Traditional Chinese medicine compound composition with nerve repairing effect, cell culture medium, traditional Chinese medicine-neural stem cell exosome compound and application

Abstract

The invention provides a traditional Chinese medicine compound composition with a nerve repairing effect, a cell culture medium, a traditional Chinese medicine-neural stem cell exosome compound and application, and belongs to the technical field of biological medicine. Based on the basic theory of traditional Chinese medicine and the combined theory of traditional Chinese medicine and western medicine, the traditional Chinese medicine compound composition is provided and comprises gastrodia elata exosomes, astragalus membranaceus exosomes and resveratrol, the traditional Chinese medicine compound composition is prepared into a cell culture medium, neural stem cells are cultured through the cell culture medium, and the neural stem cells are obtained. Therefore, the traditional Chinese medicine-neural stem cell exosome compound with an efficient nerve repairing function is prepared. The traditional Chinese medicine-neural stem cell exosome compound achieves the purpose of comprehensively repairing nerve injury by precisely targeting nerve cells, promoting neurogenesis, repairing the function of a neural circuit and reducing the inflammatory effect, and can be used for preparing related medical instruments and drugs for nerve repair.

Description

A traditional Chinese medicine compound composition with nerve repair effects, a cell culture medium, a traditional Chinese medicine-neural stem cell exosome complex, and their applications. Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a traditional Chinese medicine compound composition with nerve repair function, cell culture medium, and traditional Chinese medicine-neural stem cell exosome complex and its application. Background Technology

[0002] Nerve injury refers to damage to the structure and function of nerve tissue caused by factors such as brain diseases (e.g., stroke, brain tumor), cardiovascular diseases (e.g., cerebral vascular occlusion or hemorrhage), and trauma (e.g., craniocerebral trauma, spinal cord injury). It is one of the common and serious sequelae of these primary diseases. Nerve injury often leads to impairments in motor, sensory, and cognitive functions, severely affecting patients' quality of life. Due to the extremely limited regenerative capacity of nerve tissue and the complexity of its repair mechanisms, current treatment methods have limited effectiveness and constitute a challenge in clinical treatment. Currently, drugs used to treat nerve injury mainly include neurotrophic factors, antioxidants, blood flow-promoting drugs, and anti-inflammatory drugs, which mainly work by reducing nerve cell apoptosis, inhibiting inflammatory responses, improving local circulation, and promoting nerve regeneration. However, these treatments have significant limitations, such as single target, unstable efficacy, significant side effects, and difficulty in crossing the blood-brain barrier to effectively reach the lesion area. Therefore, the treatment of nerve injury still faces many challenges, and there is an urgent need for more efficient and safer new treatment strategies.

[0003] The advantage of traditional Chinese medicine (TCM) in nerve repair lies in its multi-target and holistic regulatory effects, which can simultaneously promote neuroprotection, anti-inflammation, and regeneration processes, thereby comprehensively enhancing the nerve repair effect. Gastrodia elata is a precious TCM used to treat neurological diseases such as stroke, headache, and dizziness. Modern research has confirmed that its main active ingredients, such as gastrodin, have multiple pharmacological effects, including antioxidant, anti-inflammatory, inhibition of nerve cell apoptosis, promotion of nerve regeneration, and improvement of cerebral blood flow, effectively alleviating the pathological processes related to nerve damage. Astragalus membranaceus is rich in polysaccharides and saponins (mainly astragaloside A), which can synergistically exert multiple pharmacological effects such as antioxidant, anti-inflammatory, axonal regeneration promotion, and neuroprotection, thereby comprehensively enhancing nerve repair capabilities. Astragalus membranaceus, Gastrodia elata, and other TCMs have advantages such as multi-target effects, low toxicity, and high safety, and have shown good efficacy in various nerve injuries and neurodegenerative diseases, making them a promising natural neuroprotective drug. However, TCMs generally suffer from low biocompatibility and low bioavailability of their active ingredients, leading to difficulties in their application in nerve repair. Summary of the Invention

[0004] This invention provides a traditional Chinese medicine compound composition with nerve repair function, a cell culture medium, a traditional Chinese medicine-neural stem cell exosome complex, and its application, which can significantly improve the bioactivity, targetability, and bioavailability of traditional Chinese medicine exosomes.

