Use of pityriacitrin derivatives for the preparation of a medicament for the treatment or prevention of nerve damage

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to application of a Pityriacitrin derivative in preparation of a medicine for treating or preventing nerve injury. The compound of structural formula 1 has a significant promoting effect on recovery of motor function after nerve injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology. In particular, this invention relates to the use of a Pityriacitrin derivative in the preparation of medicaments for treating or preventing nerve damage. Background Technology

[0002] Nerve injury refers to damage to the structure and function of nerve tissue caused by various factors such as trauma, ischemia, compression, inflammation, and degenerative changes. It can affect the central nervous system or the peripheral nervous system. Among them, central nervous system injury often leads to persistent sensory, motor, and autonomic dysfunction due to the limited regenerative capacity of nerve tissue, which seriously affects the patient's quality of life.

[0003] Spinal cord injury (SCI) is a severe type of central nervous system injury, caused by traumatic factors such as traffic accidents, falls, and mechanical compression, as well as non-traumatic factors such as tumors, infections, and degenerative diseases. Its pathological development is mainly divided into two stages: primary injury and secondary injury. Primary injury is the direct damage to neurons, axons, and blood vessels caused by mechanical impact, compression, traction, etc. Secondary injury involves the interaction of a series of pathological processes, including inflammatory responses, oxidative stress, apoptosis, and abnormal activation of glial cells, which further expand the extent of injury and hinder nerve repair.

[0004] Despite advancements in medical technology leading to increasingly effective treatments for many diseases, spinal cord injury remains a complex and unresolved problem. Clinical treatment primarily involves surgical decompression, spinal stabilization, supportive care, complication prevention and treatment, and rehabilitation training. While these measures play a crucial role in relieving mechanical compression, maintaining spinal stability, and improving the patient's general condition, they cannot reverse the damage that has already occurred. In the field of drug therapy, existing research has explored various drug and biological treatment strategies targeting pathological processes such as inflammatory responses, oxidative stress, neuronal death, and nerve regeneration. However, due to the complexity and distinct temporal and multi-target nature of the pathological process of spinal cord injury, most clinical trials have not yet achieved breakthrough progress. Drugs capable of stably improving neurological function recovery after spinal cord injury and possessing further clinical translational potential are extremely limited; to date, no drug has been officially approved globally for the treatment of spinal cord injury. Therefore, screening and developing new candidate compounds with neuroprotective and neurofunctional repair potential is of great significance.

[0005] Pityriacitrin is a class of natural alkaloids with a typical β-carboline skeleton, initially isolated from marine microorganisms. Its characteristic β-carboline heterocyclic skeleton offers ample space for structural modification; by chemically modifying its core and substituents, derivatives with different biological activities can be obtained, providing a foundation for further screening of candidate compounds with varying pharmacological activities. Previous studies have reported that pityriacitrin and its derivatives possess biological activities such as antiproliferative, antimalarial, antioxidant, and phospholipase A2 regulation. However, current research on pityriacitrin compounds mainly focuses on antitumor, anti-infective, and antioxidant activities, with limited research on their application in nerve injury, particularly spinal cord injury. Whether pityriacitrin derivatives with specific substituted structures can improve functional impairment after nerve injury and promote nerve tissue repair remains insufficiently investigated using current techniques. Therefore, further screening and research of pityriacitrin compounds are necessary to obtain candidate compounds with potential for treating nerve injuries. Summary of the Invention

[0006] The present invention aims to provide the use of a Pityriacitrin derivative having structural formula 1 in the preparation of a medicament for treating or preventing nerve damage.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] 1. Use of the compound of structural formula 1 in the preparation of a medicament for the treatment or prevention of nerve injury.

[0009] .

[0010] 2. The use as described in item 1, wherein the nerve injury is a central nervous system injury.

[0011] 3. The use as described in item 2, wherein the central nervous system injury is brain injury and / or spinal cord injury.

[0012] 4. The use as described in item 3, wherein the spinal cord injury is an acute spinal cord injury or a chronic spinal cord injury.

[0013] 5. The use as described in any one of items 1-4, wherein the compound of structural formula 1 is present in any pharmaceutically acceptable dosage form selected from the group consisting of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalers, and sprays.

