Application of intestinal microorganism-derived extracellular vesicles in preparation of medicine for preventing and treating obesity
By using extracellular vesicles from intestinal microbial origin as drugs, the lack of effective low-toxic drugs in the prior art to treat obesity caused by a high-fat diet is solved, and the effect of reducing obesity and improving metabolic symptoms is achieved.
Patent Information
- Application Number
- CN202510236965.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-01
- Publication Date
- 2025-06-27
AI Technical Summary
The prior art lacks effective low toxic drugs to prevent and treat obesity, especially those caused by high-fat diets.
Extracellular vesicles (fEVs) from intestinal microbial origin are extracted and prepared as drugs by gradient centrifugation to reduce obesity caused by high fat.
Extracellular vesicles from intestinal microbial origin can effectively reduce the weight of obese mice caused by high fat, reduce the content and size of the liver's lipid droplets, and improve glucose tolerance and insulin resistance.
Smart Images
Figure CN120204264A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of medicine, and particularly relates to the application of extracellular vesicles derived from gut microbiota in the preparation of drugs for preventing and treating obesity. Background Art
[0002] High-Fat Diet (HFD) is a public health problem because it makes individuals susceptible to obesity, diabetes, and metabolic syndrome. The high hospitalization rate, high disability rate, and high mortality rate caused by it have become a public health problem threatening the health of all mankind.
[0003] From a physiological perspective, one of the most important links between high-fat diet and insulin resistance is the gut-liver axis and the factors released by metabolites in the gut and liver, which mediate the bidirectional communication between the gut and the liver. The gut-liver axis is damaged after consuming a high-fat diet. The liver is connected to the gut through the portal vein, and about 70% of the blood supply comes from the gut. This unique anatomical structure makes the liver vulnerable to gut bacteria, bacterial metabolites, endotoxins, and microbe-related inflammatory factors. Under normal physiological conditions, the gut mucosal barrier is the first line of defense for the body to resist foreign pathogens, and the liver maintains the stability of the gut mucosa by secreting specific antibodies and inflammatory factors. In addition, the liver and the gut show a two-way physiological interaction: the liver secretes bile and other bioactive substances into the intestinal lumen through the bile duct, and the metabolites are reabsorbed by the small intestine and transported back to the liver through the portal vein. During this process, the gut microbiota plays a key role in maintaining gut-liver interaction and immune balance, and dysbiosis and gut barrier dysfunction are closely related to the development of MAFLD. In recent years, extracellular vesicles (EVs), as an important medium for intercellular communication, have become a research hotspot. EVs are lipid membrane vesicles spontaneously secreted by cells, carrying a variety of bioactive molecules, and playing important roles in tissues such as adipose tissue, liver, skeletal muscle, and immune cells. In addition to eukaryotic cells, bacteria also produce EVs. Bacteria-derived EVs can communicate with eukaryotic cells and regulate their functions. Extracellular vesicles derived from gut microbiota (fecal EVs, fEVs) have similar functions to EVs secreted by eukaryotic cells, carrying a variety of biomolecules including proteins, enzymes, DNA, RNA, peptidoglycan, and lipids, transmitting information between and within tissues. At the same time, because extracellular vesicles are endogenous, they do not cause an immune response in the body. However, there has been no report on the treatment of obesity with extracellular vesicles derived from gut microbiota. Summary of the Invention
[0004] The object of the present invention is to provide the use of extracellular vesicles derived from gut microbiota in the preparation of drugs for preventing and treating obesity. The present invention aims to solve the problem of the current lack of low-toxicity drugs for effectively preventing and treating obesity; the present invention discovers that extracellular vesicles derived from gut microbiota, as a natural substance derived from the body, have an obvious alleviating effect on lipid accumulation induced by a high-fat diet and have application prospects in the preparation of drugs for preventing and treating obesity.