[0005] This invention provides a traditional Chinese medicine compound composition, comprising traditional Chinese medicine exosomes and resveratrol; the traditional Chinese medicine exosomes include gastrodia elata exosomes and astragalus membranaceus exosomes.

[0006] In one specific embodiment of the present invention, the protein concentration ratio of Gastrodia elata exosomes and Astragalus membranaceus exosomes in the herbal exosomes is 1~5:5~4; the concentration of resveratrol is 1~200 mM, and it is fused with the herbal exosomes in a volume ratio of 1~10:1~10.

[0007] The present invention also provides a cell culture medium, which is based on serum-free cell culture medium and includes the above-mentioned traditional Chinese medicine compound composition.

[0008] In one specific embodiment of the present invention, the amount of the traditional Chinese medicine compound composition added to the cell culture medium is 1% to 15% by mass percentage.

[0009] The present invention also provides a method for preparing the above-mentioned cell culture medium, comprising the following steps: mixing Gastrodia elata exosomes and Astragalus membranaceus exosomes to form traditional Chinese medicine exosomes; diluting the traditional Chinese medicine exosomes with serum-free cell culture medium to obtain an exosome solution; dissolving resveratrol in 0.1% DMSO solution to prepare a resveratrol solution; and mixing the exosome solution and the resveratrol solution to obtain a serum-free cell culture medium containing a traditional Chinese medicine compound composition.

[0010] In one specific embodiment of the present invention, the protein concentration in the exosome solution is ≥100 μg / mL; and the concentration of the resveratrol solution is 1~200 mM.

[0011] In one specific embodiment of the present invention, the volume ratio of the exosome solution to the resveratrol solution is 1~10:1~10.

[0012] The present invention also provides a traditional Chinese medicine-neural stem cell exosome complex formed by seeding neural stem cells into the above-mentioned cell culture medium.

[0013] In one specific embodiment of the present invention, the neural stem cells are obtained by induction and differentiation of human induced pluripotent stem cells.

[0014] This invention also provides the application of the above-mentioned traditional Chinese medicine-neural stem cell exosome complex in the preparation of medical devices and pharmaceuticals for repairing nerve damage.

[0015] Beneficial Effects: This invention, based on the fundamental theories of Traditional Chinese Medicine (TCM) and the integration of TCM and Western medicine, innovatively utilizes nanotechnology and neural cell biotransformation methods to prepare a TCM-neural stem cell exosome complex with highly efficient nerve repair functions. Active ingredients from TCM, including Gastrodia elata and Astragalus membranaceus, are encapsulated within exosomes. These exosomes can rapidly penetrate the blood-brain barrier membrane, and through multi-target and precise regulation, they achieve neuroprotection and promote nerve self-repair. In the embodiments of this invention, experiments have demonstrated that the TCM-neural stem cell exosome complex comprehensively repairs nerve damage by precisely targeting nerve cells, promoting neurogenesis, repairing neural circuit function, and reducing inflammatory effects. This can be used to prepare nerve repair-related dietary supplements, medical devices, and pharmaceuticals. Detailed Implementation

[0016] This invention provides a traditional Chinese medicine compound composition, comprising traditional Chinese medicine exosomes and resveratrol; the traditional Chinese medicine exosomes include gastrodia elata exosomes and astragalus membranaceus exosomes.

[0017] The exosomes of traditional Chinese medicine described in this invention are extracted from fresh, mature medicinal plants. For example, in this embodiment, three-year-old Astragalus membranaceus and two-year-old Gastrodia elata are selected. After harvesting, the Astragalus membranaceus and Gastrodia elata are washed with running water to remove surface soil, and then rinsed with sterile water. The fresh Gastrodia elata is sliced, dried at low temperature, and then pulverized. The exosomes are extracted under low-temperature conditions (≤ 4℃) throughout the process to better protect the active macromolecular components, such as nucleic acids and proteins. The processed Astragalus membranaceus is pulverized into a fine powder and then extracted at low temperature.

[0018] This invention does not specifically limit the extraction method for the exosomes from the traditional Chinese medicine. Conventional extraction methods in the art can be used, provided that the exosome particle size is ≤200 nm, the protein concentration (as determined by BCA) is 10–100 μg / mL, and the zeta potential range is -15 to -30 mV. In one embodiment of this invention, a combination of centrifugation and tangential ultrafiltration is used to extract the exosomes, and the extraction methods for Gastrodia elata exosomes and Astragalus membranaceus exosomes are the same.

