Use of neuron TOX3 in preparation of a drug for treating neonatal hypoxic-ischemic encephalopathy

Abstract

The application discloses application of neuron TOX3 in preparation of a medicine for treating neonatal hypoxic-ischemic encephalopathy, and belongs to the technical field of biological medicine. The application finds that neuron TOX3 expression is significantly down-regulated after HIE by constructing an HIE model and neuron TOX3 knockout / overexpression mice; overexpression of TOX3 can effectively inhibit microglial cell activation, reduce demyelination and improve mouse movement and cognitive function; knockout of TOX3 leads to enhanced microglial cell activation, aggravated demyelination injury and abnormal behavior, and it is confirmed that TOX3 can regulate CSF1R expression through lactic acidification modification, maintain microglial cell homeostasis and further promote myelin repair. The application provides application of neuron TOX3 in preparation of a medicine for treating neonatal hypoxic-ischemic encephalopathy, provides a basis for precise intervention of clinical white matter demyelination related diseases, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of neuronal TOX3 in the preparation of drugs for treating hypoxic-ischemic encephalopathy in newborns. Background Technology

[0002] Cerebral palsy (CP) is a non-progressive motor disorder caused by perinatal brain injury. As one of the most devastating neurological diseases in childhood, it is also a leading cause of disability in children (Novak I, Jackman M, Finch-Edmondson M, Fahey M: Cerebral palsy. Lancet (London, England) 2025,406:174-188.). Disorders of myelin development and secondary demyelination are the core pathological mechanisms causing motor and cognitive deficits in children with CP. Hypoxic-ischemic encephalopathy (HIE) of the newborn caused by perinatal hypoxia and asphyxia not only leads to acute neonatal death (Dixon BJ, Reis C, Ho WM, Tang J, Zhang JH: Neuroprotective Strategies after Neonatal Hypoxic Ischemic Encephalopathy. International Journal of Molecular Sciences 2015, 16:22368-22401. Dumbuya JS, Chen L, Wu JY, Wang B: The role of G-CSF neuroprotective effects in neonatal hypoxic-ischemic encephalopathy (HIE): current status. Journal of Neuroinflammation 2021, 18:55.), but is also the most important risk factor for CP. Demyelination of the white matter in the brain caused by perinatal hypoxic-ischemic encephalopathy (HIE) is the core pathological link in the development of CP. Therefore, elucidating the mechanisms of HIE-induced CP-induced white matter damage and repair, and identifying key regulatory targets, has significant scientific and clinical value. Our previous studies found that neuronal TOX3 expression was significantly downregulated after HIE. By constructing TOX3 neuronal conditional knockout mice for the first time, we observed significant impairment in myelin development and repair in the white matter region of the knockout mice, accompanied by abnormal microglial activation. This suggests that neuronal TOX3 may participate in the repair process of white matter myelin damage by regulating microglial homeostasis. Furthermore, it did not have a direct and significant effect on oligodendrocytes (OLs), further indicating that its regulatory role is cell-specific.Signals originating from neurons can achieve transcellular regulation of glial cells in both healthy and diseased states (Chamera K, Trojan E, Szuster-G). uszczak M, Basta-Kaim A: The Potential Role of Dysfunctions in Neuron-Microglia Communication in the Pathogenesis of Brain Disorders. Current Neuropharmacology 2020, 18:408-430. Although the activation state of microglia is widely recognized as a core regulatory node in myelin damage and repair (Cao L, He C: Polarization of macrophages and microglia in inflammatory demyelination. NeuroscienceBulletin 2013, 29:189-198.), the molecular mechanism by which neuronal signaling precisely regulates microglia functional homeostasis and mediates anti-demyelination effects remains unclear; no relevant reports have been found domestically or internationally. TOX3 is an important transcription factor containing an HMG-box domain, and exploring its regulatory role in HIE demyelination has significant clinical value. Therefore, exploring the neuronal TOX3-mediated neuron-microglia transcellular communication mechanism is a key scientific issue for elucidating the repair mechanism of CP myelin damage and overcoming intervention bottlenecks. Summary of the Invention

[0003] Therefore, the purpose of this invention is to provide the use of neuronal TOX3 in the preparation of a medicament for treating hypoxic-ischemic encephalopathy in newborns.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] In a first aspect, the present invention provides the use of neuronal TOX3 in the preparation of a medicament for treating perinatal hypoxic-ischemic encephalopathy in newborns.

