A snx9 engineered mitochondrial vesicle, and a preparation method and application thereof in improving insulin resistance

Abstract

The application discloses a kind of SNX9 engineering mitochondria vesicles and its preparation method and application in improving insulin resistance.The application first constructs the vector of overexpression SNX9 gene, transfects cell, obtains stable overexpression SNX9 gene cell strain, culture, collects culture solution, obtains SNX9-MDVs by centrifugal separation;Then mtDNA is extracted from cell and introduced into SNX9-MDVs, to obtain the engineering mitochondria vesicles mtDNA-SNX9-MDVs rich in mtDNA.MtDNA-SNX9-MDVs are used for insulin resistance treatment, and the results show that mtDNA-SNX9-MDVs can effectively increase mitochondrial ATP and mtDNA content, enhance cell antioxidant enzyme activity, reduce ROS, improve mitochondrial network structure, increase the expression of insulin signal pathway related proteins, and show obvious mitochondrial function repair potential and insulin resistance improvement effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to SNX9 engineered mitochondrial vesicles rich in mtDNA, their preparation method, and their application in improving insulin resistance. Background Technology

[0002] Mitochondrial dysfunction is closely linked to the development of insulin resistance syndrome (IRS), which includes disorders of glucose metabolism, hyperinsulinemia, dyslipidemia, and hypertension. For example, in patients with type 2 diabetes and IR, significant mitochondrial dysfunction leads to impaired β-oxidation, resulting in fatty acid accumulation and increased reactive oxygen species (ROS), activating corresponding stress proteins and thus inhibiting insulin signaling. During oxidative metabolism, mitochondrial electrophoresis (ETC) easily leads to the reduction of oxygen molecules into reactive oxygen species (ROS) due to the leakage of large amounts of electrons. Excessive ROS leads to a decrease in mtDNA content, causing mitochondrial dysfunction, affecting mitochondrial homeostasis, and further exacerbating the progression of IR. Therefore, increasing mitochondrial DNA (mtDNA) content to promote mitochondrial function is expected to improve insulin resistance.

[0003] Mitochondrial-derived vesicles (MDVs) are important intracellular membrane vesicles formed by the outer mitochondrial membrane, or the contents of both the inner and outer membranes and the matrix. MDVs have multiple biological functions, including maintaining mitochondrial quality, enhancing antioxidant capacity, promoting intercellular communication, regulating cellular metabolism, and immune responses, playing a crucial role in normal physiological processes. They are also closely related to the occurrence and development of various diseases. MDVs contain mtDNA and mitochondrial components, making them a natural and potential mtDNA delivery carrier. By delivering MDVs secreted by normal cells to the mitochondria of damaged cells with accumulated mtDNA mutations, the proportion of mutated mtDNA can be reduced, mitochondrial function can be promoted, and insulin resistance may be improved. However, the production of naturally secreted MDVs is low, making it difficult to meet the needs of drug therapy. Summary of the Invention

[0004] The purpose of this invention is to overcome the technical problems existing in the prior art, namely, the low yield of naturally secreted MDVs under physiological conditions, which makes it difficult to meet the needs of drug therapy, and the lack of technical applications of mtDNA in improving insulin resistance. This invention provides an SNX9 engineered mitochondrial vesicle, its preparation method, and its application in improving insulin resistance.

[0005] This invention first provides a method for preparing SNX9 engineered mitochondrial vesicles, which is a prerequisite for first constructing an overexpressing mitochondrial vesicle. SNX9Lentiviral vectors of the gene are transfected into cells to obtain stable overexpression. SNX9 The cell line containing the gene was cultured, and the culture medium was collected and centrifuged to obtain mitochondrial vesicles SNX9-MDVs. Then, mitochondrial DNA was extracted from the wild-type cells of the above cell line and introduced into SNX9-MDVs to obtain SNX9-engineered mitochondrial vesicles mtDNA-SNX9-MDVs rich in mitochondrial DNA.

[0006] The formation of MDVs involves multiple proteins. This invention introduces Sorting nexin 9 (SNX9), an important intracellular membrane transport protein that plays a crucial role in various intracellular vesicle transport processes, including endocytosis, exocytosis, and intercellular exchange of substances. Our research shows that stable overexpression… SNX9 The gene's cell line mainly produces outer membrane MDVs composed of the mitochondrial outer membrane. Although it can increase the MDVs secreted by natural cells, these outer membrane MDVs only contain some mtDNA fragments, that is, the mtDNA is incomplete, making them difficult to use for treatment. Therefore, further work was done by extracting mtDNA from wild-type cells and introducing it into SNX9-MDVs to obtain SNX9-engineered mitochondrial vesicle mtDNA-SNX9-MDVs rich in complete mtDNA for subsequent treatment.

[0007] Specifically, the SNX9 The amino acid composition of the SNX9 protein encoded by the gene is shown in SEQ ID No. 2.

[0008] Preferably, the SNX9 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0009] Preferably, the lentiviral vector uses pCDH-CMV-CMV-EF1-copGFP-Puro9 (pCDH for short) as its template. SNX9 Lentiviral vectors for gene delivery SNX9 The gene was introduced into the multiple cloning site of the pCDH vector to construct the vector.

[0010] Preferably, the cells are L-O2 cells, which are derived from normal human liver tissue and have typical characteristics of hepatocytes, such as secreting albumin and synthesizing urea. They are often used to simulate the physiological or pathological processes of normal hepatocytes.

[0011] Preferably, the introduction is performed by introducing mtDNA into SNX9-MDV using lipid transfection to obtain SNX9-engineered mitochondrial vesicles rich in mtDNA-SNX9-MDVs.

[0012] As an optional implementation, the present invention constructs a pCDH-SNX9 lentiviral vector, then infects L-O2 cells, screens SNX9-L-O2, and then cultures them. The culture medium is collected, and mtDNA is extracted from L-O2 cells by iodixanol gradient density centrifugation of MDVs and introduced into SNX9-MDVs, thereby obtaining SNX9-engineered mitochondrial vesicle mtDNA-SNX9-MDVs rich in complete mtDNA.

[0013] The present invention also provides SNX9 engineered mitochondrial-derived vesicles mtDNA-SNX9-MDVs rich in mtDNA prepared by the above preparation method.

[0014] This invention applies SNX9-engineered mitochondrial vesicle mtDNA-SNX9-MDVs to a high glucose-induced insulin-resistant HepG2 cell model. The results show that SNX9-engineered mitochondrial vesicle mtDNA-SNX9-MDVs have a therapeutic effect on insulin-resistant model cells, indicating that mtDNA successfully plays a role in the treatment of insulin resistance through mitochondrial vesicle expression.

