Preparation method and application of slc25a26 binding p12 peptide engineered targeting mitochondria-derived vesicles

Abstract

The application provides a preparation method and application of SLC25A26 binding P12 peptide engineered mitochondria-derived vesicles, and relates to the technical field of biology.The SLC25A26 / P12 fusion plasmid is constructed to infect MSC cells, SLC25A26-MSC is screened and cultured, the culture solution is collected, and the SLC25A26-MDV capable of targeting insulin receptors is obtained by iodixanol gradient density centrifugation MDVs.Researches show that the engineered vesicles can significantly promote mitochondrial oxidative phosphorylation, promote the expression of cell antioxidant enzymes, improve the mitochondrial network structure, promote the recovery of mitochondrial function, treat insulin resistance cell mitochondrial dysfunction, and successfully realize the use of SLC25A26 protein in the form of mitochondria-derived vesicles for related treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for preparing SLC25A26-bound P12 peptide-engineered mitochondrial-derived vesicles and their application in improving insulin resistance. Background Technology

[0002] Insulin resistance syndrome (IRS), encompassing disorders of glucose metabolism, hyperinsulinemia, dyslipidemia, and hypertension, is a common contributing factor to diseases such as diabetes and coronary heart disease, with IR being a central link in the development of these diseases. IR is a pathological state of cells, tissues, or the body, referring to insensitivity to insulin, resulting in the inability of normal insulin levels to perform their normal physiological functions, thereby hindering glucose uptake and utilization. IR can be induced by a high-calorie diet and lack of physical activity, and is also closely linked to other physiological factors, including mitochondrial dysfunction and systemic inflammation. Increasing research confirms the link between IR and mitochondrial dysfunction; for example, in patients with type 2 diabetes and IR, there is a significant decrease in the expression levels of genes involved in mitochondrial oxidative phosphorylation and mitochondrial biogenesis.

[0003] Cellular insulin resistance easily leads to mitochondrial dysfunction, with a significant increase in mitochondrial DNA (mtDNA) mutations. Accumulated mtDNA mutations further disrupt the mitochondrial respiratory chain, increasing reactive oxygen species (ROS) production. Insulin-resistant cells have a reduced ability to process ROS, making mtDNA susceptible to oxidative damage, triggering oxidative stress, and leading to cell cycle arrest and insulin resistance. Furthermore, mtDNA mutations readily induce the GAS-STING pathway, inducing oxidative stress. These two factors are closely linked, affecting cellular function and playing a crucial role in insulin resistance-related diseases. Interventions targeting mtDNA mutations and cellular insulin resistance may provide new approaches to delaying insulin resistance and preventing disease.

[0004] Mitochondrial-derived vesicles (MDVs) are important intracellular membrane vesicles formed from the contents of the mitochondrial outer membrane, inner and outer membranes, and matrix, possessing a variety of biological functions. These functions include serving as carriers of mitochondrial DNA and are closely associated with mitochondrial DNA mutations and related diseases. Therefore, appropriate regulation of MDVs may be an important target for the treatment of mitochondrial diseases, and is of great significance for understanding mitochondrial biology and developing new disease treatment strategies. However, physiologically, MDV production is extremely low, insufficient to achieve therapeutic efficacy; therefore, increasing MDV production is a key issue for its potential as a drug.

[0005] SLC25A26 can synergistically promote the generation of inner membrane MDV with OPA1, potentially increasing MDV yield. Furthermore, since inner membrane MDV buds from the mitochondrial cristae and contains many mitochondrial matrix proteins and complete mtDNA, using inner membrane MDV can improve the production of mutated or missing mtDNA in insulin resistance due to its high mtDNA content. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of SLC25A26 engineered mitochondrial-derived vesicles rich in mtDNA in the preparation of drugs for treating mitochondrial dysfunction in insulin-resistant cells.

[0007] A second objective of this invention is to provide the use of SLC25A26 dual-gene engineered mitochondrial-derived vesicles rich in mtDNA in the preparation of medicaments for treating cellular insulin resistance caused by mitochondrial dysfunction.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention involves constructing an insulin receptor-targeting peptide, screening for peptides that tightly bind to the insulin receptor (INSR), and naming the peptide P12. A recombinant plasmid pcDNA3.1-SLC25A26-P12-C is constructed, and peptide P12 is linked to the C-terminus of the SLC25A26 protein using gene recombination technology, forming the SLC25A26-P12 peptide fusion protein expression system. Peptide P12 can help MDV target the insulin receptor on target cells. A recombinant plasmid expressing the recombinant protein SLC25A26-P12 is constructed, and the plasmid is transfected into cells for culture. The resulting SLC25A26 MDV can target the insulin receptor, promoting the binding of MDV to the target protein.

