Application of clobetasol in neurotoxic injury treatment

Abstract

The invention provides application of clobetasol in neurotoxic injury treatment, and belongs to the technical field of biological medicine. The clobetasol is proved to be capable of effectively reversing or relieving demyelination, axonal injury, neuroinflammation and depression and anxiety-like behaviors caused by neurotoxic injury, a new treatment strategy is provided for neurotoxic injury occurring after cancer patients are subjected to chemotherapy, the living quality and treatment compliance of the patients can be improved, and the clobetasol can be used for treating neurotoxic injury caused by neurotoxic injury. Meanwhile, the clinical application range of clobetasol is expanded, and a new direction is provided for development of neuroprotective drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of clobetasol in the treatment of neurotoxic injuries. Background Technology

[0002] Neurotoxic injury refers to damage to the structure and function of the nervous system caused by exogenous chemical substances. Common causes include chemotherapy drugs, industrial chemicals, heavy metals, biotoxins, and environmental pollution. This damage can manifest as pathological features such as demyelination, neuronal damage, and neuroinflammation, as well as cognitive impairment and behavioral abnormalities. In recent years, with the widespread use of chemotherapy in cancer treatment, chemotherapy-induced neurotoxic injury has received increasing attention. For example, the phenomenon of "chemotherapy brain" manifests as memory loss, poor concentration, and decreased executive function, severely impacting patients' quality of life and recovery process. Currently, interventions for neurotoxic injury are relatively limited, mainly relying on terminating chemotherapy, symptomatic supportive treatment, neurotrophic drugs, and rehabilitation training. However, the effects are often unsatisfactory, and specific therapeutic drugs are lacking.

[0003] Clobetasol is a potent corticosteroid that, since its market launch in 1985, has been primarily used to treat various refractory inflammatory skin diseases, such as psoriasis, eczema, and lichen planus. Its mechanism of action mainly involves anti-inflammation and immunosuppression, exerting its therapeutic effect by activating glucocorticoid receptors. Although clobetasol is widely used in dermatology, its role in the nervous system has not been fully explored. Recent studies have shown that clobetasol can cross the blood-brain barrier and may promote oligodendrocyte differentiation and myelin regeneration, suggesting potential central nervous system protective functions. However, whether clobetasol can be used to treat neurotoxic injuries, especially those caused by chemotherapy drugs, currently lacks clear evidence and clinical application reports. Summary of the Invention

[0004] The purpose of this invention is to provide the application of clobetasol in the treatment of neurotoxic injury. This invention solves the problem of the lack of effective treatments for neurotoxic injury caused by chemotherapy drugs (such as demyelination, axonal injury, neuroinflammation, and depressive and anxious behaviors).

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of clobetasol in the preparation of medicaments for the prevention or treatment of neurotoxic damage.

[0006] The present invention also provides the use of clobetasol in the preparation of a medicament for improving demyelination caused by neurotoxic injury.

[0007] This invention also provides the use of clobetasol in the preparation of drugs for promoting oligodendrocyte differentiation or maturation.

[0008] The present invention also provides the use of clobetasol in the preparation of a medicament for inhibiting neuroinflammation.

[0009] The present invention also provides the use of clobetasol in the preparation of a medicament for alleviating depression and / or anxiety-like behavior caused by neurotoxic injury.

[0010] Preferably, the neurotoxic injury is induced by chemotherapy drugs.

[0011] Preferably, the chemotherapy drug is temozolomide.

[0012] Preferably, the route of administration of the drug is intraperitoneal injection, intravenous injection, oral administration, or nasal administration.

[0013] Preferably, the dosage form of the drug is an injection, a lyophilized powder for injection, a tablet, or a capsule.

[0014] Preferably, the drug further comprises one or more pharmaceutically acceptable carriers, including solvents, cosolvents, solubilizers, buffers, isotonic modifiers, preservatives, antioxidants, diluents, binders, disintegrants, or lubricants.

