Application of DDC silencing in encephalatrophy model

Abstract

The invention discloses application of DDC silencing in an encephalatrophy model, and relates to the field of traumatic brain injury. According to the application, by inhibiting DDC gene expression or protein activity, GSK-3beta kinase mediated Tau protein phosphorylation is regulated and controlled, so that encephalatrophy is relieved or prevented; wherein the encephalatrophy model is an immature brain traumatic brain injury (TBI) after-encephalatrophy model, and DDC silencing is achieved through siRNA, shRNA or antisense oligonucleotide. A gene expression profile after immature brain TBI is analyzed through transcriptome sequencing, DDC is found to be remarkably up-regulated and enriched in a 5-hydroxytryptamine synaptic pathway, an in-vivo experiment proves that DDC expression and Tau phosphorylation level are synchronously increased, an in-vitro experiment shows that DDC silently inhibits GSK-3beta-mediated Tau phosphorylation, and a basis is provided for a DDC-targeted treatment strategy.

Description

Technical Field

[0001] This invention relates to the field of traumatic brain injury, and more particularly to the application of DDC silencing in a brain atrophy model. Background Technology

[0002] Traumatic brain injury (TBI) is structural or functional brain damage caused by external mechanical forces and is a major global public health problem. Epidemiological data shows that there are approximately 50 million cases of TBI annually, with a particularly high incidence and disability risk among children and adolescents due to their immature brain development. The neuroplasticity of the immature brain after TBI differs from that in adults, often manifesting as chronic progressive brain atrophy that can last for months or even years, but currently, effective interventions are limited.

[0003] Tau protein is a neuronal microtubule-binding protein, and its hyperphosphorylation is associated with various neurodegenerative diseases. Under the pathological conditions of TBI, Tau undergoes abnormal phosphorylation (such as at Ser-404 and Thr-231 sites), leading to microtubule dysfunction, neuronal death, and brain atrophy. Tau phosphorylation after TBI in immature brains is consistent with the spatiotemporal progression of brain atrophy, but its upstream regulatory mechanisms remain unclear.

[0004] Existing research has focused primarily on inflammatory responses or oxidative stress, with less attention paid to the role of neurotransmitter synthases such as dopa decarboxylase (DDC).

[0005] Therefore, revealing the role of DDC in brain atrophy is of great scientific significance.

[0006] Therefore, this invention proposes the application of DDC silencing in a brain atrophy model. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of DDC silencing in a brain atrophy model.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The application of DDC silencing in a brain atrophy model involves regulating GSK-3β kinase-mediated Tau protein phosphorylation by inhibiting DDC gene expression or protein activity, thereby alleviating or preventing brain atrophy. The brain atrophy model is an immature brain traumatic brain injury (TBI) brain atrophy model, and DDC silencing is achieved through siRNA, shRNA, or antisense oligonucleotides.

[0009] Preferably, the DDC silencing uses a siRNA sequence, and the RNA sequence is selected from any of the following: Sequence 1: 5'-GUGAUCCAUCGGCUGCAGG-3', Sequence 2: 5'-GUCGGUCCUAUCUGCAACA-3', Sequence 3: 5'-GAGCUGGGUUAAUUGGUGG-3', Or its functionally equivalent variants.

[0010] Preferably, the application includes silencing DDC expression in an in vitro or in vivo model, wherein the in vivo model is an immature rat TBI model established by controlled cortical impaction technique, with impaction parameters of: velocity 5.5 m / s, depth 1.5 mm, and duration 50 ms.

[0011] Preferably, DDC silencing is achieved by inhibiting GSK-3β phosphorylation, wherein the level of GSK-3β phosphorylation is negatively correlated with the phosphorylation level of Tau protein at Ser-404 and Thr-231 sites; and the DDC silencing can be reversed by the GSK-3β agonist FTY720.

[0012] Preferably, the application further includes detecting DDC expression levels or Tau phosphorylation levels as biomarkers to evaluate the therapeutic effect of brain atrophy; wherein the detection methods include Western blot, immunofluorescence, or RT-qPCR.

