Application of PRP-Exos in preparation of medicine for treating neuropathic pain

Abstract

The invention provides application of PRP-Exos in preparation of a medicine for treating neuropathic pain, and relates to the technical field of clinical medicine. According to the invention, the treatment effect of PRP-Exos on neuropathic pain by inhibiting a TLR4 / NF-kappa B signal channel is clarified, a new thought is provided for clinical transformation, meanwhile, an intrathecal injection delivery mode is adopted, the limitation of low bioavailability of peripheral administration is overcome, meanwhile, the treatment effect is comprehensively verified through a behavioral-histological-molecular biological multi-modal evaluation system, and the application prospect is wide. The potential of the PRP-Exos in relieving the neuropathic pain is proved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of clinical medicine, and particularly relates to application of PRP-Exos in preparation of a medicine for treating neuropathic pain. BACKGROUND

[0002] Neuropathic pain (NP) is a chronic pain syndrome caused by injury or disease of the somatosensory nervous system, and is clinically manifested as spontaneous pain, hyperalgesia and allodynia, which seriously affects the quality of life and mental health of patients. The pathogenesis of NP is complex, involving peripheral and central sensitization, neuroinflammatory response, ion channel abnormality and other links. Activation of the spinal cord level of the inflammatory signaling pathway is considered to be one of the important factors leading to persistent and aggravated pain. Studies have shown that overactivation of Toll-like receptor 4 (TLR4) and its downstream nuclear factor-κB (NF-κB) signaling pathway can promote the release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), exacerbate neuroinflammatory response, and thus participate in the occurrence and development of NP.

[0003] At present, the efficacy of non-steroidal anti-inflammatory drugs, opioid drugs and other drugs for clinically treating NP is limited, and long-term use is prone to produce tolerance and adverse reactions, so it is of great significance to explore new therapeutic targets and intervention strategies. In recent years, exosomes (Exos) have become a hot spot in regenerative medicine research due to their unique biological functions. Platelet-rich plasma-derived exosomes (PRP-Exos) are rich in growth factors, microRNAs and anti-inflammatory proteins, and have the effects of promoting tissue repair, regulating immune response and inhibiting inflammation. Studies have shown that PRP-Exos can reduce inflammatory response by regulating macrophage polarization, but whether it plays a role in NP and the mechanism are not clear.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] In order to solve the above technical problems, the present application provides application of PRP-Exos in preparation of a medicine for treating neuropathic pain and a pharmaceutical composition. PRP-Exos can significantly reduce the expression of inflammatory factors in the spinal cord tissue by inhibiting the TLR4 / NF-κB signaling pathway, thereby reducing the inflammatory response caused by nerve injury and improving the symptoms of neuropathic pain, providing a new idea for clinical transformation.

[0006] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides the use of PRP-Exos in the preparation of medicaments for treating neuropathic pain, wherein the PRP-Exos is used to inhibit the TLR4 / NF-κB pathway.

[0007] Furthermore, the PRP-Exos is also used to reduce the levels of TNF-α and IL-6 in spinal cord tissue.

[0008] Furthermore, the concentration of the drug is 50 mg / L-500 mg / L, and the dosage is 0.5 mL per administration.

[0009] Furthermore, the concentration of the drug is 200 mg / L.

[0010] Furthermore, the method of administration is intrathecal injection.

[0011] Furthermore, the neuropathic pain is caused by selective damage to the sciatic nerve branches.

[0012] Furthermore, the particle size of the PRP-Exos is between 50 nm and 150 nm; and the marker protein expressing the PRP-Exos exosomes is any one of CD63, TSG101, or CD9.

[0013] The present invention also provides a pharmaceutical composition for treating neuropathic pain, the pharmaceutical composition comprising an effective dose of PRP-Exos and a pharmaceutically acceptable carrier or excipient.

[0014] Furthermore, the PRP-Exos are exosomes isolated, purified, and identified from platelet-rich plasma.

[0015] Furthermore, the dosage forms of the pharmaceutical composition include injection, oral, and transdermal dosage forms.

