Use of taf15 as a target site in the preparation of a drug for treating stroke

Abstract

The application relates to the technical field of biological pharmacy, and particularly relates to application of TAF15 as a target site in preparation of a drug for treating cerebral stroke. The application first systematically clarifies the biological function of the gene in cerebral ischemia and reperfusion injury, confirms that the gene is significantly up-regulated in a MCAO / R model brain tissue, and down-regulation of the expression of the gene can significantly improve neuromotor dysfunction, reduce cerebral infarction volume, reduce neuron pathological injury and apoptosis; the application first discloses a molecular mechanism that TAF15 participates in the pathological process of ischemic cerebral stroke by regulating an iron death signal path. Down-regulation of TAF15 can reduce Fe 2+ , MDA contents in the brain tissue, increase SOD activity and GSH content, down-regulate the expression of a pro-iron death gene, up-regulate the expression of an anti-iron death gene, thereby inhibiting iron death and playing a neuroprotective role.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to the application of TAF15 as a target site in the preparation of drugs for treating stroke. Background Technology

[0002] Ischemic stroke (IS) is an acute cerebrovascular disease caused by stenosis or occlusion of cerebral arteries, leading to insufficient blood supply to the local brain tissue and resulting in neuronal damage, necrosis, and neurological dysfunction. It is characterized by high incidence, high disability rate, high mortality rate, and high recurrence rate. Currently, clinical treatment for ischemic stroke mainly includes intravenous thrombolysis, endovascular mechanical thrombectomy, and antiplatelet therapy, as well as measures to improve cerebral circulation and neuroprotection. However, existing revascularization treatments are limited by strict treatment time windows, indications, and bleeding risks. Even after reperfusion is achieved, some patients may still experience significant secondary brain tissue damage.

[0003] Following cerebral ischemia, brain tissue energy metabolism rapidly becomes unbalanced, inducing oxidative stress, inflammatory responses, mitochondrial dysfunction, excitatory amino acid toxicity, and various forms of cell death. After blood flow is restored, these damaging responses may further intensify, forming a complex process of cerebral ischemia-reperfusion injury. This process is influenced by a multi-level molecular network involving transcriptional regulation, post-transcriptional regulation, and epigenetic regulation. Among these, RNA-binding proteins (RBPs) recognize and bind to specific RNA molecules, participating in RNA splicing, stability maintenance, nucleoplasmic transport, localization, and translation, playing a crucial role in maintaining cellular homeostasis and regulating stress responses. Increasing research indicates that abnormal post-transcriptional regulation mediated by RNA-binding proteins is closely related to neuronal damage, inflammatory responses, and cell death, thus potentially serving as an important molecular basis for intervention in ischemic cerebral diseases.

[0004] TAF15 (TATA-box binding protein associated factor 15) is a nucleic acid-binding protein capable of binding to both RNA and DNA, belonging to the FET protein family. TAF15 contains an RNA recognition-associated domain and participates in various biological processes, including RNA metabolism, gene transcription regulation, RNA processing, and RNA stability regulation. When cells are subjected to hypoxia, oxidative stress, and other damaging stimuli, the expression level, subcellular localization, and interaction with target RNA of RNA-binding proteins may change, thereby affecting cellular stress response and survival. As an important RNA-binding regulator, TAF15 has the potential to participate in disease progression by regulating the stability and expression levels of target gene RNA.

[0005] Current research on TAF15 primarily focuses on RNA metabolism regulation, tumor development, and certain neurological diseases. However, the expression changes, functional roles, and molecular regulatory mechanisms of TAF15 in ischemic stroke, particularly in cerebral ischemia-reperfusion injury, remain largely unclear. Furthermore, existing technologies lack effective techniques to target TAF15 as a therapeutic target for ischemic stroke and to mitigate brain tissue damage and protect neuronal function by regulating its expression or activity. Therefore, clarifying the role of TAF15 in the development of ischemic stroke and developing therapeutic drugs or interventions targeting TAF15 holds potential value for the prevention and treatment of ischemic stroke. Summary of the Invention

[0006] Given the lack of existing technologies that target TAF15 for the treatment of ischemic stroke, and the lack of clarity regarding the expression changes, functional roles, and molecular regulatory mechanisms of TAF15 in cerebral ischemia-reperfusion injury, the present invention aims to provide the application of TAF15 as a target site in the preparation of drugs for treating stroke, clarify the pro-injury role of TAF15 in the occurrence and development of ischemic stroke, and develop therapeutic drugs or interventions targeting TAF15, thereby providing new molecular targets and technical solutions for the prevention and treatment of ischemic stroke.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The application of an inhibitor targeting TAF15 in the preparation of a drug for treating stroke, wherein the nucleotide sequence of the TAF15 gene is shown in SEQ ID NO:1.

[0009] Furthermore, the TAF15 inhibitor is a substance that can reduce TAF15 expression levels, inhibit TAF15 activity, or block TAF15-related biological functions.