[0015] The beneficial effects of this invention are: This invention demonstrates that clobetasol can effectively reverse or alleviate demyelination, axonal damage, neuroinflammation, and depression and anxiety-like behaviors caused by neurotoxicity. It provides a new treatment strategy for neurotoxicity damage after chemotherapy in cancer patients, helps improve patients' quality of life and treatment adherence, expands the clinical application of clobetasol, and provides a new direction for the development of neuroprotective drugs. Attached Figure Description

[0016] Figure 1 The diagram illustrates the method for preparing a neurotoxic injury model using TMZ treatment and the subsequent medication regimen for CBT therapy; Figure 2 LFB staining of brain tissue showed that CBT effectively reversed TMZ-induced demyelinating damage. Images showed that TMZ treatment led to a significant reduction in myelin, a component of the myelin sheath, which was significantly restored after CBT treatment. Figure 3 Black-Gold staining of brain tissue showed that CBT effectively reversed TMZ-induced demyelinating damage. Images showed that TMZ treatment led to a significant decrease in protein content in the myelin sheath, which was significantly restored after CBT treatment. Figure 4Immunofluorescence staining of brain tissue showed that CBT effectively reversed TMZ-induced reduction in mature oligodendrocytes. Images showed that the proportion of mature oligodendrocytes in the corpus callosum region of the TMZ-treated group was significantly decreased, while the proportion of mature oligodendrocytes in the TMZ+CBT group was significantly increased. Figure 5 Transmission electron microscopy (TEM) images of the optic nerve showed that CBT effectively reversed TMZ-induced demyelination damage. (A) The figure shows a representative TEM image of the optic nerve. Black asterisks indicate normal axons, red asterisks indicate unmyelinated axons, and orange asterisks indicate degenerated axons. (B) The figure shows the proportion of axons with demyelination in the mouse optic nerve; Figure 6 Transmission electron microscopy (TEM) images of the optic nerve show that CBT can alleviate TMZ-induced axonal damage. (A) is a representative TEM image of the optic nerve. Black asterisks indicate normal axons, and orange asterisks indicate degenerated axons. (B) shows the number of axons per unit area in the mouse optic nerve. (C) shows the changes in axonal diameter in the mouse optic nerve. (D) shows the proportion of degenerated axons in the mouse optic nerve; Figure 7 Immunofluorescence staining of brain tissue showed that CBT treatment effectively reversed TMZ-induced microglial activation. The staining images showed that after CBT treatment, the number of Iba1+ microglia in the corpus callosum of TMZ-treated mice was significantly reduced. Figure 8 Immunofluorescence staining of brain tissue showed that CBT treatment effectively reversed TMZ-induced astrocyte activation. The staining images showed that after CBT treatment, the number of GFAP+ reactive astrocytes in the corpus callosum of TMZ-treated mice was significantly reduced. Figure 9 Tail suspension assays showed that CBT tended to alleviate TMZ-induced depressive-like behavior in mice. Statistical graphs showed that, compared with the TMZ group, the CBT treatment group (TMZ+CBT) had a shorter immobility time. Figure 10 Open-field experiments showed that CBT tended to alleviate TMZ-induced anxiety-like behavior in mice. (A) Figure is a schematic diagram of the open-field experiment. (B) Figure shows the distance the mouse traveled across the entire field. (C) Figure shows the percentage of distance the mouse traveled in the central area versus the entire field. (D) Figure shows the duration of mouse activity. (E) Figure shows the duration the mouse remained still. Figure 11 Bulk RNA-seq analysis showed that CBT treatment could reverse TMZ-induced gene pathway alterations. Detailed Implementation

[0017] This invention provides the application of clobetasol in the preparation of medicaments for the prevention or treatment of neurotoxic injury. Neurotoxic injury refers to damage to the structure and function of the nervous system caused by exogenous chemicals. Its causes are diverse, stemming from adverse drug reactions, exposure to environmental toxins, contact with industrial chemicals, or metabolic diseases, among other factors. Pathologically, this type of injury can manifest as various forms of cellular dysfunction, such as neuronal degeneration, glial cell activation, and oxidative stress, ultimately leading to abnormal nerve conduction and behavioral changes. Currently, clinical treatments for neurotoxic injury are limited, primarily relying on symptomatic supportive care and neurotrophic drugs, lacking specific targeted therapies. Clobetasol, as a potent glucocorticoid, has not yet been fully explored in the treatment of neurological diseases. Expanding the application of marketed drugs to new therapeutic areas is a crucial strategy in modern drug development. This "drug repurposing" approach can significantly shorten drug development cycles, reduce R&D costs, and provide new options for disease treatment.

