Application of NSUN3 in promotion of peripheral nerve injury repair

Abstract

The invention discloses an application of NSUN3 in promotion of peripheral nerve injury repair. Intermittent hypoxia induction is adopted to promote peripheral nerve injury repair, and it is found for the first time that intermittent hypoxia affects peripheral nerve injury repair by inducing lactylation of key protein NSUN3. The NSUN3 disclosed by the invention can be used as a brand-new treatment target, and a new research angle and a theoretical basis are provided for expanding clinical treatment of peripheral nerve injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of NSUN3 in promoting the repair of peripheral nerve damage. Background Art

[0002] Peripheral nerve injury (PNI) is one of the most disabling conditions worldwide, commonly occurring in situations such as trauma, obstetrical paralysis, limb amputation, and tumor resection. However, repairing peripheral nerve injury presents significant challenges. Unlike other tissues, nerve tissue repair cannot rely on scar tissue replacement but must instead regenerate itself to reconstruct its structure and function. Currently, epineurial or perineurial suturing is a common clinical approach to treat peripheral nerve injury.

[0003] Currently, the commonly used epineurial or perineurial suturing in clinical practice can restore nerve continuity, but the effect of restoring nerve function after nerve injury is not ideal. This is mainly related to two factors: first, the lack of precision in the anastomosis of the broken ends during nerve repair; second, nerve injury repair involves complex nerve-distal effect interactions, including the synthesis of regeneration-related proteins in neurons and Schwann cells and the initiation of neuronal apoptosis. These signals that promote and inhibit regeneration are interwoven into 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 important scientific value and clinical application prospects. Summary of the Invention

[0004] In light of the shortcomings of existing technologies, this study utilizes intermittent hypoxia to promote the repair of peripheral nerve injury. The study also demonstrates for the first time that intermittent hypoxia influences peripheral nerve repair by inducing lactic acidification of the key protein NSUN3. NSUN3 could serve as a novel therapeutic target, providing a theoretical basis for developing new clinical treatment approaches for peripheral nerve injury.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides an application of methyltransferase NSUN3, which includes any of the following: 1) for preparing a product for promoting peripheral nerve injury repair; 2) for promoting peripheral nerve injury repair for non-therapeutic purposes in vitro.

[0007] In the present invention, NSUN3, also known as MST077, COXPD48, MSTP077, and NOP2 / Sun RNA methyltransferase 3, encompasses wild-type, mutant forms, or fragments thereof. The term encompasses full-length, unprocessed NSUN3, as well as any form of NSUN3 derived from cellular processing. The term also encompasses naturally occurring variants of NSUN3 (e.g., splice variants or allelic variants). For example, the term encompasses the NSUN3 gene, 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 the present invention, NSUN3 is a human gene with Gene ID 63899.

[0008] For the purposes of this invention, the term "peripheral nerves" refers to all nerves outside the brain and spinal cord, including ganglia, nerve trunks, nerve plexuses, and nerve terminal devices. Peripheral nerves can be divided into cranial nerves connected to the brain and spinal nerves connected to the spinal cord, depending on where they connect to 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, depending on where they are distributed. Somatic nerves are distributed to the body surface, bones, joints, and skeletal muscles, while visceral nerves are distributed to the internal organs, cardiovascular system, smooth muscles, and glands.

[0009] In this context, peripheral nerve injury refers to sensory, motor, and nutritional disorders in the area innervated by the nerves due to various causes. Causes of injury include traction injury, cutting injury, compression injury, firearm injury, ischemic injury, electrical burns, radiation burns, drug injection injury, and other iatrogenic injuries.

[0010] In the present invention, the term "repair" refers to the process by which the body repairs and restores nerve function after nerve damage caused by external factors, resulting in motor, sensory, or other dysfunction. Repair can fully or partially restore nerve function. In some embodiments, the severity or duration of peripheral nerve damage in a patient is reduced by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to a patient who had not been administered an active ingredient of the present invention (e.g., an agent that enhances NSUN3 expression or function).

