A small molecule peptide and application thereof in preparing an obesity improvement product

Abstract

The application discloses a kind of small molecule peptides and application in preparation of improving obesity product, a kind of small molecule peptide, its amino acid sequence is: leucine-lysine-leucine-lysine-leucine-leucine, is abbreviated as LKLKLL, experiment proves, the small molecule peptide LKLKLL of the application can significantly promote the secretion of intestinal incretin (GLP-1 and GIP), to help regulate energy metabolism.New target is provided for the treatment of obesity, reduce body weight and improve insulin sensitivity.Small molecule peptide LKLKLL in promoting the effect of intestinal incretin secretion is significantly better than existing positive control, such as glutamine, shows stronger biological activity.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine or health food, specifically a small molecule peptide and its application in the preparation of products that improve obesity. Background Technology

[0002] With the high incidence of obesity and metabolic diseases worldwide, developing effective strategies for treating and preventing obesity has become an important research direction. Incretin hormones play a crucial role in regulating energy metabolism and controlling blood glucose levels, and are currently a hot topic in metabolic disease research. Incretins include glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP), which significantly enhance glucose-dependent insulin secretion by acting on pancreatic β-cells. Studies show that incretins contribute approximately 50%-65% to postprandial insulin secretion, with GIP accounting for about two-thirds of this contribution.

[0003] In obesity and related metabolic disorders, the secretion and function of incretins are often impaired, manifesting as a reduced incretin effect. The delayed peak insulin secretion following oral glucose administration indicates weakened incretin regulation. This reduced effect may be related to insufficient incretin secretion or decreased sensitivity. Therefore, enhancing incretin secretion or improving its function is considered a potential strategy for alleviating obesity and related metabolic problems.

[0004] Recent studies have revealed the crucial role of gut microbiota in host metabolic regulation, particularly certain probiotics such as Akkermansia muciniphila (AKK). AKK bacteria significantly improve host metabolism by modulating the intestinal environment, enhancing intestinal barrier function, and boosting incretin secretion. For instance, its metabolites (such as short-chain fatty acids) can promote GLP-1 secretion; its membrane protein Amuc_1100 regulates the gut microbiota by enhancing intestinal barrier integrity, thus contributing to increased GLP-1 secretion; furthermore, its secretory protein P9 can directly induce GLP-1 secretion and enhance thermogenesis in brown adipose tissue. These studies demonstrate the significant potential of gut microbiota and their metabolites in improving metabolic disorders.

[0005] However, research on specific active molecules that promote incretin secretion, especially small peptides, remains limited. No studies have reported that the small peptide LKLKLL improves obesity and related metabolic disorders by enhancing GLP-1 and GIP secretion. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a small molecule peptide.

[0007] The second objective of this invention is to provide an application of small molecule peptides in the preparation of products that improve obesity.

[0008] A third objective of this invention is to provide a composition in which the aforementioned small molecule peptide is the sole active ingredient.

[0009] A fourth object of the present invention is to provide the use of the above composition in the preparation of products for improving obesity.

[0010] The technical solution of this invention is summarized as follows:

[0011] A small molecule peptide, wherein the amino acid sequence of the small molecule peptide is: leucine-lysine-leucine-lysine-leucine, abbreviated as LKLKLL.

[0012] The above-mentioned small molecule peptide is used in the preparation of products to improve obesity.

[0013] The above products are either health foods or medicines.

[0014] For health food products, oral health food products are preferred; for medicines, oral medicines are preferred.

[0015] A composition in which one of the above-mentioned small molecule peptides is the sole active ingredient.

[0016] The application of the above composition in the preparation of products for improving obesity.

[0017] The above-mentioned products are preferably health foods or medicines.

[0018] The above-mentioned health food products are preferably oral health food products; the medicines are preferably oral medicines.