[0019] In one embodiment of the present invention, fresh Astragalus membranaceus is pulverized into a fine powder and stirred evenly with 10 times its volume of PBS buffer at a stirring rate of 10,000 rpm for 5 minutes to obtain a homogeneous suspension. The obtained homogeneous suspension is then filtered, including sequential gauze filtration and centrifugal filtration. The centrifugation force is 3000×g to filter large particle precipitates, followed by a second filtration at 8000×g. The supernatant is collected and filtered through a 0.22 μm filter membrane. After filtration, TFF ultrafiltration is performed using a 300 kDa MWCO membrane, and the retentate is enriched with exosome-like vesicles.

[0020] The protein concentration ratio of Gastrodia elata exosomes and Astragalus membranaceus exosomes in the exosomes of traditional Chinese medicine described in this invention is 1~5:5~1; the resveratrol is fused with the exosomes of traditional Chinese medicine in a volume ratio of 1~10:1~10.

[0021] The present invention also provides a cell culture medium, which is based on serum-free cell culture medium and includes the above-mentioned traditional Chinese medicine compound composition.

[0022] This invention does not specifically limit the type of serum-free cell culture medium, including commonly used cell culture media such as Rohto AOF medium, Thermo Fisher's CTS series, and StemPro® NSC SFM medium. In one embodiment of this invention, Rohto AOF medium is used as an example, but it should not be considered as representing the entire scope of protection of this invention.

[0023] Before use, the cell culture medium described in this invention needs to undergo microbial, mycoplasma, and endotoxin testing to ensure negative colony and mycoplasma tests and an endotoxin content of <0.5 EU / mL. The traditional Chinese medicine compound composition in the cell culture medium of this invention is negative for both colony and mycoplasma tests.

[0024] The present invention also provides a method for preparing the above-mentioned cell culture medium, comprising the following steps: mixing Gastrodia elata exosomes and Astragalus membranaceus exosomes to form traditional Chinese medicine exosomes; diluting the traditional Chinese medicine exosomes with serum-free cell culture medium to obtain an exosome solution; dissolving resveratrol in 0.1% DMSO solution to prepare a resveratrol solution; and mixing the exosome solution and the resveratrol solution to obtain a serum-free cell culture medium containing a traditional Chinese medicine compound composition.

[0025] This invention mixes Gastrodia elata exosomes and Astragalus membranaceus exosomes, and dilutes the protein concentration to 100 μg / mL with serum-free cell culture medium. Resveratrol is separately dissolved in 0.1% DMSO solution, adjusting the concentration to 1-200 mM, such as 1 mM, 10 mM, 20 mM, 50 mM, 100 mM, and 200 mM. The concentrations of resveratrol solutions shown in this invention are exemplary concentrations and can be any concentration between any two. One embodiment uses 100 mM as an example, but this should not be considered as the entire scope of protection of this invention. The traditional Chinese medicine exosomes and resveratrol solution (1:1, v / v) of this invention are used to prepare a serum-free cell culture medium containing traditional Chinese medicine compound exosomes. When diluting the traditional Chinese medicine exosomes using the serum-free cell culture medium, in one embodiment, premixing is performed on a shaker at 37°C and 50 rpm for 4 h.

[0026] The present invention also provides a traditional Chinese medicine-neural stem cell exosome complex formed by seeding neural stem cells into the above-mentioned cell culture medium.

[0027] The neural stem cells described in this invention are derived from human induced pluripotent stem cells through induced differentiation, and to ensure cell viability, only neural stem cells from passages 3 to 5 are collected for use. In this invention, the pH of the cell culture medium is adjusted to neutral, neural stem cells are inoculated, and static culture is performed at 37 °C with a CO2 concentration of 5% for 48 hours.

[0028] In this invention, after static culture, the supernatant is centrifuged twice, with a centrifugal force of 3000×g for 10 min each time, to remove precipitate. Then, the supernatant is centrifuged again at 8000×g for 10 min to remove precipitate. The supernatant is then subjected to tangential flow ultrafiltration to obtain a component with a particle size ≤200 nm, yielding a traditional Chinese medicine-neural stem cell exosome complex.