[0015] Specifically, this invention incorporates the SNX9-engineered mitochondrial vesicle mtDNA-SNX9-MDVs into a high-glucose-induced insulin resistance cell model, evaluating its effects on ATP content, mitochondrial membrane potential, ROS levels, and mtDNA content in model cells, and detecting the expression levels of insulin signaling pathway proteins. The results show that the SNX9-engineered mitochondrial vesicle mtDNA-SNX9-MDVs constructed in this invention significantly improve mitochondrial function, promote the expression of antioxidant enzymes in animal cells, and improve mitochondrial network structure, thus promoting mitochondrial functional recovery, regulating the biological effects of various mitochondrial metabolic enzymes, and enhancing their antioxidant capacity. Therefore, it has application value in treating insulin resistance caused by mitochondrial dysfunction.

[0016] Therefore, the present invention also provides the application of the aforementioned mtDNA-rich SNX9 engineered mitochondrial vesicle mtDNA-SNX9-MDVs in the preparation of drugs for treating insulin resistance.

[0017] Furthermore, the insulin resistance is caused by mitochondrial dysfunction resulting from mtDNA mutations.

[0018] Furthermore, the drug internalizes MDVs rich in normal mtDNA into the subject's cells, regulates the activity of mitochondrial antioxidant enzymes to clear ROS, increases ATP and mtDNA levels, enhances mitochondrial network generation and restores mitochondrial function, increases the expression of insulin signaling pathway-related proteins, and improves insulin resistance, thereby achieving the treatment of insulin resistance.

[0019] Furthermore, the subjects were selected from mammals.

[0020] Furthermore, the mammals mentioned are selected from rats, cats, dogs, pigs, cattle, horses, sheep, monkeys, and humans.

[0021] Furthermore, the drug also includes other pharmaceutically acceptable excipients.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides an SNX9 engineered mitochondrial vesicle, its preparation method, and its application in improving insulin resistance. This invention involves first constructing an overexpressing... SNX9 Lentiviral vectors of the gene are transfected into cells to obtain stable overexpression. SNX9 A cell line containing the gene was developed; this cell line was then cultured, and the culture medium was collected and centrifuged to obtain mitochondrial vesicles SNX9-MDVs; then, mtDNA was extracted from the cells and introduced into SNX9-MDVs to prepare engineered mitochondrial vesicles rich in mtDNA, namely mtDNA-SNX9-MDVs. The mtDNA-SNX9-MDVs were used for insulin resistance cell therapy. Results showed that mtDNA-SNX9-MDVs could be internalized into the patient's cells, effectively increasing mitochondrial ATP and mtDNA levels, enhancing cellular antioxidant enzyme activity and reducing ROS, improving mitochondrial network structure, and increasing the expression of insulin signaling pathway-related proteins, demonstrating significant potential for mitochondrial function repair and improvement of insulin resistance. This invention successfully realizes the use of mtDNA in the form of MDVs for the treatment of insulin resistance, providing a scientific strategy for its application in clinical drugs for the treatment of insulin resistance. Attached Figure Description

[0024] Figure 1 In Example 1 SNX9 PCR amplification results of the gene (M: DNA Marker).

[0025] Figure 2 This is a colony PCR of pSNX9 from Example 1.

[0026] Figure 3 This is a partial sequencing result of the pSNX9 vector sequence in Example 1.

[0027] Figure 4 The results of pSNX9 vector digestion identification in Example 1 show that after pSNX9 digestion, a band appeared below 2000bp, which is consistent with the size of the SNX9 gene of 1788bp.

[0028] Figure 5 The relative mRNA expression level of SNX9 in Example 1 (n=3, ***: p<0.001).

[0029] Figure 6 The results of Western blot analysis of SNX9 protein expression levels in Example 1 are shown.

[0030] Figure 7 The results show the expression levels of MDV marker proteins TOM20 and TIMM44 in 20% iodixanol concentration in Example 1. (n=3, ns: p>0.05, ***: p<0.001).

[0031] Figure 8 The image shows the diameter results of WT-MDVs, Empty-MDVs, and SNX9-MDVs extracted using a Nanosight particle size analyzer in Example 1.

[0032] Figure 9 The expression of 13 protein genes encoded by mtDNA in SNX9-MDVs was detected by PCR and RT-qPCR in Example 1.

[0033] Figure 10 This represents the changes in glucose uptake by insulin-resistant cells in Example 2. (n=3, **: p<0.01).

[0034] Figure 11 The images show fluorescence images of high glucose-HepG2 cells in Example 2 taking up EvLINK505-labeled mtDNA-SNX9-MDVs at 24, 48, 72, and 96 h.

[0035] Figure 12 The fluorescence images of high glucose-HepG2 cells taking up EvLINK505-labeled mtDNA-SNX9-MDVs at different time points in Example 2 were quantitatively analyzed (n=3, *: p<0.05, **: p<0.01, ***: P<0.001).

[0036] Figure 13 The relative mtDNA levels in high-glucose-HepG2 cells after internalization of different MDVs were analyzed in Example 2 (n=3, **: p<0.01, ***: p<0.001, ns: no significant).

[0037] Figure 14 The change in glucose uptake after treatment with mtDNA-SNX9-MDVs in Example 2 (n=3, **: p<0.01).

[0038] Figure 15 For ATP level detection in Example 2 (n=3, ***: p<0.001).

[0039] Figure 16 ROS level detection in Example 2 (n=3, **: p<0.01).

[0040] Figure 17 This is a diagram showing the fluorescence detection of the mitochondrial network structure (Mito-Tracker Green fluorescent probe) in Example 2.

[0041] Figure 18 The results show the detection of insulin signaling pathway-related proteins in Example 2. (n=3, **: p<0.01, ***: p<0.001). Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0043] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0044] 1. Experimental Materials

[0045] Table 1. Cell and vector sources

[0046]

[0047] 2. Experimental Methods

[0048] 2.1 Cell resuscitation

[0049] Remove the cryovials from the liquid nitrogen container and quickly place them in a 37°C water bath to thaw completely. Then centrifuge at 800 rpm for 5 minutes, discard the supernatant, and gently resuspend the cells in 1 mL of DMEM complete cell culture medium by pipetting. Add the cell suspension to a culture dish containing room-temperature complete culture medium. The complete culture medium consists of DMEM, fetal bovine serum (FBS), and penicillin-antibody in a ratio of 100:10:1. Incubate the culture dishes in a 37°C, 5% CO2 cell culture incubator.

[0050] 2.2 Cell passage and cryopreservation

[0051] (1) Cell passage: Observe the cell state. When the cell density reaches 80%, discard the culture medium, rinse once with sterile PBS at room temperature, add an appropriate amount of trypsin, shake the culture dish to ensure that the trypsin is in full contact with the cells, and then place it in a 37℃ incubator for 1-2 min for digestion. Add an appropriate amount of culture medium containing 10% FBS to stop digestion, transfer the cell suspension to a 4mL centrifuge tube, centrifuge at 800rpm for 5 min at room temperature, discard the supernatant, resuspend the cells with fresh culture medium, and transfer them to a new culture dish at a ratio of 1:2. Place the newly seeded cells in a 37℃, 5% CO2 incubator for culture.

