CN113616782B - Application of irisin in treating epilepsy - Google Patents
Application of irisin in treating epilepsy Download PDFInfo
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- CN113616782B CN113616782B CN202110639354.0A CN202110639354A CN113616782B CN 113616782 B CN113616782 B CN 113616782B CN 202110639354 A CN202110639354 A CN 202110639354A CN 113616782 B CN113616782 B CN 113616782B
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Abstract
The invention provides an application of tectoridin in treating epilepsy, and belongs to the technical field of biological medicines. The invention proves for the first time that the tectorigenin treatment has remarkable inhibition effect on neuronal injury, cognitive defect and epileptic seizure induced by acute and chronic epileptic. Also, its protective effect may occur through the BDNF/UCP2 pathway. Therefore, the tectorigenin treatment is a potential method for treating epilepsy, and provides a new thought for researching the action targets of epilepsy medicaments and clinically treating epilepsy-related diseases, thereby having important clinical application value.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of tectoridin in treating epilepsy.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Epilepsy is the second largest disease of the nervous system and can be manifested as many different forms of seizures with serious consequences in pathology and psychology. Currently, the clinical treatment of epilepsy is mainly drug therapy. However, antiepileptic drugs are generally resistant and cannot achieve radical cure. Limitations of drug therapy are closely related to the complex and ambiguous pathogenesis of epilepsy. Thus, research on the pathogenesis of epilepsy, and finding new and effective targets and means for treating epilepsy are urgent problems to be solved at present.
Irisin is formed after hydrolysis of fibronectin domain protein 5. In animal experiments, irisin can promote the transformation of white adipose tissue into brown adipose tissue, increase energy consumption, improve glucose and lipid metabolism, reduce insulin resistance, and gently reduce weight, has close correlation with endocrinologic metabolic diseases, cardiovascular diseases, tumors and the like, but has different results in human body and clinical researches. Therefore, the mechanism of action of irisin and its relationship to disease also require more intensive research to confirm.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides the application of tectoridin in treating epilepsy. The invention proves for the first time that the tectorigenin treatment has remarkable inhibition effect on neuronal injury, cognitive defect and epileptic seizure induced by acute and chronic epileptic. Also, its protective effect may occur through the BDNF/UCP2 pathway. Therefore, the tectorigenin treatment is a potential method for treating epilepsy, and provides a new thought for researching the action targets of epilepsy medicaments and clinically treating epilepsy-related diseases, thereby having important clinical application value.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the invention, there is provided the use of irisin in the manufacture of a medicament for the treatment of epilepsy.
According to the invention, the concept of "treatment" means any relevant measure suitable for the treatment of epilepsy, either for the prophylactic treatment of such a represented disease or of a represented symptom, or for the avoidance of recurrence of such a disease, for example after the end of a treatment period or for the treatment of a symptom of a disease that has already developed.
More specifically, the epilepsy includes acute or chronic epilepsy.
In the present invention, the epilepsy is acute or chronic epilepsy induced by alginic acid.
The treatment of epilepsy is characterized by having at least any one or more of the following uses:
a) Increasing the level of brain-derived neurotrophic factor and/or uncoupling protein-2 expression in the brain;
b) Reducing apoptosis caused by epilepsy;
c) Reducing neuronal damage caused by epilepsy;
d) Reversing epileptic-induced learning and/or memory disorders;
e) Alleviating seizures;
f) Reducing the level of oxidative stress in the brain.
Wherein, in said a), said brain comprises hippocampal and cortical areas;
In said e), the reduction of seizures is embodied as a reduction of seizure frequency and/or a reduction of seizure duration;
The f) reduces the level of oxidative stress in the brain, which is specifically characterized by reducing the level of DCF and/or Mito-SOX expression in the hippocampus and cortex.
According to the invention, not only is the use of irisin in the manufacture of a medicament for the treatment of epilepsy disclosed, but it is also disclosed that this effect may be enhanced when a combination of irisin with at least one other pharmaceutically active ingredient is administered. Alternatively or in addition to other pharmaceutically active ingredients, irisin may also be used in combination with other non-pharmaceutically active ingredients.
In view of this, the second aspect of the present invention provides a pharmaceutical composition for the treatment of epilepsy, consisting of irisin with at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.
In the sense of the present invention, the pharmaceutical composition of the present invention represents a substance containing irisin having an obvious therapeutic effect on epilepsy.
In a third aspect of the invention, there is provided the use of tectoridin in the preparation of a protein promoter:
wherein the proteins include, but are not limited to, brain-derived neurotrophic factor and uncoupling protein-2.
In a fourth aspect of the invention, there is provided a method of treating epilepsy, comprising administering to a subject a therapeutically effective amount of the above-described irisin and/or pharmaceutical composition.
The beneficial technical effects of one or more of the technical schemes are as follows:
The technical scheme provides an intervention target point for preparing the anti-epileptic drug, and the irisin is applied to drug development related to epileptic treatment so as to provide a better preparation method of the epileptic therapeutic drug. In the spontaneous model of acute and chronic rat epilepsy induced by alginic acid, exogenous tectorigenin is administered indoors, the level of brain-derived neurotrophic factor and uncoupling protein-2 in the brain of animals is obviously increased, and apoptosis, neuron injury, cognitive dysfunction and epileptic seizure are obviously reduced. The neuroprotection of irisin was partially reversed while the levels of UCP2 were reduced in the brain by injection of genipin into the lateral ventricle.
