Use of sarcosine as the sole active ingredient in the preparation of drugs for preventing and treating obesity and / or hyperlipidemia

Abstract

The present invention belongs to the field of drug application, and specifically discloses the use of sarcosine as the only active ingredient in the preparation of drugs for preventing and treating obesity and / or hyperlipidemia. The present invention uses sarcosine to treat elderly mice, and confirms that sarcosine can reduce the total body mass and fat mass, and reduce the body fat rate without affecting the absolute lean body mass. Through tissue staining and biochemical analysis, it is determined that sarcosine can reduce the cross-sectional area of ​​visceral adipose tissue (VAT) cells, upregulate the expression of UCP1, a marker of brown adipose tissue in visceral adipose tissue, and significantly reduce the level of triglycerides in plasma. Sarcosine has practical application value in the treatment of obesity and hyperlipidemia.

Description

Technical Field

[0001] The invention belongs to the field of drug application, and specifically relates to the use of sarcosine as the only active ingredient in the preparation of drugs for preventing and treating obesity and / or hyperlipidemia. Background Art

[0002] Obesity is a state in which excessive body fat accumulates, causing body weight to exceed the normal range. Obese people usually consume too many calories, which are not effectively converted into energy and consumed, but converted into fat accumulation in the body, especially the accumulation of abdominal fat. This increase in adipose tissue releases a variety of cytokines and hormones, interfering with the normal lipid metabolism process. For example, adipocytes can promote the liver to synthesize and secrete more very low-density lipoprotein cholesterol (VLDL-C), which is one of the important factors leading to increased blood lipids. In addition, obese people often have insulin resistance, which further affects lipid metabolism, increases triglyceride levels, and reduces high-density lipoprotein cholesterol (HDL-C) levels, thereby causing hyperlipidemia.

[0003] As people age, their basal metabolic rate gradually decreases, their muscle mass decreases and their fat mass increases, which reduces the amount of energy consumed by the body and easily leads to weight gain. In addition, due to physiological changes and comorbidities, the elderly are more likely to have high blood lipids.

[0004] Sarcosine, CAS No.: 107-97-1, chemical formula: C3H7NO2, is a non-protein amino acid and an intermediate in the natural metabolism of choline. It has a wide range of applications in the biomedical field. Studies have shown that it can act as a competitive inhibitor of glycine transporter-1 (GlyT1) and a co-agonist of NMDA receptors, enhancing glycine receptor function, thereby improving intelligence, and has potential applications in the treatment of neurodegenerative diseases and heart disease. In addition, sarcosine is also used in sports nutrition supplements to improve muscle strength, endurance and recovery.

[0005] There are currently no reports on the use of creatine in preventing and treating obesity and hyperlipidemia.

[0006] The structural formula of sarcosine is . Summary of the invention

[0007] To solve the above problems, the present invention provides the use of sarcosine as the only active ingredient in the preparation of a drug for preventing and / or treating obesity.

[0008] The structural formula of sarcosine is .

[0009] Furthermore, the drug has the effect of reducing body weight.

[0010] Furthermore, the drug has the effect of reducing the mass of white fat.

[0011] Furthermore, the drug has the effect of reducing body fat percentage.

[0012] Furthermore, the drug has the effects of increasing the level of uncoupling protein 1 in visceral adipose tissue, promoting the browning of white fat, and reducing the cross-sectional area of ​​visceral adipose tissue cells.

[0013] Finally, the present invention provides the use of sarcosine as the only active ingredient in preparing a medicine for preventing and / or treating hyperlipidemia.

[0014] Furthermore, the drug has the effect of reducing the triglyceride level in plasma.

[0015] Furthermore, the drug is a preparation prepared by using creatine as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0016] Furthermore, the preparation is an oral preparation.

[0017] Furthermore, the oral preparation is a solution, granules, paste, emulsion, pills, capsules or tablets.

[0018] The sarcosine of the present invention can effectively prevent and treat obesity and / or hyperlipidemia as the only active ingredient. Through the sarcosine treatment experiment on elderly mice, it is confirmed that sarcosine can reduce the total body mass and white fat mass and reduce the body fat rate without affecting the absolute lean body mass. Through tissue staining and biochemical analysis, it is determined that sarcosine can reduce the cross-sectional area of ​​visceral adipose tissue (VAT) cells, upregulate the expression of UCP1, a marker of brown adipose tissue in visceral adipose tissue, promote the browning of white fat, and significantly reduce the level of triglycerides in plasma. The application of sarcosine in the treatment of obesity and hyperlipidemia, especially hypertriglyceridemia, has practical value.

