Application of exogenous NO donor in preparation of medicine for treating and / or preventing epilepsy

Abstract

The invention discloses application of an exogenous NO donor in preparation of a medicine for treating and / or preventing epilepsy, and belongs to the technical field of biological medicine. According to the invention, the inhibition effect of long-term application of the exogenous NO donor on epilepsy is confirmed for the first time. A mouse is intervened by using an exogenous NO donor DETA / NONOate during the epilepsy occurrence period and after the epilepsy pathology is formed, so that the formation of epilepsy can be blocked when the application is performed during the epilepsy occurrence period, and the epilepsy seizure can be blocked and complications such as cognition and depression can be improved when the application is performed after the epilepsy pathology is formed; the invention further verifies the prevention and treatment effects of DETA / NONOate on epilepsy after long-term application, and further researches find that an exogenous NO donor slowly releases NO in the brain of a mouse so as to inhibit the generation of an abnormal neural circuit of dentate gyrus granulosa cells and finally inhibit the occurrence of chronic epilepsy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of an exogenous NO donor in the preparation of a drug for treating and / or preventing epilepsy. Background Art

[0002] Epilepsy is a chronic brain disease characterized by recurrent seizures in which local neurons in the brain discharge abnormally at high frequencies and spread to surrounding normal tissues, causing disorders of the nervous system. Temporal lobe epilepsy is one of the most common types of epilepsy in clinical practice. Epidemiological survey reports indicate that epilepsy is the second most common central nervous system disease in the world, with approximately 50 million patients. Epilepsy patients in my country account for approximately 7‰ of the country's total population, with approximately 9 million epilepsy patients, and approximately 400,000 new diagnoses each year. Epilepsy has a long course and is painful during attacks. Clinical treatment is mainly through long-term drug treatment, and surgical treatment is ultimately required, which seriously affects the patient's quality of life and also poses a huge challenge to the patient's family and society.

[0003] The main lesion area of ​​epilepsy is in the hippocampus. After the onset of the disease, abnormal basal dendrites are formed, ectopic granule cells appear, glial scars are produced, and abnormal neuronal excitability is caused. The existing anti-epileptic drug strategy still remains at the level of controlling symptoms, and there is no effective intervention in the mechanism of epileptic seizures, which leads to the occurrence of refractory epilepsy. Neurogenic nitric oxide synthase (nNOS) is a subtype of nitric oxide synthase, which mainly exists in neurons and plays the function of catalyzing L-Arginine to produce nitric oxide (NO). A large number of studies have shown that nNOS in the brain is closely related to various diseases such as stroke, depression, fear memory and epilepsy. The gaseous signal molecule NO is widely distributed in various tissues of the body, especially in the central nervous system. It not only has the dual effects of neurotoxicity and neuroprotection, but also can induce the excitation of neurons and glial cells. Studies have found that a decrease in the number of GABAergic inhibitory neurons was observed in the hippocampus of patients with temporal lobe epilepsy and animal models. Interneurons containing nNOS are a subtype of GABAergic neurons in the gate area. Knockout of nNOS leads to a significant decrease in NO in the hippocampus and an increase in excitatory input, which ultimately leads to the occurrence of epilepsy. However, there are currently no relevant studies reporting whether regulating exogenous NO can also affect the occurrence of epilepsy. Summary of the invention

[0004] The purpose of the present invention is to provide an application of an exogenous NO donor in the preparation of a drug for treating and / or preventing epilepsy, so as to solve the problems existing in the above-mentioned prior art. The present invention confirms for the first time the inhibitory effect of long-term application of an exogenous NO donor on epilepsy. Mouse experiments confirm that after long-term application of DETA / NONOate, the abnormal neural circuit formation of granule cells in the dentate gyrus is inhibited by slowly releasing NO in the mouse brain, thereby ultimately inhibiting the occurrence of chronic epilepsy, and at the same time, it can improve the cognitive damage caused by the formation of epileptic pathology.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of an exogenous NO donor in preparing a medicine for treating and / or preventing epilepsy.

[0007] Furthermore, the exogenous NO donor is DETA / NONOate.

[0008] Furthermore, the epilepsy includes chronic epilepsy.

[0009] Furthermore, the chronic epilepsy includes temporal lobe epilepsy.

[0010] Furthermore, the exogenous NO donor plays a role in preventing epilepsy by blocking the formation of epilepsy.

[0011] Furthermore, the exogenous NO donor plays a role in treating epilepsy by blocking the onset of epilepsy and improving cognitive damage caused by the formation of epileptic pathology.

