Use of sh-slk in the preparation of a preparation for alleviating cerebral ischemia-reperfusion injury

Abstract

The application discloses application of sh-SLK in preparation of a preparation for relieving cerebral ischemia-reperfusion injury, and relates to the technical field of gene targeting drugs. An effective active component of the preparation comprises short hairpin RNA sh-SLK capable of targeting and specifically silencing STE20-like kinase gene expression. The preparation is loaded in a recombinant virus carrier, and is formulated into a lateral cerebral ventricle injection dosage form; the preparation is delivered to brain tissues through a brain stereotactic technique, so that high-efficiency expression of sh-SLK is established in ischemic brain tissues, and then the activity of SLK is specifically inhibited.

Description

Technical Field

[0001] This invention relates to the field of gene-targeted drug technology, and more particularly to the application of sh-SLK in the preparation of formulations for alleviating cerebral ischemia-reperfusion injury. Background Technology

[0002] Stroke is one of the leading public health challenges worldwide, and the second leading cause of death and the third leading cause of disability. Among all types of stroke, acute ischemic stroke (AIS) is the most common. This disease is primarily caused by occlusion of cerebral arteries, leading to severe insufficiency of oxygen and nutrient supply to brain tissue, resulting in ischemic injury and widespread neuronal damage. In clinical treatment, timely restoration of cerebral blood flow, i.e., reperfusion, is crucial for salvaging brain tissue in the ischemic penumbra. However, reperfusion itself initiates a series of complex cascade pathophysiological processes, including apoptosis, necrosis, oxidative stress, excessive inflammatory response, autophagy disorders, intracellular calcium overload, blood-brain barrier disruption, extracellular matrix remodeling, and abnormal angiogenesis. These reactions collectively exacerbate the original brain damage, ultimately leading to an increase in infarct volume, cerebral edema, hemorrhagic transformation, and progressive neuronal loss. This complex pathological process is known as cerebral ischemia-reperfusion injury (CIRI).

[0003] Currently, clinical interventions for cerebral ischemia-reperfusion injury remain very limited. Traditional drugs often struggle to penetrate the blood-brain barrier or lack targeted lesion control, resulting in unsatisfactory treatment outcomes. In recent years, with the rapid development of genomics, approximately 4,500 genes in the human genome have been defined as druggable genes. The proteins they encode can specifically interact with drugs to regulate downstream signaling pathways. In research on related pathological mechanisms, STE20-like kinase (SLK) has gradually attracted attention. SLK, a serine-threonine kinase widely expressed in mammalian tissues and cells, plays a central role in regulating apoptosis and stress responses.

[0004] However, current technologies for the treatment of cerebral ischemia-reperfusion injury lack clinically available methods that can directly and precisely intervene in the SLK target in local brain tissue, especially lacking targeted drug formulations that can specifically silence this target at the gene expression level to block severe neurological damage. There is an urgent need in this field to develop innovative formulations that can penetrate deep into ischemic brain lesions and precisely inhibit the activity of key pathogenic targets. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of sh-SLK in the preparation of formulations for alleviating cerebral ischemia-reperfusion injury.

[0006] This invention proposes the application of sh-SLK in the preparation of a formulation for alleviating cerebral ischemia-reperfusion injury. The effective active ingredient of the formulation includes short hairpin RNA sh-SLK, which can target and specifically silence the expression of the STE20-like kinase gene. The formulation is prepared for targeted delivery to the brain tissue of the target subject to establish the expression of short hairpin RNA sh-SLK locally in the brain tissue, thereby specifically inhibiting the activity of STE20-like kinase.

[0007] Preferably, the STE20-like kinase is a serine-threonine kinase that is widely expressed in mammalian tissues and cells; the formulation targets and intervenes in the STE20-like kinase to block or slow down the cascade pathophysiological processes initiated by cerebral blood flow reperfusion, including apoptosis, necrosis, oxidative stress, excessive inflammatory response, autophagy disorder, intracellular calcium overload, blood-brain barrier disruption, extracellular matrix remodeling and abnormal angiogenesis.

[0008] Preferably, the short hairpin RNA sh-SLK is loaded into a viral vector to form a recombinant viral solution, which is used as the core pharmacodynamic component of the formulation; the recombinant viral solution is formulated to rescue the ischemic penumbra of the brain after being injected into the ventricular system of the target.

[0009] Preferably, the formulation is prepared as a lateral ventricle injection dosage form; the dosage titer concentration of the recombinant virus solution in the formulation is set to 1×10¹² vg / ml, and the baseline dosage volume parameter for a single lateral ventricle injection is set to 5 μl per lateral ventricle.

[0010] Preferably, the formulation is prepared to be compatible with a micro-injection device for slow, uniform injection at a fixed flow rate of 0.5 μl / min during stereotactic administration to the lateral ventricle; and the administration procedure specifies that the formulation must remain in place for 5 minutes after the fluid injection is completed to ensure sufficient diffusion of the viral fluid in the target area of ​​the ventricle and to avoid sudden changes in cerebrospinal fluid pressure and fluid backflow caused by the injection.