[0016] Furthermore, the dosage form of the pharmaceutical composition includes any one of tablets, capsules, oral liquids, injections, and powder injections.

[0017] Furthermore, the drug combination includes any one of various analgesics, anti-inflammatory drugs, and psychotropic drugs.

[0018] Furthermore, the drug combination is for treating neuropathic pain.

[0019] The present invention has the following technical effects: This invention elucidates the therapeutic effect of PRP-Exos on neuropathic pain by inhibiting the TLR4 / NF-κB signaling pathway, providing a new approach for clinical translation. Furthermore, the intrathecal delivery method overcomes the limitation of low bioavailability associated with peripheral administration. The therapeutic efficacy was comprehensively validated through a multimodal evaluation system encompassing behavioral, histological, and molecular biology, demonstrating the potential of PRP-Exos in alleviating neuropathic pain. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 Transmission electron micrograph of PRP-Exos; Figure 2 : Nanoparticle tracking analysis of PRP-Exos; Figure 3 Western blot identification results of PRP-Exos marker proteins; Figure 4 Changes in mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in rats of different groups; Figure 5 Comparison of toluidine blue staining pathological morphology of sciatic nerve in rats of different groups (400×), where A is blank group; B is model group; C is PRP-Exos group; Figure 6 : Expression levels of TNF-α and IL-6 in the spinal cord tissue of rats in each group; Figure 7 Western blot electrophoresis images of TLR4 and NF-κBp65 proteins in the spinal cord tissues of rats in each group; Figure 8 Relative expression levels of TLR4 and NF-κBp65 proteins in the spinal cord tissues of rats in each group. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0023] In a first aspect, the present invention provides the use of PRP-Exos in the preparation of medicaments for treating neuropathic pain, wherein the PRP-Exos is used to inhibit the TLR4 / NF-κB pathway.

[0024] In some embodiments, the PRP-Exos is also used to reduce TNF-α and IL-6 levels in spinal cord tissue.

[0025] In some embodiments, the concentration of the drug is 50 mg / L-500 mg / L, and the dose is 0.5 mL per administration.

[0026] In some embodiments, the concentration of the drug is 200 mg / L.

[0027] In some embodiments, the administration method is intrathecal injection.

[0028] In some embodiments, the neuropathic pain is caused by selective damage to the sciatic nerve branches.

[0029] In some embodiments, the particle size of the PRP-Exos is between 50 nm and 150 nm; and the marker protein expressing the PRP-Exos exosomes is any one of CD63, TSG101, or CD9.

[0030] Secondly, the present invention also provides a pharmaceutical composition for treating neuropathic pain, the pharmaceutical composition comprising an effective dose of PRP-Exos and a pharmaceutically acceptable carrier or excipient.

[0031] In some embodiments, the PRP-Exos are exosomes isolated, purified, and identified from platelet-rich plasma.

[0032] In some embodiments, the dosage forms of the pharmaceutical composition include injectable, oral, and transdermal dosage forms.

[0033] In some embodiments, the dosage form of the pharmaceutical composition includes, but is not limited to, tablets, capsules, oral liquids, injections, and powder injections.

[0034] In some embodiments, the drug combination includes, but is not limited to, various analgesics, anti-inflammatory drugs, and psychotropic drugs.

[0035] In some embodiments, the drug combination is for treating neuropathic pain.

[0036] The following is a detailed explanation using specific embodiments: Example 1: Constructing an animal model 30 male Sprague-Dawley rats, weighing 250 - 300 g, were purchased from Spiberfu (Beijing) Biotechnology Co., Ltd. The animal certificate number was SCXK (Beijing) 2019 - 0010, and the use permit number was SYXK (Tianjin) 2020 - 0001. PRP-Exos were prepared and identified by the laboratory of Zhu Xianyi Memorial Hospital, Tianjin Medical University. Hematoxylin-eosin staining solution was purchased from Guangzhou Vigorous Company. TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) kits, SDS-PAGE gel preparation kits were purchased from Shanghai Beyotime Biotechnology Company. Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG was purchased from Wuhan Boster Company. HRP-labeled goat anti-mouse IgG was purchased from Beijing Zhongshan Jinqiao Company. 4% paraformaldehyde, 2.5% glutaraldehyde, RIPA lysis buffer (containing PMSF), BCA kit, 10× electrotransfer buffer were purchased from Beijing Solarbio Company. TLR4 (96KD), NF-κB p65 (65KD), GAPDH (37KD) antibodies were purchased from Wuhan Sanying Biotechnology Company.