[0018] In the embodiments of this invention, the factors inducing neurotoxic damage are diverse and extensive, including not only temozolomide but also various other chemotherapeutic drugs, such as platinum-based drugs, antimetabolites, anthracyclines, and other conventional chemotherapeutic drugs, as well as various molecularly targeted therapies; in addition, they include therapeutic drugs such as the anti-tuberculosis drug isoniazid, the antibacterial drug metronidazole, and various immunosuppressants; industrial chemicals such as industrial alcohol, narcotics, pesticide formulations, radioactive elements, hazardous chemicals, and harmful industrial gases; heavy metal pollution such as common neurotoxic metals such as lead, mercury, arsenic, and aluminum; biotoxins including snake venom and scorpion venom from animal sources, aconitine and ricin from plant sources, and botulinum toxin from microbial sources; and various environmental pollutants such as atmospheric particulate matter, persistent organic pollutants, and endocrine disruptors. These different types of neurotoxic substances can cause damage to the nervous system through multiple mechanisms, providing a wealth of research models for evaluating the neuroprotective effects of clobetasol.

[0019] Regarding the types of neurotoxic injury, this invention includes both central nervous system (CNS) and peripheral nervous system (PNS) damage. CNS damage can involve different brain regions such as the cerebral cortex, basal ganglia, hippocampus, cerebellum, and brainstem, as well as CNS pathways such as the optic nerve and spinal cord. Peripheral nerve damage can manifest as dysfunction of sensory, motor, and autonomic nerves, commonly seen in chemotherapy-induced peripheral neuropathy. This broad range of applications gives clobetasol a wider prospect for application in the field of neuroprotection.

[0020] In terms of sample selection, in addition to the cerebral cortex and body callosity, the following can also be included: hippocampus—a brain region closely related to learning and memory; cerebellum—responsible for motor coordination and balance; brainstem—regulating basic life activities; optic nerve—a special component of the central nervous system; spinal cord—an important structure connecting the central and peripheral nervous systems; and representative samples from peripheral nerves such as the sciatic nerve. This diverse range of samples provides ample technical support for a comprehensive evaluation of the neuroprotective effects of clobetasol.

[0021] This invention also provides the application of clobetasol in the preparation of a drug for improving demyelination caused by neurotoxicity. Demyelination refers to the pathological process of damage or loss of the myelin sheath membrane of nerve fibers. The myelin sheath, a lipid structure surrounding nerve axons, is formed by oligodendrocytes in the central nervous system, and its function is to ensure rapid nerve impulse conduction. Demyelination lesions can lead to slowed nerve conduction velocity or even conduction block, causing a series of neurological dysfunctions. Clinically, demyelinating diseases include various types such as multiple sclerosis and acute disseminated encephalomyelitis, and secondary demyelination caused by chemotherapy drugs is receiving increasing attention. Currently, treatments to promote myelin regeneration are limited, and there is an urgent clinical need for drugs that can effectively promote myelin repair. Clobetasol, as a drug with the ability to cross the blood-brain barrier, has potential in promoting myelin repair that deserves further exploration.

[0022] This invention also provides the application of clobetasol in the preparation of drugs for promoting oligodendrocyte differentiation or maturation. Oligodendrocytes are an important type of glial cell in the central nervous system, primarily responsible for forming and maintaining myelin sheath structures. Oligodendrocyte precursor cells undergo differentiation and maturation to eventually form mature oligodendrocytes with branched processes; this process is precisely regulated by various internal and external factors. Disorders in oligodendrocyte differentiation or impaired maturation lead to abnormal myelin formation, thereby affecting the normal function of the nervous system. Current research indicates that multiple signaling pathways are involved in regulating oligodendrocyte development and differentiation, including classic pathways such as Notch, Wnt, and Hedgehog. Finding drugs that can promote oligodendrocyte differentiation and maturation is of great significance for the treatment of demyelinating diseases. Clobetasol, as a glucocorticoid receptor agonist, may provide a new means of promoting oligodendrocyte maturation through its role in regulating cell differentiation.

