Application of earthworm extract in medicine for treating spinal cord injury

Abstract

The invention relates to the technical field of biological medicine, in particular to application of earthworm extract in medicine for treating spinal cord injury. The invention provides an application of an earthworm extract in preparation of a medicine for treating spinal cord injury, an application of the earthworm extract in preparation of a medicine for promoting spinal cord injury repair and an application of the earthworm extract in preparation of a medicine for promoting exercise ability recovery after spinal cord injury. The treatment medicine disclosed by the invention has a relatively excellent treatment effect on spinal cord injury. The treatment medicine disclosed by the invention can promote functional recovery after spinal cord injury; the activity of thrombin after spinal cord injury is inhibited; the inflammatory reaction is reduced; the reduction of neurons after spinal cord injury is protected; the formation of fibrosis at the damaged part is inhibited.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to the application of earthworm extract in drugs for treating spinal cord injury. Background Art

[0002] Spinal cord injury refers to the injury caused by external force or disease invasion to the spinal cord, which usually leads to nerve dysfunction, including impairment of limb movement, sensation and autonomic nerve function. The reason for such severe symptoms after spinal cord injury is the vulnerability and difficult regeneration of spinal cord nerve cells. Research shows that the factors and pathological mechanisms leading to poor prognosis after spinal cord injury are relatively complex, including the change of local inflammatory microenvironment after injury, which will cause damage to surrounding tissues and cells, releasing a series of inflammatory mediators, such as cytokines, chemokines and inflammatory mediators. At the same time, it will also cause damage to surrounding blood vessels, and the vascular endothelial cells are stimulated to release inflammatory mediators, resulting in vasodilation and increased permeability, thus further triggering an inflammatory response. In the acute phase, the damaged spinal cord tissue will cause a strong inflammatory response, leading to local edema and infiltration of inflammatory cells, and then a large number of cell apoptosis, causing dysfunction in all aspects. In the chronic phase, the degeneration and loss of neurons and nerve fibers lead to further impairment of nerve conduction function, the proliferation of glial cells and the formation of scar tissue, hindering nerve regeneration and repair. At the same time, fibrosis, glial scar formation and angiodestructive changes hinder the extension of regenerated axons to the distal end and seriously affect the functional recovery of patients. Such changes in the pathological environment of this injury greatly affect the repair of spinal cord tissue and also affect the repair effect of intervention measures, but there is currently no systematic and complete mechanism theory. Due to the combined effects of multiple factors on cells after injury, there is currently a lack of specific therapeutic intervention measures, so the achieved therapeutic effect is not ideal. The treatment and management of spinal cord injury repair have always been a major challenge to the medical community.

[0003] Earthworm is one of the animal drugs recorded in "Shennong Ben Cao Jing". Compared with plant drugs, mineral drugs and fungal drugs, earthworm, as an animal drug, has strong active ingredients, more significant curative effects and lower toxic and side effects. The earthworm extract contains biological drugs such as protease, lumbrokinase and earthworm fibrinolytic enzyme, and is considered to have various medicinal values, including anti-inflammatory, anti-fibrotic and anti-thrombotic effects. However, there is no report on the use of earthworm extract for spinal cord injury. Summary of the Invention

[0004] Based on the complex pathological mechanism of spinal cord injury, in order to solve the problem of nerve repair after spinal cord injury, the present invention provides the application of earthworm extract in drugs for treating spinal cord injury.

[0005] Specifically, the present invention relates to the following technical solutions:

[0006] On the one hand, the present invention relates to the use of earthworm extract in the preparation of a medicament for treating spinal cord injury.

[0007] On the other hand, the present invention relates to the use of earthworm extract in the preparation of a medicament for promoting the repair of spinal cord injury.

[0008] Still on the other hand, the present invention relates to the use of earthworm extract in the preparation of a medicament for promoting the recovery of motor ability after spinal cord injury.

[0009] In some embodiments, the recovery of motor ability is manifested as an improvement in motor ability.

[0010] In some embodiments, the active substance in the earthworm extract is earthworm protein.