The technical scheme shows that: the tectorigenin treatment has remarkable inhibition effect on neuronal injury, cognitive defect and epileptic seizure induced by acute and chronic epilepsia. Also, its protective effect may occur through the BDNF/UCP2 pathway. The inhibition of tectorigenin treatment on epileptic-induced nerve injury and cognitive defect is an effective target for treating epilepsia, can be used as a new epilepsia treatment drug or drug target for development, and provides a new method for treating epilepsia, so that the method has good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
Antiepileptic and neuroprotective effects of first fraction of irisin in alginic acid-induced chronic epileptic
FIG. 1 shows that exogenous irisin can reduce spontaneous seizures induced by KA in an embodiment of the invention. Wherein (a) the cumulative onset time of each group at class I-V of 30d-170d (ka+saline group: n=16; ka+irisin group: n=8); (B) Representative EEG and PSD analysis of the average cumulative time of onset at class I-III of 30D-170D for each group (C) and average cumulative time of onset at class I-III of 30D-170D (D) 170D; (G) water maze latency for each group; (H) percentage of time the target quadrant is located; (I) percentage of time in which the para-quadrant is located; (J) number of platform passes. (K) representative trace plot.
FIG. 2 illustrates that irisin reduces KA-induced apoptosis and neuronal degeneration in accordance with embodiments of the invention. Wherein, (A-F): immunoreactivity (n=5) of each of the groups caspase-3 and ACTIVED CASPASE-3 at 3d and 170d post-KA treatment; (G-M): the FJB positive signals of each group were counted at 3d EC and hippocampal CA 2.
FIG. 3 shows that tectorigenin treatment increases BDNF and UCP2 expression in the brain in an embodiment of the present invention. Wherein, (A-B): immunoreactivity of UCP2 and BDNF for each group 3d and 15d after KA treatment; (C-F): protein level comparison at 3d and 15d for each group of UCP2 and BDNF; (G-I): immunoreactivity of each group of BDNF; (J): immunoreactivity comparison of BDNF for each group.
FIG. 4 shows that exogenous irisin reduces the increase in DCF/Mito-SOX levels caused by KA treatment in an embodiment of the invention. Wherein, (A-B): DCF level expression at 24h and 3d cortex and hippocampus for each group (n=5/group); (C-D): mito-SOX levels expressed in 24h and 3d cortex and hippocampus for each group; (E): mito-SOX at 3d for each group represents the streaming detection results.
FIG. 5 shows increased UCP2 levels from genipin reversal irisin treatment in accordance with embodiments of the present invention. Wherein, (A-B): UCP2 protein level expression at 3d and 15d (n=5/group); (C-D): UCP2 immunoreactivity at 3d and 15 d.
FIG. 6 shows that genipin reverses the inhibition of KA-induced epilepsy by exogenous tectorigenin in accordance with an embodiment of the present invention. Wherein, (A): the cumulative onset time of grade I-V30 d-170d after KA injection (ka+saline group: n=16; ka+irisin group: n=8; ka+irisin+genipin group: n=10); b: average cumulative onset time for each group I-III at 30d-170d after KA injection; c: the average number of the attacks of the I-III levels of each group is 30d-170d after KA injection; d: EEG and EEG analysis of KA+ saline group at 170 d; e: EEG at 170d for KA+Irin+saline group; f: EEG of KA+irisin+genipin group at 170 d; (G) water maze latency for each group Mirros; (H) percentage of time the target quadrant is located; (I) percentage of time in which the para-quadrant is located; (J) number of platform passes; (K) representative trace plot.
FIG. 7 shows that genipin reverses the inhibition of KA-induced apoptosis and neuronal degeneration by irisin in accordance with an embodiment of the present invention. Wherein, (A-C): caspase-3 and ACTIVED CASPASE-3 apoptosis-related protein content expression at 3d (n=5/group); (D-I): FJB staining experiments to examine neuronal degenerative changes in the rat brain at 3d for each group (J): the FJB positive signals of each group were counted at 3d EC and hippocampal CA 2.
FIG. 8 shows that genipin reverses the effect of irisin on reducing DCF and mito-SOX levels in accordance with an embodiment of the present invention. Wherein, (A-B): DCF level expression at 24h and 3d in the cortex and hippocampus of each group of rats (n=5/group); (C-D): mito-SOX level expression at 24h and 3d in the rat cerebral cortex and hippocampus of each group (n=5/group); (E): flow-through Mito-SOX representative expression patterns at 3d in the hippocampus of rats of each group.
Neuroprotection of irisin in the second fraction of alginic acid-induced acute seizure
FIG. 9 shows that tectorigenin treatment increases BDNF and UCP2 expression in the brain in an embodiment of the present invention. Wherein, (A-B): immunoreactivity of UCP2 and BDNF for each group 24h and 3d after KA treatment; (C-F): protein level comparison at 24h and 3d for each group of UCP2 and BDNF; (G-L): immunoreactivity of each group of BDNF; (M): immunoreactivity comparison of BDNF for each group.
FIG. 10 illustrates that irisin reduces KA-induced apoptosis and neuronal degeneration in accordance with embodiments of the invention. Wherein, (A-E): immunoreactivity (n=5) of each of the groups Bax, bcl-2, caspase-3 and ACTIVED CASPASE-3 at 3d post KA treatment; (F-L): the FJB positive signals of each group were counted at 3d EC and hippocampal CA 2.
FIG. 11 shows that exogenous irisin reduces the increase in DCF/Mito-SOX levels caused by KA treatment in examples of the present invention. Wherein, (A-B): DCF level expression at 24h and 3d cortex and hippocampus for each group (n=5/group); (C-D): mito-SOX levels expressed in 24h and 3d cortex and hippocampus for each group; (E-H): mito-SOX representative flow assay results at 24h and 3d for each group.