[0014] Beneficial effects of the invention

[0015] Through treatment experiments on animals with spinal cord injury models, this invention found that the compound of formula 1 can significantly improve the BBB score of the treated animals and significantly improve their motor function, indicating that the compound of formula 1 has a significant promoting effect on the recovery of motor function after nerve injury. Attached Figure Description

[0016] Figure 1 This is a line graph of the BBB score obtained in Example 2.

[0017] Figure 2 The bar chart shows the results of the grid experiment obtained in Example 3. From left to right, the results are: sham surgery group, control group, and experimental group.

[0018] Figure 3 The bar chart shows the hot plate experiment results obtained in Example 4. From left to right, the results are: sham surgery group, control group, and experimental group.

[0019] Figure 4 The bar chart shows the swimming test score results obtained in Example 5. From left to right, the results are: sham surgery group, control group, and experimental group.

[0020] Figure 5 The image shows the Catwalk footprints obtained in Example 6, with different colors representing the footprints of the right forelimb, left forelimb, right hindlimb, and left hindlimb, respectively. From top to bottom, they represent the sham surgery group, control group, and experimental group.

[0021] Figure 6 The graphs and bars showing the MEP experimental results obtained in Example 7 are shown. In the graph, from top to bottom, the groups are the sham surgery group, the control group, and the experimental group. In the bar chart, from left to right, the groups are the sham surgery group, the control group, and the experimental group.

[0022] Figure 7 The image shows the results of the immunofluorescence experiment obtained in Example 8. Green represents NeuN, red represents GFAP, and blue represents DAPI. From top to bottom, the images represent the sham-operated group, the control group, and the experimental group.

[0023] Figure 8 The bar chart shows the quantitative results of the immunofluorescence GFAP experiment obtained in Example 8. From left to right, the results are: sham operation group, control group, and experimental group.

[0024] Figure 9 The bar chart shows the results of the immunofluorescence NeuN quantification experiment obtained in Example 8. From left to right, the results are: sham-operated group, control group, and experimental group.

[0025] Figure 10The image shows the results of the immunofluorescence experiment obtained in Example 9. Green represents GFAP, red represents Iba-1, and blue represents DAPI. From top to bottom, the images represent the sham-operated group, the control group, and the experimental group.

[0026] Figure 11 The bar chart shows the quantitative results of the immunofluorescence GFAP experiment obtained in Example 9. From left to right, the results are: sham-operated group, control group, and experimental group.

[0027] Figure 12 The bar chart shows the quantitative results of the immunofluorescence Iba-1 assay obtained in Example 9. From left to right, the results are: sham-operated group, control group, and experimental group.

[0028] Note: In the figure, an asterisk represents p < 0.05, a double asterisk represents p < 0.01, a triple asterisk represents p < 0.001, and a quadruple asterisk represents p < 0.0001, all of which indicate that the difference is statistically significant. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The compound of structural formula 1 of this invention is a known compound, namely compound I-7 described in patent document CN113444087 B, the chemical structure of which and its preparation method are disclosed in that patent document.

[0030] The inventors of this application discovered through treatment experiments on an animal model of spinal cord injury that the compound of structural formula 1 can significantly improve the BBB score of treated animals, improve motor function, sensory function, and nerve conduction function in rats after spinal cord injury, reduce nerve cell apoptosis, inhibit excessive glial cell activation, improve the glial microenvironment after spinal cord injury, thereby inhibiting neuroinflammation, promoting nerve cell survival, and achieving functional repair after nerve injury. Therefore, the following invention is provided.

[0031] This invention provides the use of the compound of structural formula 1 in the preparation of a medicament for treating or preventing nerve damage.

[0032] The nerve injury can be any nerve injury, such as central nervous system injury, preferably brain injury or spinal cord injury, preferably acute or chronic spinal cord injury.

[0033] The nerve injury can be caused by factors such as violence, mechanical compression, bleeding, electrolyte leakage, and / or ischemic perfusion.

[0034] The compound of structural formula 1 can be formulated into any pharmaceutically acceptable dosage form, such as tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), suppositories, inhalers, or sprays.