[0011] In some embodiments, the content of the active substance is 5000U / mg ± 10%.

[0012] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

[0013] In some embodiments, the earthworm extract is obtained by the following method, which includes:

[0014] Rinsing off the impurities on the surface of fresh earthworms with clean water;

[0015] Soaking the earthworms in salt water;

[0016] Adding water in a certain proportion, adjusting the pH value to 7.8 - 8.2, heating to 60°C, and continuing for 1 hour to dissolve the earthworms, then standing for 24 hours;

[0017] Filtering the solution after standing, and collecting the supernatant;

[0018] Putting the supernatant into an alcohol precipitation device, adding 75% alcohol to make the concentrated solution precipitate for 4 - 5 hours;

[0019] Collecting the supernatant, spray drying, pulverizing, and sieving to obtain the earthworm extract.

[0020] Beneficial technical effects

[0021] The therapeutic medicament of the present invention has a relatively excellent therapeutic effect on spinal cord injury. The therapeutic medicament of the present invention can promote the functional recovery after spinal cord injury; inhibit the activity of thrombin after spinal cord injury; reduce the inflammatory response; protect against the reduction of neurons after spinal cord injury; inhibit the formation of fibrosis at the injury site. Description of the drawings

[0022] Figure 1 Shows the BBB scores of rats in each group at various time periods one week after immediately administering earthworm extract after spinal cord injury.

[0023] Figure 2 Shows the results of the grid test of rats in each group 6 weeks after immediately administering earthworm extract for one week after spinal cord injury.

[0024] Figure 3 Shows the results of the hot plate test of rats in each group 6 weeks after immediately administering earthworm extract for one week after spinal cord injury.

[0025] Figure 4 Shows the results of the swimming test of rats in each group 6 weeks after immediately administering earthworm extract for one week after spinal cord injury.

[0026] Figure 5 Shows the Catwalk analysis of rats in each group 6 weeks after immediately administering earthworm extract for one week after spinal cord injury, where RF (Right Front) represents the right front limb, LF (Left Front) represents the left front limb, RH (Right Hind) represents the right hind limb, and LH (Left Hind) represents the left hind limb.

[0027] Figure 6 Shows the MEP results of rats in each group 6 weeks after immediately administering earthworm extract for one week after spinal cord injury.

[0028] Figure 7 Shows the KEGG enrichment results detected by RNA sequencing in the control group and the experimental group 1 week after immediately administering earthworm extract for one week after spinal cord injury.

[0029] Figure 8 Shows the results of the immunofluorescence experiment of rats in each group 6 weeks after immediately administering earthworm extract for one week after spinal cord injury.

[0030] Figure 9 Shows the results of the immunofluorescence GFAP quantification experiment of rats in each group 6 weeks after immediately administering earthworm extract for one week after spinal cord injury.

[0031] Figure 10 Shows the results of the immunofluorescence NeuN quantification experiment of rats in each group 6 weeks after immediately administering earthworm extract for one week after spinal cord injury. Specific embodiments

[0032] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further elaborates on the present invention in detail with reference to specific embodiments and the accompanying drawings. The embodiments provided below can serve as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0033] For the experimental methods in the following examples, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0034] Experimental animals

[0035] The experimental animals used were 6 - 8-week-old female Wistar rats weighing 200 g, purchased from: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0036] Rearing conditions for rats: They were reared in an environment with a 12 / 12-h light-dark cycle (light between 7:00 and 19:00), at a temperature of 20 - 25 °C and a humidity of 40 - 60%. They had free access to standard laboratory food and tap water.