[0019] Advantages of this invention:

[0020] Experiments have demonstrated that the small molecule peptide (LKLKLL) of this invention can significantly promote the secretion of incretins (GLP-1 and GIP), thereby helping to regulate energy metabolism. It also provides a new target for the treatment and prevention of obesity, reducing body weight and improving insulin sensitivity. The small molecule peptide LKLKLL is significantly more effective than existing positive controls, such as glutamine, in promoting incretin secretion, exhibiting stronger biological activity. Attached Figure Description

[0021] Figure 1 An experiment was conducted to investigate the intervention of a small molecule peptide, LKLKLL, and glutamine (Gln) in STC-1 cells using CCK-8 assays (A: LKLKLL; B: Gln).

[0022] Figure 2 The incretin secretion function of a small molecule peptide, LKLKLL, was detected by ELISA, wherein:

[0023] A: LKLKLL (LK for short) promotes GLP-1 secretion;

[0024] B: LKLKLL promotes GIP secretion;

[0025] C: LKLKLL promotes GLP-1 secretion in a gradient;

[0026] D: LKLKLL promotes GIP secretion in a gradient.

[0027] Figure 3 The effect of a small peptide, LKLKLL, on incretin gene expression, wherein:

[0028] A: qPCR method for detecting GIP mRNA expression;

[0029] B: qPCR method for detecting proglucagon (PRO) mRNA expression;

[0030] C: qPCR method for detecting PCSK1 / 3 mRNA expression of endonucleases;

[0031] D: qPCR method for detecting PCSK2 mRNA expression;

[0032] E: qPCR method was used to detect the expression of GIP transcription factor PDX1 mRNA;

[0033] F: qPCR method was used to detect the expression of GIP transcription factor GATA4 mRNA.

[0034] Figure 4 For the targeted metabolomics and tolerability evaluation of a small molecule peptide LKLKLL, wherein:

[0035] A: Targeted metabolomics;

[0036] B: Tolerance test for artificial gastric juice (referred to as gastric juice);

[0037] C: Tolerance test for artificial intestinal fluid (hereinafter referred to as intestinal fluid).

[0038] Figure 5 A small molecule peptide, LKLKLL, was developed to improve obesity induced by a high-fat diet.

[0039] Figure 6 A small molecule peptide, LKLKLL, inhibits fat accumulation induced by a high-fat diet, wherein:

[0040] A: White adipose tissue of the epididymis;

[0041] B: White adipose tissue in the groin.

[0042] Figure 7A small molecule peptide, LKLKLL, was developed to improve the morphological changes in adipose tissue induced by a high-fat diet. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0044] The reagents or formulations used in the following examples:

[0045] BHI liquid medium: Brain Heart Extract (BHI) broth medium, 0.4% mucin, 3 g / L threonine, 2.5 g / L N-acetyl-D-glucosamine, 0.05% L-cysteine ​​hydrochloride anhydrous. Solid medium is liquid medium with 2% agar added.

[0046] Complete cell culture medium: 10% fetal bovine serum (FBS), 1% penicillin-streptomycin solution, and 44.5 mL of DMEM high glucose medium.

[0047] High-glucose complete culture medium: 10% fetal bovine serum (FBS), 1% penicillin-streptomycin solution, 55.5 mmol / L glucose, and 44.5 mL DMEM medium.

[0048] Mouse intestinal endocrine cell line (STC-1 cells): purchased from BeiNa Biotechnology Co., Ltd.

[0049] Akkermansia muciniphila ATCC BAA-835, abbreviated as AKK, was purchased from Beina Biotechnology Co., Ltd.

[0050] The ELISA kit for measuring GLP-1 / GIP content was purchased from Wuhan Elite Biotechnology Co., Ltd.; the CCK-8 kit was purchased from Shanghai Taoshu Biotechnology Co., Ltd.; the total RNA extraction kit was purchased from Tiangen Biotech Co., Ltd.; and the SYBR green fluorescent polymerase chain reaction kit and reverse transcription kit were purchased from Shandong Cisco Biotechnology Co., Ltd.

