Use of human umbilical cord mesenchymal stem cell-derived small extracellular vesicles in preparation of a medicament for treating diabetes combined with liver injury

Abstract

The application provides an application of human umbilical cord mesenchymal stem cell-derived small extracellular vesicles in preparation of a medicine for treating diabetes combined with liver injury, and belongs to the technical field of biological medicine; it is found for the first time that the human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hucMSC-sEVs) have the effect of down-regulating the D-lactylation modification level of a T2DM liver, and can be used for developing the medicine for treating diabetes combined with liver injury, opening up a new application field for the small extracellular vesicles, and providing a new action target and treatment strategy for the treatment of T2DM liver injury, filling the blank of the abnormal regulation means of T2DM related D-lactylation modification in the prior art, and having good practicability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of small extracellular vesicles derived from human umbilical cord mesenchymal stem cells in the preparation of drugs for treating diabetes complicated with liver damage. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by insulin resistance and progressive failure of pancreatic β-cell function. It is influenced by genetic factors and environmental factors such as obesity and high-fat, high-sugar diets. Currently, the number of people with type 2 diabetes worldwide exceeds 500 million, and the disease is increasingly affecting younger people. T2DM can cause metabolic disorders and functional damage in multiple organs. As a core metabolic organ, the liver is prone to pathological changes such as inflammatory infiltration, which further aggravates insulin resistance and creates a vicious cycle.

[0003] Type 2 diabetes mellitus (T2DM) involves disordered glucose metabolism, which produces a large amount of lactic acid, a metabolic byproduct that can mediate organ damage by participating in post-translational modifications of proteins, thus becoming an important link between metabolic abnormalities and tissue pathological changes.

[0004] Lactic acidification is a novel modification type that has attracted much attention in recent years. It refers to the covalent binding of lactate molecules to specific amino acid residues in proteins, thereby altering the protein's structure and function. D-lactation is an important subtype of lactation modification, mediated by D-lacate molecules. Proteins modified by D-lactation are widely involved in physiological and pathological processes such as cellular metabolism and inflammation regulation. Currently, the role of D-lactation in type 2 diabetes mellitus (T2DM)-related liver lesions remains unclear.

[0005] Small extracellular vesicles (EVs) are membrane-bound vesicles with a diameter of less than 200 nm that can carry bioactive substances such as proteins, nucleic acids, and lipids to regulate target cell function. Studies have shown that EVs from different sources can participate in the regulation of the pathological process of metabolic diseases, can serve as disease diagnostic biomarkers, and have advantages such as good biocompatibility and strong targeting. However, there are no reports on the regulatory role of EVs on D lactation levels in the liver of type 2 diabetes mellitus (T2DM) mice. Summary of the Invention

[0006] To address some shortcomings in existing technologies, this invention provides an application of human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hucMSC-sEVs) in the preparation of drugs for treating diabetes mellitus complicated with liver injury. This invention is the first to discover that human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hucMSC-sEVs) can downregulate the level of D-lactation modification in the liver of patients with type 2 diabetes mellitus (T2DM), enabling their use in the development of drugs for treating diabetes mellitus complicated with liver injury. This opens up new application areas for small extracellular vesicles and provides new targets and treatment strategies for the treatment of T2DM liver injury, filling the gap in existing technologies for regulating abnormal D-lactation modification related to T2DM, and demonstrating significant practicality.

[0007] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0008] This invention first provides the application of small extracellular vesicles derived from human umbilical cord mesenchymal stem cells in the preparation of drugs for treating liver injury.

[0009] Preferably, the liver injury is liver metabolic injury caused by abnormal D-lactation modification.

[0010] Preferably, the liver injury is diabetic liver metabolic injury.

[0011] Preferably, the diabetes is type 2 diabetes.

[0012] Preferably, the application includes reducing the level of D-lactation modification in liver tissue.

[0013] Preferably, the applications include improving body weight, metabolic disorders in diabetes, and pathological damage to liver tissue.