[0011] Furthermore, the product includes an agent that enhances the expression or function of NSUN3 and / or an agent that enhances the level of NSUN3 lactylation.

[0012] Furthermore, the product is selected from probe sets, primer sets, kits, chips, test strips, high-throughput sequencing, systems, equipment, and devices.

[0013] Furthermore, the reagents for enhancing NSUN3 expression or function include overexpression vectors containing NSUN3 encoding genes, NSUN3 mimics, and NSUN3 agomir.

[0014] In the context of the present invention, "mimics" refers to small, double-stranded miRNAs designed and synthesized to target the mature form of a miRNA targeting a specific gene. They act similarly to the mature miRNA, upregulating the target gene's level in cells. "Agomirs" refer to chemically modified RNA molecules that have the same sequence as the target miRNA and are engineered to have enhanced stability and transfection efficiency, thereby overexpressing the target gene. Given the identifier of a specific gene (i.e., its gene ID, such as NSUN3 in the present invention, whose gene ID is 63899), those skilled in the art are able to search for miRNAs that may regulate the gene using bioinformatics databases (e.g., miRBase, TargetScan, miRanda, etc.). They can then design and synthesize mimics and agomirs that are compatible with regulating the target gene's function using online websites or bioinformatics software (e.g., ClustalW). Furthermore, they are able to add modifying groups to appropriate positions within the mimic and agomir sequences based on the principles and experience of chemical modification.

[0015] Furthermore, the agent that enhances the expression or function of NSUN3 and / or the agent that enhances the lactylation level of NSUN3 is used to inhibit neural cell aging and / or promote neural cell proliferation.

[0016] Furthermore, the nerve cells are nerve cells of peripheral nerves, and the peripheral nerves include cranial nerves, spinal nerves, and visceral nerves.

[0017] 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.

[0018] Furthermore, the spinal nerves include cervical nerves, thoracic nerves, lumbar nerves, sacral nerves, and coccygeal nerves.

[0019] Furthermore, the visceral nerves include visceral motor nerves and visceral sensory nerves.

[0020] Furthermore, the peripheral nerve is a dorsal root ganglion.

[0021] Furthermore, the agent that enhances the expression or function of NSUN3 and / or the agent that enhances the lactation level of NSUN3 promotes the repair of peripheral nerve injury by any one of the following or any combination thereof:

[0022] 1) Promote the upregulation of m5C methylation modification levels; 2) Promote the upregulation of the expression levels of neural repair factors NGF, BDNF and / or GDNF; 3) Promote the downregulation of the expression levels of cell senescence markers p21 and p16, the upregulation of the expression level of β-galactosidase and / or the upregulation of the proliferation marker Lamin B1; 4) Promote the upregulation of the expression levels of mitochondrial generation-related mRNAs such as Ak2, Fh1, Prc1, and Atp5f1b.

[0023] In some embodiments, the neural repair factor can be nerve growth factor (NGF), brain-derived nerve growth factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 / 5 (NT4 / 5), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), insulin-like growth factor (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), monosialotetrahexosylganglioside sodium (GM-1), etc.

[0024] In some embodiments, markers promoting cell senescence can be β-galactosidase, p21, p16, p53 and phospho-p53, senescence-associated secretory phenotype (SASP), retinoblastoma protein (Rb) and phospho-Rb, gamma-H2A.X, 53BP1, Ki-67, etc.

[0025] In some embodiments, the proliferation marker can be Ki-67 nuclear protein, PCNA, EDU, BrdU, Cyclins, and phosphorylated forms of H3 histones, among others.

[0026] In some embodiments, the mitochondrial biogenesis-related mRNA can be Ak2, Fh1, Prc1, Atp5f1b, Tfam, COXIV, TOM20, TIM23, HSP60, ND1, ND4, ATP6, etc.

