Small molecule peptide and application of small molecule peptide in preparation of products for improving obesity

Abstract

The invention discloses a small molecule peptide and application thereof in preparation of products for improving obesity, the amino acid sequence of the small molecule peptide is leucine-lysine-leucine-lysine-leucine-leucine, LKLKLL for short, and experiments prove that the small molecule peptide LKLKLL can significantly promote secretion of enterotryptophan (GLP-1 and GIP), and can be used for improving obesity. Therefore, energy metabolism is helped to be adjusted. A new target spot is provided for obesity treatment, the body weight is reduced, and the insulin sensitivity is improved. The effect of the small molecule peptide LKLKLL in promoting secretion of the enterotrypsin is obviously better than that of the existing positive control, such as glutamine, and the small molecule peptide LKLKLL shows higher biological activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine or health food, and specifically relates to a small molecule peptide and its application in the preparation of a product for improving obesity. Background Art

[0002] With the high incidence of obesity and metabolic diseases worldwide, developing effective strategies to treat and prevent obesity has become an important research direction. Incretin hormones play a key role in regulating energy metabolism and controlling blood sugar levels, and are one of the current hot spots in metabolic disease research. Incretins include glucagon-like peptide-1 (GLP-1, Glucagon-Like Peptide-1) and gastric inhibitory peptide (GIP, Gastric Inhibitory Polypeptide), which significantly enhance glucose-dependent insulin secretion by acting on pancreatic beta cells. Studies have shown that incretins contribute about 50%-65% to postprandial insulin secretion, of which GIP accounts for about two-thirds.

[0003] In obesity and related metabolic disorders, the secretion and function of incretin are often affected, which is manifested as a reduction in the effect of incretin. The delay in the peak of insulin secretion after oral glucose in patients indicates that the regulatory effect of incretin is weakened. This weakened effect may be related to insufficient secretion of incretin or decreased sensitivity. Therefore, enhancing the secretion of incretin or improving its function is regarded as a potential strategy to alleviate obesity and related metabolic problems.

[0004] In recent years, studies have found that intestinal microorganisms play an important role in regulating host metabolism, especially certain probiotics, such as Akkermansia muciniphila (AKK). AKK bacteria significantly improve host metabolism by regulating the intestinal environment, improving intestinal barrier function, and enhancing incretin secretion. For example, its metabolites (such as short-chain fatty acids) can promote the secretion of GLP-1; its membrane protein Amuc_1100 regulates the intestinal flora by enhancing the integrity of the intestinal barrier, which helps to increase the secretion of GLP-1; in addition, its secretory protein P9 can directly induce GLP-1 secretion and enhance the thermogenic effect of brown adipose tissue. These studies show that intestinal microorganisms and their metabolites have important potential in improving metabolic disorders.

[0005] However, the research on specific active molecules that promote incretin secretion, especially small molecule peptides, is still relatively limited. No study has reported that the small molecule peptide LKLKLL improves obesity and related metabolic disorders by improving GLP-1 and GIP secretion. Summary of the invention

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

[0007] The second object of the present invention is to provide an application of a small molecule peptide in the preparation of a product for improving obesity.

[0008] The third object of the present invention is to provide a composition with the above-mentioned small molecule peptide as the only active ingredient.

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

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

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

[0012] The application of the above-mentioned small molecule peptide in the preparation of products for improving obesity.

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

[0014] The health food is preferably an oral health food; the medicine is preferably an oral medicine.

[0015] A composition with the above-mentioned small molecule peptide as the only active ingredient.

[0016] Application of the composition in preparing products for improving obesity.

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

[0018] The above-mentioned health food is preferably an oral health food; and the medicine is preferably an oral medicine.

[0019] Advantages of the present invention:

[0020] Experiments have shown that the small molecule peptide (LKLKLL) of the present invention can significantly promote the secretion of incretin (GLP-1 and GIP), thereby helping to regulate energy metabolism. It also provides a new target for the treatment and prevention of obesity, reduces body weight and improves insulin sensitivity. The effect of the small molecule peptide LKLKLL in promoting incretin secretion is significantly better than existing positive controls, such as glutamine, and shows stronger biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a CCK-8 experiment in STC-1 cells using a small molecule peptide LKLKLL and glutamine (Gln) (A: LKLKLL; B: Gln);

[0022] Figure 2 Elisa method is used to detect the incretin secretion function of a small molecule peptide LKLKLL, in which:

