Use of SKP-SC-EVs in preparation of drugs for treating DPN

Abstract

The application discloses application of SKP-SC-EVs in preparation of DPN treatment drugs and belongs to the technical field of biological medicine.The SKP-SC-EVs in the application can be internalized by SCs, and can directly repair and protect damaged SCs in multiple dimensions, laying a good cell foundation for nerve regeneration, in contrast to other source EVs.In addition, through treatment of DPN model rats with the SKP-SC-EVs, it is proved that the SKP-SC-EVs can effectively improve the nerve sensory and motor functions of the DPN rats, promote the regeneration of the nerve structure and myelin sheath of the rats, enhance the nerve internal microcirculation blood flow, delay the atrophy and degenerative changes of the target muscle, and realize systematic repair of the three functional units of "nerve-vascular-muscle".The DPN treatment scheme provided by the application effectively overcomes the defects in the prior treatment schemes, i.e., single repair effect dimension and difficulty in realizing fundamental reconstruction of nerve functions and keeping long-term stability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of SKP-SC-EVs in the preparation of drugs for treating DPN. Background Technology

[0002] Diabetic peripheral neuropathy (DPN) is the most common and disabling chronic complication of diabetes, affecting approximately 50% of diabetic patients worldwide. Its main clinical manifestations include numbness, pain, decreased or absent sensation in the limbs. In advanced stages, it can lead to foot ulcers, infections, and even amputation, severely impacting patients' quality of life and imposing a heavy socioeconomic burden. The pathogenesis of DPN is complex, involving multiple pathological processes such as activation of the polyol pathway induced by long-term hyperglycemia, oxidative stress, chronic inflammation, microvascular complications, and dysfunction of glial cells (SCs), leading to axonal degeneration, demyelination, and overall damage to the neuromuscular unit. SCs, as key glial cells in the peripheral nervous system, play a crucial role in maintaining myelin structure, supporting axonal survival, and guiding nerve regeneration.

[0003] EVs are nanoscale membrane vesicles actively secreted by cells, carrying various bioactive molecules such as proteins, lipids, and nucleic acids, and are key carriers mediating intercellular communication. In recent years, EVs derived from stem cells, progenitor cells, or functional cells have shown broad application prospects in the field of tissue repair and regenerative medicine as a "cell-free therapy" strategy. Regarding DPN treatment, the current representative treatment regimens mainly include mesenchymal stem cell (MSC)-derived EVs, SC-derived EVs, and engineered / modified EVs. Among them, MSC-derived EVs mainly focus on anti-inflammation and angiogenesis, with limited direct repair and myelin regeneration effects on SCs, and rely on modifying parental cells (overexpressing immunomodulatory factors) to achieve synergistic effects. Although SC-derived EVs can promote synaptic growth (including axonal growth and myelin repair), there is a lack of evidence for improving intraneural microcirculation perfusion and delaying target muscle atrophy. Moreover, this regimen is highly dependent on the functional status of SCs, and the use of SCs under pathological conditions such as high glucose can inhibit the nerve repair process. Engineered / modified EVs have strong targeting, and are mostly loaded with specific drugs or nucleic acids to repair or enhance the function of a specific unit. The technology is complex and costly, and the repair dimension is relatively singular. In summary, current treatments for DPN using EVs from different sources still suffer from limited repair dimensions and fail to address the synergistic repair of the three core damaged units: nerve conduction, blood supply support, and muscle maintenance. As a result, the therapeutic effects are mostly limited to symptom relief.

[0004] SKP-SC is a Schwann cell (SC) derived from skin progenitor cells (SKP). It is abundant and easily obtained, and readily cultured and expanded in vitro. Chinese invention patent application CN112402697A discloses SKP-SC-derived EVs (SKP-SC-EVs) as tissue-engineered neural grafts capable of repairing nerve defects. However, as a targeted transplantation graft, it primarily bridges the damaged nerve and only locally promotes nerve regeneration and repair within the transplanted area. Whether it can effectively repair nerve damage in cases of DPN, a chronic complication affecting the entire peripheral nervous system, remains unknown, let alone its potential for multidimensional repair.

[0005] In summary, there is an urgent need for a DPN treatment plan that can achieve multi-dimensional repair and has high safety and stability, so as to fundamentally rebuild and stabilize nerve function in the long term. This is of great significance for improving the overall efficacy of DPN and its clinical translation. Summary of the Invention

[0006] 1. The problem to be solved To address the shortcomings of existing DPN treatments, which often rely on EVs from different sources to improve one or a few pathological aspects of DPN (including pain and conduction velocity) by regulating a single core pathway (including inflammation, oxidative stress, or a certain pro-regenerative pathway), resulting in limited efficacy and symptom relief, this invention provides Schwann extracellular vesicles (SKP-SC-EVs) derived from skin progenitor cells. These SKP-SC-EVs can be effectively taken up (internalized) by SCs. When used to treat DPN model rats, they can achieve synergistic repair of the three core functional units of "nerve-vascular-muscle," effectively overcoming the limitations of existing DPN treatments in terms of their singular repair efficacy and inability to achieve multi-dimensional comprehensive repair.

[0007] 2. Technical Solution To solve the above problems, the technical solution adopted by the present invention is as follows: This invention provides the application of SKP-SC-EVs in the preparation of drugs for treating DPN. It should be noted that this invention verifies the in vitro biological functions of SKP-SC-EVs, finding that they can be effectively taken up (internalized) by SCs and directly repair SC damage induced by a high-glucose environment through multiple mechanisms, including enhancing SC cell viability, promoting proliferation and migration, and inhibiting apoptosis. The above reveals the key differences and advantages of SKP-SC-derived EVs compared to other EV sources (such as MSC-EVs) in treating DPN: other EV sources mainly focus on regulating pathways such as inflammation and oxidative stress, with indirect mechanisms of action and insufficient regulation of SCs, the core executive cells of neural repair; SKP-SC-EVs can directly act on damaged SCs, effectively repairing DPN. This repair effect, which directly targets the "executors" of neural regeneration (SCs), is key to overcoming the single dimension of repair efficacy in existing DPN treatments. This invention has also demonstrated through in vivo animal model experiments that SKP-SC-EVs treatment in DPN model rats effectively improves rat nerve sensory and motor functions, promotes the regeneration of rat nerve structures and myelin sheaths, enhances intraneural microcirculation blood flow, and delays target muscle atrophy and degenerative changes, achieving systematic repair of the three major damaged functional units of "nerve-blood vessel-muscle".

[0008] The present invention also provides a pharmaceutical composition for treating DPN, the pharmaceutical composition comprising SKP-SC-EVs and acceptable pharmaceutical excipients.

[0009] Furthermore, the aforementioned SKP-SC-EVs are granular and can be internalized by SC cells.