[0010] Furthermore, the TAF15 inhibitor is selected from at least one of shRNA, siRNA, antisense oligonucleotide, gene editing vector nucleic acid aptamer, and small molecule inhibitors that target TAF15.

[0011] Furthermore, the drug is an oral formulation, an injectable formulation, or other pharmaceutically acceptable drug formulation.

[0012] Furthermore, the drug is administered after ischemic stroke or during cerebral ischemia-reperfusion injury. Furthermore, the target sequence of the shRNA is the nucleotide sequence shown in SEQ ID NO:2, or is transcribed from the nucleotide sequence shown in SEQ ID NO:2.

[0013] The present invention also includes the application of the TAF15 gene overexpression reagent in the preparation of in vitro cell models of ischemic stroke.

[0014] The present invention also includes the TAF15 gene overexpression reagent described above, which is used to promote the release of lactate dehydrogenase and accelerate PC12 cell apoptosis.

[0015] This invention also includes the TAF15 gene overexpression reagent for increasing Fe 2+ Content and MDA content.

[0016] The present invention also includes the TAF15 gene overexpression reagent used to inhibit SOD activity and GSH content.

[0017] The present invention has the following beneficial effects.

[0018] This invention systematically elucidates for the first time the biological function of the TAF15 gene in cerebral ischemia-reperfusion injury. By constructing a rat middle cerebral artery embolism-reperfusion (MCAO / R) model, it was confirmed that TAF15 is significantly upregulated in brain tissue after cerebral ischemia-reperfusion injury. TAF15 overexpression significantly aggravates PC12 cell damage, manifested as decreased cell viability, increased LDH release, and increased apoptosis rate. Furthermore, this application reveals the molecular mechanism by which TAF15 participates in the pathological process of ischemic stroke by regulating the ferroptosis signaling pathway. In vitro and in vivo experiments confirmed that TAF15 overexpression significantly increases intracellular Fe... 2+ Accumulation, MDA levels, and ROS production, along with decreased SOD activity and GSH levels, indicate that TAF15 can exacerbate oxidative stress and lipid peroxidation damage. Furthermore, at the cellular level, TAF15 overexpression significantly aggravates OGD / R-induced PC12 cell damage, apoptosis, and abnormalities in ferroptosis-related biochemical indicators. At the animal level, intraventricular injection of AAV-shTAF15 to silence TAF15 expression significantly improved neuromotor function (significantly reduced mNSS score) and significantly reduced cerebral infarction volume in MCAO / R rats. These in vitro and in vivo results corroborate each other, suggesting that inhibitors targeting TAF15 can effectively alleviate cerebral ischemia-reperfusion injury, demonstrating good drug development potential and clinical application prospects. Attached Figure Description

[0019] Figure 1 The image shows the results of the CCK-8 assay for detecting the viability of PC12 cells after OGD / R intervention following TAF15 overexpression.

[0020] Figure 2 The figure shows the results of quantitative analysis of LDH content, a marker of PC12 cell damage induced by OGD / R after TAF15 overexpression.

[0021] Figure 3The figure shows the results of flow cytometry analysis of OGD / R-induced apoptosis in PC12 cells after TAF15 overexpression; in the figure, A is a scatter plot of flow cytometry and B is a bar chart of statistical analysis of cell apoptosis rate.

[0022] Figure 4 To detect OGD / R-induced Fe after TAF15 overexpression using the kit 2+ Figure 1 shows the analysis results of the content of SOD, MDA and GSH, indicators of oxidative stress damage; A in the figure represents Fe. 2+ A) Content results chart, B) MDA content results chart, C) SOD content results chart, D) GSH content results chart.

[0023] Figure 5 The figure shows the results of ROS content analysis induced by OGD / R after TAF15 overexpression, detected by ROS probes; in the figure, A is a fluorescence microscope image and B is a quantitative analysis of fluorescence intensity.

[0024] Figure 6 The graph shows the modified neurological severity score (mNSS) results of rats in each group after MCAO / R surgery.

[0025] Figure 7 This is a graph showing the quantitative analysis of cerebral infarction volume using TTC staining. Detailed Implementation

[0026] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0027] Unless otherwise stated, each feature disclosed in this specification is merely one example of a series of equivalent or similar features.

[0028] Example 1.

[0029] This example uses the TAF15 nucleotide sequence:

[0030] The nucleotide sequence of TAF15 of the present invention is shown in SEQ ID NO:1.

[0031] Example 2

[0032] This example illustrates an animal experiment using the TAF15 gene as a drug, as detailed below:

[0033] Rat adrenal pheochromocytoma PC12 cells (Procell, CL-0480, Wuhan, China) were used in the experiment. After cell resuscitation, cells were cultured in DMEM high-glucose complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, and placed in a cell culture incubator at 37℃, 5% CO2, and saturated humidity. During culture, cell growth was observed every 1-2 days, and fresh complete medium was replaced according to the color of the culture medium and cell growth. When the cells reached approximately 80% confluence, they were passaged. At passage, the original culture medium was discarded, and the cells were washed twice with sterile PBS, digested with an appropriate amount of trypsin digestion solution, and digestion was stopped by adding complete medium after the cells became rounded and detached. The cells were gently pipetted and a homogeneous single-cell suspension was prepared. After centrifugation and resuspending, the cells were passaged at an appropriate ratio. PC12 cells in the logarithmic growth phase and in good growth condition were used for subsequent experiments.