[0035] The compound of structural formula 1 can be administered by any suitable route of administration, such as oral, parenteral, rectal, pulmonary, transdermal, or topical administration to patients or subjects in need. When administered orally, the compound of structural formula 1 can be formulated into any pharmaceutically acceptable oral dosage form, such as oral solid dosage forms, like tablets, capsules, pills, granules, etc.; or oral liquid dosage forms, such as oral solutions, oral suspensions, syrups, etc. When formulated into oral dosage forms, the oral dosage forms may also contain suitable fillers, binders, disintegrants, lubricants, etc. When administered parenterally, the compound of structural formula 1 can be formulated into injectable preparations, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When formulated into injectable preparations, the injectable preparations can be manufactured using suitable methods in the existing pharmaceutical field. When formulating injectable preparations, the injectable preparations may be prepared without excipients or with suitable excipients. The injectable preparations can be administered by suitable injection methods, such as intravenous administration, intramuscular administration, subcutaneous administration, and intradermal administration. When used for rectal administration, the compound of structural formula 1 can be formulated into suppositories, etc. When used for pulmonary administration, the compound of structural formula 1 can be formulated into inhalers or sprays, etc., for administration by inhalation or nasal administration. When used for transdermal administration, the compound of structural formula 1 can be formulated into patches, gels, creams, etc. The compound of structural formula 1 can be present in the drug in therapeutic and / or preventive effective amounts. The compound can be present in the drug in unit dose form. The term "effective amount" as used in this invention refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, "effective amount for disease prevention" refers to an amount sufficient to prevent, stop, or delay the onset of a disease; "effective amount for disease treatment" refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such effective amounts is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of drug administration, and other concurrent treatments, etc. In some preferred embodiments, the patient or subject may be a vertebrate, such as a mammal, such as a bovine, equine, sheep, swine, canine, feline, rodent, or primate, preferably a human.

[0036] The effective amount of the compound can be from 0.1 mg / kg to 100 mg / kg body weight, for example, 10 mg / kg.

[0037] The compound of Formula 1 can be administered at any suitable frequency, such as once or multiple times daily, for a period of at least one day, such as about 3, 7, or 14 days, or until a plateau in functional recovery is observed. Alternatively, it can be administered daily during the acute phase, followed by every-other-day or weekly administration. Preferably, administration should begin immediately after injury and continue for 7 days.

[0038] The following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples were performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0039] Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures described herein are standard procedures widely used in their respective fields.

[0040] Example

[0041] laboratory animals

[0042] Female Wistar rats aged 6-8 weeks and weighing 200g were used as experimental animals and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0043] Rats were housed in an environment with a 12 / 12-h light-dark cycle (lighting between 7:00 and 19:00), at a temperature of 20-25℃ and a humidity of 40-60%. They had free access to standard laboratory food and tap water.

[0044] Main reagents and instruments

[0045] The compound of structural formula 1 (referred to as I-7 in the following examples) was prepared by Tianjin Normal University.

[0046] Instruments: IMPACTOR MODEL-Ⅱ impactor; constant temperature cryostat.

[0047] Statistical methods

[0048] Statistical data were collected using Graph Pad Prism 9.5.1 software. Data are expressed as mean ± standard deviation. All data were tested for Shapiro-Wilk normality and were found to be normally distributed. For multiple groups of continuous repeated measures data at multiple time points (e.g., BBB scores), two-way repeated measures ANOVA was used, with Tukey's test used for post-hoc multiple comparisons. For data from multiple groups at a single time point, one-way ANOVA combined with Tukey's post-hoc test was used for inter-group comparisons. A p-value < 0.05 was considered statistically significant.

[0049] Example 1: Establishment of a rat model of spinal cord injury

[0050] An acute spinal cord injury experimental animal model was established using the IMPACTOR MODEL-II spinal cord injury strike system. Eighteen 6-8 week old, 200g female Wistar rats were selected and divided into three groups of six each: a sham-operated group (simple removal of the vertebral laminae), a control group (received solvent after spinal cord injury), and an experimental group (received I-7 after spinal cord injury). After anesthesia, the lamina of the 10th thoracic vertebra was surgically removed to expose the corresponding segment of the spinal cord. The rat was then fixed under the striker, and a 10g weight (2.5mm in diameter) was dropped from a height of 25mm (10g × 25mm). Following the strike, local congestion and edema occurred in the injured spinal cord. The rats exhibited brief convulsions and twitches in their hind limbs, followed by tail wagging, and then paralysis of both hind limbs. A BBB score of 0 on the day of injury indicated successful model establishment. I-7 was dissolved in 10 μL of DMSO at a dose of 10 mg / kg body weight, and then dissolved in 1 ml of 0.9% physiological saline. The experimental group rats were then administered the drug via intraperitoneal injection, while the control group rats were given physiological saline containing an equal volume of DMSO to ensure that both groups of animals received the same solvent treatment. The administration time was immediately after the impact injury, once daily for 7 consecutive days.