[0013] Preferably, it is also applied to neurodegenerative disease models, which are associated with Tau protein hyperphosphorylation, including Alzheimer's disease or Parkinson's disease; and DDC silencing affects the dopamine system through serotonergic synaptic pathways.

[0014] A pharmaceutical composition comprising a DDC silencer and a pharmaceutically acceptable carrier, wherein the DDC silencer is an siRNA sequence for the application of DDC silencing in a model of brain atrophy, and the composition is intended to prevent or treat brain atrophy, which is progressive brain atrophy following immature brain TBI.

[0015] Preferably, the composition further comprises a GSK-3β modulator, which is an agonist or an inhibitor, and the composition is administered by intravenous injection, topical administration or oral administration.

[0016] A method for preparing a DDC silencing agent includes synthesizing an siRNA sequence for the application of DDC silencing in a brain atrophy model and packaging it via liposomes or a viral vector for transfection of cells or tissues; wherein the preparation method includes in vitro transfection using Lipofectamine 3000 reagent.

[0017] A brain atrophy treatment system includes a DDC silencing module and a GSK-3β regulation module, wherein the DDC silencing module implements the application of DDC silencing in a brain atrophy model, and the GSK-3β regulation module uses FTY720; the system is used for combined treatment of chronic brain atrophy after TBI in immature brains.

[0018] The beneficial effects of this invention are as follows: This invention analyzes the gene expression profile of immature brain TBI through transcriptome sequencing and finds that DDC is significantly upregulated and enriched in 5-hydroxytryptamine synaptic pathways. In vivo experiments confirm that DDC expression and Tau phosphorylation levels increase synchronously, and in vitro experiments show that DDC silencing inhibits GSK-3β-mediated Tau phosphorylation, providing a basis for a therapeutic strategy targeting DDC. Attached Figure Description

[0019] Figure 1 This is a diagram of differentially expressed genes after TBI according to the present invention, wherein: A is a volcano plot showing the distribution of differentially expressed genes; B is a hierarchical clustering heatmap of differentially expressed genes, showing the differences in expression patterns between the TBI group and the Sham group; Figure 2 This is a GO functional enrichment analysis diagram of differentially expressed genes in this invention; Figure 3 This is a diagram showing the KEGG pathway enrichment analysis of differentially expressed genes in this invention. Figure 4 This is a diagram of neurotransmitter signaling pathways and the role of DDC in brain atrophy after TBI, as shown in the present invention. A is a diagram of the 5-hydroxytryptaminergic synaptic signaling pathway; B is a diagram of the dopaminergic synaptic signaling pathway; Figure 5 This is a schematic diagram of the upregulation of DDC expression after immature traumatic brain injury (TBI) according to the present invention. Western blot analysis shows the DDC protein expression level at different time points after TBI (sham control group, day 1 and day 3). GAPDH was used as an internal reference. The bar chart represents the quantitative analysis of DDC expression relative to the sham control group. **p<0.01, n=3. Figure 6 This is a schematic diagram illustrating the increased phosphorylation levels of DDC and Tau proteins after TBI in immature brains according to the present invention, wherein: A is a representative immunofluorescence image showing DAPI, DDC (and combined staining) in the sham group and TBI group; the bar chart represents the quantitative analysis of DDC expression relative to the sham control group. B is a representative immunofluorescence image showing DAPI, p(Thr-231)Tau and combined staining in the sham group and TBI group. The bar chart represents the quantitative analysis of p(Thr-231)Tau expression relative to the sham control group. **p<0.01. Scale bar = 50μm; Figure 7 This is a schematic diagram of the co-localization analysis of DDC and Tau after TBI in this invention; Figure 8 This is a schematic diagram illustrating the expression level of DDC mRNA in SH-SY5Y cells under different treatment conditions using RT-qPCR. Figure 9 This is a schematic diagram illustrating the phosphorylation of Tau protein mediated by the DDC of the present invention via GSK-3β. Detailed Implementation