[0034] The OGD / R process was used to simulate an in vitro ischemia-reperfusion injury model. The specific procedures are as follows:

[0035] (1) Pretreatment and preparation: Before the experiment, PC12 cells were gently washed twice with glucose-free Dulbecco's Modified Eagle's Medium (DMEM) to remove residual serum and sugar sources.

[0036] (2) Hypoxia-glucose deprivation treatment (OGD): Immediately after washing, the cells were immersed in glucose-free DMEM, and the cell culture plates were placed in a pre-set anaerobic incubator. The gas environment of the anaerobic incubator was adjusted to 5% CO2 and 95% N2, the temperature was maintained at a constant 37°C, and the humidity was maintained under standard culture conditions. The cells were incubated in this environment for 4 hours to induce neurons to enter a hypoxic-glucose deprivation state, thereby simulating the process of cerebral ischemia.

[0037] (3) Reperfusion Recovery Treatment: After 4 hours of oxygen-glucose deprivation treatment, PC12 cells were immediately removed from the hypoxic culture environment. The glucose-free medium was discarded and replaced with DMEM complete medium containing normal concentrations of glucose and serum to simulate the reoxygenation process after blood flow recovery. The cells were then placed back into a cell culture incubator at 37°C, 5% CO2, and normal oxygen concentration for continued culture. A 24-hour reoxygenation time point was set, and cells or culture supernatant were collected after the end of culture for subsequent detection. PC12 cells in the normal control group (Control group) underwent synchronous culture and medium replacement, but did not receive oxygen-glucose deprivation treatment. Control group cells were always cultured at 37°C, 5% CO2, and normal oxygen concentration, and were synchronously treated for the same time using DMEM medium containing normal concentrations of glucose to eliminate the non-specific effects of medium replacement, culture time, and operation process on the experimental results.

[0038] Example 2

[0039] This example describes the PC12 cell grouping and TAF15 overexpression transfection experiment, as detailed below:

[0040] PC12 cells were extracted and cultured and then randomly divided into three groups: blank control group (Control), OGD / R model + overexpression negative control group (OGD / R+oe-NC), and OGD / R model + overexpression TAF15 group (OGD / R+oe-TAF15).

[0041] Administration method: Neurons were seeded at approximately 60-70% confluence in Poly-D-lysine-coated culture plates and transfected after stable adhesion. Once the cells had stabilized and reached suitable confluence, viral vectors carrying either a negative control sequence or a TAF15 overexpression sequence were added to the OGD / R+oe-NC group and the OGD / R+oe-TAF15 group, respectively, according to the viral infection conditions determined in preliminary experiments. After transduction, the cells were cultured until TAF15 expression reached a stable level, and the TAF15 overexpression efficiency was verified using qRT-PCR and Western blot. Subsequently, OGD / R treatment was performed according to the experimental groups, and cells and culture supernatants were collected for subsequent functional and biochemical assays.

[0042] Example 3

[0043] This example demonstrates a cell viability assay (CCK-8 assay), as detailed below:

[0044] Healthy PC12 cells were seeded at an appropriate density in 96-well plates, with multiple replicates per group. After cell adhesion and stable growth, transduction with either a negative control lentivirus (oe-NC) or a TAF15 overexpression lentivirus (oe-TAF15) was performed according to the experimental groups. Once viral transduction was complete and stable TAF15 overexpression was confirmed, OGD / R treatment was applied to the corresponding experimental groups. Control group cells were cultured under normal conditions without OGD / R treatment or viral transduction; OGD / R+oe-NC group cells were transduced with the negative control lentivirus and then treated with OGD / R; OGD / R+oe-TAF15 group cells were transduced with the TAF15 overexpression lentivirus and then treated with the same OGD / R treatment.

[0045] After completing the appropriate treatment, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C and 5% CO2 for 1 hour. After incubation, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The relative cell viability of each experimental group was calculated using the control group as a reference.

[0046] The results are attached. Figure 1 As shown, compared with the Control group, the viability of PC12 cells in the OGD / R+oe-NC group was significantly decreased, indicating that OGD / R treatment can induce significant cell damage. Compared with the OGD / R+oe-NC group, the cell viability in the OGD / R+oe-TAF15 group was further reduced, indicating that TAF15 overexpression can aggravate OGD / R-induced PC12 cell damage. These experimental results suggest that increased TAF15 expression levels can further reduce the survival ability of PC12 cells under OGD / R conditions, implying that TAF15 plays a pro-cell damage role in ischemia-reperfusion-related cell injury.

