Use of nsun3 in promoting peripheral nerve injury repair

Abstract

The application discloses application of NSUN3 in promoting peripheral nerve injury repair. The application adopts intermittent hypoxia induction to promote peripheral nerve injury repair, and for the first time finds that intermittent hypoxia promotes peripheral nerve injury repair by inducing protein expression of a key protein NSUN3. The application discloses that NSUN3 can be used as a brand-new treatment target, and provides a new research angle and theoretical basis for expanding clinical treatment of peripheral nerve injury.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of NSUN3 in promoting the repair of peripheral nerve injuries. Background Technology

[0002] Peripheral nerve injury (PNI) is one of the leading causes of disability worldwide, commonly occurring after trauma, birth paralysis, limb amputation, and tumor resection. As a populous country, my country has approximately 20 million people suffering from PNI, with about 2 million new cases each year, making it one of the countries with the largest number of PNI patients globally. However, the repair of PNI faces significant challenges. Unlike other tissues, nerve tissue repair cannot rely on scar tissue replacement; it must achieve structural and functional reconstruction through the nerve tissue's own regeneration. Currently, the common clinical treatment for PNI is epineurial or perineurial suture.

[0003] Currently, while commonly used epineurial or perineurial suture techniques can reconstruct nerve continuity, the functional recovery from nerve injury is not ideal. This is mainly related to two factors: first, insufficient precision in the anastomosis of nerve ends during nerve repair; and second, the complex neuro-distal effect interactions involved in nerve injury repair, including the synthesis of regeneration-related proteins in neurons and Schwann cells, as well as the initiation of neuronal apoptosis. These signals that promote and inhibit regeneration intertwine to form a complex dynamic network, significantly affecting the process of nerve repair. Therefore, in-depth exploration of the potential mechanisms of peripheral nerve injury repair and the search for new therapeutic targets and methods have significant scientific value and promising clinical applications. Summary of the Invention

[0004] In view of the shortcomings of existing technologies, this invention utilizes intermittent hypoxia induction to promote the repair of peripheral nerve injury, and for the first time discovers that intermittent hypoxia promotes peripheral nerve injury repair by inducing the protein expression of the key protein NSUN3. NSUN3 can serve as a novel therapeutic target, providing a theoretical basis for expanding new clinical treatment approaches for peripheral nerve injury.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides the use of reagents that enhance NSUN3 expression or function in the preparation of products that promote the repair of peripheral nerve injuries.

[0006] In this invention, NSUN3 is also known as MST077, COXPD48, MSTP077, and NOP2 / Sun RNA methyltransferase 3. NSUN3 includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed NSUN3, as well as any form of NSUN3 derived from cell-processed sources. The term encompasses naturally occurring variants of NSUN3 (e.g., splice variants or allelic variants). The term encompasses, for example, the NSUN3 gene, the NSUN3 protein, human NSUN3, and NSUN3 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in this invention, NSUN3 is a human gene with gene ID 63899.

[0007] In this invention, the term "peripheral nerve" refers to all nerves outside the brain and spinal cord, including ganglia, nerve trunks, nerve plexuses, and nerve terminals. Peripheral nerves can be divided into cranial nerves (connected to the brain) and spinal nerves (connected to the spinal cord) based on their location within the central nervous system. There are 12 pairs of cranial nerves and 31 pairs of spinal nerves. Peripheral nerves can also be divided into somatic nerves and visceral nerves based on their distribution areas. Somatic nerves are distributed to the body surface, bones, joints, and skeletal muscles, while visceral nerves are distributed to internal organs, cardiovascular systems, smooth muscles, and glands.

[0008] In this invention, peripheral nerve injury refers to sensory, motor, and nutritional disturbances in the area innervated by the nerve caused by various reasons. Causes of injury include traction injuries, lacerations, compression injuries, gunshot wounds, ischemic injuries, electrical burns, radiation burns, drug injection injuries, and other iatrogenic injuries.

[0009] In this invention, the term "repair" refers to the process by which the body repairs and restores function after nerves have been damaged by external factors, leading to functional impairments such as motor and sensory disturbances. Repair can completely or partially restore nerve function. In some embodiments, compared to not applying the active ingredient described in this invention (such as a reagent that enhances NSUN3 expression or function), the severity or duration of peripheral nerve injury in patients is reduced by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[0010] In an optional embodiment, the reagent that enhances NSUN3 expression or function is an overexpression vector containing the NSUN3 encoding gene.