[0014] Preferably, the method for obtaining the small extracellular vesicles includes: separating and purifying the culture supernatant of human umbilical cord mesenchymal stem cells by ultrafiltration combined with ultracentrifugation to obtain small extracellular vesicles derived from human umbilical cord mesenchymal stem cells.

[0015] The present invention also provides a product for treating liver damage, wherein the product uses small extracellular vesicles derived from human umbilical cord mesenchymal stem cells as the active ingredient.

[0016] Preferably, the liver injury includes: liver injury complicated by diabetes.

[0017] Preferably, the product includes a drug.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention reveals that in diabetic patients, the level of D-lactation modification in liver tissue is significantly elevated. Intervention with hucMSC-sEVs significantly reduces liver D-lactation levels and simultaneously improves metabolic abnormalities and liver tissue damage in diabetic animals. Furthermore, by comparing D-lactation levels in multiple organs, including the heart, liver, spleen, lungs, and kidneys, it was found that hucMSC-sEVs have a significant advantage in D-lactation remodeling of liver tissue, exhibiting organ-specific regulatory characteristics. This invention is the first to discover that hucMSC-sEVs can downregulate the level of D-lactation modification in the liver of patients with type 2 diabetes mellitus (T2DM), opening up a new application area for small extracellular vesicles. It also provides a new target and treatment strategy for the treatment of liver injury in T2DM, filling the gap in existing technologies for regulating abnormal D-lactation modification related to T2DM, and providing a new application direction for developing novel biotherapeutic products based on extracellular vesicles.

[0020] This invention is the first to systematically compare the D-lactation modification characteristics of diabetic heart, liver, spleen, lung, and kidney tissues, establishing the distribution pattern of D-lactation in multiple organs of diabetic patients, and providing new experimental evidence for related research. Furthermore, this invention was validated using both hereditary diabetes models (db / db mice) and induced diabetes models (ICR mice), improving the reliability and applicability of the technical solution. Attached Figure Description

[0021] Figure 1 This image shows the isolation, purification, and identification of hucMSC-sEVs. In the figure, A is an electron micrograph of hucMSC-sEVs, scale bar 100 μm; B is a graph showing the particle size and number of hucMSC-sEVs detected by nanoparticle tracking analysis; C is a graph showing the expression of protein markers of hucMSC-sEVs detected by Western blot.

[0022] Figure 2 For the construction and validation of animal models; in the figure, A is the flowchart of the db / db model construction; B is the weight graph of db / db mice; C is the blood glucose change graph of db / db mice; D is the flowchart of the ICR model construction; E is the weight graph of ICR mice; F is the blood glucose change graph of ICR mice.

[0023] Figure 3 HE staining of mouse liver; in the figure, A is HE staining of db / db mouse liver; B is HE staining of ICR mouse liver. Figure 4 Fluorescent staining for D-lactation modification of liver; In the figure, A is the result of fluorescent staining for D-lactation modification of liver in db / db mice; B is the result of fluorescent staining for D-lactation modification of liver in ICR mice (blue: Hoechst, red: Kd-la).

[0024] Figure 5 Fluorescent staining for D-lactation modification of multiple organs; Figure A shows the fluorescent staining results of D-lactation modification of heart, spleen, lung, and kidney of db / db mice; Figure B shows the fluorescent staining results of D-lactation modification of heart, spleen, lung, and kidney of ICR mice (blue: Hoechst, red: Kd-la). Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0026] The technical solutions of the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0028] Example 1: Isolation, purification and identification of hucMSC-sEVs

[0029] (1) Isolation and culture of human umbilical cord mesenchymal stem cells:

[0030] Fresh umbilical cord tissue was obtained from the Fourth Affiliated Hospital of Jiangsu University (Zhenjiang Maternal and Child Health Hospital) (ethics approval number 202301), and washed in sterile saline to remove surface blood and connective tissue; the umbilical cord vessels were removed and the tissue was cut into 1-2 mm pieces. 3 Tissue blocks were placed in culture dishes and inverted to adhere to the surface. After adhesion, αMEM (gibco, C12571500BT) medium containing 15% fetal bovine serum and 1% penicillin was added and cultured in a 37°C, 5% CO2 incubator, changing the medium every 2-3 days. When mesenchymal stem cells emerged and the cell confluence reached 80%-90%, trypsin was added to digest the cells. After digestion, αMEM medium containing 10% fetal bovine serum (Novizan, F101-03) was added to terminate digestion and the cells were passaged. Cells in the logarithmic growth phase of passages 3-5 were selected for subsequent experiments.

[0031] (2) Preparation of small extracellular vesicles:

[0032] Human umbilical cord mesenchymal stem cells (HMSCs) of passages 3-5 were seeded in culture dishes. When the cell confluence reached 80%-90%, the original culture medium was discarded, and the cells were washed twice with PBS buffer (Sempercapto, BC-BPBS-01). The culture medium was then replaced with αMEM conditioned medium containing no vesicles, and cultured for another 48 hours. The supernatant was collected and centrifuged at 2000g for 30 minutes at 4°C, and the precipitate was discarded. The supernatant was then centrifuged at 12000g for 30 minutes at 4°C, and the precipitate was discarded. The supernatant was poured into an ultrafiltration tube and centrifuged at 2000g for 30 minutes at 4°C. The concentrated supernatant was collected and transferred to an ultrafiltration tube. The tube was then ultracentrifuged at 100000g for 2 hours, and the supernatant was discarded. The supernatant was resuspended in PBS buffer and centrifuged again at 100000g for 2 hours, and the supernatant was discarded. The precipitate was the purified small extracellular vesicles (hucMSCs-sEVs). After resuspending in PBS, the precipitate was stored at -80°C for later use.

[0033] (3) Identification of small extracellular vesicles:

[0034] 10 μL of EV suspension was dropped onto a copper grid and allowed to stand at room temperature for 5 min. Excess liquid was then absorbed with filter paper. 2% phosphotungstic acid solution was added for staining for 5 min, the stain was absorbed with filter paper, and the mixture was allowed to dry naturally before observation under a transmission electron microscope. The results showed that the prepared EVs exhibited a typical round or cup-shaped bilayer membrane structure with a particle size distribution between 30-150 nm, consistent with the morphological characteristics of small extracellular vesicles. Figure 1 A).

[0035] A suitable amount of purified small extracellular vesicle suspension was diluted to an appropriate concentration with sterile PBS buffer. The particle size distribution of the small extracellular vesicles was detected using a nanoparticle tracking analyzer (model: ZetaView Twin pmx220). The diluted sample was added to the detection sample cell, and the instrument automatically captured Brownian motion video of the particles. The particle size and concentration were calculated using analysis software. Each group of samples was tested three times, and the average value was taken. The test results showed that the particle size of the small extracellular vesicles prepared in this invention was mainly distributed below 150 nm, and the average particle size conformed to the typical particle size characteristics of small extracellular vesicles, proving that the isolated and purified product is a small extracellular vesicle. Figure 1 B).

[0036] Specific surface markers of hucMSC-sEVs were detected using Western blotting. Total protein was extracted from hucMSC-sEVs, subjected to SDS-PAGE electrophoresis, and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h, then incubated overnight at 4°C with primary antibody. After washing three times with TBST, a fluorescent secondary antibody was added, and the membrane was incubated at room temperature for 1 h. After washing three times with TBST, the membrane was developed using chemiluminescence. The results showed that the prepared hucMSC-sEVs specifically expressed CD81 (Proteintech, 27855-1-AP), TSG101 (Proteintech, 28283-1-AP), and Alix (CST, 92880S), but did not express Calnexin (Proteintech, 10427-2-AP), confirming that they are small extracellular vesicles. Figure 1 C).

[0037] In summary, small extracellular vesicles derived from human umbilical cord mesenchymal stem cells were successfully obtained in this experiment.