[0010] Furthermore, the above-mentioned pharmaceutical composition formulation includes a liquid formulation, and the pharmaceutical excipients include a diluent.

[0011] Furthermore, the concentration of SKP-SC-EVs in the above-mentioned liquid formulation is ≥10. 11 particles / mL.

[0012] Furthermore, the particle size distribution of the aforementioned SKP-SC-EVs is between 80 and 700 nm.

[0013] Furthermore, the particle size distribution of the aforementioned SKP-SC-EVs is between 100 and 300 nm.

[0014] 3. Beneficial effects Compared with the prior art, the advantages of this invention are as follows: (1) This invention provides the application of SKP-SC-EVs in the preparation of drugs for treating DPN. In vitro cell experiments have verified that SKP-SC-EVs in this invention have direct and powerful multidimensional repair and protection functions for damaged SCs: SKP-SC-EVs can be efficiently internalized by SCs; after treatment, SCs show significantly improved cell viability and a significant increase in the proportion of EdU-positive cells (proliferating cells). Scratch assays show that SKP-SC-EVs treatment significantly promotes the migration of damaged SCs and accelerates scratch healing. Flow cytometry analysis shows that SKP-SC-EVs can significantly reduce the apoptosis rate of SCs induced by high glucose. Western blot results further confirm that it can upregulate the anti-apoptotic protein Bcl-2 and downregulate the pro-apoptotic protein Bax, thereby reversing the apoptotic trend. In summary, the SKP-SC-EVs provided by this invention can precisely target and directly repair the core damaged SCs of DPN, laying a solid cellular foundation for nerve regeneration by enhancing their survival, proliferation, migration, and anti-apoptotic abilities.

[0015] (2) This invention provides the application of SKP-SC-EVs in the preparation of drugs for treating DPN. In vivo animal model experiments have confirmed that SKP-SC-EVs in this invention can achieve overall and synergistic repair and functional recovery of the three major functional units of "nerve-blood vessel-muscle": ① Significantly improves neurological function: Behavioralally, it effectively increases mechanical and thermal pain thresholds and reverses hyperalgesia; improves motor coordination (shortening the time to pass over the balance beam); Electrophysiologically, it significantly increases the amplitude of sciatic nerve NCV and CMAP.

[0016] ②Promoting nerve structure regeneration: Histological studies showed that the fluorescence signals of axonal (NF200) and myelin sheath (S100) markers of the sciatic nerve were significantly enhanced in the treatment group, and TEM confirmed that the axonal diameter of myelinated nerve fibers increased and the myelin sheath structure improved.

[0017] ③ Improves nerve blood supply: Laser Doppler and micro-CT have confirmed that the treatment can significantly enhance blood perfusion and vascular density in the sciatic nerve region, providing key microenvironmental support for nerve repair.

[0018] ④ Effective protection of target organs: Significantly increases the cross-sectional area of ​​target muscle fibers such as TA and GN, reduces muscle fibrosis, and promotes the morphological recovery of NMJ.

[0019] ⑤ Good biocompatibility: HE staining of major organs (heart, liver, lungs, kidneys, and brain) revealed no significant pathological changes, and serum and urine biomarkers and routine blood indicators were within the normal range, indicating that the treatment has good safety. In summary, this invention provides a comprehensive DPN treatment plan that integrates the synergistic repair of the three major functional units of "nerve-vascular-muscle," effectively overcoming the limitations of existing plans that have single efficacy and fail to comprehensively promote neural structure regeneration, improve local blood perfusion, and protect target organ function.

[0020] (3) This invention provides the application of SKP-SC-EVs in the preparation of drugs for treating DPN. The DPN treatment regimen of this invention has stable efficacy, does not depend on pathological parental cells, and does not require complex genetic engineering modification, effectively solving the efficacy uncertainty and potential safety risks caused by the use of pathological cells or engineering modification in existing technologies. At the same time, the SKP-SC-EVs of this invention are easy to obtain, have a stable cell source that can be expanded, and can achieve standardized and large-scale production, with good prospects for clinical translation. Attached Figure Description

[0021] Figure 1 Figure A shows the characterization of SKP-SC-EVs in Example 1 of this invention. Figure B shows the particle size distribution of SKP-SC-EVs using NTA. Figure C shows the TEM image of SKP-SC-EVs, bar=100 nm. Figure D shows the expression of EV markers (CD81, TSG101, Alix) in SKP-SC-EVs using Western Blot analysis.

[0022] Figure 2 Figure A shows the results of SCs purity identification in Example 2 of this invention. Figure B shows the morphological observation of SCs under bright field, bar=100 nm. Figure B shows the IF staining of SCs specific markers S100 (green) and GFAP (red), and the cell nuclei are counterstained with DAPI (blue), bar=20 μm.

[0023] Figure 3 This is a graph showing the results of SCs internalizing SKP-SC-EVs in Example 2 of the present invention. In the graph, S100 (green) marks SCs, PKH26 (red) marks EVs, and DAPI (blue) marks the cell nucleus, with bar=50 μm.

[0024] Figure 4The results of SCs cell viability assays in different treatment groups in Example 2 of this invention are shown in Figure A: SCs activity after treatment with 50, 75, 100, 125 and 150 mM glucose for 24 h; Figure B: SCs activity after treatment with 50, 75, 100, 125 and 150 mM glucose for 48 h; Figure C: SCs activity after glucose-damaged treatment with different concentrations of SKP-SC-EVs.

[0025] Figure 5 Figure A shows the proliferation results of glucose-damaged SCs after SKP-SC-EVs treatment in Example 2 of this invention. Figure B shows an electron micrograph of glucose-damaged SCs after SKP-SC-EVs treatment, detected by EdU staining (bar=50 μm). Figure C shows a histogram of the percentage of EdU-positive cells.

[0026] Figure 6 This is a graph showing the migration results of glucose-damaged SCs after SKP-SC-EVs treatment in Example 2 of the present invention, where Figure A shows the results of scratch test at 16×10⁻⁶. 8 Particles / ml SKP-SC-EVs treatment glucose-damaged SCs migration map, bar=200 μm; Figure B: SCs migration area statistical histogram.

[0027] Figure 7 Figure A shows the apoptosis results of glucose-damaged SCs after SKP-SC-EVs treatment in Example 2 of this invention. Figure B shows the apoptosis of SCs after glucose-damaged SKP-SC-EVs treatment detected by flow cytometry (Annexin V-FITC labeled apoptotic cells, PI labeled necrotic cells); Figure B shows the statistical histogram of SC apoptosis rate.