[0011] In an optional embodiment, the reagent that enhances NSUN3 expression or function is used to inhibit neuronal senescence and / or promote neuronal proliferation.

[0012] In an optional embodiment, the nerve cells are nerve cells of peripheral nerves, including cranial nerves, spinal nerves, and visceral nerves.

[0013] Furthermore, the cranial nerves include the olfactory nerve, optic nerve, oculomotor nerve, trochlear nerve, trigeminal nerve, abducens nerve, facial nerve, vestibulocochlear nerve, glossopharyngeal nerve, vagus nerve, accessory nerve, and hypoglossal nerve.

[0014] Furthermore, the spinal nerves include cervical nerves, thoracic nerves, lumbar nerves, sacral nerves, and coccygeal nerves.

[0015] Furthermore, the visceral nerves include visceral motor nerves and visceral sensory nerves.

[0016] Furthermore, the peripheral nerve is the dorsal root ganglion.

[0017] In an optional embodiment, the agent that enhances NSUN3 expression or function promotes peripheral nerve injury repair through any one or any combination of the following: 1) Promotes upregulation of m5C methylation modification level; 2) Promotes upregulation of expression levels of neural repair factors NGF, BDNF and / or GDNF; 3) Promotes downregulation of expression levels of cell senescence markers p21 and p16, upregulation of expression levels of β-galactosidase and / or upregulation of expression levels of proliferation marker Lamin B1; 4) Promotes upregulation of expression levels of mitochondrial generation-related mRNAs such as Ak2, Fh1, Prc1 and Atp5f1b.

[0018] In optional embodiments, the product is selected from probe sets, primer sets, kits, chips, and test strips.

[0019] A second aspect of the present invention provides a method for promoting the repair of peripheral nerve injury in vitro without therapeutic purpose, the method comprising the steps of overexpressing NSUN3 in the injured nerve region or in the injured nerve cells.

[0020] Furthermore, the method includes constructing an overexpression vector containing the NSUN3 encoding gene, wherein the overexpression vector can achieve overexpression of NSUN3 in the damaged nerve region or damaged nerve cells; and introducing the overexpression vector into the damaged nerve region or damaged nerve cells to overexpress NSUN3 in the damaged nerve region or damaged nerve cells, thereby promoting the repair of peripheral nerve injury.

[0021] Furthermore, the methods of introduction include viral transduction, electroporation transfection, liposome delivery, polymer carriers, chemical carriers, lipid complexes, polymeric complexes, dendritic polymers, nanoparticles, natural endocytosis or phagocytosis pathways, cell-penetrating peptides, microinjection, microneedle delivery, and particle bombardment.

[0022] Furthermore, the overexpression vector containing the NSUN3 encoding gene is obtained by ligating the NSUN3 encoding gene with a plasmid vector.

[0023] Furthermore, the step of constructing an overexpression vector containing the NSUN3 encoding gene includes: using NSUN3 cDNA as a template, amplifying and cloning it into a plasmid vector, and then ligating it using DNA ligase.

[0024] Furthermore, the plasmid vector is selected from at least one of pLVX, pCDNA, pLV, pCMV, pBABE, and pTrip.

[0025] Furthermore, the plasmid vector is pLVX.

[0026] Advantages and beneficial effects of the present invention: 1) This invention uses intermittent hypoxia induction to promote the repair of peripheral nerve injury. It is simple and easy to implement, and provides an innovative treatment strategy with good clinical application prospects for the clinical treatment of peripheral nerve injury, promotion of nerve regeneration and functional recovery. It has good clinical translational significance.

[0027] 2) This invention proposes and demonstrates that intermittent hypoxia induction promotes peripheral nerve injury repair by regulating neuronal senescence and mitochondrial dysfunction. It also comprehensively explores the relationship between intermittent hypoxia induction, neuronal senescence, mitochondrial dysfunction, and the mechanism of peripheral nerve injury repair, providing a new research perspective on the molecular mechanism of peripheral nerve regeneration and repair.