[0047] Example 4

[0048] This example describes an experiment to determine the release level of lactate dehydrogenase (LDH), as detailed below:

[0049] PC12 cells in good growth condition were seeded at an appropriate density (5 × 10³ cells / well) in 96-well plates. After the cells adhered stably and reached a suitable confluence, they were divided into three groups according to the experimental design: Control, OGD / R+oe-NC, and OGD / R+oe-TAF15. The OGD / R+oe-NC and OGD / R+oe-TAF15 groups were transduced using a negative control lentivirus and a TAF15 overexpression lentivirus, respectively. After viral transduction was complete and TAF15 expression was stable, OGD / R treatment was performed. Before OGD treatment, the original cell culture medium was discarded, and the cells were gently washed with sugar-free medium. Then, sugar-free medium was added, and the cells were placed under hypoxic conditions for oxygen-glucose deprivation treatment for 4 hours. Immediately after OGD treatment, the sugar-free medium was discarded and replaced with normal sugar-containing complete medium. The cells were then cultured again at 37°C, 5% CO2, and standard oxygen concentration for 24 hours to simulate the reperfusion process caused by the restoration of blood flow after ischemia. Cells in the Control group were cultured under normal conditions throughout and underwent synchronized medium replacement.

[0050] After 24 hours of reoxygenation, 50 μL of cell culture supernatant was collected from each well and transferred to a new 96-well plate for LDH release level detection. The corresponding working solution was prepared according to the LDH detection kit instructions. 50 μL of the reaction solution was added to each well, gently mixed, and incubated at room temperature in the dark for 30 minutes. After the reaction was complete, the stop solution was added to terminate the reaction as required by the kit, and the absorbance of each well at the corresponding wavelength was measured using a microplate reader. Background values ​​from blank wells were subtracted according to the method specified in the kit, and the relative LDH release levels for each group were calculated.

[0051] The results are attached. Figure 2As shown, compared with the Control group, the LDH release level in the culture supernatant of PC12 cells in the OGD / R+oe-NC group was significantly increased, indicating that OGD / R treatment led to impaired cell membrane integrity and caused significant cell damage. Compared with the OGD / R+oe-NC group, the LDH release level in the OGD / R+oe-TAF15 group was further increased, indicating that TAF15 overexpression can significantly aggravate OGD / R-induced PC12 cell damage. The above experimental results show that the increased TAF15 expression level is positively correlated with the degree of OGD / R-induced cell damage.

[0052] Example 5

[0053] This example demonstrates an experiment for detecting apoptosis in PC12 cells, as detailed below:

[0054] Apoptosis of PC12 cells in each group was detected by Annexin V-FITC / PI double staining combined with flow cytometry. PC12 cells in good growth condition were arranged at an appropriate density (2.5 × 10⁻⁶). 5 Cells were seeded at 1000 cells / well in 6-well plates. After the cells adhered stably and grew to a suitable confluence (80%), the experiment was divided into three groups: Control, OGD / R+oe-NC, and OGD / R+oe-TAF15. The OGD / R+oe-NC and OGD / R+oe-TAF15 groups were transduced using a negative control lentivirus and a TAF15-overexpressing lentivirus, respectively. After viral transduction was complete and TAF15 expression was stable, OGD / R treatment was performed, i.e., cells were deprived of oxygen and glucose for 4 hours, then returned to normal glucose-containing medium and conventional culture conditions for reoxygenation for 24 hours. Cells in the Control group were maintained under normal culture conditions throughout, without receiving OGD / R treatment or viral transduction.

[0055] After processing, suspended cells from the culture supernatant and adherent cells from the culture plates were collected separately. Adherent cells were digested using an appropriate method and then combined with suspended cells from the corresponding culture supernatant for collection. After centrifugation, the cells were gently washed twice with pre-cooled PBS, and then resuspended in binding buffer according to the Annexin V-FITC / PI apoptosis detection kit instructions. An appropriate amount of cell suspension was taken, and 5 μL of Annexin V-FITC and 10 μL of PI staining solution were added. After gentle mixing, the cells were incubated at room temperature in the dark for 20 min. After staining, flow cytometry was used to detect the staining within the specified time, and the Annexin V-FITC and PI fluorescence signals were analyzed. Cell states were differentiated based on Annexin V-FITC and PI staining: Annexin V - / PI - The cells are living cells, Annexin V+ / PI - The cells were early apoptotic cells, Annexin V + / PI + The cells were mainly late-stage apoptotic or secondary-stage necrotic cells. The total apoptosis rate was calculated as the sum of the proportions of early-stage and late-stage apoptotic cells.

