Application of fluoxetine in preparation of medicine for treating epileptic seizure or brain injury

Abstract

The invention discloses application of fluoxetine in preparation of a medicine for treating epileptic seizure or brain injury. In-vivo and in-vitro experiments find that fluoxetine can inhibit epilepsy-induced ferroptosis by inhibiting ubiquitination of GPX4 protein so as to play a role in treating epileptic seizure or brain injury for the first time, and a new technical means is provided for treatment of epilepsy or brain injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to the use of fluoxetine in treating epilepsy or brain damage caused by epilepsy. Background Art

[0002] Epilepsy is a chronic brain disorder caused by a variety of etiologies, characterized by sudden, recurrent, and transient central nervous system dysfunction due to excessive neuronal discharges. Epidemiological studies show that approximately 65 million people worldwide suffer from epilepsy, with a prevalence of 4-10‰, and approximately 2.4 million new cases each year. In my country, approximately 9 million people suffer from epilepsy, of whom 6 million have active forms, with an estimated annual incidence of approximately 400,000 new cases. Recurrent epileptic seizures can lead to mental and psychological impairment, reduced ability to function, and even suicide, placing a significant financial and emotional burden on society and families. The pathogenesis of epilepsy is complex and diverse, and remains largely unexplained. Currently, clinical treatment for epilepsy primarily relies on medication, and there is currently no effective and fundamental treatment for post-injury damage. Effectively preventing epilepsy, intervening in and reversing epilepsy-induced brain damage, promoting post-injury repair, and improving prognosis are major challenges in neurology, and the search for effective antiepileptic and neuroprotective drugs remains a key research priority.

[0003] Therefore, it is crucial to provide an anti-epileptic drug and a neuroprotective drug. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the present invention aims to provide the use of fluoxetine in the preparation of a drug for treating epilepsy or brain damage caused by epilepsy.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: A first aspect of the present invention provides use of fluoxetine or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating epileptic seizures or brain damage.

[0006] Furthermore, fluoxetine reduces ferroptosis by inhibiting GPX4 ubiquitination to treat epileptic seizures or brain damage.

[0007] Furthermore, the fluoxetine or a pharmaceutically acceptable salt thereof is used as the sole active ingredient or one of the active ingredients of a drug for treating epileptic seizures or brain damage.

[0008] Furthermore, the epilepsy is convulsion or status epilepticus, and the brain damage is induced by epilepsy.

[0009] Furthermore, the dosage form of the drug includes an oral dosage form, a parenteral dosage form and / or a topical dosage form.

[0010] Furthermore, the dosage form of the pharmaceutical preparation includes solutions, sustained-release preparations, suspensions, granules, tablets, capsules, powders, effervescent preparations, emulsions, syrups, drops and / or chewable preparations.

[0011] Furthermore, the administration of the drug includes oral, subcutaneous, intravenous, intramuscular, intraarterial, intranasal, intrathecal, mucosal, intrapulmonary and / or rectal administration.

[0012] In the present invention, treatment refers to the improvement, prevention, or reversal of a disease or condition or at least one identifiable symptom thereof. In certain specific embodiments, the treatment refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or condition to be treated, and the parameter is not necessarily identifiable in mammals or recognized by mammals. In some embodiments, the treatment refers to the inhibition or slowing of a disease or disease course, and this inhibition or slowing can be physical, such as certain identifiable adverse symptoms. The "treatment" used in the present invention covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease. (b) Inhibiting the disease, such as blocking the development of the disease. Or (c) Alleviating the disease, such as alleviating the symptoms associated with the disease.

[0013] In the present invention, the fluoxetine described herein may be fluoxetine itself or in the form of a pharmaceutically acceptable salt of fluoxetine. Pharmaceutically acceptable salts refer to acidic salts formed with inorganic and / or organic acids and basic salts formed with inorganic and / or organic bases. Furthermore, when the compound contains a basic moiety (such as, but not limited to, pyridine or imidazole) and an acidic moiety (such as, but not limited to, a carboxylic acid), zwitterions may be formed, and such zwitterions are included in the pharmaceutically acceptable salts described herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, but other salts are also useful. Pharmaceutically acceptable salts of the compounds described herein can be formed, for example, by reacting the compound with an amount of an acid or base in a medium, such as a medium in which the salt precipitates or an aqueous medium (followed by lyophilization).

[0014] Specific pharmaceutically acceptable salts include those salts that are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts (pharmaceutically acceptable salts) are well known in the art. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids and inorganic and organic bases.