[0052] (2) Cell cryopreservation: Digest cells in good growth condition and transfer them to centrifuge tubes for centrifugation. Discard the supernatant and add an appropriate amount of cell cryopreservation solution. Mix gently with a pipette tip. Record the date, cell name and passage number and aliquot into 1.5 mL cell cryopreservation tubes. First, place them in a programmed cooling box and perform programmed cooling in an ultra-low temperature freezer at -80℃. After 24 h, transfer them to a liquid nitrogen tank for cryopreservation.

[0053] Example 1: Preparation of SNX9 engineered mitochondrial vesicles rich in mtDNA and isolation and characterization of MDVs

[0054] I. Experimental Methods

[0055] 1. Construction of SNX9 overexpression vector

[0056] 1.1 DNA Extraction

[0057] According to the mammalian genomic DNA extraction kit (purchased from Beijing Zhuangmeng International Biotechnology): after completely digesting L-O2 cells with a growth confluence of 80-90% with trypsin, centrifuge, add 1 mL PBS, resuspend by pipetting, centrifuge at 10000 rpm for 1 min, and discard the supernatant. Add 250 μL of buffer A to the precipitate and vortex until completely suspended. Add 10 μL of proteinase K, mix well, then add 250 μL of buffer B, vortex to mix, and place in a 70°C oven for 10 min until the solution becomes clear. Add 250 μL of anhydrous ethanol to the solution and gently vortex for 15 s. At this point, a small amount of suspended flocculent precipitate can be observed in the solution. Transfer this precipitate to the adsorption column and place the adsorption column in a collection tube. Centrifuge at 12000 rpm for 30 s, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Add 500 μL of buffer C to the adsorption column, centrifuge at 12000 rpm for 30 s, discard the waste liquid, add 700 μL of wash buffer W2 (wash buffer W2 needs to be diluted with anhydrous ethanol beforehand), centrifuge, and repeat this step once. Discard the waste liquid and centrifuge at 12000 rpm for 2 min. Let the adsorption column stand at room temperature for several minutes to completely dry any remaining wash buffer. Finally, the adsorption column was transferred to a clean 1.5 mL centrifuge tube, and 150 μL of elution buffer TE was added dropwise to the middle of the adsorption membrane. The adsorption column was allowed to stand at room temperature for several minutes to allow the elution buffer to fully wet the adsorption column precipitate. The column was then centrifuged at 12,000 rpm for 2 minutes. The solution obtained after centrifugation was the genomic DNA extract. After the concentration was detected by an ultra-micro UV spectrophotometer, the extract was stored at -20°C for later use.

[0058] 1.2 PCR amplification

[0059] According to NCBI SNX9 The transcript NM_016224.5 of (Gene ID: 51429) was obtained, and primers were designed based on Primer Blast for amplification. The primers were synthesized by Qingke Biotechnology, and the primer sequences are shown in Table 2. NheⅠ and XhoⅠ restriction sites were introduced at both ends of the SNX9 gene. PCR was performed using whole DNA from L-O2 cells as a template, according to the systems and reaction conditions described in Tables 3 and 4.

[0060] Table 2 PCR amplification primers

[0061]

[0062] Table 3 Amplification System

[0063]

[0064] Table 4 Amplification Procedure

[0065]

[0066] The PCR products were recovered by gel extraction after 1% agarose gel electrophoresis.

[0067] 1.3 Agarose gel electrophoresis

[0068] (1) Electrophoresis: Weigh 0.5 g of agarose and dissolve it in 50 mL of electrophoresis buffer. Heat the solution in a microwave oven or a heated magnetic stirrer until the agarose is completely dissolved. Cool the dissolved agarose solution to 60-70℃ and add 5 μL of SYBRSafe. Pour the agarose solution into a gel mold, insert a comb to ensure it is flat and free of air bubbles, and let it stand for 10-15 minutes until the gel is completely solidified. During this time, prepare 1×TAE electrophoresis buffer and inject it into the electrophoresis tank. Place the gel wells of the gel plate against the negative electrode of the electrophoresis tank. Mix the SNX9 gene amplification sample with 10×Loading Buffer, and load the DNA Marker and the mixed samples. Perform electrophoresis at 140 V for 30 min. After electrophoresis, place the gel in a gel imaging system and take pictures for recording.

[0069] (2) Fragment recovery: The target gene was cut and transferred to a centrifuge tube under UV light. After weighing, an equal volume of Binding Buffer was added, and the mixture was melted in a 56°C water bath for 10 min to accelerate the melting into a DNA-agarose solution. The solution was transferred to an adsorption column, allowed to stand for 1 min, and then placed in a collection tube. The tube was centrifuged at 12000 rpm for 1 min, and the lower layer was discarded. This step was repeated. Wash buffer diluted with anhydrous ethanol was added, and the tube was centrifuged again and the subsoil was discarded. This step was repeated once to remove any possible residual ethanol solution. The adsorption column was placed in a centrifuge tube, 50 μL of EB elution buffer was added, and the tube was centrifuged at 12000 rpm for 1 min to collect the DNA. The DNA solution was detected using the Nucleic acid function of an ultra-micro UV-Vis spectrophotometer. When the OD value was... 260 / OD 280 A concentration between 1.8 and 2.0 indicates suitable DNA purity, yielding DNA concentration data in ng / μL. Store at -20℃ for later use.

[0070] 1.4 Recombinant Ligation of Overexpression Vectors

[0071] (1) Vector linearization: pCDH-CMV-MCS-EF1-copGFP-Puro vector (pCDH for short) was digested with BstXⅠ / NotⅠ or NheⅠ / BamHⅠ. The reaction systems are shown in Table 5. QuickCut NheⅠ and QuickCut XhoⅠ were used to construct pCDH-SNX9, and agarose gel electrophoresis and recovery were performed.

[0072] Table 5 pCDH digestion system

[0073]

[0074] The reaction system was gently mixed and placed in a 37°C water bath for 1 hour, then transferred to a 70°C water bath for 10 minutes.

[0075] (2) Ligation: pCDH-SNX9 (abbreviated as pSNX9) was constructed according to the Trelief™ SoSoo Cloning Kit (purchased from Qingke Biotechnology). The ligation reaction is shown in Table 6, and the mixture was incubated at 50℃ for 25 min. Transformation and bacterial coating experiments were then performed.

[0076] Table 6 Connection Reaction System

[0077]

[0078] 1.5 Extraction and Identification of the Vector

[0079] (1) Preparation of Escherichia coli DH5α competent cells: After thawing frozen E. coli from liquid nitrogen, the cells were inoculated onto LB agar plates and allowed to grow stably for 24 h. Single colonies with good morphology were picked and inoculated into 20 mL of LB liquid medium, and cultured in a constant temperature shaking incubator for 3 h. When the bacterial culture OD... 600 When the concentration of the precipitate is 0.4–0.6, transfer it to a pre-chilled centrifuge tube and centrifuge at 4000 rpm for 10 min at 4°C, discarding the supernatant. Add 5 mL of pre-chilled 0.1 mol / L CaCl2 hypotonic solution to the precipitate, resuspend the strain for 30 min, centrifuge, and discard the supernatant. Add 1 mL of ice-cold glycerol containing 0.1 mol / L CaCl2 to the precipitate to resuspend the strain, incubate on ice for 24 h, and obtain *E. coli* DH5α competent cells, which can be stored at -80°C for later use.