[0029] The traditional Chinese medicine exosomes and the traditional Chinese medicine-neural stem cell exosome complex described in this invention contain small molecule active ingredients such as gastrodin and astragaloside A, as well as active macromolecules such as nucleic acids, proteins, and neural cell targeting proteins. Furthermore, the traditional Chinese medicine exosomes undergo neural stem cell biotransformation treatment, resulting in increased bioactivity, stronger targeting of nerve cells, and enhanced biocompatibility, thus achieving a highly efficient effect in repairing nerve damage.

[0030] The traditional Chinese medicine-neural stem cell exosome complex of the present invention can be preserved in the form of lyophilized powder. It can be lyophilized by adding a cryoprotectant to the traditional Chinese medicine-neural stem cell exosome complex. The cryoprotectant includes trehalose, glycerol, sodium chloride, sodium ganglioside, and human serum albumin. The lyophilization temperature is -40°C and the time is 24 h.

[0031] This invention also provides the application of the above-mentioned traditional Chinese medicine-neural stem cell exosome complex in the preparation of medical devices and pharmaceuticals for repairing nerve damage.

[0032] The traditional Chinese medicine-neural stem cell exosome complex described in this invention can significantly repair nerve damage caused by diseases such as stroke, traumatic brain injury, neurodegenerative diseases, and diabetes by precisely targeting nerve cells, promoting neurogenesis, repairing neural circuit function, and reducing inflammatory effects. It can also be used to prepare medical devices and drugs related to nerve repair.

[0033] The present invention does not have any particular limitation on the dosage form of the drug. The traditional Chinese medicine-neural stem cell exosome complex can be prepared into lyophilized powder injection, hydrogel injection, nebulized inhalation, oral capsule, oral coated tablet, oral granule or oral liquid preparation, and the administration method can also be various, such as local injection, intravenous injection, nebulization and oral administration.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a traditional Chinese medicine compound composition with nerve repair function, a cell culture medium, a traditional Chinese medicine-neural stem cell exosome complex, and their applications, but these should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1: Preparation of a traditional Chinese medicine-neural stem cell exosome complex (Ga-NSC-EX) with nerve repair function (1) Freshly collected Gastrodia elata was pulverized and made into fine powder. PBS buffer was added at 10 times the volume and stirred thoroughly. The resulting mixture was placed in a homogenizer and homogenized at 10,000 rpm for 5 min to obtain a uniform Gastrodia elata suspension. The suspension was first filtered through gauze to remove coarse impurities; then centrifuged at 3000×g and 8000×g to remove large precipitates stepwise. The supernatant was finally collected and filtered through a 0.22μm filter membrane for sterilization. The supernatant was concentrated and purified by tangential flow ultrafiltration (TFF) to retain components with a particle size less than 200 nm, obtaining Gastrodia elata exosomes. Astragalus membranaceus exosomes were prepared using the same method.

[0036] (2) Gastrodia elata and Astragalus membranaceus exosomes were mixed at a protein concentration of 1:1, and the protein concentration was diluted to 100 μg / mL with serum-free DMEM / F12 cell culture medium. Resveratrol was dissolved in 0.1% DMSO solution and the concentration was adjusted to 100 mM. Cell culture medium containing Chinese medicine compound exosomes and resveratrol solution (1:1, v / v) was prepared. The culture medium was placed on a shaker and gently premixed at 37 ℃ and 50 rpm for 4 h. The activated neural stem cells were transferred to the culture medium containing Chinese medicine exosomes and cultured statically at 37 ℃ and 5% CO2 for 48 h. The supernatant was centrifuged twice. After centrifugation at 3000×g for 10 min, the precipitate was removed. The supernatant was then centrifuged at 8000×g for 10 min to remove the precipitate. The supernatant was tangentially ultrafiltered to remove particles with a particle size ≤200 nm to obtain the Chinese medicine-neural stem cell exosome complex.

[0037] The morphology of the samples was observed under a transmission electron microscope. The zeta potential range, particle size, and particle number of the samples were analyzed using a Zetasizer Nano ZS dynamic light scattering and nanoparticle tracking analyzer. The protein concentration of the samples was determined using a BCA kit.

[0038] The results showed that the traditional Chinese medicine-neural stem cell exosome complex exhibited a cup-shaped structure under transmission electron microscopy, consistent with the characteristics of exosomes. Its zeta potential ranged from -20.3 ± 3.65 mV. The median particle size was 175.7839 nm, and the particle concentration was 2.9 × 10⁻⁶. 10 The concentration of the protein was 11.05 μg / mL.

