGA active peptide group (CMGA active peptide group). The mice in the above 2 groups were administered once a day at 2 p.m., and the specific administration method is as follows: each mouse in the normal saline group was intraperitoneally injected with 0.1 mL of 0.9% normal saline per day; each mouse in the CMGA active peptide group was intraperitoneally injected with 0.1 mL of CMGA active peptide solution (the solute is CMGA active peptide, the solvent is 0.9% normal saline, and the final concentration is 100 μg / mL) per day, that is, the administration dose is 10 μg CMGA active peptide per mouse. The administration by intraperitoneal injection was continued for 7 days, and then the glucose tolerance and insulin resistance capabilities of the mice were tested.

[0089] 2. Determination of mouse function

[0090] 1) Determination of mouse metabolic indicators

[0091] The metabolic cage was tested using a Columbia 950NORTH HAGUE AVENUE OHI0 43204USA 8-channel metabolic analyzer with the following parameters: carbon dioxide concentration 0.05%, oxygen concentration 20.9%, flow rate 0.5 LPM, and detection pressure 800 mmHg.

[0092] Metabolic cages were used to detect the metabolism of mice: the caloric output of the mice was measured continuously for 24 hours, and the data were directly output and analyzed by software. The results of the mouse metabolic detection are as follows: Figure 5 As shown, the results indicated that within 24 hours after administration, the caloric output of mice in the CMGA active peptide group was significantly higher than that in the saline group. The cumulative daytime and nighttime caloric output of the mice were analyzed, and the results are as follows: Figure 6 As shown, the active peptide group produced more heat at night than the saline group. In summary, this active peptide can promote thermogenesis and enhance metabolism in mice.

[0093] 2) Glucose tolerance test (GTT) in mice

[0094] After the above administration method was completed, a glucose tolerance test was performed on mice. The animals were fasted for 12 hours before the experiment. A 50% glucose solution was injected intraperitoneally into the mice at a dose of 2 g / kg body weight. The fasting blood glucose (FBG) value of the mice was measured before intraperitoneal injection, and blood was collected from the tail vein at 0, 15, 30, 45, 60, and 120 minutes after glucose administration, and the blood glucose concentration was measured using a glucometer.

[0095] Statistical analysis: Data are expressed as mean ± SD and analyzed using t-tests. GraphPadPrism 9 software was used for multiple comparisons to determine differences; P < 0.05 was considered statistically significant.

[0096] The results are as follows Figure 7 As shown, under normal eating conditions, the rate of blood glucose rise was consistent in both the CMGA active peptide group and the saline group after intraperitoneal injection of glucose, reaching its peak at 15 minutes post-injection. Subsequently, blood glucose concentrations began to decrease in both groups, with the CMGA active peptide group showing a faster rate of decline than the saline group. At 45 minutes post-injection, the CMGA active peptide group exhibited the most significant decrease in blood glucose compared to the saline group. In conclusion, CMGA active peptides can more quickly lower blood glucose concentration when it rises, maintaining blood glucose at normal levels and improving glucose tolerance.

[0097] 3) Insulin Resistance Test (ITT) in Mice

[0098] After the above administration method was completed, the mice underwent an insulin resistance test. Mice were fasted for 6 hours prior to the experiment, and insulin (Beyotime insulin P3376-100 IU) was administered intraperitoneally at a dose of 0.75 IU / kg. Fasting blood glucose levels were measured before intraperitoneal injection, and blood was collected from the tail vein at 0, 15, 30, 45, 60, and 120 minutes after intraperitoneal administration. Blood glucose concentrations were measured using a glucometer.

[0099] Statistical analysis: Data are expressed as mean ± SD and analyzed using t-tests and one-way repeated measures ANOVA. GraphPad Prism 9 software was used for multiple comparisons to determine differences; p < 0.05 was considered statistically significant.

[0100] The results are as follows Figure 8 As shown, under normal feeding conditions, using the initial glucose concentration as a baseline, the subsequent changes in glucose were normalized. The experimental results showed that the rate of glucose decline in the CMGA active peptide group mice was consistent with the control group in the first 15 minutes, and did not affect the insulin sensitivity of the mice. Under autoregulation, the rate of glucose recovery was higher than that in the saline group. At 120 minutes, the blood glucose concentration in the CMGA active peptide group was significantly higher than that in the saline group. In summary, this indicates that CMGA active peptides can regulate the concentration of glucose in the blood, and the rapidly declining glucose level in the body under the interference of external insulin can be regulated to a normal level by CMGA active peptides.

[0101] This invention screened novel peptides that regulate metabolism and blood glucose. Experiments using the C57BL / 6 mouse model demonstrated that these bioactive peptides regulate body weight, significantly increasing caloric production and slowing weight gain in mice under normal feeding conditions. Continuous intraperitoneal injection of the CMGA bioactive peptide resulted in increased blood glucose regulation in mice, effectively controlling blood glucose homeostasis.

[0102] Example 4: Conservation Analysis of CMGA Active Peptides

[0103] Based on the above research, the conservation of the amino acid sequence and the conservation of the coding sequence of this peptide in different species were analyzed. The results are as follows... Figure 9 As shown, the amino acid sequence of this peptide ( Figure 9 The peptide (top and middle figures) exhibits high conservation in human, mouse, bovine, and equine cultures; further analysis revealed the conservation of the encoding gene, specifically the gene coding sequence of this peptide (…). Figure 9 The lower part of the image (in the middle section) is conservative.

[0104] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. SEQUENCE LISTING <110> Beijing Proteome Research Center Military Medical Research Institute of the Academy of Military Sciences of the Chinese People's Liberation Army <120> An endogenous bioactive peptide and its application <130> GNCJF221423 <140> 202210696220.7 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <400> 1 Met Asp Gln Leu Ala Lys Glu Leu Thr Ala Glu 1 5 10

Claims

1. The use of a polypeptide or its pharmaceutical salt in the preparation of a medicament for maintaining glucose homeostasis in animal blood, characterized in that, The polypeptide is a CMGA active peptide, and the polypeptide has at least one of the following properties: A1. Slows down the rate of weight gain in animals. A2. Promotes increased heat production. A3. Improves glucose tolerance in animals. A4. Increases the rate at which glucose levels in animal blood decrease. The amino acid sequence of the polypeptide is shown in SEQ ID No. 1 in the sequence listing.

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