FIG. 12 shows increased UCP2 levels by the reverse tectoridin treatment with genipin according to the examples of the present invention. Wherein, (A-C): each group of BDNF and UCP2 proteins were expressed at 24 h; (D-F): each group BDNF and UCP2 protein level expressed at 3d (n=5/group).
FIG. 13 shows that genipin reverses the inhibition of KA-induced apoptosis and neuronal degeneration by irisin in accordance with an embodiment of the present invention. Wherein, (A-E): bax, bcl-2, caspase-3 and ACTIVED CASPASE-3 apoptosis-related protein content expression at 3d (n=5/group); (F-K): FJB staining experiments detect degenerative changes of neurons in the rat brain at 3d of each group; (L): the FJB positive signals of each group were counted at 3d EC and hippocampal CA 2.
FIG. 14 shows that exogenous irisin reduces the increase in DCF/Mito-SOX levels caused by KA treatment in examples of the present invention. Wherein, (A-B): DCF level expression at 24h and 3d cortex and hippocampus for each group (n=5/group); (C-D): mito-SOX levels expressed in 24h and 3d cortex and hippocampus for each group; (E-H): mito-SOX representative flow assay results at 24h and 3d for each group.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention will be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not to be construed as limiting the invention. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the sales company; materials, reagents and the like used in the examples were commercially available unless otherwise specified.
In the invention, some of the terms used herein are expressed as follows:
alginic acid (KAINIC ACID, KA);
Brain-derived neurotrophic factor (brain derived neurotrophic factor, BDNF);
uncoupling protein-2 (uncoupling protein, UCP 2).
As mentioned above, research on the pathogenesis of epilepsy, and finding new and effective targets and means for treating epilepsy are urgent problems to be solved.
Reactive oxygen species accumulation is an important mechanism for seizures and neuronal damage. During seizures, increases in ROS can lead to neuronal damage and even apoptosis. Furthermore, mitochondrial dysfunction is closely related to ROS production. Mitochondria are the major sites of ROS accumulation during seizures, playing an important role in neuronal excitability. Mitochondrial dysfunction and oxidative stress injury are obvious pathological changes after epilepsy.
BDNF is a neurotrophic factor expressed primarily in the central nervous system. Exogenous irisin or a precursor of irisin, FNDC5, can promote BDNF gene expression and promote growth and survival of mouse brain neurons.
BDNF may further promote UCP2 expression. UCP2 has remarkable neuroprotection, reduces the generation of ROS mediated by mitochondria through uncoupling, increases ATP level, and reduces mitochondrial damage caused by free radicals. Elevated levels of UCP2 can reduce excitotoxic cell death caused by epilepsy and can combat pathological changes in neurodegenerative diseases such as epilepsy and Alzheimer's disease. In conclusion, UCP2 is involved in BDNF-regulated protection.
Thus, the present invention contemplates that irisin may reduce oxidative stress through the BDNF/UCP2 pathway, exerting neuronal protection and antiepileptic effects. In the present invention, the expression of BDNF and UCP2 in KA-induced acute and chronic epilepsy, as well as the levels of oxidative stress and neuronal damage, were studied. And further verifies the protective action and possible mechanism of the irisin through exogenous irisin treatment and genipin treatment.
In view of the above, in one embodiment of the present invention, there is provided the use of irisin in the preparation of a medicament for the treatment of epilepsy.
According to the invention, the concept of "treatment" means any relevant measure suitable for the treatment of epilepsy, either for the prophylactic treatment of such a represented disease or of a represented symptom, or for the avoidance of recurrence of such a disease, for example after the end of a treatment period or for the treatment of a symptom of a disease that has already developed.
In yet another embodiment of the present invention, the epilepsy is acute or chronic epilepsy.
In yet another embodiment of the present invention, the epilepsy is acute or chronic epilepsy induced by alginic acid.
In yet another embodiment of the invention, the treatment of epilepsy is at least characterized by any one or more of the following uses:
a) Increasing the level of brain-derived neurotrophic factor and/or uncoupling protein-2 expression in the brain;
b) Reducing apoptosis caused by epilepsy;
c) Reducing neuronal damage caused by epilepsy;
d) Reversing epileptic-induced learning and/or memory disorders;
e) Alleviating seizures;
f) Reducing the level of oxidative stress in the brain.
Wherein, in said a), said brain comprises hippocampal and cortical areas;
In said e), the reduction of seizures is embodied as a reduction of seizure frequency and/or a reduction of seizure duration;
The f) reduces the level of oxidative stress in the brain, which is specifically characterized by reducing the level of DCF and/or Mito-SOX expression in the hippocampus and cortex.
According to the invention, not only is the use of irisin in the manufacture of a medicament for the treatment of epilepsy disclosed, but it is also disclosed that this effect may be enhanced when a combination of irisin with at least one other pharmaceutically active ingredient is administered. Alternatively or in addition to other pharmaceutically active ingredients, irisin may also be used in combination with other non-pharmaceutically active ingredients.
In view of this, in yet another embodiment of the present invention, a pharmaceutical composition for the treatment of epilepsy is provided, which is composed of tectorigenin and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.
In the sense of the present invention, the pharmaceutical composition of the present invention represents a substance containing irisin having an obvious therapeutic effect on epilepsy.
In still another embodiment of the present invention, the pharmaceutically inactive ingredient may be a carrier, excipient, diluent, etc. generally used in pharmacy. Further, the composition can be formulated into various dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injectable solutions according to a usual method.