[0056] The results are attached. Figure 3 As shown in the figure, A is a scatter plot of Annexin V-FITC / PI double staining flow cytometry, showing the distribution of PC12 cells in the Control group, OGD / R+oe-NC group, and OGD / R+oe-TAF15 group in four quadrants (lower left quadrant represents surviving cells, lower right quadrant represents early apoptotic cells, upper right quadrant represents late apoptotic / necrotic cells, and upper left quadrant represents mechanically damaged cells). B is a quantitative statistical bar chart of the total apoptosis rate of PC12 cells in each group (the sum of early and late apoptosis). It can be seen from the figure that compared with the Control group, the apoptosis rate of PC12 cells in the OGD / R+oe-NC group was significantly increased, indicating that OGD / R treatment can effectively induce significant apoptotic damage in PC12 cells. Compared with the OGD / R+oe-NC group, the apoptosis rate of cells in the OGD / R+oe-TAF15 group was further increased, indicating that TAF15 overexpression can promote cell apoptosis under OGD / R conditions. Flow cytometry results showed that cells in the Control group were mainly concentrated in the viable cell region, while the proportion of Annexin V positive cells increased significantly after OGD / R treatment. Under TAF15 overexpression, the proportion of early and late apoptotic cells further increased, suggesting that elevated TAF15 expression levels can exacerbate OGD / R-induced apoptosis in PC12 cells.

[0057] Example 6

[0058] This example uses a ROS fluorescent probe and Fe... 2+ The detection experiments for MDA, SOD, and GSH are as follows:

[0059] PC12 cells in the logarithmic growth phase and growing well were divided into three groups according to the experimental design: Control, OGD / R+oe-NC, and OGD / R+oe-TAF15. The OGD / R+oe-NC group and the OGD / R+oe-TAF15 group were transduced using a negative control lentivirus and a TAF15 overexpressing lentivirus, respectively. After the virus transduction was completed and TAF15 expression was stable, OGD / R treatment was performed, i.e., oxygen and glucose deprivation for 4 hours, followed by restoration to normal sugar-containing medium, and continued reoxygenation culture for 24 hours under standard culture conditions.

[0060] After treatment, the level of reactive oxygen species in cells of each group was detected using ROS fluorescent probes. The working solution of ROS fluorescent probes was added to the cells according to the kit instructions and incubated at 37°C in the dark. After washing away the free probes that did not enter the cells, the cells were observed and images were acquired using a fluorescence microscope, and the average fluorescence intensity was quantitatively analyzed.

[0061] Simultaneously, cells from each group were collected, washed with pre-cooled PBS, lysed, and centrifuged to collect the supernatant. Intracellular Fe was then measured according to the instructions of the corresponding detection kits. 2+ Content, MDA level, SOD activity, and GSH content. Results for each indicator were obtained according to the standard curve or the calculation method specified in the kit, and normalized based on the total protein concentration of the sample.

[0062] The results are attached. Figure 4 and attached Figure 5 As shown, Figure 4 To detect the Fe induced by OGD / R after TAF15 overexpression using the kit 2+ Figure 1 shows the analysis results of the content of SOD, MDA and GSH, indicators of oxidative stress damage; A in the figure represents Fe. 2+ A) Content results diagram, B) MDA content results diagram, C) SOD content results diagram, D) GSH content results diagram; As can be seen from the diagrams, compared with the Control group, the Fe content in the OGD / R+oe-NC group... 2+ The content and MDA level of SOD were significantly increased, while the SOD activity and GSH content were significantly decreased. Compared with the OGD / R+oe-NC group, the ROS level and Fe content of the OGD / R+oe-TAF15 group were significantly increased. 2+ The content and MDA level further increased, while SOD activity and GSH content further decreased; Figure 5 The figure shows the results of ROS content analysis induced by OGD / R after TAF15 overexpression, detected by ROS probe. In the figure, A represents representative ROS fluorescence microscopy images of PC12 cells after DCFH-DA probe staining in each group; B is a quantitative statistical bar chart of the average ROS fluorescence intensity in each group. As can be seen from the figure, compared with the Control group, the ROS fluorescence signal of PC12 cells in the OGD / R+oe-NC group was significantly enhanced. The bar chart shows that the average fluorescence intensity of the OGD / R+oe-NC group was significantly higher than that of the Control group, and the average fluorescence intensity of the OGD / R+oe-TAF15 group was further significantly higher than that of the OGD / R+oe-NC group. This indicates that TAF15 overexpression can exacerbate OGD / R-induced oxidative stress and Fe2+. 2+ Accumulation and lipid peroxidation damage further weaken the cell's antioxidant defense capabilities.

[0063] Example 7

[0064] This example studies the expression changes of TAF15 after cerebral ischemia-reperfusion injury in rats, as detailed below:

[0065] 1. This example describes the preparation of a focal cerebral ischemia-reperfusion animal model: a rat model of middle cerebral artery occlusion (MCAO) was established using the modified Longa suture occlusion method. Six- to eight-week-old male SD rats weighing 220-250g were selected and housed in a clean-grade environment.