[0051] LKLKLL was synthesized by Beijing BaiTai Biotechnology Co., Ltd.

[0052] 1. Identification and synthesis of small molecule peptides: The amino acid sequence of the small molecule peptide was extracted and identified as LKLKLL from ATCCBAA-835 Akkermansia myxophilus, and its pure product was obtained by chemical synthesis.

[0053] 2. Bioactivity assessment: The bioactivity of LKLKLL was systematically assessed using in vitro experiments (STC-1 cells) and animal models, with a focus on its promoting effect on the secretion of incretins (GLP-1 and GIP) and its inhibitory effect on obesity induced by a high-fat diet.

[0054] 3. Mechanism study: The specific mechanism by which LKLKLL promotes incretin secretion will be analyzed using molecular biology techniques.

[0055] 4. Animal studies: Further validate the effectiveness of LKLKLL in improving metabolic status and reducing body weight in animal models, laying the foundation for future clinical applications.

[0056] Example 1

[0057] A small molecule peptide, LKLKLL, promotes GIP / GLP-1 secretion.

[0058] 1. Preparation of Small Molecule Peptides: After anaerobic culture of AKK bacteria in complete cell culture medium for 2 hours, non-targeted metabolomics analysis revealed a significant increase in the content of a small molecule peptide (the amino acid sequence of the small molecule peptide is: leucine-lysine-leucine-lysine-leucine, abbreviated as LKLKLL). Subsequently, it was synthesized chemically. 1 mg of LKLKLL was removed from a -20℃ freezer and allowed to recover its activity at room temperature for 1 hour. It was then added to high-glucose complete culture medium to prepare a 5 mM stock solution. After mixing, it was sonicated for 5 minutes to aid dissolution and then briefly centrifuged for later use.

[0059] 2. Resuscitation of mouse intestinal endocrine cell line (STC-1 cells)

[0060] Remove STC-1 cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath. Transfer 1 mL of the cell suspension to a 15 mL centrifuge tube and add 2 mL of complete cell culture medium. Centrifuge at 800 rpm for 5 minutes, discarding the supernatant. Add 6 mL of complete cell culture medium and gently pipette to mix the cells. Transfer the mixture to a culture flask and incubate at 37°C in a cell culture incubator containing 5% CO2. Depending on the cell condition, change the complete cell culture medium every 1-2 days until the cells reach confluence.

[0061] 3. Cell viability assay (CCK-8)

[0062] The cell suspension obtained in step 2 was seeded into 96-well plates. After adhesion, STC-1 cells were treated with a concentration gradient of LKLKLL obtained in step 1 and commercially available Gln for 24 hours, after which the culture medium was discarded. Cell viability was assessed using a CCK-8 cell counting kit: 100 μL of complete cell culture medium and 10 μL of CCK-8 reagent were added to each well, and the cells were cultured for another 0.5 hours. Cell viability was assessed by measuring absorbance at 450 nm using a multi-mode microplate reader. (See [link to relevant documentation]). Figure 1 . Figure 1 An experiment was conducted to investigate the intervention of a small peptide, LKLKLL, and glutamine (Gln) in STC-1 cells using CCK-8 assays (A: LKLKLL; B: Gln).

[0063] 4. The in vitro incretin activity of the small molecule peptide LKLKLL was determined.

[0064] STC-1 cells were starved with HBSS for 1 hour and then treated with LKLKLL and Gln for 24 hours, respectively. A blank control group was set up. After the intervention, the cell supernatant was collected, centrifuged at 1000 rpm for 20 min, and the supernatant was collected. The in vitro incretin activity of the small molecule peptide LKLKLL was measured using an ELISA kit. The ELISA plate was set up with blank wells, standard wells, and sample wells. 100 μL of sample diluent was added to the blank wells, and 100 μL of standard or sample was added to the remaining wells. Cover the microplate with a membrane and incubate at 37°C for 90 min; (prepare biotinylate antibody working solution 15 min in advance), agitate dry, add 100 μL of biotinylate antibody working solution to each well, cover the microplate with a membrane, and incubate at 37°C for 1 h; (prepare washing buffer and HRP working solution in advance), discard the liquid in the wells, agitate dry, wash the plate 3 times with 350 μL of washing buffer for 1 min each time, agitate dry and pat dry on absorbent paper; add 100 μL of HRP working solution to each well, cover with a membrane, and incubate at 37°C for 30 min; discard the liquid in the wells and agitate dry.