The non-pharmaceutically active ingredients, such as carriers, excipients and diluents, which may be included, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.
In yet another embodiment of the present invention, the carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like.
In yet another embodiment of the invention, the medicament of the invention may be administered to the body in a known manner. For example, by intravenous systemic delivery or local injection into the tissue of interest. Alternatively via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via single or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.
In yet another embodiment of the present invention, the subject to which the pharmaceutical composition is administered may be human or non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.
In yet another embodiment of the present invention, there is provided the use of tectoridin in the preparation of a protein promoter:
wherein the proteins include, but are not limited to, brain-derived neurotrophic factor and uncoupling protein-2.
In yet another embodiment of the present invention, a method of treating epilepsy is provided, comprising administering to a subject a therapeutically effective amount of the above-described irisin and/or pharmaceutical composition.
The subject of the present invention is an animal that has been the subject of treatment, observation or experiment, and may be human or non-human mammals such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.
The therapeutically effective amount of the present invention refers to that amount of the active compound or pharmaceutical formulation, including the compound of the present invention, which results in a biological or medical response of the tissue system, animal or human being sought by the researcher, veterinarian, medical doctor or other medical personnel, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.
The range of therapeutically effective amounts that can be used is known to researchers, veterinarians, doctors, or other medical personnel in the art from clinical trials or other means known in the art.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Examples
1. Materials and methods
1.1 Animals and surgery
Male Sprague-Dawley rats (280-300g,No.SCXK 2017-0002, jinan Pond Yue laboratory animal Breeding Co., china) were used for the experiment. All experiments were in compliance with national institutes of health laboratory animal care and use guidelines (national institutes of health publication No. 80-23, 1996 revision) and with littoral medical institutes of laboratory animal center animal ethics Specification (approval No. 2017003), and were not preregistered. The number and pain of animals are reduced as much as possible. Animals were free to feed in separate cages. All experiments were performed between 9:00 and 17:00. Animals were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.; CAS,57-33-0, western asia reagent, china) and fixed on a stereotactic instrument (Anhua zhenghua biosystems, inc., china). A stainless steel cannula (Ruiwod, china) was implanted into the lateral ventricle (AP) of each rat (directly posterior (1.0 mm), transverse (L) 1.8mm, ventral (V) 3.6 mm) and CA3 region (AP 5.3mm, L5 mm, V6 mm) respectively. The tip of each electrode was stripped of 0.5mm insulation and then implanted into the right cortex (AP: -3.2mm; L:3.0mm; V: -2 mm) for electroencephalogram (EEG) recordings using the PowerLab system (AD Instruments, australia). The implanted electrode was connected to a microelectrode socket, and the electrode and microelectrode socket were connected to the skull bone with dental cement. Rats were allowed to recover for 7 days post-surgery.
1.2 Drug treatment and chronic epileptic recording
KA was injected through an implantable stainless steel cannula into the CA3 region of the hippocampus to induce spontaneous seizures. Rats were randomly grouped and the KA+irisin group (n=8), and irisin (3 μg/brain, 5 μl, national treasures biotechnology Co., ltd.) was injected into the ventricles 30min before KA injection, 1 time every 3 days through stainless steel cannula until day 15. The ka+ saline group (n=16) replaced irisin with saline. The control group (n=5) was injected with physiological saline instead of KA into CA3.
Genipin (0.5 μg/μl,5 μl, CAS,6902-77-8, ala Ding Shiji, china) was injected as a UCP 2-specific inhibitor into the lateral ventricle 30 minutes before administration of irisin (60 minutes before KA injection) in the ka+irisin+genipin group (n=10). Genipin and irisin were injected through stainless steel cannulas every 3 days after surgery until day 15. The ka+irisin+saline group (n=8) replaced genipin with saline.
From 30d after administration of KA to the CA3 zone of the hippocampus, spontaneous seizure behavior of the animals was observed, and EEG was recorded at multiple observation time points (30 d, 40d, 50d, 60d, 70d, 80d, 90d, 100d, 110d, 120d, 130d, 140d, 150d, 170 d). Each observation time point was recorded for 3 days in continuous observation. The spectrum and power spectral density (power spectrum density, PSD) of an electroencephalograph (electroencephalo-graph, EEG) was analyzed using a PowerLab biorecorder system. The severity of epileptic seizures was assessed by counting the number of seizure accumulation times, and recording EEG, respectively, for each level of seizure in animal epileptics. Animal epileptic behaviour is classified as 1-5 according to the Racine standard. Grade 1-3 is a focal episode including strabismus, sustained indiscriminate chewing, head shaking, and unilateral forelimb lifting. Grade 4-5 is systemic spasticity, including bilateral forelimb lifting, wet dog-like tremors, systemic spastic tics, and impossibility of falling.
1.3 Drug treatment and acute epileptic recording
KA was injected into the lateral ventricle through an implantable stainless steel cannula to induce acute seizures. Rats were randomly grouped and the KA+irisin group (n=20) and irisin (3 μg/brain, 5 μl, china Xingbao biosciences) was injected into the ventricles 30min prior to KA injection (1.25 mg/ml,3.25×10 -3 mg/kg). The ka+ saline group (n=20) replaced irisin with saline.
Genipin (0.5 μg/μl,5 μl, CAS,6902-77-8, ala Ding Shiji, china) was injected as a UCP 2-specific inhibitor into the lateral ventricle 30 minutes before administration of irisin (60 minutes before KA injection) in the ka+irisin+genipin group (n=20). The ka+irisin+saline group (n=20) replaced genipin with saline.
