Application of AMSCs-derived exosome in preparation of medicine for treating diabetic vasculopathy

Abstract

The invention discloses application of an AMSCs-derived exosome in preparation of a medicine for treating diabetic vasculopathy, and relates to the technical field of medicines. The exosome is separated and extracted from the AMSCs, the HUVECs under the high glucose condition are intervened, it is proved for the first time that the exosome derived from the AMSCs can improve the HUVECs autophagy obstacle induced by the high glucose, it is also proved that the exosome derived from the AMSCs can weaken the activation effect of the high glucose on AKT / mTOR, meanwhile, an AKT / mTOR signaling pathway activator MHY1485 can block the activation of the exosome on the HUVECs autophagy, and therefore the HUVECs autophagy obstacle induced by the high glucose is inhibited. It is prompted that the exosome derived from the AMSCs and the AKT / mTOR signal channel inhibitor have a certain curative effect on the vascular endothelial cell autophagy disorder related diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to exosomes derived from AMSCs, and more specifically to the application of AMSCs-derived exosomes in the preparation of drugs for treating diabetic vascular complications. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia, often accompanied by multivascular damage. Diabetic vascular complications are among the most common complications of diabetes. Common diabetic vascular complications include diabetic nephropathy, diabetic retinopathy, and diabetic cardiomyopathy. Epidemiological data shows that diabetic vascular complications are the leading cause of death in patients with type 2 diabetes. Statistics indicate that 50% of newly diagnosed type 2 diabetes patients have vascular complications, and approximately 75-80% of these patients die from them. Although many drugs have vascular protective effects, the unclear pathogenesis of diabetic vascular complications leads to poor treatment outcomes. Therefore, there is an urgent need to find new treatment methods.

[0003] Normal vascular endothelial cells release various active mediators to regulate vasodilation, coagulation, and inflammatory responses. However, long-term hyperglycemia, glucose and lipid metabolism disorders, and oxidative stress can all cause damage to vascular endothelial cells in diabetic patients, leading to vascular lesions. Current research has demonstrated that vascular endothelial cell dysfunction is closely related to autophagy. For example, Bai X et al. found that autophagy flux in vascular endothelial cells is blocked under hyperglycemic conditions, and activating autophagy can improve endothelial cell function. Rezabakhsh et al. used autophagy activators to intervene in vascular endothelial cells under hyperglycemic conditions, significantly improving the cell state of vascular endothelial cells and alleviating oxidative stress damage. This suggests that promoting autophagy is an effective strategy for alleviating vascular endothelial cell dysfunction.

[0004] Currently, stem cell therapy is considered one of the most promising treatments for diabetic vascular complications. Among them, adipose-derived mesenchymal stem cells (AMSCs) have attracted widespread attention due to their multipotent differentiation potential, low immunogenicity, and immune clearance properties. They exert their effects through the secretion of cytokines or exosomes. Therefore, this invention aims to investigate whether AMSC exosomes can affect autophagy in vascular endothelial cells (HUVECs) under high glucose conditions and to explore the specific mechanisms of action, thereby providing new insights for the development of new drugs for diabetic complications. Summary of the Invention

[0005] The purpose of this invention is to provide the application of AMSCs-derived exosomes in the preparation of drugs for treating diabetic vascular complications.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] On the one hand, this invention provides the application of AMSCs-derived exosomes in the preparation of drugs for treating diabetic vascular complications.

[0008] On the other hand, the present invention also provides the application of the AKT / mTOR signaling pathway as a target of AMSCs-derived exosomes in the preparation of drugs for treating diabetic vascular complications.

[0009] Preferably, the drug is an AMSCs-derived exosome or an AKT / mTOR signaling pathway inhibitor.

[0010] Preferably, the method for extracting AMSCs-derived exosomes includes the following steps:

[0011] S1. Culture AMSCs in serum-free medium. After 48 hours, collect the cell supernatant into a centrifuge tube and centrifuge at 4-10℃ and 800-1200g for 10-15 minutes. Collect the supernatant.

[0012] S2. Centrifuge the obtained supernatant at 4~10℃ and 3500~4500g for 15~25min, and collect the supernatant;

[0013] S3. Centrifuge the supernatant at 4~10℃ and 18000~22000g for 30~35min, filter the supernatant through a sterile filter membrane, and collect the filtrate.