[0023] A: LKLKLL (abbreviated as LK) promotes GLP-1 secretion;

[0024] B: LKLKLL promotes GIP secretion;

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

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

[0027] Figure 3 Effects of a small molecule peptide LKLKLL on incretin gene expression, including:

[0028] A: qPCR method was used to detect GIP mRNA expression;

[0029] B: qPCR method was used to detect the expression of proglucagon (PRO) mRNA;

[0030] C: qPCR method was used to detect the expression of endonuclease PCSK1 / 3 mRNA;

[0031] D: qPCR method was used to detect the expression of endonuclease PCSK2 mRNA;

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

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

[0034] Figure 4 This is a targeted metabolomics and tolerability evaluation of a small molecule peptide, LKLKLL, which includes:

[0035] A: Targeted metabolomics;

[0036] B: Artificial gastric juice (referred to as gastric juice) tolerance test;

[0037] C: Artificial intestinal fluid (abbreviated as intestinal fluid) tolerance test.

[0038] Figure 5 A small molecule peptide LKLKLL improves obesity induced by high-fat diet.

[0039] Figure 6 It is a small molecule peptide LKLKLL that inhibits fat accumulation induced by a high-fat diet, including:

[0040] A: epididymal white adipose tissue;

[0041] B: Inguinal white adipose tissue.

[0042] Figure 7LKLKLL is a small molecule peptide that improves the morphological changes of adipose tissue induced by a high-fat diet. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

[0045] BHI liquid medium: brain heart infusion (BHI) broth medium, 0.4% mucin, 3g / L threonine, 2.5g / L N-acetyl-D-glucosamine, 0.05% L-cysteine ​​hydrochloride anhydrous. Solid medium: add 2% agar to the liquid medium.

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

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

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

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

[0050] Elisa kits for measuring GLP-1 / GIP content were purchased from Wuhan Eliaright Biotechnology Co., Ltd.; CCK-8 kits were purchased from Shanghai Taoshu Biotechnology Co., Ltd.; total RNA extraction kits were purchased from Tiangen Biochemical Technology Co., Ltd.; SYBR green fluorescence polymerase chain reaction kits and reverse transcription kits were purchased from Shandong Sikoje Biotechnology Co., Ltd.

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

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

[0053] 2. Evaluation of biological activity: The biological activity of LKLKLL was systematically evaluated using in vitro experiments (STC-1 cells) and animal models, with a focus on its effects on promoting the secretion of incretin hormones (GLP-1 and GIP) and inhibiting obesity induced by a high-fat diet.

[0054] 3. Mechanism research: Analyze the specific mechanism of LKLKLL in promoting incretin secretion through molecular biology technology.

[0055] 4. Animal research: Further verify 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 2 hours of anaerobically culturing AKK bacteria in complete cell culture medium, non-targeted metabolomics analysis was performed, and a small molecule peptide (the amino acid sequence of the small molecule peptide is: leucine-lysine-leucine-lysine-leucine-leucine, referred to as LKLKLL) content was found to be significantly increased. Thereafter, it was synthesized by chemical synthesis, and 1 mg of LKLKLL was taken out of the -20℃ refrigerator and restored to activity at room temperature for 1 hour. High-glucose complete culture medium was added to prepare a 5mM mother solution, mixed, sonicated for 5 minutes to assist melting, and separated instantly for use.

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

[0060] Take out STC-1 cells from the liquid nitrogen tank, quickly put them into a 37℃ water bath to thaw quickly, draw 1mL of cell suspension into a 15mL centrifuge tube and add 2mL of complete cell culture medium, centrifuge at 800rpm for 5min, discard the supernatant; add 6mL of complete cell culture medium, blow with a sterile dropper until the cells are mixed, transfer to a culture bottle, and place in a cell culture incubator at a constant temperature of 37℃ and 5% CO2 for culture. According to the cell status, replace the complete cell culture medium every 1-2 days until the cells are full.