[0037] A. Preparation and identification of PRP-Exos 1. Extraction of platelet-rich plasma (1) Ten healthy Sprague-Dawley rats were anesthetized by inhaled isoflurane, and then 100 ml of venous blood was drawn through the renal vein by laparotomy. The drawn venous blood was shaken well in a centrifuge tube containing 10 ml of sodium citrate anticoagulant. (2) PRP was prepared by the two-step centrifugation method. For the first step, centrifuge at 1500 r / min for 10 min, and aspirate the upper supernatant to 2 mm below the interface and transfer it to another centrifuge tube. For the second step, centrifuge at 5000 r / min for 10 min, and a total of 20 mL of PRP was obtained.

[0038] 2. Extraction of exosomes (PRP-exos) Equipment: Ultracentrifuge, fixed-angle rotor (such as Type 70 Ti, 90 Ti) or vertical rotor, ultracentrifuge tube, high-speed centrifuge, pipette and pipette tips, sterile PBS buffer (pH 7.4), 0.22 μm filter (for filtering PBS) Detailed steps of the ultracentrifugation method Step 1: Serum pretreatment (removing cell debris and large particles) 1. Thawing and dilution: Slowly thaw the frozen serum at \(4^{\circ}C\). After thawing, dilute the serum with an equal volume of pre-cooled PBS (pH 7.4). This helps to reduce the serum viscosity and improve the subsequent centrifugation efficiency.

[0039] 2. Low-speed centrifugation: Aliquot the diluted serum into high-speed centrifuge tubes and centrifuge at 4°C and 2,000×g for 20 minutes.

[0040] 3. Collect the supernatant: Carefully aspirate the supernatant, avoiding touching the sediment at the bottom (cell debris, large particles).

[0041] 4. High-speed centrifugation: Transfer the supernatant to a new ultracentrifuge tube and centrifuge at 4°C and 10,000-12,000×g for 30 minutes.

[0042] 5. Collect and filter the supernatant: Carefully aspirate the supernatant again and filter it through a 0.22 μm filter to remove any microbubbles and large protein aggregates that may be present.

[0043] At this point, the supernatant mainly contains exosomes, soluble proteins, and lipoproteins (such as LDL / HDL).

[0044] Step 2: Ultracentrifugation (precipitation of exosomes) 1. Dispensing: Dispense the filtered supernatant into ultracentrifuge tubes that match the ultracentrifuge rotor, and ensure precise balance.

[0045] 2. First departure: Conditions: Centrifuge at 4°C, 100,000-120,000×g for 70-90 minutes.

[0046] Objective: This centrifugal force is sufficient to precipitate exosomes to the bottom of the tube.

[0047] 3. Discard the supernatant: After centrifugation, very carefully discard the supernatant (containing soluble proteins and lipoproteins). At this point, there should be tiny, invisible precipitates at the bottom of the tube.

[0048] Step 3: Cleaning (to improve purity) 1. Resuspension: Gently resuspend the precipitate in a large volume (e.g., 10-15 mL) of pre-cooled sterile PBS and mix by pipetting.

[0049] Objective: To wash away contaminants (such as proteins) that are co-precipitated with exosomes.

[0050] 2. Second transcendence: Conditions: Centrifuge again at 4°C, 100,000-120,000×g for 70-90 minutes.

[0051] Final result: Discard the supernatant completely and resuspend the precipitate in a small amount (e.g., 50-200 μL) of sterile PBS or a specific buffer (e.g., 25 mM sucrose solution or PBS).