[0006] Based on the above technical solution, the ischemic hypoxic encephalopathy further includes ischemic hypoxic white matter injury.

[0007] Based on the above technical solution, the active ingredient of the drug further includes TOX3 protein and substances that can upregulate the expression level of TOX3 protein or TOX3 mRNA.

[0008] Based on the above technical solution, the substances that can upregulate the expression level of TOX3 protein or TOX3 mRNA include gene drugs, peptide drugs and small molecule agonists that can upregulate the expression level of TOX3 protein or TOX3 mRNA.

[0009] Based on the above technical solution, the gene-based drug further includes an adenovirus vector overexpressing TOX3 and a liposome vector overexpressing TOX3.

[0010] Based on the above technical solution, the drug further includes an effective amount of the above-mentioned active ingredient and pharmaceutically acceptable excipients.

[0011] Based on the above technical solution, the pharmaceutically acceptable excipients further include fillers, diluents, binders, disintegrants, and emulsifiers.

[0012] Based on the above technical solution, the drug is further prepared into a pharmaceutically permissible dosage form.

[0013] Based on the above technical solution, the dosage forms further include tablets, granules, oral liquid preparations, drops, injectable preparations, and capsule preparations.

[0014] Based on the above technical solution, the drug is further prepared in the form of a single-dose drug.

[0015] Based on the above technical solution, the single-dose drug further comprises 1~1000 mg of active ingredient.

[0016] Based on the above technical solution, the drug can further inhibit demyelination of the white matter in perinatal hypoxic-ischemic encephalopathy, promote myelin repair, significantly increase the expression of myelin functional proteins NF200 and MBP, and improve motor ability and cognitive dysfunction.

[0017] Based on the current state of research on cerebral palsy (CP) and hypoxic-ischemic encephalopathy (HIE) in newborns, this invention has the following advantages compared to traditional techniques and existing research systems: (1) Novel target Current research on HIE-induced white matter demyelination and cerebral palsy mainly focuses on single-cell levels such as oligodendrocytes, inflammatory factors, and microglia, with few studies exploring transcellular regulatory mechanisms from the neuronal origin. This invention is the first to clearly identify neuronal TOX3 as a key regulatory molecule for the repair of white matter myelin damage, and there are currently no reports on the use of TOX3 for the regulation of neuronal damage in cerebral palsy. The target is original and rare, breaking through the limitations of existing research on the pathological mechanism of demyelination.

[0018] (2) The mechanism of action is clear and the regulatory specificity is strong. Existing neuroprotective targets generally suffer from broad-spectrum cellular effects and poor targeting, easily leading to non-specific neural interference. This invention demonstrates that neuronal TOX3 has strict cell specificity; it does not directly act on oligodendrocytes, but rather improves demyelination of brain white matter by maintaining microglia functional homeostasis and inhibiting abnormal microglia activation. This neuronal-microglia transcellular regulatory mechanism is clearly defined, with a well-defined pathway, avoiding the shortcomings of broad-spectrum targets such as off-target effects and significant side effects.

[0019] (3) It closely matches the real clinical pathological model and has high reliability. This invention focuses on the mechanism of perinatal hypoxic-ischemic encephalopathy (HIE), the most important cause of cerebral palsy in clinical practice. It uses a conditional knockout mouse model of neurons to verify gene function. Compared with ordinary cell models and non-specific animal models, it is more consistent with the real pathological process of neonatal HIE-induced cerebral palsy. The experimental results have good reproducibility and high clinical relevance, providing a solid animal experimental basis for clinical translation.

[0020] (4) Possesses strong potential for early prevention Current treatments for cerebral palsy mainly involve post-natal rehabilitation and symptomatic treatment, lacking early intervention targets during the perinatal period and failing to prevent the progressive deterioration of brain injury. This invention targets the core pathological process of early white matter demyelination in HIE, and regulates the expression of neuronal TOX3. It can inhibit inflammatory activation and promote myelin repair in the early stage of neonatal brain injury, achieving early intervention for cerebral palsy and making up for the technical shortcomings of the current lack of early clinical intervention methods.