[0010] This invention incorporates the engineered mitochondrial-derived vesicles SLC25A26 / P12-MDV into a high-glucose-induced mitochondrial dysfunction-induced insulin resistance cell model. The effects on ATP content, mitochondrial membrane potential, ROS levels, and mtDNA content in the model cells were evaluated. The expression levels of proteins related to the insulin resistance signaling pathway were also detected. The results show that the engineered mitochondrial-derived vesicles SLC25A26 / P12-MDV significantly promote mitochondrial oxidative phosphorylation and mitochondrial function recovery, regulate the levels of intracellular insulin-related proteins, and enhance their antioxidant capacity. Therefore, it has application value in treating cellular insulin resistance caused by mitochondrial dysfunction.

[0011] Therefore, the present invention first provides the application of the above-mentioned mtDNA-rich SLC25A26 engineered targeted mitochondrial-derived vesicles in the preparation of drugs that improve mitochondrial dysfunction in insulin-resistant cells.

[0012] The present invention also provides the use of the mtDNA-rich SLC25A26 engineered targeting mitochondrial-derived vesicles in the preparation of medicaments for treating cellular insulin resistance associated with mitochondrial dysfunction.

[0013] Furthermore, the cellular insulin resistance is caused by mitochondrial dysfunction.

[0014] Furthermore, the subjects were selected from mammals.

[0015] Furthermore, the mammals mentioned are selected from rats, cats, dogs, pigs, cattle, horses, sheep, monkeys, and humans.

[0016] Furthermore, the drug also includes other pharmaceutically acceptable excipients.

[0017] Furthermore, a recombinant plasmid overexpressing SLC25A26 was constructed. After transfecting MSC cells (engineered cells), mitochondrial-derived vesicles were extracted. SLC25A26-enriched engineered mitochondrial-derived vesicles were successfully constructed.

[0018] Furthermore, the amino acid sequence of the polypeptide P12 is shown in SEQ ID No. 1: CGERGFFYTPKA. This invention has constructed three polypeptide sequences capable of binding to the recombinant insulin receptor protein. ELISA detection showed that the polypeptide with the sequence CGERGFFYTPKA exhibits stronger binding affinity.

[0019] Furthermore, the template plasmid of the recombinant plasmid is pcDNA3.1, that is, the pcDNA3.1-SLC25A26-P12 recombinant plasmid is constructed.

[0020] Furthermore, the cells are human mesenchymal stem cells.

[0021] Furthermore, the mitochondrial-derived vesicles are extracted using ultracentrifugation.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of engineered SLC25A26-targeting mitochondrial-derived vesicles (SLC25A26-MDV) rich in mtDNA in the preparation of drugs for treating mitochondrial dysfunction in insulin-resistant cells. By constructing an SLC25A26 / P12 fusion plasmid to infect MSC cells, SLC25A26-MSCs were screened and cultured. The culture medium was collected and centrifuged with iodixanol at a gradient density to obtain SLC25A26-MDVs. Studies have shown that these engineered vesicles significantly promote mitochondrial oxidative phosphorylation, stimulate the expression of cellular antioxidant enzymes, improve mitochondrial network structure, and promote mitochondrial function recovery, thus treating mitochondrial dysfunction in insulin-resistant cells. This successfully enables the use of SLC25A26 protein in the form of mitochondrial-derived vesicles for related therapies. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 For example 1, INSR affinity peptides were screened using ELISA; (n=3, P < 0.05 P < 0.01 P < 0.001, ns: no significant difference compared with wild type; Figure 2 The results of Western blot analysis of SLC25A26 protein expression level and mRNA expression level (n=3) in transfected MSCs in Example 1 are shown. P < 0.05 P < 0.01 (P < 0.001, ns: no significant difference compared with MSC); Figure 3 The relative expression level of flag in transfected MSCs in Example 1 (n=3, P < 0.05 P < 0.01 (P < 0.001, ns: no significant difference compared with MSC); Figure 4 The particle size of MDVs was detected by nanoparticle tracking analysis in Example 1; Figure 5 The expression levels of related proteins and mtDNA in each group of MDVs in the Western Blot of Example 1 (n=3, P < 0.05 P < 0.01 (P < 0.001, ns: no significant difference compared with WT-MDV). Figure 6 DEP thermal images of SLC25A26 / P12-MDV and WT-MDV in Example 1; Figure 7 The image shows the KEGG and GO analysis results in the SLC25A26 / P12-MDV in Example 1. Figure 8 Western blot analysis of the expression levels of insulin signaling pathway-related proteins in HepG2 cells after high glucose induction in Example 2; Figure 9 The internalization level of cells in each group in Example 2 (n=3, (P<0.05); Figure 10 To detect the relative mitochondrial membrane potential of JC-1 cells after internalizing MDVs in each group in Example 2 (n=3, P < 0.05 P < 0.01 (P < 0.001, ns: no significant difference compared with the normal group). Figure 11 The ATP levels of cells after internalizing MDVs in each group in Example 2 (n=3, P < 0.05 P < 0.01 (P < 0.001, ns: no significant difference compared with the normal group). Figure 12 The relative ROS levels of cells in each group in Example 2 (n=3, P < 0.05 P < 0.01 (P < 0.001, ns: no significant difference compared with the normal group). Figure 13 The relative mtDNA content (n=3) of HepG2 cells after treatment with SLC25A26 / P12-MDVs in Example 2. P < 0.05 P < 0.01 (P < 0.001, ns: no significant difference compared with the normal group). Figure 14 The expression concentrations of insulin resistance-related proteins in cells after internalization of MDVs in each group in Example 2 (n=3, P < 0.05 (P < 0.01, ns: no significant difference compared with the normal group). Figure 15 The effect of internalizing MDVs on glucose uptake in cells after each group in Example 2 (n=3, P < 0.05 P < 0.01, ns: no significant difference compared with the normal group. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the 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.