[0019] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0020] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1: Creatine treatment reduces DXA results in aged mice; a: Schematic diagram of the 4-month creatine treatment regimen for 18-month-old male mice; b: Representative DXA images of mice in the control and creatine-treated groups; cd: Statistical analysis of the absolute values ​​(c) and changes (d) in body weight, lean mass, fat mass, lean percentage, and fat percentage of mice in the control and creatine-treated groups compared with baseline (n = 8 in each group).

[0022] Figure 2 : Statistical analysis of the relative weights of skeletal muscle (a) and adipose tissue (b) in the control and creatine-treated mice (n = 8 per group).

[0023] Figure 3 : Representative images of H&E staining (a) and statistical evaluation of adipocyte cross-sectional area (b) of visceral adipose tissue (VAT) of control and creatine-treated mice (n = 7 per group; more than 450 adipocytes were measured for each sample).

[0024] Figure 4 : Sarcosine treatment upregulates the expression of UCP1 in white fat; ab, Immunoblotting (a) and statistical analysis (b) of UCP1 in VAT of sarcosine-treated and untreated mice (n = 5 for each group); cd, Immunohistochemistry (c) and statistical analysis (d) of UCP1 in VAT of sarcosine-treated and untreated mice (n = 6 for each group).

[0025] Figure 5 : Results of blood triglyceride biochemical analysis of mice in the control and sarcosine-treated groups; n = 8 (control group) or n = 11 (sarcosine-treated group).

[0026] In the figures, ns, not significant; *, p < 0.05; two-tailed unpaired t-test; **, p < 0.01. DETAILED DESCRIPTION

[0027] The raw materials and equipment used in the specific embodiments of the present invention are all known products and can be obtained by purchasing commercial products.

[0028] Example 1 Study on weight loss and blood lipid reduction with sarcosine

[0029] 1. Experimental Methods

[0030] 1. Drug configuration

[0031] Sarcosine was purchased from Sigma-Aldrich, catalog number 131776-100G.

[0032] 2. Animal model construction

[0033] The animal model used in the present invention is a naturally aged mouse model purchased from Enswell Company (Chongqing). The mice were allowed to eat and drink freely and were grown in an SPF environment until they were 18 months old.

[0034] 3. Creatine efficacy experiment

[0035] The experiment was divided into a control group and a Sarcosine treatment group. The Sarcosine treatment group was given a feed supplemented with Sarcosine (0.75 g of Sarcosine per kg of feed), and the control group was given the same feed without Sarcosine. Both groups of mice were free to eat. The total duration of the experiment was 4 months.

[0036] 4. Detection indicators and detection methods

[0037] 1) DXA detection of mouse body composition

[0038] Before the experiment, all mice were tested using DXA (dual-energy X-ray absorptiometry, instrument model InAlyzer, manufacturer: MEDIKORS, South Korea), and the measured indicators included weight, lean mass, fat mass, lean percent (%), and fat percent (%). According to the test results, the mice were randomly divided into two groups, a control group and a Sarcosine treatment group. After the start of the experiment, DXA tests were performed every two months. The specific DXA test method is as follows:

[0039] a. Mice were fasted for approximately 4-6 hours to ensure that gastrointestinal contents did not significantly affect the measurements.

[0040] b. Anesthetize mice: Place mice in an anesthesia chamber and use isoflurane for anesthesia. Select chamber mode in the anesthesia chamber, set the initial concentration of isoflurane to 3-5%, maintain the concentration to 1-2%, and connect the anesthesia chamber trachea to the DXA scanning table.

[0041] c. Open the InAlyzer software, log in to your account, select the Maintenance mode, and perform instrument preheating and maintenance. Before running the Maintenance, you need to place the equipped scanning paper and mold on the DAX instrument scanning table. The mold should be placed in the position and orientation specified in the manual, and wait for the Maintenance to end. After the Maintenance is completed, enter the Registry interface and set the mouse information: date, name, age, etc. After setting, click Scan and select the Scan mode: mouse with mask. The first step is to follow the instrument prompts, not put in the mouse, but only scan the mask. After the mask scan is completed, put in the mouse in a fully anesthetized state (no pain reflex). At this time, adjust the anesthesia chamber mode to mask, set the maintenance concentration, and gently place the mouse on the scanning table of the DXA instrument, with the mouse's head in the mask, the back against the scanning table, and the limbs stretched naturally to avoid overlap and ensure a clear scan. Use sterile gauze to gently fix the mouse to prevent movement.

[0042] d. After the scan is completed, check the results, manually remove the ear tags, and export the data.

[0043] 2) Weighing of mouse muscle and adipose tissue

[0044] a. At the end of the experiment, mice were killed by cervical dislocation.