[0021] (5) High safety and controllable side effects TOX3 is an endogenous transcription factor in the human body and is physiologically expressed in normal brain tissue. This invention regulates the neural microenvironment by restoring endogenous TOX3 expression, without the need for the introduction of exogenous toxic genes or highly stimulating drugs. It has good biocompatibility and, compared with chemical neuroprotective drugs, has lower immunotoxicity and brain side effects, making it safer for clinical application. Attached Figure Description

[0022] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0023] Figure 1 A schematic diagram of the construction of a conditional knockout mouse for TOX3.

[0024] Figure 2Figures showing the comparison of neuronal TOX3 knockout efficiency, body size, and weight between TOX3 knockout mice and wild-type mice. In the figure, AC represents the genotype identification of neuronal TOX3 conditional knockout mice constructed using agarose gel electrophoresis and the gene knockout efficiency detection using protein immunoblotting, respectively; DF represents the comparison of body weight and brain weight between TOX3 knockout mice and control mice; n=3-12. P<0.01; t-test analysis; all values ​​are expressed as mean ± SD of at least three independent experiments.

[0025] Figure 3 The study investigated the effect of neuronal TOX3 deficiency on inhibiting myelin formation in mice. A showed changes in white matter in the corpus callosum region after serial sectioning of brain tissue from knockout and wild-type mice. BC showed the expression of MBP protein after TOX3 knockout using immunofluorescence staining. D analyzed the cellular status and thickness changes in the corpus callosum region of knockout mice using HE and LFB staining techniques. EF used transmission electron microscopy to observe changes in the number of myelinated nerves and myelin sheath thickness in the corpus callosum region of knockout mice (n=3-5). P<0.05, P<0.0001; t-test analysis, all values ​​are expressed as mean ± SD of at least three independent experiments.

[0026] Figure 4 The figure shows the effect of neuronal TOX3 on oligodendrocyte fate regulation. A represents the effect of qPCR on the expression of mouse corpus callosum oligodendrocyte development-related marker genes CC1, SOX10, and PDGFR-α, as detected by neuronal TOX3 knockout; BC represents the effect of Western blotting on the expression of mouse oligodendrocyte development-related marker proteins CC1, SOX10, and PDGFR-α, as detected by neuronal TOX3 knockout; DG represents the expression of SOX10 and PDGFR-α using immunofluorescence assays (n=3). Compared with the cKO group... P<0.05, P<0.01, P<0.001, P < 0.0001; one-way ANOVA or t-test, all values ​​are expressed as mean ± SD of at least three independent experiments.

[0027] Figure 5To illustrate the protective effect of TOX3 on neurons in HIE injury, A represents a schematic diagram of intraventricular injection of AAV-TOX3 overexpressing virus into P2 mouse pups using stereotactic brain imaging; B and E represent analyses of myelin repair after TOX3 overexpression in neurons using LFB staining, qPCR, and WB techniques, respectively; F and K represent analyses of changes in motor and cognitive function in TOX3-overexpressing mice using novel object recognition, open field, and fatigue rotarod experiments; n=3-9; compared with the sham-operated group... P<0.05, P<0.01, P<0.001, P<0.0001; compared with the HIE group, ## P<0.01, ### P<0.001, #### P<0.0001; one-way ANOVA, all values ​​are expressed as mean ± SD of at least three independent experiments.

[0028] Figure 6 To investigate the effects of TOX3 knockout on demyelination and long-term behavior in HIE mice, the study included: A) a schematic diagram of HIE construction in mice; B) analysis of brain damage after HIE using HE staining; C) detection of myelin damage after HIE using LFB staining; D) detection of the effect of TOX3 knockout on cerebral infarction using TTC staining; and E) analysis of motor and cognitive abilities in knockout mice after HIE injury using behavioral experiments such as open field, fatigue rotundus, Morris water maze, and novel object recognition. n=9-10; compared with the sham-operated group... P<0.001, P<0.0001; compared with the HIE group, ## P<0.01, ### P<0.001, #### P < 0.0001; one-way ANOVA. All values ​​are expressed as mean ± SD of at least three independent experiments.