1.4 Immunohistochemistry
At various time points following KA dosing, 5 rats per group were withdrawn on a randomized basis to administer pentobarbital sodium deep anesthesia. The anesthetized rats were placed on an operating table and perfused with 4% paraformaldehyde in normal saline and PBS. Sections of 12 μm were prepared with a cryostat (CM 3050s, leica, germany). Subsequently, brain sections were washed with 0.01M phosphate buffered saline (phosphate buffer saline, PBS) and incubated with 10% bovine serum albumin for 1 hour at 37 ℃. mu.L of mouse primary monoclonal anti-mouse BDNF (1:20 00,Abcam,ab205067) was added to each plate, and the plate was washed 3 times with 0.01M PBS at 4℃overnight. Incubation with fluorescein isothiocyanate (fluorescein isothiocyanate, FITC,1:200, A0562, beyotime, china) for 1 hour at 37 ℃. After washing 3 times, 50. Mu.L of DAPI (C1005, beyotime, china) was added and incubated for 15min at room temperature. After washing with 0.01M PBS, the plates were sealed. Fluorescence intensity of the hippocampal cortex was observed with a laser confocal microscope (LSM 880, zeiss, germany).
1.5Fluoro-Jade B (FJB) staining
FJB is a fluorescein derivative dye that specifically binds to degenerative neurons. Preparing brain tissue slices of 5 rats in each group by anesthesia with pentobarbital sodium, soaking the slices in 1% NaOH/80% ethanol for 5min, then in 70% ethanol for 2min, and flushing with distilled water for 2min; placing in 0.06% potassium permanganate solution for 15min to ensure the same background; washing with distilled water for 2min. Placing in 0.0004% FJB staining solution (prepared from 0.01% FJB stock solution 4mL and 0.1% glacial acetic acid 96 mL), incubating for 20min in dark place, washing with distilled water for 1min after incubation, drying in oven at 50deg.C for 10min, soaking in xylene for 10min, and sealing with neutral resin. The tissue sections were observed under a fluorescence microscope (CX 41, olympus, japan) with a blue (450 nm) excitation filter. FJB positive signals were counted manually and quantitatively analyzed.
1.6 Oxidative stress detection
Changes in the levels of 2',7' -dichlorofluorescein (2 ',7' -dichlorofluorescin, DCF) in each group were examined to assess the level of oxidative stress.
After KA treatment, 5 rats were randomly extracted and treated with pentobarbital sodium (after deep anesthesia at 24h and 3 d. Brain was removed and cortex and hippocampus were separated on ice. Separate cortex and hippocampus were added to 0.01M PBS, nylon mesh was filtered to prepare single cell suspensions, cell suspensions were centrifuged, supernatant was removed, 500. Mu.L of 2',7' -dichlorofluorescein diacetate (2 ',7' -dichlorodihydrofluorescein diacetate, DCFH-DA,1:1000,Beyotime,S0033, china) was added, and incubated at 37℃for 40min in the dark, centrifuged, supernatant was removed, cells were washed, and impurities in single cell suspensions were filtered. Fluorescence intensities of each group were analyzed with a fluoroenzyme-labeled instrument (Thermo, USA) using excitation and emission wavelengths of 488nm and 525nm, respectively.
1.7Mito-SOX fluorescence intensity and flow cytometry evaluation of mitochondrial oxidative stress
Following KA treatment, 5 rats were randomly withdrawn at different time points and sacrificed after deep anesthesia with sodium pentobarbital. The brain was removed from the head and cortex and hippocampus were separated. Mitochondrial ROS levels in the cortex and hippocampus were detected using Mito-SOX (M36008, thermo Fisher, USA). After immersing the isolated cortex and hippocampus in 0.01M PBS, a single cell suspension was prepared. 1mL of 5. Mu.M Mito-SOX working solution was added, and the cells were incubated at 37℃for 10min in the dark. An appropriate amount of 0.01M PBS was added to wash 3 times. Measured with a fluorescence microplate reader (Thermo, synergy H1, USA) and a flow cytometer (Becton, dickinson and Company USA) at an excitation wavelength of 510nm and an emission wavelength of 580 nm.
1.8 Cognitive function test
The cognitive ability of rats 170d was assessed using Morris water maze (ZS-001, beijing Zhongdi Ind technologies development Co., china). Rats of ka+saline group (n=16), ka+irisin group (n=8), ka+irisin+saline group (n=8), ka+irisin+genipin group (n=10) were subjected to experiments. The water maze experiment consists of a positioning navigation experiment and a space exploration experiment. The rats were placed in the pool to swim freely for 2min prior to the experiment, familiar with the surrounding environment. In the 4d positioning navigation experiment, rats were placed on the pool wall in any quadrant and the time required for the rats to find the platform was recorded. If the platform is not reached within 60s, the rat is instructed to swim onto the platform and stay on it for 10s. Thereafter, a space exploration experiment was performed on day 5. The platform was removed and the rats were allowed to swim freely for 60s and the number of passes across the platform was recorded. Rats were assessed for learning memory by assessing platform latency, by number of platforms, residence time in target quadrant and contralateral quadrant.