[0011] Preferably, when the formulation is applied to non-human mammalian target subjects for pre-efficacy validation, the applicable pre-administration conditions include fasting the target subject for 12 hours and withholding water for 4 hours, and administering intraperitoneal anesthesia with 1% sodium pentobarbital at a dose of 50 mg / kg; and the targeted administration coordinates on the stereotaxic instrument are set with the anterior fontanelle of the target subject as a reference as follows: 1.2 mm anterior to the anterior fontanelle, 1.2 mm lateral to the midline, and 3.0 mm below the surface of the skull.

[0012] Preferably, cerebral ischemia-reperfusion injury includes acute ischemic stroke caused by occlusion of the middle cerebral artery; the preparation is formulated to effectively antagonize severe brain injury caused by the restoration of blood flow and oxygen supply after occlusion of the middle cerebral artery for up to 60 minutes.

[0013] Preferably, the formulation has pharmacodynamic characteristics that reduce the infarct volume of cerebral ischemia-reperfusion injury; specifically, when targeting brain tissue that has undergone cerebral ischemia-reperfusion injury, brain tissue sections are extracted 24 hours after the reperfusion process and stained with triphenyltetrazolium chloride for evaluation. Compared with the control group that received the same amount of saline, the brain tissue treated with the formulation showed a significantly reduced percentage of cerebral infarction area.

[0014] Preferably, the formulation has behavioral intervention characteristics that improve neurological function impairment scores in cerebral ischemia-reperfusion injury; specifically, when the body is evaluated using a modified neurological function impairment scoring system 24 hours after the reperfusion process, the target subjects who received the formulation intervention showed a significantly lower score compared to the control group that received the same amount of saline intervention.

[0015] Preferably, the clinical and behavioral scoring ranges set by the modified neurological impairment scoring system are as follows: 1 to 6 points indicate mild neurological impairment, 7 to 12 points indicate moderate neurological impairment, and 13 to 18 points indicate severe neurological impairment; the formulation has the specific pharmacological effect of reversing the neurological impairment score of the target subject from the severe or moderate impairment range to the low score range and reducing motor deficits.

[0016] Beneficial effects: This invention provides the application of sh-SLK in the preparation of formulations for alleviating cerebral ischemia-reperfusion injury. By directing the effective active ingredient that specifically silences STE20-like kinase expression into the brain tissue of the target, this invention achieves precise targeted therapy of ischemic brain tissue, effectively overcoming the technical limitations of existing technologies such as limited clinical treatment methods, unclear intervention targets, and lack of effective targeted gene preparations. By establishing high-efficiency expression of sh-SLK in the brain tissue, this invention can specifically inhibit SLK activity, effectively blocking or slowing down a series of cascade pathophysiological processes initiated by cerebral blood flow reperfusion, such as apoptosis, oxidative stress, and excessive inflammatory response. Compared with conventional drugs that lack targeting, the formulation of this invention is specially formulated for brain-directed delivery, capable of directly penumbra in the ischemic brain to salvage damaged neurons, greatly reducing the infarct volume of brain tissue caused by acute ischemic stroke. Attached Figure Description

[0017] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This invention provides a quantitative statistical comparison chart of brain tissue slice staining images and the percentage of cerebral infarction volume.

[0018] Figure 2 This is a statistical comparison chart of the scores of the neurological function impairment scores of mice in each experimental group of the present invention. Detailed Implementation

[0019] To more clearly illustrate the technical solutions in the embodiments of the invention or the prior art, the invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the drawings is only some embodiments of the invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. It should be noted that the description of these embodiments is for the purpose of helping to understand the invention, but does not constitute a limitation on the invention.

[0020] To more clearly illustrate the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Those skilled in the art should understand that the following description of the structure and experimental steps is merely a specific embodiment of the present invention, and other derivative embodiments can be obtained based on these embodiments without creative effort. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0021] Unless otherwise specified, the reagents, materials, and instruments used in the embodiments of this invention can all be purchased through conventional commercial channels. Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional molecular biology, cell biology, or medical animal experimental methods in the field. The embodiments of this invention propose the application of sh-SLK in the preparation of a formulation for alleviating cerebral ischemia-reperfusion injury. The effective active ingredient of the formulation includes short hairpin RNA sh-SLK, which can target and specifically silence the expression of the STE20-like kinase gene. The formulation is prepared for targeted delivery to the brain tissue of the target subject to establish local expression of short hairpin RNA sh-SLK in the brain tissue, thereby specifically inhibiting the activity of STE20-like kinase.

[0022] During the formulation construction and preparation, the vector system required for constructing recombinant adeno-associated virus (AAV) included the shuttle backbone plasmid pAAV-U6-shRNA-CMV-EGFP containing a green fluorescent protein (EGFP) reporter gene driven by the U6 promoter and CMV promoter, the helper plasmid pAAV-RC providing viral capsid proteins, and the plasmid pHelper providing adenovirus helper functions. All plasmids were extracted and purified using an endotoxin-free plasmid extraction kit. After determining the concentration and purity (OD260 / 280 ratio between 1.8 and 2.0) using a UV spectrophotometer, they were stored at -20℃ for later use. The human embryonic kidney 293T cell line (HEK293T) was used as the host working cell line for packaging the recombinant adeno-associated virus. Cell culture was performed using Durbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), 0.25% Trypsin-EDTA digestion solution, Penicillin-Streptomycin (P / S) solution, and Phosphate-Buffered Saline (PBS). Cell transfection was performed using Lipofectamine 3000 high-efficiency liposome transfection reagent.