[0029] Figure 7To investigate the mechanism by which neuronal TOX3 regulates microglial homeostasis during demyelination, the following steps were performed: A and B, respectively, using RNA-Seq analysis to screen for new candidate genes in the corpus callosum tissue of conditionally knockout mice; C, using Western blotting (WB) to detect the expression of LDHA, GLUT1, and CSF1R in the corpus callosum tissue of knockout mice; D, using a colorimetric method to detect lactate content in the corpus callosum tissue of knockout mice; EF, using WB to detect the total histone lactation level in the corpus callosum and microglia after neuronal TOX3 knockout; G, using a Co-IP experiment to verify the regulatory effect of histone lactation modification on CSF1R mediated by neuronal TOX3; and H, using WB to detect the expression of H3K18la, H3K14la, H3K12la, and H3K9la after neuronal TOX3 knockout. The expression of classic histone lactation modification sites was analyzed; n=3-4; compared with the cKO group, P<0.01, P<0.001; one-way ANOVA; all values ​​are expressed as mean ± SD of at least three independent experiments. Detailed Implementation

[0030] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0031] Example 1 1. Animal Model Construction 1.1 Neonatal mouse hypoxia-ischemia (HIE) model Seven-day-old C57BL / 6J mice were anesthetized with isoflurane, and a midline cervical incision was made. The right common carotid artery was separated, ligated, cut, and the muscles and skin were sutured to establish a hypoxic-ischemic encephalopathy (HIE) model. After surgery, the mice were placed next to their mothers for 1 hour of recovery, and then placed in a closed hypoxic chamber (8% O2, 5% CO2). The hypoxia was repeated 6 times according to the rule of 15 minutes of hypoxia followed by 1 minute of rest, maintaining a total hypoxic time of 90 minutes. After surgery, the mice were kept in the same littermates as their mothers and fed normally. On the third day after surgery, corpus callosum tissue was collected for molecular biological experiments. On the 28th day after surgery, behavioral tests were performed, and relevant indicators were detected at the histological, molecular biological, and animal behavioral levels. After successful establishment of the HIE model, TTC staining showed that the ischemic brain tissue of the mice appeared white, while the normal brain tissue appeared red.

[0032] 1.2 Construction of Gene Overexpression Mice The TOX3 overexpression plasmid and adenovirus (AAV) packaging were both completed by Shanghai Jikai Gene Medical Technology Co., Ltd. Newborn C57BL / 6J mice aged 2 days were injected with AAV-TOX3 overexpression virus into the lateral ventricle; HIE modeling was performed on mice at 7 days of age; behavioral tests were conducted on mice 28 days post-surgery.

[0033] 1.3 Construction of gene knockout mice With the assistance of the Southern Model Animal Center, a TOX3 knockout mouse, Tox3tm1a(KOMP)Mbp, was successfully constructed. Through crossbreeding with other tool mice, conditional knockout of TOX3 was further achieved. A schematic diagram of the construction is shown below. Figure 1 As shown.

[0034] 2. Real-time quantitative PCR (qPCR) Total RNA was extracted from mouse corpus callosum or microglia and reverse transcribed to obtain cDNA. Gene-specific primers and SybrGreen Mix were added to prepare a PCR reaction solution, which was then placed on a PCR instrument for PCR reaction. β-Actin was used as an internal control. Primer sequence information is shown in Table 1.

[0035] Table 1 qPCR primer sequences

[0036] 3. Protein immunoblotting After collecting proteins from corpus callosum tissue or microglia, the protein concentration was measured, and then SDS-PAGE electrophoresis was performed. The proteins on the gel were transferred to a PVDF membrane, blocked with 5% skim milk prepared with 1× TBST, incubated with primary antibody at 4°C overnight, and then hybridized with HRP-labeled secondary antibody. Finally, the signal was detected using an ECL kit.