[0014] S4. Centrifuge the collected filtrate at 100,000~150,000g for 80~120 min. The precipitate is the exosome.

[0015] Preferably, the concentration of the exosomes is 20~500 μg / mL.

[0016] Preferably, the diameter of the exosomes is 50~400 nm.

[0017] Preferably, the exosomes contain exosome marker proteins CD9, CD63, and TSG101.

[0018] On the other hand, the present invention also provides a medicament for treating diabetic vascular complications, including AMSCs-derived exosomes or AKT / mTOR signaling pathway inhibitors.

[0019] Preferably, it also includes a pharmaceutically acceptable carrier or excipient.

[0020] Preferably, the concentration of the AMSCs-derived exosomes is 20-500 μg / mL, and the diameter of the AMSCs-derived exosomes is 50-400 nm.

[0021] Preferably, the concentration of the AMSCs-derived exosomes is 20-200 μg / mL, and the diameter of the AMSCs-derived exosomes is 100-200 nm.

[0022] Preferably, the AMSCs-derived exosomes contain exosome marker proteins CD9, CD63, and TSG101.

[0023] Preferably, the drug is formulated as an injection, capsule, tablet, powder, ointment, or spray.

[0024] Preferably, the pharmaceutically acceptable carrier includes phosphate buffer and / or physiological saline.

[0025] Compared with existing technologies, the beneficial effects of this solution are:

[0026] This invention isolates and extracts exosomes from AMSCs and intervenes in HUVECs under high glucose conditions. It is the first time that AMSC-derived exosomes can improve high glucose-induced autophagy disorders in HUVECs. It also demonstrates that AMSC-derived exosomes can weaken the activation of AKT / mTOR by high glucose. At the same time, the AKT / mTOR signaling pathway activator MHY1485 can block the activation of autophagy in HUVECs by exosomes, suggesting that AMSC-derived exosomes and AKT / mTOR signaling pathway inhibitors have certain therapeutic effects on diseases related to vascular endothelial cell autophagy disorders. Attached Figure Description

[0027] Figure 1 High glucose-induced autophagy disorder in HUVECs (A: transmission electron microscopy observation of autophagosomes in HUVECs cells (black arrows indicate autophagosomes and autolysosomes); B: Western blot detection of the expression levels of LC3-I, LC3-II, P62, and ATG5 in HUVECs cells).

[0028] Figure 2 Characterization and identification of AMSCs exosomes (A: electron micrograph of exosomes (red arrows indicate exosomes); B: diameter measurement of exosomes; C: Western blot analysis of the expression levels of CD9, CD63, and TSG101 in AMSCs cell supernatant and exosome samples; compared with the supernatant, **P<0.01, ***P<0.001).

[0029] Figure 3AMSCs-derived exosomes promote autophagy in HUVECs (A: transmission electron microscopy observation of autophagosomes in HUVECs cells (black arrows indicate autophagosomes and autolysosomes); B: Western blot detection of the expression levels of LC3-I, LC3-II, P62, and ATG5 in HUVECs cells; compared with the control group, **P<0.01, ***P<0.001; compared with the model group, ##P<0.01, ###P<0.001).

[0030] Figure 4 To enable AMSCs-derived exosomes to mediate the AKT / mTOR signaling pathway (A: Western blot analysis of AKT and mTOR phosphorylation levels in HUVECs cells; B: Western blot analysis of LC3-I, LC3-II, P62, and ATG5 expression levels in HUVECs cells; ***P<0.001 compared with the control group; ##P<0.01 and ##P<0.001 compared with the model group; &&P<0.01 compared with the exosome group). Detailed Implementation

[0031] To facilitate understanding of the present invention by those skilled in the art, the technical solution of the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] The experimental materials and methods used in the following embodiments are as follows:

[0036] 1. Experimental cells

[0037] AMSCs were purchased from Wuhan Pronosai Biotechnology Co., Ltd., batch number: CP-H202, and were incubated using AMSCs complete culture medium; HUVECs were purchased from Wuhan Pronosai Biotechnology Co., Ltd., batch number: CL-0675, and were cultured using HUVECs complete culture medium at 37 ℃ in a cell culture incubator with 5% CO2.