[0020] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0021] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0022] Example 1: 1. Materials and Methods 1.1 Laboratory Animals and Grouping This invention utilizes SPF-grade male SD rats, approximately 9g in weight, aged 3-4 days, purchased from Silek Jingda Laboratory Animal Co., Ltd. Animals were housed in an environment with controlled temperature (22±2℃) and humidity (55±5%), maintaining a 12-hour light / dark cycle. Mother rats had free access to clean water and standard feed. After acclimatizing to the experimental environment for 6 days, male pups aged 9-10 days (postnatal day, PND9-10) were selected for the experiment. The rats were randomly divided into a sham surgery group and a traumatic brain injury (TBI) group, with 6 rats in each group. All experimental protocols were approved by the Animal Ethics Committee of Chongqing Medical University.

[0023] 1.2 Establishment of the TBI Model A brain injury model in immature rats was established using controlled cortical impact (CCI) technology. Surgical instruments were sterilized 24 hours in advance. Young rats were anesthetized with 1.5% tribromoethanol (0.01 ml / g) via intraperitoneal injection. After confirming the anesthesia was effective, the tongue was pulled back to maintain airway patency. A 1.5 cm incision was made along the midline of the skull to expose the skull. A bone window of approximately 3 mm in diameter was drilled between the coronal and lambdoid sutures, 2 mm to the left of the sagittal line, avoiding damage to the dura mater. The young rats were fixed in the trauma instrument, and the brain tissue was impacted with a 3 mm probe at a speed of 4.5 m / s, a depth of 1.5 mm, and a duration of 50 ms. Immediately after impact, the young rats were removed, airway patency was confirmed again, hemostasis was achieved, bone fragments were reinserted, and the scalp was sutured. The area was disinfected with iodine. Postoperatively, the rats were placed on a warming blanket and monitored until awakening, then returned to their mothers. The Sham group underwent only craniotomy without impact; the remaining procedures were the same as the trauma group.

[0024] 1.3 Transcriptome Sequencing and Analysis Workflow On day 3 after TBI, cortical tissue from the injured side of each group of young mice was collected, flash-frozen in liquid nitrogen, and stored at -80℃. Total RNA was extracted using a modified TRIzol method, and purified by tissue homogenization, chloroform layering, isopropanol precipitation, and ethanol washing. Finally, the concentration, purity, and integrity were determined using NanoDrop 2000 and Agilent 2100, respectively.

[0025] Qualified samples were sent to MegiGene for transcriptome sequencing. First, rRNA was removed using the Ribo-Zero kit, and linear RNA was digested with RNase R to enrich circular RNA. Subsequently, the RNA was fragmented and reverse transcribed into double-stranded cDNA, which underwent end repair, A-addition, adapter ligation, and PCR amplification to construct a library. Finally, PE150 sequencing was performed on the Illumina Novaseq 6000 platform, yielding approximately 60M reads per sample.

[0026] In bioinformatics analysis, raw data, after quality control and adapter removal, were aligned to the rat reference genome using Hisat2, and transcript assembly and expression quantification were performed using StringTie and RSEM. Differentially expressed genes (|log2FC|>1 and P<0.05) were functionally enriched using GO and KEGG. Furthermore, circRNAs, miRNAs, and lncRNAs were identified using CIRI2 and miRDeep2 software, and their differential expression was analyzed.