[0015] Examples of pharmaceutically acceptable nontoxic acid addition salts are salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or salts formed with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Salts formed using conventional methods in the art, such as ion exchange methods, are also included. Other pharmaceutically acceptable salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, hemisulfate, heptanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, and ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like.

[0016] The second aspect of the present invention provides the use of fluoxetine or a pharmaceutically acceptable salt thereof in the preparation of a product having any one or more of the following effects: (1) reducing the frequency of epileptic seizures; (2) reducing the grade of epileptic seizures; (3) reducing the inflammatory response of hippocampal tissue; (4) reducing cognitive impairment induced by epilepsy; (5) reducing neuronal ferroptosis; and (6) inhibiting the ubiquitination and degradation of GPX4 protein.

[0017] A third aspect of the present invention provides a pharmaceutical composition for treating epileptic seizures or brain damage.

[0018] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of fluoxetine or a pharmaceutically acceptable salt thereof.

[0019] Furthermore, the pharmaceutical composition also includes a second therapeutic agent.

[0020] Furthermore, the second therapeutic agent is other drugs used to treat epileptic seizures or brain damage.

[0021] Preferably, the other drugs for treating epileptic seizures or brain damage include sodium valproate, carbamazepine, phenobarbital, phenytoin sodium, ethosuximide, oxcarbazepine, lamotrigine, topiramate, levetiracetam, and lacosamide.

[0022] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0023] Furthermore, the auxiliary materials include adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators and / or buffers, solvents, surfactants or emulsifiers.

[0024] Furthermore, the diluent includes but is not limited to lactose, sodium chloride, glucose, urea, starch, water, etc. The binder includes but is not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, etc. The surfactant includes but is not limited to sodium lauryl sulfate, monoglyceride stearate, cetyl alcohol, etc. The lubricant includes but is not limited to zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric sucrose, magnesium lauryl sulfate, etc. The filler includes but is not limited to mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, starch, etc. The disintegrant includes but is not limited to cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soy polysaccharide, etc.

[0025] The term "pharmaceutical composition" refers to any composition comprising at least one biologically active agent. As used herein, the term "pharmaceutical composition" also refers to a composition comprising an active pharmaceutical ingredient to be delivered to a subject, e.g., to achieve a therapeutic, prophylactic, diagnostic, preventive, or prognostic effect.

[0026] In the present invention, the term "therapeutically effective amount" refers to an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent, used alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disease, disorder or condition. The term "therapeutically effective amount" can include an amount that improves overall treatment, reduces or avoids the symptoms or causes of a disease or condition, or enhances the therapeutic effect of other therapeutic agents. In a specific embodiment of the present invention, the disease, disorder or condition is epilepsy or brain damage caused by epilepsy. In a specific embodiment of the present invention, the therapeutic agent is fluoxetine or a pharmaceutically acceptable salt thereof.

[0027] In some embodiments, the drugs, pharmaceutical compositions or pharmaceutical preparations provided herein can be administered in a variety of ways, including but not limited to: oral, subcutaneous, intravenous, intraarterial, intracoronary, intranasal, intrathecal, transdermal, mucosal, topical (e.g., gels, ointments, lotions, creams, etc.), intraperitoneal, intramuscular, intrapulmonary (e.g., using inhalable technology or pulmonary delivery systems), vaginal, parenteral, rectal or intraocular.

[0028] Those skilled in the art will appreciate that, although the pharmaceutical composition or drug mentioned above in the present invention may further comprise pharmaceutically acceptable excipients and / or vehicles, when fluoxetine or its pharmaceutically acceptable salt and / or at least one of the other drugs for treating epileptic seizures or brain damage are used as drugs for humans or animals, they may also be administered by themselves, that is, the present invention can be achieved without adding any of the above-mentioned pharmaceutically acceptable excipients and / or vehicles.

[0029] When using the pharmaceutical composition provided by the present invention, its administration regimen and dosage regimen can be selected according to a variety of factors, including the type, species, age, weight, sex and type of disease being treated by the subject; the severity of the disease being treated; the route of administration; the patient's renal and liver function; and the specific compound or salt thereof used. A dosing / dosage regimen can be used, for example, to prevent the disease, inhibit (completely or partially) the disease, or stop the development of the disease. In a specific embodiment of the present invention, the disease specifically refers to epileptic seizures or brain damage.