[0023] In this experiment, 1% sodium pentobarbital injection was used as a general anesthetic for non-human mammalian subjects. 2,3,5-Triphenyltetrazolium chloride (TTC) powder was used for staining. 4% paraformaldehyde (PFA) solution was used for gross perfusion fixation and tissue section fixation. Total RNA extraction kits, reverse transcription kits, and quantitative real-time polymerase chain reaction (qPCR) premixes were used for gene detection. Radioimmunoprecipitation assay (RIPA) tissue and cell lysates, bicinchoninic acid (BCA) protein concentration assay kit, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit, and enhanced chemiluminescence (ECL) chromogenic solution were used for protein detection.

[0024] The primary antibodies (first antibodies) specifically designed to validate cascade pathophysiological processes include: anti-STE20-like kinase antibody, anti-B-cell lymphoma-2 (Bcl-2) antibody, anti-Bcl-2 associated X protein (Bax) antibody, anti-cleaved caspase-3 antibody, anti-receptor-interacting protein kinase 1 / 3 (RIPK1 / RIPK3) antibody, anti-mixed lineage kinase domain-like protein (MLKL) antibody, and anti-microtubule-associated protein 1 light chain 3B antibody. Antibodies against 3B (LC3B), anti-ubiquitin-binding protein p62 (SQSTM1 / p62), anti-zonulaoccludens-1 (ZO-1), anti-ocludin, anti-macroproteinase-2 / 9 (MMP-2 / MMP-9), anti-CD31, anti-vascular endothelial growth factor (VEGF), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and anti-β-actin antibodies used as internal controls. Horseradish peroxidase (HRP)-labeled goat anti-mouse or goat anti-rabbit secondary antibodies (secondary antibodies), and fluorescein-labeled secondary antibodies were used for colorimetric or luminescent development. Superoxide dismutase (SOD) activity assay kit, malondialdehyde (MDA) content assay kit, reactive oxygen species (ROS) fluorescent probe (DCFH-DA), and intracellular free calcium ion fluorescent probe (Fluo-4 AM) are used for biochemical detection.An enzyme-linked immunosorbent assay (ELISA) kit for mouse tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) is used for the detection of inflammatory factors. A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptosis detection kit is used for histopathological examination.

[0025] The main equipment involved in the experiment included: a small animal stereotaxic apparatus; a microinjection pump with a precision controller and microinjectors of different sizes; a fully automated small animal physiological monitor and rectal body temperature maintenance system; a laser Doppler flowmeter for real-time monitoring of cerebral blood flow changes; a real-time quantitative PCR instrument; an ultracentrifuge and rotor for virus gradient purification; a fully automated gel imaging analysis system; a laser scanning confocal microscope; a full-wavelength microplate reader; a flow cytometer; a paraffin microtome and a cryostat; a mouse brain slice mold (Brain Matrix); and an ultrapure water preparation system.

[0026] To establish efficient and specific expression of the short hairpin RNA sh-SLK in local brain tissue, multiple candidate short hairpin RNA sequences were designed using online design software targeting the conserved coding region of the mouse STE20-like kinase gene. After homology alignment to eliminate sequences that might produce off-target effects, the core sequence with the highest knockdown efficiency and strongest specificity was selected as the target short hairpin RNA sh-SLK. Simultaneously, a scramble sequence with no significant homology to the mouse, rat, and human genomes was designed as a negative control sequence (sh-con). Related oligonucleotide single strands were chemically synthesized by a commercial biotechnology company. The synthesized sh-SLK and sh-con oligonucleotide single strands (sense and antisense strands) containing the correct sticky ends were dissolved in annealing buffer and annealed in a PCR instrument (95°C for 5 minutes, then slowly cooled to 25°C at a rate of 0.1°C / second) to form double-stranded DNA fragments with sticky ends. The pAAV-U6-shRNA-CMV-EGFP vector backbone was linearized by double restriction endonucleases BamHI and EcoRI. The annealed double-stranded DNA fragment was ligated to the linearized AAV backbone plasmid using T4 DNA ligase overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing ampicillin, and incubated upside down at 37°C for 12–16 hours. Single colonies were picked and inoculated into liquid LB medium for expansion. Plasmids were extracted and identified by double restriction endonuclease digestion and verified by Sanger bidirectional sequencing. Sequencing results confirmed that the inserted sh-SLK and sh-con sequences were 100% correct in length, sequence composition, and orientation, with no mutations or deletions, successfully obtaining the recombinant plasmid pAAV-sh-SLK and the control plasmid pAAV-sh-con.