[0028] 3) This invention elucidates the important role of upregulating NSUN3 protein expression in the repair of peripheral nerve injury induced by intermittent hypoxia, providing a theoretical basis for expanding new clinical treatment approaches for peripheral nerve injury. Attached Figure Description

[0029] Figure 1 The images show the results of intermittent hypoxia promoting DRG neuron proliferation and inhibiting neuronal senescence. A represents lactate concentration; B represents neuronal cell viability; CD represents ROS levels detected by the DCFH-DA probe; C represents representative fluorescence staining images; D represents statistical graphs of fluorescence staining results (scale bar: 50 µm); EF represents β-galactosidase staining analysis; E represents representative β-galactosidase staining images; F represents statistical graphs of β-galactosidase staining results; GH represents representative images of p21(G) and LaminB1(H) expression detected by immunofluorescence (scale bar: 100 µm).

[0030] Figure 2The results of intermittent hypoxia promoting lactate production and regulating nerve repair via NSUN3 are shown in Figure A, where A is a statistical graph of lactate concentration detection; BC is a dot blot analysis of m5C and MB, B is a representative graph of dot blot analysis, C is a statistical graph of dot blot analysis results; D is a heatmap analysis of the expression level of m5C-related enzymes; EG is a statistical graph of the results of RT-PCR analysis of nerve repair factors NGF (E), BDNF (F), and GDNF (G).

[0031] Figure 3 The images show the results of lactation modification of NSUN3 protein promoted by intermittent hypoxia. A is an immunohistochemical detection image (scale bar: 100 µm); BC shows the expression level of Pan Kla detected by Western blot (B is a representative image, C is a statistical image); DE shows the potential lactation modification sites of NSUN3 protein predicted by the CPLM database (D is the prediction score image, E is the potential lactation modification site of NSUN3); FG shows the results of immunoprecipitation analysis of NSUN3 protein showing lactation modification (F is a representative image of immunoprecipitation analysis, G is a statistical image).

[0032] Figure 4 The graphs show the NSUN3 overexpression transcriptome sequencing analysis, where A is a differential clustering circos heatmap; B is a differential gene volcano plot; and C is a Reactome enrichment analysis graph.

[0033] Figure 5 The results of intermittent hypoxia regulated neuronal senescence and mitochondrial dysfunction by NSUN3 are shown in the figure. AD represents Western blot detection and quantitative analysis of protein expression, A is a representative figure, and BD is a statistical graph of quantitative analysis of p16 (B), p21 (C), and Lamin B1 (D). E is a representative figure of mitochondria fluorescently labeled with Mito-Tracker, with a scale bar of 20 µm. FI is a statistical graph of changes in mitochondrial production-related mRNAs detected by RT-PCR, including Ak2 (F), Atp5f1b (G), Fh1 (H), and Prc1 (I).

[0034] Figure 6 The results of NSUN3 overexpression promoting the expression of neural repair factors in DRG neurons are shown in Figure A, where Western blot analysis shows representative images of BDNF, GDNF, and NGF protein expression; and quantitative analysis is shown in Statistical graph B.

[0035] Figure 7 The image shows the results of NSUN3 overexpression promoting axonal growth in DRG neurons. A is a representative image of Tuj1 immunofluorescence staining, with a scale bar of 50 µm; B is a statistical graph of quantitative analysis of axonal length.

[0036] Figure 8 The graph shows the results of NSUN3 overexpression inhibiting the expression of DRG neuronal aging markers. In the graph, A is a representative graph of p21 and p16 protein expression detected by Western blot; B is a statistical graph of quantitative analysis.

[0037] Figure 9 The image shows the results of NSUN3 overexpression promoting mitochondrial generation in DRG neurons. In the image, A is a representative image of Mito-Tracker fluorescent labeling, with a scale bar of 20 µm; B is a statistical graph of quantitative fluorescence intensity analysis.