[0030] The term "subject" may refer to a human and a non-human animal. Preferably, the term "subject" refers to a human. The term "subject" may also refer to a fully grown human, a fully grown non-human animal, a pre-developed human, and / or a pre-developed non-human animal. A fully grown human may be an adult. A pre-developed human may be a child. Preferably, the term "subject" refers to a fully grown human. Further preferably, the term "subject" refers to a pre-developed human. Most preferably, the term refers to a fully grown human and / or a pre-developed human.

[0031] The fourth aspect of the present invention provides an in vitro non-therapeutic method for reducing the level of ROS and Fe in neuronal cells. 2+ Ways to level up and / or improve the level of GPX4.

[0032] Furthermore, the method comprises the step of administering fluoxetine or a pharmaceutically acceptable salt thereof to the neuronal cells.

[0033] Furthermore, the neuronal cells are PC12 or HT22 cells.

[0034] The terms "preferably," "more preferably," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are unsuitable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0035] In the description of the present invention, the term "and / or" includes all and any combinations of one or more related listed items.

[0036] In the description of the present invention, the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the exemplary expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0037] Advantages and beneficial effects of the present invention: The present invention discovered for the first time through in vivo and in vitro experiments that fluoxetine can inhibit the ubiquitination of GPX4 protein, thereby inhibiting epilepsy-induced ferroptosis, thereby playing a role in treating epileptic seizures or brain damage. The present invention provides a new technical means for the treatment of epilepsy or brain damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Fluoxetine (FLX) can reduce the frequency, grade and pathological damage of epileptic seizures in mice induced by kainate (KA); Figure 1 A is the result graph of epileptic seizure frequency in mice; Figure 1 B is the result of evaluating epileptic seizures in mice using the Racine scale; Figure 1 C is the expression results of inflammatory factors (IL-1β, IL-6, TNF-α) detected by qPCR; Figure 1 D. Figure 1 E are the results of HE and Nissl staining of pathological damage in the hippocampal CA1 and CA3 regions; Figure 2 The figure shows that FLX can reduce the cognitive impairment of epileptic mice induced by kainate (KA); Figure 2 A. Figure 2 B are the results and experimental trajectory diagrams of the effect of FLX on habituation memory in epileptic mice induced by kainate (KA); Figure 2 C. Figure 2 D are the results and experimental trajectory diagrams of the effect of FLX on the novel object recognition ability of epileptic mice induced by kainate (KA); Figure 2 E is the result graph showing the effect of FLX on the escape latency of the Morris water maze positioning navigation test in epileptic mice induced by kainate (KA); Figure 2 F. Figure 2 G. Figure 2 H. Figure 2I shows the experimental trajectory, the number of crossing the target quadrant, the time spent in the target quadrant, and the speed of the Morris water maze spatial exploration test of epileptic mice induced by kainate (KA). Figure 3 The figure shows that FLX can upregulate GPX4 protein expression and reduce ferroptosis of neurons in epileptic mice induced by kainate (KA) and neuronal cell death induced by glutamate; Figure 3 A is the effect of FLX on GPX4 protein expression in hippocampus of epileptic mice induced by kainate (KA); Figure 3 B. Figure 3 C shows the effect of FLX on glutamate (Glu)-induced GPX4 protein expression in HT22 cells and PC12 cells; Figure 3 D is the result of the effect of FLX on iron ion deposition in the hippocampus of epileptic mice induced by kainate (KA); Figure 3 E. Figure 3 F is the effect of FLX on ROS and Fe in HT22 cells and PC12 cells induced by glutamate (Glu), respectively. 2+ , the results of the effect of lipid peroxidation level; Figure 4 Figure 2 shows that FLX can inhibit the ubiquitination and degradation of GPX4 protein; Figure 4 A. Figure 4 B is the result diagram of the effect of FLX on the stability of GPX4 protein in HT22 cells and PC12 cells; Figure 4 C. Figure 4 D are the results of the effects of FLX on different post-translational degradation pathways of GPX4 protein in HT22 cells and PC12 cells; Figure 4 E is the result of the effect of FLX on the K48-linked ubiquitination modification of GPX4 protein in the hippocampus of epileptic mice induced by kainate (KA); Figure 4 F. Figure 4 G is the result of the effect of FLX on glutamate (Glu)-induced K48-linked ubiquitination modification of GPX4 protein in HT22 cells and PC12 cells; Figure 4 H is the result of transfecting Flag-ACSL4 and HA-Ub-K48 plasmids into HEK-293T cells to verify the effect of FLX on the ubiquitination modification of GPX4 protein. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific examples, which are intended only to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these examples without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods in the following examples, for which specific conditions are not specified, are generally tested under conventional conditions or as recommended by the manufacturer.