[0027] 1.4 Western Blot Analysis Following TBI, 1-, 3-, 7-, and 30-day-old mice were rapidly decapitated after deep anesthesia. Brain tissue was extracted, rinsed in ice-cold PBS, and the cortical tissue (approximately 3mm × 3mm × 3mm) from the injured side was quickly dissected and separated on ice. Samples were flash-frozen in liquid nitrogen and stored at -80°C. For protein extraction, tissue was added to pre-chilled RIPA lysis buffer containing protease and phosphatase inhibitors at a weight-to-volume ratio of 1:10. After homogenization using a tissue homogenizer and incubation on ice, the supernatant was obtained by centrifugation at 13,000 rpm for 13 minutes at 4°C. Cell samples were washed with PBS and then directly lysed and centrifuged. Protein concentration was determined using the BCA method. An equal volume of protein (30-50 μg) was mixed with loading buffer and denatured by boiling at 95°C. Subsequently, SDS-PAGE electrophoresis was performed (target protein was separated using an 11% separating gel), and the sample was transferred to a PVDF membrane at a constant current of 300 mA on ice for 120 minutes. After membrane blocking, the membrane was incubated overnight at 4°C with primary antibody, followed by incubation at room temperature for 1 hour with HRP-labeled secondary antibody. Finally, the membrane was developed using ECL chemiluminescence reagent, imaged using the ChemiDoc XRS+ system, and the grayscale ratio of the target protein to the internal control GAPDH was analyzed using Image Lab software.

[0028] 1.5 Immunofluorescence staining Young mice 1, 3, 7, and 30 days after TBI were randomly selected and subjected to cardiac perfusion under deep anesthesia. The tissues were first rinsed with physiological saline, followed by perfusion with 4% paraformaldehyde until rigidity was achieved. Brain tissues were then removed and fixed in paraformaldehyde of the same concentration for 24 hours, rinsed with PBS, and then dehydrated in 30% sucrose solution until set. After dehydration, the tissues were embedded in OCT, flash-frozen at -80°C, cut into 25 μm thick sections using a cryostat, and stored in cryoprotectant for later use.

[0029] For immunofluorescence staining, sections were washed with PBS, permeabilized with 0.3% Triton X-100, and blocked with 5% BSA. They were then incubated overnight at 4°C with the primary antibody, followed by incubation at room temperature in the dark for 2 hours with the corresponding secondary fluorescent antibody. Finally, cell nuclei were counterstained with DAPI, and sections were mounted with an anti-quenching agent. All sections were imaged using a Leica SP8 confocal microscope at the same parameters, and the average fluorescence intensity of the target protein was analyzed using ImageJ software.

[0030] 1.6 SH-SY5Y cell culture and transfection SH-SY5Y cells were resuscitated and cultured routinely at 37°C and 5% CO2. When the cells reached 80–90% confluence, they were passaged using 0.25% trypsin. 24 hours before the experiment, cells in the logarithmic growth phase were passaged at 2 × 10⁻⁶ cells / year. 5Cells were seeded at a density of / wells in 6-well plates. Transfection was performed using Lipofectamine 3000 reagent: 5 μl of transfection reagent and 20 nM mixed siRNA (three sequences targeting the DDC gene or a negative control, sequences shown in the table) were diluted separately in 125 μl of Opti-MEM, mixed, and incubated at room temperature for 15 minutes to form a complex. Cells were then replaced with serum-free medium, and after incubation with the transfection complex for 6 hours, the medium was replaced with complete medium and cultured for another 18 hours.

[0031] siRNA sequence 24 hours after transfection, the following treatment groups were set up: Control (routine culture), LPS (1 μg / ml), siRNA-NC+LPS, siRNA-DDC+LPS, siRNA-DDC+FTY720+LPS (pretreated with 2 μM FTY720 for 2 hours) and FTY720 alone. Each treatment lasted for 24 hours.

[0032] 1.7 Real-time quantitative PCR Total RNA was extracted from brain tissue or cells using the TRIzol method. After TRIzol lysis and chloroform separation centrifugation, the upper aqueous phase was collected and RNA was precipitated with isopropanol. The precipitate was then washed with 75% ethanol, dried, and dissolved in DEPC water. The concentration and purity were determined using a NanoDrop 2000. 1 μg of total RNA was used to synthesize cDNA using a GoScript reverse transcription kit. The reaction volume was 20 μL, and the program was set to 25℃ for 5 minutes, 42℃ for 60 minutes, and 70℃ for 15 minutes.

