Application of activin A in preparation of nerve regeneration medicine

Abstract

The invention discloses an application of activin A in preparation of a nerve regeneration medicine. The invention proposes and verifies that the activin A can promote the repair and regeneration of nerve injury through the synergistic regulation of Schwann cells and neurons for the first time, and provides a new direction for the treatment of diseases caused by nerve injury.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to the application of activin A in the preparation of nerve regeneration drugs. Background Technology

[0002] Peripheral nerve injury is a common disabling disease in clinical practice, often caused by factors such as trauma, traction, or compression. It can lead to permanent motor, sensory, and autonomic dysfunction, severely impacting patients' quality of life. Although the peripheral nervous system has a certain regenerative capacity compared to the central nervous system, functional recovery is still far from satisfactory for patients with large nerve defects, chronic injuries, or comorbidities.

[0003] The core reason for the failure of regeneration and repair after injury lies in the limited function of Schwann cells, which constitute the myelin sheath. After injury, Schwann cells undergo a reprogramming process similar to the epithelial-mesenchymal transition, transforming into a repair phenotype and constructing a supportive microenvironment for axonal regeneration. In this complex regulatory network, the transforming growth factor-β signaling pathway is a key regulatory hub. Activin A, an important member of the TGF-β superfamily, has been found to be significantly upregulated after nerve injury and can promote the proliferation and migration of Schwann cells. However, whether Activin A can further contribute to nerve regeneration remains unclear. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide an application of activin A in the preparation of neuroregeneration drugs through the synergistic regulation of Schwann cells and neurons.

[0005] Technical solution: The application of activator A described in this invention in the preparation of nerve regeneration drugs.

[0006] Preferably, the amino acid sequence of activin A in rats is shown in UniProtKB_P18331, and its encoding gene sequence is shown in NCBI Reference Sequence: NM_017128.2.

[0007] Preferably, the application is in the preparation of a treatment for diseases caused by nerve damage.

[0008] Preferably, the diseases caused by nerve damage include diseases caused by physical and / or physiological damage to the peripheral nerves.

[0009] Preferably, the drug uses recombinant activin A protein or its derivatives as the active ingredient; more preferably, the derivatives include pharmaceutically acceptable C-terminal and / or N-terminal and / or side chain and / or intermediate residue modifications of recombinant activin A protein, or salts, solvates, or hydrates of recombinant activin A protein; even more preferably, the C-terminal and / or N-terminal and / or side chain and / or intermediate residue modifications are any one or more of acylation, amidation, alkylation, esterification, glycosylation, and phosphorylation.

[0010] Preferably, the drug has an activator A expression enhancer as its active ingredient; more preferably, the activator A expression enhancer is an overexpression vector containing an activator A encoding gene expression cassette; even more preferably, the overexpression vector is a viral vector or a non-viral vector.

[0011] Preferably, the dosage form of the drug includes tablets, capsules, granules, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to propose and verify that activin A can promote the repair and regeneration of nerve damage through the synergistic regulation of Schwann cells and neurons, providing a new direction for the treatment of diseases caused by nerve damage. Attached Figure Description

[0013] Figure 1 The image shows the results of neuronal neurite length detection after co-culture with Schwann cells. A is a representative image with a scale bar of 100 μm, and B is a statistical graph of relative total axonal length. Figure 2 The image shows the effect of recombinant Activin A protein on Schwann cell migration after sciatic nerve injury. A is a representative image with a scale bar of 1000 μm, and B is a statistical graph of relative fluorescence intensity. Figure 3 The image shows the effect of recombinant Activin A protein on the regeneration of damaged nerves after sciatic nerve injury. A is a representative image with a scale bar of 1000 μm, and B is a statistical graph of the average axonal regeneration length. Detailed Implementation

[0014] The technical solution of the present invention will be further described below.