[0037] 4. Immunofluorescence Tissue sections were washed three times with 1×PBST; antigen retrieval was performed at 95°C for 15 min with citrate buffer; permeabilization and blocking were performed for 3 h (components: 0.5% PBST / 10% goat serum); primary antibody was incubated overnight at 4°C; 1×PBST was used for washing; fluorescent secondary antibody was incubated at room temperature in the dark for 60 min; 1×PBST was used for washing; and the sections were mounted with anti-fluorescence quenching agent and observed and photographed under a fluorescence microscope.

[0038] 5. HE staining After anesthetizing the experimental animals with isoflurane, the brains were harvested via perfusion with 4% PFA and fixed overnight. The tissues were then dehydrated, embedded in paraffin, and sectioned using a paraffin microtome. Paraffin sections were dewaxed by soaking in xylene, followed by gradient hydration with alcohols (100%, 95%, 90%, 80%, 70%, 50%, 30%). After washing with distilled water, they were stained with hematoxylin; after rinsing with distilled water until they turned blue, they were separated by 0.5% hydrochloric acid-alcohol treatment and rinsed with running distilled water. Gradient ethanol dehydration was performed (70%, 80%, 90%), followed by eosin staining, and then twice rinsed with 100% ethanol and cleared with xylene. The sections were air-dried, mounted with neutral resin, and observed and photographed under an optical microscope.

[0039] 6. Co-IP experiment Mouse corpus callosum tissue was collected, and a mixture of IP lysis buffer and protease inhibitor was added. 50 μL of each sample was then used as input for normal protein extraction. 400 μL of supernatant was collected from the remaining sample, and 4 μL of Pan-Kla antibody and 40 μL of agarose beads were added. The mixture was incubated overnight at 4°C on a shaker. The sample was then centrifuged at 1000g for 2 min at 4°C, and the supernatant was discarded. The sample was washed with PBS and centrifuged at 1000g for 2 min at 4°C, and the supernatant was discarded. Sufficient loading buffer was added to each sample, and the sample was boiled at 95°C for 5 min and stored at -20°C for later use.

[0040] 7. Transmission electron microscopy The tissue blocks of the corpus callosum were fixed with glutaraldehyde, dehydrated with graded alcohol, embedded, processed into ultrathin sections, stained with lead salts, and finally observed and analyzed by transmission electron microscopy to determine the thickness, quantity and distribution of myelin sheath.

[0041] 8. RNA transcriptome sequencing RNA-seq is one of the most widely used high-throughput sequencing technologies and is a transcriptomics research method based on next-generation sequencing technology. RNA was extracted from the corpus callosum tissue of WT and CKO mice, and transcriptome amplification and library construction were performed. BGISEQ RNA-seq data were then collected and analyzed to identify and confirm differentially expressed gene profiles in order to select candidate genes.

[0042] 9. LFB staining Frozen sections were thawed at room temperature for 30 min and washed with PBS for 15 min. They were then dehydrated using a gradient of alcohols (70%, 80%, 90%), followed by washing with PBS. The sections were then placed in LFB staining solution and stained in an oven at 60℃ for 6 h. Afterward, they were cooled to room temperature. The destaining process involved a gradient of alcohols: 95% ethanol for 2 min; lithium carbonate for 30 s; 70% ethanol for 2 min; 80% ethanol for 2-3 min; 90% ethanol for 5 min; and 100% ethanol for 5 min. The sections were then air-dried, mounted with neutral resin, and observed and photographed under a microscope.

[0043] 10. TTC staining Three days after HIE injury, mice were anesthetized with isoflurane and decapitated. The mouse brain tissue was cut into 2mm thick coronal sections and stained in TTC staining solution at 37°C for 30 minutes in the dark. After incubation, the coronal sections were fixed in 4% PFA for 24 hours. The infarct area was analyzed using ImageJ software; infarct area (%) = infarct area / total area × 100.

[0044] 11. Isolation and culture of neurons and astrocytes Primary astrocytes: Mouse heads were taken and soaked in 75% ethanol for 1 min. The hippocampus was removed, and the cortical portion was retained. The meninges were dissected in a sterile operating table, the tissue was shredded, transferred to centrifuge tubes, and digested with 0.25% trypsin. The supernatant was removed by centrifugation, and the cell pellet was placed in a culture flask and cultured under 37°C and 5% CO2 conditions for 10-14 days. The cells were then cultured in glial cell culture medium, with the medium changed every 3 days. The mixed glial cells were then placed in a constant temperature shaker at 180 rpm and shaken for 4 h to obtain purified astrocytes.