[0061] 3. Cell activity detection (CCK-8)

[0062] The cell suspension obtained in step 2 was inoculated into a 96-well plate. After adherence, LKLKLL obtained in step 1 and commercial Gln were set to a concentration gradient to intervene in STC-1 cells for 24 hours, and the culture medium was discarded. The cell viability was detected using a CCK-8 cell counting kit. The method was: add 100 μL of complete cell culture medium and 10 μL of CCK-8 reagent to each well and continue to culture for 0.5 hours. Use a multifunctional microplate reader to measure the absorbance at 450 nm to evaluate cell activity, see Figure 1 . Figure 1 A small molecule peptide LKLKLL and glutamine (Gln) intervene in the CCK-8 experiment of STC-1 cells (A: LKLKLL; B: Gln)

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

[0064] After STC-1 cells were starved with HBSS for 1 hour, they were intervened with LKLKLL and Gln for 24 hours, and a blank control group was set up. After the intervention, the cell supernatant was collected and centrifuged at 1000rpm for 20 minutes. The supernatant was retained and the in vitro incretin capacity of the small molecule peptide LKLKLL was determined by the Elisa method using an Elisa kit. The ELISA plate was set with blank wells, standard wells, and sample wells to be tested. 100μL of sample diluent was added to the blank well, and 100μL of standard or sample to be tested was added to the remaining wells. Cover the ELISA plate with film and incubate at 37°C for 90 min; (prepare biotin antibody working solution 15 min in advance), spin dry, add biotin antibody working solution to each well, 100 μL / well, cover the ELISA plate with film and incubate at 37°C for 1 h; (prepare washing solution and HRP working solution in advance), discard the liquid in the well, spin dry, wash the plate 3 times with washing solution at 350 μL / well, soak for 1 min each time, spin dry and pat dry on absorbent paper; add 100 μL HRP working solution to each well, cover the plate with film and incubate at 37°C for 30 min; discard the liquid in the well, spin dry,

[0065] Wash the plate 5 times with washing solution; add 90μL of substrate solution to each well, cover the plate and incubate at 37℃ in the dark for 15min; add 50μL of stop solution to each well to stop the reaction, and the blue color will turn yellow immediately; immediately use a microplate reader to measure the optical density (OD value) of each well at a wavelength of 450nm. Note: The intervention concentration of LKLKLL and Gln in this experiment is 800μmol / L. Results are shown in Figure 2 .

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

[0067] A control group, LKLKLL experimental group and Gln positive control group were set up, and the cells were intervened for 24 hours. After the treatment, the total RNA of the cells was extracted, and the expression levels of the pancreatic hormone genes (GIP (Gene ID: 14607), Proglucagon (Proglucagon) (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 instructions of the kit, the cells were treated with lysis buffer, and then the supernatant was collected by centrifugation. The RNA precipitate was resuspended in RNase-free water, and its concentration and purity were determined. cDNA synthesis: Total RNA was taken and reverse transcription reaction was performed using SPARKscriptⅡRT Plus Kit (With gDNA Eraser). First, incubate with gDNA Eraser to remove the genome, then add 2×SPARKscript II RTPlus Master Mix, react at 50°C for 15 minutes, and then incubate at 85°C for 5 minutes to inactivate the reverse transcriptase, and finally obtain cDNA. qRT-PCR reaction setup: Prepare the qRT-PCR reaction system and use the SYBR green fluorescent polymerase chain reaction kit for amplification (see Table 1, Table 2 and Table 3).

[0068] Table 1: PCR reaction system

[0069]

[0070] Table 2: PCR reaction program

[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 methods, and it was found that a small molecule peptide LKLKLL can promote the secretion of incretin in a gradient, and its effect is better than that of 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: The present invention uses triple quadrupole mass spectrometry (Triple Quadrupole MS / MS) as a detection method for small molecule peptide targeted metabolomics, which has high sensitivity and high selectivity and is widely used in the qualitative and quantitative analysis of small molecule compounds in biological samples. The specific steps include: first, collecting the metabolic supernatant of Akkermansia muciniphila (AKK bacteria) cultured in high-sugar complete medium, and collecting and filtering after culturing for 2 hours and 6 hours, respectively. Then, add a 20% acetonitrile methanol solution extract (1.5mL), vortex mix for 3 minutes, and then centrifuge at 12000r / min for 10 minutes at 4°C. After centrifugation, the supernatant (500μL) is aspirated and transferred to an injection bottle. The chromatographic conditions are to use a Waters Atlantis T3 Column column, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, the column temperature is set to 40°C, the flow rate is 0.4mL / min, and the injection volume is 3μL. The separated components were detected by triple quadrupole mass spectrometry, with the first two quadrupoles selecting specific parent ions and daughter ions to enhance the selectivity and sensitivity of the signal. Finally, the mass spectrometry data was analyzed to quantify the abundance of small molecule peptides and compared with the control group to verify the detection results.