[0026] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field.

[0027] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0028] 1. Experimental Materials Table 1. Cell and plasmid sources

[0029] 2. Experimental Methods (1) Cell Culture: Quickly remove the frozen sample from the liquid nitrogen container, place it in a 37°C constant temperature water bath to thaw, and gently shake for 60 seconds. After observing the cell state with an optical microscope, centrifuge at 8000 rpm for 5 minutes to precipitate the cells. Discard the supernatant, resuspend the cells in culture medium, and then seed them into 25ml culture flasks and place them in a 37°C constant temperature incubator for amplification culture. The purpose of centrifugation is to separate the cells from the cryopreservation solution, while resuspending provides a suitable growth environment for the cells.

[0030] (2) Cell proliferation and passage: When the cell coverage in the culture dish reaches 70%-80%, the cells enter the passage stage. At this time, the cells are growing vigorously, and passage can ensure the continuous healthy growth of the cells. Carefully remove the old culture medium and wash the cells twice with phosphate-buffered saline (PBS) to remove residual culture medium and metabolic products. Then add 1 ml of 0.25% trypsin-EDTA mixture and digest at 37°C for 3 minutes. This allows the cells to detach from the surface of the culture dish. Microscopic examination confirms changes in cell morphology. When the cells detach from the surface, it indicates that the digestion is appropriate. Add 3 ml of DMEM medium containing 10% fetal bovine serum to stop the digestion and prepare a single-cell suspension. Fetal bovine serum can provide the nutrients required for cell growth and at the same time stop the digestion of trypsin, protecting the cells from the effects of over-digestion. Place the culture container in an incubator with 5% carbon dioxide and 37°C. Change the medium for the first time after 48 hours, and then change the medium every 2-3 days thereafter. Changing the medium can provide fresh nutrients for the cells and remove metabolic waste, ensuring a stable growth environment for the cells. Finally, well-growing cells were selected for experiments and cryopreservation.

[0031] Example 1 Preparation of engineered mitochondrial-derived vesicles rich in mtDNA, SLC25A26, and isolation and characterization of MDVs. I. Experimental Methods 1. Construction of targeted peptides Based on the insulin-binding domain of the insulin receptor, three insulin receptor-targeting peptides with binding capacity were constructed. The interaction between the target and the peptide was simulated using molecular docking software (AutoDock), and peptide sequences capable of forming hydrogen bonds, hydrophobic interactions, or cation-π interactions with key residues of the target were screened. Sequence optimization through mutation and amino acid residue substitution improved affinity and specificity. AlphaFold was used to predict and design the secondary structure and three-dimensional conformation of the peptides, ensuring they had suitable conformations for target binding. The peptides were synthesized using solid-phase synthesis or genetic engineering methods, and their purity and structure were verified.