[0027] A second aspect of the present invention provides a composition for promoting the repair of peripheral nerve damage, wherein the composition comprises an agent that enhances the expression or function of NSUN3 and / or an agent that enhances the lactylation level of NSUN3.

[0028] As used herein, the term "composition" refers to a composition comprising at least one biologically active compound (e.g., an agent for enhancing NSUN3 expression or function as described herein). The compositions of the present invention can be administered orally, parenterally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The compositions of the present invention may contain any commonly used non-toxic, pharmaceutically acceptable carriers, excipients, or vehicles. In some cases, pharmaceutically acceptable acids, bases, or buffers may be used to adjust the pH of the formulation to enhance the stability of the formulated compound or its dosage form. Parenteral administration, as used herein, includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may be administered to the recipient by any route that reaches the target tissue.

[0029] Furthermore, the reagents for enhancing NSUN3 expression or function include overexpression vectors containing NSUN3 encoding genes, NSUN3 mimics, and NSUN3 agomir.

[0030] Furthermore, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0031] In the present invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies that are harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions can include fluids such as water, saline, glycerol and ethanol. Auxiliary substances such as wetting agents or emulsifiers, pH buffering substances, etc. may also be present in such vehicles. "Pharmaceutically acceptable excipients" are substances that have been reasonably evaluated for safety and are included in pharmaceutical preparations in addition to the active ingredient. In addition to excipients, acting as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of drugs.

[0032] Furthermore, the pharmaceutically acceptable carrier or excipient includes a buffer, a stabilizer, a preservative, an osmotic pressure regulator, a diluent, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a surfactant, a coating material, a colorant, an antioxidant or an antibacterial agent.

[0033] The third aspect of the present invention provides an in vitro method for promoting the repair of peripheral nerve damage for non-therapeutic purposes, the method comprising overexpressing NSUN3 in damaged nerve regions or damaged nerve cells.

[0034] Furthermore, the method includes the steps of constructing an overexpression vector containing the NSUN3 encoding gene, wherein the overexpression vector can achieve overexpression of NSUN3 in the damaged nerve area or damaged nerve cells; introducing the overexpression vector into the damaged nerve area or damaged nerve cells, so that NSUN3 is overexpressed in the damaged nerve area or damaged nerve cells, thereby promoting the repair of peripheral nerve damage.

[0035] Furthermore, the introduction method includes viral transduction, electroporation transfection, liposome delivery, polymer carriers, chemical carriers, lipid complexes, polymer complexes, dendrimers, nanoparticles, natural endocytosis or phagocytosis pathways, cell penetrating peptides, microinjection, microneedle delivery, and particle bombardment.

[0036] Furthermore, the overexpression vector containing the NSUN3 encoding gene is obtained by connecting the NSUN3 encoding gene with a plasmid vector.

[0037] Furthermore, the step of constructing an overexpression vector containing the NSUN3 coding gene includes: using NSUN3 cDNA as a template, amplifying and cloning it into a plasmid vector, and connecting it with DNA ligase.

[0038] Furthermore, the plasmid vector is selected from at least one of pLVX, pCDNA, pLV, pCMV, pBABE, and pTrip.

[0039] Furthermore, the plasmid vector is pLVX.

[0040] Advantages and beneficial effects of the present invention:

[0041] 1) The present invention uses intermittent hypoxia induction to promote the repair of peripheral nerve injury, which is simple and easy to implement. It provides an innovative therapeutic strategy with good clinical application prospects for the clinical treatment of peripheral nerve injury, promoting nerve regeneration and functional recovery, and has good clinical translational significance.

[0042] 2) This study proposes and demonstrates that intermittent hypoxia promotes peripheral nerve repair by regulating neuronal aging and mitochondrial dysfunction. This study comprehensively explores the relationship between intermittent hypoxia, neuronal aging, mitochondrial dysfunction, and the mechanisms of peripheral nerve repair, providing a new perspective on the molecular mechanisms of peripheral nerve regeneration and repair.