[0039] Application Example 1: Examination of the Effect of Brain Nerve Repair after Traumatic Brain Injury 1. Experimental Animals and Grouping: Seventy-two healthy adult male SPF-grade SD rats, weighing 250±20 g, were randomly divided into 6 groups of 12 rats each. The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL / kg) and fixed in a stereotaxic apparatus. The rats were connected to a ventilator, and after their breathing stabilized, they were routinely disinfected and shaved to expose the bone window. A small animal cranial slapper was used to simulate moderate cortical injury, thus establishing a traumatic brain injury (TBI) model. The blank control group only had the bone window exposed without slapping. After successful model establishment, the drug was administered once daily for 14 consecutive days.

[0040] Blank control group: sham surgery without nerve damage, intracranial injection of 1 μL saline; TBI model group: intracranial injection of 1 μL saline; Low-dose group of the traditional Chinese medicine-neural stem cell exosome complex described in Example 1: intracranial injection of 1 μL, 1×10 9 particles / rat; Medium-dose group of the traditional Chinese medicine-neural stem cell exosome complex described in Example 1: Intracranial injection of 1 μL, 1×10 10 particles / rat; High-dose group of the traditional Chinese medicine-neural stem cell exosome complex described in Example 1: Intracranial injection of 1 μL, 1×10 11 particles / rat; Edaravone group: 3 mL intraperitoneal injection, 3 mg / kg rat.

[0041] 2. Detection indicators and methods (1) Neurological function score (postoperative days 1, 7 and 14) mNSS score (modified neurological severity score): assesses motor, sensory, reflex and balance functions, with a score of 0-18. The higher the score, the more severe the damage.

[0042] Water maze test (day 14 post-surgery): Record the time spent in the target quadrant and the number of times the platform was crossed to assess learning and memory abilities.

[0043] (2) Detection of inflammatory factors (ELISA method): Brain tissue homogenate supernatant was collected, and changes in IL-1β were detected using a mouse interleukin-1β (IL-1β) ELISA kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., Shanghai). Changes in TNF were detected using a mouse tumor necrosis factor-α (TNF-α) kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., Shanghai). After incubating the samples with reagents, free components were washed away, and the OD value was measured at 450 nm using an ELISA reader. The color intensity was directly proportional to the concentration of the analyte in the sample.

[0044] (3) Pathway protein detection: The expression levels of PI3K, AKT, and mTOR proteins in brain tissue were detected by Western blotting, with β-Actin and GAPDH as a combined internal control. Brain tissue was ground with liquid nitrogen, and total protein was extracted using RIPA lysis buffer containing inhibitors. Quantification was performed using the BCA method. After denaturation of an equal volume of protein, separation was performed using 10% SDS-PAGE, followed by wet transfer to a PVDF membrane (AKT / PI3K: 300mA, 70min; mTOR: 350mA, 120min). After transfer, the membrane was blocked with 5% BSA at room temperature for 1 hour. Subsequently, the membrane was incubated overnight at 4°C with the corresponding primary antibodies (rabbit anti-PI3K, AKT, mTOR, and GAPDH antibodies, diluted according to the manufacturer's recommended ratio). After washing with TBST, the membrane was incubated with the corresponding HRP-labeled secondary antibody at room temperature for 1 hour. Finally, the membrane was visualized on a chemiluminescence imaging system using ECL chemiluminescence substrate. ImageJ software was used to analyze the gray values ​​of the target band and the GAPDH band, and the relative expression level was expressed as the gray value ratio of the target protein to GAPDH.

[0045] (4) Proteomic and transcriptomic analysis: Rat brain tissue was rinsed with pre-cooled PBS, blotted dry, and flash-frozen in liquid nitrogen within 5 minutes. It was then stored in RNase-free cryovials. The processed brain tissue samples were sent to BGI Genomics for proteomic and transcriptomic analysis.