[0032] 2. Affinity verification To verify the binding affinity and specificity of the selected insulin receptor-targeting peptide P12 to INSR, this invention employs an enzyme-linked immunosorbent assay (ELISA). INSR was dissolved in 0.1 M NaHCO3 (pH 8.6) to prepare a 150 μL INSR protein solution with a concentration of 100 μg / mL. This solution was added to a 96-well plate and incubated gently at 4°C for 30 minutes to allow the INSR protein to initially attach to the well walls. Then, it was incubated overnight at 4°C to further strengthen the protein's adhesion to the well walls. 2 × 10⁻⁶ 9 CFU-purified targeting peptides were added to blocking buffer, followed by the addition of the blocking buffer containing the targeting peptides to a 96-well plate coated with INSR protein, and incubated at room temperature for 1 hour. After incubation, to remove unbound material, the plate was washed three times with PBS containing 0.5% Tween 20, and then three times with PBS to ensure the plate was clean and free of residual impurities. After washing, the plate was incubated at room temperature with alkaline phosphatase-conjugated anti-fd targeting peptide IgG (Abcam, MA, USA) for 1 hour to form a "targeting peptide-antibody-enzyme" complex. After washing the plate again, the substrate p-nitrophenol phosphate was added to the wells, and finally, the absorbance was measured at 405 nm using a plate reader.

[0033] 3. Plasmid synthesis and transfection This invention uses pcDNA3.1 plasmid as a template and designs KpnI and XbaI restriction endonuclease sites at both ends of the SLC25A26-Flag-P12 sequence to construct a recombinant plasmid named pcDNA3.1-SLC25A26-P12-C (i.e., pcDNA3.1-SLC25A26-Flag-P12); the host bacterium for plasmid construction is E. coli DH5α. The transfection procedure was as follows: MSC cells were evenly seeded into 6-well plates and cultured until the cell density reached 30%-50%. In a 1.5 mL centrifuge tube, 200 μL of serum-free medium and 3 μg of plasmid were added and mixed. In another centrifuge tube, 200 μL of serum-free medium and 6 μL of transfectant were mixed. Lipofectamine 2000-mediated transfection was used. After reacting for 5 minutes at room temperature, the contents of the two tubes were combined, and the reaction was continued for another 20 minutes. The transfection complex was added to a pre-layered 6-well plate containing 2 mL of serum-free medium and gently mixed. The plate was incubated in a cell culture incubator for 5 hours, then replaced with complete medium and cultured for 48 hours. Cells were then collected for subsequent experiments. Cells transfected with pcDNA3.1-SLC25A26-P12-C were named C+ (same as MSC-SLC25A26).

[0034] 4. Extraction of extracellular vesicles by ultracentrifugation Seed cells in 75 cm² culture flasks and collect them when the cell density reaches 80-90%. Centrifuge at low speed: -4°C, 300 g for 20 min, collect the supernatant; centrifuge at 4°C, 2000 g for 20 min, collect the supernatant; centrifuge at high speed: 4°C, 10000 g for 20 min, seal the supernatant and store at -80°C. Thaw the supernatant, balance, and centrifuge at 100000 g for 70 min using an SW70Ti rotor, discard the supernatant, and resuspend in PBS. After transfer, centrifuge at 4°C, 10000 g for 70 min using an SW41Ti rotor, discard the supernatant. Resuspend in pre-cooled PBS, aliquot into sterile EP tubes, and store at -80°C, avoiding repeated freeze-thaw cycles.

[0035] 5. Preparation of gradient solutions for MDV separation using iodixanol gradient density separation Pre-equilibrate 60% iodixanol solution using a Tris-HCl buffer system to prepare solutions A, OWS, and B. Add different concentrations of iodixanol solution sequentially to centrifuge tubes, followed by extracellular vesicle solution. After balancing, centrifuge at 4°C and 200,000 g for 16 h using an SW41Ti rotor. Aliquot each layer, bring the 20% layer to volume, centrifuge at 4°C and 100,000 g for 70 min, and discard the supernatant. Resuspend the MDVs precipitate in pre-cooled PBS, aliquot, and store at -80°C, avoiding repeated freeze-thaw cycles.

[0036] 6. Western Blot analysis of MDV marker proteins isolated from cells stably overexpressing MDVs MDVs were isolated from MSCs and SLC25A26 / P12-MSCs. MDVs were grouped as follows: MDVs secreted by MSCs were WT-MDVs; MDVs secreted by SLC25A26 / P12-MSCs were SLC25A26 / P12-MDVs.

[0037] The concentration of collected samples was determined using the BCA method. Loading buffer was added to the protein sample at the concentration to be determined in a ratio (sample volume: 5 × loading buffer = 5:1) and placed in a 100 °C metal bath for 10 min to complete sample preparation. Western blotting was then used to detect the expression levels of INSR, a marker of MDVs, and INSR protein.

[0038] 7. Nanoparticle size detection Inject the extracted MDVs suspension into the sample cell using a 1mL syringe, ensuring full coverage of the liquid surface. Set the parameters, adjust the focus of the microscope to make the particles clearly visible, and select to observe and calculate the particle size of the sample in the standard operating procedure.