[0033] Using cDNA as a template, amplification was performed using GoTaq qPCR premixed reagents. The total reaction volume was 20 μL, containing 10 μL of 2× Master Mix, 0.4 μL each of forward and reverse primers, and 2 μL of cDNA. IL-18, IL-1β, and GAPDH-specific primers (sequences shown in Table 2-2) were used, and the reaction was run at 95℃ for 10 minutes, followed by 95℃ for 15 seconds and 60℃ for 1 minute, for a total of 40 cycles.

[0034] Primer sequences used in qPCR 1.8 Statistical Analysis All experiments were independently repeated at least three times. Data are expressed as mean ± standard error (Mean ± SEM). Statistical analysis was performed using GraphPad Prism 9.0 software. Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups, followed by Tukey's multiple comparison test. P < 0.05 was considered statistically significant, with the following significance levels: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. In figures and tables, ns indicates no statistical difference (P > 0.05).

[0035] 2. Results 2.1 Overall Changes in Cerebral Cortical Gene Expression Profiles After TBI To elucidate the molecular mechanisms of brain atrophy following tender endothelial brain injury (TBI) in immature brains, this invention performed transcriptome sequencing analysis on brain tissue from rats in the TBI and Sham groups. The raw data were filtered for quality control before transcriptome analysis, identifying 43,875 mRNA transcripts. Using |log2FC|>1 and P<0.05 as the threshold, 644 differentially expressed genes were screened between the TBI and Sham groups, including 339 upregulated genes and 305 downregulated genes. Heatmap clustering analysis of the differentially expressed genes showed that the TBI and Sham group samples were clearly distinguishable based on gene expression patterns, indicating that systematic changes occurred in the transcriptome level after TBI in immature brains, and these changes may be closely related to the occurrence and development of brain atrophy.

[0036] 2.2 Differentially expressed genes reveal TBI's influence on neural development and serotonergic synaptic pathways To investigate the biological functions of differentially expressed genes, this invention performed GO and KEGG enrichment analyses. GO analysis showed that differentially expressed genes were mainly enriched in cell surface receptor signaling pathways, Wnt signaling pathways, and developmental processes in terms of biological processes; concentrated in local focal adhesion, cell matrix junctions, and cell processes in terms of cellular components; and involved in adenosine monophosphate binding and protein domain-specific binding in terms of molecular function, suggesting that TBI significantly affects neuronal development, synaptic plasticity, and intercellular communication. KEGG pathway analysis further revealed that, in addition to the Wnt signaling pathway, the serotonergic synaptic pathway was also significantly enriched. This pathway is involved in important processes such as neuronal differentiation, migration, axonal guidance, and synapse formation, further indicating that TBI may affect the recovery of neural function and the reconstruction of neural circuits by regulating key signaling networks such as serotonergic synapses.

[0037] 2.3 Discovery of the DDC gene in the 5-hydroxytryptaminergic synaptic pathway To screen for key genes potentially involved in brain atrophy following tender brain injury (TBI) in immature brains, this invention focuses on analyzing differentially expressed genes in the serotonergic synaptic pathway. This invention found that the expression level of the dopa decarboxylase (DDC) gene was significantly higher in the TBI group than in the Sham group, with a fold change of up to 172.28. Furthermore, this gene is located at a key node in this pathway and participates in the synthesis of the neurotransmitters serotonin (5-HT) and dopamine (DA). According to the serotonergic synaptic signaling pathway, DDC can catalyze the generation of 5-HT from 5-HTP in serotonergic neurons, which is stored in vesicles. These vesicles fuse with the presynaptic membrane, releasing 5-HT into the synaptic cleft via exocytosis. 5-HT then binds to 5-HT receptors (5-HTR) on the postsynaptic membrane of neurons, regulating downstream pathways. Meanwhile, the binding of 5-HT and 5-HTR can stimulate dopaminergic neurons to release DA. According to the dopaminergic synapse signaling pathway, DA binds to the D2 receptor and regulates GSK-3β (GSK-3β is a key enzyme in the hyperphosphorylation of Tau protein), thereby promoting the hyperphosphorylation of Tau protein.