[0038] Example 2: Validation of the application of hucMSC-sEVs in improving liver metabolic damage and regulating D-lactation modification in db / db diabetic mice

[0039] (1) Experimental grouping and intervention:

[0040] Seven-week-old male db / db mice (spontaneous T2DM model mice) and db / m mice (normal control mice) with the same background were purchased from Jicui Pharmaceutical Co., Ltd. The db / db mice were randomly divided into two groups, and the db / m mice were kept as a separate group, with six mice in each group. After one week of acclimatization, fasting blood glucose was measured in all mice. Before testing, the mice were fasted for 12 hours. A fasting blood glucose level ≥16.7 mmol / L in db / db mice confirmed the establishment of the T2DM model, while the fasting blood glucose level in db / m mice remained within the normal range (3.9-6.1 mmol / L), serving as the normal control. All mice were fed standard feed throughout the process, with free access to water, and kept in a consistent environment (temperature 22-25℃, humidity 50%-60%, 12h light / 12h dark cycle).

[0041] Using db / m mice as the normal control group and db / db mice as the T2DM model group, this study investigated the effects of hucMSC-sEVs on improving liver metabolic damage and regulating D-lactation modification in db / db diabetic mice. The groups are as follows:

[0042] hucMSC-sEVs intervention group (hucMSC-sEVs group): In week 12, when the T2DM model was confirmed, hucMSC-sEVs prepared in Example 1 were injected via the tail vein at a dose of 1×10⁻⁶. 10 Particles per particle, injected once a week, for 4-12 weeks.

[0043] Normal control group (NC group): db / m mice were injected with the same amount of PBS buffer as the hucMSC-sEVs group, and the intervention frequency and time were the same as those of the hucMSC-sEVs group.

[0044] T2DM model group (T2DM group): db / db mice were injected with the same amount of PBS buffer as the hucMSC-sEVs group, and the intervention frequency and time were the same as the hucMSC-sEVs group.

[0045] During the intervention period, fasting blood glucose levels in mice were monitored weekly, and changes in mental state, diet, and weight were observed. Figure 2 (A, B, C). As shown in the figure, compared with the NC group, the T2DM group mice had significantly higher body weight and blood glucose levels, indicating a successful establishment of the diabetes model. Compared with the T2DM group, the hucMSC-sEVs intervention resulted in lower body weight and blood glucose levels in the mice. These results demonstrate that hucMSC-sEVs can effectively improve hyperglycemia and metabolic disorders in db / db diabetic mice, thereby alleviating diabetes-induced damage and possessing potential application value for the prevention or treatment of diabetes and its related complications.

[0046] After the intervention, the mice were fasted but allowed free access to water for 12 hours. After weighing, blood was collected from the eyeballs and the serum was separated by centrifugation for later use. The mice were then euthanized and quickly dissected. Liver, heart, spleen, lung, and kidney tissues were collected. Each tissue was divided into two parts: one part was fixed in 4% paraformaldehyde for pathological examination; the other part was stored at -80℃ for the detection of D lactation level.

[0047] (2) Hematoxylin-eosin (HE) staining of liver tissue:

[0048] Liver tissue fixed in 4% paraformaldehyde was dehydrated with graded ethanol, embedded in paraffin, and sectioned to a thickness of 4–5 μm. Paraffin sections were dewaxed in xylene, rehydrated with graded ethanol, stained with hematoxylin for 5–10 min, and rinsed with tap water. They were then differentiated with 1% hydrochloric acid ethanol for a few seconds, rinsed with running water to regain blue color, and stained with eosin for 30 s–2 min. After staining, the sections were dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. Pathological changes in each group of liver tissue were observed under an optical microscope, and photographs were taken and recorded. The results are as follows: Figure 2 As shown.