[0066] The specific procedure is as follows: First, the hair on the rat's neck is cleaned with a shaver to expose the intact skin tissue. Then, the skin is carefully cut open with surgical scissors, making a 1.5cm longitudinal incision in the midline of the neck. At the same time, the surgical microscope is turned on, and under the microscope, the tissues around the rat's neck are carefully opened with relatively large forceps to avoid mechanical damage. The muscles below the trachea are separated to expose the common carotid artery (CCA). The muscles and fascia adhering to the common carotid artery are further dissected with microforceps. The bifurcation of the CCA can be seen upwards, and the internal carotid artery (ICA) and external carotid artery (ECA) can be seen further forward. At this time, the proximal end of the CCA and the ECA near the bifurcation are ligated. Since it is possible to avoid accidental suture embolism into the pterygopalatine artery (PPA), the PPA is not ligated. Next, a small incision was made in the blood vessel near the CCA ligation site using microscissors. A 0.3mm silicone-coated nylon suture was inserted into the CCA through the small incision. To prevent massive arterial bleeding during suture insertion, a slipknot was tied at the middle of the vessel before insertion. The slipknot was then loosened, and the suture was gently pushed forward along the course of the internal carotid artery. After passing the intersection of the ECA and ICA, it continued to be inserted along the course of the ICA until it reached the middle cerebral artery (MCA) of the rat. The insertion was stopped when slight resistance was felt and the suture could not be pushed further. Calculated from the bifurcation, the suture had entered the MCA approximately 12-14mm, essentially blocking the blood flow to the brain supplied by the middle cerebral artery. After 120 minutes of ischemia, the suture was slowly withdrawn, and the external carotid artery was ligated. Then, the suture at the common carotid artery was loosened to restore blood flow to the right carotid artery. The incision was sutured, disinfected with iodine, and the rat was placed in a cage and kept at 25°C. The MCAO model was then completed, thus establishing the cerebral ischemia-reperfusion model. Throughout the procedure, laser Doppler flowmeter was used to monitor local cerebral blood flow to ensure successful model establishment. From the start of surgery until the animals regained consciousness, a thermometer was used to maintain the rats' body temperature at 36.5℃±1℃. In the sham-operated group, after anesthesia, only the bifurcation of the internal and external carotid arteries was exposed; no suture embolism was inserted.

[0067] 2. Experimental grouping and drug administration:

[0068] ① Experimental grouping: After weighing, male SD rats were randomly divided into 3 groups: sham operation group (Sham), model + intervention negative control group (MCAO / R+AAV-NC), and TAF15 intervention treatment group (MCAO / R+AAV-shTAF15).

[0069] ② Construction of the therapeutic expression plasmid AAV-shTAF15: A recombinant expression plasmid targeting TAF15 was constructed using 5'-ATTGACTGGTTTGATGGAA-3' (SEQ ID NO:2) as the target sequence. The shRNA expression cassette is composed of the following elements connected in sequence: the positive strand coding sequence, whose nucleotide sequence is shown in SEQ ID NO:2; the hairpin circular structure sequence TTCAAGAGA; the antisense strand coding sequence, which is the reverse complementary sequence 5'-TTCCATCAAACCAGTCAAT-3' of the nucleotide sequence shown in SEQ ID NO:2; and the RNA polymerase III termination sequence TTTTTT. A sticky end sequence GATC (BamHI restriction site) was introduced at the 5' end of the positive strand coding sequence, and a base matching the EcoRI restriction site was introduced at the 3' end of the termination sequence; a base matching the BamHI restriction site was introduced at the 3' end of the antisense strand coding sequence, and a sticky end sequence AATT (EcoRI restriction site) was introduced at the 5' end of the complementary strand of the termination sequence. The corresponding forward and reverse oligonucleotides were synthesized, wherein the nucleotide sequence of the forward oligonucleotide is 5'-GATCCATTGACTGGTTTGATGGA.

[0070] ATTCAAGAGATTCCATCAAACCAGTCAATTTTTTTG-3', the nucleotide sequence of the reverse oligonucleotide is 5'-AATTCAAAAAAATTGACTGGTTTGATGGAATCTCTTGAATTCCATCAAACCAGTCA

[0071] ATG-3'. Annealing of shRNA oligonucleotides: The synthesized forward and reverse oligonucleotides were dissolved in nuclease-free water to a concentration of 100 μmol / L. Equal molar amounts of the forward and reverse oligonucleotides were mixed in annealing buffer (10 mmol / L Tris-HCl pH 8.0, 50 mmol / L NaCl, 1 mmol / L EDTA), denatured at 95°C for 5 min, annealed at 72°C for 10 min, and slowly cooled to 4°C at a rate of 1°C per min to ensure complete annealing of the sense and antisense strands, forming a double-stranded shRNA expression cassette DNA fragment with BamHI and EcoRI sticky ends. Construction and validation of recombinant plasmids: The pAAV-U6-shRNA-CMV-EGFP vector was double-digested with BamHI and EcoRI restriction endonucleases at 37°C for 2 h, and the linearized vector fragment was recovered by agarose gel electrophoresis. Annealed double-stranded shRNA expression cassette DNA fragments were mixed with linearized vectors at a molar ratio of 3:1, and ligation was performed at 16°C for 4 hours or overnight at 4°C using T4 DNA ligase to construct the recombinant plasmid pAAV-U6-shTAF15-CMV-EGFP. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing ampicillin (100 μg / mL). After incubation at 37°C for 12–16 hours, single colonies were picked for colony PCR identification. The upstream primer was located in the U6 promoter region, and the downstream primer was located downstream of the shRNA insertion site, with an expected amplified fragment size of approximately 250 bp. Plasmids from PCR-positive clones were extracted and verified by Sanger sequencing, confirming the correct shTAF15 expression cassette sequence and insertion orientation. The verified recombinant plasmid was extracted using an endotoxin-free extraction kit, digested with restriction endonucleases BamHI / EcoRI, and sequenced again for verification. The plasmid concentration was then determined and stored at -20°C for later use.