[0037] Main reagents and instruments

[0038] Earthworm Extract (EE) was prepared by the following conventional method: 1. Fresh earthworms were rinsed with clean water to remove impurities on the surface of the earthworms; 2. The earthworms were soaked in salt water for 1 h; 3. Water weighing 80% of the weight of the earthworms was added in a ratio of 1:0.8, the pH value was adjusted to 7.8 - 8.2, heated to 60 °C, and the earthworms were dissolved for 1 hour, then left to stand for 24 hours; 4. After standing, the solution was filtered, and the supernatant was collected; the supernatant was put into an alcohol precipitation device, 75% alcohol was added to precipitate the concentrated solution for 4 - 5 hours, the supernatant was collected, spray-dried, pulverized, and sieved to obtain the earthworm extract. The obtained earthworm extract was a standardized product, and its main active substance was earthworm protein, with a content of 5000 U / mg ± 10%.

[0039] GFAP was purchased from (bs-0199 R, Bioss, China)

[0040] DAPI was purchased from (AB104139, Abcam, UK)

[0041] NeuN was purchased from NeuN (ab 177487, Abcam, UK)

[0042] Instruments: IMPACTOR MODEL-II impactor; cryostat

[0043] Statistical methods

[0044] GraphPad Prism 9.5.1 software was used to statistically analyze the data. The data were expressed as mean ± standard deviation. One-way ANOVA with Tukey's post hoc test was used for multiple group comparisons. A P value < 0.05 was considered statistically significant.

[0045] Example 1: Establishment of an animal model of spinal cord injury in rats

[0046] An acute spinal cord injury experimental animal model was established using the IMPACTOR MODEL-Ⅱ spinal cord injury impact system. Female Wistar rats aged 6-8 weeks and weighing 200 g were selected. Eighteen rats were divided into 3 groups, with 6 rats in each group, namely the sham operation group (simply removing the vertebral lamina of the spine), the control group (spinal cord injury group), and the experimental group. After anesthetizing the rats, the vertebral lamina of the 10th thoracic vertebra was surgically removed to expose the corresponding segment of the spinal cord, which was fixed under the impactor. The impact force was a 10 g weight, 2.5 mm in diameter, falling from a height of 25 mm (10 g×25 mm). After the impact, the local spinal cord was congested and edematous. The hind limbs of the rats twitched spasmodically briefly, and after the tail swung, the hind limbs were paralyzed. The BBB score on the day after the injury was 0, indicating successful model establishment by impact. After the impact injury, the previously extracted earthworm extract was dissolved in 1 ml of 0.9% physiological saline at a dose of 2 g / kg body weight, and then the experimental group of rats was administered by gavage.

[0047] Example 2. Evaluation of the hind limb motor function of experimental animals using the BBB score

[0048] The BBB (Basso Beattie Bresnahan locomotor rating scale) score is one of the internationally recognized objective criteria for evaluating nerve function after spinal cord injury in rats. In this project, double-blind and independent observation and recording by two people were used. The rats were placed in the observation field and allowed to move freely for 3-5 minutes to adapt to the environment. Then, the observers observed the movement of the hind limbs of the rats from various angles at an appropriate distance (usually 1-2 meters) from the rats and performed statistical analysis according to the BBB scoring criteria. The hind limb motor function of the rats was evaluated 1 day before the injury, on the day of the injury, and weekly from 1 week to 6 weeks after the injury. The results are as Figure 1 shown.

[0049] Scoring criteria:

[0050] 0 points: No visible hind limb movement;

[0051] 1 point: Slight movement of one or two joints, usually the hip and / or knee joint;

[0052] 2 points: One joint has a large range of movement or one joint has a large range of movement and another joint has slight movement;

[0053] 3 points: Two joints have a large range of movement;

[0054] 4 points: Slight movement of all three joints of the hind limb;

[0055] 5 points: Slight movement of two joints and a large range of movement of the third joint;

[0056] 6 points: Two joints move significantly, and the third joint can move slightly.

[0057] 7 points: All three joints of the hind limbs can move significantly.

[0058] 8 points: Can land on the palmar surface of the paw under non-weight-bearing conditions.

[0059] 9 points: The sole is only in the weight-bearing position, or walks with the dorsal surface of the foot occasionally / frequently / constantly, without walking with the sole in the weight-bearing position. Weight-bearing: When the sole is in the weight-bearing position or only when the posterior trunk is elevated, the extensor digitorum longus muscle contracts.