[0043] 3) This study proposes and clarifies for the first time the important role of NSUN3 protein lactylation in the repair of peripheral nerve injury induced by intermittent hypoxia, providing a theoretical basis for developing new clinical treatment approaches for peripheral nerve injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The figures show that intermittent hypoxia promotes DRG neuron proliferation and inhibits neuronal aging, where A is the lactate concentration detection; B is the neuronal cell viability detection; CD are the ROS level detection using the DCFH-DA probe, C is a representative fluorescence staining image, and D is a statistical graph of the fluorescence staining results. The scale of the image is 50 µm; EF is the β-galactosidase staining analysis, E is a representative β-galactosidase staining image, and F is a statistical graph of the β-galactosidase staining results; GH is a representative immunofluorescence detection of p21 (G) and LaminB1 (H) expression. The scale of the image is 100 µm.

[0045] Figure 2 Figure 3: Intermittent hypoxia promotes lactate production and regulates neural repair through NSUN3. A is a statistical graph of lactate concentration detection; BC are m5C and MB dot blot analysis, B is a representative graph of dot blot analysis, and C is a statistical graph of dot blot analysis results; D is a heat map analysis of the expression levels of m5C-related enzymes; EG are statistical graphs of RT-PCR analysis of neural repair factors NGF (E), BDNF (F), and GDNF (G).

[0046] Figure 3 Figure 3 shows the results of intermittent hypoxia promoting the lactic acid modification of NSUN3 protein, where A is the immunohistochemistry detection image, and the image scale is 100 µm; BC is the expression level of Pan Kla detected by Western blot, B is a representative image, and C is the result statistical graph; DE is the potential lactic acid modification site of NSUN3 protein predicted by the CPLM database, D is the prediction result score graph, and E is the potential lactic acid modification site of NSUN3; FG are the results of immunoprecipitation analysis of NSUN3 protein for lactic acid modification, F is a representative image of immunoprecipitation analysis, and G is the result statistical graph.

[0047] Figure 4 The following are the transcriptome sequencing analysis diagrams of NSUN3 overexpression, where A is the differential clustering circos heat map; B is the differential gene volcano map; and C is the Reactome enrichment analysis diagram.

[0048] Figure 5 Figure 1 shows the results of intermittent hypoxia regulating neuronal aging and mitochondrial dysfunction through NSUN3. A and B are Western blot analysis and quantitative analysis of protein expression, A is a representative figure, and B and B are statistical figures of quantitative analysis of p16 (B), p21 (C), and Lamin B1 (D). E is a representative figure of mitochondria labeled with mito-tracker fluorescence, and the scale is 20 µm. FI is a statistical figure of changes in mitochondrial-related mRNA generated by RT-PCR. The related mRNAs include Ak2 (F), Atp5f1b (G), Fh1 (H), and Prc1 (I). DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0050] Example

[0051] 1. Experimental Materials

[0052]

[0053] 2. Experimental Design

[0054] 1. Effects of intermittent hypoxia on damaged DRG neurons

[0055] 1) Intermittent Hypoxia Induction: Cells were cultured in a cell culture incubator at 37°C, 0.5% O₂, 5% CO₂, and a balanced N₂ atmosphere for intermittent hypoxia (5 hours of hypoxia / 7 hours of normoxia). Damaged DRG neurons were subjected to intermittent hypoxia for varying durations (0, 12, 24, 48, and 72 hours). Lactate production in each group was measured using a lactate detection kit.

[0056] 2) Based on the experimental groupings described above, use the CCK-8 assay kit to measure changes in cell viability in each group. Select an appropriate time point (48 hours of intermittent hypoxia induction) based on cell viability for subsequent experiments.

[0057] 3) DRG neuron grouping and processing:

[0058] CTL: control group, no processing is performed.

[0059] ACR: Acrylamide treatment causes neuronal damage.

[0060] ACR+Hypoxia: Acrylamide treatment causes neuronal damage + intermittent hypoxia induction.