[0049] As shown in the figure, the normal control group had intact liver tissue structure, regular hepatic cord arrangement, normal hepatocyte morphology, and clear hepatic sinusoidal structure, with no obvious inflammatory cell infiltration or tissue damage. Compared with the normal control group, the T2DM group showed significant pathological changes in liver tissue, including increased hepatocyte volume, loose cytoplasm, disordered hepatocyte arrangement, focal vacuolar degeneration, and inflammatory cell infiltration, suggesting that diabetes causes significant metabolic damage to liver tissue. After treatment with hucMSC-sEVs, the pathological damage to liver tissue was significantly improved, the hepatocyte arrangement was more regular, vacuolar degeneration and inflammatory cell infiltration were significantly reduced, and the overall structure of liver tissue tended to be normal, indicating that hucMSC-sEVs can effectively alleviate the pathological damage to liver tissue caused by diabetes. Figure 3 A).

[0050] (3) Detection of D-lactation modification level:

[0051] The level of D-lactation modification in various tissues was detected by immunofluorescence. The steps were as follows: Tissues from various organs stored at -80℃ were paraffin-embedded and sectioned (section thickness 4-5 μm). The paraffin sections were baked in a 70℃ oven for 6 hours. After dewaxing to water, the sections were sequentially immersed in xylene I and xylene II for 60 min each, followed by anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 80% ethanol, and 70% ethanol for 5 min each. The sections were then washed twice with distilled water for 5 min each time. Antigen retrieval was performed by immersing the sections in 0.01 mmol / L citrate buffer. In the washing solution, heat in a boiling water bath for 40 min, cool naturally to room temperature, wash three times with PBS buffer for 5 min each time; block with 5% bovine serum albumin (BSA) at room temperature for 60 min, discard the blocking solution, add D-lactation-specific primary antibody (Jingjie Biotechnology, PTM-1429RM) (dilution ratio 1:100), and incubate overnight at 4℃; the next day, wash three times with PBS for 5 min each time, add fluorescently labeled secondary antibody (dilution ratio 1:200), and incubate at room temperature in the dark for 1 h; after washing three times with PBS, stain the nuclei with Hoechst 33342 (Sigma, B2261) for 5 min, and wash three times with PBS again; finally, add anti-fluorescence quencher (Yisheng Biotechnology, 36307ES08), and mount the slide. Observe and acquire images under a fluorescence microscope. The results are as follows. Figure 4 As shown.

[0052] As shown in the figure, compared with the NC group, the fluorescence intensity of D lactation modification in the liver of mice in the T2DM group was significantly increased; compared with the T2DM group, the fluorescence intensity of D lactation modification in the liver of mice in the hucMSC-sEVs group was significantly decreased, approaching the level of the NC group. Figure 4 A).

[0053] In summary, this embodiment used a db / db diabetic mouse model to verify the application effect of hucMSC-sEVs in metabolic damage of diabetic liver. The results showed that hucMSC-sEVs could improve abnormal body weight and hyperglycemia in diabetic mice and significantly reduce the level of D-lactation modification in liver tissue. This indicates that hucMSC-sEVs can improve metabolic damage by remodeling D-lactation modification in diabetic liver, providing experimental evidence for its potential as a novel therapeutic strategy for diabetic liver disease.

[0054] Example 3: Validation of the application of hucMSC-sEVs in improving liver metabolic damage and regulating D-lactation modification in ICR diabetic mice

[0055] This embodiment uses a high-fat diet combined with STZ induction to establish an ICR diabetic mouse model, and further verifies the application effect of hucMSC-sEVs in improving liver metabolic damage and regulating D-lactation modification in ICR diabetic mice. The specific steps are as follows:

[0056] Three-week-old ICR mice were purchased from Jicui Pharmaceutical Co., Ltd., and after one week of acclimatization feeding, they were randomly divided into three groups: a normal control group (NC group), a T2DM model group (T2DM group), and a hucMSC-sEVs intervention group (hucMSC-sEVs group), with six mice in each group. Modeling was performed on mice in the T2DM model group and the hucMSC-sEVs intervention group. The modeling steps were as follows: starting from week four, mice were fed a 60% high-fat diet for six weeks. In week ten, mice were intraperitoneally injected with 50 mg / kg streptozotocin (STZ) for five consecutive days. Fasting blood glucose was measured 72 hours after the last STZ injection. Mice with two consecutive fasting blood glucose levels ≥11.1 mmol / L were considered to have successfully established a diabetic model. Other feeding conditions were the same as in Example 2.