[0028] Figure 8 Figure 2 shows the expression results of Bcl-2 and Bax proteins in glucose-damaged SCs after SKP-SC-EVs treatment (β-actin is used as an internal reference). Figure A: Bcl-2 and Bax protein expression bands in glucose-damaged SCs; Figures BC: Histograms of gray values ​​of Bcl-2 (B) and Bax (C) protein expression; Figure D: Histogram of Bcl-2 / Bax ratio.

[0029] Figure 9 This is a flowchart of the STZ modeling, DPN development period, and SKP-SC-EVs treatment period in Embodiment 3 of the present invention.

[0030] Figure 10 These are graphs showing the changes in blood glucose and body weight of rats at different stages in Example 3 of the present invention, wherein Figure A: Blood glucose changes in rats during the modeling period; Figure B: Body weight changes in rats during the modeling period; Figure C: Blood glucose changes in rats during the treatment period; and Figure D: Body weight changes in rats during the treatment period.

[0031] Figure 11 The figures are the results of the motor function assessment of the treated rats in Example 3 of this invention, wherein Figure A: results of rat motor coordination measurement; Figure B: results of rat exercise endurance measurement.

[0032] Figure 12 The results are the sensory function assessment results of the treated rats in Example 3 of the present invention, wherein Figure A: statistical results of mechanical pain threshold of rats; Figure B: statistical results of thermal pain threshold of rats.

[0033] Figure 13 The results of nerve conduction function and hindlimb blood flow measurement in treated rats in Example 3 of this invention are shown in Figure A: bilateral sciatic nerve CMAP waveforms of rats in each group, with a stimulation intensity of 1 mA; Figures B and D: statistical histograms of CMAP amplitude (Figure B), NCV (Figure C), and latency (Figure D) of each group; Figure E: laser Doppler blood flow imaging showing the hindlimb blood flow distribution of each group; Figure F: statistical histogram of region of interest (ROI) for hindlimb blood flow; Figure G: three-dimensional reconstruction of hindlimb vascular network by microCT angiography, bar=100 mm; Figure H: statistical histogram of hindlimb vascular area.

[0034] Figure 14 These are morphological observation images of the sciatic nerve in treated rats in Example 3 of this invention. Figure A: IF staining of a cross-section of the sciatic nerve showing the distribution and density of nerve fibers; NF200 (red) labeling of axons, S100 (green) labeling of myelin sheaths, and DAPI (blue) labeling of cell nuclei, bar=100 μm; Figure B: Histogram of NF200 positive fluorescence intensity in each group (n=4); Figure C: Histogram of S100 positive fluorescence intensity in each group (n=4); Figure D: TEM observation of representative ultrastructures of the sciatic nerve in each group, bar=5 μm (top) and 500 nm (bottom); Figure E: Histogram of axonal area (n=3).

[0035] Figure 15These are morphological observation images of target muscles in rats treated in Example 3 of this invention. Figure A: Gross view of TA and GN in each group of rats 8 weeks after treatment; Figure B: Histogram of wet weight of the three target muscles (TA, GN, Sol) in each group of rats (n=8); Figure C: Representative images of TA cross-section IF staining (Laminin labeling, top) and Masson trichrome staining (bottom) in each group of rats, bar=50 μm; Figure D: Histogram of TA muscle fiber cross-sectional area in each group (n=3); Figure E: Representative images of TA ultrastructure observed by TEM, bar=2 μm (top) and 1 μm (bottom); Figure F: Representative images of longitudinal NMJ morphology of TA in each group observed by IF staining, with α-BTX (red) labeling acetylcholine receptors, NF200 (green) labeling nerve axons, and DAPI (blue) labeling cell nuclei, bar=50 μm.

[0036] Figure 16 The results of the in vivo biocompatibility assessment of SKP-SC-EVs in this invention are shown in Figure A: representative HE staining images of heart, liver, lung, kidney and brain tissues of rats in each group 8 weeks after treatment, bar=100 μm; Figure B: levels of neurotransmitters (bradykinin, serotonin) and vasoactive substances (histamine, β-endorphin); Figure C: platelet count; Figure D: quantification of urinary protein. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0039] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0040] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable. As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof. Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0041] Example 1 This embodiment provides the preparation and characterization of SKP-SC-EVs.

[0042] (1) Primary culture of SKPs (skin progenitor cells) ① Take 1-day-old Sprague-Dawley (SD) rats, disinfect them with 75% ethanol, and aseptically peel off the skin from their backs and place them in pre-cooled dissection solution. Remove subcutaneous tissue on ice, rinse three times with PBS, and then cut them into pieces about 1 mm in size. 3 ① Add 2 mL of type XI collagenase at a concentration of 1 mg / mL, incubate at 37℃ in a 5% CO2 incubator, and intermittently pipette until the tissue digestion results in a white, cloudy cell suspension. ② Add 10 mL of DMEM / F12 (3:1) medium containing 10% FBS to terminate digestion, and centrifuge at 4℃ and 1300 rpm for 10 min. Discard the supernatant and resuspend in 10 mL of the same medium, filter through a 400-mesh sieve, centrifuge again, and resuspend for counting. ③ Adjust the cell density to 2.5 × 10⁴ cells using SKPs proliferation medium. 4 Cells / mL. The culture medium consisted of DMEM / F12 (3:1) supplemented with 2% B27, 20 ng / mL human epidermal growth factor (EGF), 40 ng / mL basic fibroblast growth factor (bFGF), and 1% penicillin-streptomycin antibiotics. ⑤ Half-volume medium replacement was performed every 3 days. After 12 days of culture, when cells aggregated into spheroids and the central region became dense and dark, passage was performed. ⑥ Cell spheroids were collected, centrifuged at 500 rpm for 5 min at room temperature, and the supernatant was discarded. Accutase enzyme (cell digestion enzyme) was added to digest and the cells were pipetted into a single-cell suspension. Digestion was terminated by adding DMEM / F12 (3:1) medium containing 10% FBS, and the cells were resuspended after centrifugation. Cells were reseeded in fresh SKP proliferation medium at a ratio of 1:3 to obtain P1 generation SKPs. Cells were cultured for another 12 days and then passaged to the P2 generation.

[0043] (2) Directed differentiation of SKP-SCs (Schwan cells derived from skin progenitor cells) Induced differentiation of S1 and SKP-SCs: ① Take the SKPs cell spheres cultured to P2 generation in step (1) above, digest them into a single-cell suspension with Accutase enzyme, and then... 4 Cells were seeded at a density of [number] cells / mL in cell culture dishes coated with poly-D-lysine (PDL, 10 µg / mL) and laminin (2 µg / mL). ② After cell adhesion, the medium was replaced with SKP-SCs induction and differentiation initiation medium, consisting of DMEM / F12 (3:1), supplemented with 10% FBS, 2% B27, 20 ng / mL EGF, 40 ng / mL bFGF, and 1% penicillin antibiotics, and cultured for 3 days. ③ The medium was then replaced with SKP-SCs maintenance medium, consisting of DMEM / F12 (3:1), supplemented with 3% FBS, 5 µM Forskolin, 50 ng / mL heregulin-β1, 2% N2 supplement, and 1% penicillin antibiotics, with half the medium changed every 3 days. Bipolar spindle-shaped cell colonies were observed after 3 weeks of induction.