[0038] Figure 10 The graph shows the expression results of mRNAs related to mitochondrial generation promoted by overexpression of NSUN3. A and D are statistical graphs of the expression levels of Ak2 (A), Fh1 (B), Prc1 (C), and Atp5f1b (D) detected by RT-PCR, respectively. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1 I. Experimental Materials

[0041] II. Experimental Design 1. Effects of intermittent hypoxia on damaged DRG neurons 1) Intermittent hypoxia induction treatment: Cells were cultured in a cell culture incubator with parameters of 37℃, 0.5% O2, 5% CO2, and N2 balance for intermittent hypoxia treatment (5 hours of hypoxia / 7 hours of normoxic). Damaged DRG neurons were subjected to intermittent hypoxia induction for different durations (0, 12, 24, 48, and 72 hours). Changes in lactate production levels in each group were detected using a lactate assay kit.

[0042] 2) Based on the experimental groups described above, the changes in cell viability in each group were detected using a CCK-8 assay kit. Subsequent experiments were conducted at appropriate time points (48 hours of intermittent hypoxia induction) based on cell viability.

[0043] 3) DRG neuron grouping and processing: CTL: control group, no processing.

[0044] ACR: Acrylamide treatment causes neuronal damage.

[0045] ACR+Hypoxia: Acrylamide treatment causes neuronal damage and induces intermittent hypoxia.

[0046] ACR+Hypoxia+2-DG: Acrylamide treatment caused neuronal damage + intermittent hypoxia induction + 2-DG (lactate production inhibitor) treatment.

[0047] 4) The generation of reactive oxygen species (ROS) in each group of cells was detected using the DCFH-DA probe kit according to the grouping in 3).

[0048] 5) The cellular senescence status of each group was detected by galactosidase staining according to the grouping in 3).

[0049] 6) The expression of aging marker p21 and proliferation marker Lamin B1 was detected using immunofluorescence according to the groups in 3).

[0050] 2. Effects of intermittent hypoxia on a rat model of peripheral nerve injury (PNI) 1) Establish a rat peripheral nerve injury model (PNI) and subject it to intermittent hypoxia induction treatment, with specific groupings: PNI: PNI model group.

[0051] PNI+Hypoxia: PNI+intermittent hypoxia treatment.

[0052] 2) Collect damaged nerve tissue from each group of animals and test the lactate level in the damaged nerve tissue using a lactate detection kit.

[0053] 3) Based on lactate levels, the samples were divided into a high lactate group (Lac... High >0.5 mmol / g prot) and low lactate group (Lac Low The total m5C methylation level in the two groups of nerve tissues was detected by dot blot assay (≤ 0.5 mmol / gprot), and the role of intermittent hypoxia-induced lactate upregulation in the m5C modification level of genes in nerve cells was explored.

[0054] 4) DRG neuron grouping and processing: Control: Do not process.

[0055] LA: Exogenous lactic acid (LA) treatment.

[0056] 5) The expression of major m5C modification-related methyltransferases (NSUN1~NSUN7, DNMT1~DNMT2), demethylases (ALYREF, YBX1), and recognition proteins (TET1~TET2) in the two groups of cells was detected by RT-PCR. The results showed that the expression of methyltransferase NSUN3 was upregulated by the greatest extent (approximately 8-fold) after exogenous lactate treatment.

[0057] 6) DRG neuron cell grouping: Control: Do not process.

[0058] ACR, ACR+Hypoxia: Same as above.

[0059] ACR+Hypoxia+sh-NSUN3: Acrylamide treatment caused neuronal damage + intermittent hypoxia induction + sh-NSUN3 knockdown of NSUN3 expression.

[0060] The expression of neural repair factors NGF, BDNF, and GDNF in cells of each group was detected by PCR.

[0061] 3. Lactic acidification modification of NSUN3 1) The high lactate group obtained in 2 (Lac) High >0.5 mmol / g prot) and low lactate group (Lac Low The expression of pan-lactation antibody (Pan Kla) was detected in neural tissue samples with a concentration of ≤ 0.5 mmol / gprot by immunohistochemistry.

[0062] 2) DRG neuron grouping: CTL, ACR, ACR+Hypoxia: Same as above.

[0063] Western blot analysis was used to detect Pan Kla expression in cells of each group, with Histone H3 as an internal control.

[0064] 3) Bioinformatics analysis of NSUN3 protein was performed using the CPLM database (https: / / cplm.biocuckoo.cn / index.php), and 8 potential lactation modification sites (K6, K8, K27, K67, K87, K111, K207, K319) were successfully predicted.

[0065] 4) DRG neuron grouping and processing: CTL, ACR, ACR+Hypoxia: Same as above.