[0046] 3. Experimental Results: The traditional Chinese medicine-neural stem cell exosome complex showed a significant dose-dependent improvement effect on the recovery of neurological function in rats with nerve injury. The results are shown in Table 1. The model group's mNSS score remained at a high level (9.5±1.0) for 14 days post-surgery, indicating persistent neurological deficit. However, the exosome treatment group showed significant improvement as early as day 7, especially the high-dose group, where the score decreased from 12.6±1.3 to 3.8±0.5 (P<0.01), an improvement of 70%, significantly better than the edaravone group (5.5±0.6). This result indicates that the exosome complex can accelerate the recovery of neurological function, its effect is manifested in the early stage of injury, and the high-dose group has the best effect.

[0047] Table 1 Comparison of mNSS scores among rats in each group

[0048] The water maze test showed that exosome treatment significantly improved spatial learning and memory abilities, as shown in Table 2. The target quadrant dwell time in the high-dose group (25.2±2.0 s) was close to the normal level (28.5±2.1 s), significantly better than the model group (12.3±1.5 s). Nissl staining further confirmed the neuroprotective effect of exosomes; the number of Nissl-positive neurons in the high-dose group (39.1±3.0 neurons / field of view) recovered to 86.5% of normal, and the morphological integrity of neurons was significantly better than that in the edaravone group (30.8±2.7 neurons / field of view). These results indicate that exosome complexes not only promote neuronal survival but also effectively improve cognitive function.

[0049] Table 2 Comparison of the number of Nissl positive neurons in the water maze experiment

[0050] The results are shown in Table 3. Exosome treatment significantly inhibited the neuroinflammatory response. The high-dose group showed a 50.2% and 48.4% decrease in IL-1β (22.7±2.5 pg / mL) and TNF-α (30.1±3.0 pg / mL) levels compared to the model group, exhibiting a dose-dependent effect. Simultaneously, PI3K / AKT / mTOR pathway protein expression analysis revealed that the high-dose exosome group significantly activated this pathway (PI3K / GAPDH: 0.84±0.07; mTOR / GAPDH: 0.80±0.07), reaching 1.35 times that of the edaravone group. This molecular-level result reveals a dual mechanism by which exosomes exert their therapeutic effect by regulating inflammatory signaling pathways and promoting neuroregeneration-related pathways.

[0051] Table 3 Comparison of inflammatory factors and protein expression levels

[0052] Transcriptomic and proteomic results indicated that the high-dose exosome group possesses potential functions in regulating neural repair, mitochondrial function, and DNA damage. Transcriptomic results showed that, compared to the model group, the high-dose exosome group contained 96 differentially expressed miRNAs, of which 53 were upregulated and 43 were downregulated. Combined GO enrichment and KEGG pathway analysis revealed that the differentially expressed miRNAs were enriched in pathways related to nucleotide excision repair, oxidative phosphorylation, p53 signaling, neurogenesis, neuronal differentiation, Wnt signaling, PI3K-Akt signaling, and wound healing.

[0053] Proteomic results showed that, compared with the model group, the high-dose exosome group contained 286 differentially expressed proteins, of which 226 proteins were upregulated and 60 proteins were downregulated. Combined GO enrichment analysis and KEGG pathway analysis revealed that the differentially expressed proteins were enriched in areas including neural development, receptor internalization in the classical Wnt signaling pathway, the p53 signaling pathway, oxidative phosphorylation pathway, extracellular matrix structural components, neurotrophic factor signaling pathway, and the PI3K-Akt signaling pathway.

[0054] Experiment Example 2: Examination of the Repair Effect after Sciatic Nerve Injury 1. Experimental Animals and Grouping: Seventy-two healthy male SD rats (200-250g) were randomly divided into 6 groups of 12 rats each. After anesthesia with intraperitoneal injection of sodium pentobarbital (30mg / kg), the right sciatic nerve was exposed, and a 10mm defect was created above the bifurcation point. The blank control group only exposed the nerve. After successful model establishment, the drug was administered once daily for 28 days.

[0055] Blank control group: Sham surgery did not cause nerve damage; 1 μL of normal saline was injected around the exposed nerve. Model group: 1 μL of normal saline was injected around the exposed nerve. Low-dose group of traditional Chinese medicine-neural stem cell exosome complex: 1 μL of 1×10⁻⁶ m²⁻¹ was injected around the exposed nerve. 9 particles / rat; Medium-dose group of traditional Chinese medicine-neural stem cell exosome complex: 1 μL injected around the nerve exposure, 1×10 10 particles / rat; High-dose group of traditional Chinese medicine-neural stem cell exosome complex: 1 μL injected around the nerve exposure, 1×10 11 particles / rat; Edaravone group: 3 mL intraperitoneal injection, 3 mg / kg rat.