1.9Western Blotting
At various time points after KA injection, 5 rats were harvested from each group, anesthetized broken heads were harvested from the brain and cortex and hippocampus were isolated. After treatment with RIPA lysate (1 mg brain tissue: 10. Mu.L lysis buffer), the tissue was subsequently homogenized by sonication to prepare a protein supernatant. Proteins in tissue samples were isolated by electrotransformation with 12% sodium dodecyl sulfate polyacrylamide gel. Transfer to polyvinylidene fluoride (polyvinylidene fluoride, PVDF) membrane and incubation with mouse anti-BDNF monoclonal antibodies (1:1000, abc5067, abcam, UK), anti-rabbit UCP2 (1:2000, ab97931, abcam, UK), anti-rabbit caspase-3 (1:1000, 9662, cell signaling techniques, USA), anti-rabbit ACTIVED CASPASE-3 (1:1000, ab2302, abcam, UK) or glyceraldehyde-3-phosphate dehydrogenase (GLYCERALDEHYDE-3-phosphate dehydrogenase, GAPDH,1:1000,AB-P-R001, kangchen China) at 4℃overnight. The immunoreactive bands were incubated with horseradish peroxidase-conjugated IgG secondary antibodies. Images were obtained from different gels under the same electrophoresis conditions using an imaging analyzer (Image QuantLAS, beijing spring business trade company, usa) and the results were expressed as the gray value ratio of the target band to GAPDH band.
1.10 Statistical analysis
All data were obtained blindly and expressed as mean ± SEM. Statistical analysis was performed in Windows using SPSS 25.0 (SPSS inc., chicago, IL, USA). The cumulative number and duration of stages 1-3 between KA+ saline and KA+ irisin were analyzed using a non-parametric MANN WHITNEY U test. The cumulative seizure duration and platform latency were analyzed using a two-factor variance (analysis of variance, ANOVA). Other parameters were analyzed by one-way anova followed by a Dunnett's T post-test. In all analyses, the differences were considered significant at P < 0.05.
2. Results of the study
2.1 Exogenous Iretin can alleviate KA-induced chronic spontaneous epileptic seizure
At 170 days post KA treatment, spontaneous seizure behavior was observed in rats and EEG was recorded for each group. In the ka+irisin group (n=8), the cumulative duration of onset at various time points over 170 days after KA injection was significantly shorter than in the ka+saline group (n=16) (P <0.001; fig. 1A). Also, irisin-treated rats had lower seizure numbers and shorter seizure duration than normal saline-treated rats (fig. 1B and C). Representative EEG and PSD analyses thereof are shown in FIGS. 1D-F. Epileptic seizure and EEG results indicate that exogenous irisin has significant inhibitory effects on KA-induced chronic epileptic seizures.
2.2 Protection of irisin against learning and memory in the induction of chronic epilepsy
The difference in learning and memory capacity between KA treatment and irisin treatment was evaluated by a water maze test. The latency, quadrant time percentage, and platform crossing time of each animal to reach the platform at 170 days were analyzed to explore the learning and memory capabilities of each rat. The results showed that the incubation period was significantly prolonged for KA treated rats (n=16) and physiological saline (n=5, p <0.001, fig. 1G). In addition, the time in the target quadrant of the KA treated rat was reduced (p=0.005, fig. 1H), the time in the para-quadrant was longer (P <0.001, fig. 1I), and the number of crossing stages was reduced (p=0.005, fig. 1J). Tectorigenin treatment partially reversed the KA-induced cognitive deficit. A representative trace is shown in fig. 1K. The results indicate that irisin may reverse the learning and memory impairment of seizures caused by KA.
2.3KA induces chronic epilepsy, iris reduces apoptosis and neuronal damage
The western blotting experiment analyzed changes in apoptosis-related proteins caspase-3 and ACTIVED ASPASE-3 at 3d and 170d after KA administration (n=5/group) and evaluated the effect of irisin on apoptosis. The results indicate that KA administration significantly increased the level of cortex and hippocampus ACTIVED CASPASE-3 (3D, cortex, P <0.001; hippocampus, P <0.001; FIGS. 2A and D;170D, cortex, P <0.001; hippocampus, P <0.001; FIGS. 2B and F), and decreased caspase-3 levels (FIGS. 2A-C and E). However, the KA+ tectoridin group ACTIVED CASPASE-3 levels were significantly reduced compared to the KA+ saline group. The result shows that the irisin has stronger anti-apoptosis effect on KA-induced apoptosis.
Neuronal degeneration was further analyzed with FJB staining (n=5/group). FJB positive signals were observed in 24h and 3d rats after KA administration. The positive signal for hippocampal FJB was significantly increased (CA 2, P <0.001, fig. 2H and M) and EC (P <0.001, fig. 2K and M) after KA administration for 3d compared to the control group (fig. 2g, j and M). Irisin significantly reduced the number of FJB positive signals in CA2 (P <0.001, fig. 2I and M) and EC (P <0.001, fig. 2L and M). Similar changes in FJB signal were observed at 24h (data not shown). FJB staining results indicate that KA treatment can significantly cause neuronal degeneration in CA2 and entorhinal cortex (entorhinal cortex, EC) and tectorigenin reverses this injury. The result shows that the exogenous tectorigenin treatment has obvious neuroprotective effect on the epilepsy caused by KA.
2.4 KA-induced chronic epilepsy, iris treatment increases BDNF and UCP2 expression in brain
BDNF and UCP2 expression was assessed at each group 3d and 15d (n=5/group). western blotting results showed that BDNF and UCP2 expression decreased at 3D (FIGS. 3A, C and D) and 15D (FIGS. 3B, E and F) in both the hippocampus and cortex of KA group. Immunohistochemical results also showed reduced immunoreactivity of BDNF following KA administration (EC, fig. 3H and J, P < 0.001).