[0045] Primary neurons: The isolation steps for primary neurons are the same as those for astrocytes described above; after the neurons adhere to the culture medium for 24 hours, the medium is replaced with complete medium (DMEM / F12+2%B27), and the neurons are cultured for 3 days to observe their growth; then, the medium is replaced with cytarabine medium at a final concentration of 2.5 μg / mL (only half the medium needs to be replaced) for 3 days to inhibit the growth of glial cells and other cells, thus obtaining purified primary neurons; then, the complete medium is replaced every 3 days, with only half the medium replaced each time.

[0046] 12. Construction of in vitro experimental cell models Conditioned culture medium group: Primary neuronal culture medium was collected, and the supernatant was collected by centrifugation to obtain neuronal conditioned culture medium; purified astrocytes were seeded into well plates, and wild-type primary astrocytes were cultured in conditioned culture medium separately. The medium was changed every other day, and the cells were collected by digestion with 0.25% EDTA trypsin.

[0047] 13. Behavioral Experiments Open field test: This test assesses the voluntary movement ability of mice. In short, mice are placed in a 35cm×35cm×25cm blue plexiglass arena and allowed to explore freely for 10 minutes. The total distance moved within 10 minutes is recorded and used as the basis for analyzing the voluntary movement ability of mice.

[0048] Fatigue rotundus test: To assess the motor coordination and endurance of mice, all experimental mice were trained on a rotundus bar for 30 s (5~10 r / min) before the formal test. After all mice had adapted, the experimental mice were subjected to the rotundus test at a uniform acceleration of 5~30 r / min for 300 s, and the time the mice spent on the bar was recorded.

[0049] Water maze: This test assesses the spatial localization learning and memory abilities of mice. The water tank is divided into four quadrants. During the acquisition training phase, a platform is hidden in the center of one quadrant. Mice are randomly placed in a quadrant with their heads facing the tank wall, and the time and trajectory for finding the platform within 60 seconds are recorded. If the mouse still cannot find the platform within 60 seconds, it is guided to the platform and stays there for 60 seconds. Training is conducted once per quadrant per day for a total of 5 days. During the exploration phase, the platform is removed, and the mouse is placed in the quadrant containing the platform and the quadrant opposite it. Exploration training is then conducted for 60 seconds each time, and the time and number of times the mouse finds the platform are recorded.

[0050] The novel object recognition experiment primarily tested the fine and sensitive behavioral aspects of mouse recognition memory. On the first day of testing, mice were allowed 10 minutes to adapt to the testing area. On the second day, two objects of the same shape and color were placed in the testing area, and the mice's contact with the two objects was recorded using a tracking device. On the third day, one of the objects was moved, and the mice's contact with the two objects was recorded again. Then, one of the objects was placed in the same position but replaced with another object of different shape and color, and the mice's contact with the two objects was recorded again. Finally, the percentage (%) of the total recognition time for the novel object was used as the basis for analyzing the mice's recognition memory ability.

[0051] 14. Experimental Results 14.1 Construction of TOX3 neuron-conditioned mice Our previous research results showed that neuronal TOX3 may be involved in the pathogenesis of HIE and is associated with white matter damage in mice. To further explore the important role of TOX3 in white matter development and function, we crossbred fluxed-TOX3 mice with CaMKII-Cre tool mice and finally screened out TOX3. fl / fl CaMKII-Cre neuron-specific conditional knockout mice ( Figure 1 The genotypes of the constructed neuronal TOX3 conditional knockout mice were detected using agarose gel electrophoresis, and the gene knockout efficiency was detected using protein immunoblotting. Changes in body weight and brain weight were also detected in TOX3 knockout mice and control mice.

[0052] Experimental results are as follows Figure 2As shown in the Western blot results, compared with WT mice, knockout mice showed significantly reduced brain TOX3 expression and significant knockout efficiency; there was no significant difference between knockout mice and WT mice in terms of body weight and brain weight.