[0065] Wash the plate 5 times with washing buffer; add 90 μL of substrate solution to each well, cover the plate with a membrane, and incubate at 37°C in the dark for 15 min; add 50 μL of stop solution to each well to stop the reaction, at which point the blue color immediately turns yellow; immediately measure the optical density (OD value) of each well at 450 nm using a microplate reader. Note: The concentrations of LKLKLL and Gln in this experiment were 800 μmol / L. Results are shown below. Figure 2 .

[0066] 5. Effects of the small molecule peptide LKLKLL on incretin gene expression

[0067] A control group, an LKLKLL experimental group, and a Gln-positive control group were set up. Cells were treated for 24 hours. After treatment, total RNA was extracted from the cells, and the expression levels of trypsin genes (GIP (Gene ID: 14607), Proglucagon (Pro (Gene ID: 14526), ​​PCSK1 / 3 (Gene ID: 18548), PCSK2 (Gene ID: 18549), PDX1 (Gene ID: 18609), GATA4 (Gene ID: 14463)) were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). First, total RNA was extracted from the treated STC-1 cells using a total RNA extraction kit. According to the kit instructions, the cells were treated with lysis buffer, and the supernatant was collected by centrifugation. The resulting RNA precipitate was resuspended in RNase-free water, and its concentration and purity were determined. cDNA synthesis: Total RNA was used for reverse transcription using the SPARKscript II RT Plus Kit (With gDNA Eraser). First, the DNA was incubated with a gDNA Eraser to remove the genome, then 2×SPARKscript II RTPlus Master Mix was added, and the reaction was carried out at 50°C for 15 min, followed by incubation at 85°C for 5 min to inactivate the reverse transcriptase, finally yielding cDNA. qRT-PCR reaction setup: The qRT-PCR reaction system was prepared, and amplification was performed using the SYBR Green Fluorescent Polymerase Chain Reaction Kit (see Tables 1, 2, and 3).

[0068] Table 1: PCR reaction system

[0069]

[0070] Table 2: PCR reaction procedure

[0071]

[0072] Table 3: Primer sequences

[0073]

[0074] 6. Test Results

[0075] The expression levels of GIP and GLP-1 after LKLKLL intervention were detected by ELISA and qPCR. It was found that a small peptide, LKLKLL, can promote incretin secretion in a gradient manner, with an effect superior to the positive control glutamine. Figure 2 , Figure 3 .

[0076] Example 2: Targeted metabolomics and tolerability evaluation of a small molecule peptide LKLKLL

[0077] 1. Targeted metabolomics of LKLKLL

[0078] (1) Experimental Method: This invention employs triple quadrupole mass spectrometry (Triple Quadrupole MS / MS) as a detection method for small molecule peptide-targeted metabolomics, which has high sensitivity and selectivity and is widely used in the qualitative and quantitative analysis of small molecule compounds in biological samples. Specific steps include: First, collecting the metabolic supernatant of Akkermansia muciniphila (AKK) bacteria cultured in high-glucose complete medium, collecting and filtering after 2 hours and 6 hours of culture, respectively. Then, adding 1.5 mL of 20% acetonitrile methanol extract, vortexing for 3 minutes, followed by centrifugation at 12000 r / min for 10 minutes at 4℃, and then transferring the supernatant (500 μL) to a sample vial. Chromatographic conditions: using a Waters Atlantis T3 Column, mobile phase A being 0.1% formic acid aqueous solution, mobile phase B being 0.1% formic acid acetonitrile solution, column temperature set at 40℃, flow rate at 0.4 mL / min, and injection volume at 3 μL. The separated components were detected using triple quadrupole mass spectrometry. The first two quadrupoles were used to select specific precursor and daughter ions to enhance signal selectivity and sensitivity. Finally, the mass spectrometry data were analyzed to quantify the abundance of small molecule peptides and compared with a control group to verify the detection results.