[0012] Furthermore, blocking the onset of epilepsy includes blocking the construction of the excitatory circuit of dentate gyrus granule cells.

[0013] The present invention discloses the following technical effects:

[0014] The present invention is the first to confirm the inhibitory effect of long-term application of exogenous NO donors on epilepsy. Mouse experiments have confirmed that after long-term application of DETA / NONOate, NO is slowly released in the mouse brain, thereby inhibiting the generation of abnormal neural circuits in the dentate gyrus granule cells, thereby inhibiting the occurrence of chronic epilepsy, and at the same time improving cognitive damage caused by the formation of epileptic pathology.

[0015] The present invention uses exogenous NO donor DETA / NONOate to intervene in mice during epilepsy and after epilepsy pathology is formed, and finds that its application during epilepsy can block the formation of epilepsy, and its application after epilepsy pathology is formed can block the onset of epilepsy and improve cognitive damage, thereby verifying the preventive and therapeutic effects of long-term application of DETA / NONOate on epilepsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The experimental results of using exogenous NO donors to block the formation of chronic epilepsy during epilepsy; A is a schematic diagram of the experimental process; B is a schematic diagram of the EEG detection of mice in different treatment groups; C is the statistical results of the number of epileptic spikes (SPKs) and spontaneous recurrent seizures (SRS) detected in mice in different treatment groups; D is the results of the nitric oxide concentration detection in the dentate gyrus of mice in different treatment groups;

[0018] Figure 2 The results of the experiment of using exogenous NO donors to block the formation of chronic epilepsy in the late stage of epilepsy; A is a schematic diagram of the experimental process; B is a schematic diagram of the EEG detection of epilepsy model mice and normal saline control mice after they were given normal saline and NO donors respectively; C is the statistical results of the number of epileptic spikes (SPKs) and spontaneous recurrent seizures (SRS) detected in mice in different treatment groups;

[0019] Figure 3 The results of the experiment of using exogenous NO donor to block chronic epileptic seizures after epilepsy pathology is formed; A is a schematic diagram of the experimental process; B is a schematic diagram of the EEG detection of chronic epileptic mice after one month of treatment with saline and NO donor drugs; C is the statistical results of the number of epileptic spikes (SPKs) and spontaneous recurrent seizures (SRS) in mice in different treatment groups;

[0020] Figure 4 The results of the experiment of using exogenous NO donor to block the excitatory circuit of dentate gyrus granule cells after epilepsy pathology is formed; A is a schematic diagram of the experimental process; B is a schematic diagram of the changes in the excitatory circuit of the dentate gyrus of mice in different treatment groups; C is the statistical results of the number of excitatory input cells in the dentate gyrus of mice in different treatment groups;

[0021] Figure 5The figures are the experimental results of exogenous NO donor improving cognitive impairment in temporal lobe epilepsy; A is a schematic diagram of the Morris water maze behavioral test process; B is the statistical results of the target area residence time and movement speed of mice in different treatment groups in the water maze test; C is a schematic diagram of the trajectory of mice in different treatment groups in the water maze test; D is the statistical results of the time required for mice in different treatment groups to find the platform from the beginning of swimming in the pool; E is a schematic diagram of the Y maze behavioral test process; F is the statistical results of the Y maze cognitive ability test of mice in different treatment groups; G is a schematic diagram of the new object recognition behavioral test process; H is the statistical results of the position memory and object discrimination memory test of mice in different treatment groups. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The exogenous NO donor used in the examples of the present invention is diethylenetriamine / nitric oxide adduct (DETA / NONOate): [2,2'-(hydroxynitrosohydrazono)bis-(ethanamine)], CAS No.: 146724-94-9, and the molecular structure is as follows:

[0028]

[0029] The public can purchase it through regular channels based on the above information.

[0030] Example

[0031] 1. Construction and detection of epilepsy model

[0032] (1) Construction of a mouse model of chronic spontaneous epilepsy induced by pilocarpine

[0033] C57BL / 6J mice aged 6-8 weeks and weighing 22-24g were selected to induce epilepsy model. Scopolamine, atropine, and terbutaline were first injected into the mice intraperitoneally in sequence, and the dose of the drugs was 1mg / kg. Thirty minutes after the injection, pilocarpine (310mg / kg) was injected subcutaneously into the mice. After 2-3 hours of induction, diazepam (10mg / kg) was injected intraperitoneally to stop the aggravation and attack of epilepsy. The severity of the mouse's seizure was determined according to the Racine grading standard.