[0057] The interventions were performed on the normal control group (NC group), the T2DM model group (T2DM group), and the hucMSC-sEVs intervention group (hucMSC-sEVs group) according to the description in Example 2. The steps are as follows:

[0058] hucMSC-sEVs intervention group (hucMSC-sEVs group): hucMSC-sEVs prepared in Example 1 were administered via tail vein injection at a dose of 1×10⁻¹² at week 12, confirming successful establishment of the diabetes model. 10 Particles per particle, injected once a week, for 4-12 weeks.

[0059] T2DM model group (T2DM group): Mice with successful modeling were injected with the same amount of PBS buffer as the hucMSC-sEVs group, and the intervention frequency and time were the same as those of the hucMSC-sEVs group.

[0060] Normal control group (NC group): ICR mice were injected with the same amount of PBS buffer as the hucMSC-sEVs group, and the intervention frequency and time were the same as those of the hucMSC-sEVs group.

[0061] During the intervention period, fasting blood glucose levels in mice were monitored weekly, and changes in mental state, diet, and weight were observed. Figure 2 (D, E, F). As shown in the figures, compared with the NC group, the T2DM group mice had significantly higher body weight and blood glucose levels, indicating a successful establishment of the diabetes model. Compared with the T2DM group, the hucMSC-sEVs intervention resulted in lower body weight and blood glucose levels in the mice. These results demonstrate that hucMSC-sEVs can effectively improve hyperglycemia and metabolic disorders in ICR diabetic mice, thereby alleviating diabetes-induced damage and possessing potential application value for the prevention or treatment of diabetes and its related complications.

[0062] After the intervention, the mice were fasted but allowed free access to water for 12 hours. After weighing, blood was collected from the eyeballs and the serum was separated by centrifugation for later use. The mice were then euthanized and quickly dissected. Liver, heart, spleen, lung, and kidney tissues were collected. Each tissue was divided into two parts: one part was fixed in 4% paraformaldehyde for pathological examination; the other part was stored at -80℃ for the detection of D lactation level.

[0063] Following the method described in Example 2, the mice after the intervention were subjected to hematoxylin-eosin (HE) staining of liver tissue and detection of D lactation levels in various organs. The results are as follows: Figure 3 and Figure 4 As shown.

[0064] from Figure 3 As can be seen, the normal control group showed intact liver tissue structure and regular hepatocyte arrangement; the diabetic model group showed pathological changes such as disordered hepatocyte arrangement, cytoplasmic vacuolation, and inflammatory cell infiltration; after treatment with hucMSC-sEVs, the pathological damage of liver tissue was significantly improved, the hepatocyte arrangement became more regular, and vacuolation and inflammatory response were significantly reduced, indicating that hucMSC-sEVs can effectively improve the pathological damage of liver tissue in diabetic ICR mouse models. Figure 2 B).

[0065] from Figure 4 As can be seen, the D-lactation fluorescence signal in the liver tissue of the diabetic model group was significantly enhanced; after intervention with hucMSC-sEVs, the D-lactation fluorescence intensity in the liver tissue was significantly reduced, indicating that hucMSC-sEVs can effectively remodel the D-lactation modification state of the liver in the ICR mouse model of diabetes. Figure 4 B).

[0066] In summary, hucMSC-sEVs can improve metabolic indicators such as body weight and blood glucose in induced diabetic animals, alleviate pathological damage to liver tissue, and significantly reduce the level of D-lactation modification in liver tissue. Combined with the results of Example 2, it is clear that the technical solution of this invention is applicable not only to hereditary type 2 diabetes models but also to induced type 2 diabetes models, exhibiting good stability, reproducibility, and broad applicability. This provides further experimental evidence for hucMSC-sEVs to improve metabolic damage by remodeling D-lactation modification in diabetic livers.