[0023] Regarding routes of administration and dosage forms, in addition to intraperitoneal injection, this invention also includes various conventional routes of administration such as intravenous injection, oral administration, and nasal administration, as well as innovative drug delivery systems based on biomaterials such as local sustained-release gels, nanoparticles, hydrogels, and microspheres. These novel delivery systems can improve drug bioavailability, prolong drug action time, enhance targeting, and reduce systemic side effects. Furthermore, clobetasol can be combined with radiotherapy, chemotherapy, immunotherapy, electric field therapy, and other molecularly targeted drugs to form various combination treatment regimens, enhancing efficacy and reducing toxic side effects through synergistic effects, providing more individualized treatment options for clinical practice.

[0024] Regarding the timing of treatment, clobetasol can exert its therapeutic effect both after neurotoxic injury has occurred and simultaneously with the injury, providing more options for clinical medication. Early intervention may prevent further development of the injury, while post-injury treatment helps promote repair and functional recovery.

[0025] This invention also provides the use of clobetasol in the preparation of drugs for inhibiting neuroinflammation. Neuroinflammation is a defensive response of the nervous system to various damaging factors, characterized by the activation of microglia and astrocytes, the release of inflammatory cytokines, and the production of inflammatory mediators. Moderate neuroinflammation has a protective effect, but excessive or persistent neuroinflammatory responses can lead to nerve cell damage and dysfunction. Neuroinflammation plays an important role in many neurological diseases, such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Currently used anti-inflammatory drugs include various types such as nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, but drugs that specifically act on the central nervous system and have fewer side effects are still limited. Clobetasol, as a potent anti-inflammatory drug, is widely used in dermatology, and its central anti-inflammatory effects warrant further development.

[0026] This invention also provides the use of clobetasol in the preparation of a medicament for alleviating depression and / or anxiety-like behavior caused by neurotoxic injury. Neurotoxic injury not only causes changes in tissue structure but is also often accompanied by behavioral abnormalities, among which depression and anxiety-like behavior are among the most common manifestations. These behavioral changes may be related to multiple mechanisms such as neurotransmitter system dysregulation, abnormal neural circuit function, and alterations in neuroplasticity. Clinically, chemotherapy-related cognitive and behavioral disorders are known as "chemotherapy brain," which seriously affects patients' quality of life. Currently, the treatment for these behavioral abnormalities mostly uses traditional antidepressants and anti-anxiety drugs, but the effects are often unsatisfactory. Finding drugs that can simultaneously improve structural and functional damage from a neuroprotective perspective has important clinical significance. As a pleiotropic drug, the role of clobetasol in improving neurotoxic injury-related behavioral abnormalities deserves further investigation.

[0027] In a preferred embodiment of the invention, the neurotoxic injury is induced by chemotherapy drugs. Chemotherapy is one of the main methods of cancer treatment, but many chemotherapy drugs, while killing tumor cells, also have toxic effects on normal tissues, with the nervous system being a common site of involvement. Chemotherapy-induced neurotoxicity can manifest in various forms, such as peripheral neuropathy and central nervous system damage, severely impacting patients' quality of life and treatment adherence. With the increasing survival time of cancer patients, the management of chemotherapy-related neurotoxicity is becoming increasingly important.

[0028] More preferably, the chemotherapy drug is temozolomide. Temozolomide is an oral alkylating agent antitumor drug that can cross the blood-brain barrier and is mainly used to treat central nervous system tumors such as malignant gliomas. It has shown good antitumor effects in clinical applications, but may also cause a series of neurological side effects. Exploring protective drugs to reduce the neurotoxicity of temozolomide is of great significance for improving treatment efficacy and patients' quality of life.