[0052] Step 4: Storage The resuspended exosome suspension was aliquoted and stored at -80°C for a long period of time to avoid repeated freeze-thaw cycles.

[0053] PRP-exos observed under an electron microscope (TEM) Take 10 μL of exosome sample, drop it onto a copper grid, and let it stand at room temperature for 5 min. Then, blot away the liquid with filter paper. Add staining solution (uranyl acetate) and stain for 5 min, then blot away the liquid with filter paper. Wash 2-3 times with PBS, blot away excess liquid with filter paper, air dry, and observe under a transmission electron microscope.

[0054] Detection of specific marker molecules on the surface of PRP-exos using Western blotting The extracted PRP-exos were simply resuspended by pipetting at 4°C and centrifuged at 12,000 rpm for 10 min at 4°C. After centrifugation, the supernatant (total extracted protein) was collected and placed in a sterile tube and stored at -4°C.

[0055] The total protein extracted was analyzed to determine its concentration according to the instructions of the BCA Protein Quantitative Reagent Kit.

[0056] Add 1.2 ml of protein standard preparation solution to a sterile tube containing protein standard (30 mg BSA), dissolve it completely, and prepare a protein standard solution with a concentration of 25 mg / ml.

[0057] Dilute the prepared protein standard solution to a final concentration of 25 mg / ml.

[0058] Prepare BCA working solution by mixing reagents A and B in a 50:1 ratio at room temperature, ensuring thorough mixing.

[0059] The wells were pre-marked in the 96-well plate, and 0, 1, 2, 4, 8, 12, 16, and 20 μl of standard were added sequentially. Then, the wells were filled to 20 μl with standard diluent.

[0060] Add 10 μl of PRP-exos to a 96-well plate, and add 10 μl to 20 μl of standard dilution solution.

[0061] Add 200 μl of BCA working solution to each well at 37°C and let stand for 30 minutes.

[0062] The wavelength was set to 562 nm, and the absorbance of each well was measured using a microplate reader. A standard curve was plotted based on the measurement results, and the protein concentration of PRP-exos was calculated. Then, the protein was diluted according to the concentration requirements of the experimental design.

[0063] The SDS-PAGE related experiments on PRP-exos are as follows: 1) Electrophoresis Mix an appropriate amount of PRP-exos with an appropriate amount of 5X SDS-PAGE protein loading buffer, centrifuge, and then place in a boiling water bath for 8 minutes to allow the protein to denature fully. Cool at room temperature for later use.

[0064] Prepare SDS-PAGE gel.

[0065] Based on the protein concentration of PRP-exos, the optimal loading volume was determined to be 40 μg. PRP-exos and marker were added to the wells of the SDS-PAGE gel, followed by electrophoresis buffer. Electrophoresis was started after setting the voltages for the stacking and separating gels. Electrophoresis was stopped when bromophenol blue was observed reaching the bottom of the gel.

[0066] 2) Transfer After electrophoresis, the gel is removed, cut, and soaked in transfer buffer at room temperature for about 10 minutes.

[0067] Cut the PVDF membrane to resemble the cut electrophoresis gel, then soak it in methanol for 15 seconds and rinse it with distilled water for 5 minutes. Cut the filter paper in the same way to resemble the cut electrophoresis gel, then soak the cut filter paper in transfer buffer for at least 10 minutes.

[0068] Place the gel, PVDF membrane, filter paper, and sponge pad into the transfer device in sequence, then add an appropriate amount of transfer solution, set the current, and begin the transfer.

[0069] 3) Blocking Immediately after transfer, rinse the protein membrane in TBST for 2 minutes to remove as much transfer buffer as possible.

[0070] Remove the TBST washing solution, immediately add the blocking solution, and then place it on a shaker and shake slowly; block at room temperature for about 1 hour.

[0071] 4) Immune response Remove the blocking solution, immediately add the diluted primary antibody, and incubate at room temperature for about 2 hours.

[0072] Remove the primary antibody, wash for 10 minutes, and repeat 3 times.

[0073] Immediately after washing, add the diluted secondary antibody and incubate at room temperature for 2 hours.