[0067] Example 4: hucMSC-sEVs remodel the distribution characteristics of D-lactation in multiple organs of diabetes and their liver-specific regulatory effects

[0068] This embodiment is the first to systematically compare the tissue distribution characteristics of D-lactation modification in the heart, liver, spleen, lungs, and kidneys under diabetic conditions, and verifies the regulatory effect of hucMSC-sEVs on D-lactation modification in multiple organs. The specific steps are as follows:

[0069] Heart, liver, spleen, lung, and kidney tissues from db / db mice and ICR mice in Examples 2 and 3 were fixed in 4% paraformaldehyde for 24 hours, then routinely embedded in paraffin, serially sectioned, and sectioned to a thickness of 4–5 μm for subsequent immunofluorescence detection. All organ tissues were processed under the same experimental conditions to ensure comparability of detection results between different tissues.

[0070] The lactation modification was detected according to the method described in Example 2, and the results are as follows: Figure 5 As shown.

[0071] As shown in the figure, the heart and spleen in the NC group showed basic D-lactation modification expression, while the overall fluorescence signal in the lungs and kidneys was weak. In the T2DM group, the D-lactation modification levels in all organs were increased to varying degrees compared with the normal group, with the most significant increase in liver tissue. This suggests that D-lactation modification in diabetes exhibits differential expression characteristics in different organs, and the liver may be the main target organ for abnormal accumulation of D-lactation. After treatment with hucMSC-sEVs, there were significant differences in different tissues. The fluorescence intensity of D-lactation in liver tissue was significantly reduced, while it was not significant in other organs, suggesting that hucMSC-sEVs have a more significant regulatory effect on liver D-lactation. Figure 4 A, B).

[0072] The results showed that hucMSC-sEVs significantly remodeled D-lactation in diabetic livers while having relatively little impact on other organs, demonstrating a clear liver-specific regulatory effect. This embodiment further confirms that the technical solution of hucMSC-sEVs in improving metabolic damage by remodeling D-lactation in diabetic livers has good stability and biological effects, providing sufficient experimental support for this invention.

[0073] In summary, the hucMSC-sEVs described in this invention can improve blood glucose, body weight, and liver tissue pathological damage in different diabetic animal models. In particular, in liver tissue, hucMSC-sEVs exhibit a more significant D-lactation regulation ability and have a liver-specific D-lactation remodeling effect.

[0074] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of small extracellular vesicles derived from human umbilical cord mesenchymal stem cells in the preparation of drugs for treating liver injury.

2. The application according to claim 1, characterized in that, The liver damage described is metabolic liver damage caused by abnormal D-lactation modification.

3. The application according to claim 1, characterized in that, The liver damage mentioned is diabetic liver metabolic damage.

4. The application according to claim 3, characterized in that, The diabetes mentioned is type 2 diabetes.

5. The application according to claim 1, characterized in that, The application includes reducing the level of D-lactation modification in liver tissue.

6. The application according to claim 1, characterized in that, The applications include improving metabolic abnormalities in diabetes and pathological damage to liver tissue.

7. The application according to claim 1, characterized in that, The method for obtaining the small extracellular vesicles includes: separating and purifying the culture supernatant of human umbilical cord mesenchymal stem cells by ultrafiltration combined with ultracentrifugation to obtain small extracellular vesicles derived from human umbilical cord mesenchymal stem cells.

8. A product for treating liver damage, characterized in that, The product uses small extracellular vesicles derived from human umbilical cord mesenchymal stem cells as its active ingredient.

9. The product for treating liver injury according to claim 8, characterized in that, The liver injury mentioned includes: liver injury complicated by diabetes.

10. The product for treating liver injury according to claim 8, characterized in that, The products include pharmaceuticals.

Images