[0040] Example: Study on Fluoxetine (FLX) for the Treatment of Epilepsy and Brain Injury 1. Main instruments, reagents and materials used in the present invention 1. Instruments A high-speed cryogenic centrifuge and a high-speed cryogenic tissue grinder were purchased from Wuhan Sewell Biotechnology Co., Ltd.; a microinjector and a stereotaxic apparatus were purchased from Shenzhen Reward Life Science Co., Ltd.; and a chemiluminescence imaging system was purchased from Shanghai Qinxiang Scientific Instrument Co., Ltd. Open-field and water maze equipment were purchased from Beijing Zhongshi Dichuang Technology Development Co., Ltd.

[0041] 2. Reagents Kainic acid (KA) and FLX were purchased from MCE, USA; reactive oxygen species detection kit and lipid peroxidation detection kit (BODIPY 581 / 591 C11) were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; FerroOrange Fe 2+ The detection kit was purchased from Dojindo Chemical Research Institute, Japan; the GPX4 antibody was purchased from Thermo Fisher Scientific, USA.

[0042] 3. Experimental mice 6- to 8-week-old male C57BL / 6J mice were purchased from Beijing Spefoc Biotechnology Co., Ltd.

[0043] 2. Experimental methods used in the present invention 1. Epilepsy mouse model and construction 1) Anesthesia: Inject 1% sodium pentobarbital (10 μL / g) intraperitoneally into C57BL / 6J mice. Pinch the mouse's tail with your finger. If there is no response, anesthesia is successful. This should last approximately 10 minutes.

[0044] 2) Fixation: After successful anesthesia, place the mouse on a digital stereotaxic apparatus. Hang the mouse's teeth on the front incisor clamp of the apparatus. Keep the mouse's head and body in a straight line and on a horizontal plane. Insert the two fixing rods into the mouse's ears to fix the head so that it does not shake. Adjust the mouse's head to the center position. Pull the mouse's tongue outward to avoid airway obstruction. Always monitor the mouse's breathing and heart rate.

[0045] 3) Skin preparation: Use curved scissors to trim the mouse hair 1 cm on each side of the sagittal line and disinfect the exposed scalp with iodine.

[0046] 4) Expose the bregma: Cut the skin along the sagittal line to a size of about 2 cm. The bregma will be visible. Wipe the bregma with 1% hydrogen peroxide and remove the periosteum.

[0047] 5) Positioning: Place a microsyringe containing 1 μL of KA on a digital stereotaxic instrument. Move the syringe needle to the bregma point. Using the bregma point as the origin, move the needle tip so that it is 1.8 mm behind the bregma point and 2 mm to the right of the sagittal line. Mark this point with a marker.

[0048] 6) Drill the skull at the marked point using an electric miniature handheld cranial drill and stop after the dura mater is exposed.

[0049] 7) Brain microinjection: Move the microinjector needle back to the marked point and advance the needle at a rate of 1 mm / min. Stop advancing after approximately 2.3 mm and inject 1 μL of KA or saline into the hippocampus at a rate of 0.02 μL / min. Keep the mouse scalp moist with a cotton ball dipped in saline. After the injection, leave the needle in place for 5 minutes and slowly remove the syringe needle to prevent backflow of the injection solution.

[0050] 8) Skin suturing: After observing that there is no active bleeding, suture the mouse scalp and disinfect the scalp with iodine.

[0051] 2. Experimental Design The experimental mice were divided into three groups: sham group: mice were injected with an equal volume of normal saline into the hippocampus; KA group: mice were injected with 1uL (0.3ug) KA into the hippocampus; KA+FLX group: mice were injected with 1uL KA into the hippocampus and FLX was injected intraperitoneally.

[0052] HT22 cells and PC12 cells were plated and divided into three groups: Control group: no treatment; Glutamate group: 5 mM Glutamate was added to the culture medium; Glutamate+FLX group: Glutamate (5 mM) and FLX (5 ug / mL) were added to the culture medium.

[0053] 3. Evaluation of epileptic seizure grading in mice One hour after intracerebroventricular injection of KA, mice were assessed for seizure activity using the Racine scale. The Racine scale includes the following criteria: Grade 0: no seizures; Grade 1: oral and facial twitching, including jaw clonus; Grade 2: Grade 1 plus rhythmic nodding; Grade 3: Grade 2 plus unilateral forelimb twitching; Grade 4: bilateral forelimb twitching with standing; and Grade 5: generalized tonic-clonic convulsions with falling while standing. Sustained occurrence of Grade 4 or higher (≥2 times within 30 minutes) was considered status epilepticus.