[0039] II. Experimental Results 1. Three polypeptide sequences were constructed that exhibit affinity for the INSR recombinant protein. These polypeptide sequences and their binding frequencies are shown in Table 2. ELISA analysis revealed that the polypeptide with the sequence CGERGFFYTPKA exhibited stronger binding affinity, as shown in the results. Figure 1 As shown. This polypeptide sequence is named P12.

[0040] Table 2. Constructed peptide sequences and peptide binding frequencies to INSR

[0041] 2. After confirming that P12 can specifically bind to INSR, the P12 sequence, Flag tag sequence, and SLC25A26 protein-coding sequence were sequentially inserted into the recombinant plasmid to overexpress the SLC25A26-P12 fusion protein in cells, thereby achieving the targeted localization of the fusion protein to INSR. Western blot analysis of MSCs transfected with the recombinant plasmid (SLC25A26 / P12-MSCs) revealed a significantly higher SLC25A26 protein content. RNA was extracted from stable cell lines for RT-qPCR to detect the overexpression efficiency. Compared with the control group, the mRNA level of SLC25A26 in stably overexpressing cells was significantly increased, indicating the successful construction of a stable MSC cell line overexpressing the SLC25A26 gene. This demonstrates that the recombinant plasmid was successfully transfected into cells (…). Figure 2 ).

[0042] The P12 polypeptide coding sequence (36 bp) is shown in SEQ ID No. 4: TGCGGTGAAC GTGGTTTCTT CTACACCCG AAAGCG; The protein coding sequence of SLC25A26 (561 bp) is shown in SEQ ID No. 5: ; SLC25A26 The cDNA sequence of the gene (2154 bp) (NM_001164796.1) is shown in SEQ ID No. 6.

[0043] 3. The expression level of flag in the transfected cells was also detected, and the results are as follows: Figure 3 As shown, the flag was highly expressed after the recombinant plasmid was transfected into the cells, further proving that the transfection was successful.

[0044] 4. MDVs particle size analysis The diameters of the extracted WT-MDVs and SLC25A26 / P12-MDVs were determined using a Nanosight particle size analyzer. The results are as follows: Figure 4 As shown, the particle size range is mainly 50-200 nm, with the peak particle size appearing at (97.57±12.28 nm), which is consistent with the morphological characteristics of MDVs.

[0045] 5. Western Blot of MDV-related proteins and SLC25A26 Western blotting analysis was performed on the MDV obtained above. The Western blotting analysis is as follows: Figure 5 As shown, SLC25A26 / P12-MDVs highly express mitochondrial inner membrane marker proteins TIM23 and SLC25A26, confirming that this MDV is an inner membrane type. Analysis of mtDNA levels in each group of MDVs revealed that the mtDNA level in SLC25A26 / P12-MDV was higher than that in the WT-MDV group.

[0046] 6. MDV sequencing This study performed whole-genome transcriptome sequencing analysis on wild-type and SLC25A26-derived MDV (WT-MDV, SLC25A26 / P12-MDV) samples. DEP heatmap results showed that SLC25A26 / P12-MDV was enriched with SLC25A26 and OPA1, a fusion protein closely related to SLC25A26 in mitochondrial biosynthesis. Figure 6 ). GO and KEGG analysis results show ( Figure 7 In the BP (Proteinized Biotinylate) group, oxidative phosphorylation and mitochondrial respiratory chain complex assembly were significantly enriched, suggesting that SLC25A26 / P12-MDV treatment enhances core mitochondrial energy metabolism processes. CC (Carbon Dioxide) analysis showed significant enrichment in both the mitochondrial matrix and inner mitochondrial membrane, as well as the mitochondrial respiratory chain. MF (Fluorescence Mechanism) analysis showed significant upregulation of electron transport activity and NADH dehydrogenase activity, indicating that MDV promotes the functional activity of the respiratory chain complex. Simultaneously, increased ATP binding capacity suggested a decrease in cellular energy supply capacity. KEGG analysis showed significant upregulation of the tricarboxylic acid cycle and oxidative phosphorylation pathways in the control group, indicating that SLC25A26 / P12-MDV directly promotes core mitochondrial energy metabolism pathways.