[0060] 10 points: Occasionally, the palmar surface bears weight and moves, without coordinated movements of the fore and hind limbs.

[0061] 11 points: Can be seen more frequently with the palmar surface bearing weight and moving, but without coordinated movements of the fore and hind limbs.

[0062] 12 points: Can be seen more frequently with the palmar surface bearing weight and moving, and occasionally with coordinated movements of the fore and hind limbs.

[0063] 13 points: Frequently seen with the palmar surface bearing weight and moving, and frequently with coordinated movements of the fore and hind limbs.

[0064] 14 points: There is continuous palmar surface weight-bearing movement and coordinated movements of the fore and hind limbs, or there are common surface movements, continuous coordinated movements of the fore and hind limbs, and occasionally dorsal movement of the paw.

[0065] 15 points: Continuous palmar surface movement and continuous coordinated movements of the fore and hind limbs. During the forward movement of the forelimb, there is no or occasionally when initially contacting the ground, the position of the active paw is parallel to the body.

[0066] 16 points: In the gait, continuous palmar surface movement and continuous coordinated movements of the fore and hind limbs can be seen. During the forward movement of the forelimb, the paw frequently grasps the ground. When initially contacting, the position of the active paw is parallel to the body, and it rotates after weight transfer.

[0067] 17 points: In the gait, continuous palmar surface movement and continuous coordinated movements of the fore and hind limbs can be seen. During the forward movement of the forelimb, it is common.

[0068] The paw grasps the ground. When initially contacting and after weight transfer, the position of the active paw is parallel to the body.

[0069] 18 points: In the gait, continuous palmar surface movement and continuous coordinated movements of the fore and hind limbs can be seen. During the forward movement of the forelimb, the paw can continuously grasp the ground. When initially contacting, the position of the active paw is parallel to the body, and it rotates after weight transfer.

[0070] 19 points: In the gait, continuous palmar surface movement and continuous coordinated movements of the fore and hind limbs can be seen. During the forward movement of the forelimb, the paw can continuously grasp the ground. When initially contacting and after weight transfer, the position of the active paw is parallel to the body, and the tail is sometimes or always down.

[0071] 20 points: Persistent palmar movement, persistent coordinated gait, continuous toe gripping, the position of the active paw is parallel to the body both at initial contact and after weight transfer, unstable trunk, and persistent tail elevation;

[0072] 21 points: Persistent palmar movement, persistent coordinated gait, continuous toe gripping, the position of the active paw is always parallel to the body during movement, persistent trunk stability, and persistent tail elevation.

[0073] Figure 1 The BBB scores of each group at different times after injury are shown. At the 5th week after injury, significant statistical differences began to appear when the experimental group was compared with the control group (SCI group) (p < 0.05).

[0074] Example 3. Evaluation of the hindlimb motor function of experimental animals using a grid experiment

[0075] The rat grid experiment is a commonly used experimental method for evaluating the sensory and motor function recovery of rats after spinal cord injury. At the 6th week after injury, the rats in the sham operation group, control group, and experimental group were respectively trained to walk on a grid. By observing the walking ability of each rat on the grid, the proportion of the number of steps that the hindlimbs of each rat took wrong or missed and the total number of steps was mainly used to evaluate its motor function. In this project, double-blind and three-person independent observation records were used, and finally statistical analysis was carried out to evaluate the hindlimb motor function of rats at the 6th week after injury.

[0076] From Figure 2 it can be seen that 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 drug treatment can significantly promote the motor function recovery of rats after spinal cord injury.

[0077] Example 4. Evaluation of the hindlimb motor function of experimental animals using a hot plate experiment

[0078] The rat hot plate experiment is a commonly used experimental method for evaluating the pain perception and conduction of rats after spinal cord injury. In this experiment, the changes in pain perception and conduction were evaluated by observing the reaction time of rats on the hot plate. The rats were placed on a hot plate with a preset temperature of 52 °C, and the reaction time of each rat was observed, including reactions such as licking the feet, jumping, or scratching the feet, to evaluate its pain perception and conduction. In this project, double-blind and two-person independent observation records were used, and finally statistical analysis was carried out to evaluate the hindlimb pain sensation function of rats at the 6th week after injury.