[0054] 4. Detection of epileptic seizure frequency in mice After KA was injected into the lateral ventricle of mice, the epileptic seizure status of the mice was continuously recorded within 24 hours using a video monitoring and acquisition system, with the presence of a seizure being recorded as 1 and the absence of a seizure being recorded as 0.

[0055] 5. Detection of inflammatory factor expression in mouse hippocampus tissue After anesthetizing mice and performing cardiac perfusion, the hippocampus was carefully dissected out. TRIzol was added and homogenized using a tissue grinder. Tissue RNA was extracted and analyzed for the expression of inflammatory factors using qPCR.

[0056] 6. HE staining of mouse hippocampus tissue After anesthetizing mice and performing cardiac perfusion, the brain tissue was carefully excised and fixed in 4% paraformaldehyde for 48 hours. The sections were dehydrated, embedded, frozen, sectioned, stained with hematoxylin, differentiated, bluing, and eosin stained, dehydrated, and mounted. The sections were then observed under a light microscope and photographed for preservation.

[0057] 7. Nissl staining of mouse hippocampal tissue After anesthetizing mice and performing cardiac perfusion, the brain tissue was carefully excised and fixed in 4% paraformaldehyde for 48 hours. The brain tissue was then dehydrated, embedded, frozen sectioned, stained with toluidine blue, differentiated, dehydrated, and mounted. The tissue was then observed under a light microscope and photographed for preservation.

[0058] 8. Prussian blue staining of mouse brain tissue After anesthetizing mice and performing cardiac perfusion, the brain tissue was carefully excised and fixed in 4% paraformaldehyde for 48 hours. Paraffin sections were then dewaxed, stained with Prussian blue, stained for nuclei, and dehydrated for mounting. Finally, microscopic examination and image analysis were performed.

[0059] 9. Behavioral experiments The behavioral experiment used the ANY-maze system to record and analyze the behavior of epileptic mice and control mice.

[0060] (1) Open field test The open field test consisted of a square, open box measuring 50 cm long, 50 cm wide, and 40 cm high, along with data recording equipment and analysis software. Mice in the sham, KA+Saline, and KA+FLX groups were removed from the animal room and placed in the behavioral laboratory 1 hour before the experiment to allow them to acclimate to the environment, eliminate fear, and prevent environmental factors and stress reactions from interfering with the experimental results. The experimental environment was kept quiet, with uniform and relatively stable lighting, and the temperature maintained between 22 and 25°C. A 50 × 50 cm square, opaque white box (the open field) was divided into 16 equal-sized square areas. Each mouse was gently placed in a fixed position in the open field. After acclimation for 2 minutes, they were allowed to freely explore the area for 5 minutes (training). During this period, the number of line crossings was recorded using Fusion software (ANY-MAZE) to reflect the mouse's motor activity. After training, the mice were returned to their original cages. After 24 hours, the mice were tested again, and their threading time within 5 minutes was recorded (test). Changes in threading times between the training and testing phases were compared to characterize the mice's motor skills and memory retention.

[0061] (2) Novel object recognition experiment The novel object recognition experiment was designed to test the memory and cognitive functions of epileptic mice. On the first day, mice underwent acclimation training in a 50×50 cm open field. With no objects in the field, each mouse was gently placed in a fixed, uniform location on the open field and allowed to freely explore for 5 minutes to acclimate. On the second day, two identical objects, labeled A1 and A2, were placed at diagonal corners of the field (approximately 10 cm from the wall). The mice were allowed to freely explore the field for 5 minutes. 24 hours later, the mice underwent a long-term recognition and memory test. Object A1 was placed in the same location as the previous day, while A2 was replaced by another object, B, of completely different shape, color, and size. The mice were allowed to freely explore the open field for 5 minutes. The recognition index (RI) was calculated as: RI = novel object / (new object + old object) × 100%. The recognition indexes of the test and training tests were compared to reflect the mice's memory ability.

[0062] (3) Water maze experiment The water maze consists of a circular pool with an inner diameter of 120 cm and a height of 50 cm. A platform with a diameter of 6–9 cm is placed within the pool, along with corresponding data recording equipment and analysis software. Before the experiment, the pool is filled with water to a depth of approximately 30–40 cm, with the water surface slightly above the platform. The temperature is maintained at 22–25°C. The pool is divided into four equal quadrants. Each quadrant is marked with a distinctive, brightly colored, and shaped graphic marker on the sidewall. These markers remain fixed in position and size throughout the experiment to help mice distinguish and establish spatial memory. A platform is placed in quadrant IV and marked as the platform area in the system software. The platform is slightly submerged below the water surface, and an appropriate amount of titanium dioxide is poured into the water to conceal the platform.