Irisin treatment significantly increased BDNF and UCP2 expression (cortex and hippocampus, 3D, FIGS. 3A and D;15D, FIGS. 3B and F) and increased the immune response of BDNF observed in immunohistochemistry compared to the KA+ saline group. Taken together, the results indicate that exogenous irisin increases the expression of hippocampal and cortical BDNF and UCP2 in the early stages of KA-induced chronic epilepsy.
2.5 KA-induced chronic epilepsy, irisin reduces KA-induced elevation of DCF/Mito-SOX levels
The level of oxidative stress in the hippocampus and cortex was assessed 24h and 3d after KA administration by DCF/Mito-SOX assay (n=5/group). DCF levels (cortex (FIG. 4A) and hippocampus (FIG. 4B) and Mito-SOX levels (FIG. 4C, D) increased significantly in the KA+ iris group, in contrast, DCF and Mito-SOX levels (FIG. 4A, B, C, D) decreased significantly in the KA+ iris group compared to the KA+ saline group.
2.6KA induced chronic epilepsy, genipin reverses tectoridin-treated increased UCP2 levels
The expression of UCP2 was measured for each group (n=5/group) at 3d and 15d after KA administration using the western blotting method. The results showed that UCP2 expression was significantly higher in tectoridin-treated rats (KA+tectoridin+saline) than in KA+saline (P <0.001, FIGS. 5A-D). In contrast, genipin administration (ka+irisin+genipin) reduced the expression of UCP2 in the hippocampus and cortex at 3D (P <0.001, fig. 5A and C) and 15D (P <0.001, cortex; p=0.001, hippocampus, fig. 5B and D) compared to the ka+irisin+saline group.
2.7KA induces chronic epilepsy, genipin reverses the anti-epileptic effect of irisin
Spontaneous seizure behavior and electroencephalogram were recorded between 30d and 170d in the ka+irisin+genipin group (n=10), the cumulative seizure duration at stages 1-5 was significantly longer than in the ka+irisin+saline group (n=8) at all observation time points (P <0.001, fig. 6A). The number and duration of epileptic seizures treated with genipin were significantly higher than those of the ka+irisin+saline group (fig. 6B and C). Representative EEG and PSD analysis thereof are shown in FIGS. 6D-F. The results show that genipin can reverse the antiepileptic effect of exogenous irisin.
2.8KA induced chronic epilepsy, genipin reverses the repair of irisin to learning and memory
The difference in learning and memory ability was evaluated by a water maze test. The results showed that genipin treated rats (n=10) and ka+irisin+saline rats (n=8, p <0.001, fig. 6G). In addition, the target quadrant (p=0.002, fig. 6H) and target area frequency (p=0.012, fig. 6J) times decrease, and the bit quadrant time percentage increases (P <0.001, fig. 6I). The results show that genipin can partially reverse the protective effect of irisin on cognitive deficits caused by KA. Representative trajectories for each group of rats are shown in fig. 6K.
2.9KA induces chronic epilepsy, genipin reverses the neuroprotective effects of irisin
Changes in apoptosis-related proteins caspase-3 and ACTIVED CASPASE-3 were observed by western blotting 3d after KA treatment to assess the effect of genipin on the anti-apoptotic effect of irisin. Tectorigenin significantly reduced cortical and hippocampal ACTIVED CASPASE-3 levels (fig. 7A and C). However, expression of ACTIVED CASPASE-3 in rats treated with genipin (ka+irisin+genipin group, n=10) was significantly increased compared to ka+irisin+normal saline rats (fig. 7A and C). The results indicate that genipin can reverse the anti-apoptotic effect of irisin in KA-induced apoptosis.
FJB analysis of neuronal degeneration, the FJB positive signal was significantly increased in both the ka+irisin+genipin group and the ka+irisin+saline group (CA 2, fig. 7E and J; EC, fig. 7H and J) hippocampus (CA 2, P <0.001, fig. 7F and J) and EC (P <0.001, fig. 7I and J). Similar changes were also observed at 24h (data not shown). The results indicate that irisin can reduce KA-induced neuronal damage, while the intervention drug genipin reverses the neuroprotective effects of irisin.
2.10KA induced chronic epilepsy genipin reverses the role of irisin in reducing DCF and mito-SOX levels
Hippocampal and cortical oxidative stress levels (n=5 per group) were examined 24h and 3d after KA administration with DCF/Mito-SOX. The results indicate that genipin can raise DCF levels in the cortex (24 h, P <0.001, FIG. 8A;3d, P <0.001, FIG. 8A) and hippocampus (24 h, P <0.001, FIG. 8B;3d, P <0.001, FIG. 8B). In addition, mito-SOX levels were increased in cortex (24 h, FIG. 8C;3D, FIG. 8C) and Hippocampus (24 h, FIG. 8D;3D, FIG. 8D) compared to rats treated with physiological saline (KA+irisin+physiological saline group). Representative Mito-SOX flow cytometer detection results are shown in FIG. 8E. The results indicate that irisin reduces the oxidative stress levels in epileptic rats, while UCP2 inhibitors reverse the antioxidant stress effects of irisin.
2.11 Treatment with irisin increases brain BDNF and UCP2 expression in the induction of acute epilepsy
BDNF and UCP2 expression was assessed at 24h and 3d (n=5/group) per group. western blotting results showed that BDNF and UCP2 expression decreased at 24h (FIGS. 9A, C and D) and 3D (FIGS. 9B, E and F) in both the hippocampus and cortex of KA group. Immunohistochemical results also showed reduced immunoreactivity of BDNF following KA administration (EC, fig. 9H and K, M, P < 0.001).