[0056] 2. Detection Indicators and Methods 2.1 Behavioral Evaluation (Postoperative Days 1, 7, 14, and 28) Behavioral evaluations (0-20) were conducted on postoperative days 1, 7, 14, and 28. The rat model was assessed for neurological repair behavior by comprehensively evaluating mechanical pain, thermal pain, catwalk gait analysis, and the sciatic nerve function index. Higher scores indicated better recovery of motor function.

[0057] 2.2 Histological and Immunofluorescence Analysis: Sciatic nerve tissue from rats was harvested 28 days post-surgery and prepared into frozen sections. Hematoxylin and eosin (H&E) staining was used to observe the structure of the damaged area. Immunofluorescence was used to label neurons (NeuN), astrocytes (GFAP), and axons (NF-200). Mitotracker Red was used to label mitochondria in exosomes, and their uptake in neurons was observed. Image analysis software was used to quantify neuronal density and glial scar area.

[0058] 2.3 Biochemical Factor Detection: Using the same method, rat sciatic nerve tissue was homogenized thoroughly with the kit's lysis buffer. After high-speed centrifugation, the supernatant was passed through a gDNA removal column to remove genomic DNA. The resulting filtrate was adjusted for binding conditions with anhydrous ethanol and then transferred to an RNA adsorption column to specifically bind RNA to the membrane. The adsorption column was thoroughly washed with protein removal buffer and rinsing buffer to remove impurities. Finally, an appropriate amount of RNase-free water was added to the center of the adsorption membrane, followed by centrifugation and elution to obtain high-purity total RNA. The RNA concentration and purity were measured at A260 / A280 using a micro-spectrophotometer. cDNA was synthesized using a reverse transcription kit containing gDNA removal functionality. Specific primers were designed for the target genes (TNF-α, IL-6, BDNF, NT-3) and the internal reference gene (GAPDH) (see Table 4). A qPCR reaction system was prepared by mixing the cDNA template, specific primers, and SYBR Green premix. Three c replicates were set for each sample, and a template-free control was included. After amplification using a real-time quantitative PCR instrument, the normalized expression level of the target gene relative to the internal reference gene is calculated.

[0059] Weighed rat sciatic nerve tissue was collected and homogenized thoroughly on ice with pre-chilled RIPA lysis buffer containing protease and phosphatase inhibitors. The homogenate was incubated on ice for 30 minutes, with intermittent vortexing. Subsequently, it was centrifuged at 12,000×g for 15 minutes at 4°C, and the supernatant was carefully aspirated to obtain the total protein sample. The entire extraction process must be carried out at low temperatures, and the obtained protein solution should be immediately quantified using the BCA method (BCA protein concentration assay kit, Shanghai Beyotime Biotechnology Co., Ltd.). The rat total protein sample was mixed with loading buffer and boiled for denaturation. 20-50 μg of the sample was loaded and separated by electrophoresis on a 12-15% SDS-PAGE gel. The protein was then transferred to a PVDF membrane using a wet transfer method and blocked with 5% skim milk or BSA at room temperature for 1 hour. Subsequently, the membrane was incubated overnight at 4°C with specific primary antibodies (rabbit anti-LC3, P62, Bax, Bcl-2, and internal control GAPDH antibodies). After washing with TBST, it was incubated with HRP-labeled secondary antibody at room temperature for 1 hour. Finally, it was developed on an imaging system using ECL chemiluminescent substrate. The LC3-II / I ratio, P62 level, and Bax / Bcl-2 ratio were calculated by analyzing the gray values ​​of the target band and the internal control band. Table 4 shows the rat qPCR primers.

[0060] 3. Analysis of Detection Indicators and Experimental Results 3.1 Analysis of Behavioral Detection Results During the 28-day functional assessment, the traditional Chinese medicine-neural stem cell exosome treatment group showed a significant dose-dependent recovery advantage. The high-dose group achieved a score of 15.7±1.1 points at 28 days, significantly better than the edaravone positive control group's 11.9±1.0 points and the model group's 5.3±0.7 points. The high-dose exosome group showed the fastest recovery rate in the mid-recovery phase (7-14 days), with the score jumping from 6.3 points to 11.2 points. It maintained a stable improvement trend in the later stage (14-28 days), ultimately achieving a recovery level close to functional walking ability. The medium- and low-dose groups achieved scores of 12.4 and 8.2 points, respectively, fully demonstrating the significant efficacy advantage of this therapy in promoting nerve regeneration and functional recovery.