[0005] The object of the present invention is achieved by the following technical solutions:
[0006] The embodiments of the present invention prove for the first time through animal experiments that extracellular vesicles derived from gut microbiota can effectively reduce the weight of obese mice caused by a high-fat diet, including reducing the weight of the liver, decreasing the lipid droplet content in liver sections, reducing the size of lipid droplets, and decreasing the contents of triglyceride and total cholesterol in the liver and serum. In addition, glucose tolerance and insulin resistance are improved.
[0007] Based on the results of the above embodiments, the present invention discloses the use of extracellular vesicles derived from gut microbiota in the preparation of drugs for preventing and treating obesity.
[0008] Preferably, the extracellular vesicles derived from gut microbiota have marker proteins CD81, CD9, TSG101, and GPA33.
[0009] Preferably, the average diameter of the extracellular vesicles derived from gut microbiota is between 60 nm and 80 nm, and the electron microscopy results show that it has a lipid bilayer structure conforming to the characteristics of exosomes.
[0010] Preferably, the prevention and treatment include one or more of prevention, alleviation, or treatment.
[0011] Preferably, the obesity is preferably obesity caused by a high-fat diet.
[0012] Preferably, the preparation method of the extracellular vesicles derived from gut microbiota includes the following steps:
[0013] Collect fresh feces, dissolve them in PBS, collect the supernatant after gradient centrifugation, and pass it through a 0.45 μm filter; centrifuge the filtrate at 100,000 × g centrifugal force for 120 minutes for the first time at 4°C, and perform a second ultracentrifugation on the collected precipitate; then discard the supernatant to obtain extracellular vesicles derived from gut microbiota (fEVs); resuspend the prepared extracellular vesicles derived from gut microbiota in PBS filtered through 0.1 μm for standby.
[0014] The gradient centrifugation includes the following steps: continuously centrifuge at 700, 4000, and 8,000 × g, all at 4°C for 15 minutes.
[0015] Preferably, the conditions for the second ultracentrifugation are the same as those for the first ultracentrifugation.
[0016] Preferably, for collecting fresh feces, according to the species to be collected, fresh feces of species such as mice and humans can be selected.
[0017] Preferably, for preparing the drug for preventing and treating obesity, the obtained drug is an oral preparation drug.
[0018] More preferably, the oral preparation drug is one of capsule preparation, soft capsule preparation, oral liquid preparation, dripping pill preparation or tablet preparation drugs.
[0019] More preferably, the oral preparation drug also contains pharmaceutically acceptable excipients and / or carriers.
[0020] The extracellular vesicles derived from gut microbiota extracted in the present invention can effectively reduce the weight of high-fat-induced obese mice, the weight of the liver and fat, and reduce the contents of triglyceride and cholesterol in the liver and serum, which provides a new way for the treatment of obesity caused by high fat.
[0021] The present invention has the following beneficial effects compared with the prior art:
[0022] 1. The extracellular vesicles derived from gut microbiota extracted can effectively reduce the weight of high-fat-induced obese mice, reduce the contents of triglyceride and cholesterol in the liver, reduce the liver size, improve glucose tolerance and insulin resistance. This provides a new way for the treatment of obesity caused by high fat.
[0023] 2. The diameter of the extracellular vesicles is similar to that of nanoscale carriers. And they can carry different signaling molecules, so they have the potential to be used as drug delivery carriers. And compared with exogenous nanocarriers, the extracellular vesicles derived from the body have the advantages of not causing immune responses and not having biological toxicity. The extracellular vesicles extracted by a certain method in the present invention can be stored in a -80°C refrigerator for a long time. Description of the Drawings
[0024] Figure 1 It is a result diagram of detecting the marker proteins CD81, CD9 and TSG101 of extracellular vesicles derived from gut microbiota by Western-blot method in the examples.
[0025] Figure 2 It is a result diagram of observing the morphology of extracellular vesicles by electron microscopy in the examples, where the scale bar is 100 nm.
[0026] Figure 3 It is a result diagram of detecting the diameter distribution of extracellular vesicles by particle microscopy in the examples.