[0027] AAV virus was packaged in HEK293T cells using either calcium phosphate transfection or liposome transfection. HEK293T cells in logarithmic growth phase after resuscitation were seeded in 150 mm cell culture dishes in high-glucose DMEM containing 10% FBS and 1% P / S. Cells were cultured at 37°C in a humidified incubator with 5% CO2. Transfection was performed when cells adhered and reached 70%-80% confluence. In this embodiment, short hairpin RNA sh-SLK was loaded into a viral vector to form a recombinant viral solution, which served as the core pharmacodynamic component of the formulation. Specifically, purified pAAV-sh-SLK (or pAAV-sh-con), helper plasmid pAAV-RC, and pHelper were mixed in equimolar proportions, and Lipofectamine 3000 transfection reagent was added to prepare a liposome-DNA complex, which was then uniformly added to the cell culture dish. After 72 hours of transfection and culture, the cell status was observed under an inverted fluorescence microscope. When more than 80% of the cells expressed bright EGFP green fluorescence, it indicated that the plasmid co-transfection efficiency had reached the required level and viral assembly was at its peak. The cell supernatant and cell layer were collected from the culture dish. HEK293T cells were scraped off with a cell scraper and transferred to a centrifuge tube along with the supernatant. The cells were centrifuged at 1000 rpm for 5 minutes, discarding most of the supernatant and retaining the cell pellet. An appropriate amount of sterile cell lysis buffer was added to the cell pellet. The resuspended cell suspension was rapidly frozen in a dry ice-anhydrous ethanol bath, then rapidly thawed in a 37°C water bath. This freeze-thaw cycle was repeated 3 to 4 times to utilize the mechanical shear force of the ice crystals to thoroughly disrupt the cell membrane and nuclear membrane, releasing the assembled recombinant AAV virus particles. Subsequently, a totipotent nuclease was added to the lysate at a final concentration of 50 U / ml, and the mixture was incubated at 37°C for 30 to 45 minutes to thoroughly digest and degrade residual genomic DNA and RNA in the lysate and reduce the solution viscosity. After processing, the lysate was centrifuged at 4000 rpm for 30 minutes at 4°C, and the clear supernatant rich in recombinant virus particles was collected.

[0028] To obtain a high-purity formulation suitable for stereotactic brain injection, discontinuous density gradient ultracentrifugation with iodixanol was used to remove contaminating proteins and empty capsids. In sterile ultracentrifuge tubes, iodixanol gradient solutions of 60%, 40%, 25%, and 15% concentrations were slowly layered from bottom to top. The coarsely extracted viral supernatant was carefully layered on top of the 15% iodixanol solution. The tubes were symmetrically balanced and placed in the horizontal rotor of a Beckman ultracentrifuge. Centrifugation was performed at 18°C ​​and 60,000 rpm for 2 hours. After centrifugation, a clear band enriched with intact, infectious, solid AAV viral particles was carefully aspirated from the 40% and 60% iodixanol concentrations by puncturing the side wall of the tube. The aspirated viral suspension contained a high concentration of iodixanol and required desalting and buffer replacement. The collected virus solution was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa. Sterile PBS buffer containing 0.001% Pluronic F-68 surfactant was added to prevent virus particles from adsorbing onto the tube wall or aggregating. The solution was concentrated by centrifugation at 3000 rpm at 4°C. The elution and displacement process was repeated 3 to 4 times until iodixanol was completely removed.

[0029] The physical titer of the concentrated recombinant virus was determined using qPCR absolute quantification. A small amount of single-stranded DNA was extracted from the purified virus as a template, and qPCR amplification was performed using specific primers targeting the inverted terminal repeat (ITR) sequence of the AAV vector. Absolute quantification was performed by plotting a standard curve using a standard plasmid with known copy numbers. For subsequent administration to non-human mammalian targets, the formulation was prepared for intraventricular injection. Based on the initial titer determined by qPCR, the recombinant virus solution was diluted with sterile PBS containing Pluronic F-68. In this embodiment, the dosage titer concentration of the recombinant virus solution in the formulation was set at 1 × 10⁻⁶. 12 vg / ml. This dosage concentration ensures that a sufficient number of viral particles are provided in subsequent single microinjections to transduce neurons in the ischemic penumbra, while avoiding carrier toxicity or nonspecific neuroinflammation caused by excessively high titers. The prepared AAV-sh-SLK formulation and AAV-sh-con control formulation were aseptically aliquoted and stored at -80°C for later use.

[0030] Healthy, specific pathogen-free (SPF) adult male C57BL / 6J mice were used as non-human mammals for preliminary efficacy validation. Mice were 8 to 10 weeks old and weighed between 22 and 25 grams. All mice were housed in a standard SPF-grade animal facility with individually ventilated cages, maintaining a 12-hour light-12-hour dark circadian rhythm. The ambient temperature was maintained at 22±2℃, and the relative humidity at 50±10%. Animals had free access to standard rodent complete pelleted feed and sterilized drinking water, and underwent at least one week of acclimatization before the experiment. All animal experimental procedures complied with the review requirements of the Laboratory Animal Welfare and Ethics Committee and relevant laws and regulations. When a formulation is applied to non-human mammalian targets for preliminary efficacy validation, to minimize the risks of gastric reflux, aspiration pneumonia, and suffocation caused by anesthesia or surgery, and to ensure the stability of the animal's internal environmental indicators and blood glucose levels during surgery, the applicable pre-administration conditions include fasting for 12 hours and withholding water for 4 hours. After completing the fasting and water withholding treatment, anesthesia is administered via intraperitoneal injection of 1% sodium pentobarbital at a dose of 50 mg / kg. During the procedure, a 1 ml sterile syringe with a 29G disposable needle is used, inserted into the lower third of the mouse's abdomen, slightly off-center from the midline. After aspirating to ensure no blood or intestinal fluid is found, the 1% sodium pentobarbital solution is slowly injected into the peritoneal cavity. After injection, the mouse is placed in a separate observation cage, and its respiratory rate and muscle relaxation are closely monitored. Once the mouse's righting reflex disappears, and there is no obvious pain reflex when the hind limb toes are gently pinched with tweezers, and the corneal reflex is sluggish and breathing is deep and steady, the depth of anesthesia is determined to be appropriate, and the mouse can then be placed in the stereotaxic instrument for subsequent targeted drug delivery.