[0079] (2) Test Results

[0080] This invention successfully detected the content of the small peptide LKLKLL in the supernatant of *AKK* bacteria using triple quadrupole mass spectrometry. The results showed that the abundance of LKLKLL in the *AKK* bacteria supernatant was significantly higher than that in the control group, and the content at 6 h was higher than that at 2 h. Figure 4 A.

[0081] 2. Evaluation of tolerance to LKLKLL artificial gastric juice

[0082] (1) Test methods

[0083] LKLKLL was dissolved in 1 mL of artificial gastric fluid (pH=2) and 1 mL of ddH2O to prepare a 1000 ng / mL solution. After thorough mixing, the solution was placed at 37℃ for 1.5 h. The content of LKLKLL was immediately detected by triple quadrupole mass spectrometry, and the degradation rate was calculated.

[0084]

[0085] Where A0 is the content of LKLKLL dissolved by ddH2O, and An is the content of LKLKLL dissolved by artificial gastric juice.

[0086] (2) Test Results

[0087] The degradation of LKLKLL in artificial gastric fluid is as follows: Figure 4 B. Under simulated gastric fluid conditions at pH 2, the content of the small molecule peptide LKLKLL was almost unaffected, with a degradation rate of 2%.

[0088] 3. Evaluation of tolerance to LKLKLL artificial intestinal fluid

[0089] (1) Test methods

[0090] LKLKLL was dissolved in 1 mL of artificial intestinal fluid and 1 mL of ddH2O to prepare a 1000 ng / mL solution. After thorough mixing, the solution was placed in an environment of 37℃ for 4 h. The content of LKLKLL was immediately detected by triple quadrupole mass spectrometry, and the degradation rate was calculated.

[0091]

[0092] (2) Test Results

[0093] The degradation of LKLKLL in artificial intestinal fluid is as follows: Figure 4 C. Under artificial intestinal fluid conditions, the content of the small molecule peptide LKLKLL was almost unaffected, with a degradation rate of 0.2%.

[0094] Example 3: Effects of a small molecule peptide, LKLKLL, on weight gain and adipose tissue morphology induced by a high-fat diet.

[0095] 1. Test Methods

[0096] (1) Six-week-old male C57BL / 6 mice were selected and after 1 week of acclimatization, the mice were randomly divided into three groups: normal control group (normal diet, n=12), high-fat diet group (HFD, high-fat diet, n=16) and high-fat diet + LKLKLL group (experimental group, n=16).

[0097] The normal control group was given standard feed;

[0098] Both the high-fat diet group and the experimental group were given a high-fat diet with 60% fat content;

[0099] The experimental group was administered LKLKLL (25 mg / kg body weight) orally daily. The experiment lasted for 8 weeks, during which the body weight changes of the mice were recorded. The weight of the mice was measured at fixed times each week, and the body weight changes of each group were compared.

[0100] (2) Determination of adipose tissue weight: At the end of the experiment, the mice were sacrificed and the inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) were collected. The weights of iWAT and eWAT of each group of mice were weighed and statistically analyzed.

[0101] (3) HE (hematoxylin-eosin) staining of adipose tissue: Tissue fixation and embedding: Collected adipose tissue was fixed in 10% neutral buffered formaldehyde for 24 hours, followed by dehydration, embedding, and cutting into 4-6 micrometer thin sections. HE staining: Sections were stained with hematoxylin-eosin. Hematoxylin was used to stain cell nuclei, and eosin was used to stain cytoplasm and extracellular matrix. After staining, the morphology of adipocytes was observed under a microscope. Image analysis: The area and size of adipocytes were measured using image analysis software to evaluate the inhibitory effect of LKLKLL on adipocyte hypertrophy.