[0079] (2) Test results

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

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

[0082] (1) Test method

[0083] LKLKLL was dissolved in 1 mL of artificial gastric juice with pH = 2 and 1 mL of ddH2O to prepare a 1000 ng / mL solution, and the solution was thoroughly mixed and placed in an environment of 37°C for 1.5 h. The content of LKLKLL was immediately detected by triple quadrupole mass spectrometry, and the degradation rate was calculated.

[0084]

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

[0086] (2) Test results

[0087] Degradation of LKLKLL in artificial gastric fluid Figure 4 B. In the artificial gastric fluid environment of pH=2, the content of the small molecule peptide LKLKLL was almost unaffected, and the degradation rate was 2%.

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

[0089] (1) Test method

[0090] LKLKLL was dissolved in 1 mL of artificial intestinal fluid and 1 mL of ddH2O to prepare a 1000 ng / mL solution, and the solution was thoroughly mixed and placed at 37°C 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] Degradation of LKLKLL in artificial intestinal fluid Figure 4 C. In the artificial intestinal fluid environment, the content of the small molecule peptide LKLKLL was almost unaffected, and the degradation rate was 0.2%.

[0094] Example 3 Effect of a small molecule peptide LKLKLL on high-fat diet-induced weight gain and adipose tissue morphology

[0095] 1. Test methods

[0096] (1) Six-week-old male C57BL / 6 mice were selected and, after one week of adaptive feeding, 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 given LKLKLL (25 mg / kg body weight) by oral administration every day. The experiment lasted for 8 weeks, during which the weight changes of the mice were recorded. The mice were weighed at a fixed time point every week, and the weight changes of each group were compared.

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

[0101] (3) HE (Hematoxylin-Eosin) staining of adipose tissue: Tissue fixation and embedding: The collected adipose tissue was fixed with 10% neutral buffered formaldehyde for 24 hours, then dehydrated, embedded, and cut into 4-6 μm slices. HE staining: The slices were stained with hematoxylin-eosin. Hematoxylin was used to stain the cell nucleus, and eosin was used to stain the 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 by 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: By monitoring the weight levels of C57BL / 6 male mice during an 8-week experiment, it was found that the weight of mice in the high-fat diet (HFD) group increased significantly, and the blood glucose level was significantly higher than that in the normal diet group. In contrast, the weight gain of mice given LKLKLL intervention (experimental group) was significantly suppressed, indicating that LKLKLL has the effect of inhibiting high-fat diet-induced weight gain. Figure 5 .

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

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

[0106] In summary, the small molecule peptide LKLKLL provided by the present invention can significantly promote the secretion of incretin (GIP / GLP-1), thereby regulating metabolic function. In addition, LKLKLL can also effectively inhibit the weight gain and lipid accumulation induced by a high-fat diet, showing a significant fat-reducing effect. Through in vitro experiments and animal model verification, the small molecule peptide LKLKLL of the present invention has shown good application prospects in promoting incretin secretion, improving metabolic diseases and inhibiting obesity.

[0107] Based on the biological functions of LKLKLL, relevant health foods or medicines are prepared to provide new options for the treatment of obesity and metabolic diseases.

[0108] The health food is prepared into convenient oral dosage forms such as tablets, capsules, solutions or granules by conventional technical means.

[0109] The drug is prepared into convenient oral dosage forms such as tablets, capsules, solutions or granules by conventional techniques.

[0110] A composition with the above-mentioned small molecule peptide as the only active ingredient.

[0111] The composition is used in preparing products for promoting incretin secretion and improving obesity.

[0112] The above product is preferably a health food or a medicine.

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

[0114] The dosage form of the above-mentioned oral health food is tablet, capsule, solution or granule.

[0115] The above-mentioned oral medicine is in the form of tablets, capsules, solutions or granules.

[0116] The embodiments of the present invention are 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 of these embodiments are made without departing from the principles and spirit of the present invention, and 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. Use of a small molecule peptide according to claim 1 in the preparation of products for improving obesity.

3. The use according to claim 2, characterized in that The product is a health food or medicine.

4. The use according to claim 3, characterized in that The health food is an oral health food; the medicine is an oral medicine.

5. A composition comprising the small molecule peptide of claim 1 as the sole active ingredient.

6. Use of the composition of claim 5 in preparing products for improving obesity.

7. The use according to claim 6, characterized in that The product is a health food or medicine.

8. The use according to claim 7, characterized in that The health food is an oral health food; the medicine is an oral medicine.

Images