[0034] The Racine grading standard is as follows: Grade I, facial muscle twitching such as chewing, blinking, and whisker erection; Grade II, neck muscle twitching mainly with nodding movements; Grade III, unilateral forelimb spasm and twitching; Grade IV, bilateral forelimb spasm and twitching accompanied by hindlimb uprightness; Grade V, bilateral hindlimb rigidity, dorsal flexion rigidity, falling with whole-body clonus.

[0035] The criteria for judging a successful temporal lobe epilepsy model are that mice have grade III seizures lasting more than 40 minutes during the modeling period, and after a 4-5 week quiescent period, they have spontaneous seizures and are in good condition during the chronic epileptic period.

[0036] (2) Electrode implantation and EEG monitoring in mouse brain

[0037] Mice were anesthetized with 2.5% tribromoethanol (0.2 mL / 10 g, ip) and fixed in a stereotaxic apparatus. For hippocampal recordings, bipolar stainless steel electrodes were implanted bilaterally in the hippocampus (left hippocampus [LH]; right hippocampus [RH]; 3.4 mm posterior to bregma; 2.7 mm lateral to the raphe; 2.9 mm below the dura). Stainless steel screws were implanted in the frontal cortex bilaterally (left frontal lobe [LF]; right frontal lobe [RF], 0.5 mm posterior to bregma and 2.8 mm lateral to the raphe). Another screw was placed on the right side of the frontal sinus as a reference electrode [REF]. The electrodes were then connected to a plastic plug that connected the electrodes and fixed to the skull with dental cement. Before EEG testing, mice were allowed to move freely for 24-72 hours to recover from surgery. Digital EEG recordings were performed using EEG2100 software, and EEG testing was performed for 3 to 4 consecutive days per mouse. Seizure-like spikes were defined as bursts of electrical activity of 20–150 ms with an amplitude at least 5 times that of the background EEG activity. Mice had free access to food and water throughout the EEG recordings.

[0038] 2. Exogenous NO donors are used during epilepsy to block the formation of chronic epilepsy.

[0039] Mice were induced by pilocarpine or injected with saline (Vehicle). Seven days later, DETA / NONOate (1 mg / kg, intraperitoneal injection, once a day) or an equal volume of saline was given for two months. The number of epileptic spikes (SPKs) and spontaneous recurrent seizures (SRS) in mice was detected by implanted electrodes. Figure 1 The result is as follows Figure 1 BD showed that DETA / NONOate administration can inhibit the formation of chronic epilepsy induced by pilocarpine.

[0040] 3. Exogenous NO donors are used in the late stage of epilepsy to block the formation of chronic epilepsy.

[0041] After pilocarpine or saline injection (Vehicle), mice were given DETA / NONOate (1 mg / kg, intraperitoneal injection, once a day) or an equal volume of saline (Vehicle) for one month starting 37 days later. The number of epileptic spikes (SPKs) and spontaneous recurrent seizures (SRS) in mice was detected by implanted electrodes. Figure 2 The result is as follows Figure 2As shown in BC, DETA / NONOate can inhibit the formation of chronic epilepsy induced by pilocarpine when applied in the late stage of epilepsy.

[0042] 4. Exogenous NO donors are used after epilepsy pathology is formed to block chronic epileptic seizures.

[0043] Mice were induced by pilocarpine or injected with saline (Vehicle). After 2.5 months, DETA / NONOate (1 mg / kg, intraperitoneal injection, once a day) or an equal volume of saline (Vehicle) was given for one month. The number of epileptic spikes (SPKs) and spontaneous recurrent seizures (SRS) in mice was detected by implanted electrodes. Figure 3 The result is as follows Figure 3 As shown in BC, it shows that the use of DETA / NONOate also inhibits chronic epileptic seizures after chronic epilepsy pathology has been established.

[0044] 5. Exogenous NO donors are used after epilepsy pathology is formed, blocking the construction of the excitatory circuit of granule cells in the dentate gyrus and exerting an anti-epileptic effect

[0045] After pilocarpine or saline injection (Vehicle), mice were injected with LV-Syn-GTRgp virus in the dentate gyrus of the hippocampus 7 days later, and DETA / NONOate (1 mg / kg, intraperitoneal injection, once a day) or an equal volume of saline (Vehicle) was given for two months. After that, Enva-Rbv-Rgp-MCh virus was injected in situ in the dentate gyrus. The brain was removed 7 days later to observe the formation of abnormal neural circuits in mice marked by the virus tracking system ( Figure 4 The result is as follows Figure 4 As shown in BC, DETA / NONOate can inhibit the formation of abnormal hyperexcitable neural circuits in pilocarpine-induced epilepsy.