[0029] In terms of the pharmaceutical formulation of the present invention, the drug can be administered via various routes, including intraperitoneal injection, intravenous injection, oral administration, or nasal administration. Intraperitoneal injection is a commonly used route of administration in animal experiments, characterized by its simplicity and stable absorption; intravenous injection allows the drug to directly enter the circulatory system, rapidly achieving an effective blood concentration; oral administration is the most common clinical method, offering advantages such as ease of use and high patient compliance; nasal administration allows absorption through the nasal mucosa directly into the central nervous system, bypassing the blood-brain barrier. Different routes of administration can be selected based on clinical needs and drug characteristics.

[0030] Regarding dosage forms, the drugs include common forms such as injections, lyophilized powder for injection, tablets, and capsules. Injections can be divided into various forms such as solutions, emulsions, and suspensions, which can be selected according to the requirements of drug solubility and stability; lyophilized powder for injection is prepared using freeze-drying technology, which can improve drug stability and facilitate storage and transportation; tablets are the most common oral solid dosage forms, and drug release behavior can be controlled by adjusting excipients and process parameters; capsules can mask unpleasant drug odors and improve patient medication adherence. These dosage forms are all mature technologies in the pharmaceutical field and can be selected and optimized according to specific needs.

[0031] Regarding the pharmaceutical composition, the drug further comprises one or more pharmaceutically acceptable carriers. These carriers include common pharmaceutical excipients such as solvents, cosolvents, solubilizers, buffers, isotonic modifiers, preservatives, antioxidants, diluents, binders, disintegrants, or lubricants. Solvents may include water for injection, physiological saline, vegetable oils, etc.; cosolvents include ethanol, propylene glycol, polyethylene glycol, etc.; solubilizers may use surfactants such as Tween and Span derivatives; buffers may include phosphate, acetate, citrate, etc.; isotonic modifiers include sodium chloride, glucose, mannitol, etc.; preservatives may include parabens, benzoic acid, sorbic acid, etc.; antioxidants may include sulfites, vitamin C, vitamin E, etc.; diluents may include starch, lactose, microcrystalline cellulose, etc.; binders may include hydroxypropyl methylcellulose, polyvinylpyrrolidone, etc.; disintegrants may include croscarmellose sodium, low-substituted hydroxypropyl cellulose, etc.; lubricants may include magnesium stearate, talc, etc. These excipients are widely used in pharmaceutical preparations, and their properties and functions are well known to those skilled in the art.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Example

[0033] Preparation of neurotoxic injury model and clobetasol dosing regimen.

[0034] Six-week-old adult wild-type C57 / BL6 mice were randomly assigned to each group. All mice were housed in an environment with a 12-hour light cycle and free access to food and water. Animals were housed under specific temperature-controlled, pathogen-free (SPF) conditions. No other treatments were given to the animals except as reported for each specific experimental group, and there was no prior drug exposure or surgery.

[0035] A neurotoxicity model was induced by intraperitoneal injection of temozolomide (TMZ) (25 mg / kg), a first-line chemotherapy drug for glioma. Following a clinical chemotherapy regimen for glioma, TMZ treatment was administered in three cycles, each lasting four days, with a three-day interval between cycles. Starting four days after the end of the TMZ treatment cycle, some mice received daily intraperitoneal injections of cBT (2 mg / kg) for 14 days to assess its neuroprotective effect. Behavioral tests were performed at specified time points, and mice were sacrificed at the experimental endpoint for tissue collection and analysis. A schematic diagram of the specific drug administration regimen is shown below. Figure 1As shown in the figure. The study included four groups: a control group (Mock) receiving intraperitoneal injection of saline as a CBT solvent at the same time; a CBT group treated with clobetasol or temozolomide alone; and a TMZ+CBT group treated with TMZ followed by CBT. In this study, female mice were primarily used to assess whether chemotherapy drugs cause neurological damage. This choice was based on previous research finding that female mice are more prone to central nervous system degeneration, particularly white matter degeneration. Therefore, this invention focuses on evaluating whether female mice exhibit higher sensitivity to temozolomide (TMZ)-induced white matter damage and whether clobetasol (CBT) can more effectively mitigate these effects.