[0047] Example 2 The effect of MDVs on improving cellular insulin resistance induced by high glucose-induced mtDNA mutations I. Experimental Methods 1. Establishment of cell model HepG2 cells were seeded in 6-well plates containing DMEM medium (10% FBS) and cultured at 37°C and 5% CO2. When confluence reached 90%, cells were washed with PBS, digested with trypsin, and transferred to 6-well plates containing fresh medium for further culture. Cells were treated with 35 mmol / L glucose solution for 24 h. HepG2 cells were then seeded in 6-well plates containing 35 mmol / L high-glucose DMEM complete medium and cultured for 3 days to induce insulin-resistant HepG2 cells. After induction, cells were seeded in DMEM complete medium without high-glucose for scale-up culture. The medium was changed every 2 days, and subsequent experiments were performed after 5 days.

[0048] The cell experiments were divided into four groups: normal group, HepG2 group after high glucose induction (IR), HepG2 group with internalized WT-MDV (WT-MDV), and HepG2 group with internalized SLC25A26 / P12-MDV (SLC25A26 / P12-MDV).

[0049] 2. Western blot analysis of expression levels of insulin resistance-related proteins in the insulin signaling pathway. The expression levels of insulin resistance-related proteins and insulin signaling pathway-related proteins were detected in cells from both the IR group and the normal group.

[0050] 3. Internalization detection of MDVs MDVs were extracted using the iodixanol gradient density method. Following the kit instructions, 100 μg of MDVs were labeled with 5 μL of EvLINK505, and the mixture was gently mixed and incubated for 30 minutes in the dark at room temperature. After labeling, the purified sample was collected for subsequent assessment of cellular MDV uptake. Cells were seeded in confocal culture dishes and cultured for 24 hours to allow for stable cell growth. HepG2 and PBMC cells were then incubated with EvLINK505-labeled MDVs for 24, 48, 72, and 96 hours, respectively. After each incubation, the cells were washed with PBS to remove unuptaken MDVs. To clearly visualize the cell membrane, HepG2 and PBMC cells were labeled with Cel-ILINK 555 and incubated for 30 minutes in the dark at room temperature. After incubation, the cells were washed again with PBS, and then fixed with 4% paraformaldehyde for 30 minutes to fix their morphology and structure. Following fixation, the cells were stained with DAPI for 5 minutes. DAPI binds to DNA in the cell nucleus, clearly revealing its location. The internalization of the labeled MDVs was observed using a laser confocal scanning microscope.

[0051] 4. Detection of mtDNA content Cells were digested using trypsin. After digestion, the cells were centrifuged at 12,000 rpm for 2 minutes at 4°C. 250 μL of solutions A and B were added sequentially and mixed thoroughly. The mixture was incubated on ice for 6 minutes. 350 μL of solution C (on ice) was added, and the mixture was shaken until a white precipitate formed. The mixture containing the precipitate was then incubated on ice for another 25 minutes to allow for a more complete reaction. The supernatant and precipitate were separated. The supernatant was carefully transferred to an adsorption column and allowed to stand for 5 minutes to allow sufficient time for the mtDNA to adsorb onto the column. The column was then centrifuged at 12,000 rpm for 1 minute, and the eluent was discarded. 500 μL of wash buffer was added, and the column was centrifuged at 12,000 rpm for 1 minute. The centrifugation was repeated once, and the remaining liquid was removed to obtain the mtDNA extract.

[0052] Genomic DNA was extracted according to the DNA extraction kit. mtDNA was quantified using qPCR. Primers were designed based on NCBI and synthesized by Qingke Biotechnology. mtDNA was quantified using the ND1 subunit gene of NADH dehydrogenase, and nDNA was quantified using the β-actin gene. Each group was added to SYBR Green Master Mix and the corresponding primer preparation system for qPCR, with β-actin as an internal control gene. The corresponding primer sequences and PCR reaction conditions are shown in Tables 3 and 4. Three independent biological replicates were performed. Calculate the relative gene expression levels.

[0053] Table 3 PCR amplification primers

[0054] Table 4 PCR reaction conditions

[0055] 5. ATP level detection Equilibrate the kit to room temperature for 30 minutes. Perform serial dilutions of the standards (e.g., 0, 0.1, 0.5, 1, 5, 10 μM). Prepare the working solution (luciferin + luciferase) fresh and protect from light. Add samples to 96-well black / white plates. Add 100 μL of lysis buffer to blank wells, 100 μL of standard to standard wells, and 100 μL of sample supernatant to sample wells. Add 100 μL of working solution to each well and mix immediately (to remove air bubbles). Incubate at room temperature in the dark for 5-10 minutes, then measure the RLU value using a chemiluminescence analyzer. Calculate the ATP concentration.