[0031] The recombinant viral solution was formulated to salvage the ischemic penumbra of the brain after injection into the ventricular system of the target patient. Mice under deep anesthesia were placed prone on the worktable of a small animal stereotaxic apparatus. Incisor bars were inserted into the mouse's mouth to stabilize the maxilla, and the height of the incisor bars was adjusted. Ear rods with blunt, conical heads were inserted into both external auditory canals, and the ear rod screws were tightened symmetrically to ensure the mouse's head was firmly fixed and the skull was horizontal. Hair in the surgical area of ​​the head was shaved using clippers, and the skin was disinfected at least three times in a circular motion from the inside out using povidone-iodine solution and 75% medical ethanol. A 1.5 cm incision was made along the midsagittal line of the skull using a sterile surgical blade. A miniature retractor was used to pull the skin open to expose the skull. The skull surface was gently wiped with 3% hydrogen peroxide solution and sterile cotton swabs to remove the periosteum and soft tissue, making the cranial sutures clearly visible. The tip of the micromanipulation three-dimensional moving arm of the positioning instrument is aligned with the anterior fontanelle, and its X, Y, and Z axis scales are zeroed as reference points. Its targeted drug delivery coordinates on the stereotaxic instrument are set with the anterior fontanelle as the reference: 1.2 mm anterior to the anterior fontanelle, 1.2 mm lateral to the midline, and 3.0 mm below the skull surface. The manipulator arm is moved horizontally to the corresponding coordinate point, and a micro-skull drill is used to drill a micro-hole approximately 0.8 mm in diameter at the corresponding vertical position on the skull. Sterile saline solution is intermittently dripped during drilling for cooling until the skull is penetrated and the dura mater is exposed. Careful operation is necessary to prevent damage to the blood vessels in the brain parenchyma.

[0032] The baseline dosing volume for a single lateral ventricle injection of the formulation was set at 5 μl per lateral ventricle. Using a 10 μl microsyringe connected to a microinfusion pump, 5 μl of AAV-sh-SLK formulation, thawed at -80°C and stored on ice protected from light, was drawn up. The injection needle was positioned directly above a microforamen in the skull and slowly lowered, penetrating the dura mater and cortex until the needle tip reached the set depth, 3.0 mm below the skull surface, accurately entering the right lateral ventricle. During stereotactic lateral ventricle administration, the formulation was prepared for a microinfusion device adapted for a constant, slow injection at a fixed flow rate of 0.5 μl / min. The microinfusion pump program was activated, and the device continuously injected 5 μl of the viral formulation into the target area over 10 minutes at a constant rate of 0.5 μl / min. Furthermore, the administration protocol specifies that the needle must remain in place for 5 minutes after the fluid injection to ensure sufficient diffusion of the viral fluid within the ventricular target area and to avoid sudden changes in cerebrospinal fluid pressure and fluid backflow caused by the injection. During the 5-minute in-situ needle retention period, the infusion pump remains off. After the retention period, the needle is withdrawn upwards at a slow, uniform speed of approximately 1 mm / min to prevent negative pressure from drawing out the medication. After needle withdrawal, the skull borehole is sealed tightly with sterile bone wax, the wound is rinsed with sterile saline, and the subcutaneous tissue and scalp are sutured layer by layer with non-absorbable surgical sutures. A small amount of erythromycin ointment is applied to the wound to prevent infection. Post-operatively, the mice are placed on a 37°C constant-temperature recovery mat until they awaken and regain spontaneous activity, after which they are returned to their original cages. Following ventricular injection of the viral preparation, the mice are fed routinely for 21 days. This incubation period allows adeno-associated virus particles to diffuse into the brain parenchyma, enter neurons, and complete uncoating, genome entry into the nucleus, and transcription and translation processes, thereby establishing stable and efficient expression of short hairpin RNA sh-SLK in the local brain tissue.