[0080] (2) Transformation of the vector: 5 μL of DNA vector was added to 200 μL of DH5α competent bacterial culture, and after being pipetted and mixed evenly, it was placed on an ice box for 30 min. Then it was transferred to a 42℃ water bath for 90 s and then placed on ice for 3 min to cool. 1 mL of sterile antibiotic-free LB medium was added and mixed with the vector bacterial culture. The culture was then incubated at 250 rpm and 37℃ for 1 h with shaking. 100 μL of colonies was centrifuged at 3000 rpm for 2 min. The precipitated bacterial cells were inoculated onto an LB culture plate containing Amp (50 μg / mL, 1:100). The plate was incubated upright for 1 h until the bacterial culture was completely absorbed. The plate was then incubated upside down at 37℃ for 24 h. Then, a single colony was picked with an inoculation loop and inoculated into LB liquid medium containing Amp. The plate was then incubated at 37℃ with shaking for 12 h.

[0081] (3) Colony PCR identification: The pCDH-FP and pCDH-RP primers on pCDH (Table 7) were used for bacterial PCR detection. The reaction system and procedure are shown in Tables 8 and 9. The PCR products were detected by agarose gel electrophoresis to screen positive colonies. The colonies were cultured overnight at 37°C in a shaker. After extracting the vector from the overnight bacterial culture, it was sent to Qingke Biotechnology for sequencing. If the sequencing results were correct, the vector was successfully constructed.

[0082] Table 7. Primers for bacterial culture PCR

[0083]

[0084] Table 8. Bacterial PCR Reaction System

[0085]

[0086] Table 9. PCR reaction procedure for bacterial culture

[0087]

[0088] 2. Construction of L-O2 cell line stably overexpressing SNX9

[0089] Vector extraction: Follow the instructions of the DNA plasmid miniprep kit (DP103) (purchased from Tiangen Biotech): Take 30 mL of fresh bacterial culture cultured for 12-16 h, centrifuge at 12000 rpm for 10 min at 4℃; equilibrate the column during this time by placing the CP3 adsorption column into a collection tube, adding 500 μL of equilibration buffer BL, centrifuging at 12000 rpm for 1 min, discarding the waste liquid in the collection tube, and returning the adsorption column to the collection tube. After centrifugation of the bacterial culture, discard the supernatant, leaving the bacterial cell precipitate. Add 500 μL of P1 solution (with RNase A added) to the bacterial precipitate and resuspend thoroughly. Then, add 500 μL of P2 solution to the resuspended solution and gently invert several times to fully lyse the bacterial cells until the bacterial solution becomes clear. Then add 700 μL of P3 solution and immediately gently invert thoroughly to mix until a white flocculent precipitate appears. P3 should be mixed immediately after addition to avoid local precipitation. Centrifuge at 12000 rpm for 10 min and collect the supernatant. Transfer the supernatant collected in the previous step to the CP3 adsorption column in several batches. Centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Add 600 μL of wash buffer PW (with anhydrous ethanol added) to the CP3 adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and repeat the operation once. Return the adsorption column to the collection tube, centrifuge at 12000 rpm for 2 min to remove the residual wash buffer in the adsorption column, open the cap of the CP3 adsorption column, and place it at room temperature for several minutes to completely dry the wash buffer. Finally, place the CP3 adsorption column in a clean centrifuge tube, add 100 μL of eluent EB to the middle of the adsorption membrane, place at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and the solution in the centrifuge tube after centrifugation is the solution containing the carrier. After determining the concentration of the carrier solution, store it at -20℃.

[0090] 3. Screening for positive cell lines

[0091] Inoculate 5×10⁶ cells into 6-well plates 5 Infected cells were cultured for 24 h. The following day, the original culture medium was aspirated, and the cells were washed three times with sterile PBS. 2 mL of fresh DMEM medium containing puromycin at a final concentration of 2 μg / mL was added, and the cells were cultured for 14 days for selection. Subsequently, RNA was extracted from stable cell lines for RT-qPCR to detect knockdown efficiency, or protein was extracted from positive cell lines for Western blotting to detect overexpression efficiency.

[0092] 4. qPCR detection

[0093] 4.1 Total RNA extraction from cells

[0094] When the cell density reaches 80%–90%, the cell culture medium is aspirated and the cells are washed three times with PBS. 1 mL of TRNsol is added evenly to the cell culture dish and repeatedly pipetted. The lysate is then transferred to an EP tube, 0.2 mL of chloroform is added, and the mixture is vigorously shaken for 15 seconds. After incubating on ice for 5 minutes, the mixture is centrifuged at 12,000 rpm for 10 minutes at room temperature. The upper aqueous phase is transferred to a new EP tube, and an equal volume of isopropanol is added. The mixture is vortexed and incubated at room temperature for 10 minutes, followed by centrifugation at 12,000 rpm for 10 minutes at 4°C. The supernatant is discarded, and the RNA is washed with 75% ethanol at 10,000 rpm for 5 minutes at 4°C. This step is repeated three times. After thorough washing, the ethanol is carefully aspirated, and an appropriate amount of DEPC water is added to dissolve the RNA. The RNA concentration is then measured for reverse transcription and stored at -80°C.

[0095] 4.2 Reverse transcription of cellular RNA

[0096] RNA was processed using a reverse transcription kit (PrimeScript). TM II. Prepare reaction solution 1 and reaction solution 2 (see Tables 10 and 11) according to the instructions of the 1st Strand cDNA Synthesis Kit (purchased from Takara) and perform reverse transcription.

[0097] Table 10 RNA Reverse Transcription Reaction Solution 1

[0098]

[0099] After the above solution was treated at 65°C for 5 min, it was immediately transferred to ice for cooling. Then, reaction solution 2 was prepared according to Table 11.

[0100] Table 11 RNA Reverse Transcription Reaction Solution 2

[0101]

[0102] After slowly mixing, reverse transcription is performed by placing the mixed sample in a PCR instrument and setting the temperature to 30℃ for 10 min, 42℃ for 30 min, 95℃ for 5 min, and cooling on ice to complete the reverse transcription and obtain cDNA.

[0103] 4.3 qPCR reaction

[0104] According to the NCBI database SNX9Primers were designed based on the sequence (see Table 12), and the primers were synthesized by Qingke Biotechnology. 20 μL of sterile water was added to the cDNA for dilution, and a 20 μL qPCR reaction system was constructed in eight-tube sets according to the qPCR reagent instructions (see Table 13). Each sample was divided into three replicates, and qPCR was performed according to the conditions in Table 14. The obtained CT values ​​were expressed as 2... -△△Ct Perform the analysis.