[0015] Example 1: Activin A for the co-culture of Schwann cells and neurons 1. Isolation, culture and processing of primary Schwann cells The sciatic nerve of newborn SD rats was collected, and after removing the vascular membrane, collagenase I at a concentration of 3 mg / mL was used to digest the nerve at 37°C for 30 min. After removing the collagenase, 0.25% trypsin was added and digested at 37°C for 8 min. Then, 3 mL of DMEM complete culture medium was added to terminate the digestion.

[0016] Centrifuge at 800 rpm for 5 min, discard the supernatant, and wash twice with DMEM complete medium at 1×10⁻⁶. 6 Cells were seeded at a density of 10 cells / mL in poly-L-lysine (PLL)-coated culture dishes and cultured at 37°C in a 5% CO2 incubator for 24 h. Then, the medium was replaced with DMEM complete medium containing 10 mM cytarabine (Sigma-Aldrich, catalog number C1768) and cultured for another 36 h. After washing twice with PBS, the medium was replaced with complete medium containing 2 µM forskolin (Sigma-Aldrich, catalog number F6886) and 10 ng / mL HRG (R&D Systems, catalog number 377-HB). The medium was changed every 2 days. After the cells reached confluence, Schwann cell purification was performed.

[0017] After purification, the cells were digested, centrifuged, and resuspended to obtain a cell suspension. 0.1% volume of anti-thymidine 1.1 (Sigma-Aldrich, catalog number M7898) was added, and the cells were incubated on ice for 2 h. After centrifugation and discarding the supernatant, a mixture of 250 µL rabbit complement (One Lambda, catalog number CABC-1D) and 750 µL DMEM culture medium was added, and the cells were incubated at 37°C for 45 min. After incubation, the cells were washed twice with DMEM complete medium at a concentration of 1×10⁻⁶. 6 Cells were seeded at a density of 10 cells / mL in PLL-coated culture dishes and cultured at 37°C in a 5% CO2 incubator for 24 h. The culture medium was then replaced with DMEM complete medium containing 2 µM forsokolin and 10 ng / mL HRG. The medium was changed every 2 days. Cell purification was completed when the cells had filled the culture dishes.

[0018] Bovine serum albumin (BSA) solution was used to prepare Activin A recombinant protein solution (purchased from Abcam, catalog number ab151687). The solution was added to Schwann cell culture medium for pretreatment for 24 h. The final concentration of Activin A recombinant protein was 5 ng / mL, and the final concentration of BSA was 0.1% w / v. The control group was pretreated with only 0.1% w / v BSA.

[0019] 2. Co-culture of Schwann cells and neurons based on a microfluidic system Dorsal root ganglion (DRG) tissues were collected from 8-week-old adult SD rats. After segmentation, the cells were digested with type I collagenase at 3 mg / mL for 1.5 h, followed by digestion with 0.25% trypsin for 10 min. Then, 15% BSA solution was added to obtain a cell suspension. After centrifugation at 900 rpm for 5 min, a precipitate containing DRG neurons was obtained. The precipitate was seeded in poly-L-lysine-coated culture dishes and cultured in Neurobasal A medium containing B27 and L-glutamine to obtain adult rat DRG neurons.

[0020] A dual-compartment microfluidic chip (Xona 2-compartment SND 150, catalog number SND150) was used, pre-coated with 3 mL of 0.1 mg / mL PLL. Adult rat DRG neurons were seeded on the left side of the dual-compartment microfluidic chip, while the pre-treated Schwann cells were seeded on the right side.

[0021] DRG neurons were cultured to allow their axons to grow into the axonal chambers through the microgroove barrier, thus achieving direct contact between the axons and Schwann cells. Axons co-cultured for 3 days on the right side of the dual-chamber microfluidic chip were fixed with 4% paraformaldehyde and then incubated overnight at 4°C with a 1:1000 dilution of Tuj1 primary antibody (purchased from Abcam, catalog number ab18207). After incubation, the cells were washed with PBS buffer and then incubated with a 1:400 dilution of the fluorescent secondary antibody Alexa Fluor. TM Incubate at room temperature in the dark using 488 (purchased from Invitrogen, catalog number A-21206), rinse after incubation, stain with Hoechst 33342 (CAS No.: 23491-52-3), rinse and mount, then observe and acquire images using a fluorescence microscope.