[0045] b. Carefully remove the tibialis anterior muscle (TA), gastrocnemius muscle (GA), visceral fat (VAT) and brown fat (BAT) tissues of mice, weigh them using a precision balance, and record the data. Statistical analysis was performed after correcting the weight of the mice.

[0046] 3) H&E staining of VAT tissue

[0047] a. When dissecting mice, fix VAT tissue of appropriate size for 24 hours using adipose tissue fixative (Servicebio, Cat. No. CR2310085).

[0048] b. Use an automatic dehydrator to dehydrate and clear the tissue.

[0049] c. Embed the tissue in paraffin and slice it. Dewax the slices in xylene I and II twice, 10 minutes each time.

[0050] d. Immerse the slices in different concentrations of ethanol (100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol) for 5 minutes each time, and then change to distilled water I and II for 2 minutes each.

[0051] e. Immerse the sections in hematoxylin stain for 3 minutes, then rinse with distilled water to remove floating color.

[0052] f. Treat the sections with 1% hydrochloric acid alcohol differentiation solution for 30 seconds, then switch to tap water I and II for 3 minutes each.

[0053] g. Immerse the sections in eosin stain for 2 minutes, then rinse with distilled water to remove floating color.

[0054] h. Dehydrate with 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol for 1 minute each. Then switch to xylene I and II for 1 minute each.

[0055] i. Add an appropriate amount of neutral resin to the center of the tissue. Hold the slide in one hand and the cover glass in the other, and gently let the center of the cover glass touch the convex point of the neutral resin droplet. After the two are in contact, slowly lower the cover glass to allow the neutral resin to spread naturally under the cover glass to avoid the formation of bubbles.

[0056] j. Imaging: Slice scanning was performed using a fully automatic slide scanner (VS200).

[0057] 4) UCP1 immunohistochemical staining of VAT tissue

[0058] a. Dewax paraffin tissue sections to water (same as the H&E staining steps above).

[0059] b. Use microwave technology to perform antigen repair. The specific steps are as follows: use a plastic slice rack, place it in a plastic container, submerge the slices in sodium citrate buffer (10 mM, PH = 6.0), put in the slices, place in a microwave oven on medium heat for 8 minutes, turn off the heat for 7 minutes, and turn to low heat for 8 minutes; take out the staining box and cool to room temperature.

[0060] c. Wash twice with PBS, 5 minutes each time.

[0061] d. Use 3% hydrogen peroxide (H2O2) to block endogenous peroxidase and soak at room temperature for 10 minutes (protect from light).

[0062] e. Wash twice with distilled water, 5 minutes each time.

[0063] f. Serum blocking: Take the slices out of the staining jar, wipe off the moisture on the back of the slices and the moisture around the tissue on the front of the slices (keep the tissue moist), add rabbit serum (diluted with PBS, serum: PBS = 1:20), and incubate at 37℃ for 15 minutes. After blocking, wash the slices once with PBS.

[0064] g. Aspirate the serum, apply UCP1 (Proteintech, Cat. No. 23673-1-AP) primary antibody diluent (UCP1:PBS = 1:200) evenly on the blocked tissue sections, place in a wet box, and incubate at room temperature for 2 hours to allow the primary antibody to fully bind to the antigen.

[0065] h. Aspirate the primary antibody and wash with PBS three times, 5 minutes each time.

[0066] i. Add the diluted rabbit secondary antibody (Abcam, Cat. No. ab6702) (secondary antibody: PBS = 1:1000) onto the tissue sections and incubate at room temperature for 1 hour.

[0067] j. Aspirate the secondary antibody and wash with PBS three times, 5 minutes each time.

[0068] k. Add DAB (Merck, Catalog No. 102924) for color development, observe under a microscope, and terminate the reaction at an appropriate time.

[0069] l. Dehydrate the stained sections by soaking them in 70%, 85%, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 1 min respectively.

[0070] m. Soak the dehydrated sections in xylene twice for 1 minute each time for transparentization.

[0071] n.Put a drop of mounting medium on the tissue section and then gently cover it with a coverslip.

[0072] o. Imaging: Slice scanning was performed using a fully automatic slide scanner (VS200).

[0073] 5) Immunoblotting of UCP1 protein in VAT tissue

[0074] a. Protein extraction and concentration determination: Weigh 50 mg of white adipose tissue, add 500 μL of RIPA lysis buffer (Kangwei Century, Cat. No. CW2333S) pre-added with protease inhibitors, and crush with a tissue grinder for 30 seconds. Centrifuge at 4°C, 20,000g for 30 minutes, take the supernatant, and determine the protein concentration using the BCA method.

[0075] b. Prepare the sample to a concentration of 1 μg / μL according to the measured concentration, separate it by electrophoresis using 10% SDS-PAGE gel, and then transfer it to the membrane using the wet transfer method.