[0079] From Figure 3 it can be seen that the hot plate reaction time of the experimental group was significantly longer than that of the control group (P < 0.0001), indicating that drug treatment can significantly promote the sensory function recovery of rats after spinal cord injury.

[0080] Example 5. Evaluation of the hind limb motor function of experimental animals using the swimming experiment

[0081] The rat swimming experiment is a commonly used method for evaluating hind limb motor behavior, which is used to evaluate the motor function and coordination ability of rats after spinal cord injury. In this experiment, the recovery of motor function and coordination ability is evaluated by observing the performance of rats swimming in water. At the 6th week after injury, the rats are placed in a water pool, and their swimming postures, speeds, and coordination abilities are observed, and the performance of the rats swimming in water is recorded, including indicators such as swimming speed, swimming posture, and coordination ability. In this project, double-blind and independent observation and recording by two people are adopted. The specific scoring criteria are as follows in the table. Finally, statistical analysis is carried out to evaluate the hind limb motor function of rats at the 6th week after injury.

[0082]

[0083] From Figure 4 it can be seen that the swimming experiment score of the experimental group is significantly higher than that of the control group (P < 0.0001), indicating that drug treatment can significantly promote the recovery of motor function in rats after spinal cord injury.

[0084] Example 6. Evaluation of the hind limb motor function of experimental animals using the Catwalk experiment

[0085] The rat Catwalk experiment is a commonly used gait analysis method, which is used to evaluate the gait function and motor ability of rats after spinal cord injury. The Catwalk system is a high-resolution gait analysis system that can record the plantar footprints of rats in real time when they walk on a transparent glass plate, so as to evaluate gait characteristics and gait parameters. At the 6th week after injury, the rats are placed on the Catwalk system and allowed to walk freely. The system will record the plantar footprints and gait characteristics of the rats in real time. The gait parameters of the rats are analyzed by the software provided by the Catwalk system, including indicators such as gait cycle, stride length, and support time, to evaluate the gait function and motor ability of the rats. In this project, double-blind and independent observation and recording by two people are adopted. Finally, statistical analysis is carried out to evaluate the hind limb motor function of rats at the 6th week after injury.

[0086] From Figure 5 it can be seen that the coordination of the hind limb footprint analysis and the size of the footprint area in the Catwalk experiment of the experimental group are significantly better than those of the control group, indicating that drug treatment can significantly promote the recovery of motor function in rats after spinal cord injury.

[0087] Example 7. Evaluation of the hind limb motor function of experimental animals using the MEP experiment

[0088] Motor Evoked Potential (MEP) is an electrophysiological signal that records muscle movement responses by stimulating the cerebral cortex area. The MEP experiment is a commonly used electrophysiological experimental method for evaluating the motor function and nerve conduction of rats after spinal cord injury. At the 6th week after injury, before the experiment, electrodes were implanted into the cerebral cortex area, and then the motor cortex neurons were activated by electrical stimulation (8 uV). The MEP signals of the rats were recorded by an electrophysiological instrument, including the electrical activity responses of the muscles after stimulation. By analyzing the recorded MEP signals, the nerve conduction and motor function of the rats can be evaluated, including parameters such as latency, amplitude, and waveform. After collecting the experimental data, statistical analysis was performed to compare the differences between different groups and evaluate the recovery of nerve conduction and motor function of the rats after spinal cord injury. In this project, double-blind, independent observation and recording by two people were adopted, and finally statistical analysis was carried out to evaluate the hindlimb motor function of the rats within 6 weeks after injury.

[0089] It can be seen from Figure 6 that the amplitude of the electrophysiological experiment in the experimental group was higher than that in the control group (P < 0.05), indicating that drug administration treatment can significantly promote the recovery of motor function in rats after spinal cord injury.