[0053] 14.2 Neuronal TOX3 deficiency leads to a significant white matter lesion phenotype in mice. To further investigate the role of neuronal TOX3 in the development and function of mouse brain white matter, we performed morphological analysis on the brain tissue of TOX3 knockout mice; the results are as follows: Figure 3 As shown, serial sections of brain tissue from knockout mice revealed that, compared to WT mice, knockout mice exhibited a significantly reduced number of white matter myelin sheaths and a significantly thinner corpus callosum, characteristic of white matter damage. LFB and HE staining experiments further confirmed this conclusion. Immunofluorescence experiments further demonstrated that knockout of neuronal TOX3 primarily affected myelin formation in the corpus callosum region. Transmission electron microscopy results showed that, compared to WT mice, knockout of neuronal TOX3 resulted in a significantly reduced number and thinner myelin sheaths in the corpus callosum region. These experiments indicate that neuronal TOX3 plays a crucial role in myelin development and damage repair.

[0054] 14.3 Knocking out TOX3 in neurons inhibits oligodendrocyte maturation. Next, we conducted a series of molecular biology experiments to detect and analyze markers related to key nodes in myelin development in TOX3 knockout mice and WT mice. The results are as follows: Figure 4 As shown, knocking out TOX3 in neurons inhibits the maturation of oligodendrocytes and impairs myelin development in mice, which is similar to the myelin damage observed in the HIE group mice.

[0055] 14.4 Overexpression of neuronal TOX3 can effectively inhibit HIE demyelination and improve motor and cognitive dysfunction in mice. To further verify the specific role of TOX3 in HIE, we used stereotactic injection of AAV virus into the brain to specifically transfect neurons and overexpress TOX3. We established a littermate control group (Sham group), an HIE group, and an HIE+OE overexpression group. We used LFB staining, qPCR, WB assay, rotarod assay, open field assay, and novel object recognition assay to investigate the effect of TOX3 overexpression on HIE demyelination and the improvement of motor and cognitive dysfunction in mice.

[0056] The results are as follows Figure 5As shown, the myelin function of HIE mice after TOX3 overexpression was first examined using LFB staining. The results showed that, compared to the Sham group, the HIE group mice exhibited significantly reduced white matter myelin sheath quantity and significantly thinner corpus callosum, indicating myelin damage. Overexpression of neuronal TOX3 effectively inhibited HIE demyelination and promoted myelin repair. The expression of myelin function-related proteins NF200 and MBP was detected using qPCR and Western blotting. The results showed that, compared to the Sham group, the expression of NF200 and MBP in the HIE group was significantly reduced; however, overexpression of neuronal TOX3 significantly increased the expression of these proteins. To further clarify the long-term behavior of neuronal TOX3 in HIE mice, we examined the improvement in motor ability of TOX3-overexpressing mice using rotarod and open field tests, and the improvement in cognitive-motor ability using a water maze test. The water maze test results showed that overexpression of the neuron TOX3 effectively improved cognitive impairment in HIE mice. The open field test results showed that HIE significantly reduced motor function in mice, while overexpression of the neuron TOX3 effectively improved motor function. The rotarod test results showed that HIE reduced the time mice spent on the rotarod, while overexpression of the neuron TOX3 effectively increased the time mice spent on the rotarod.

[0057] 14.5 Knockout of TOX3 exacerbates demyelinating injury and motor and cognitive dysfunction in HIE mice To further explore the key role of neuronal TOX3 in the regulation of myelin function, we constructed a HIE mouse model of cerebral palsy and set up a control mouse Sham group and a HIE+CKO group. We investigated the effects of TOX3 knockout on demyelination and motor and cognitive functions in HIE mice through LFB staining, HE staining, TTC staining, open field test, fatigue rotarod test, Morris water maze test and novel object recognition test.

[0058] The results are as follows Figure 6 As shown, HE and LFB staining results indicate that HIE mice exhibit significant demyelination and oligodendrocyte (OL) loss phenotypes. Knockout of neuronal TOX3 exacerbates demyelination damage and motor and cognitive abnormalities in HIE mice. Therefore, knockout of TOX3 aggravates demyelination damage and motor and cognitive dysfunction in HIE mice.