[0102] 2. Experimental Results

[0103] (1) LKLKLL inhibits high-fat diet-induced weight gain: Monitoring the body weight of C57BL / 6 male mice over an 8-week experiment revealed that mice in the high-fat diet (HFD) group showed a significant increase in body weight and significantly higher blood glucose levels than the normal diet group. In contrast, mice treated with LKLKLL (experimental group) showed significantly suppressed weight gain, indicating that LKLKLL has an inhibitory effect on high-fat diet-induced weight gain. Figure 5 .

[0104] (2) LKLKLL has an inhibitory effect on adipose tissue accumulation: At the end of the experiment, the weight of adipose tissue was measured. The results showed that the iWAT and eWAT weights of mice in the experimental group were significantly lower than those in the high-fat diet group, suggesting that LKLKLL has an inhibitory effect on adipose tissue accumulation. Figure 6 AB.

[0105] (3) HE staining results further verified the effect of LKLKLL on adipocyte hypertrophy: adipocytes in mice in the high-fat diet group were significantly enlarged, showing hypertrophic adipocyte morphology. LKLKLL significantly inhibited high-fat diet-induced adipose tissue hypertrophy and lipid accumulation. In white adipose tissue, the adipocyte volume in the LKLKLL intervention group was significantly reduced; in brown adipose tissue, lipid droplet enlargement was inhibited. Muscle tissue analysis showed that the myofiber structure in the LKLKLL group remained compact, reducing damage caused by the high-fat diet. This indicates that LKLKLL has a potential protective effect against high-fat diet-induced fat accumulation and muscle damage. Figure 7 .

[0106] In summary, the small molecule peptide LKLKLL provided by this invention can significantly promote the secretion of incretin (GIP / GLP-1), thereby regulating metabolic function. Furthermore, LKLKLL can effectively inhibit weight gain and lipid accumulation induced by a high-fat diet, demonstrating a significant fat-reducing effect. Through in vitro experiments and animal models, the small molecule peptide LKLKLL of this invention shows promising application prospects in promoting incretin secretion, improving metabolic diseases, and inhibiting obesity.

[0107] Based on the biological functions of LKLKLL, related health foods or medicines can be prepared, providing new options for the treatment of obesity and metabolic diseases.

[0108] Health foods are made into convenient oral dosage forms such as tablets, capsules, solutions, or granules using conventional technical methods.

[0109] The drug is made into a convenient oral dosage form, such as tablets, capsules, solutions or granules, using conventional technical methods.

[0110] A composition in which one of the above-mentioned small molecule peptides is the sole active ingredient.

[0111] The above composition is used in the preparation of products that promote the secretion of incretins and improve obesity.

[0112] The above-mentioned products are preferably health foods or medicines.

[0113] The above products are oral health foods or oral medicines.

[0114] The dosage forms of the above-mentioned oral health food products are tablets, capsules, solutions, or granules.

[0115] The dosage forms of the above-mentioned oral medications are tablets, capsules, solutions, or granules.

[0116] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A small molecule peptide, characterized in that, The amino acid sequence of the small molecule peptide is: leucine-lysine-leucine-lysine-leucine-leucine, abbreviated as LKLKLL.

2. The application of a small molecule peptide according to claim 1 in the preparation of a product for improving obesity; the product is a pharmaceutical product.

3. The application according to claim 2, characterized in that: The medicine in question is an oral medication.

4. A composition having a small molecule peptide as the sole active ingredient as claimed in claim 1.

5. The use of the composition of claim 4 in the preparation of an obesity-improving product; said product being a pharmaceutical product.

6. The application according to claim 5, characterized in that: The medicine in question is an oral medication.

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