[0072] ③ Construction of the AAV negative control recombinant plasmid: The AAV expression vector pAAV-U6-shRNA-CMV-EGFP, identical to the AAV-shTAF15 plasmid, was used as the backbone. The target sequence was replaced with a nonsense shRNA sequence (scrambled shRNA) with the same base composition as the target sequence but with a randomly scrambled nucleotide sequence. The U6 promoter, hairpin circular structure sequence TTCAAGAGA, and transcription termination sequence remained unchanged. Scrambled shRNA oligonucleotide pairs with sticky ends of BamHI and EcoRI were synthesized, treated with the same annealing procedure, and then ligated with the pAAV-U6-shRNA-CMV-EGFP vector that had been double-digested with BamHI and EcoRI to construct the negative control recombinant plasmid pAAV-U6-shNC-CMV-EGFP. The ligation product was transformed into E. coli DH5α competent cells, and single clones were picked for colony PCR and sequencing verification to confirm that the scrambled shRNA expression cassette sequence was correct and without mutation. The control plasmid was extracted and verified using an endotoxin-free extraction kit. After enzyme digestion and sequencing verification, it was stored at −20℃ for later use.

[0073] ④ AAV Virus Packaging and Purification: AAV9 virus was packaged using a three-plasmid co-transfection method. HEK293T cells were seeded in cell culture dishes. When the cell confluence reached 70%–80%, the recombinant plasmid pAAV-U6-shTAF15-CMV-EGFP (or pAAV-U6-shNC-CMV-EGFP), the helper plasmid pHelper, and the packaging plasmid pRC / AAV9 were co-transfected into HEK293T cells at a mass ratio of 1:1:1 using liposome transfection reagent. Cells and culture supernatant were collected 72 h post-transfection. After repeated freeze-thaw cycles, DNase I digestion, iodixanol density gradient centrifugation, and ultrafiltration concentration, high-titer AAV9-shTAF15 and AAV9-shNC viral particles were obtained. The viral genome titer was determined using qPCR to ensure that the viral titer was not less than 1 × 10⁻⁶. 12 vg / mL, aliquoted and stored at −80℃.

[0074] ⑤ Stereoscopic Injection Administration: Viral injection into the lateral ventricle was performed 14 days prior to MCAO modeling. Based on rat weight, rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (4.0 mL / kg), fixed in a prone position on a stereotaxic instrument, ensuring the skull remained horizontal and midline. Hair was clipped, and the area was disinfected with iodine. The skin was incised along the midline of the skull to expose the skull. A suitable amount of H2O2 was applied to the top of the skull to expose the anterior fontanelle and the lambdoid suture. The coordinates of the left lateral ventricle were determined using a rat brain stereotaxic atlas: anteroposterior to anterior fontanelle -0.8 mm (posterior), lateral to midline ±1.4 mm (lateral to the midline), and dorsoventral to -3.5 mm (relative to the skull surface). Using a skull drill, a 1 mm diameter hole was drilled at the marked site. A suspension of AAV9-shTAF15 or AAV9-shNC virus was aspirated using a microsyringe and slowly injected into each lateral ventricle (3 μL per hemisphere) at a rate of 0.15 μL / min. The needle was left in place for 5 minutes after injection to allow for sufficient virus diffusion, then slowly withdrawn. The skull hole was sealed with bone wax, the skin incision was sutured, and the area was disinfected with iodine before being returned to the cage. Postoperative care included warming until the animals fully recovered. Rats in the sham-operated group (Sham group) received the same anesthesia and stereotactic surgical procedure, but were not injected with the virus suspension.