[0051] Example 2: Evaluation of hindlimb motor function in experimental animals using the BBB score

[0052] The BBB (Basso Beattie Bresnahan locomotor rating scale) score is one of the internationally recognized objective standards for evaluating neurological function after spinal cord injury in rats. This example establishes an acute spinal cord injury experimental animal model using the same modeling method as in Example 1, and administers medication in the same manner as in Example 1. A double-blind, two-person independent observation and recording method was used. Rats were placed in the observation area and allowed to move freely for 3-5 minutes to acclimatize. Then, observers observed the movement of the rats' hind limbs from various angles at an appropriate distance (generally 1-2 meters), and performed statistical analysis according to the BBB scoring standard. Hind limb motor function was evaluated weekly, one day before injury, on the day of injury, and from one to six weeks after injury. The results are as follows: Figure 1 As shown.

[0053] Scoring criteria:

[0054] 0 points: No visible hind limb movement;

[0055] 1 point: Slight movement of one or two joints, usually the hip and / or knee joints;

[0056] 2 points: One joint moves significantly or one joint moves significantly while the other joint moves slightly;

[0057] 3 points: Both joints move extensively;

[0058] 4 points: All three joints of the hind limbs can move slightly;

[0059] 5 points: Two joints have slight movement, while the third joint has a wide range of motion;

[0060] 6 points: Two joints have a wide range of motion, and the third joint can move slightly;

[0061] 7 points: All three joints of the hind limbs can move to a great extent;

[0062] 8 points: The paw pads can touch the ground when not under load;

[0063] 9 points: The sole of the foot is only in a weight-bearing position, or the dorsiflexion of the foot is used for walking occasionally / frequently / continuously, without sole weight-bearing. Weight-bearing: The hind limb extensor muscles contract when the sole of the foot is in a weight-bearing position or only when the hindquarters are raised;

[0064] 10 points: Occasionally, weight-bearing movement is observed on the palmar surface of the claws, but there is no coordinated movement of the forelimbs and hindlimbs;

[0065] 11 points: The palmar weight-bearing movement can be seen more often, but there is no coordinated movement of the forelimbs and hindlimbs;

[0066] 12 points: Palmar weight-bearing movement can be seen more often, and coordinated movements of the forelimbs and hindlimbs can be seen occasionally;

[0067] 13 points: Commonly seen in palmar weight-bearing movements, and can often coordinate forelimb and hindlimb movements;

[0068] 14 points: There is continuous palmar weight-bearing movement and coordinated forelimb and hindlimb movements, or common face movements, continuous coordinated forelimb and hindlimb movements, and occasional dorsal claw movements.

[0069] 15 points: Continuous palmar movement and continuous coordinated forelimb and hindlimb movements; during the forward movement of the forelimbs, there is no or occasional initial contact with the ground, and the position of the active claw is parallel to the body.

[0070] 16 points: Continuous palmar movement and continuous coordinated forelimb and hindlimb movements are visible in the gait. Claws are often seen gripping the ground during the forelimbs' forward movement. The active claw is parallel to the body at the initial contact and rotates after the weight is transferred.

[0071] 17 points: Continuous palmar movement and continuous coordinated forelimb and hindlimb movements are visible in the gait; these are common during forelimb forward movement.

[0072] The claws grip the ground, and the active claws are positioned parallel to the body during initial contact and after weight transfer.

[0073] 18 points: The gait shows continuous palmar movement and continuous coordinated forelimb and hindlimb movements. The forelimbs can continuously grip the ground with their claws during forward movement. The active claws are parallel to the body at the initial contact and rotate after the weight is transferred.

[0074] 19 points: The gait shows continuous palmar movement and continuous coordinated forelimb and hindlimb movements. The forelimbs can continuously grip the ground with their claws during forward movement. The active claws are parallel to the body during initial contact and after weight transfer. The tail is sometimes or always drooping.

[0075] 20 points: Continuous palm movement, continuous coordinated gait, continuous gripping of the toes, active paws are parallel to the body during initial contact and after weight transfer, unstable trunk, and tail is continuously raised.

[0076] 21 points: Continuous palm movement, continuous coordinated gait, continuous gripping of the toes, active paws always parallel to the body during the activity, continuous stability of the torso, and continuous tail held high.