[0056] 6. ROS level measurement Aspirate the cell culture medium and gently wash the cells 2-3 times with PBS to remove serum and other impurities that may affect probe loading. Add an appropriate amount of DCFH-DA working solution, ensuring it covers the cell surface. For example, add 1-2 mL of working solution per well for a 6-well plate and 100-200 μL per well for a 96-well plate. Incubate the cells at 37°C with 5% CO2 for 20-30 minutes to allow DCFH-DA to enter the cells. During incubation, intracellular esterases hydrolyze the acetate groups of DCFH-DA to generate DCFH (dichlorodihydrofluorescein), which can be oxidized by ROS to fluorescent DCF (dichlorofluorescein). After incubation, aspirate the DCFH-DA working solution and gently wash the cells 3-4 times with PBS to remove any un-entered DCFH-DA and reduce background fluorescence. The fluorescence intensity of intracellular DCF was detected using fluorescence microscopy (EX 488 nm, EM 525 nm).

[0057] 7. Mitochondrial membrane potential detection Aspirate the cell culture medium and gently wash the cells 2-3 times with PBS to remove serum and other impurities. Add an appropriate amount of JC-1 working solution, ensuring it covers the cell surface. For example, add 1-2 mL of working solution per well for a 6-well plate and 100-200 μL per well for a 96-well plate. Incubate the cells at 37°C with 5% CO2 for 20-30 minutes to allow JC-1 to enter the cells and accumulate in the mitochondria. After incubation, aspirate the JC-1 working solution and gently wash the cells 2-3 times with PBS to remove any JC-1 that has not entered the cells. Place the culture plate or dish under a fluorescence microscope. JC-1 forms polymers and emits red fluorescence when the mitochondrial membrane potential is high; it exists as monomers and emits green fluorescence when the mitochondrial membrane potential decreases. By observing the intensity and ratio of red and green fluorescence, changes in mitochondrial membrane potential can be determined.

[0058] 8. Enzyme-linked immunosorbent assay (ELISA) to detect the expression level of insulin resistance-related proteins. Cells were treated with lysis buffer containing PMSF, centrifuged, and the supernatant was collected and diluted. Then, standard working solutions, biotinylated antibody working solutions, HRP enzyme conjugate working solutions, and washing buffer were prepared. Samples or standards were added sequentially to the ELISA plate, incubated at 37°C, and the solutions were discarded. Biotinylated antibody working solutions were added, and the plate was incubated again. After washing, enzyme conjugate working solutions were added, incubated, and washed again. TMB substrate was added for color development in the dark. Finally, stop solution was added, and the OD value was measured at 450 nm. The sample concentration was calculated using a standard curve.

[0059] 9. 2-NBDG assay for glucose uptake Cells from each group were collected by centrifugation, cultured in sugar-free DMEM medium for 5 h, then cultured in 100 nM insulin for 20 min, and finally co-incubated in 50 nM 2-NBDG for 30 min. Cells were washed with PBS to terminate the reaction, and fluorescence values ​​were measured at 475 and 550 nm using a fluorescence microplate reader.

[0060] 10. Statistical Analysis Statistical analysis of continuous variables is expressed as mean ± standard deviation. GraphPad Prism 10.0 was used for statistical analysis. Independent samples t-tests or one-way ANOVA were used to analyze the statistical differences between groups. A p-value less than 0.05 was considered statistically significant.

[0061] II. Experimental Results 1. Insulin resistance markers: expression levels of proteins related to the insulin signaling pathway mtDNA mutations lead to cellular insulin resistance, and proteins related to the insulin signaling pathway are downregulated during cellular insulin resistance. Next, total protein was extracted from the cells and analyzed by Western blot. The results are as follows: Figure 8 As shown, the expression levels of insulin signaling pathway-related proteins Akt and GLUT4 in HepG2 cells were significantly reduced, indicating that an insulin-resistant cell model was successfully constructed.

[0062] 2. Internalization of MDVs The fluorescence intensity of HepG2 and PBMC cells co-cultured with EvLINK505-labeled MDV at 24 h, 48 h, 72 h, and 96 h was observed using laser confocal scanning microscopy. Figure 9 The results showed that P12 binding enabled MDV to effectively target cells with insulin receptors, and the internalization efficiency of SLC25A26 / P12-MDV was significantly improved in both cell types.

[0063] 3. Detection of mitochondrial membrane potential after MDVs internalized HepG2 cells in each group In insulin-resistant cells, mitochondrial membrane potential is typically significantly reduced due to decreased efficiency of oxidative phosphorylation and excessive ROS production. In this study, JC-1 staining was performed on HepG2 cells with internalized MDVs in each group. Fluorescence analysis showed a significant increase in mitochondrial red fluorescence intensity in the SLC25A26 / P12-MDV group, indicating an increase in membrane potential and suggesting recovery of mitochondrial function. The recovery was most pronounced in the SLC25A26 / P12-MDV group. Figure 10 ).