[0075] 3. Neuromotor function scoring: When performing the modified neurological severity score (mNSS), the experimental mice were given 3 days of acclimatization training before the operation to familiarize them with the testing room and the 1m long and 3cm wide wooden beam. The test was usually performed by the same operator who was unaware of the grouping on the first day after modeling to reduce subjective error. The assessment is out of 18 points, with 1 point for failure and 0 points for success. It includes four main modules: ① Motor coordination: Tail lift observation of the affected side's forelimbs and hindlimbs for flexion, and whether the animal continuously circles to one side or drags its gait during free movement (6 points); ② Sensory function: Lightly brushing the whiskers with a cotton swab and quickly bringing the hand close to the eyes, lack of avoidance response (1 point each, 2 points); ③ Brainstem reflexes: Lightly touching the cornea and tapping the table near the ear, no blinking or startle (1 point each, 2 points); ④ Balance and coordination: Placing the animal in the center of a wooden beam and recording its performance 6 times, no falls (0 points), more falls result in higher scores (maximum 6 points). If needed by the laboratory, a suspension test or inclined plane hold (2 points) can be added as supplementary tests, but the total score remains 18 points. Scores are accumulated after completing each test: 0–4 points for mild, 5–10 points for moderate, and ≥11 points for severe functional impairment. In animal experiments, animals exhibiting excessive bleeding during surgery, postoperative respiratory abnormalities, premature death, or subarachnoid hemorrhage upon euthanasia were discarded and randomly replaced in later experiments. Experimental results are as follows: Figure 6 As shown, the mNSS score of the TAF15 intervention group (MCAO / R+AAV-shTAF15) was significantly lower than that of the model + intervention negative control group (MCAO / R+AAV-NC); indicating that silencing TAF15 can significantly improve neuromotor dysfunction in rats after cerebral ischemia-reperfusion.

[0076] 4. Determination of Cerebral Infarction Volume: Rats were decapitated 24 hours after ischemia-reperfusion, and the brain tissue was quickly removed. The olfactory bulb, cerebellum, and lower brainstem were discarded, and the tissue was frozen at -20℃ for 20 min. Then, using a brain mold, the rat brain tissue was continuously sectioned in a coronal plane, starting 3 mm from the frontal pole and proceeding from anterior to posterior, into 5 equal-spaced sections. These sections were placed in 1% TTC and incubated at 37℃ for 10 min, with the sections being turned occasionally to ensure uniform staining. After TTC staining, normal tissue appeared red, and infarcted tissue appeared white. ImageJ software was used to scan the rat brain slices, and the percentage of infarct volume to the contralateral brain volume was calculated. Results are as follows: Figure 7 As shown, the TTC-corrected infarct volume in the TAF15 intervention group (MCAO / R+AAV-shTAF15) was significantly lower than that in the model + intervention negative control group (MCAO / R+AAV-NC); indicating that the silencing TAF15 treatment group significantly reduced the cerebral infarct volume compared with MCAO / R+AAV-NC.

[0077] In summary, the above experimental results indicate that TAF15 overexpression can further aggravate OGD / R-induced PC12 cell damage, apoptosis, oxidative stress, Fe2+ accumulation, and lipid peroxidation, and weaken the cell's endogenous antioxidant defense capacity, suggesting that TAF15 has a pro-damage effect in ischemia-reperfusion-related cell injury. Therefore, targeting TAF15, by reducing TAF15 expression levels, inhibiting TAF15 activity, or blocking its related biological functions, may alleviate cerebral ischemia-reperfusion-related cell injury and could be used to prepare drugs for the prevention and / or treatment of ischemic stroke.

[0078] In summary, this invention demonstrates that TAF15 is significantly upregulated and exerts a pro-injury effect in cerebral ischemia-reperfusion injury, while downregulating its expression can significantly alleviate brain tissue damage and improve neurological function by inhibiting the ferroptosis pathway. Therefore, TAF15 can serve as a molecular target for the treatment of ischemic stroke and can be used to prepare drugs for the prevention and / or treatment of ischemic stroke, showing promising clinical application prospects and translational value.

[0079] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of an inhibitor targeting TAF15 in the preparation of drugs for treating stroke, characterized in that, The nucleotide sequence of the TAF15 gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The TAF15 inhibitor is a substance that can reduce TAF15 expression levels, inhibit TAF15 activity, or block TAF15-related biological functions.

3. The application according to claim 1, characterized in that, The TAF15 inhibitor is selected from at least one of shRNA, siRNA, antisense oligonucleotide, gene editing vector nucleic acid aptamer, and small molecule inhibitors that target TAF15.

4. The application according to claim 1, characterized in that, The drug is an oral preparation, an injectable preparation, or other pharmaceutically acceptable drug preparation.

5. The application according to claim 4, characterized in that, The drug is administered after ischemic stroke or during brain ischemia-reperfusion injury.

6. The application according to claim 4, characterized in that, The target sequence of shRNA is the nucleotide sequence shown in SEQ ID NO:2, or is transcribed from the nucleotide sequence shown in SEQ ID NO:

2.

7. The application of the TAF15 gene overexpression reagent as described in claim 1 in the preparation of an in vitro cell model of ischemic stroke.

8. The TAF15 gene overexpression reagent as described in claim 1 is used to promote the release of lactate dehydrogenase and accelerate PC12 cell apoptosis.

9. The TAF15 gene overexpression reagent as described in claim 1 is used to increase Fe... 2+ Content and MDA content.

10. The TAF15 gene overexpression reagent as described in claim 1 is used to inhibit SOD activity and GSH content.

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