[0036] Clobetasol treatment can effectively reverse temozolomide-induced demyelinating damage: To assess myelin damage and recovery, this invention employs Luxol Fast Blue (LFB) staining and Black Gold (BG) staining to assess the degree of myelination in mouse brains, specifically myelin and myelin proteins. The specific procedures are as follows: Mice were anesthetized with isoflurane and perfused with PBS, followed by fixation with 4% paraformaldehyde (PFA, Sigma-Aldrich). Brain tissue was harvested and fixed overnight at 4°C with 4% PFA fixative, then dehydrated in 30% sucrose (Sigma-Aldrich) at 4°C until the brain tissue settled. The brain tissue was then embedded and fixed in Tissue-TekOCT (Sakura) containers, and frozen sections were prepared with a section thickness of 20 µm. BG staining was performed according to the manufacturer's instructions (Biosensis). LFB staining was performed according to the standard protocol, using 0.1% LFB solution (Sigma-Aldrich) and counterstained with 0.5% eosin and 0.1% cresyl violet. Both staining procedures included observation of the corpus callosum (CC) and cortex. At least three sections of the brain from each mouse were analyzed for BG and LFB staining. Staining intensity was quantified by comparing the relative intensities of stained sections from the experimental and control groups using ImageJ / FIJI software. Figure 2 LFB staining and Figure 3 Black Gold staining showed that TMZ treatment significantly reduced the strength of myelin fibers in mice, while CBT treatment significantly restored the strength of myelin fibers in mice.

[0037] To clarify the changes in mature oligodendrocytes that form the myelin sheath of the central nervous system, this invention uses co-staining with CC1 antibody (1:200, Millipore), a marker of mature oligodendrocytes, and Olig2 antibody (1:100, Millipore), a marker of oligodendrocyte lineage. The tissue immunostaining steps are as follows: Mouse brain tissue was obtained following standard anesthesia and cardiac perfusion procedures, followed by fixation overnight in 4% paraformaldehyde at 4°C, and dehydration with 30% sucrose solution. 20µm thick frozen sections were prepared for immunostaining. Sections were blocked for 1 hour at room temperature with blocking buffer containing 1% bovine serum albumin (BSA, Sigma) and 3% normal goat serum. Primary antibody was diluted with the same blocking buffer and incubated overnight at 4°C. After primary antibody incubation, sections were washed three times with PBS and then incubated for 1–2 hours at room temperature with AlexaFluor-conjugated secondary antibody (Thermo Fisher Scientific) at a dilution of 1:1000. Sections were counterstained with DAPI (Invitrogen) to label cell nuclei and mounted with anti-fluorescence quenching mounting medium. Immunofluorescence images were acquired using an S3000 confocal laser scanning microscope (HOOKE Instruments, Changchun, China). For quantitative analysis, at least three sections were analyzed for each brain tissue sample. ImageJ / FIJI software was used to measure staining intensity or count the number of positive cells to ensure that the regions of interest (ROIs) in the experimental and control groups were compared in a consistent manner.

[0038] The results are as follows Figure 4 As shown, immunofluorescence staining results of CC1 / Olig2 in the corpus callosum (CC), a classic region of mouse brain white matter, showed that compared with the TMZ group, the proportion of mature oligodendrocytes with CC1+ / Olig2+ was significantly increased in the TMZ+CBT group, suggesting that clobetasol can effectively reverse the reduction of mature oligodendrocytes induced by TMZ.

[0039] To directly observe morphological changes in brain tissue, this invention uses mouse optic nerves for transmission electron microscopy (TEM). The specific steps are as follows: Female C57BL / 6 mice were anesthetized and perfused with PBS, followed by fixation with an electron microscopy fixative containing 2.5% glutaraldehyde and 4% paraformaldehyde (PFA) (pH 7.2–7.4). The optic nerve was carefully dissected and post-fixed overnight at 4°C in the same fixative. The tissue was then washed with phosphate-buffered saline (PB) and post-fixed with 2% osmium tetroxide (OsO4) at 4°C in the dark. Subsequently, the tissue was dehydrated with a gradient of ethanol concentrations, infiltrated, and embedded to complete polymerization. Ultrathin sections (70 nm thick) were cut and stained with 2% uranium acetate and 3% lead citrate. TEM imaging was performed using a 120 kV electron microscope, "JEM-1400Flash" (JEOL, Japan). Image quantitative analysis was performed using ImageJ / FIJI software, calculating hundreds of axons in at least six random fields of view for each sample. Figure 5Transmission electron microscopy images showed that the proportion of demyelination of optic nerve axons was significantly increased after TMZ treatment, while CBT could effectively reverse it.