[0061] ACR+Hypoxia+2-DG: Acrylamide treatment causes neuronal damage + intermittent hypoxia induction + 2-DG (lactate production inhibitor) treatment.

[0062] 4) According to the grouping in 3), the DCFH-DA probe kit was used to detect the generation of reactive oxygen species (ROS) in each group of cells.

[0063] 5) According to the grouping in 3), use galactosidase staining to detect cell senescence in each group.

[0064] 6) According to the grouping in 3), the expression of senescence marker p21 and proliferation marker Lamin B1 was detected by immunofluorescence.

[0065] 2. Effects of intermittent hypoxia on the rat peripheral nerve injury (PNI) model

[0066] 1) Establish a rat peripheral nerve injury (PNI) model and perform intermittent hypoxia induction treatment. Specific groups:

[0067] PNI: PNI model group.

[0068] PNI+Hypoxia: PNI+intermittent hypoxia treatment.

[0069] 2) The damaged nerve tissues of the animals in each group were collected, and the lactate levels in the damaged nerve tissues were detected using a lactate detection kit.

[0070] 3) According to the lactate level, the samples were divided into high lactate group (Lac High , >0.5 mmol / gprot) and low lactate group (Lac Low , ≤ 0.5 mmol / gprot), and the total m5C methylation modification levels in the neural tissues of the two groups were detected by dot blot experiments to explore the m5C modification levels of genes in neural cells involved in the upregulation of lactate induced by intermittent hypoxia.

[0071] 4) DRG neuron grouping and processing:

[0072] Control: No processing is performed.

[0073] LA: exogenous lactic acid (LA) treatment.

[0074] 5) RT-PCR was used to examine the expression of major m5C modification-related methyltransferases (NSUN1–NSUN7, DNMT1–DNMT2), demethylases (ALYREF, YBX1), and recognition proteins (TET1–TET2) in both cell groups. The results showed that the expression of the methyltransferase NSUN3 was most significantly upregulated (approximately 8-fold) after exogenous lactate treatment.

[0075] 6) DRG neuron cell grouping:

[0076] Control: No processing is performed.

[0077] ACR, ACR+Hypoxia: same as above.

[0078] ACR+Hypoxia+sh-NSUN3: Acrylamide treatment causes neuronal damage + intermittent hypoxia induction + sh-NSUN3 knocks down NSUN3 expression.

[0079] PCR was used to detect the expression of nerve repair factors NGF, BDNF and GDNF in cells of each group.

[0080] 3. Lactic acid modification of NSUN3

[0081] 1) The high lactate group (Lac High , >0.5 mmol / gprot) and low lactate group (Lac Low , ≤ 0.5mmol / gprot) were used to detect the expression of pan-Kla antibody by immunohistochemistry.

[0082] 2) DRG neuron grouping:

[0083] CTL, ACR, ACR+Hypoxia: same as above.

[0084] Western blot assay was used to detect the expression of Pan Kla in cells of each group, with Histone H3 as the internal reference.

[0085] 3) Bioinformatics analysis of the NSUN3 protein was performed using the CPLM database (https: / / cplm.biocuckoo.cn / index.php), and eight potential lactylation modification sites (K6, K8, K27, K67, K87, K111, K207, and K319) were successfully predicted.

[0086] 4) DRG neuron grouping and processing:

[0087] CTL, ACR, ACR+Hypoxia: same as above.

[0088] Immunoprecipitation experiments were performed using anti-NSUN3 antibodies, and the pulled-down proteins were immunoblotted with anti-Lactyllysine to analyze the lactylation modification of protein NSUN3.

[0089] 4. The role of NSUN3 in mitochondrial biogenesis and cell aging

[0090] 1) DRG neuron grouping and processing:

[0091] NC: negative control group.

[0092] sh-NSUN3: NSUN3 knockdown group.

[0093] 2) Western blotting was used to detect changes in cell senescence markers p21 and p16, and proliferation marker Lamin B1, using GAPDH as an internal reference.

[0094] 3) Mitochondria in each group of cells were fluorescently labeled with Mito-tracker.