[0064] 4. Changes in ATP levels after MDVs internalized HepG2 cells in each group ATP is an important product of mitochondrial oxidative phosphorylation, and its level can measure mitochondrial function. By detecting the ATP levels in each group of cells, the results are as follows: Figure 11 As shown, ATP production in the SLC25A26 / P12-MDV group was significantly increased, and it was closer to that of normal HepG2 cells compared to other groups, indicating that the mitochondrial electron transport chain function in this group of cells was improved.

[0065] 5. Changes in ROS levels after internalization of HepG2 cells by MDVs in each group Mitochondrial dysfunction in insulin-resistant cells promotes increased ROS production. ROS accumulation further exacerbates cell damage. The results of internalizing MDV into HepG2 in each group are as follows: Figure 12 As shown, the ROS level in the IR group was significantly increased, while that in the treatment group was decreased. In addition, the ROS level in the SLC25A26 / P12-MDV group was the most significantly reduced.

[0066] 6. Detection of mtDNA content in cells of each group To investigate the changes in mtDNA content in cells after MDVs were internalized into HepG2 in each group, this study extracted and quantified mtDNA and nDNA from the cells, and calculated the relative mtDNA content in each group. The results are as follows: Figure 13 As shown, the mtDNA content of cells in both the WT-MDV and SLC25A26 / P12-MDV groups increased, with the increase being more pronounced in the SLC25A26 / P12-MDV group.

[0067] 7. Internalization of MDVs in each group reduced the expression levels of proteins in cellular insulin resistance-related pathways. Impairment of the insulin signaling pathway can lead to insulin resistance (IR). To further verify the effect of SLC25A26 / P12-MDV on improving IR in HepG2 cells, this invention detected the activity of proteins related to insulin signaling in each group of cells. Figure 14 Compared to normal cells, the IR group showed decreased activity of PI3K and Akt proteins and reduced expression of GLUT4 protein on the plasma membrane, indicating impaired insulin signaling and resulting in IR. After treatment with SLC25A26 / P12-MDV, the activities of PI3K and Akt were enhanced, and the expression of GLUT4 protein on the plasma membrane significantly increased. This demonstrates that SLC25A26 / P12-MDV can promote the activity of proteins related to the insulin signaling pathway, improve the sensitivity of model cells to insulin, and thus treat IR.

[0068] 8. Results of glucose uptake in HepG2 cells Cellular irritation (IR) directly manifests as abnormal glucose uptake; therefore, this invention uses the 2-NBDG method to detect glucose uptake in HepG2 cells. Compared to normal cells, glucose uptake in the IR group was significantly reduced (…). Figure 15 After treatment with SLC25A26 / P12-MDV, this change was reversed, and glucose uptake in the IR group cells significantly increased. This indicates that SLC25A26 / P12-MDV can improve cellular sensitivity to insulin.

[0069] In summary, the results indicate that SLC25A26 genetically engineered mitochondrial-derived vesicles can carry complete mtDNA into insulin-resistant cells for treatment, demonstrating a significant anti-insulin resistance effect.

[0070] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing SLC25A26-bound P12 peptide-engineered mitochondrial-derived vesicles, characterized in that, Includes the following steps: 1) The gene encoding the target insulin receptor P12 polypeptide with the amino acid sequence shown in SEQ ID No.1 was linked to the 3' end of the SLC25A26 protein encoding gene to obtain a chimeric gene; 2) Construct a recombinant plasmid capable of expressing the chimeric gene; 3) Transfect the constructed recombinant plasmid into the cells and allow the cells to express it; the cells are human mesenchymal stem cells; 4) Extract mitochondrial-derived vesicles from cells to obtain SLC25A26-bound P12 peptide-engineered targeted mitochondrial-derived vesicles.

2. The preparation method according to claim 1, characterized in that, The gene encoding the P12 polypeptide is linked to the C-terminus of the gene encoding the SLC25A26 protein via a Flag tag.

3. The preparation method according to claim 1, characterized in that, The template plasmid used for the recombinant plasmid is pcDNA3.

1.

4. The use of SLC25A26-bound P12 peptide-engineered mitochondrial-derived vesicles prepared by any of the methods described in claims 1-3 in the preparation of drugs that improve insulin resistance.

5. The application according to claim 4, characterized in that, The drug improves mitochondrial function in insulin-resistant cells by promoting ROS clearance in subject cells, protecting mitochondrial DNA, improving mitochondrial network structure, and ultimately improving mitochondrial function.

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