[0077] BBB scores of different groups at different time points after injury are as follows: Figure 1 As shown, the BBB score of the sham-operated group remained at 21 points. However, on day 21 post-injury, a significant statistical difference began to appear between the experimental group and the control group (p < 0.05). From day 21 to day 42 post-injury, the average BBB score of the control group was consistently lower than that of the experimental group, indicating that I-7 administration can significantly promote the recovery of motor function after spinal cord injury in rats.

[0078] Example 3: Evaluation of proprioception and hindlimb fine motor function in experimental animals using a grid experiment.

[0079] The rat grid test is a commonly used experimental method to assess the recovery of proprioceptive and fine motor function in rats after spinal cord injury. In this embodiment, an acute spinal cord injury animal model was established using the same modeling method as in Example 1, and medication was administered in the same manner. On day 42 post-injury, rats in the sham-operated group (simple removal of the vertebral laminae), control group (treated with solvents after spinal cord injury), and experimental group (treated with I-7 after spinal cord injury) were trained to walk on a grid. Motor function was assessed by observing each rat's ability to walk on the grid, primarily evaluating the proportion of missteps or missed steps by the hind limbs relative to the total number of steps. This project employed a double-blind, three-person independent observation and recording method, followed by statistical analysis. Hind limb motor function was evaluated on day 42 post-injury.

[0080] The results of the rat grid experiment are as follows Figure 2 As shown, from Figure 2 As can be seen, the error rate of the grid experiment in the experimental group was significantly lower than that in the control group (P<0.01), indicating that I-7 administration can significantly promote the recovery of proprioception and fine motor function of the hind limbs after spinal cord injury in rats.

[0081] Example 4: Evaluation of hindlimb sensory function in experimental animals using the hot plate test

[0082] The hot plate test in rats is a commonly used experimental method to assess pain perception and conduction in rats after spinal cord injury. In this experiment, changes in pain perception and conduction are assessed by observing the rats' reaction time on a hot plate. Rats are placed on a hot plate at a preset temperature of 52°C, and the reaction time of each rat is observed, including responses such as licking its paw, jumping, or scratching its paw, to assess its pain perception and conduction. In this embodiment, an acute spinal cord injury experimental animal model was established using the same modeling method as in Example 1, and the administration was carried out in the same manner as in Example 1. A double-blind, two-person independent observation and recording method was used, followed by statistical analysis. On day 42 post-injury, the hindlimb pain sensation function of rats in the sham-operated group (simple removal of the vertebral laminae), the control group (treated with solvents after spinal cord injury), and the experimental group (treated with I-7 after spinal cord injury) was evaluated.

[0083] The results of the hot plate test in rats are as follows: Figure 3 As shown, from Figure 3 As can be seen, the hot plate reaction time of the experimental group was significantly longer than that of the control group (P<0.001), indicating that I-7 administration can significantly improve pain hypersensitivity after spinal cord injury in rats and promote the recovery of sensory function after spinal cord injury in rats.

[0084] Example 5: Evaluation of hindlimb coordination function in experimental animals using a swimming experiment.

[0085] The rat swimming test is a commonly used behavioral assessment method for hindlimb motor function, used to evaluate the motor function and coordination of rats after spinal cord injury. In this experiment, the recovery of motor function and coordination is assessed by observing the rats' swimming performance in water. This embodiment uses the same modeling method as in Example 1 to establish an acute spinal cord injury animal model and administers drugs in the same manner as in Example 1. On day 42 post-injury, rats were placed in a pool, and their swimming posture, speed, and coordination were observed. The swimming performance of rats in the sham-operated group (simply removing the vertebral laminae), control group (receiving solvent after spinal cord injury), and experimental group (receiving I-7 after spinal cord injury) was recorded, including swimming speed, swimming posture, and coordination. This project uses a double-blind, two-person independent observation and recording method. The specific scoring criteria are shown in Tables 1-3 below. Finally, statistical analysis was performed to evaluate the hindlimb motor function of rats on day 42 post-injury.

[0086] Table 1

[0087]

[0088] Table 2

[0089]

[0090] Table 3

[0091]

[0092] The results of the rat swimming experiment are as follows Figure 4 As shown, from Figure 4 As can be seen, the swimming test score of the experimental group was significantly higher than that of the control group (P < 0.0001), indicating that I-7 administration can significantly promote the recovery of coordinated motor function after spinal cord injury in rats.