[0044] S2 and SKP-SCs propagation Colony picking and amplification: Target colonies were labeled under a microscope. The culture medium was discarded, and the cells were gently washed with PBS. A sterile cloning loop was precisely placed over the labeled colonies, and 0.25% trypsin digestion solution was added to the loop. The cells were incubated at room temperature for 1 min. Under a microscope, when the intercellular spaces within the colonies increased and the morphology became rounded, an equal volume of DMEM / F12 (3:1) complete culture medium containing 10% FBS was immediately added to terminate the digestion. The cells were gently pipetted into the area within the loop and collected into centrifuge tubes. The cells were centrifuged at 500 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in SKP-SCs maintenance medium and seeded into new PDL / Laminin-coated culture dishes for amplification culture. When the cell confluence reached 80% or higher, the cells were passaged using standard methods.

[0045] (3) Isolation, purification and characterization of SKP-SC-EVs (extracellular vesicles secreted by Schwann cells derived from skin progenitor cells) Extraction of S1 and SKP-SC-EVs: ① Collect SKP-SCs (P3-5 generations) that have passed identification and are in the logarithmic growth phase with 80% cell confluence. Replace the medium with serum-free medium and incubate at 37℃ in a 5% CO2 incubator for 48 h. ② Collect the supernatant, centrifuge at 4℃, 500 g for 10 min to remove cell debris, and filter the supernatant through a 0.22 µm filter membrane. ③ Purify using the exoEasy Maxi Kit: Mix the filtered supernatant with an equal volume of Buffer XBP, transfer to a centrifuge column, and centrifuge at 500 g for 1 min; add 10 mL of Buffer XWP for washing, and centrifuge at 5000 g for 5 min; finally, add 800 µL of Buffer XE and incubate for 1 min, centrifuge at 500×g to collect the eluent; add the eluent back to the membrane of the centrifuge column and incubate for 1 min, centrifuge at 5000 g for 5 min to collect the eluent in a new centrifuge tube, which is the SKP-SC-EVs suspension.

[0046] Characterization of S2 and SKP-SC-EVs: ①NTA detection: Dilute the SKP-SC-EVs suspension in S1 (1:1000) with PBS and mix thoroughly; after cleaning the sample cell according to the operating procedures of the nanoparticle tracking analyzer, inject the diluted sample; adjust the instrument parameters to make the background particle number less than 20 particles / frame and the scattered light intensity stable at 5~6%; then focus; inject PBS and adjust to make the background particle number (No.) less than 20; finally inject the sample for detection; collect particle size distribution and concentration data.

[0047] The results are as follows Figure 1 As shown in Figure A, the main particle size distribution peak of SKP-SC-EVs particles is around 185.2 nm, with a concentration of 5.2 × 10⁻⁶. 10 particles / mL.

[0048] ② Transmission electron microscopy (TEM) observation: 20 µL of SKP-SC-EVs suspension from S1 was dropped onto a copper grid coated with polymethyl methacrylate / carbon film and adsorbed at room temperature for 20 min; fixed with 2% PFA for 2 min; negatively stained with 2% phosphotungstic acid for 1 min; observed by TEM after drying.

[0049] The results are as follows Figure 1 As shown in Figure B, it exhibits a typical cup-shaped or circular membranous vesicle structure.

[0050] ③Western Blot Detection: Transfer the SKP-SC-EVs eluent obtained in step S1 to an Amicon® Ultra ultrafiltration tube (Millipore, molecular weight cutoff 100 kDa), and centrifuge at 14000 g for 30 min at 4°C until the sample is concentrated to 1 / 20 of its original volume. Then, invert the inner core of the ultrafiltration tube into a new collection tube, centrifuge at 1000 g for 2 min at 4°C, and collect the concentrate for later use. Add protein lysis buffer (containing 1% protease and phosphatase inhibitor) to SKP-SC-EVs concentrate, lyse on ice for 20 min, centrifuge at 12000 g for 20 min at 4℃, and collect the supernatant; determine the protein concentration according to the BCA method kit instructions, add 5× protein loading buffer to the protein sample, denature at 95℃ for 10 min, and cool for later use; prepare 10~12% SDS-PAGE gel, load 10 µg protein sample per well, electrophoresis the upper gel at 70 V for 30 min, and the separating gel at 110 V for 70~90 min; perform wet transfer at 100 V for 120 min; block on a shaker with 5% skim milk for 2 h; add primary antibodies: anti-CD81 (1:1000), anti-TSG101 (1:1000), and anti-Alix (1:1000), and incubate overnight on a shaker at 4℃; wash with TBST, and incubate at room temperature with the corresponding horseradish peroxidase (HRP) labeled secondary antibody (1:5000) for 2 hours. h. After washing, develop the protein bands using ECL chemiluminescent substrate and observe the relevant protein bands.

[0051] The results are as follows Figure 1 As shown in Figure C, SKP-SC-EVs positively express CD81, TSG101, and Alix. This conforms to the typical characteristics of EVs, confirming the successful preparation of high-purity SKP-SC-EVs.

[0052] Example 2 This embodiment provides in vitro biological function verification of SKP-SC-EVs.

[0053] (1) Effect of SKP-SC-EVs on the recovery of SC function after hyperglycemia S1. Isolation, culture and identification of primary SCs: ① Take 1-day-old newborn SD mice, disinfect with 75% ethanol, decapitate to stop bleeding, remove the skin of the hind limbs, expose and isolate the sciatic nerve, and place it in pre-cooled DMEM basal culture medium. ② Carefully peel off the epineurium, cut the nerve tissue into small pieces, digest with 3 mg / mL type I collagenase at 37℃ for 30 min, and then digest with 0.25% trypsin for 10 min. ③ Add DMEM complete medium containing 10% FBS to terminate digestion, filter through a 400-mesh sieve, and seed the single-cell suspension into PLL (poly-L-lysine) coated culture dishes; ④ Treat with differential adhesion combined with cytarabine (10 μM) for 48 h, then replace with DMEM complete medium containing 2 μM Forskolin and 10 ng / mL heregulin-β1; ⑤ When cells reach confluence, digest with 0.125% trypsin, terminate digestion, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in 1 ml of DMEM complete medium containing the fibroblast-specific antibody Thy1.1, and incubate on ice for 2 h; ⑥ After centrifugation, discard the supernatant, add supplemental resuspending, and incubate at 37℃ for 1 h; ⑦ After centrifugation, discard the supernatant, resuspend in DMEM complete medium, and seed into culture dishes; ⑧ After 1.5 days of cell seeding... After h, differential adhesion was performed, and the cells were resuspended in DMEM complete medium with added factors and seeded into PLL pre-coated culture dishes; ⑨ The purity of SCs was identified by IF staining (S100-green and GFAP-red) combined with nuclear staining.