[0038] The above results indicate that the significant upregulation of DDC expression after TBI can simultaneously affect the serotonin and dopamine systems, thereby regulating downstream GSK-3β activity. This may be a key molecular mechanism explaining brain atrophy in immature brains after TBI.

[0039] 2.4 Upregulation of DDC expression after TBI in immature brains To verify the accuracy of the transcriptome sequencing results, this invention first detected the changes in the expression of DDC at different time points after TBI in immature brains at the protein level.

[0040] 2.5 Increased phosphorylation levels of DDC and Tau proteins after TBI in immature brains Given that Tau protein hyperphosphorylation is a significant factor leading to neuronal death and brain atrophy, this invention further investigated the relationship between DDC and Tau protein phosphorylation. The expression patterns of DDC and phosphorylated Tau protein (pTau) after TBI were observed using immunofluorescence. Simultaneously, the expression of Tau protein phosphorylated at the Thr-231 site was also examined. Thr-231 is a normal site regulating Tau protein microtubule stability and axonal transport. The results showed that the fluorescence signal of pTau in the TBI group was significantly stronger than that in the Sham group. These results indicate that the trends in the expression levels of DDC and pTau are consistent, suggesting that Tau protein hyperphosphorylation after TBI in immature brains may be an important factor leading to neuronal death and brain atrophy.

[0041] To further verify the association between DDC and Tau protein phosphorylation, this invention further observed the expression patterns and distribution relationship between DDC and phosphorylated Tau protein after TBI by immunofluorescence co-staining.

[0042] 2.6 DDC mediates Tau protein phosphorylation via GSK-3β To investigate the relationship and potential mechanism between DDC and Tau protein phosphorylation, in vitro cell experiments were conducted in this invention. Using SH-SY5Y cells, multiple experimental groups were established: Control group (normal culture), LPS group (LPS induction for 24 h), siRNA-NC+LPS group (LPS induction for 24 h after transfection with non-targeting siRNA), siRNA-DDC+LPS group (LPS induction for 24 h after transfection with DDC-targeting siRNA), siRNA-DDC+FTY720+LPS group (LPS induction for 24 h after transfection with DDC-targeting siRNA and pretreatment with FTY720 for 2 h), and FTY720 group (FTY720 only).

[0043] RT-qPCR results showed that, compared with the control group, the DDC mRNA level in the LPS group was significantly increased; compared with the LPS group, the DDC mRNA expression in the siRNA-DDC+LPS group was significantly decreased, indicating that siRNA successfully inhibited DDC expression; while there was no significant difference in DDC mRNA level between the siRNA-DDC+FTY720+LPS group and the siRNA-DDC+LPS group, indicating that the GSK-3β activator FTY720 does not affect DDC expression, suggesting that GSK-3β may be located downstream of DDC.

[0044] Subsequently, the phosphorylation levels of GSK-3β and Tau proteins in each group were detected by Western blotting. The results showed that, compared with the Control group, the expression levels of p-GSK-3β, p(Ser-404)-Tau, and p(Thr-231)-Tau were significantly increased in the LPS group; compared with the LPS group, the phosphorylation level in the siRNA-DDC+LPS group was significantly decreased; while in the siRNA-DDC+FTY720+LPS group, the phosphorylation levels of GSK-3β and Tau proteins increased again. This series of changes clearly indicates that DDC silencing can effectively inhibit LPS-induced phosphorylation of GSK-3β and Tau proteins, and the GSK-3β agonist FTY720 can reverse the inhibitory effect of DDC silencing on the phosphorylation of GSK-3β and Tau proteins.

[0045] The above results strongly demonstrate that DDC affects the phosphorylation state of Tau protein by regulating GSK-3β activity.

[0046] This invention, using transcriptome sequencing technology, is the first to discover that the DDC gene is significantly upregulated after tender brain injury (TBI) in immature brains, suggesting that it may be a key regulator of brain atrophy after TBI. This discovery is highly innovative because previous studies have largely focused on processes such as inflammation, oxidative stress, and apoptosis after TBI, with less attention paid to the role of neurotransmitter synthases in brain injury. This invention provides a novel perspective, emphasizing the potential value of neurotransmitter synthesis pathways in the pathological process of TBI in immature brains.