[0063] The experiment lasted six days, with the first five days consisting of a repetitive platform-finding learning phase. At the end of training, each mouse underwent four trials of swimming to find the platform. During each training session, mice were gently lowered into the water from the pool wall and allowed to swim freely for 60 seconds. After all mice in the sham, KA+saline (normal saline) and KA+FLX groups had swum once in a quadrant, they continued swimming in the same order in the next quadrant. After each swim, the mice rested for at least 30 minutes to recover and avoid distorting the experimental results. Each time, mice were released from the same location in each of the four quadrants of the pool. The software automatically stopped recording after swimming for 60 seconds or if the mouse found the platform prematurely. At the end of each quadrant, mice that failed to find the platform were placed on the platform and allowed to rest for 10 seconds to allow them to learn the recording. After the first five days of learning and training, the platform was removed and tested on the sixth day. Each time was set for 60 seconds. The total swimming distance of the mice during the test phase, the speed of finding the platform, the time spent on finding the platform for the first time, and the exploration time of the quadrant where the platform was located were observed and recorded and analyzed to detect changes in the spatial memory ability of epileptic mice.

[0064] 10. Flow cytometry (1) Reactive oxygen species (ROS) detection experiment: A fluorescent probe (DCFH-DA) was used. DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by the esterase in the cell to produce DCFH. However, DCFH cannot penetrate the cell membrane, making it easy for the probe to be loaded into the cell. Reactive oxygen species in the cell can oxidize the non-fluorescent DCFH to produce fluorescent DCF (488nm excitation wavelength, 525nm emission wavelength). The treated HT22 cells and PC12 cells were diluted with probe diluent at a ratio of 1:1000 to a final concentration of 10 μmol / L. After the cells were collected, they were suspended in the diluted DCFH-DA at a cell concentration of 1×106 / ml and incubated in a cell culture incubator at 37°C for 20 minutes. The probe was mixed by inversion every 5 minutes to ensure full contact between the probe and the cells. The cells were washed three times with serum-free DMEM solution to fully remove the DCFH-DA that did not enter the cells. A flow cytometry machine was used to detect the intensity of fluorescence of cells in each group.

[0065] (2) FerroOrange Fe 2+ Detection experiment: FerroOrange is a specific Fe 2+ probe, with Fe 2+ Combined to form an orange fluorescent complex (543nm excitation wavelength, 580nm emission wavelength), which can be detected by flow cytometry to quantitatively analyze intracellular Fe 2+ A 1 μM concentration of FerroOrange working solution was added to the treated HT22 and PC12 cells and incubated in a 37°C, 5% CO2 incubator for 15 minutes. The fluorescence intensity of each cell group was measured using a flow cytometer.

[0066] (3) Lipid peroxidation detection experiment: BODIPY 581 / 591 C11 is a lipid-soluble ratiometric fluorescent probe. In the reduced state, the excitation / emission wavelengths are 581nm / 591nm (red fluorescence). In the oxidized state, the excitation / emission wavelengths shift to 488nm / 510nm (green fluorescence) due to the attack of the polyunsaturated butadiene moiety by lipid peroxides. The ratio of red and green fluorescence intensities can be detected by flow cytometry to quantitatively reflect the degree of intracellular lipid peroxidation. 1mL of BODIPY 581 / 591 C11 staining working solution (2uM) was added to the treated HT22 cells and PC12 cells and incubated in a 37℃, 5% CO2 incubator for 30min. The fluorescence intensity of each group of cells was detected using a flow cytometer.

[0067] 11. Western blot experiment (1) Protein sample preparation: Add RIPA lysis buffer (with 1% protease inhibitor) to the tissue or cell sample and lyse on ice for 20 min, using a shaker to increase lysis efficiency. Centrifuge at 12,000 rpm at 4°C for 15 min, and collect the supernatant. Then, add an appropriate volume of 5× loading buffer and boil for 10 min to obtain the WB protein sample.

[0068] (2) Immunoblotting: Separate protein samples using SDS-PAGE gel. After electrophoresis, transfer to a PVDF membrane. Incubate with 2.5% blocking buffer for 2 hours and then incubate with the primary antibody at 4°C overnight. The next day, wash three times with TBST, incubate with the corresponding secondary antibody at room temperature for 2 hours, and then wash the membrane three times. Then, add ECL luminescent solution to develop the image in a developer.