[0093] Example 6: Evaluation of hindlimb coordinated motor function in experimental animals using the Catwalk test

[0094] The Catwalk test in rats is a commonly used gait analysis method to assess gait function and motor ability in rats after spinal cord injury. The Catwalk system is a high-resolution gait analysis system that can record plantar footprints of rats walking across a transparent glass plate in real time, thereby assessing gait characteristics and parameters. In this embodiment, an acute spinal cord injury experimental animal model was established using the same modeling method as in Example 1, and drugs were administered in the same manner as in Example 1. On day 42 post-injury, rats in the sham-operated group (simply removing the vertebral laminae), control group (receiving solvent after spinal cord injury), and experimental group (receiving I-7 after spinal cord injury) were placed on the Catwalk system and allowed to walk freely. The system recorded the rats' plantar footprints and gait characteristics in real time. The gait parameters of the rats, including gait cycle, stride length, and support time, were analyzed using the software provided by the Catwalk system to assess gait function and motor ability. This project employed a double-blind, two-person independent observation and recording method, followed by statistical analysis. The hindlimb coordination function of the rats was evaluated on day 42 post-injury.

[0095] The results of the rat Catwalk experiment are as follows Figure 5 As shown, from Figure 5 As can be seen from the results, the experimental group showed better coordination and larger imprint area in the hind limb imprint analysis of the Catwalk test than the control group, indicating that I-7 administration can promote the recovery of coordinated motor function after spinal cord injury in rats.

[0096] Example 7: Evaluation of hindlimb motor conduction function in experimental animals using the MEP experiment.

[0097] Motor evoked potentials (MEPs) are electrophysiological signals that record muscle motor responses by stimulating regions of the cerebral cortex. The MEP experiment is a commonly used electrophysiological method to assess neural conduction in rats after spinal cord injury. This example establishes an acute spinal cord injury animal model using the same modeling method as in Example 1, and administers drugs in the same manner. On day 42 post-injury, before the experiment, stimulating electrodes were implanted in the cerebral cortex of rats in the sham-operated group (simple removal of the vertebral laminae), the control group (received solvent after spinal cord injury), and the experimental group (received I-7 after spinal cord injury). Recording electrodes were implanted in the bilateral tibialis anterior muscles. Motor cortex neurons were then activated by electrical stimulation (stimulation current intensity of 10 mA, pulse width of 0.2 ms). The MEP signals of the rats, including the electrical activity response of the muscles after stimulation, were recorded using electrophysiological instruments. Analysis of the recorded MEP signals allows for the assessment of neural conduction and motor function in rats, including parameters such as latency, amplitude, and waveform. After collecting experimental data, statistical analysis was performed to compare the differences between the sham-operated group (simply removing the vertebral laminae), the control group (receiving solvent after spinal cord injury), and the experimental group (receiving I-7 after spinal cord injury), assessing the recovery of nerve conduction and motor function in rats after spinal cord injury. This project employed a double-blind, two-person independent observation and recording method, followed by statistical analysis. Hindlimb motor function in rats was evaluated on day 42 post-injury.

[0098] The results of the rat MEP experiment are as follows Figure 6 As shown, from Figure 6 As can be seen, the amplitude of the electrophysiological experiment in the experimental group was significantly higher than that in the control group (P<0.05), indicating that I-7 administration can significantly promote the recovery of motor conduction function after spinal cord injury in rats.

[0099] Example 8: Assessment of neuronal survival in experimental animals using immunofluorescence staining