[0074] After incubation, wash three more times, 10 minutes each time, and set aside.

[0075] Protein detection involves thoroughly washing the PVDF membrane after the immune reaction, drying it, immediately mixing it with ECL reagent, incubating it, exposing it to an image using a developing device, washing it with distilled water, and then further processing the image to obtain the corresponding grayscale value.

[0076] Western blot results analysis Based on the gray values ​​of PRP-exos and the corresponding internal references, the relative expression level of the target protein was calculated after error correction.

[0077] from Figures 1-3 As can be seen, PRP-Exos has been successfully extracted and its quality meets the experimental requirements.

[0078] B. Establishing animal models SNI model rats were established by anesthesia with sevoflurane and routine disinfection. The surgical area of ​​the left hind limb was prepared and disinfected, exposing the main trunk of the sciatic nerve. The sciatic nerve was ligated using No. 4 silk suture, tightly wrapping the nerve at the ligation site, taking care not to damage the neurovascular structures. After surgery, the skin and muscle layers were closed layer by layer. The rats were placed in a warm cage and housed routinely. Two weeks post-surgery, the model was considered successfully established if the rats exhibited symptoms such as weakness in walking, hind limb eversion, significant muscle atrophy, spontaneous paw lifting, narrowed interdigital distance, and lameness. This study has been approved by the Animal Ethics Committee of Zhu Xianyi Memorial Hospital, Tianjin Medical University (Approval No.: DXBYY-IACUC-2021047). Thirty SD rats were randomly divided into three groups (n=10 / group): blank group: only the sciatic nerve was exposed, without ligation, and no intervention was given; model group: starting from day 7 after SNI modeling, 0.9% saline (0.5 mL / time) was injected intrathecally every other day for a total of 10 times; PRP-Exos group: after SNI modeling, 0.5 mL of 200 mg / L PRP-Exos was injected intrathecally, with the same administration regimen as the model group.

[0079] Pain behavioral assessment: Comparison of preoperative MWT and TWL levels among the control group, model group, and PRP-Exos group showed no statistically significant differences (P>0.05) using ANOVA. Comparison of MWT and TWL levels 6 days after modeling among the control group, model group, and PRP-Exos group showed statistically significant differences (P<0.05) using ANOVA; compared with the control group, the MWT and TWL levels in the model group and PRP-Exos group were lower on day 6 after modeling (P<0.05). Comparison of MWT and TWL levels after 10 doses among the control group, model group, and PRP-Exos group showed statistically significant differences (P<0.05) using ANOVA; compared with the control group, the MWT and TWL levels in the model group and PRP-Exos group were lower after 10 doses (P<0.05); compared with the model group, the MWT and TWL levels in the PRP-Exos group were higher after 10 doses (P<0.05). The experimental results are as follows: Figure 4 As shown, specifically, the PRP-Exos group rats had significantly higher MWT and thermal pain threshold (TWL) than the model group, indicating that repeated administration of PRP-Exos can significantly improve the decrease in MWT and TWL caused by modeling and improve the nerve function of rats.

[0080] Example 2: Sciatic Nerve Staining Currently, toluidine blue staining is used. A 10mm section of the distal sciatic nerve from a SNI rat was taken, fixed in 4% paraformaldehyde for 24 hours, graded dehydration, clearing, and paraffin embedding to prepare 5μm sections. After dewaxing and hydration, the sections were stained with hematoxylin for 5 minutes, differentiated with hydrochloric acid and ethanol, stained with eosin for 3 minutes, and rinsed with running water. Graded ethanol dehydration, xylene clearing, and mounting with neutral resin were then performed. The nerve fiber arrangement, myelin sheath integrity, and inflammatory cell infiltration were observed under a 400× optical microscope.