[0033] Cerebral ischemia-reperfusion injury includes acute ischemic stroke caused by middle cerebral artery occlusion. A mouse model of middle cerebral artery occlusion-reperfusion was established 21 days after lateral ventricle administration of medication using a modified endovascular suture method. Mice were fasted for 12 hours preoperatively. Mice were placed in an anesthesia induction box and anesthesia was induced by inhalation with a mixture of oxygen containing 2.5% to 3.0% isoflurane gas. After successful induction, mice were fixed supine on a small animal-specific temperature-controlled operating table, and anesthesia was maintained by inhalation of 1.5% to 2.0% isoflurane gas through a cone mask. Throughout the surgery, a rectal temperature probe was inserted into the mouse rectum, and the mouse's core body temperature was maintained at 37.0 ± 0.5℃ using an automatically feedback heating pad. After preparing and disinfecting the midline of the neck, a midline longitudinal incision of approximately 1.5 cm was made. Under a microscope, the neck glands and sternocleidomastoid muscle were bluntly dissected to expose the right common carotid artery, external carotid artery, and internal carotid artery. The vagus nerve attached to the vessel walls was carefully dissected. The distal end of the external carotid artery and its branches were ligated with fine sutures, and blood flow was temporarily blocked at the proximal ends of the common carotid and internal carotid arteries using miniature arterial clamps. A small incision was made between the ligated end of the external carotid artery and the bifurcation of the common carotid artery. A silicone-coated nylon monofilament suture was gently inserted through the incision in the external carotid artery. The arterial clamp on the internal carotid artery was released, and the angle of the suture was adjusted to allow it to slide smoothly into the internal carotid artery. It was then slowly advanced into the cranial cavity for approximately 9 to 11 mm. When slight resistance was felt, advancement was stopped; this position indicated that the silicone tip of the suture had crossed the anterior cerebral artery and blocked the origin of the middle cerebral artery. During this process, changes in local cerebral blood flow were monitored in real time using a laser Doppler flowmeter probe. When the monitoring data showed that the local cerebral blood flow in the blood-supply area decreased to less than 20% of the baseline blood flow value, the cerebral ischemia model was considered successful. Subsequently, the suture suture was ligated and fixed to the stump of the external carotid artery using pre-placed fine sutures to maintain the continuous ischemic state of the middle cerebral artery.

[0034] The formulation was designed to effectively antagonize severe brain injury caused by the restoration of blood flow and oxygen supply after a 60-minute occlusion of the middle cerebral artery. Therefore, after maintaining the suture occlusion for 60 minutes, the ligature of the external carotid artery was removed, and the suture was slowly and steadily pulled outwards to the external carotid incision site. At this point, the arterial clamp on the common carotid artery was removed, restoring blood perfusion to the internal carotid artery and middle cerebral artery to achieve reperfusion. Laser Doppler flowmeter signals were observed; successful reperfusion was considered achieved when local cerebral blood flow recovered to more than 80% of the pre-ischemic baseline. Finally, the external carotid artery stump was permanently ligated, the wound was rinsed with saline, and the neck skin was sutured layer by layer. The in vivo experiment was divided into three independent groups. The sham surgery group underwent only vascular dissection and did not experience cerebral ischemia, serving as a baseline healthy control. The negative control group received a stereotactic intraventricular injection of a prepared AAV-sh-con control virus preparation 21 days prior to modeling, followed by 60 minutes of cerebral ischemia and reperfusion, equivalent to receiving an equivalent amount of ineffective solvent or saline intervention as a vector control. The treatment group received a stereotactic intraventricular injection of the active ingredient, AAV-sh-SLK virus preparation, 21 days prior to modeling, followed by a similar 60-minute cerebral ischemia and reperfusion process.

[0035] The formulation exhibits behavioral intervention characteristics that improve neurological function impairment scores in patients with cerebral ischemia-reperfusion injury. Specifically, the Modified Neurological Severity Score (mNSS) was used to evaluate the patients 24 hours after the reperfusion event. Scoring was conducted in a blinded manner by two trained researchers unaware of the mice's grouping. The mNSS is a comprehensive assessment tool integrating multiple dimensions such as motor, sensory, reflex, and balance abilities, with a score range of 0 to 18, where higher scores indicate more severe neurological deficits. The established clinical and behavioral scoring intervals are: 1 to 6 indicate mild neurological impairment, 7 to 12 indicate moderate neurological impairment, and 13 to 18 indicate severe neurological impairment. Specific assessment items and scoring rules include motor function tests, sensory function tests, balance beam tests, and reflex and abnormal movement tests. In terms of motor function, the limb status of mice was observed through the tail-lift test, and scores were given based on forelimb flexion, hindlimb flexion, or trunk twisting. This was combined with flat-ground walking and gait tests, assessing whether the mouse could walk normally in a straight line, circle towards the paralyzed side, or fall. In terms of sensory function, the agility of placing the mouse's paw back on the table was evaluated through proprioception and placement reflex tests. In terms of balance, mice were placed on a suspended wooden beam, and scores were refined based on their stable walking, the duration of holding the beam, or the likelihood of falling. In terms of reflex tests, the absence of auricular reflex, corneal reflex, and startle reflex was tested, and the presence of abnormal neurological symptoms such as epilepsy was observed. The sum of all scores yielded the total mNSS score for each mouse.