[0105] The calculation formula is: 2 -（[实验组目的基因 CT 值-实验组内参基因 CT 值]-[ 对照组目的基因 CT 值-对照组内参基因 CT 值]）。

[0106] Table 12 PCR Primers

[0107]

[0108] Table 13 qPCR reaction system

[0109]

[0110] Table 14 qPCR reaction procedure

[0111]

[0112] 5. Western Blot

[0113] After washing cells with pre-chilled PBS, lysis buffer containing PMSF was added and the cells were lysed on ice for 30 minutes. The supernatant was collected and stored at -20°C. Protein concentration was then determined using the BCA method: a series of BSA standards and protein samples were reacted together with BCA working solution, and the absorbance at 562 nm was measured using a microplate reader to plot a standard curve for calculating the sample protein concentration. SDS-PAGE gel electrophoresis was then performed: a 15% separating gel and a 5% stacking gel were prepared. Denatured protein samples and protein markers were added to the lanes. Electrophoresis was first performed on the stacking gel at 90V, then on the separating gel at 160V until the bromophenol blue reached the bottom. Transfer was then performed: the gel and activated PVDF membrane were assembled into a "sandwich" structure and transferred at a constant current of 200 mA for 60 minutes on ice. After transfer, antibody incubation was performed: the membrane was blocked with 5% skim milk powder for 1 hour, washed with TBST, and incubated overnight at 4°C with primary antibody; after washing again, the membrane was incubated at room temperature for 1 hour with secondary antibody. Finally, the samples were incubated with ECL luminescent solution, exposed and imaged, and the gray values ​​of the internal control and target gene bands were analyzed using ImageJ software.

[0114] 6. Extraction of MDVs using the iodixanol gradient density method

[0115] 6.1 Extraction of extracellular vesicles by ultracentrifugation

[0116] (1) Collecting culture medium: Seed cells to a height of 75 cm 2When the cell growth density in the culture flask reaches 80-90%, collect the culture medium. Centrifuge the collected medium at 300 g for 20 min at 4°C, retain the supernatant, and then centrifuge again at 2000 g for 20 min at 4°C, retaining the supernatant and discarding any dead cells or other precipitates. Next, centrifuge the supernatant at 10000 g for 20 min at 4°C. The supernatant obtained after this centrifugation can be sealed, labeled, and stored at -80°C for subsequent large-scale extraction of extracellular vesicles.

[0117] (2) Ultra-high speed centrifugation: Thaw the stored supernatant at 4℃, then transfer the supernatant to centrifuge tubes. Balance the centrifuged samples pairwise using an analytical balance with a balancing accuracy of 0.01g. Transfer the balanced centrifuge tubes to an SW70Ti rotor and insert the adapter. Cover the tubes and place the rotors in the centrifuge chamber. After confirming that the rotors are safely in place, close the chamber door, set the parameters, and centrifuge at 100,000g for 70 min. After centrifugation, discard the supernatant, resuspend the precipitate in the centrifuge tubes with PBS, and then transfer the resuspended liquid to a 13 mL ultra-clean centrifuge tube. Balance the centrifuge tubes and carefully transfer them to an SW41Ti rotor. Centrifuge at 4℃, 10,000g for 70 min. Discard the supernatant, resuspend the precipitate in pre-cooled PBS, and aliquot into sterile EP tubes. Store in a -80℃ freezer for subsequent experiments. Avoid repeated freeze-thaw cycles when handling the tubes.

[0118] 6.2 Iodixanol gradient density separation of MDVs

[0119] (1) Preparation of Iodixanol gradient solutions: Prepare density gradient solutions of iodixanol according to Table 15. The commercial OptiPrep solution is an aqueous solution containing 60% (wt / vol) iodixanol and needs to be pre-equilibrated in a Tris-HCl buffer system before use. Dissolve 0.17 g of sucrose in 2 mL of 60 mM Tris-HCl pH 7.4 (final sucrose concentration = 0.25 M) to prepare solution A. Add 1 mL of solution A to 5 mL of OptiPrep to obtain 6 mL of OptiPrep working solution (OWS, equivalent to 50% iodixanol in 10 mM Tris-HCl pH 7.4). Dissolve 1.28 g of sucrose in 15 mL of 10 mM Tris-HCl pH 7.4 to prepare solution B (final sucrose concentration = 0.25 M).

[0120] Table 15 Preparation of Iodixanol Gradient Concentrations

[0121]

[0122] Ultracentrifugation: First, add 40% iodixanol solution to the centrifuge tube. Then, add iodixanol solutions of decreasing concentrations sequentially on top of the solution: first 20%, then 15%, 13%, 11%, 9%, and 7%. Finally, add the collected extracellular vesicle solution. Balance the centrifuge tube and place it in an SW41Ti rotor. Set the parameters to 4°C, 200,000 g, and centrifuge for 16 h. After centrifugation, aliquot the different concentration layers into EP tubes. Make up the volume of the 20% layer solution to 10 mL with sterile PBS and centrifuge again at 4°C, 100,000 g, for 70 min. Discard the supernatant after centrifugation. Resuspend the MDVs precipitate in 1 mL of pre-cooled PBS and aliquot into sterile EP tubes. Store at -80°C for subsequent experiments. Avoid repeated freeze-thaw cycles during handling.

[0123] 6.3 Western Blot analysis of MDV marker proteins isolated from cells stably overexpressing MDVs

[0124] MDVs were isolated from L-O2, Empty-L-O2, and SNX9-L-O2 cells. MDVs were grouped as follows: MDVs secreted by L-O2 cells were WT-MDVs; MDVs secreted by Empty-L-O2 cells were Empty-MDVs; and MDVs secreted by SNX9-L-O2 cells were SNX9-MDVs. mtDNA was introduced into the MDV group: mtDNA-SNX9-MDVS.

[0125] 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 according to the ratio (sample volume: 5 × loading buffer = 5:1) and placed in a 100°C metal bath for 10 min to complete sample preparation. Then, Western blotting was used to detect the protein expression levels of the MDV markers TOMM20 and TIMM44.

[0126] 6.4 Nanoparticle Size Detection

[0127] 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.

[0128] 6.5 Detection of mtDNA Integrity in SNX9-MDVs

[0129] We used PCR to test the integrity of the mtDNA extracted from SNX9-MDVs, and designed primers for amplification based on the 13 gene sequences encoded by the mtDNA. These include ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COⅠ, COⅡ, COⅢ, ATPase8, ATPase6, and CYTB.

[0130] 7. mtDNA introduction into SNX9-MDVs

[0131] 7.1 mtDNA preparation:

[0132] Using an mtDNA extraction kit, L-O2 cells were digested with trypsin and centrifuged at 12,000 rpm for 2 min at 4°C. The cell pellet was collected. 250 μL of solution A (ice-bathed) was added, and the pellet was dispersed. Then, 250 μL of solution B (room temperature) was added, and the mixture was stirred. The mixture was incubated on ice for 6 min. 350 μL of solution C (ice-bathed) was added, and the mixture was stirred until a white precipitate formed. The mixture was incubated on ice for 25 min. The mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant was transferred to an adsorption column. After incubation for 5 min, the column was centrifuged at 12,000 rpm for 1 min, and the waste liquid was discarded. 500 μL of wash buffer was added, and the column was centrifuged at 12,000 rpm for 1 min. The centrifugation was repeated once, and the residual liquid was removed to obtain the mtDNA extract.