[0059] 14.6 Neuronal TOX3 can regulate CSF1R expression in microglia through histone lactation modification, thereby inhibiting HIE demyelination injury. To further explore the mechanism, we performed RNA-seq transcriptome sequencing analysis and screened for key differentially expressed genes between the control and knockout groups. Screening was based on |(Fold Change, FC)| ≥ 1.5. P The standard was ≤ 0.05; and gene function enrichment analysis was performed using GO and KEGG, with results as follows: Figure 7 As shown, GO analysis revealed that differentially expressed genes are mainly involved in multiple biological processes, including microglia activation and inflammatory responses. Heatmap enrichment analysis showed that knockout of neuronal TOX3 upregulates the expression of microglia homeostasis-related genes (such as CSF1R and CD68) and lactate metabolism-related lactate dehydrogenase A (LDHA) and glucose transporter 1 (GLUT1). Microglia CSF1R signaling plays a dual role in the nervous system: it is a key signal for microglia survival and proliferation, and also an important pathway driving demyelination. CSF1R gene mutations are closely related to white matter damage. Based on this, we believe that microglia CSF1R may be a key target for neuronal TOX3 to exert its anti-demystification effect. Lactic acid is an end product of glycolysis and can regulate multiple biological processes, including tumors, neuronal excitability, and inflammation, by mediating lactation modification. Under inflammatory conditions, upregulation of GLUT1 expression promotes glucose uptake in microglia, increases LDHA expression, and raises lactate levels. In vivo experiments showed that knocking out TOX3 in neurons upregulates the expression of GLUT1 and LDHA in the corpus callosum and increases lactate levels. Western blotting experiments also confirmed that knocking out TOX3 in neurons significantly increased the level of lactation modification in mouse corpus callosum and microglia. Co-IP experiments further revealed that neuronal TOX3-mediated histone lactation modification has a clear regulatory role on CSF1R expression, which further suggests that the regulation of microglia activation by neuronal TOX3 may be achieved through mediating histone lactation modification mechanisms. To further clarify which histone lactation modification sites are specifically regulated by neuronal TOX3, we conducted a preliminary analysis of several classic histone lactation modification sites after neuronal TOX3 knockout using Western blotting experiments. The results showed that neuronal TOX3 knockout significantly upregulated H3K18la expression, while having no significant effect on H3K14la, H3K12la, and H3K9la expression. ChIP-qPCR experiments further demonstrated that, compared to the control group, neuronal conditioned medium treatment promoted the enrichment of H3K18la in the CSF1R promoter region of microglia. These results suggest that studying the neuron-lactate metabolism / lactation modification-microglia regulatory network is key to revealing the neuronal TOX3 anti-demyelination mechanism.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of neuronal TOX3 in the preparation of drugs for treating perinatal hypoxic-ischemic encephalopathy in newborns.

2. The application according to claim 1, characterized in that, The active ingredient of the drug includes TOX3 protein and substances that can upregulate the expression level of TOX3 protein or TOX3 mRNA.

3. The application according to claim 2, characterized in that, The substances that can upregulate the expression level of TOX3 protein or TOX3 mRNA include gene therapy drugs, peptide drugs, and small molecule agonists.

4. The application according to claim 3, characterized in that, The gene-based drugs include adenovirus vectors that overexpress TOX3 and liposome vectors that overexpress TOX3.

5. The application according to claim 1, characterized in that, The drug comprises an effective amount of the active ingredient and pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients include fillers, diluents, binders, disintegrants, and emulsifiers.

7. The application according to claim 1, characterized in that, The drug is prepared into a pharmaceutically permissible dosage form.

8. The application according to claim 7, characterized in that, The dosage forms include tablets, granules, oral liquid preparations, drops, injectable preparations, and capsule preparations.

9. The application according to claim 8, characterized in that, The drug is prepared in the form of a single-dose drug, wherein the single-dose drug contains 1 to 1000 mg of active ingredient.

10. The application according to claim 1, characterized in that, The drug can significantly inhibit demyelination of the white matter in perinatal hypoxic-ischemic encephalopathy, promote myelin repair, significantly increase the expression of myelin functional proteins NF200 and MBP, and improve motor ability and cognitive dysfunction.

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