[0066] Immunoprecipitation experiments were performed using anti-NSUN3 antibody. The extracted protein was then subjected to anti-Lactyllysine immunoblotting to analyze the lactation modification of the protein NSUN3.

[0067] 4. The role of NSUN3 in mitochondrial formation and cellular senescence 1) DRG neuron grouping and processing: NC: Negative control group.

[0068] sh-NSUN3: NSUN3 knockdown group.

[0069] 2) Western blot was used to detect changes in cell senescence markers p21 and p16 and proliferation marker Lamin B1, with GAPDH as an internal control.

[0070] 3) Mito-Tracker fluorescent labeling of mitochondria in each group of cells.

[0071] 4) PCR detection of changes in mitochondrial-related mRNAs (Ak2, Fh1, Prc1, Atp5f1b) in each group of cells.

[0072] III. Experimental Results 1. Intermittent hypoxia promotes lactate production, which in turn promotes the proliferation of DRG neurons in a concentration-dependent manner and inhibits neuronal cellular senescence.

[0073] Intermittent hypoxia induction at different durations (0, 12, 24, 48, 72 h) gradually increased lactate production levels in neurons with prolonged hypoxia. Figure 1 (A in the text), and regulates neuronal cell activity in a concentration-dependent manner ( Figure 1 (B in the original text). We chose to induce hypoxia for 48 hours for subsequent experiments. Further treatment of DRG neurons with the lactate production inhibitor 2-DG showed that intermittent hypoxia induction significantly suppressed the level of reactive oxygen species (ROS) in neurons (…). Figure 1 CD), reducing β-galactosidase (SA-βgal) expression ( Figure 1 EF), inhibiting the upregulation of the cell senescence marker p21 and promoting the expression of the cell proliferation marker Lamin B1 ( Figure 1 (GH in the sample). The results showed that the lactate inhibitor 2-DG could reverse the effects of intermittent hypoxia and significantly promote neuronal cellular senescence.

[0074] 2. m5C methyltransferase NSUN3 is an important regulatory factor that promotes lactate production and regulates nerve repair under intermittent hypoxia.

[0075] Compared with the PNI model group, the lactate concentration in the nerve tissue of the intermittent hypoxia treatment group was significantly increased. Figure 2 (A) Based on the different levels of lactate in the damaged nerve tissue, the samples were divided into a high lactate group (Lac) and a low lactate group (Lac). High >0.5 mmol / g prot) and low lactate group (Lac Low The total m5C methylation level in the two groups of neural tissues was detected by dot blot analysis, ≤ 0.5 mmol / gprot. The results showed that the m5C methylation level was significantly upregulated in neural tissues with high lactate levels (≤ 0.5 mmol / gprot). Figure 2 (BC in the text). Further RT-PCR analysis was used to analyze the expression of major m5C modification-related methyltransferases (NSUN1~NSUN7, DNMT1~DNMT2), demethylases (ALYREF, YBX1), and recognition proteins (TET1~TET2). The results showed that the expression of methyltransferase NSUN3 was upregulated the most significantly (approximately 8-fold) after exogenous lactate treatment. Figure 2 (D in the text). Knockdown of NSUN3 expression in neurons significantly reduced the levels of neural repair factors NGF, BDNF, and GDNF. Figure 2 (EG in the middle).

[0076] 3. Intermittent hypoxia induces upregulation of lactate production and promotes lactation modification of NSUN3 protein.

[0077] The above (Lac) was detected by immunohistochemical experiments. High >0.5 mmol / g prot) and low lactate group (Lac Low The expression of pan-lactic acidified Pan Kla in neural tissue (≤0.5 mmol / gprot) was investigated, and the results showed that the total lactation modification level was significantly upregulated in the high lactate group. Figure 3 A in the text. Next, we used Western blot to analyze PanKla expression in in vitro neurons, and the results showed that intermittent hypoxia induction significantly increased the level of total lactation modification (A). Figure 3 Based on the above findings, we innovatively utilized the CPLM database (https: / / cplm.biocuckoo.cn / index.php) to perform bioinformatics analysis on the NSUN3 protein, successfully predicting eight potential lactation modification sites (K6, K8, K27, K67, K87, K111, K207, K319). Figure 3 The DE in the text is missing. To further validate the prediction results, we performed immunoprecipitation with an NSUN3-specific antibody and detected it with a lactyllysine antibody. The results showed that hypoxia treatment significantly upregulated the lactylation level of NSUN3 protein. Figure 3 (FG in the middle).