[0069] 12. Ubiquitination detection experiment Collect tissue or cell samples in 1.5 mL EP tubes and add 200 μL of CO-IP lysis buffer (supplemented with 1% protease inhibitors, DTT, and NaVO₃). Lyse on ice for 20 minutes, using ultrasonication to enhance lysis efficiency. Centrifuge at 12,000 rpm at 4°C for 15 minutes, and collect the supernatant. Add GPX4 IP antibody and incubate with rotation for 6 hours. Then, add 20 μL of Protein A+G Agarose and rotate overnight. The next day, wash the magnetic beads using high-salt and low-salt wash buffers, using a suspension for 5 minutes each wash. After each wash, add 20 μL of 2× loading buffer and boil for 15 minutes to obtain the CO-IP sample. K48-linked ubiquitination levels can be assessed by western blot.

[0070] 3. Experimental Results The epileptic seizure status of mice was continuously recorded within 24 hours by a video monitoring and acquisition system. Figure 1 As shown in the figure, compared with the KA+Saline (normal saline) group, the frequency of epileptic seizures in mice in the KA+FLX group was significantly reduced and there was a significant difference, which indicates that FLX significantly reduced the frequency of epileptic seizures in mice induced by kainate (KA) ( Figure 1 A). The Racine scale was used to evaluate the epileptic seizures in mice. Figure 1As shown in Figure B, compared with the KA+Saline group, the epilepsy level in the KA+FLX group was reduced by about one-half, indicating that FLX can reduce the grade of epileptic seizures induced by kainate (KA) in mice. After anesthetizing the mice and performing cardiac perfusion, the hippocampal tissue of the mice was carefully dissected out, TRIzol was added, and the tissue was homogenized using a tissue grinder. Tissue RNA was extracted and the expression of inflammatory factors was examined by qPCR. The degree of inflammation in the hippocampus of mice was evaluated using inflammatory factors (IL-1β, IL-6, TNF-α) in the hippocampus. The results are shown in Figure 2. Figure 1 As shown in C, FLX can reduce the inflammation level in the hippocampus of epileptic mice induced by kainate (KA). HE staining and Nissl staining results also showed that FLX can reduce the pathological damage of CA1 and CA3 regions of the hippocampus in epileptic mice induced by kainate (KA). Figure 1 D. Figure 1 E).

[0071] like Figure 2 As shown in the open field test, the results showed that there was no significant difference in the number of movement threading between the training and test periods in the epileptic mice in the KA+Saline (normal saline) group, indicating that the habituation memory was impaired; while the mice in the KA+FLX group showed significantly fewer threading times during the test period compared with the training period, indicating that the habituation memory was preserved ( Figure 2 A and Figure 2 B), which shows that FLX significantly reduced the habituation memory impairment induced by kainate (KA) in mice. The cognitive ability of mice was evaluated using the novel object recognition test. The results are shown in Figure 2. Figure 2 C and Figure 2 As shown in D, during the training period, mice in each group showed no preference for the same objects (A1 and A2); during the test period, epileptic mice in the KA+Saline (normal saline) group showed no preference for new objects (B), while mice in the KA+FLX group showed a clear preference for new objects, indicating that FLX significantly reduced the cognitive impairment induced by kainate (KA) in mice. The results of the water maze test showed that mice in the KA+FLX group showed a shorter escape latency in the navigation test than mice in the KA+Saline (normal saline) group ( Figure 2 E). In addition, the number of times the KA+FLX group mice crossed the target quadrant in the spatial exploration test ( Figure 2 F and Figure 2 G) and the dwell time in the target quadrant ( Figure 2 H) were significantly higher than those in the KA+Saline (normal saline) group, but there was no significant difference in swimming speed between the two groups of mice ( Figure 2 I), which indicated that FLX significantly reduced the impairment of learning and memory ability of mice induced by kainate (KA).

[0072] Figure 3As shown, FLX can upregulate the expression of GPX4 protein (whose function is to inhibit ferroptosis) and inhibit epilepsy-related neuronal ferroptosis. In particular, FLX upregulates the expression of GPX4 protein in the hippocampus of epileptic mice induced by kainate (KA). Figure 3 A). In HT22 cells and PC12 cells induced by glutamate (Glu) Figure 3 B and Figure 3 C), FLX upregulates GPX4 protein expression; Figure 3 As shown in Figure D, compared with the mice in the KA+Saline (normal saline) group, the iron deposition in the hippocampus of the mice in the KA+FLX group was significantly reduced, indicating that FLX can inhibit epilepsy-related ferroptosis. Further detection of ferroptosis-related markers in HT22 cells and PC12 cells induced by glutamate (Glu) showed that the iron deposition in the hippocampus of the mice in the KA+FLX group was significantly reduced, indicating that FLX can inhibit epilepsy-related ferroptosis. Figure 3 E and Figure 3 As shown in F, FLX can reduce the Fe 2+ , intracellular ROS and lipid ROS (C11-BODIPY staining). These results indicate that FLX inhibits epilepsy by upregulating GPX4 protein expression and alleviating neuronal ferroptosis.