[0133] 7.2 mtDNA loading:

[0134] Purified mtDNA was mixed with Lipofectamine 3000 at a ratio of 5:1 and incubated at room temperature for 30 min to form an mtDNA-liposome complex (RDL). Then, RDL was mixed with SNX9-MDVs at a particle ratio of 1:2 and incubated at room temperature for 30 min. After freezing in liquid nitrogen for 15 min, the mixture was thawed at 37°C for 20 min, and this freeze-thaw cycle was repeated three times to promote membrane fusion. Finally, the fusion product was separated by ultracentrifugation (100,000×g, 4°C) and resuspended in sterile PBS to obtain mtDNA-loaded mitochondrial vesicles mtDNA-SNX9-MDVs. Finally, RT-qPCR was used to analyze the ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COⅠ, COⅡ, COⅢ, ATPase8, ATPase6, and CYTB genes in mtDNA-SNX9-MDVs.

[0135] II. Experimental Results

[0136] 1. Identification of the pSNX9 vector

[0137] 1.1 PCR amplification of the SNX9 gene fragment

[0138] The SNX9 gene is 1788 bp in size. A fragment of the SNX9 gene was amplified by PCR, and the amplified fragment was subjected to agarose gel electrophoresis. The electrophoresis results are as follows: Figure 1 As shown, the position of the electrophoretic bands is close to the expected gene size, indicating that the gene amplification was successful.

[0139] 1.2 Colony PCR Identification of the Vector

[0140] After transforming the pSNX9 vector into competent E. coli cells, PCR was performed on the colony lysate. Figure 2 The PCR results of colony lysates of the transfected vector pSNX9 show a band around 2000 bp, indicating that the vector was successfully transformed into E. coli.

[0141] 1.3 Recombinant vector sequencing

[0142] The recombinant vector colonies were sent for sequencing. The sequencing results were compared with the target gene nucleotide cDNA coding sequence in the NCBI database using SnapGene version 6.0.2 software. The results showed that the target gene expression sequence in the recombinant vector was consistent with that in the database. Figure 3 This indicates that the pSNX9 recombinant vector was successfully constructed.

[0143] 1.4 Enzyme digestion identification of the vector

[0144] The constructed pSNX9 vector was subjected to double enzyme digestion for identification. The results of NheI and XhoI enzyme digestion of the pSNX9 vector are shown below. Figure 4 As shown, after pSNX9 digestion, bands appeared below 2000 bp, consistent with the SNX9 gene size of 1788 bp. The digestion results were all as expected, indicating that the gene was successfully cloned into the pCDH vector.

[0145] 2. Identification of L-O2 cell lines stably overexpressing SNX9

[0146] 2.1 Relative mRNA expression level of SNX9

[0147] After screening for stable overexpression cells, RNA was extracted from the stable cell lines and RT-qPCR was used to detect the overexpression efficiency. The results are as follows: Figure 5 As shown, compared with the control group, the mRNA level of SNX9 in the stably overexpressing cells was significantly increased, indicating that the L-O2 cell line with stable overexpression of the SNX9 gene was successfully constructed.

[0148] 2.2 Protein expression level of SNX9

[0149] To further verify whether the stable overexpression cells were successfully constructed, we also extracted proteins from the stable cell line and performed Western blot analysis to detect protein expression levels. The results are as follows: Figure 6 As shown, the protein expression level of SNX9 in the stably overexpressing cells was higher than that in the control group, further demonstrating that the L-O2 cell beads stably overexpressing NX9 (referred to as SNX9-L-O2) were successfully constructed.

[0150] 3. Western Blot analysis of MDV content and marker proteins TOMM20 and TIMM44

[0151] SNX proteins are known to be closely related to the production and secretion of MDVs, but it is unclear which specific type of MDV they promote. Therefore, we extracted L-O2 and SNX9-L-O2 MDVs using iodixanol density gradient centrifugation, and performed Western blot analysis on the 20% iodixanol concentration layer (containing the highest concentration of MDVs) for the outer membrane marker protein TOMM20 and the inner mitochondrial membrane protein TIMM44. The Western blot analysis is as follows: Figure 7 As shown, the TOMM20 band was the darkest in the SNX9-MDVs group, while the TIMM44 band was the lightest, almost identical to that in WT-MDVs, indicating that the SNX9 protein may primarily promote the production of outer membrane MDVs composed of the mitochondrial outer membrane (OMM). Furthermore, compared to the SNX9-MDVs group, the TOMM20 and TIMM44 protein levels in the WT-MDVs and Empty-MDVs groups were abnormally low, indicating that wild-type L-O2 cells and Empty-L-O2 cells produced very few MDVs.

[0152] 4. MDVs particle size analysis

[0153] The diameters of the extracted WT-MDVs, Empty-MDVs, and SNX9-MDVs were determined using a Nanosight particle size analyzer. The results are as follows: Figure 8 As shown, the particle size range is mainly 50-200nm, with the peak particle size appearing at around 100nm, which is consistent with the morphological characteristics of MDVs.

[0154] 5. Detection of mtDNA integrity within MDVs

[0155] Studies have shown that MDVs carry a certain amount of mtDNA, which encodes genes for 13 proteins related to oxidative phosphorylation. To investigate the integrity of the mtDNA extracted from the MDVs in this invention, PCR was used to detect the gene expression of the 13 protein genes encoded by the mtDNA. The results are as follows: Figure 9As shown in Figure A, only lanes ND6, COⅢ, and ATP6 showed clear bands in SNX9-MDVs, and some genes even lacked bands. This indicates that the mtDNA in the outer membrane MDVs generated by SNX9-MDVs is incomplete. Therefore, we extracted mtDNA from wild-type L-O2 cells; then, we used lipid transfection to introduce the mtDNA into SNX9-MDVs to obtain SNX9-engineered mitochondrial vesicle mtDNA-SNX9-MDVs rich in complete mtDNA. The RT-qPCR results are shown below. Figure 9 As shown in B, all 13 protein genes encoded by mtDNA are highly expressed, indicating that mtDNA-SNX9-MDVs rich in complete mtDNA have been prepared, thus solving the above problems.

[0156] Example 2: Effects of MDVs on Improving the HepG2 Insulin Resistance Model In Vitro Study

[0157] I. Experimental Methods

[0158] 1. Establishment of an insulin-resistant cell model

[0159] HepG2 cells were loaded at 5 × 10 3 Cells were seeded at a density of [number] cells / mL in 6-well plates, and DMEM complete medium supplemented with 10% fetal bovine serum was added. The plates were then transferred to a 5% CO2 incubator for routine culture. After full cell attachment, the cells were gently washed three times with pre-cooled PBS buffer to remove residual culture medium. Subsequently, hypertonic glucose treatments of 0, 5, 20, 35, and 50 mmol / L were prepared using serum-free and antibiotic-free DMEM basal medium (the specific concentrations were adjusted according to the experimental design). Each concentration of treatment was added to the corresponding well, and after 24 hours of incubation, the relative cell viability was measured using a CCK-8 assay kit to determine the optimal treatment concentration for high glucose induction. Finally, the model was validated by quantitative analysis of cellular glucose uptake. When the uptake rate in the experimental group was significantly higher than that in the control group (p<0.05), the insulin resistance cell model was considered successfully established. Successfully established cells were cultured at 2.0 × 10⁶ cells / well. 6 Cells / 100 mm 2 The culture dish was inoculated at a certain density and then incubated with SNX9-MDVs or mtDNA-SNX9-MDVs.