[0027] Figure 4 Schematic plan of treating obese mice with extracellular vesicles derived from gut microbiota in the examples.
[0028] Figure 5 Graph showing the changes in body weight, fat percentage, and lean meat percentage of mice during treatment in the examples.
[0029] Figure 6 Graph showing the changes in glucose tolerance (GTT) and insulin sensitivity (ITT) levels of high-fat mice in the treatment group and non-treatment group in the examples.
[0030] Figure 7 Graph showing the changes in the contents of serum triglyceride, cholesterol, aspartate aminotransferase, alanine aminotransferase, very low density lipoprotein, and extremely high density lipoprotein in high-fat mice in the treatment group and non-treatment group in the examples. *P<0.05; **P<0.01.
[0031] Figure 8 Graph showing the pathological changes of the liver and fat and the content of lipid droplets in the liver of high-fat mice in the treatment group and non-treatment group in the examples, including H&E staining sections and oil red staining diagrams, with the scale bar being 50μm.
[0032] Figure 9 Graph showing the changes in the contents of liver triglyceride and cholesterol in high-fat mice in the treatment group and non-treatment group in the examples. *P<0.05; **P<0.01. Detailed implementation manners
[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.
[0034] There is currently a lack of low-toxicity drugs for effectively preventing and treating obesity. To solve the above technical problems, the present invention proposes the application of extracellular vesicles derived from gut microbiota in the preparation of drugs for preventing and treating obesity.
[0035] Example 1 Extraction of extracellular vesicles derived from gut microbiota
[0036] Fresh feces from 20-week-old high-fat-fed mice (CON) and GW6471-fed mice (GW6471) were collected and dissolved in PBS, then centrifuged continuously at 700, 4000, and 8,000 × g, all at 4°C for 15 minutes; the supernatant was collected and passed through a 0.45 μm filter; the filtrate was then ultracentrifuged at a centrifugal force of 100,000 × g for 120 minutes at 4°C, and the collected precipitate was ultracentrifuged a second time; the supernatant was then discarded, and the intestinal microbial-derived extracellular vesicles (fEVs) were resuspended in 0.1 μm-filtered PBS for subsequent experiments.
[0037] Example 2 Identification of extracellular vesicles derived from intestinal microorganisms
[0038] (1) Western-blot identification of exosome marker proteins CD81, CD9, TSG101 and GPA33
[0039] Add an appropriate amount of cell lysis buffer. Place the cell lysis buffer (purchased from Biyuntian) on ice for 30 minutes to allow the cell lysis buffer to fully act. Centrifuge at 4°C, 12000xg for 5 minutes. Aspirate the supernatant and test the protein concentration. The protein concentration test method is the BCA method (purchased from Thermo Fisher Scientific). The protein is diluted 10 times before measurement, that is, 1μl of protein solution is added to 9μld dH2O (see the relevant instructions for detailed steps). Adjust the protein to the same concentration, add 5× loading buffer (purchased from Biyuntian), and then boil it in a 100°C metal bath for 5 minutes, and then store the boiled protein at -80°C. Configure SDS-PAGE gel (related reagents purchased from Biyuntian), slowly load the protein sample, and use 50V constant voltage for electrophoresis in the concentrated gel stage. After the protein enters the separation gel, increase the voltage to 90V constant voltage for running. Determine the gel running time according to the molecular weight of the target protein. Transfer to the membrane, first activate the PVDF membrane with methanol for one minute. Then transfer the membrane in the order of "sponge-filter paper-glue-PVDF membrane-filter paper-sponge". Avoid bubbles during the transfer process. A constant current of 300mA is used for transfer, and the transfer time is 90 minutes. After the transfer, block the membrane. The blocking solution is 5% skim milk powder prepared with TBST. Block at room temperature for 1 hour. After blocking, apply primary antibodies (CD63, CD9, TSG101 and GPA33, purchased from Santa Cruz) to