[0078] 4. Lactic acidification of NSUN3 protein and cell senescence.

[0079] For the construction of the NSUN3 overexpression vector, the NSUN3 cDNA (the sequence of which can be obtained from NCBI, gene ID 63899) was amplified and cloned into the overexpression vector (pLVX), and ligated using T4 DNA ligase. DH5α competent cells were transformed, and positive clones were screened by plating on LB plates containing Amp / Kan. We overexpressed NSUN3 in DRG neurons and performed transcriptome sequencing analysis. Reactome pathway enrichment analysis revealed a significant enrichment of the cellular senescence pathway (…). Figure 4 This discovery is related to Figure 1 The results shown in the study, which indicated that intermittent hypoxia inhibited cellular senescence in damaged nerve cells, corroborate each other and further confirm the important role of cellular senescence in the NSUN3 regulatory network.

[0080] 5. Intermittent hypoxia regulates neuronal aging and mitochondrial dysfunction through NSUN3.

[0081] In a hypoxic DRG neuron model, we performed NSUN3 knockdown and detected the expression of cellular senescence markers p21 and p16, as well as the proliferation marker Lamin B1, using Western blot. The results showed that NSUN3 knockdown significantly upregulated the expression of p21 and p16, while inhibiting the expression of Lamin B1. Figure 5 (AD in the text). Subsequently, we combined Mito-Tracker fluorescent labeling technology to analyze mitochondrial generation and RT-PCR to analyze changes in mitochondrial generation-related mRNAs (Ak2, Fh1, Prc1, Atp5f1b). The results showed that NSUN3 knockdown significantly inhibited mitochondrial generation (AD in the text). Figure 5 (EI in the middle).

[0082] Example 2 This embodiment aims to verify the necessary extension of the technical effects in the aforementioned inhibition experiment (Example 1). Based on the dose-dependent relationship between gene expression levels and functional phenotypes in molecular biology, and combined with the symmetry of the same technical logic, those skilled in the art can reasonably expect that overexpression of NSUN3 should lead to a phenotype completely opposite to knockdown of NSUN3, namely, significantly promoting the expression of neural repair factors (BDNF, GDNF, NGF), promoting neuronal axon growth, inhibiting the expression of cellular senescence markers (p21, p16), promoting mitochondrial generation, and upregulating the expression of mitochondrial generation-related mRNAs (Ak2, Fh1, Prc1, Atp5f1b). This experiment uses the same detection method as described above, only supplementing the verification of the overexpression group, without introducing any new technical features.

[0083] I. Experimental Design 1. Construction of NSUN3 overexpression vector The NSUN3 cDNA (the sequence of which can be obtained from NCBI, gene ID 63899) was amplified and cloned into an overexpression vector (pLVX), and ligated using T4 DNA ligase. DH5α competent cells were transformed, and positive clones were screened by plating on LB plates containing Amp / Kan. Plasmid extraction and sequencing were performed to verify correctness, yielding the NSUN3 overexpression vector (pLVX-NSUN3). An empty plasmid (pLVX-empty) served as a negative control.

[0084] 2. DRG neuron grouping and transfection The primary cultured DRG neurons were divided into the following four groups for treatment: Control group: Normally cultured DRG neurons, without any treatment; ACR group: DRG neurons were treated with acrylamide (final concentration 50 μM) for 24 h, causing neuronal damage; ACR+empty Vector group: DRG neurons were transfected with empty vector plasmid (pLVX-empty) before acrylamide treatment (50 μM, 24 h); ACR+NSUN3 OE group: DRG neurons were transfected with NSUN3 overexpression vector (pLVX-NSUN3) before acrylamide treatment (50 μM, 24 h).

[0085] Transfection procedure: Using Lipofectamine 3000 transfection reagent, follow the instructions. Collect cells for subsequent detection 48 h after transfection.