[0061] Table 5 Comparison of Behavioral Scores

[0062] 3.2 Immunofluorescence analysis of neural tissue showed that the traditional Chinese medicine-neural stem cell exosome treatment group exhibited a significant dose-dependent repair effect. Histological analysis showed that the neuronal density in the high-dose exosome group was 950±71 cells / mm². 2 The cell count recovered to 76% of the control group, which was significantly better than the edaravone group (680±58 cells / mm). 2 Meanwhile, the area of ​​glial scars (11.3±1.4%) was reduced by 70.8% compared with the model group (38.7±3.2%), and the fluorescence intensity of the axonal regeneration marker NF-200 (136±13) reached 87.7% of the normal level. Mechanistic studies have shown that exosomes can improve energy metabolism by delivering functional mitochondria to damaged neurons, thereby synergistically promoting neuronal protection, inhibiting glial scar formation, and axonal structural reconstruction.

[0063] Table 5 Comparison of neural tissue states

[0064] 3.3 Biochemical Factor Analysis Molecular-level analysis showed that traditional Chinese medicine-neural stem cell exosomes significantly improved the microenvironment after spinal cord injury through a multi-target mechanism. Regarding inflammation regulation, the high-dose exosome group reduced TNF-α mRNA expression from 3.85±0.32 in the model group to 1.23±0.12 (a reduction of 68.1%), which was superior to the edaravone group (2.14±0.21); simultaneously, it promoted the expression of the neurotrophic factor BDNF to 1.14±0.10, exceeding the level in the control group. The LC3II / I ratio, a mitophagy marker, increased to 1.48±0.11, suggesting that it functions through a synergistic anti-inflammatory and pro-autophagy pathway.

[0065] Table 6 Comparison of the number of biochemical factors

[0066] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine compound composition, characterized in that, It includes exosomes of traditional Chinese medicine and resveratrol; the exosomes of traditional Chinese medicine include exosomes of Gastrodia elata and exosomes of Astragalus membranaceus.

2. The traditional Chinese medicine compound composition according to claim 1, characterized in that, The protein concentration ratio of Gastrodia elata exosomes to Astragalus membranaceus exosomes in the herbal exosomes is 1~5:5~1; the resveratrol concentration is adjusted to 1~200 mM and fused with the herbal exosomes in a volume ratio of 1~10:1~10.

3. A cell culture medium, characterized in that, The base culture medium is serum-free cell culture medium, and it also includes the traditional Chinese medicine compound composition as described in claim 1 or 2.

4. The cell culture medium according to claim 3, characterized in that, The amount of the traditional Chinese medicine compound composition added to the cell culture medium is 1% to 15% by mass percentage.

5. The method for preparing the cell culture medium according to claim 3 or 4, characterized in that, Includes the following steps: Gastrodia elata exosomes and Astragalus membranaceus exosomes were mixed to form traditional Chinese medicine exosomes. The traditional Chinese medicine exosomes were diluted with serum-free cell culture medium to obtain an exosome solution. Resveratrol was dissolved in 0.1% DMSO solution to prepare a resveratrol solution. The exosome solution and the resveratrol solution were mixed to obtain a serum-free cell culture medium containing the traditional Chinese medicine compound composition.

6. The preparation method according to claim 5, characterized in that, The protein concentration in the exosome solution is ≥100 μg / mL; the concentration of the resveratrol solution is 1~200 mM.

7. The preparation method according to claim 6, characterized in that, The volume ratio of the exosome solution to the resveratrol solution is 1~10:1~10.

8. A traditional Chinese medicine-neural stem cell exosome complex formed after inoculating neural stem cells into the cell culture medium of claim 3 or 4.

9. The traditional Chinese medicine-neural stem cell exosome complex according to claim 8, characterized in that, The neural stem cells were obtained by induction and differentiation of human induced pluripotent stem cells.

10. The application of the traditional Chinese medicine-neural stem cell exosome complex according to claim 8 or 9 in the preparation of medical devices and pharmaceuticals for repairing nerve damage.

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