[0160] The cell experiments were divided into four groups: HepG2 group (Control), high glucose-induced HepG2 group (high glucose-HepG2), high glucose-induced HepG2 group followed by SNX9-MDVs group (SNX9-MDVs), and high glucose-induced HepG2 group followed by mtDNA-SNX9-MDVs group (mtDNA-SNX9-MDVs). 100 μL of MDVs solution was added to each well.

[0161] 2. Determination of cell viability

[0162] First, the high-glucose solution used to induce HepG2 cells was aspirated, followed by thorough washing with PBS. Then, 10 μL of CCK-8 solution was added to each well and incubated for 2 hours. The CCK-8 solution was then aspirated using a pipette. The absorbance was measured at 490 nm using a microplate reader.

[0163] 3. The validation model for glucose uptake determination using 2-NBDG was successfully established.

[0164] 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 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.

[0165] 4. Detection of internalized MDVs in insulin-resistant cells

[0166] Following the kit instructions: 100 μg of MDVs were labeled with 5 μL of EvLINK 505 and gently incubated in the dark at room temperature for 30 min. Purified samples were collected for subsequent experiments to assess cellular uptake of MDVs. Cells were seeded in confocal culture dishes and cultured for 24 h. Subsequently, cells were incubated with EvLINK 505-labeled MDVs for 12, 24, 36, and 48 h and washed with PBS. To visualize the cell membrane, cells were labeled with Cel-ILINK 555 and incubated in the dark at room temperature for 30 min. After incubation, cells were washed with PBS and fixed with 4% formaldehyde for 30 min. Then, they were stained with DAPI for 5 min. Internalization of labeled MDVs was observed using a laser confocal scanning microscope.

[0167] 5. Detection of mtDNA content after internalization of MDVs in cells

[0168] Total DNA was extracted from cells according to the kit instructions. mtDNA was amplified based on the ND1 gene, which encodes the ND1 subunit of NADH dehydrogenase. β-actinnDNA was amplified using qPCR on a LightCycler 480 II real-time PCR system. Primers used are shown in Table 16, reaction systems in Table 17, and reaction conditions in Table 18. (Based on 2...) -△△CT Calculate the relative content of genes, with 3 replicates per group, and calculate the average value.

[0169] Table 16 Primers for ND1 gene qPCR

[0170]

[0171] Table 17 PCR Reaction System

[0172]

[0173] Table 18 PCR Reaction Conditions

[0174]

[0175] 6. Effects of MDVs internalization on glucose uptake

[0176] Cells from each group were collected, cultured in sugar-free DMEM medium for 5 h, then cultured in 100 nM insulin for 20 min, and finally 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.

[0177] 7. ATP level detection

[0178] Following the instructions of the ATP kit, first aspirate the culture medium, add 200 μL of lysis buffer to each well of a 6-well plate, repeatedly pipette to lyse, then centrifuge at 12000g for 5 minutes at 4ºC. Collect the supernatant for subsequent assays. Then, thaw the reagents on ice, diluting the ATP standard solution with ATP assay lysis buffer to prepare concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM. Add 100 µL of ATP assay working solution to a light-protected 96-well plate and incubate at room temperature for 3-5 minutes to consume all background ATP, thus reducing background levels. Add 20 µL of the test solution or standard solution to each well, mix quickly, and then use the luminometer function of a full-wavelength multi-mode microplate reader to detect the chemiluminescence value. Plot a standard curve and calculate the ATP content of each group of cells based on the chemiluminescence value.

[0179] 8. ROS testing

[0180] mtDNA-SNX9-MDVs at a final concentration of 1 mg / mL were added to high glucose-induced HepG2 cells for co-incubation. Intracellular ROS production was quantitatively measured using the cell-permeable, oxidation-sensitive fluorescent probe CM-H2DCFDA (molecular probe). Cells from each group were cultured at 2.5 × 10⁶ cells / well. 5 Cells were seeded at a density of [number] cells per well in complete culture medium and pretreated with DMEM containing 1 μM CM-H2DCFDA at 37°C for 30 min. Cells were harvested, washed twice with DMEM, resuspended in DMEM, and analyzed using a FACSCalibur flow cytometer (BD Biosciences). Cells were cultured in 11 cm [cells]. 2 Cells were cultured in standard medium in BDBiosciences dishes and treated with 1 μM CM-H2DCFDA for 30 minutes. Cells were washed three times in standard medium and then analyzed using a Leica laser scanning confocal microscope at 37°C with an excitation wavelength of 488 nm and an emission wavelength of 500–540 nm.

[0181] 9. Fluorescence detection of mitochondrial network structure (Mito-Tracker Green fluorescent probe)

[0182] Mito-Tracker Green solution was prepared into a 1 mM stock working solution using anhydrous DMSO and stored at -20°C protected from light for later use; cells in good growth condition were incubated at 1×10⁻⁶ mM. 5 The cells were transferred to culture dishes and cultured in a conventional manner. After 24 h of culture, the culture medium was removed, and Mito-Tracker Green staining working solution preheated to 37 °C was added and incubated for 2 h. After the incubation was completed, the staining solution was replaced with fresh culture medium, and the mitochondrial network structure was observed under a laser confocal microscope.

[0183] 10. Expression detection of proteins related to the insulin signaling pathway

[0184] The expression levels of PI3K and GLUT4 proteins in each group of cells were detected according to the ELISA kit instructions.

[0185] 11. Statistical Analysis

[0186] 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.

[0187] II. Experimental Results

[0188] 1. Selection of glucose concentration

[0189] To determine the optimal glucose concentration for inducing insulin resistance (IR) in HepG2 cells, this experiment used glucose solutions of 0, 5, 10, 20, 35, and 50 mmol / L to co-incubate HepG2 cells for 24 h, and cell viability was measured (Table 19). Within the range of 0–50 mmol / L, the viability of HepG2 cells decreased with increasing glucose concentration. When the glucose concentration exceeded 35 mmol / L, cell viability was significantly reduced. Therefore, a 35 mmol / L glucose solution was selected for treating cells for 24 h as the construction protocol for the insulin resistance model.

[0190] Table 19 Effect of glucose on HepG2 cell survival (x±s, n=5)

[0191]

[0192] 2. Validation of successful model cell construction

[0193] Insulin resistance in cells directly manifests as abnormal glucose uptake; therefore, we used the 2-NBDG method to detect glucose uptake in HepG2 cells. Compared with the normal HepG2 cell group, glucose uptake in the high-glucose-induced HepG2 group was significantly reduced ( Figure 10 This indicates that the insulin resistance model of HepG2 cells has been successfully established.