[0081] Experimental results are as follows Figure 5 As shown, the toluidine blue staining results indicate that the blank group ( Figure 5 A) The sciatic nerve fibers are tightly arranged, the axons are regularly shaped, the myelin sheath is intact without vacuolar degeneration, the epineurium is continuous, and no inflammatory cell infiltration is observed; Model group ( Figure 5 B) Disordered nerve fiber structure, with Wallerian degeneration, axonal atrophy and breakage, myelin sheath disintegration forming vacuoles, Schwann cell proliferation, partial rupture of the outer membrane, and local inflammatory cell infiltration; PRP-Exos group ( Figure 5 C) The nerve fibers were arranged more regularly than those in the model group, the number of vacuoles was reduced, there were obvious signs of axonal regeneration, the myelin sheath structure was partially repaired, Schwann cells proliferated in an orderly manner, and inflammatory infiltration was reduced.

[0082] Example 3: Effects of PRP-Exos on the expression of TNF-α and IL-6 in the rat spinal cord 1. Sample processing: Tissue from the lumbar enlargement segment of the spinal cord of rats in each group was taken, weighed, and then a suitable amount of pre-cooled protein lysis buffer was added and homogenized thoroughly on ice.

[0083] Centrifuge the homogenate at 4°C and 12,000 rpm for 15 minutes, carefully aspirate the supernatant (i.e., the total protein solution), aliquot and store at -80°C for later use.

[0084] 2. Protein concentration determination: The concentration of all protein samples was determined using the BCA method and adjusted to the same concentration to ensure consistency in sample loading.

[0085] 3. ELISA test: Strictly follow the instructions for the rat TNF-α and IL-6 ELISA kit. The main steps are as follows: Sample addition: Add the standards to the wells after serial dilution, and set up replicates. Add the protein samples to be tested to the pre-coated microplate wells.

[0086] Incubation: Incubate at 37℃ for 90 minutes.

[0087] Add biotinylated antibody: Discard the liquid in the well, wash 3 times, add biotinylated antibody working solution, and incubate at 37°C for 60 minutes.

[0088] Add enzyme conjugate: Discard the liquid in the well, wash 3 times, add enzyme conjugate working solution, and incubate at 37°C in the dark for 30 minutes.

[0089] Add substrate: Discard the liquid in the well, wash 5 times, add substrate solution (TMB), and develop color at 37°C in the dark for 15-20 minutes.

[0090] Termination and measurement: Add the stop solution, mix gently, and immediately measure the absorbance (OD value) of each well at a wavelength of 450 nm using an ELISA reader.

[0091] 4. Data Processing: A bar chart was drawn based on the experimental results.

[0092] The levels of TNF-α and IL-6 in the spinal cord were compared among the control group, model group, and PRP-Exos group. Analysis of variance showed statistically significant differences (P<0.05). Compared with the control group, the levels of TNF-α and IL-6 in the model group and PRP-Exos group were increased (P<0.05); compared with the model group, the levels of TNF-α and IL-6 in the PRP-Exos group were decreased (P<0.05). The experimental results are shown in […]. Figure 6Specifically, the levels of inflammatory factors TNF-α and IL-6 in the spinal cord of rats in the PRP-Exos group were significantly lower than those in the model group, indicating that PRP-Exos can effectively inhibit spinal cord inflammation and improve neurological function.

[0093] Example 4: Effects of PRP-Exos on the expression of TLR4 / NF-κB pathway-related proteins in rat spinal cord tissue 1. Protein sample preparation: Tissue from the lumbar enlargement segment of the spinal cord of rats in each group was collected, and pre-cooled RIPA lysis buffer was added on ice and homogenized thoroughly.

[0094] Centrifuge at 4℃ and 12000 rpm for 15 minutes, and take the supernatant as the total protein solution.

[0095] Protein concentration was determined using the BCA method, and all sample concentrations were adjusted to be consistent using lysis buffer.

[0096] Add 5× SDS-PAGE protein loading buffer, mix well, and boil in a 100℃ metal bath for 10 minutes to fully denature the protein. Aliquot and store at -20℃.

[0097] 2. SDS-PAGE electrophoresis: Prepare a 10% or 12% separating gel and a 5% stacking gel.

[0098] The total protein load per well is 30-50 μg (determined based on preliminary experiments). Pre-stained protein markers are also added.