[0036] The formulation exhibits pharmacodynamic characteristics in reducing infarct volume in cerebral ischemia-reperfusion injury. Specifically, targeting brain tissue that has undergone cerebral ischemia-reperfusion injury, brain tissue sections were extracted 24 hours after the reperfusion process and evaluated using triphenyltetrazolium chloride staining. Mice were euthanized immediately after 24 hours of reperfusion and following behavioral assessment by intraperitoneal injection of a deep coma dose of sodium pentobarbital. The brain was rapidly decapitated and completely removed, and the surface blood was washed away with cold saline. To obtain smooth brain sections, the whole brain was briefly frozen at -20°C for 15 to 20 minutes to harden it without freezing. Using a mouse brain sectioning mold, the olfactory bulb and lower brainstem were removed. Using the optic chiasm as the anatomical reference point, the brain was cut into five consecutive coronal sections from anterior to posterior at a constant thickness of 2 mm. The sections were gently transferred to culture dishes and completely immersed in freshly prepared 2% triphenyltetrazolium chloride staining solution. Incubate the culture dish in a 37°C shaker in the dark for 20 to 30 minutes, gently rotating the brain slice every 5 minutes to ensure uniform staining on both sides. Mitochondrial succinate dehydrogenase in normal living brain tissue cells is active, reducing colorless triphenyltetrazolium chloride to a water-insoluble red formazan compound, resulting in a deep red color for normal tissue. However, in areas of ischemia-reperfusion injury leading to cell necrosis, mitochondrial enzyme activity is irreversibly lost, preventing the reduction reaction and leaving these areas pale white. After staining, gently wash away excess staining solution with PBS, then immerse the brain slice in 4% paraformaldehyde solution and fix overnight at 4°C in the dark to enhance tissue contrast. The next day, photograph both sides of the brain slice and import the images into ImageJ image analysis software to precisely delineate the red normal hemisphere area and the white infarct area area in each slice. The percentage of infarct volume was calculated using an indirect measurement method. The formula was: Percentage of infarct volume in a single-slice brain slice = [(Area of ​​the contralateral normal hemisphere - Area of ​​the non-infarcted area on the affected side) / (Area of ​​the contralateral normal hemisphere × 2)] × 100%. The weighted average of the infarct percentages from the coronal slices yielded the overall percentage of cerebral infarction in the mice.

[0037] The formulation targets and intervenes in STE20-like kinase to block or slow down the cascade pathophysiological processes initiated by cerebral reperfusion, including apoptosis, necrosis, oxidative stress, excessive inflammatory response, autophagy disorder, intracellular calcium overload, blood-brain barrier disruption, extracellular matrix remodeling, and abnormal angiogenesis. To confirm the above mechanism, molecular biological analysis was performed on ischemic cerebral cortex and striatum tissues 24 hours after reperfusion. For Western blotting of proteins, brain tissue was excised and placed in a pre-chilled glass homogenizer, homogenized with RIPA strong lysis buffer containing protease and phosphatase inhibitors, centrifuged at high speed, and the supernatant was extracted and protein concentration was determined. After denaturation, the protein samples were separated by electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocking, primary antibody and HRP-labeled secondary antibody were added sequentially for incubation. After washing, chemiluminescence imaging and grayscale analysis were performed. The results showed that the SLK protein level in the treatment group treated with the formulation was lower than that in the control group, confirming that the short hairpin RNA sh-SLK successfully achieved specific gene expression silencing. Further analysis revealed that in the model control group, pro-apoptotic protein Bax, apoptosis-inducing Cleaved Caspase-3, and necrosis-related proteins RIPK1, RIPK3, and phosphorylated MLKL were upregulated, while anti-apoptotic protein Bcl-2 was downregulated. However, the formulation intervention reversed these indicators, restoring Bcl-2 expression and inhibiting the abnormal expression of apoptosis and necrosis markers, demonstrating the blockade of the apoptosis and necrosis cascade. Biochemical assays showed that the activity of the antioxidant enzyme SOD decreased due to depletion while the lipid peroxidation product MDA increased in the control group; the formulation group restored SOD activity and reduced MDA accumulation, mitigating oxidative stress damage. ELISA analysis showed a significant release of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6 in reperfusion-injured brain tissue; targeted administration of the formulation suppressed the concentration of inflammatory factors, confirming its mitigation of excessive neuroinflammatory responses. Analysis of autophagy-related proteins revealed that the formulation group maintained the LC3B-II / I ratio at a moderate physiological level and reduced the accumulation of substrate p62, restoring autophagy homeostasis. Fluorescent probe staining revealed that the intensity of calcium fluorescence signal in the cytoplasm of neurons in the formulation group was lower than that in the control group, confirming that it inhibited intracellular calcium overload. Furthermore, the core proteins ZO-1 and Occludin, which maintain the blood-brain barrier structure, were protected after formulation application, confirming that it blocked blood-brain barrier disruption. The formulation intervention also downregulated the expression of MMP-2 and MMP-9, slowing down extracellular matrix remodeling; simultaneously, it standardized the regulation of microvascular leakage and abnormal angiogenesis, resulting in a more complete and continuous capillary morphology.

[0038] All quantitative data obtained from the experiments are expressed as mean ± standard error. Statistical software was used to analyze the data. One-way ANOVA combined with Tukey's multiple comparison post-hoc test was employed for significance analysis; P < 0.05 was considered statistically significant. (Example: Triphenyltetrazolium chloride staining results) Figure 1 As shown, the sham-operated group exhibited a uniform deep red color throughout the brain, while the negative control group showed a large, well-defined, pale white infarct lesion in the right cerebral hemisphere, accompanied by cerebral edema. In contrast, the targeted brain tissue sections treated with the sh-SLK agent showed a reduction in the pale ischemic necrosis area, with most of the cortex and penumbra remaining deep red. Quantitative analysis confirmed that the average infarct volume percentage in the negative control group was approximately 33.5%; while the brain tissue treated with the agent, compared to the control group treated with the same amount of saline, showed a reduced percentage of infarct area, with the infarct volume reduced to approximately 8.2% (P<0.001), confirming the pharmacodynamic characteristics of the agent in reducing cerebral infarct volume in cases of cerebral ischemia-reperfusion injury. Furthermore, neurological function impairment scores were also observed. Figure 2 As shown, the sham-operated group mice all scored 0 points; the negative control group mice exhibited typical defects such as unilateral paralysis and circling, with an average total mNSS score of approximately 12.8 points, falling into the severe or moderate neurological injury range, confirming the brain damage caused by 60 minutes of occlusion followed by restoration of blood flow and oxygenation; while the target subjects treated with the formulation showed improvement in various behavioral abilities, with the average mNSS score reduced to approximately 5.6 points. These results demonstrate that the formulation has the efficacy of reversing the neurological injury score of target subjects from the severe or moderate injury range to the lower range and reducing motor defects; the target subjects receiving the formulation intervention showed a reversal trend of decreasing scores compared to the control group, confirming its beneficial behavioral intervention characteristics.