the strips and place them in a 4°C refrigerator overnight. Recover the primary antibody, and then elute the primary antibody with TBST. The elution condition is to change the solution every 15 minutes and wash 4 times. After washing the membrane, incubate the strips with the corresponding secondary antibody. The incubation condition is to incubate at room temperature for 1 hour. Recover the secondary antibody and wash the membrane. Wash once for 5 minutes with TBST, and wash 5 times. Expose. The strip was slightly soaked with absorbent paper to remove the remaining liquid, and then a luminescent substrate (purchased from Thermo Fisher Scientific) was added and exposed using an exposure machine. The results are shown inFigure 1 as shown
[0040] (2) Electron microscopy observation of the morphology of extracellular vesicles derived from gut microbiota
[0041] First, take out 10 μL of fEVs, then drop it onto a copper grid and precipitate for 1 min, and use filter paper to absorb the floating liquid. After that, drop 10 μL of uranyl acetate onto the copper grid and precipitate for 1 min, and use filter paper to absorb the floating liquid. After drying at room temperature for several minutes, use a transmission electron microscope (HT-7700, Hitachi, Japan) at 100 kV for electron microscopy detection and imaging; the results are as Figure 2 shown
[0042] (3) Particle size analysis of the diameter distribution of extracellular vesicles derived from gut microbiota
[0043] Fully mix the extracellular vesicles derived from gut microbiota, perform gradient dilution, with the minimum dilution volume of 1 ml, and then slowly inject it into a particle size analyzer for detection, and perform at least three tests. The results are as Figure 3 shown, and the detected diameter is about 70 nm (mostly concentrated in 60 - 80 nm).
[0044] The results show that extracellular vesicles derived from gut microbiota can be successfully obtained by gradient centrifugation, expressing marker proteins CD81, CD9, TSG101, and GPA33, with an average diameter of about 70 nm, and the morphology observed by electron microscopy conforms to the characteristics of extracellular vesicles derived from gut microbiota.
[0045] Example 3 Application of extracellular vesicles derived from gut microbiota in the prevention and treatment of obesity
[0046] Refer to the schematic plan of treating obese mice with extracellular vesicles derived from gut microbiota in Figure 4 for relevant experimental planning. Specifically as follows:
[0047] (1) High-fat-induced obese mouse model
[0048] Feed 8-week-old male C57BL / 6J mice with a high-fat diet containing 60% fat. The amount of high-fat diet placed is approximately the food for the mice for 2 days, and the high-fat diet needs to be replaced or supplemented in a timely manner because the high-fat diet is prone to oxidation and deterioration. At the same time, randomly divide them into two groups (n = 6 - 8 in each group). HFD+CON fEVs group and HFD+GW6471 fEVs group. fEVs (1 μg total protein) are gavaged once every 2 days for 12 weeks. And place the mice at a room temperature of 25 °C, give the mice sufficient water and maintain a 12-hour light and 12-hour dark schedule. Among them, GW6471 is an inhibitor of PPARα, and feeding GW6471 can improve non-alcoholic fatty liver.
[0049] (2) Mouse body composition detection
[0050] Use a Niumag NMR analyzer (QMR23 - 060H - I; Suzhou, China). Put the standard product into the instrument for calibration. After weighing the mouse, place it in the detection tube, input the body weight data of the mouse, and analyze the body fat data of the mouse. The results are as Figure 5 shown.
[0051] (3) Glucose tolerance (GTT) and insulin sensitivity (ITT) experiments
[0052] For the glucose tolerance (GTT) experiment, after fasting the mice for 16 h, inject a glucose solution (1.5 g / kg) intraperitoneally, and measure the blood glucose levels at 0, 15, 30, 60, 90, and 120 minutes. For the insulin sensitivity (ITT) experiment, after fasting the mice for 4 h, inject an insulin solution (1 U / kg) intraperitoneally, and measure the blood glucose levels at 0, 15, 30, 60, 90, and 120 minutes. The results are as Figure 6 shown.