[0076] c. After transfer, wash once with PBST for 5 minutes, and then block the PVDF membrane with 4% milk (dissolved in PBST) at room temperature for 1 hour.

[0077] d. After blocking, discard the blocking solution, wash once with PBST, add UCP1 (Proteintech, Cat. No. 23673-1-AP) primary antibody diluent (UCP1:5% BSA (in PBST) = 1:2000), and incubate at 4°C overnight.

[0078] e. Recover the primary antibody dilution solution and wash three times with PBST, 5 minutes each time.

[0079] f. Add the secondary antibody (Abcam, Catalog No. ab6702) of the corresponding species (secondary antibody: 5% BSA (in PBST) = 1:10000) and incubate at room temperature for 1 hour.

[0080] g. Discard the secondary antibody dilution solution and wash with PBST three times, 5 minutes each time.

[0081] h. Development: Use ECL Western Blotting developer (Merck, catalog number WBKLS0500) for imaging.

[0082] 5. Data processing

[0083] 1) Calculation of adipocyte area in VAT tissue

[0084] a. Using CaseViewer software, three fields of view were randomly captured from each slice.

[0085] b. Import the captured image into FIJI software and use the Adiposoft plug-in to calculate the area of ​​each fat cell.

[0086] c. Perform data statistics based on the fat cell area distribution of each sample.

[0087] 2) Quantification of UCP1 immunohistochemical staining in VAT tissue

[0088] a. Using CaseViewer software, three fields of view were randomly captured from each slice.

[0089] b. Import the captured images into ImagePro Plus software and obtain the average optical density value of UCP1 for each image.

[0090] c. Perform data statistics based on the average optical density value of each sample.

[0091] 3) Quantification of UCP1 protein immunoblotting in VAT tissue

[0092] a. Import the protein immunoblot bands into FIJI software and calculate the intensity of each band.

[0093] b. The expression level of Vinculin protein was used as an internal reference.

[0094] c. The UCP1 expression level of each sample was corrected for the internal reference expression level and then data statistics were performed.

[0095] 2. Experimental Results

[0096] Aged mice (18 months old) were supplemented with creatine for four months, and body composition was measured every two months using DXA ( Figure 1 a). DXA results showed that creatine supplementation reduced total mass and white fat mass without affecting absolute lean body mass ( Figure 1 b, c). Compared with the control group, the lean body mass percentage of mice treated with creatine was significantly increased, while the fat percentage was significantly decreased ( Figure 1 cd; Figure 2 a). In addition, the weight of brown adipose tissue (BAT) was significantly increased in mice treated with creatine ( Figure 2 ab). After sarcosine treatment, the cross-sectional area of ​​visceral adipose tissue (VAT) cells was significantly reduced ( Figure 3 ab). Uncoupling protein 1 (UCP1), a marker of BAT, was significantly upregulated in VAT after sarcosine treatment ( Figure 4 ad), indicating that sarcosine has the effect of increasing UCP1 levels in visceral adipose tissue and promoting the browning of white fat. In addition, the level of plasma triglycerides in mice treated with sarcosine was significantly reduced ( Figure 5 ). The above results indicate that creatine itself has the effect of reducing body fat percentage, body weight and blood lipid levels in elderly mice.

[0097] In summary, the present invention has confirmed that sarcosine can reduce the total body mass and white fat mass, and reduce the body fat rate without affecting the absolute lean body mass through the sarcosine treatment experiment on elderly mice. Through tissue staining and biochemical analysis, it was determined that sarcosine can reduce the cross-sectional area of ​​visceral adipose tissue (VAT) cells, upregulate the expression of UCP1, a marker of brown adipose tissue in visceral adipose tissue, promote the browning of white adipose tissue, and significantly reduce the level of triglycerides in plasma. The drug made of sarcosine as the only active substance of the present invention can effectively treat obesity and hyperlipidemia (especially hypertriglyceridemia), and has good prospects for clinical application.

Claims

1. Use of sarcosine as the sole active ingredient in the preparation of a drug for preventing and / or treating obesity, characterized in that: The structural formula of sarcosine is 2. Use of sarcosine as the sole active ingredient in the preparation of a drug for preventing and / or treating hyperlipidemia.

3. The use according to claim 2, characterized in that: The medicine is a medicine for preventing and / or treating hypertriglyceridemia.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine is a preparation prepared by taking sarcosine as the active ingredient and adding excipients or auxiliary ingredients acceptable to medicines.

5. The use according to claim 4, characterized in that: The preparation is an oral preparation.

6. The use according to claim 5, characterized in that: The oral preparation is a solution, granule, paste, emulsion, pill, capsule or tablet.

Images