[0086] 3. Western blot analysis of the protein expression levels of neural repair factors BDNF, GDNF, and NGF. Four groups of DRG neurons were collected, and total protein was extracted using RIPA lysis buffer. Protein quantification was performed using the BCA method. Equal amounts of protein were subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk powder for 1 h, and incubated overnight at 4°C with anti-BDNF antibody, anti-GDNF antibody, and anti-NGF antibody (all diluted 1:1000). The next day, HRP-labeled secondary antibody (1:5000) was added and incubated at room temperature for 1 h. ECL chemiluminescence imaging was performed, and grayscale analysis was performed using ImageJ software with GAPDH as an internal control.

[0087] 4. Immunofluorescence staining to detect the axonal growth length of DRG neurons The four groups of DRG neurons were seeded in confocal culture dishes coated with poly-L-lysine. After transfection and ACR treatment, the culture medium was discarded, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 10 min, blocked with 5% BSA for 1 h, and incubated overnight at 4°C with Anti-Tuj1 antibody (1:500). The next day, fluorescently labeled secondary antibody (1:1000) was added and incubated at room temperature in the dark for 1 h. The nuclei were counterstained with DAPI. Images were acquired using a confocal fluorescence microscope. At least 50 neurons were randomly selected from each well, and the axonal path was manually traced along the neurite direction using the NeuronJ plugin in ImageJ software. The length of the longest axon of each neuron was measured, and the average axonal length of each group was calculated.

[0088] 5. Western blot analysis of the expression levels of cellular senescence markers p21 and p16. The four groups of DRG neurons were collected and Western blot experiments were performed according to the method in step 3. Anti-P21 antibody (1:1000) and Anti-p16 antibody (1:500) were added respectively and incubated overnight at 4°C. GAPDH was used as an internal control to detect the protein expression levels of aging markers p21 and p16 in each group of cells.

[0089] 6. Mito-Tracker fluorescent labeling for the detection of mitochondrial formation The four groups of DRG neurons were seeded in confocal culture dishes. After transfection and ACR treatment, the culture medium was discarded, and serum-free medium containing Mito-Tracker probe (final concentration 200 nM) was added. The mixture was incubated at 37°C in the dark for 30 min. After washing three times with PBS, images were observed and acquired using a confocal fluorescence microscope. The excitation wavelength was 488 nm, and the emission wavelength was 516 nm. The mean fluorescence intensity of each group of cells was quantitatively analyzed using ImageJ software.

[0090] 7. RT-PCR detection of changes in the expression of mitochondrial production-related mRNAs Four groups of DRG neurons were collected, and total RNA was extracted using Trizol reagent. RNA concentration and purity were measured. RNA was reverse transcribed into cDNA using a reverse transcription kit. Real-time quantitative PCR was performed using SYBR Green qPCR Mix with cDNA as a template. Primer sequences were the same as in Example 1. GAPDH was used as an internal reference gene, and 2... -ΔΔCT The relative expression levels of each gene are calculated using this method.

[0091] II. Experimental Results 1. Overexpression of NSUN3 significantly promotes the protein expression of neural repair factors in DRG neurons. Western blot results showed that, compared with the control group, ACR injury treatment significantly reduced the protein expression levels of neural repair factors BDNF, GDNF, and NGF; while compared with transfection with the empty vector, transfection with the NSUN3 overexpression vector significantly increased the protein expression levels of BDNF, GDNF, and NGF, alleviating ACR damage to DRG neurons. Figure 6 (AB in the middle).

[0092] 2. Overexpression of NSUN3 significantly promotes axonal growth in DRG neurons. Immunofluorescence staining (Tuj1) analysis of DRG neuron axon growth length showed that, compared with the control group, ACR injury treatment significantly inhibited DRG neuron axon growth; while, compared with transfection with the empty vector, transfection with the NSUN3 overexpression vector significantly promoted DRG neuron axon growth and alleviated ACR-induced damage to DRG neuron cells. Figure 7 (AB in the middle).

[0093] 3. Overexpression of NSUN3 significantly inhibited the expression of aging markers in DRG neurons. Western blot results showed that, compared with the control group, ACR injury treatment significantly promoted the protein expression levels of aging markers p21 and p16; while compared with transfection with an empty vector, transfection with the NSUN3 overexpression vector significantly inhibited the expression levels of p21 and p16, alleviating the senescent state of DRG neurons induced by ACR injury. Figure 8 (AB in the middle).