[0040] The above results indicate that temozolomide TMZ induces a significant reduction in mature oligodendrocytes and demyelination damage, which can be effectively reversed by clobetasol CBT.

[0041] Clobetasol treatment can alleviate temozolomide-induced nerve axonal damage: Further statistical analysis of the axonal morphology in the obtained electron microscopic images of the optic nerve revealed that, for example... Figure 6 As shown in Figure 6A, this is a representative transmission electron microscope image of the optic nerve. Black asterisks represent normal axons, and orange asterisks represent degenerated axons. TMZ treatment reduced the number of axons per unit area in the mouse optic nerve, and the increased axon diameter indicated axonal swelling. Simultaneously, the proportion of degenerated axons increased. Compared to the TMZ-only group, the TMZ+CBT group showed an increase in the number of axons per unit area in the optic nerve, a slight decrease in axon diameter, and a lower proportion of degenerated axons. Notably, although there was no statistically significant difference between the TMZ+CBT group and the TMZ group, the salvage effect of CBT (TMZ+CBT group) was comparable to the control group (Mock), suggesting that CBT can effectively salvage axonal damage in the adult mouse brain.

[0042] Clobetasol treatment can effectively reverse temozolomide-induced neuroinflammation: This invention utilizes immunofluorescence staining of tissues with specific marker antibodies for microglia (anti-Iba1, 1:200, Wako) and astrocytes (anti-GFAP, 1:200, Millipore) to evaluate the efficacy of CBT in treating TMZ-induced neuroinflammation. Figure 7 and Figure 8 As shown, the number of neuroinflammatory cells, specifically Iba1+ microglia and GFAP+ astrocytes, was significantly increased in the brains of mice treated with TMZ, while both types of inflammatory cells were significantly reduced in the brain tissue of the TMZ+CBT group. This indicates that CBT can effectively reverse TMZ-induced activation of neuroinflammatory cells.

[0043] Clobetasol treatment can alleviate temozolomide-induced depression and anxiety-like behavior in mice. This invention assessed the behavior of mice treated with TMZ and CBT, focusing on depressive and anxiety-like behavioral abnormalities. All behavioral tests were conducted during the light phase of a 12-hour light-dark cycle. The researchers remained blinded to the treatment groups during the assessment and data analysis. The specific methods are as follows: Tail suspension test: To assess depressive-like behaviors, mice underwent a tail suspension test. Each mouse's tail was secured with tape and suspended from a worktable approximately 50 cm above the surface. Video tracking was recorded and analyzed using a video tracking system (Techman, Chengdu, China) for 300 seconds, analyzing the time spent at rest during this period. Rest was defined as a state of no movement in the limbs or body.

[0044] Open field test: The open field test was used to assess anxiety-like behavior and general motor skills. Mice were placed in the center of an open field (50×50×40cm) and allowed to explore freely for 5 minutes. The field was divided into a central area and a peripheral area. Movement / stationary time, total distance traveled, and time spent in the central area were recorded using a video tracking system (Techman, Chengdu, China). During the experimental intervals, the field was cleaned with 70% ethanol to eliminate odor cues.

[0045] like Figure 9 As shown, through the tail suspension test, this invention observed that the immobility time of mice treated with TMZ was prolonged, indicating the emergence of depressive-like behavior. Conversely, the immobility time of mice treated with CBT was shortened. To further support this finding, this invention conducted an open-field test, i.e., a mining test, on TMZ-treated mice to assess their motor abilities and anxiety levels. Figure 10 As shown, the proportion of mice in the TMZ-treated group who remained in the central region was reduced, and CBT treatment alleviated this trend to some extent. These behavioral results suggest that CBT has a potential antidepressant and anxiety-like effect on mice treated with TMZ.