[0047] Through systematic in vitro and in vivo experiments, this invention, for the first time, demonstrates the molecular mechanism by which DDC affects Tau protein phosphorylation by regulating GSK-3β activity after TBI in immature brains, revealing the core role of the DDC-GSK-3β-Tau signaling axis in brain atrophy after TBI. Experiments show that DDC silencing significantly inhibits LPS-induced GSK-3β phosphorylation, and the GSK-3β agonist FTY720 can reverse this effect, proving that DDC is upstream of GSK-3β and can positively regulate its activity. Since GSK-3β phosphorylation level is negatively correlated with its activity, DDC may enhance its activity by promoting GSK-3β dephosphorylation, ultimately leading to excessive Tau protein phosphorylation. This mechanism is supported by multiple pieces of evidence: first, Kow et al. found that DDC gene deletion can inhibit Tau protein toxicity in a *C. elegans* model; second, GSK-3β has been identified as one of the main kinases for Tau protein phosphorylation; furthermore, this invention observes a spatiotemporal consistency between DDC upregulation and increased Tau protein phosphorylation levels in an immature brain TBI model.

[0048] Based on transcriptome sequencing results showing that DDC is significantly upregulated in the serotonergic synaptic signaling pathway, this invention explores the key role of serotonin in connecting DDC and GSK-3β. Studies have found that DDC-regulated serotonin metabolism may regulate the activation of the NLRP3 inflammasome by affecting cAMP levels, and there is a known signaling interaction between NLRP3 and GSK-3β

[20] . This suggests that DDC overexpression may activate the NLRP3 inflammasome by increasing serotonin synthesis, thereby affecting GSK-3β activity and ultimately leading to the complete signaling pathway of Tau protein hyperphosphorylation.

[0049] In-depth research on the functional characteristics of DDC revealed that it exhibits a significant dual role and spatiotemporal specificity. In the acute phase of injury, DDC upregulation may be a compensatory protective mechanism, maintaining neuronal function by increasing neurotransmitter synthesis; however, long-term high expression may lead to neurotransmitter metabolism imbalance, triggering excitotoxicity through excessive receptor activation

[21] . Especially in the special developmental stage of the immature brain, DDC upregulation is more likely to represent a pathological response rather than a protective mechanism, which explains why the brain atrophy process after TBI in immature brains differs significantly from that in adults.

[0050] From a clinical application perspective, the DDC-GSK-3β-Tau signaling axis discovered in this invention has significant translational medical value. DDC, as a drug-targetable enzyme, has inhibitors such as carbidopa and benserazide widely used in the treatment of Parkinson's disease, demonstrating good safety and efficacy. The experiments in this invention confirm that inhibiting DDC expression significantly reduces GSK-3β activation and Tau protein phosphorylation, providing a new potential target for the treatment of TBI in immature brains. However, considering the significant differences between immature and mature brains in neural development, synaptic density, and drug responsiveness, special attention needs to be paid to the timing of administration, dosage selection, and long-term safety when developing therapeutic strategies targeting DDC to ensure that neuroprotective effects are achieved while avoiding adverse effects on normal brain development.

[0051] This invention, by integrating transcriptomics, molecular biology, and functional experiments, systematically elucidates for the first time the crucial role and molecular mechanism of dihydrotestosterone (DDC) in brain atrophy following transient ischemic attack (TBI) in immature brains. This not only deepens our understanding of the pathological process of TBI in immature brains but also provides theoretical basis and experimental support for developing new treatment strategies. Future research will focus on optimizing the dosing regimen of DDC inhibitors and further exploring the correlation between DDC and other neurodegenerative processes to promote the translation of this discovery into clinical applications.

[0052] Transcriptome sequencing analysis revealed that dopa decarboxylase (DDC) was significantly upregulated in immature brains after transurethral resection of brain (TBI). As a key enzyme in the serotonergic synaptic pathway, DDC participates in neurotransmitter synthesis and metabolism, and may be involved in brain atrophy following TBI in immature brains by regulating Tau protein phosphorylation.