[0090] Example 8. Detection of gene expression in rats after drug administration by RNA sequencing

[0091] RNA sequencing of rats is a method for studying gene expression changes after spinal cord injury, which can help reveal the molecular mechanisms of spinal cord injury and potential therapeutic targets. In the experimental group, gavage administration was performed immediately after injury until the 7th day. On the 7th day, the sham operation group, the control group, and the experimental group were respectively sampled, and tissues about 0.5 cm around the injury center were taken, added with tissue preservation solution, stored in liquid nitrogen, and sent to Hangzhou Lianchuan Biotechnology Co., Ltd. for sequencing. Bioinformatics analysis was performed on the raw data obtained from sequencing, including data quality control, alignment to the reference genome, differential expression gene analysis, enrichment analysis, etc. According to the results of bioinformatics analysis, the changes in gene expression of rats in each group can be found, and key signaling pathways and regulatory factors can be identified.

[0092] It can be seen from Figure 7 that some genes in the experimental group were up-regulated, and the Complement and coagulation cascades signaling pathway was significantly up-regulated, indicating that drug administration treatment may act on this pathway.

[0093] Example 9. Detection of the expression of glial fibrillary acidic protein (GFAP) by frozen section immunofluorescence histochemistry

[0094] At the 6th week after injury, the rats in the sham operation group, the control group and the experimental group were sacrificed. Before the experiment, the rats were anesthetized, and then perfused with 100 ml of PBS and 100 ml of 4% paraformaldehyde. The spinal cord tissue was dissected, dehydrated with a sucrose gradient, embedded with OCT, then sectioned, and GFAP immunofluorescence staining was performed on the spinal cord tissue. In the sham operation group, less red fluorescence (GFAP) was visible and was evenly distributed; in the damaged area of the control group, a large amount of red fluorescence was visible, indicating a large number of astrocytes proliferating and aggregating; in the experimental group, only less red fluorescence was observed, indicating only mild astrocyte proliferation, and the number of glial scars formed was significantly reduced compared with the control group (see Figure 8 ). Through further statistical analysis, the GFAP expression in the experimental group was significantly lower than that in the control group (P < 0.01). The above experimental results indicate that compared with the control group, drug treatment can significantly reduce the formation of cavities and glial scars after spinal cord injury.

[0095] Example 10. Detection of the expression of neuronal nuclear antigen (NeuN) by immunofluorescence histochemistry of frozen sections

[0096] At the 6th week after injury, the rats in the sham operation group, the control group and the experimental group were sacrificed. Before the experiment, the rats were anesthetized, and then perfused with 100 ml of PBS and 100 ml of 4% paraformaldehyde. The spinal cord tissue was dissected, dehydrated with a sucrose gradient, embedded with OCT, then sectioned, and NEUN immunohistochemical staining was performed on the rat spinal cord tissue. The staining results showed that a large number of NEUN(+) neurons were shown in the sham operation group; the tissue in the control group was ruptured and no obvious NEUN(+) neurons were present; the tissue in the experimental group was ruptured and a moderate amount of NEUN(+) neurons were present (see Figure 8 ). Through further statistical analysis, the NEUN expression in the experimental group was higher than that in the control group (P < 0.05) (see Figure 10 ). The above experimental results indicate that compared with the control group, the survival rate of neuronal cells in the experimental group is higher than that in the control group.

[0097] The specific embodiments described above have further elaborated on the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of earthworm extract in the preparation of a drug for treating spinal cord injury.

2. Use of earthworm extract in the preparation of a drug for promoting spinal cord injury repair.

3. Use of earthworm extract in the preparation of a drug for promoting the recovery of motor ability after spinal cord injury.

4. The use according to claim 3, characterized in that, The recovery of the motor ability is manifested as an improvement in motor ability.

5. The use according to any one of claims 1 to 4, characterized in that, The active substance in the earthworm extract is earthworm protein.

6. The use according to claim 5, characterized in that, The content of the active substance is 5000U / mg ± 10%.

7. The use according to any one of claims 1-6, characterized in that, The drug further comprises a pharmaceutically acceptable carrier or excipient.

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