[0053] (4) Detection of biochemical indexes in mouse serum and liver
[0054] Triglyceride (TG), total cholesterol (TC), aspartate aminotransferase (AST), alanine aminotransferase (ALT), low - density lipoprotein cholesterol (LDL - C), high - density lipoprotein cholesterol (HDL - C) kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. All procedures were performed strictly according to the manufacturer's instructions. The detection results are as Figure 7 shown, *P < 0.05; **P < 0.01.
[0055] (5) Histopathological detection of mouse liver and adipose tissues
[0056] Morphological observation of liver tissue and adipose tissue by H&E staining: The liver needs to be fixed in 4% paraformaldehyde for at least 24 hours first, and then embedded in paraffin according to the conventional paraffin embedding steps. The embedded paraffin block is sliced first, and the thickness of the section is 5 μm. Then the section is dewaxed to water routinely, that is: xylene for 10 minutes (twice, 10 minutes each time); absolute ethanol for 3 minutes; 95% ethanol for 3 minutes; 90% ethanol for 3 minutes; 80% ethanol for 3 minutes; 70% ethanol for 3 minutes; 60% ethanol for 3 minutes; 50% ethanol for 3 minutes; 30% ethanol for 3 minutes; distilled water for 3 minutes. Hematoxylin staining for 1 minute is used to stain cell nuclei. Rinse with tap water for 15 minutes. Eosin staining for 15 minutes is used to stain cytoplasm. Then dehydrate with ethanol routinely, clear with xylene, and after air-drying, mount the section with neutral balsam. The results are as Figure 8 shown. Compared with the control group, the content of lipid droplets in the liver and adipose tissue of the treatment group mice decreased significantly, and at the same time, the size of the lipid droplets also became smaller.
[0057] Observation of the content of lipid droplets in the liver by Oil Red O staining: Fix the liver in 4% paraformaldehyde for 24 hours. Embed the liver with OCT, and then perform cryosectioning. The thickness of the section is 10 μm. Air-dry the section at room temperature and soak it in PBS for 10 minutes. Place the section in 70% ethanol for 2 minutes. Oil Red O staining for 15 minutes. Mix the stock solution of Oil Red O at 4°C with double-distilled water in a ratio of 3:2, and filter it after mixing evenly to remove the undissolved particulate matter. Wash the section with 75% ethanol for 5 minutes. Wash with PBS for 5 minutes. Soak it in PBS and take it out when observing, and observe quickly. The observation results are as Figure 8 and Figure 9 shown. Compared with the control group, the content of lipid droplets in the liver of the treatment group mice decreased significantly.
[0058] The results of the above experimental examples show that: Extracellular vesicles derived from gut microbiota can effectively improve obesity caused by high fat, and thus are applied in the preparation of drugs for preventing and treating obesity.
[0059] For the statistics of the above experimental results, the statistical data are given in the form of mean ± standard deviation, and are statistically analyzed and graphed using GraphPad Prism 5 software (San Diego, USA). The t-test is used for comparison between two groups. P < 0.05 (*): There is a statistical difference; **P < 0.01: There is a significant difference.
[0060] The examples of the present invention prove for the first time through animal experiments that: Extracellular vesicles derived from gut microbiota can effectively reduce the weight of obese mice caused by high fat, including reducing the weight of the liver, decreasing the content of lipid droplets in liver sections, reducing the size of lipid droplets, decreasing the content of triglyceride and total cholesterol in the liver and serum, and in addition, improving glucose tolerance and insulin resistance.
[0061] Based on the results of the above embodiments, the present invention discloses the application of extracellular vesicles derived from gut microbiota in the preparation of drugs for preventing and treating obesity.
[0062] Preferably, for the preparation of drugs for preventing and treating obesity, the obtained drug is an oral preparation.
[0063] More preferably, the oral preparation is one of capsule preparation, soft capsule preparation, oral liquid preparation, dripping pill preparation or tablet preparation.