[0039] Although embodiments of the present invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the claims and their equivalents.

Claims

1. The application of sh-SLK in the preparation of formulations for alleviating cerebral ischemia-reperfusion injury, characterized in that, The active ingredient of the formulation includes a short hairpin RNA sh-SLK that can target and specifically silence the expression of the STE20-like kinase gene; the formulation is formulated for targeted delivery to the brain tissue of a target to establish the expression of the short hairpin RNA sh-SLK locally in the brain tissue, thereby specifically inhibiting the activity of the STE20-like kinase.

2. The application according to claim 1, characterized in that, The STE20-like kinase is a serine-threonine kinase that is widely expressed in mammalian tissues and cells. The formulation targets and intervenes in the STE20-like kinase to block or slow down the cascade pathophysiological processes initiated by cerebral blood flow reperfusion, including apoptosis, necrosis, oxidative stress, excessive inflammatory response, autophagy disorder, intracellular calcium overload, blood-brain barrier disruption, extracellular matrix remodeling, and abnormal angiogenesis.

3. The application according to claim 1, characterized in that, The short hairpin RNA sh-SLK is loaded into a viral vector to form a recombinant viral solution, which serves as the core pharmacodynamic component of the formulation. The recombinant viral solution is formulated to rescue the ischemic penumbra of the brain after being injected into the ventricular system of the target.

4. The application according to claim 3, characterized in that, The formulation is prepared for intraventricular injection; the dosage titer of the recombinant virus solution in the formulation is set to 1 × 10⁻⁶. 12 The standard dosing volume was set at vg / ml, and the baseline dosing volume for a single intraventricular injection was set at 5 μl per ventricle.

5. The application according to claim 4, characterized in that, During stereotactic administration to the lateral ventricle, the formulation is prepared to be compatible with a micro-injection device for slow, uniform injection at a fixed flow rate of 0.5 μl / min. Furthermore, the administration protocol of the formulation specifies that the fluid should remain in place for 5 minutes after the injection is completed to ensure sufficient diffusion of the viral fluid in the target area of ​​the ventricle and to avoid sudden changes in cerebrospinal fluid pressure and fluid backflow caused by the injection.

6. The application according to claim 5, characterized in that, When the formulation is applied to non-human mammalian targets for pre-efficacy validation, the applicable pre-administration conditions include fasting the target for 12 hours and withholding water for 4 hours, and administering intraperitoneal anesthesia with 1% sodium pentobarbital at a dose of 50 mg / kg; and the targeted administration coordinates on the stereotaxic instrument are set with the anterior fontanelle of the target as the reference: 1.2 mm anterior to the anterior fontanelle, 1.2 mm lateral to the midline, and 3.0 mm below the surface of the skull.

7. The application according to any one of claims 1 to 6, characterized in that, The cerebral ischemia-reperfusion injury includes acute ischemic stroke caused by occlusion of the middle cerebral artery; the preparation is formulated to effectively antagonize severe brain injury caused by the restoration of blood flow and oxygen supply after occlusion of the middle cerebral artery for up to 60 minutes.

8. The application according to claim 7, characterized in that, The formulation exhibits pharmacodynamic characteristics in reducing the infarct volume of brain tissue in response to the cerebral ischemia-reperfusion injury. Specifically, when targeting brain tissue that has undergone the cerebral ischemia-reperfusion injury, brain tissue sections are extracted 24 hours after the reperfusion process and stained with triphenyltetrazolium chloride for evaluation. Compared with the control group that received an equal amount of saline, the brain tissue treated with the formulation showed a significantly reduced percentage of cerebral infarction area.

9. The application according to claim 7, characterized in that, The formulation exhibits behavioral intervention characteristics that improve neurological function impairment scores in the cerebral ischemia-reperfusion injury. Specifically, when the body is evaluated using a modified neurological function impairment scoring system 24 hours after the reperfusion process, the target subjects who received the formulation intervention showed a significantly lower score compared to the control group that received the same amount of saline intervention.

10. The application according to claim 9, characterized in that, The modified neurological impairment scoring system is defined by clinical and behavioral scoring ranges as follows: 1 to 6 points indicate mild neurological impairment, 7 to 12 points indicate moderate neurological impairment, and 13 to 18 points indicate severe neurological impairment. The formulation has the specific pharmacological effect of reversing the neurological impairment score of the target subject from the severe or moderate impairment range to the low score range and reducing motor deficits.

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