[0054] The results are as follows Figure 1 Figures A and B are shown in the figure. Figure A shows the morphology of SCs under bright field, and Figure B shows SCs stained with S100 and GFAP double positive staining. It can be seen that the cells are typical bipolar spindle-shaped.

[0055] S2 and SCs internalize SKP-SC-EVs: ① Add 4 μL of PKH26 dye to a centrifuge tube containing 500 μL of Diluent C and mix gently; ② Add 100 μL of SKP-SC-EVs (1×10⁻⁶) to a centrifuge tube containing 500 μL of Diluent C and mix gently; 11Add (particles / mL) to another centrifuge tube containing 500 μL Diluent C and mix gently; ③ Quickly mix the liquids from steps ① and ②, let stand at room temperature for 5 min, and mix periodically in between; ④ Add 1 mL of vesicle-free serum to stop staining, transfer the mixture to an ultrafiltration column, and centrifuge at 14000 g for 20 min to remove free dye; ⑤ Wash with PBS and centrifuge again; ⑥ Invert the filter column into a collection tube and centrifuge at 1000 g for 2 min; ⑦ Resuspend the EVs precipitate in SCs complete culture medium and filter through a 0.22 μm filter for sterilization; ⑧ Treat SCs with 500 μL / well PKH26-labeled SKP-SC-EVs (PBS washing solution as a control); ⑨ Co-culture the labeled SKP-SC-EVs and SCs for 6 h and then perform IF staining and observe under a laser confocal microscope.

[0056] The results are as follows Figure 3 As shown, distinct red fluorescent dot signals (PKH26) appeared in the cytoplasm of SCs, co-localizing with the green fluorescent (S100) labeled SCs cell bodies, while the control group (treated with PBS elution buffer) did not show this signal. This indicates that SKP-SC-EVs were effectively taken up (internalized) by SCs.

[0057] S3, Construction and intervention of the high-glucose-damaged SCs model: SCs were divided into three groups: a control group (DMEM medium containing 25 mM glucose, 1% vesicle-free FBS, 2 μmol / L Forskolin, and 10 ng / mL HRG), a high glucose injury group (DMEM medium containing 100 mM glucose, 1% vesicle-free FBS, 2 μmol / L Forskolin, and 10 ng / mL HRG), and an SKP-SC-EVs intervention group (high glucose injury group medium + 16×10⁻⁶ HRG). 8 32×10 8 particles / mL of SKP-SC-EVs).

[0058] (a) Cell viability (CCK-8 assay): ① With 1.5×10 5① 100 μL of SCs were seeded into 96-well plates pre-coated with PLL at a density of 1 / mL (6 replicates / group); ② After cell adhesion, the original culture medium was aspirated and replaced with culture medium supplemented with 1% vesicle-free FBS and maintenance factor for the control group, high glucose damage group, and SKP-SC-EVs intervention group, respectively; ③ After culturing for 24 and 48 h, the culture medium was replaced with 100 μL of DMEM basal medium containing 10% CCK-8; ④ The culture plates were incubated in a 37℃, 5% CO2 incubator for 2 h; ⑤ The 96-well plates were removed and the absorbance at 450 nm was measured using a microplate reader.

[0059] The results are as follows Figure 4 As shown in Figures A-B, cell viability significantly decreased after treatment with 100, 125, and 150 mM glucose for 24 h and 48 h. Therefore, treatment with 100 mM glucose for 24 h was selected as the condition for constructing the damage model. Based on this, 16 × 10⁻⁶ cells were used... 8 and 32×10 8 Intervention with particles / mL SKP-SC-EVs, CCK-8 results showed that SKP-SC-EVs could increase the viability of damaged SCs in a concentration-dependent manner. Figure 4 (Figure C in the middle)

[0060] (b) Cell proliferation assay (EdU method): ① With 1×10 5 SCs were seeded at a density of 1 / mL in PLL-pre-coated 96-well plates. After cell adhesion, the medium was replaced with treatment medium containing 1% vesicle-free FBS and maintenance factors (the glucose concentration in the damage and intervention groups was 100 mM), and cultured for 24 h. ② EdU solution was diluted with the treatment medium (final concentration 10 μM), and 100 μL of EdU medium was added to each well for incubation for 4 h. ③ The medium was discarded, and the cells were washed twice with PBS (5 min / wash), followed by incubation with 4% PFA at room temperature for 15 min. ④ 100 μL of permeabilizer (PBS containing 0.3% Triton X-100) was added and incubated for 10 min, then aspirated and washed with PBS for 5 min. ⑤ 100 μL of staining reaction solution (Alexa Fluor 594) was added and incubated at room temperature in the dark for 30 min. ⑥ After aspiration, the cells were washed three times with PBS containing 3% BSA (10 min / wash). ⑦ 100 μL of Hoechst staining solution was added and incubated at room temperature in the dark for 10 min. min, aspirate and discard, then wash 3 times with PBS (10 min / wash); ⑧ Take pictures with a fluorescence microscope and count the percentage of EdU positive cells in each group.

[0061] The results are as follows Figure 5As shown in Figures A and B, EdU staining revealed that the proportion of EdU-positive cells in the high glucose group was significantly lower than that in the control group. The proportion of EdU-positive cells in the EVs-treated group was significantly higher than that in the high glucose group, indicating that SKP-SC-EVs can promote the proliferation of SCs under high glucose damage.

[0062] (c) Cell migration (Ibidi wound healing assay): ① Cells were pretreated for 48 h according to the experimental groups (control group, high glucose injury group, and SKP-SC-EVs intervention group). ② After pretreatment, cells from each group were digested and collected, and resuspended in DMEM / F12 medium (glucose concentration 25 mM or 100 mM depending on the group) without EVs and containing 1% vesicle-free FBS and the same maintenance factors, and the density was adjusted to 4 × 10⁶ cells / cm². 5 / mL. ③ Adhere the Ibidi plug to the PLL pre-coated small dish, add 100 μL of SCs suspension to each well, and incubate in a 37℃, 5% CO2 incubator; ④ After the cells adhere to the wall after 4 h of culture, replace with fresh grouped culture medium (same formula as the pretreatment stage, but without EVs, glucose concentration maintained at 25 mM or 100 mM), and continue to culture for 24 h until the cells grow into a confluent monolayer. After the cells have grown to a confluent monolayer, the insert is carefully removed with sterile forceps, and the cells are gently washed twice with PBS. The PBS is then discarded, and the grouped culture medium (with corresponding glucose concentration) without EVs is added. The scratches are observed under a microscope and photographed (recorded as 0 h). The cells were returned to the incubator for further culture, and observed and photographed again after 24 hours; the cell migration area was counted using ImageJ software.