[0053] DDC (diuretic dystrophy) participates in brain atrophy following tender brain injury (TBI) in immature brains by activating GSK-3β and promoting Tau protein phosphorylation. In vitro experiments have confirmed that DDC silencing effectively inhibits LPS-induced GSK-3β and Tau protein phosphorylation, while GSK-3β agonists can reverse the effect of DDC silencing.

[0054] These results indicate that the DDC-GSK-3β-Tau signaling axis plays an important role in brain atrophy following TBI in immature brains, providing a theoretical basis for developing novel treatment strategies.

[0055] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of DDC silencing in a brain atrophy model, characterized by, The application aims to reduce or prevent brain atrophy by inhibiting DDC gene expression or protein activity and regulating GSK-3β kinase-mediated Tau protein phosphorylation. The brain atrophy model is an immature brain traumatic brain injury (TBI) brain atrophy model, and DDC silencing is achieved through siRNA, shRNA, or antisense oligonucleotides.

2. The application of DDC silencing in a brain atrophy model according to claim 1, characterized in that, The DDC silencing uses an siRNA sequence, which is selected from any of the following: Sequence 1: 5'-GUGAUCCAUCGGCUGCAGG-3', Sequence 2: 5'-GUCGGUCCUAUCUGCAACA-3', Sequence 3: 5'-GAGCUGGGUUAAUUGGUGG-3', Or its functionally equivalent variants.

3. The application of DDC silencing in a brain atrophy model according to claim 1, characterized in that, The application includes silencing DDC expression in an in vitro or in vivo model, wherein the in vivo model is an immature rat TBI model established by controlled cortical impaction technique with impaction parameters of 5.5 m / s velocity, 1.5 mm depth and 50 ms duration.

4. The application of DDC silencing in a brain atrophy model according to claim 1, characterized in that, DDC silencing is achieved by inhibiting GSK-3β phosphorylation, where the level of GSK-3β phosphorylation is negatively correlated with the phosphorylation level of Tau protein at Ser-404 and Thr-231 sites; and the DDC silencing can be reversed by the GSK-3β agonist FTY720.

5. The application of DDC silencing in a brain atrophy model according to claim 1, characterized in that, The application also includes detecting DDC expression levels or Tau phosphorylation levels as biomarkers to evaluate the efficacy of brain atrophy treatment; wherein the detection methods include Western blot, immunofluorescence, or RT-qPCR.

6. The application of DDC silencing in a brain atrophy model according to claim 1, characterized in that, It is also used in neurodegenerative disease models, which are associated with Tau protein hyperphosphorylation, including Alzheimer's disease or Parkinson's disease; and DDC silencing affects the dopamine system through serotonergic synaptic pathways.

7. A pharmaceutical composition, characterized in that, The composition comprises a DDC silencer and a pharmaceutically acceptable carrier, wherein the DDC silencer is the siRNA sequence of claim 2 in the application of DDC silencing in a model of brain atrophy, and the composition is used to prevent or treat brain atrophy, which is progressive brain atrophy following immature brain TBI.

8. The pharmaceutical composition according to claim 7, characterized in that, It also contains a GSK-3β modulator, which is an agonist or inhibitor, and the composition is administered by intravenous injection, topical administration or oral administration.

9. A method for preparing a DDC silencer, characterized in that, The method includes synthesizing the siRNA sequence of claim 2 for the application of DDC silencing in a brain atrophy model, and packaging it via liposomes or viral vectors for transfection of cells or tissues; wherein the preparation method includes in vitro transfection using Lipofectamine 3000 reagent.

10. A brain atrophy treatment system, characterized in that, The system includes a DDC silencing module and a GSK-3β regulation module, wherein the DDC silencing module implements the application of DDC silencing as described in any one of claims 1-6 in a brain atrophy model, and the GSK-3β regulation module uses FTY720; the system is used for combined treatment of chronic brain atrophy after TBI in immature brains.

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