[0038] 2. Experimental reagents

[0039] Fetal bovine serum was purchased from Suzhou Yikesai Biotechnology Co., Ltd., batch number: FND500; glucose solution was purchased from Sigma-Aldrich, USA, batch number: G7021; MHY1485 solution, CCK-8 assay kit, BCA protein assay kit, and Trizol lysis buffer were purchased from Shanghai Beyotime Biotechnology Co., Ltd., batch numbers: SC0042, C0105M, C0037, P0011, and R0016; 2.5% glutaraldehyde solution was purchased from Shaanxi Proanti Biotechnology Development Co., Ltd., batch number: 10217-1; uranium acetate solution was purchased from Shenzhen Zhenqiang Biotechnology Co., Ltd., batch number: RG-A; rabbit monoclonal antibody was also purchased. The cloning antibodies LC3-I, LC3-II, P62, ATG5, CD9, CD63, TSG101, AKT, p-AKT, mTOR, p-mTOR, and GAPDH were purchased from Wuhan Sanying Biotechnology Co., Ltd., with batch numbers of 14600-1-AP, 14600-1-AP, 18420-1-AP, 10181-2-AP, 20597-1-AP, 25682-1-AP, 28283-1-AP, 10176-2-AP, 66444-1-Ig, 28273-1-AP, 80596-1-RR, and 60004-1-IG, respectively.

[0040] 3. Experimental Apparatus

[0041] The microplate reader was purchased from PE Corporation, USA, model: VICTOR NIVO; the complete set of electrophoresis apparatus, electrophoresis tank, and standard wet transfer device for Western blot experiments was purchased from Bio-Rad Laboratories, USA, model: 1681130B; the centrifuge was purchased from Shanghai Luxiangyi Centrifuge Instrument Co., Ltd., model: TDZ4-WS; the transmission electron microscope was purchased from Hitachi, Japan, model: HT7700; and the CO2 cell incubator was purchased from Thermo Fisher Scientific, USA, model: 311.

[0042] 4. Western blot detection of protein levels

[0043] Cell or exosome samples were lysed using enhanced RIPA lysis buffer containing protease activator, and protein concentration was determined using a BCA protein assay kit. Protein samples were mixed with loading buffer, boiled for 5–10 min, and then separated by polyacrylamide gel electrophoresis. Subsequently, proteins were transferred to PVDF membranes using wet blotting. After blocking with skim milk powder for 2 h, the membranes were washed with TBST solution containing 0.1% Tween-20 and incubated overnight at 4 °C with primary antibodies diluted in TBST (LC3-I, LC3-II, P62, ATG5, CD9, CD63, TSG101, AKT, p-AKT, mTOR, p-mTOR), with GAPDH as the internal control. After washing three times in TBST, HRP-labeled goat anti-rabbit secondary antibody IgG was added, and the membranes were incubated at room temperature for 1 h, followed by development with ECL working solution. Gray-scale quantification of each band in the Western blot images was performed using Image Pro Plus 6.0, with each experiment repeated three times.

[0044] Example 1: High glucose induces autophagy disorder in HUVECs

[0045] In this embodiment, HUVECs were divided into two groups: a control group and a model group. The control group was treated with a 5.5 mmol / L glucose solution for 48 h, while the high glucose model group was treated with a 30 mmol / L glucose solution for 48 h. After establishing a diabetic vascular lesion cell model by high glucose stimulation of HUVECs, autophagosomes in the cells were observed using transmission electron microscopy. Western blot was used to detect protein levels in HUVECs cells.

[0046] Transmission electron microscopy (TEM) detection of autophagosomes: Cells were scraped off directly, added to PBS to make a cell suspension, centrifuged at 1000 g for 5 min, the supernatant was discarded, washed twice with PBS and centrifuged again, fixed with 2.5% glutaraldehyde solution and 2% osmium tetroxide solution, dehydrated and embedded, and then ultrathin sections were prepared. The sections were stained with uranium acetate and lead citrate, and photographed for recording.

[0047] like Figure 1 As shown in Figure A, the control group exhibited significant autophagy, with the formation of autophagosomes containing undigested organelles within the cytoplasm, and the formation of autolysosomes by binding with lysosomes. Partial cytoplasmic degradation was also observed. In contrast, the model group showed only minimal autophagy. Western blot analysis of the expression levels of autophagy-related proteins LC3-I, LC3-II, P62, and ATG5 revealed that, compared to the control group, the expression levels of LC3-II / LC3-I and ATG5 were significantly decreased in the model group, while the expression level of P62 was significantly increased. Figure 1 (All values ​​in B are P < 0.001). These results indicate that high glucose-induced autophagy impairment exists in HUVECs.