[0099] The initial voltage was 80V. After the bromophenol blue entered the separating gel, the voltage was changed to 120V until the bromophenol blue reached the bottom of the gel and electrophoresis was stopped.

[0100] 3. Transfer: Wet transfer was used. The PVDF membrane was activated with methanol for 15 seconds and then immersed in transfer buffer along with the gel and filter paper.

[0101] Assemble the "sandwich" structure (cathode-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-anode) and eliminate air bubbles.

[0102] Under ice bath conditions, a constant current of 300 mA was applied for 60-90 minutes for membrane transfer.

[0103] 4. Immune response: Sealing: After the transfer is complete, immerse the PVDF membrane in 5% skim milk (prepared by TBST) and seal it on a shaker at room temperature for 1 hour.

[0104] Primary antibody incubation: Dilute the primary antibody with TBST (TLR4, 1:1000; NF-κB p65, 1:1000; GAPDH, 1:5000). Incubate the membrane with the corresponding primary antibody overnight at 4°C on a shaker.

[0105] Wash the membrane: Wash the membrane 3 times with TBST, 10 minutes each time.

[0106] Secondary antibody incubation: Dilute the corresponding HRP-labeled secondary antibody with TBST (1:5000) and incubate on a shaker at room temperature for 1 hour.

[0107] Wash the membrane: Wash the membrane three more times with TBST, 10 minutes each time.

[0108] 5. Chemiluminescence and Development: Mix equal volumes of ECL chemiluminescence solution A and solution B, and drop the mixture onto the PVDF membrane. Allow the mixture to react for 1-2 minutes.

[0109] Images were acquired using a chemiluminescence imaging system, and bar charts were plotted based on the experimental results.

[0110] The relative expression levels of TLR4 / NF-κB pathway-related proteins in spinal cord tissues were compared among the control group, model group, and PRP-Exos group. Analysis of variance showed statistically significant differences (P<0.05). Compared with the control group, the relative expression levels of TLR4 and NF-κB p65 proteins were increased in both the model group and PRP-Exos group (P<0.05). Compared with the model group, the relative expression levels of TLR4 and NF-κB p65 proteins were decreased in the PRP-Exos group (P<0.05). The experimental results are as follows: Figure 7 , Figure 8 As shown, specifically, PRP-Exos treatment can reduce the expression of TLR4 and NF-κB p65, suggesting that PRP-Exos has anti-inflammatory or protective effects, and may alleviate spinal cord inflammation or injury progression by inhibiting the TLR4 / NF-κB signaling pathway.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. The application of PRP-Exos in the preparation of drugs for treating neuropathic pain, characterized in that, The PRP-Exos is used to inhibit the TLR4 / NF-κB pathway; The concentration of the drug is 50 mg / L-500 mg / L, and the dosage is 0.5 mL per administration.

2. The application of PRP-Exos according to claim 1 in the preparation of a medicament for treating neuropathic pain, characterized in that, The concentration of the drug is 200 mg / L.

3. The application of PRP-Exos according to claim 1 in the preparation of a medicament for treating neuropathic pain, characterized in that, The particle size of the PRP-Exos is between 50 nm and 150 nm; and the marker protein expressing the PRP-Exos exosomes is any one of CD63, TSG101 or CD9.

4. The application of PRP-Exos according to claim 1 in the preparation of a medicament for treating neuropathic pain, characterized in that, The neuropathic pain is caused by selective damage to the branches of the sciatic nerve.

5. A pharmaceutical composition, said pharmaceutical composition being used in any one of the applications described in claims 1-4, characterized in that, The pharmaceutical composition comprises an effective dose of PRP-Exos and a pharmaceutically acceptable carrier or excipient.

6. The pharmaceutical composition according to claim 5, characterized in that, The PRP-Exos are exosomes isolated, purified, and identified from platelet-rich plasma.

7. The pharmaceutical composition according to claim 5, characterized in that, The dosage forms of the pharmaceutical composition include injection, oral, and transdermal dosage forms.

8. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the pharmaceutical composition includes any one of tablets, capsules, oral liquids, injections, and powder injections.

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