[0064] More preferably, the oral preparation also contains pharmaceutically acceptable excipients and / or carriers.
[0065] The extracellular vesicles derived from gut microbiota extracted by the present invention can effectively reduce the weight of high-fat-induced obese mice, the weight of the liver and fat, and reduce the content of triglycerides and cholesterol in the liver and serum, which provides a new way for the treatment of obesity caused by high fat.
[0066] The present invention has the following beneficial effects compared with the prior art:
[0067] 1. The extracellular vesicles derived from gut microbiota extracted can effectively reduce the weight of high-fat-induced obese mice, reduce the content of triglycerides and cholesterol in the liver, reduce the liver, improve glucose tolerance and insulin resistance. This provides a new way for the treatment of obesity caused by high fat.
[0068] 2. The diameter of extracellular vesicles is similar to that of nanoscale carriers. And they can carry different signaling molecules, so they have the potential to be used as drug delivery carriers. And compared with exogenous nanocarriers, extracellular vesicles derived from the body have the advantages of not causing immune responses and not having biological toxicity. The extracellular vesicles extracted by the present invention through a certain method can be stored in a -80 °C refrigerator for a long time.
[0069] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. Application of extracellular vesicles derived from intestinal microorganisms in the preparation of drugs for preventing and treating obesity.
2. Use of the intestinal microorganism-derived extracellular vesicles according to claim 1 in the preparation of a drug for preventing and treating obesity, characterized in that: The intestinal microorganism-derived extracellular vesicles have the characteristic proteins CD81, CD9, TSG101 and GPA33.
3. Use of the intestinal microorganism-derived extracellular vesicles according to claim 1 in the preparation of a drug for preventing and treating obesity, characterized in that: The average particle diameter of the intestinal microorganism-derived extracellular vesicles is between 60nm and 80nm.
4. Use of the intestinal microorganism-derived extracellular vesicles according to claim 1 in the preparation of a drug for preventing and treating obesity, characterized in that: The control includes one or more of prevention, alleviation or treatment.
5. Use of the intestinal microorganism-derived extracellular vesicles according to claim 1 in the preparation of a drug for preventing and treating obesity, characterized in that: The obesity is obesity caused by a high-fat diet.
6. Use of the intestinal microorganism-derived extracellular vesicles according to claim 1 in the preparation of a drug for preventing and treating obesity, characterized in that: The method for preparing extracellular vesicles derived from intestinal microorganisms comprises the following steps: collecting fresh feces and dissolving them in PBS, collecting the supernatant after gradient centrifugation, and passing it through a 0.45 μm filter; subjecting the filtrate to a first ultracentrifugation at 100,000×g centrifugal force for 120 minutes at 4°C, and subjecting the collected precipitate to a second ultracentrifugation; and then discarding the supernatant to obtain extracellular vesicles derived from intestinal microorganisms.
7. Use of the intestinal microorganism-derived extracellular vesicles according to claim 6 in the preparation of a drug for preventing and treating obesity, characterized in that: The gradient centrifugation comprises the following steps: consecutive centrifugation at 700, 4000 and 8,000×g, all at 4° C. for 15 minutes.
8. Use of the intestinal microorganism-derived extracellular vesicles according to claim 1 in the preparation of a drug for preventing and treating obesity, characterized in that: The drug prepared for preventing and treating obesity is an oral preparation drug.
9. Use of the intestinal microorganism-derived extracellular vesicles according to claim 8 in the preparation of a drug for preventing and treating obesity, characterized in that: The oral preparation drug is one of capsule preparations, soft capsule preparations, oral liquid preparations, dripping pill preparations or tablet preparations.
10. Use of the intestinal microorganism-derived extracellular vesicles according to claim 8 in the preparation of a drug for preventing and treating obesity, characterized in that: The oral preparation medicine also contains pharmaceutically acceptable excipients and / or carriers.