[0048] Example 2 Extraction and Characterization of AMSCs Exosomes

[0049] 1. Experimental Methods

[0050] 1.1 Extraction of AMSCs exosomes

[0051] In this embodiment, serum-free culture medium was used to culture AMSCs. After 48 h, the cell supernatant was collected in centrifuge tubes and centrifuged at 1000 g for 10 min at 4 °C. The supernatant was then collected and centrifuged at 4 °C for 4000 g for 20 min. The supernatant was then collected and centrifuged at 20000 g for 30 min at 4 °C. The supernatant was then filtered through a 0.22 μm sterile filter membrane, and the filtrate was collected. The collected filtrate was centrifuged at 120000 g for 90 min, and the precipitate, which was AMSCs-derived exosomes, was collected. The precipitate was then resuspended and aliquoted. The entire separation process was performed on ice.

[0052] 1.2 Observation of exosome micromorphology by transmission electron microscopy

[0053] The exosome samples were fixed with 2% paraformaldehyde for 30 min and then dropped onto a carbon-coated copper grid. The mixture was counterstained twice with 1% uranium acetate, and microscopic images of the exosomes were acquired at 120 kV.

[0054] 1.3 Nanoparticle tracking system for analyzing exosome size

[0055] The collected exosomes were diluted with PBS to a concentration of 1×10⁻⁶. 6 The size of exosomes was analyzed by injecting them into a nanoparticle tracking analysis (NTA) system using a 1 mL syringe.

[0056] 2. Experimental Results

[0057] The samples isolated from the culture supernatant of AMSCs were characterized and identified. The first step was to observe the morphology of exosomes using transmission electron microscopy, such as... Figure 2 As shown in Figure A, a large number of exosomes are visible in the sample, appearing as concave hemispherical vesicles; NTA results show that the diameter of the exosomes is approximately 134.5 nm, which is within the normal range. Figure 2 (B in the text); further, Western blot was used to detect the expression levels of exosome marker proteins CD9, CD63, and TSG101. The results showed that compared with the supernatant, the expression levels of CD9, CD63, and TSG101 were significantly upregulated in the exosome samples. Figure 2 The above data (C < 0.01) indicates that the extracted sample contains a large number of exosomes with high purity.

[0058] Example 3: AMSCs-derived exosome intervention promotes HUVEC autophagy

[0059] To investigate whether AMSCs-derived exosomes affect autophagy in HUVECs under high glucose conditions, this study divided HUVECs into five groups: a control group, a model group, a low-concentration exosome group, a medium-concentration exosome group, and a high-concentration exosome group. The control group and the high-glucose model group were identical to those in Example 1. The low, medium, and high-concentration exosome groups were prepared by treating cells with a 30 mmol / L glucose solution for 48 h, followed by culturing with AMSCs-derived exosomes at concentrations of 50, 100, and 150 μg / mL, respectively, for 24 h.

[0060] After intervening in a hyperglycemic cell model with low, medium, and high concentrations of exosomes, autophagy-related proteins and autophagosomes were detected. Compared with the model group, low, medium, and high concentrations of exosomes increased the number of autophagosomes and autolysosomes, while the expression levels of LC3-II / LC3-I and ATG5 significantly increased, and the expression level of P62 significantly decreased; among them, the medium concentration of exosomes showed the best effect. Figure 3 (All P < 0.01). This indicates that AMSCs-derived exosomes improve autophagy in HUVECs in a high-glucose environment.

[0061] Example 4: AMSCs-derived exosomes mediate the AKT / mTOR signaling pathway.

[0062] The AKT / mTOR signaling pathway is an important pathway regulating autophagy. In this example, the AKT / mTOR signaling pathway was used as a downstream target of exosomes. Since the results of Example 3 showed that 100 μg / mL of exosomes had the best intervention effect, this concentration was used for subsequent experiments.

[0063] HUVECs were divided into four groups: control group, model group, medium-concentration exosome group, and medium-concentration exosome + MHY1485 (AKT / m-TOR signaling pathway activator) group. The control group, model group, and medium-concentration exosome group were exactly the same as those in Example 3. The exosome + MHY1485 group was the same as the exosome (100 μg / mL) group, but with the addition of 10 μM MHY1485 solution.