[0063] The results are as follows Figure 6 As shown in Figures A and B, the scratch assay revealed that the wound healing area in the high-glucose group was significantly smaller than that in the control group. Conversely, the EVs-treated group showed a significant recovery in cell migration ability, with a significantly larger wound healing area than the high-glucose group.

[0064] (d) Apoptosis assay: ① Flow cytometry: Collect cells from each group (including suspension and adherent cells) and wash twice with pre-cooled PBS. Stain using the Annexin V-FITC / PI apoptosis detection kit. 1×10⁻⁶ cells were then added to the container. 5 Each cell was resuspended in 100 μL binding buffer, and 5 μL of Annexin V-FITC dye was added. The cells were incubated at room temperature in the dark for 15 min. Then, 10 μL of PI solution was added, and the mixture was gently mixed and incubated at room temperature in the dark for 5 min. 400 μL of binding buffer was added, and the cells were analyzed by flow cytometry within 1 h. The proportion of Annexin V-positive cells (early apoptosis + late apoptosis) to the total number of cells was calculated as the apoptosis rate.

[0065] The results are as follows Figure 7 As shown in Figures A and B, the apoptosis rate of SCs in the high glucose group (Annexin V) + The apoptosis rate of cells was significantly higher in the EVs-treated group than in the control group, and the apoptosis rate was significantly lower in the EVs-treated group than in the glucose-treated group.

[0066] ② Apoptosis-related protein expression (Western Blot): Total protein was extracted from cells in each group, and the expression levels of Bcl-2 and Bax were detected by Western Blot. The Bcl-2 / Bax ratio was calculated.

[0067] The results are as follows Figure 8 As shown in Figures A through D, the high glucose group showed upregulation of the pro-apoptotic protein Bax and downregulation of the anti-apoptotic protein Bcl-2, resulting in a decreased Bcl-2 / Bax ratio. SKP-SC-EVs treatment significantly upregulated Bcl-2, downregulated Bax, and restored the Bcl-2 / Bax ratio.

[0068] In summary, this embodiment demonstrates at the in vitro cellular level that SKP-SC-EVs can be efficiently internalized by target SC cells and directly repair SC damage induced by a high-glucose environment through multiple mechanisms, including enhancing cell viability, promoting proliferation and migration, and inhibiting apoptosis. This mechanistically reveals the key difference / advantage of the SKP-SC-derived EVs of this invention compared to EVs from other sources (such as MSC-EVs): SKP-SC-EVs can directly act on and potently repair the core damaged SCs of the DPN, meaning its repair effect is directly targeted at the "executors" of neural regeneration.

[0069] Example 3 This embodiment provides a method for applying SKP-SC-EVs in the treatment of DPN.

[0070] (1) Preparation of therapeutic drug formulations The SKP-SC-EVs prepared in Example 1 were diluted with PBS buffer to prepare a homogeneous suspension that could be directly administered via tail vein injection in rats (SKP-SC-EVs concentration was 10). 11 (particles / mL).

[0071] (2) Establishment of DPN animal model, treatment and efficacy evaluation S1. Establishment of the DPN model: Healthy male SD rats aged 5-6 weeks and weighing 180-220 g were selected. After one week of acclimatization, a single intraperitoneal injection of freshly prepared streptozotocin solution (STZ, 65 mg / kg, dissolved in 0.1 mol / L, pH 4.5 pre-cooled sodium citrate buffer) was administered. 72 hours after injection, blood was collected from the tail vein to measure non-fasting blood glucose. Rats with two consecutive blood glucose values ​​≥16.7 mmol / L were considered to have established a diabetic model and were fed for another 8 weeks to induce stable DPN.

[0072] S2. Grouping and Treatment: DPN model rats were randomly divided into a normal control group (Ctrl group), a model group (DPN group), and an SKP-SC-EVs treatment group (DPN+EVs group). The treatment group received the SKP-SC-EVs preparation prepared in step (1) via tail vein injection. The treatment regimen for the treatment group was: once a week, with a single dose of 2 × 10⁻⁶ mg / L. 10 Each particle EV was administered continuously for 8 weeks. The model group and the control group were injected with the same volume of PBS during the same period.

[0073] The above operation procedure is as follows: Figure 9 As shown.

[0074] (3) Behavioral assessment: ① Blood glucose and body weight: Non-fasting blood glucose and body weight of all rats were measured at fixed times each week.

[0075] Modeling period ( Figure 10 (A~B) and treatment period ( Figure 10 In both the C-D group and the DPN+EVs group, rats consistently exhibited hyperglycemia and slow weight gain, with no statistically significant difference between the two groups, indicating that the treatment did not affect the overall glucose metabolism status.

[0076] ② Motor function assessment: Record the time (s) required for the rat to pass through a square wooden strip 100 cm long and 2.5 cm wide. Take the average value of 3 tests. Place the rat on an automatic acceleration rotating bar and accelerate it linearly from 5 rpm to 100 rpm for 5 min. Record the latency (s) from the start to the fall. The upper limit is 300 s. Take the best result of 3 tests.

[0077] The results are as follows Figure 11 As shown in Figures A and B, the DPN group experienced prolonged passing time, indicating impaired motor coordination; the EVs treatment group showed significantly shorter passing time from week 10, approaching normal levels (Figure A). In the rotating bar test, the DPN group showed a shortened fall latency, indicating decreased motor coordination and endurance, but there was no significant difference between the EVs treatment group and the DPN group, possibly due to the influence of body weight (Figure B).

[0078] ③ Sensory function assessment: The mechanical pain withdrawal threshold of the hind limbs was measured using an electronic Von Frey analgesic device (three tests were conducted on each hind limb, and the average value was taken). The plantar part of the rat's hind limb was immersed in a constant temperature water bath (55.0±0.5℃) and the time was recorded. The time until the rapid withdrawal response appeared was the thermal pain withdrawal latency (s), and 10 s was set as the cutoff time. Three tests were conducted on each hind limb, and the average value was taken.

[0079] The results are as follows Figure 12 As shown in Figures A and B, the DPN group developed mechanical pain and thermal hypersensitivity from 6 to 8 weeks onwards; SKP-SC-EVs treatment significantly improved the pain threshold from 12 to 16 weeks onwards, and gradually recovered to near the control group level.