[0094] 4. Overexpression of NSUN3 significantly enhances mitochondrial fluorescence intensity in DRG neurons. The results of the mitochondrial labeling experiment showed that, compared with the control group, ACR damage treatment significantly reduced the Mito-Tracker fluorescence intensity and inhibited mitochondrial generation; while, compared with transfection with the empty vector, transfection with the NSUN3 overexpression vector significantly increased the Mito-Tracker fluorescence intensity and promoted mitochondrial generation. Figure 9 (AB in the middle).

[0095] 5. Overexpression of NSUN3 significantly promotes the expression of mitochondrial synthesis-related mRNAs. RT-PCR results showed that, compared with the control group, ACR damage treatment significantly inhibited the expression of mitochondrial production-related mRNAs Ak2, Fh1, Prc1, and Atp5f1b; while compared with transfection with the empty vector, transfection with the NSUN3 overexpression vector significantly promoted the expression of Ak2, Fh1, Prc1, and Atp5f1b. Figure 10 (AD in the text).

[0096] This embodiment directly verified the key role of NSUN3 in the repair of peripheral nerve injury by constructing an NSUN3 overexpression vector and transfecting it into damaged DRG neurons. The experimental results showed that NSUN3 overexpression significantly: (1) upregulated the protein expression of nerve repair factors BDNF, GDNF, and NGF; (2) promoted the regeneration and growth of damaged neuronal axons; (3) inhibited the expression of cell senescence markers p21 and p16; and (4) enhanced mitochondrial fluorescence intensity and upregulated the expression of mitochondrial production-related mRNAs (Ak2, Fh1, Prc1, Atp5f1b). The above results are completely opposite to the NSUN3 knockdown (loss-of-function) phenotype in Example 1, which fully confirms the core regulatory role of NSUN3 in promoting the repair of peripheral nerve injury and provides direct and strong experimental evidence for NSUN3 as a clinical therapeutic target.

[0097] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of reagents that enhance NSUN3 expression or function in the preparation of products that promote the repair of peripheral nerve injuries.

2. The application according to claim 1, characterized in that, The reagent used to enhance NSUN3 expression or function is an overexpression vector containing the NSUN3 encoding gene.

3. The application according to claim 2, characterized in that, The reagents that enhance NSUN3 expression or function are used to inhibit neuronal senescence and / or promote neuronal proliferation.

4. The application according to claim 3, characterized in that, The nerve cells are nerve cells of the peripheral nerves, which include cranial nerves, spinal nerves, and visceral nerves.

5. The application according to claim 4, characterized in that, The peripheral nerve in question is the dorsal root ganglion.

6. The application according to claim 1, characterized in that, The reagent that enhances NSUN3 expression or function promotes peripheral nerve injury repair by any one or any combination of the following: 1) Promotes upregulation of m5C methylation modification levels; 2) Promotes the upregulation of expression levels of neural repair factors NGF, BDNF, and / or GDNF; 3) Promotes downregulation of cellular senescence markers p21 and p16, upregulation of β-galactosidase expression, and / or upregulation of proliferation marker Lamin B1 expression; 4) Promotes the upregulation of expression levels of mitochondrial-related mRNAs such as Ak2, Fh1, Prc1, and Atp5f1b.

7. The application according to claim 1, characterized in that, The products are selected from probe sets, primer sets, reagent kits, chips, and test strips.

8. A method for promoting the repair of peripheral nerve injury in vitro without therapeutic target, characterized in that, The steps of the method include overexpressing NSUN3 in the damaged nerve region or in the damaged nerve cells.

9. The method according to claim 8, characterized in that, The method includes constructing an overexpression vector containing the NSUN3 encoding gene, which can achieve overexpression of NSUN3 in damaged neural regions or damaged neural cells. The overexpression vector is introduced into the damaged nerve region or damaged nerve cells, so that NSUN3 is overexpressed in the damaged nerve region or damaged nerve cells, thereby promoting the repair of peripheral nerve damage.

10. The method according to claim 9, characterized in that, The overexpression vector containing the NSUN3 encoding gene is obtained by ligating the NSUN3 encoding gene with a plasmid vector.

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