[0100] Immunofluorescence staining is a fluorescent labeling technique based on the specific binding of antigen and antibody. It can precisely locate the expression distribution and level of specific proteins in spinal cord tissue and is a core experimental method for assessing the remodeling of the neural microenvironment, neuronal survival, and glial cell activation after spinal cord injury. In this embodiment, an acute spinal cord injury experimental animal model was established using the same modeling method as in Example 1, and the drugs were administered in the same manner as in Example 1. On day 42 after injury, rats in the sham-operated group (simply removing the vertebral laminae), control group (receiving solvent after spinal cord injury), and experimental group (receiving I-7 after spinal cord injury) were anesthetized, their hearts were perfused with physiological saline and fixed with 4% paraformaldehyde, the injured segment and adjacent spinal cord tissue were separated, and 12μm thick frozen sections were prepared after OCT embedding. After washing with PBST (PBS containing 0.1% Tween-20), slides were blocked with immunostaining blocking solution (Beyotime, P0260), followed by incubation overnight at 4°C with rabbit-derived NeuN polyclonal antibody (abcam, ab104225) and goat-derived GFAP polyclonal antibody (abcam, ab53554). The next day, after washing, slides were incubated for 1 hour at room temperature in the dark with Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody (abcam, ab150061) and Alexa Fluor 555-labeled donkey anti-goat secondary antibody (abcam, ab150130). Finally, the slides were stained with DAPI and mounted with an anti-fluorescence quencher. Fluorescence images of the central and peripheral areas of the spinal cord injury were acquired using a Leica laser confocal microscope. One field of view on the cephalic side of the injury center was selected for quantification on each slide. At least three rats were counted in each group. ImageJ software was used for image analysis: the number of NeuN-positive cells was counted and the fluorescence integral density of GFAP-positive astrocytes was measured.

[0101] The results of the rat immunofluorescence staining NeuN+GFAP experiment are as follows: Figure 7 , Figure 8 , Figure 9 As shown, compared with the control group, the number of NeuN positive cells in the experimental group was significantly higher than that in the control group (P<0.05), and the relative fluorescence integral density of GFAP was significantly lower than that in the control group (P<0.05). This indicates that I-7 administration can improve the survival level of neurons after spinal cord injury, inhibit astrocyte activation, and improve the glial microenvironment after spinal cord injury.

[0102] Example 9: Assessment of glial cell activation in experimental animals using immunofluorescence staining

[0103] Immunofluorescence staining is a fluorescent labeling technique based on the specific binding of antigen and antibody. It can precisely locate the expression distribution and level of specific proteins in spinal cord tissue and is a core experimental method for assessing the remodeling of the neural microenvironment, neuronal survival, and glial cell activation after spinal cord injury. In this embodiment, an acute spinal cord injury experimental animal model was established using the same modeling method as in Example 1, and the drugs were administered in the same manner as in Example 1. On day 42 after injury, rats in the sham-operated group (simply removing the vertebral laminae), control group (receiving solvent after spinal cord injury), and experimental group (receiving I-7 after spinal cord injury) were anesthetized, their hearts were perfused with physiological saline and fixed with 4% paraformaldehyde, the injured segment and adjacent spinal cord tissue were separated, and 12μm thick frozen sections were prepared after OCT embedding. After washing with PBST (PBS containing 0.1% Tween-20), the slides were blocked with immunostaining blocking solution (Beyotime, P0260), and then incubated overnight at 4°C with mouse-derived Iba-1 monoclonal antibody (abcam, ab283319) and goat-derived GFAP polyclonal antibody (abcam, ab53554). The next day, after washing, the slides were incubated with Alexa Fluor 488-labeled donkey anti-goat secondary antibody (abcam, ab150129) and Alexa Fluor 555-labeled donkey anti-mouse secondary antibody (abcam, ab150106) at room temperature in the dark for 1 hour. Finally, the slides were stained with DAPI and mounted with anti-fluorescence quencher. Fluorescence images of the central and peripheral areas of spinal cord injury were acquired using a Leica laser confocal microscope. One field of view on the cephalic side of the injury center was selected for quantification in each slice. At least three rats were included in each group. ImageJ software was used for image analysis to measure the fluorescence integral density of Iba-1 positive microglia and GFAP positive astrocytes.

[0104] The results of the rat immunofluorescence staining Iba-1+GFAP experiment are as follows: Figure 10 , Figure 11 , Figure 12 As shown, compared with the control group, the number of Iba-1 positive cells in the experimental group was significantly lower than that in the control group (P<0.05), and the relative fluorescence integral density of GFAP was significantly lower than that in the control group (P<0.05). This indicates that I-7 administration can significantly reduce the activation of microglia and astrocytes after spinal cord injury in rats and improve the glial microenvironment after spinal cord injury.

[0105] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of the compound of structural formula 1 in the preparation of a medicament for the treatment or prevention of spinal cord injury. 。 2. The use as described in claim 1, wherein the spinal cord injury is an acute spinal cord injury or a chronic spinal cord injury.

3. The use as described in claim 1 or 2, wherein the compound of structural formula 1 is present in an injectable dosage form.

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