[0046] Bulk RNA sequencing analysis showed that CBT treatment could reverse TMZ-induced gene pathway alterations: This invention employs batch RNA transcriptome sequencing analysis to further explore the mechanism by which clobetasol inhibits temozolomide-induced neurotoxicity and to identify altered gene profiles. The specific methods are as follows: A tissue RNA extraction kit (Foregene) was used according to the manufacturer's instructions. Total RNA was extracted from cortical tissues of mice in the control group, TMZ-treated group, and CBT+TMZ group. RNA concentration and purity were detected using a NanoDrop spectrophotometer (Thermo Fisher Scientific) to ensure the quality of downstream analysis. Sequencing libraries were prepared using the NEBNext® Ultra™ RNA library preparation kit (Illumina®, New England Biolabs) according to the manufacturer's provided protocol. Subsequently, Tianjin Novogene Co., Ltd. sequenced the libraries using the Illumina platform and performed in-depth analysis of the sequencing data to identify differentially expressed genes and pathway enrichment, highlighting changes in gene profiles and signaling pathways related to TMZ-induced neurotoxicity and CBT treatment efficacy.

[0047] like Figure 11 Bulk RNA sequencing (BRNA-seq) analysis showed that CBT treatment could reverse TMZ-induced gene pathway alterations. The heatmap illustrates the BRNA-seq results, showing that TMZ treatment induces upregulation of genes in pathways such as JAK-STAT and NF-κB while downregulating genes in pathways related to cell-cell interactions; these gene changes were effectively reversed after CBT treatment. Gene Ontology (GO) enrichment and KEGG analysis revealed that in TMZ-treated samples, pathways related to stress and inflammation were upregulated, while pathways related to cell proliferation and differentiation, such as PI3K / AKT, were downregulated; these gene changes could also be reversed by CBT.

[0048] In summary, this invention provides a novel use of clobetasol (CBT) as a treatment for neurotoxic injury, demonstrating that clobetasol (CBT) can effectively rescue neurotoxic injury induced by temozolomide (TMZ) and degenerative changes in oligodendrocytes, microglia, and neurons, effectively reverse demyelination, neuroinflammation, and changes in related pathway genes, and alleviate nerve axonal damage and depressive and anxiety-like behavioral abnormalities.

[0049] As can be seen from the above embodiments, the present invention provides a new use of clobetasol in the treatment of neurotoxic injury, and confirms that clobetasol can effectively improve demyelination, axonal damage, neuroinflammation, and depression and anxiety-like behavior caused by chemotherapy drugs, providing a new drug option for the treatment of neurotoxic injury.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of clobetasol in the preparation of a medicament for preventing or treating neurotoxic injury.

2. Use of clobetasol in the preparation of a medicament for ameliorating demyelination caused by neurotoxic injury.

3. Use of clobetasol in the preparation of a medicament for promoting differentiation or maturation of oligodendrocytes.

4. Use of clobetasol in the preparation of a medicament for inhibiting neuroinflammation.

5. Use of clobetasol in the preparation of a medicament for alleviating depression and / or anxiety-like behavior caused by neurotoxic injury.

6. Use according to claim 1 or 5, characterized in that, The neurotoxic injury is induced by a chemotherapeutic drug.

7. Use according to claim 6, characterized in that, The chemotherapeutic drug is temozolomide.

8. Use according to any one of claims 1 to 5, characterized in that, The administration route of the medicament is intraperitoneal injection, intravenous injection, oral administration or nasal administration.

9. Use according to claim 8, characterized in that, The dosage form of the medicament is injection solution, freeze-dried powder injection, tablet or capsule.

10. The use according to any one of claims 1 to 5, characterized in that, The medicament further comprises one or more pharmaceutically acceptable carriers, which include solvents, cosolvents, solubilizers, buffers, isotonicity adjusting agents, preservatives, antioxidants, diluents, binders, disintegrants or lubricants.

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