[0080] In summary, SKP-SC-EVs can effectively improve motor coordination and reverse sensory nerve hypersensitivity in DPN rats without altering blood glucose levels, demonstrating clear neuroprotective and repair effects.

[0081] (4) Electrophysiological and blood flow assessment: ① Neuroelectrophysiology: After 8 weeks of treatment, the neuroelectrophysiology of rats in each group was detected using a full-function electromyography evoked potential instrument. After deep anesthesia, the rats underwent routine skin preparation and disinfection with iodine. The skin was incised and the tissue was separated to fully expose the proximal and distal ends of the sciatic nerve transplant segment. The recording electrode needle was inserted into the belly of the gastrocnemius muscle, the interference electrode was clamped at the incised skin, and the stimulation electrode was hooked onto the proximal and distal ends of the nerve transplant segment 2 mm apart. The stimulation intensity was set to 1 mA and the interval was 1 s. The instrument was clicked to detect the nerve. The amplitude, latency, and distance between the two electrodes of the compound muscle action potential (CMAP) were recorded, and the instrument automatically calculated the motor nerve conduction velocity (NCV).

[0082] The results are as follows Figure 13 As shown in Figures A-D, compared with the control group, the CMAP amplitude and NCV of the sciatic nerve in the DPN group rats were significantly reduced, indicating impaired nerve conduction function; after treatment with SKP-SC-EVs, both CMAP amplitude and NCV were significantly increased, indicating improved nerve function.

[0083] ② Nerve blood supply: Rats were anesthetized, and the hind limbs were shaved. A laser Doppler blood flow imaging system was used to scan the hind limb region to obtain pseudo-color images of blood perfusion. Regions of interest were delineated along the sciatic nerve pathway, and the average blood perfusion units were quantitatively analyzed. Micro-CT 3D vascular reconstruction: Hind limb tissue from treated rats was perfused with contrast agent and then subjected to high-resolution micro-CT scanning. Three-dimensional vascular reconstruction and quantitative analysis were performed using the accompanying software to calculate the vascular area in the hind limb muscles and surrounding nerve regions.

[0084] The results are as follows Figure 13As shown in Figures E-H, laser Doppler blood flow imaging revealed that the blood flow perfusion signal in the hind limbs (especially the sciatic nerve pathway) was significantly weakened in the DPN group, while the blood flow signal in this area was significantly enhanced in the treatment group. Figure 13 Figures E-F). MicroCT vascular 3D reconstruction further confirmed that hindlimb vascular density decreased in the DPN group, while EVs treatment significantly improved vascular density. Figure 13 (G~H diagram).

[0085] In summary, this indicates that SKP-SC-EVs can not only restore the electrophysiological function of damaged nerves, but also improve local blood perfusion by promoting angiogenesis, providing blood supply support for nerve repair, thereby synergistically promoting the structural and functional recovery of DPN.

[0086] (5) Evaluation of tissue morphology and ultrastructure: ① Sample Collection: After the efficacy assessment, rats were deeply anesthetized and perfused with physiological saline and 4% PFA via the left ventricle for systemic fixation. Bilateral sciatic nerves, tibialis anterior (TA), gastrocnemius (GN), and soleus (Sol) muscles were rapidly removed. Some tissues were immediately flash-frozen in liquid nitrogen; others were fixed in 4% PFA for paraffin or frozen sectioning; and some (approximately 1 mm) were... 3 2.5% glutaraldehyde was added to TEM.

[0087] ② Nerve Structure: Frozen transverse sections (10 μm) of the mid-sciatic nerve were prepared and co-stained with NF200 (axonal marker) and S100 (SCs / myelin marker). The average fluorescence intensity was quantitatively analyzed using ImageJ software under a laser confocal microscope. Fresh sciatic nerve tissue was double-fixed with glutaraldehyde and osmium tetroxide, dehydrated, embedded in epoxy resin, and then ultrathin sections (70 nm) were prepared and double-stained with uranium acetate and lead citrate. The morphology of myelinated nerve fibers and the lamellar structure of the myelin sheath were observed under TEM, and the axonal diameter and myelin sheath thickness of the myelinated fibers were statistically analyzed.

[0088] ③ Muscle and neuromuscular junction: The target muscle was weighed, and frozen cross-sections were prepared for laminin immunofluorescence staining to delineate muscle fiber boundaries. The cross-sectional area (μm) of muscle fibers was randomly measured using ImageJ software. 2 Paraffin sections of the target muscle were stained with Masson's trichrome stain; under the microscope, collagen fibers appeared blue, and muscle fibers appeared red. Image analysis software was used to calculate the percentage of collagen fiber area and assess the degree of interstitial fibrosis. Longitudinal frozen sections (20 μm) of the gastrocnemius muscle were prepared and stained with NF200 (nerve endings) IF, and acetylcholine receptors were labeled with α-BTX (α-cobra venom). The morphology and number of neuromuscular junctions (NMJs) were observed under a confocal microscope.

[0089] The results are as follows Figure 14As shown, after 8 weeks of treatment, IF staining of the sciatic nerve transverse section revealed strong positive signals for NF200 (axon) and S100 (myelin sheath) in the control group, with dense nerve fiber arrangement and intact structure. In the DPN group, fluorescence intensity was significantly weakened, and the fiber structure was loose and sparse, suggesting degenerative changes in both axons and myelin sheath. After treatment with SKP-SC-EVs, the fluorescence intensity of NF200 and S100 was significantly enhanced compared to the DPN group, and the fiber arrangement tended to be more compact and orderly, indicating that SKP-SC-EVs can effectively promote sciatic nerve fiber regeneration and structural repair in DPN rats. Figure 14 Figures A-C). TEM ultrastructural observation showed that in the control group, myelinated nerve fibers had regular morphology, full axons, and dense myelin sheaths; in the DPN group, axonal area was reduced, morphology was irregular, and myelin sheath structure was disordered; in the treatment group, nerve fiber morphology returned to regularity, axonal area significantly increased, and myelin sheath structure improved (Figures A-C). Figure 14 (Figures D-E). Histological and ultrastructural results consistently indicate that SKP-SC-EVs can effectively promote sciatic nerve axon regeneration and myelin sheath repair in DPN rats.

[0090] ④ Hematoxylin-eosin (HE) staining of major organs: Paraffin sections of major organs such as the heart, liver, lungs, kidneys, and brain were dewaxed with xylene and rehydrated with a series of ethanol solutions (100%, 95%, 85%, 75%). Routine hematoxylin staining of the nuclei (5-10 min), differentiation with 1% hydrochloric acid ethanol, and rehydration with tap water were then performed, followed by eosin staining of the cytoplasm (1-3 min). The sections were then dehydrated with a series of ethanol solutions, cleared with xylene, and finally mounted with neutral resin. Under a microscope, the nuclei appeared blue-purple, while the cytoplasm and stroma appeared pink, used to assess the basic histological structure of each major organ.