[0064] First, the protein levels of the AKT / mTOR signaling pathway were measured. Compared with the control group, the p-AKT / AKT and p-mTOR / mTOR values ​​in the model group were significantly upregulated, indicating that the AKT / mTOR signaling pathway negatively regulates autophagy in HUVECs. Exosome intervention in AMSCs can partially reverse the activation of the AKT / mTOR pathway by the high-glucose environment, thereby activating autophagy in HUVECs. Further validation with the addition of the AKT / mTOR signaling pathway activator MHY1485 showed that, compared with the medium-concentration exosome group, the exosome + MHY1485 group had a significantly higher degree of AKT / mTOR pathway activation (see...). Figure 4 The results (A, P < 0.001) indicate that the activator MHY1485 can inhibit autophagy in HUVECs by activating the AKT / mTOR pathway, thus blocking the activation of HUVEC autophagy by exosomes. Next, the expression of autophagy-related proteins was detected, and the results are as follows: Figure 4 As shown in Figure B, compared with the exosome group, the expression levels of LC3-II / LC-I and ATG5 in HUVECs cells were significantly decreased and the expression level of P62 was significantly increased after treatment with MHY1485 (all P < 0.01). This reveals that exosomes promote autophagy by upregulating AKT / mTOR.

[0065] The above experimental results demonstrate that AMSCs-derived exosomes can weaken the activation effect of high glucose on AKT / mTOR, while the AKT / mTOR signaling pathway activator MHY1485 can block the activation of HUVECs autophagy by exosomes.

[0066] In summary, AMSCs-derived exosomes can promote autophagy in HUVECs under high glucose conditions by downregulating the AKT / mTOR signaling pathway, thereby alleviating diabetic vascular complications. This invention explores the mechanism of action of AMSCs-derived exosomes in treating autophagy impairment in vascular endothelial cells under high glucose conditions through in vitro cell experiments, providing new clues and ideas for the clinical application of stem cell therapy and the development of new drugs for diabetic vascular complications.

[0067] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. Application of AMSCs-derived exosomes in the preparation of drugs for treating diabetic vascular complications.

2. Application of the AKT / mTOR signaling pathway as a target of AMSCs-derived exosomes in the preparation of drugs for treating diabetic vascular complications.

3. The application according to claim 1 or 2, characterized in that, The drug is an AMSCs-derived exosome or an AKT / mTOR signaling pathway inhibitor.

4. The application according to claim 3, characterized in that, The method for extracting AMSCs-derived exosomes includes the following steps: S1. Culture AMSCs in serum-free medium. After 48 hours, collect the cell supernatant into a centrifuge tube and centrifuge at 4-10℃ and 800-1200g for 10-15 minutes. Collect the supernatant. S2. Centrifuge the obtained supernatant at 4~10℃ and 3500~4500g for 15~25min, and collect the supernatant; S3. Centrifuge the supernatant at 4~10℃ and 18000~22000g for 30~35min, filter the supernatant through a sterile filter membrane, and collect the filtrate. S4. Centrifuge the collected filtrate at 100,000~150,000g for 80~120 min. The precipitate is the exosome.

5. The application according to claim 4, characterized in that, The concentration of the exosomes is 20~500 μg / mL.

6. The application according to claim 4, characterized in that, The diameter of the exosomes is 50~400nm.

7. The application according to claim 4, characterized in that, The exosomes contain exosome marker proteins CD9, CD63, and TSG101.

8. A drug for treating diabetic vascular complications, characterized in that, This includes AMSCs-derived exosomes or AKT / mTOR signaling pathway inhibitors.

9. The medicament according to claim 8, characterized in that, It also includes pharmaceutically acceptable carriers or excipients.

10. The medicament according to claim 8, characterized in that, The concentration of AMSCs-derived exosomes is 20-500 μg / mL, and the diameter of the AMSCs-derived exosomes is 50-400 nm.

11. The medicament according to claim 8, characterized in that, The AMSCs-derived exosomes contain exosome marker proteins CD9, CD63, and TSG101.

12. The medicament according to claim 8, characterized in that, The drug is prepared as an injection, capsule, tablet, powder, ointment, or spray.

13. The medicament according to claim 8, characterized in that, Pharmaceutically acceptable carriers include phosphate buffer and / or physiological saline.

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