[0095] 4) PCR was used to detect the changes in mitochondrial biogenesis-related mRNA (Ak2, Fh1, Prc1, Atp5f1b) in each group of cells.

[0096] 3. Experimental Results

[0097] 1. Intermittent hypoxia promotes lactate production, promotes DRG neuron proliferation in a concentration-dependent manner, and inhibits neuronal cell senescence.

[0098] Neuronal cells were subjected to intermittent hypoxia induction for different durations (0, 12, 24, 48, 72 h), and the lactate production level gradually increased with the extension of hypoxia time ( Figure 1 A in the figure) and regulates the viability of neuronal cells in a concentration-dependent manner ( Figure 1 B in the figure). We chose to conduct subsequent experiments after hypoxia induction for 48 hours. We further treated DRG neurons with the lactate production inhibitor 2-DG. The results showed that intermittent hypoxia induction significantly inhibited the level of reactive oxygen species (ROS) in neurons ( Figure 1 CD in the ), reducing β-galactosidase (SA-βgal) expression ( Figure 1 EF in the mitochondria), inhibiting the upregulation of the cell senescence marker p21 and promoting the expression of the cell proliferation marker Lamin B1 ( Figure 1 The results showed that the lactate inhibitor 2-DG could reverse the effects of intermittent hypoxia and significantly promote neuronal senescence.

[0099] 2. m5C methyltransferase NSUN3 is an important regulatory factor that promotes lactate production and regulates neural repair under intermittent hypoxia.

[0100] Compared with the PNI model group, the lactate concentration in the neural tissue of the intermittent hypoxia treatment group was significantly increased ( Figure 2 According to the different levels of lactate in damaged nerve tissue, the samples were divided into high lactate group (Lac High , >0.5 mmol / gprot) and low lactate group (Lac Low , ≤ 0.5 mmol / gprot, and the total m5C methylation modification level in the neural tissues of the two groups was detected by dot blot experiment. The results showed that the m5C methylation modification level in the neural tissues with high lactate level was significantly upregulated ( Figure 2BC in). RT-PCR experiments were further 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 the most upregulated (about 8 times) after exogenous lactate treatment ( Figure 2 D in the figure). After knocking down NSUN3 expression in neurons, the levels of the neural repair factors NGF, BDNF, and GDNF were significantly decreased ( Figure 2 EG in).

[0101] 3. Intermittent hypoxia induces upregulation of lactate production and promotes lactic acid modification of NSUN3 protein.

[0102] Immunohistochemical assay was used to detect the above (Lac High , >0.5 mmol / gprot) and low lactate group (Lac Low , ≤0.5 mmol / gprot) in neural tissues. The results showed that the total lactic acid modification level in the high lactate group was significantly upregulated ( Figure 3 We then used Western blot to analyze the expression of PanKla in in vitro neuronal cells and found that intermittent hypoxia induced a significant increase in the total lactylation modification level ( Figure 3 Based on the above findings, we innovatively used the CPLM database (https: / / cplm.biocuckoo.cn / index.php) to perform bioinformatics analysis on the NSUN3 protein and successfully predicted 8 potential lactylation modification sites (K6, K8, K27, K67, K87, K111, K207, K319) ( Figure 3 To further verify the prediction results, we used NSUN3-specific antibodies for immunoprecipitation and detected it with lactyllysine antibodies. The results showed that hypoxia treatment significantly upregulated the lactylation modification level of NSUN3 protein ( Figure 3 FG in ).

[0103] 4. Lactic acid modification of NSUN3 protein and cell senescence.

[0104] To construct the NSUN3 overexpression vector, the NSUN3 cDNA (available through 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 that the cellular senescence pathway was significantly enriched ( Figure 4 ), this finding is consistent with Figure 1 The results shown in

[15] that intermittent hypoxia inhibits cell senescence of damaged nerve cells corroborate each other, further confirming the important role of cell senescence in the NSUN3 regulatory network.