[0091] Eight weeks after treatment, the morphological and weight changes of TA, GN, and Sol in rats of each group were examined. The results are as follows: Figure 15 As shown, compared with the control group, the wet weight of all three target muscles in DPN rats was significantly decreased, indicating significant diabetic muscle atrophy; although EV treatment did not significantly restore the wet weight of the target muscles ( Figure 15 (Figures A-B in the image), but histological and ultrastructural analyses revealed significant structural recovery. IF staining (labeled with laminin) showed that the DPN group had a reduced myofiber cross-sectional area, exhibiting typical atrophy characteristics; while the EVs treatment group showed a significant increase (…). Figure 15 (Figures C (top) and D); Masson staining showed increased collagen deposition in the DPN group, indicating significant interstitial fibrosis, while the degree of fibrosis was reduced in the treatment group. Figure 15 (Figure C below). TEM observation showed that the DPN group exhibited disordered myofibril arrangement, blurred Z-lines, and mitochondrial swelling and vacuolation, while the treatment group showed significant improvement in ultrastructure. Figure 15(Figure E). To further assess the recovery of nerve innervation, the morphology of the NMJ was observed using α-BTX combined with IF staining. Results showed that the NMJs in the normal control group were well-formed and clearly defined; the number of NMJs in the DPN group was reduced and their morphology was elongated; and the number of NMJs in the treatment group increased, with a more complete structure. Figure 15 (Figure F in the middle)

[0092] In summary, SKP-SC-EVs treatment not only repairs nerves but also delays target muscle atrophy, reduces fibrosis, protects muscle cell structure, and promotes NMJ reconstruction, thereby achieving overall functional recovery of the neuromuscular unit.

[0093] (6) Serum biomarker monitoring: Enzyme-linked immunosorbent assay (ELISA) was used to detect relevant biomarkers in rat serum and urine.

[0094] ① Serum detection: Using species-specific ELISA kits, strictly follow the kit instructions to quantitatively detect the levels of bradykinin (BK), serotonin (5-HT), histamine, and β-endorphin (β-EP) in rat serum.

[0095] ②Routine blood test: The peripheral blood routine indicators of rats were measured using a fully automated blood analyzer.

[0096] ③ Urine test: The content of urinary protein in rat urine was detected using a urinary protein (UP) quantitative ELISA kit.

[0097] Histopathological analysis of major organs (heart, liver, lungs, kidneys, brain) showed that no treatment-related pathological structural changes were observed in the SKP-SC-EVs treatment group. Figure 16 Figure A shows that the treatment did not cause observable toxic damage to vital organs. Serum biomarker detection further revealed the regulatory effect of the treatment on systemic pathological states. Regarding neurotransmitters and vasoactive substances, SKP-SC-EVs treatment significantly reversed the abnormal increase in serotonin (5-HT) induced by the DPN model, while upregulating the level of β-endorphin (β-EP), which has endogenous analgesic effects; the moderate increase in bradykinin (BK) and histamine suggests that the treatment may have affected local microvascular regulatory mechanisms. Figure 16 (Figure B). Hematological and renal function analyses provided evidence of metabolic syndrome-related changes. Compared to the control group, the DPN model group exhibited characteristic changes of significantly reduced platelet count and decreased urinary protein excretion. Figure 16 (Figure C). Thrombocytopenia may be related to the hypercoagulable state associated with diabetes and increased platelet activation and consumption; abnormal urinary protein excretion reflects the characteristic pathophysiological changes of glomerular hyperfiltration in the early stages of diabetes. Figure 16 (D diagram).

[0098] Based on the above multi-system assessment results, SKP-SC-EVs demonstrated good biocompatibility during the 8-week treatment period, did not cause organic damage to vital organs, and showed certain regulatory potential for multi-system dysfunction in diabetic patients.

[0099] Results: This embodiment comprehensively validates the superior efficacy and good safety of SKP-SC-EVs in treating DPN at the whole animal level. Its effects are not singular but systemic: it simultaneously improves neurosensory and motor function, promotes the regeneration of neural structures and myelin sheaths, enhances intraneural microcirculation blood flow, and effectively protects target muscles from atrophy and degenerative changes. This achieves holistic repair of the "neurovascular-muscle" functional unit. This comprehensive efficacy far surpasses existing EV therapies that target only a single pathological link, completely resolving the core deficiency of the prior art: "the repair efficacy is one-dimensional, making it difficult to achieve holistic functional recovery of the neurovascular-muscle unit." Furthermore, the treatment did not cause systemic toxicity and demonstrated good safety.

[0100] In summary, the above specific implementation methods, through three coherent examples, fully demonstrate the entire process of the present invention, "Application of SKP-SC-EVs in the Treatment of DPN," from product preparation and mechanism verification to efficacy evaluation. Experimental data fully demonstrate that the SKP-SC-EVs preparation method provided by this invention is stable and reliable, and the product possesses clear biological characteristics; it can directly and efficiently repair damaged SCs; in DPN animal models, it can achieve multi-dimensional synergistic treatment, comprehensively reversing pathological changes of the disease, and exhibits high safety. This invention effectively overcomes the shortcomings of existing DPN treatments, which have a single efficacy dimension and only focus on symptom relief, lacking the ability to achieve multi-dimensional synergistic repair such as substantial regeneration and reconstruction of neural structures, fundamental restoration of blood flow, and effective protection of target muscles. It provides a novel and highly promising strategy for the clinical treatment of DPN.

Claims

1. Application of SKP-SC-EVs in the preparation of drugs for treating DPN.

2. A pharmaceutical composition for treating DPN, characterized in that, The pharmaceutical composition includes SKP-SC-EVs and acceptable pharmaceutical excipients.

3. The pharmaceutical composition for treating DPN according to claim 2, characterized in that, The SKP-SC-EVs are granular and can be internalized by SCs cells.

4. The pharmaceutical composition for treating DPN according to claim 2 or 3, characterized in that, The pharmaceutical composition dosage form includes a liquid formulation, and the pharmaceutical excipients include a diluent.

5. The pharmaceutical composition for treating DPN according to claim 4, characterized in that, The concentration of SKP-SC-EVs in the liquid formulation is ≥10. 11 particles / mL.

6. The pharmaceutical composition for treating DPN according to claim 5, characterized in that, The particle size distribution of the SKP-SC-EVs is between 80 and 700 nm.

7. The pharmaceutical composition for treating DPN according to claim 6, characterized in that, The particle size distribution of the SKP-SC-EVs is between 100 and 300 nm.

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