[0194] 3. Results of cell internalization of MDVs

[0195] High glucose-HEPG2 cells were co-cultured with mtDNA-SNX9-MDVs labeled with EvLINK505, and their values ​​were observed at 24 h, 48 h, 72 h, and 96 h. Figure 11 and Figure 12 After co-culturing for 72 h, mtDNA-SNX9-MDVs reached the peak of internalization in cells.

[0196] 4. Effect of internalization of mtDNA-SNX9-MDVs on mtDNA content in insulin-resistant cells

[0197] To investigate the effect of mtDNA-SNX9-MDVs on mtDNA levels in mammalian cells, we added either SNX9-MDVs or mtDNA-SNX9-MDVs to model cell cultures. The results are as follows: Figure 13As shown, compared with the HepG2 group, the mtDNA content in the high glucose-HepG2 group was significantly decreased; after treatment with SNX9-MDVs, the mtDNA content in the high glucose-HepG2 group remained almost unchanged (no statistical difference); however, after treatment with mtDNA-SNX9-MDVs, the mtDNA content significantly increased (p < 0.001). This indicates that SNX9-MDVs had almost no effect on the mtDNA content of insulin-resistant cells, while mtDNA-SNX9-MDVs significantly increased the mtDNA content of insulin-resistant cells, further demonstrating that mtDNA-SNX9-MDVs contains abundant intact mtDNA. Therefore, we will use mtDNA-SNX9-MDVs in our subsequent experiments.

[0198] 5. Effects of mtDNA-SNX9-MDVs on glucose uptake in insulin-resistant cells

[0199] like Figure 14 After treatment with mtDNA-SNX9-MDVs, compared with the high glucose-HepG2 group, the glucose uptake rate of the mtDNA-SNX9-MDVs group was significantly increased, indicating that mtDNA-SNX9-MDVs can improve the glucose uptake of cells.

[0200] 6. mtDNA-SNX9-MDVs promote oxidative phosphorylation and increase ATP levels.

[0201] Because mitochondrial dysfunction leads to impaired oxidative phosphorylation and enhanced glycolysis, resulting in decreased ATP levels, we investigated whether mtDNA-SNX9-MDVs increase oxidative phosphorylation and thus raise ATP levels. To this end, mtDNA-SNX9-MDVs were transfected into insulin-resistant cells. ATP levels in the cells were measured using an ATP assay kit. The results are as follows: Figure 15 As shown, the ATP level in insulin-resistant cells is significantly increased, indicating that mtDNA-SNX9-MDVs enhance cellular oxidative phosphorylation, increase ATP content in insulin-resistant cells, and help improve insulin resistance.

[0202] 7. ROS level measurement results

[0203] like Figure 16 After treatment with mtDNA-SNX9-MDVs, compared with the high glucose-HepG2 group, the ROS level of the mtDNA-SNX9-MDVs group was significantly reduced, indicating that mtDNA-SNX9-MDVs can increase the antioxidant enzyme activity of insulin-resistant cells, thereby reducing ROS.

[0204] 8. mtDNA-SNX9-MDVs improve mitochondrial network structure

[0205] Mitochondrial dysfunction leads to disruption of the mitochondrial network structure. After staining each group of cells with Mito-Tracker Green fluorescence, we observed the following under a fluorescence microscope: Figure 17 As shown, the mitochondria in HepG2 cells induced by high glucose were fragmented and structurally incomplete. However, when mtDNA-SNX9-MDVs were transferred into high glucose-HepG2 cells, the mitochondria were found to be interconnected and formed a network, indicating that mtDNA-SNX9-MDVs can significantly improve the mitochondrial network in insulin-resistant cells, and mitochondrial dysfunction is restored. This invention successfully realizes the use of mtDNA in the form of MDVs to improve the mitochondrial network structure of insulin-resistant cells.

[0206] 9. Expression of proteins related to the insulin signaling pathway

[0207] Insulin irritation (IR) can manifest as a decrease in the expression of proteins related to insulin signaling. To further investigate whether mtDNA-SNX9-MDVs, rich in mtDNA, can improve the IR state of HepG2 cells, we co-incubated HepG2 cells with mtDNA-SNX9-MDVs and then measured the expression levels of PI3K and GLUT4 proteins. The results are as follows: Figure 18 The results showed that, compared with HepG2 insulin-resistant cells under high glucose conditions, the levels of PI3K protein and the content of GLUT4 protein on the plasma membrane were significantly increased after treatment with mtDNA-SNX9-MDVs. This indicates that mtDNA-SNX9-MDVs can enhance the activity of proteins related to the insulin signaling pathway, improve the sensitivity of IR cells to insulin, and has application value in the treatment of insulin resistance.

[0208] In summary, the mtDNA-SNX9-MDVs prepared in this invention can improve insulin resistance by delivering mtDNA into insulin-resistant cells, increasing the mtDNA content of the cells, restoring mitochondrial function, and increasing the expression level of insulin pathway proteins.

Claims

1. A method for preparing SNX9 engineered mitochondrial vesicles, characterized in that, First, a lentiviral vector overexpressing the SNX9 gene was constructed, using pCDH-CMV-MCS-EF1-copGFP-Puro as the template. L-O2 cells were then transfected to obtain a cell line stably overexpressing the SNX9 gene. This cell line was then cultured, and the culture medium was collected and centrifuged using an iodixanol gradient density centrifugation method to obtain mitochondrial vesicles SNX9-MDVs. Finally, mitochondrial DNA was extracted from wild-type cells of the above cell line and introduced into SNX9-MDVs to obtain SNX9-engineered mitochondrial vesicles (mtDNA-SNX9-MDVs) rich in mitochondrial DNA. The nucleotide sequence of the SNX9 gene is shown in SEQ ID No.

1.

2. The SNX9-engineered mitochondrial vesicles mtDNA-SNX9-MDVs rich in mitochondrial DNA prepared by the preparation method of claim 1.

3. The use of the SNX9-engineered mitochondrial vesicle mtDNA-SNX9-MDVs rich in mitochondrial DNA as described in claim 2 in the preparation of a medicament for treating insulin resistance.

4. The application according to claim 3, characterized in that, The drug treats insulin resistance by internalizing MDVs rich in normal mtDNA into the subject's cells, regulating the activity of mitochondrial antioxidant enzymes to clear ROS, increasing ATP and mtDNA levels, enhancing mitochondrial network generation and restoring mitochondrial function, increasing the expression of insulin signaling pathway-related proteins, and improving insulin resistance.

5. A drug for treating insulin resistance, characterized in that, The drug contains SNX9 engineered mitochondrial vesicles mtDNA-SNX9-MDVs rich in mitochondrial DNA as described in claim 2.

6. The drug according to claim 5, characterized in that, It also contains pharmaceutically acceptable excipients.

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