[0073] Figure 4 As shown, FLX enhances the stability of GPX4 protein by inhibiting K48-linked ubiquitination and degradation of GPX4. Figure 4 A and Figure 4 (B) FLX enhanced the stability of GPX4 protein in HT22 and PC12 neuronal cells in the presence of cycloheximide (CHX, which inhibits protein synthesis in ribosomes). Figure 4 C and Figure 4 D shows that the decrease in GPX4 induced by glutamate (Glu) can be reversed by FLX. GPX4 expression is further enhanced only by the proteasome inhibitor MG132, while neither the lysosomal inhibitor chloroquine nor the autophagy inhibitor 3-MA further enhances GPX4 expression, indicating that FLX can inhibit the proteasome degradation of GPX4 induced by glutamate (Glu) in neuronal cells. Figure 4 E shows that FLX can inhibit the K48-linked ubiquitination level of GPX4 protein in the hippocampus of epileptic mice induced by kainate (KA). Figure 4 F and Figure 4 G shows that FLX inhibits glutamate (Glu)-induced K48-linked ubiquitination of GPX4 in HT22 and PC12 cells. FLX's ability to inhibit K48-linked ubiquitination of GPX4 was further verified in HEK-293T cells. These results suggest that FLX enhances GPX4 protein stability by inhibiting its ubiquitination and degradation.

[0074] In summary, the above results indicate that fluoxetine can inhibit epilepsy-induced ferroptosis by inhibiting the ubiquitination of GPX4 protein, thereby playing a role in treating epileptic seizures or brain damage.

[0075] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. Use of fluoxetine or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating epileptic seizures or brain damage.

2. The use according to claim 1, characterized in that Fluoxetine reduces ferroptosis by inhibiting GPX4 ubiquitination to treat epileptic seizures or brain damage.

3. The use according to claim 1, characterized in that The fluoxetine or its pharmaceutically acceptable salt is used as the sole active ingredient or one of the active ingredients of a drug for treating epileptic seizures or brain damage; The epilepsy is convulsion or status epilepticus, and the brain damage is induced by epilepsy.

4. The use according to claim 1, characterized in that The dosage form of the drug includes oral dosage form, parenteral dosage form and / or topical dosage form; More preferably, the dosage form of the pharmaceutical preparation includes solution, sustained-release agent, suspension, granule, tablet, capsule, powder, effervescent, emulsion, syrup, drops and / or chewable; Preferably, the administration of the drug includes oral, subcutaneous, intravenous, intramuscular, intraarterial, intranasal, intrathecal, mucosal, intrapulmonary and / or rectal administration.

5. Use of fluoxetine or a pharmaceutically acceptable salt thereof in the preparation of a product having any one or more of the following effects: (1) reducing the frequency of epileptic seizures; (2) reducing the grade of epileptic seizures; (3) reducing the inflammatory response of hippocampal tissue; (4) reducing cognitive impairment induced by epilepsy; (5) reducing neuronal ferroptosis; and (6) inhibiting the ubiquitination and degradation of GPX4 protein.

6. A pharmaceutical composition for treating epileptic seizures or brain damage, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of fluoxetine or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further includes a second therapeutic agent; Preferably, the second therapeutic agent is another drug used to treat epileptic seizures or brain damage; More preferably, the other drugs for treating epileptic seizures or brain damage include sodium valproate, carbamazepine, phenobarbital, phenytoin sodium, ethosuximide, oxcarbazepine, lamotrigine, topiramate, levetiracetam, and lacosamide.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the auxiliary materials include adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH adjusting and / or buffering agents, solvents, surfactants or emulsifiers.

9. A method for reducing the level of ROS and Fe in neuronal cells for non-therapeutic purposes in vitro 2+ A method for leveling and / or improving GPX4 level, characterized in that, The method includes the step of administering fluoxetine or a pharmaceutically acceptable salt thereof to neuronal cells.

10. The method according to claim 9, characterized in that The neuronal cells are PC12 or HT22 cells.

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