[0022] The results are as follows Figure 1 As shown, neurons co-cultured with Schwann cells pretreated with BSA exhibited longer neurite lengths than those co-cultured with Schwann cells pretreated with Activin A recombinant protein, indicating that Schwann cells treated with Activin A promote neuronal neurite growth.

[0023] Example 2: Treatment of a rat model of sciatic nerve clipping with activator A Eight-week-old male SD rats weighing 180-220 g were anesthetized and their skin prepared. The sciatic nerve was exposed using surgical instruments. A 3 mm pinch was made on the left hind limb at the bifurcation of the tibial and common nerves, 1 cm above the sciatic nerve, for 30 seconds, to establish a pinch injury model. Subsequently, a microsyringe was used to inject Matrix-Gel... TMActivin A recombinant protein (purchased from Beyotime, catalog number C0376) at a final concentration of 1000 ng / mL or an equal volume of 0.1% w / v BSA was injected into the epineurium of the pressure injury site. The injection volume was 5 μL per animal, and 3 animals were treated for each method.

[0024] Sciatic nerve tissue was collected 3 days after sciatic nerve injury. After freezing and sectioning, it was blocked with immunostaining blocking solution for 50 min, and then incubated overnight at 4°C with either 1:500 diluted SCG10 primary antibody (Novus, catalog number NBP1-49461) or 1:500 diluted S100 primary antibody (Abcam, catalog number ab52642). After incubation, the tissue was washed with PBS buffer, followed by incubation with 1:400 diluted Alexa Fluor secondary antibody. TM 488 (purchased from Invitrogen, item number A-21206) was incubated at room temperature in the dark. After incubation, the slides were rinsed, mounted, and observed and images were acquired using a fluorescence microscope.

[0025] The migration of Schwann cells after sciatic nerve contusion is as follows: Figure 2 As shown, Schwann cells migrate from both ends of the injury site towards each other. The S100 fluorescence intensity in the Activin A recombinant protein treatment group was higher than that in the control group, indicating that Activin A promotes the migration of Schwann cells after sciatic nerve injury. The regeneration of damaged nerves after sciatic nerve contusion is as follows: Figure 3 As shown, the damaged nerve itself has a certain regenerative capacity. At the same time, compared with the control group, the regenerated nerve length labeled with SCG10 in the Activin A recombinant protein treatment group was longer, indicating that Activin A promotes the regeneration of damaged axons after sciatic nerve injury.

Claims

1. The application of activin A in the preparation of nerve regeneration drugs.

2. The application according to claim 1, characterized in that, The application is in the preparation of drugs for the treatment of diseases caused by nerve damage.

3. The application according to claim 2, characterized in that, The diseases caused by nerve damage include those caused by physical and / or physiological damage to the peripheral nerves.

4. The application according to any one of claims 1 to 3, characterized in that, The drug uses recombinant activin A protein or its derivatives as its active ingredient.

5. The application according to claim 4, characterized in that, The derivatives include pharmaceutically acceptable C-terminal and / or N-terminal and / or side chain and / or intermediate residue modifications of activin A recombinant protein, or salts, solvates, and hydrates of activin A recombinant protein.

6. The application according to claim 5, characterized in that, The modification of the C-terminus and / or N-terminus and / or side chain and / or intermediate residues is any one or more of acylation, amidation, alkylation, esterification, glycosylation, and phosphorylation.

7. The application according to any one of claims 1 to 3, characterized in that, The drug uses an activator A expression enhancer as its active ingredient.

8. The application according to claim 7, characterized in that, The activin A expression enhancer is an overexpression vector containing an activin A encoding gene expression cassette.

9. The application according to claim 8, characterized in that, The overexpression vector can be a viral vector or a non-viral vector.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

Images