Irisin treatment significantly increased BDNF and UCP2 expression (cortex and hippocampus, 24h, FIGS. 9A, C and D;3D, FIGS. 9B, E and F) and increased the immune response to BDNF observed in immunohistochemistry compared to the KA+ saline group. Taken together, the results indicate that exogenous irisin increases the expression of hippocampal and cortical BDNF and UCP2 in KA-induced acute seizures.
2.12KA induces acute epilepsy, iris reduces apoptosis and neuronal injury
The results of western blotting experiments showed that KA administration significantly increased the levels of cortex and hippocampus ACTIVED CASPASE-3 (24 h, cortex, P <0.001; hippocampus, P <0.001; FIGS. 10A and D;3D, cortex, P <0.001; hippocampus, P <0.001; FIGS. 10A and C), caspase-3 (FIGS. 10A and B) and BCl-2 (FIGS. 10A, E) and decreased. However, the KA+ irisin group ACTIVED CASPASE-3 levels were significantly reduced compared to the KA+ saline group (FIGS. 10A, C). The result shows that the irisin has stronger antagonism on KA-induced apoptosis.
Neuronal degeneration was further analyzed with FJB staining (n=5/group). The hippocampal FJB positive signal was significantly increased (CA 2, P <0.001, fig. 10G and L) and EC (P <0.001, fig. 10J and L) after KA administration for 3d compared to the control group (fig. 10f, i). Irisin significantly reduced the number of FJB positive signals in CA2 (P <0.01, figures 10H and L) and EC (P <0.001, figures 10K and L). Similar changes in FJB signal were observed at 24h (data not shown). FJB staining results indicate that KA treatment can significantly cause neuronal degeneration in CA2 and entorhinal cortex (entorhinal cortex, EC) and tectorigenin reverses this injury. The result shows that the exogenous tectorigenin treatment has obvious neuroprotective effect on the epilepsy caused by KA.
In contrast, in the KA+ tectoridin group, DCF and Mito-SOX levels (FIG. 11A, B, C, D) were significantly reduced compared to the KA+ saline group, as shown in FIGS. 11E-H, results of flow cytometry detection of each group of Mito-SOX showed that exogenous tectoridin treatment significantly reversed the KA-induced elevation of oxidative stress levels.
2.14 Increasing UCP2 levels in KA-induced acute epilepsy by treatment with genipin reversing tectoridin
The expression of UCP2 was measured for each group (n=5/group) by western blotting 24h and 3d after KA administration. The results showed that UCP2 expression was significantly higher in tectoridin-treated rats (KA+tectoridin+saline) than in KA+saline (FIGS. 12A-F). In contrast, genipin administration (KA+irisin+genipin) reduced the expression of hippocampal and cortical UCP2 compared to the KA+irisin+saline group (FIGS. 12A-F).
2.15KA induces acute epilepsy, genipin reverses the neuroprotective effects of irisin
Changes in apoptosis-related proteins Bax, bcl-2, caspase-3 and ACTIVED CASPASE-3 were observed by western blotting 3d after KA treatment to assess the effect of genipin on the anti-apoptotic effects of irisin. Tectorigenin significantly reduced the cortical and hippocampal Bax, ACTIVED CASPASE-3 levels (FIGS. 13A, C and D), while caspase-3 and Bcl-2 levels were significantly elevated (FIGS. 13A, B, E). However, expression of apoptosis-related proteins was significantly reversed in rats treated with genipin (ka+irisin+genipin group, n=10) compared to ka+irisin+normal saline rats (fig. 13A-E). The results indicate that genipin can reverse the neuroprotective effects of irisin.
Similar changes were found in FJB, with treatment with irisin significantly reduced neuronal damage, while genipin intervention reversed the neuroprotective effects of irisin (fig. 13F-L).
2.16KA induces acute epilepsy, genipin reverses the effect of irisin in reducing DCF and mito-SOX levels
Oxidative stress experimental results indicate that genipin treatment can raise DCF levels in the cortex (24 h, p <0.001;3d, p <0.001; fig. 14A) and hippocampus (24 h, p <0.001;3d, p <0.001; fig. 14B). In addition, mito-SOX levels were also significantly increased in the cortex and hippocampus compared to rats treated with physiological saline (KA+irisin+physiological saline group) (FIGS. 14A, B). The Mito-SOX flow cytometer detection results are shown in FIGS. 14E-H. The results indicate that irisin reduces the oxidative stress levels in epileptic rats, while UCP2 inhibitors reverse the antioxidant stress effects of irisin.
Taken together, the results of the present invention demonstrate the antiepileptic and neuroprotective effects of early tectorigenin treatment in KA-induced epileptic models. Research shows that exogenous irisin increases the expression of BDNF and UCP2 in brain, reduces oxidative stress and neurodegeneration, reduces cognitive dysfunction, and inhibits spontaneous epileptic seizure induced by KA. In addition, the UCP 2-specific inhibitor genipin partially reverses the neuronal protection and antiepileptic effects of irisin. This further suggests that activation of the BDNF/UCP2 pathway may be a potential mechanism for irisin protection.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (1)
1. The use of irisin as the sole active ingredient in the manufacture of a medicament for the treatment of chronic idiopathic epilepsy represented by the following uses:
increasing the level of expression of brain-derived neurotrophic factors and uncoupling protein-2 in hippocampal and cortical areas;
Reducing apoptosis caused by chronic spontaneous epilepsy;
Reducing neuronal damage caused by chronic spontaneous epilepsy;
reducing the number of and duration of chronic spontaneous epileptic seizures;
reversing elevated levels of oxidative stress.
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