[0105] 5. Intermittent hypoxia regulates neuronal aging and mitochondrial dysfunction through NSUN3.

[0106] In a hypoxia-treated DRG neuron model, we knocked down NSUN3 and detected the expression of cell senescence markers p21 and p16, as well as the proliferation marker Lamin B1, by Western blot. The experimental 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 figure). Subsequently, we combined mito-tracker fluorescence labeling technology to analyze mitochondrial biogenesis and RT-PCR to analyze the changes in mitochondrial biogenesis-related mRNA (Ak2, Fh1, Prc1, Atp5f1b). The results showed that NSUN3 knockdown significantly inhibited mitochondrial biogenesis ( Figure 5 in EI).

[0107] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be implemented over a wide range under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides embodiments, it will be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any variations, uses, or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the disclosed scope of this application.

Claims

1. Application of methyltransferase NSUN3, characterized in that, The application includes any of the following: 1) Used to prepare products that promote the repair of peripheral nerve damage; 2) Used for non-therapeutic purposes in vitro to promote the repair of peripheral nerve damage.

2. The use according to claim 1, characterized in that The product includes an agent that enhances NSUN3 expression or function and / or an agent that enhances NSUN3 lactylation levels; Preferably, the product is selected from the group consisting of probe sets, primer sets, kits, chips, test strips, high-throughput sequencing, systems, equipment, and devices.

3. The use according to claim 2, characterized in that The reagents for enhancing NSUN3 expression or function include overexpression vectors containing NSUN3 encoding genes, NSUN3 mimics, and NSUN3 agomir.

4. The use according to claim 2, characterized in that The agent that enhances the expression or function of NSUN3 and / or the agent that enhances the lactylation level of NSUN3 is used to inhibit neural cell aging and / or promote neural cell proliferation.

5. The use according to claim 4, characterized in that The nerve cells are nerve cells of peripheral nerves, and the peripheral nerves include cranial nerves, spinal nerves, and visceral nerves; Preferably, 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; Preferably, the spinal nerves include cervical nerves, thoracic nerves, lumbar nerves, sacral nerves, and coccygeal nerves; Preferably, the visceral nerves include visceral motor nerves and visceral sensory nerves; Preferably, the peripheral nerve is a dorsal root ganglion.

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

7. A composition for promoting the repair of peripheral nerve damage, characterized in that: The composition includes an agent that enhances NSUN3 expression or function and / or an agent that enhances NSUN3 lactylation levels; Preferably, the reagent for enhancing the expression or function of NSUN3 includes an overexpression vector containing the NSUN3 encoding gene, NSUN3 mimics, or NSUN3 agomir.

8. The composition according to claim 7, characterized in that The composition further comprises a pharmaceutically acceptable carrier or excipient; Preferably, the pharmaceutically acceptable carrier or excipient includes a buffer, a stabilizer, a preservative, an osmotic pressure regulator, a diluent, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a surfactant, a coating material, a colorant, an antioxidant or an antibacterial agent.

9. A method for promoting the repair of peripheral nerve damage in vitro for non-therapeutic purposes, characterized in that: The method comprises the steps of overexpressing NSUN3 in the damaged nerve area or damaged nerve cells.

10. The method according to claim 9, characterized in that The method comprises the steps of constructing an overexpression vector comprising a NSUN3 encoding gene, wherein the overexpression vector can achieve overexpression of NSUN3 in damaged nerve areas or damaged nerve 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; Preferably, the overexpression vector comprising the NSUN3 encoding gene is obtained by connecting the NSUN3 encoding gene with a plasmid vector; Preferably, the step of constructing an overexpression vector containing the NSUN3 encoding gene comprises: using NSUN3 cDNA as a template, amplifying and cloning into a plasmid vector, and ligating by DNA ligase; Preferably, the plasmid vector is selected from at least one of pLVX, pCDNA, pLV, pCMV, pBABE, and pTrip; Preferably, the plasmid vector is pLVX.

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