[0046] 6. Application of exogenous NO donor improves cognitive impairment caused by temporal lobe epilepsy

[0047] After pilocarpine modeling or saline injection (Vehicle), mice were given DETA / NONOate (1 mg / kg, intraperitoneal injection, once a day) or an equal volume of saline (Vehicle) 7 days later for two months. The mice were then tested in Morris water maze, Y maze and novel object recognition to analyze changes in their cognitive functions.

[0048] The water maze behavioral test was performed as follows:

[0049] Spatial learning was assessed using the hidden platform version of the MWM behavioral test. The test apparatus consisted of a large circulating pool (120 cm diameter, 70 cm depth) filled with water (25-26 °C) to a depth of 40 cm. The water was made opaque with white food dye to prevent the animals from seeing a circular platform (12 cm diameter) submerged 1 cm below the water surface and located at a fixed position in one of the quadrants 20 cm from the pool wall. The pool was divided into four quadrants and different graphic cues were fixed to the pool wall. 24 h before the start of training, all mice were acclimated to swimming in the pool by performing a 60 s swim without the platform. During the next 5 days, mice were trained to find the hidden platform in the fixed quadrant. Mice received 1 training session per day, which consisted of 3 trials. Each trial was separated by 15 min. For each trial, the mouse was gently released into the pool facing the wall. The mouse was given a maximum of 60 s to find the platform. After finding the platform, it was left there for 10 seconds and then placed in the mouse cage until the next trial began. Animals that failed to find the platform within 60 seconds were placed on the platform and left to rest for 10 seconds. The latency and swimming speed of the mice to reach the platform were collected and counted. After the 5-day training was completed, the mice were returned to their cages until 24 hours later when the quadrant exploration test was performed. The quadrant exploration test was a 60-second free swimming time for the mice, during which there was no platform in the pool, and the percentage of mice swimming in the target quadrant was recorded and counted.

[0050] The Y-maze test is as follows:

[0051] The Y-maze was used to assess the short-term spatial memory of mice. The Y-maze apparatus consisted of three opaque plastic walls (length × width × height = 30 cm × 6 cm × 15 cm). When testing the novel exploration experiment, the three arms were randomly divided into (A) the starting arm; (B) the other arms, which were always kept open; (C) the novel arm, which was kept closed during training and opened during testing. During the training phase, the mice were placed in the starting arm and allowed to freely explore the starting arm and other arms for 10 minutes. The test was performed 2 hours later, the novel arm was opened, and the time spent exploring each arm and the proportion of novel arm preference were recorded.

[0052] When testing the alternating behavior experiment, the three arms were marked as arms I, II, and III, and the mice were placed in the center of the maze and allowed to explore freely for 8 minutes. The criterion for the mouse to enter each arm was that all four limbs entered, and when the mouse entered three different arms in turn, it was recorded as an alternation. The alternation score of the mouse was calculated. In the above experiments, the maze was wiped with 75% alcohol between the experiments of both mice, and the alcohol was completely evaporated to ensure that the smell would not interfere with the experiment.

[0053] The novel object recognition test is as follows:

[0054] The experimental apparatus was a closed square container (length × width × height = 40 cm × 40 cm × 40 cm) to ensure that the animals would not be disturbed externally during the experiment. In the test, two identical objects were placed in the experimental apparatus and the mice were allowed to explore freely for 5-10 minutes. One day after training, the mice were allowed to explore the two objects again. For object location memory (OLM), one of the identical objects was moved to a new location while the other was not. For object recognition memory (ORM), one object was replaced by a new object, but the location remained unchanged. The discrimination index was used to represent the relative exploration time.

[0055] The results of the above three tests are as follows Figure 5 As shown, Figure 5 It shows that DETA / NONOate can improve cognitive impairment caused by pilocarpine-induced epileptic seizures.

[0056] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of an exogenous NO donor in the preparation of a drug for treating and / or preventing epilepsy.

2. The use according to claim 1, characterized in that: The exogenous NO donor is DETA / NONOate.

3. The use according to claim 1, characterized in that: The epilepsy includes chronic epilepsy.

4. The use according to claim 3, characterized in that: The chronic epilepsy includes temporal lobe epilepsy.

5. The use according to claim 1, characterized in that: The exogenous NO donor plays a role in preventing epilepsy by blocking the formation of epilepsy.

6. The use according to claim 1, characterized in that: The exogenous NO donor plays a role in treating epilepsy by blocking the onset of epilepsy and improving cognitive damage caused by the formation of epileptic pathology.

7. The use according to claim 6, characterized in that: Blocking the onset of epilepsy includes blocking the establishment of the excitatory circuit of dentate gyrus granule cells.

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