KR20110006545A - Method for screening of agent for treating and diagnosing prenatal neuronal disease in maternal epilepsy - Google Patents
Method for screening of agent for treating and diagnosing prenatal neuronal disease in maternal epilepsy Download PDFInfo
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- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
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Abstract
Description
The present invention relates to a method for providing information on neurological dysfunction diseases caused by epilepsy of a pregnant mother and a screening method of a therapeutic agent for treating the disease, specifically in fetal hippocampal neurons of individuals suspected of epilepsy and normal individuals After confirming the expression pattern of the GABA B1 receptor, PKA, cytochrome-c or caspase-3, the candidate protein is administered to a method of providing information on neurological disorders of the fetus and a model of maternal epilepsy, and then the protein The present invention relates to a method for screening a therapeutic agent related to neurological dysfunction by confirming whether or not the change in expression.
Epilepsy is repetitive despite the absence of physical factors that can cause seizures, such as electrolyte imbalances, acid-base abnormalities, uremia, alcohol withdrawal, and severe sleep deprivation. Group of chronic diseases that cause seizures (at least two times apart, at least 24 hours apart), or pathological changes that may cause epilepsy in brain imaging (brain MRI, etc.) even if the seizure occurs only once. Means disease. The incidence and prevalence of epilepsy is highest within one year, then sharply lowered, while maintaining a low incidence during adolescence and adulthood, and then rapidly increasing in older people over 60 years old.
Epilepsy with this tendency is composed of various syndromes whose causes and clinical characteristics are difficult to accurately determine the course of the disease and the outcome of treatment. What is known so far is that epilepsy in a large number of patients gradually decreases over time, and in about 70%, antiepileptic drugs can relieve symptoms over time. In general, 30 to 40% of patients with epilepsy can be easily controlled by a small amount of monotherapy, and if there is no seizure for a long time, it is known that a remedy without recurrence occurs even if the drug treatment is stopped. However, about 30% of the symptoms are controlled by monotherapy but relapse when the drug is discontinued, and about 20% of the symptoms can be controlled by active drug treatment, but seizures are known to recur during medication. About 20% of patients develop intractable epilepsy, which does not respond well to medication and continues to have seizures.
In particular, epilepsy causes neuronal damage, and it is known that these effects can continue if seizures are not controlled. In particular, epilepsy often causes neuronal death, axonal sprouting, and dendritic change. , Receptor changes, cellular molecular changes, etc. The most well known symptom is the death of neurons. As a method for suppressing seizures causing neuronal cell death, studies have been made on modulators to control the inhibition of GABA, a type of neurotransmitter. Nevertheless, there are still few effective palliative or therapeutic agents for epilepsy, and there is no known effect on the fetus due to the epilepsy of the mother during pregnancy and the treatment for the fetus.
On the other hand, a neurotransmitter is a series of substances released from nerve cells in the body, including the brain, and transmits information to adjacent nerve cells. The aminotransmitters are mainly amino acids (acetylcholine, glycine, aspartic acid, glutamate). ), Amines (dopamine, adrenaline (epinephrine), (noradrenaline), peptides (vasopressin), and fatty acids (histamine, serotonin), among which GABA (gamma-aminobutyric acid) is inhibitory ( ingibiytory) A neurotransmitter that produces post-synaptic potentials, and is present in neurons that act as inhibitors, forming synapses with other nerve cells or proximal axons. In particular, GABA-A opens Cl - channels and negatively charged chlorine enters the cell. The change in the embryo leads to the inhibition of intracellular excitability, which leads to a variety of neurological disorders due to abnormalities in GABA or GABA receptors, or inhibitory mechanisms in the absence of these molecules. It is known that abnormalities in the GABA family cause diseases such as depression and epilepsy.
The other GABA receptors are largely divided into GABA-A and GABA-B receptors. GABA-B receptors are composed of GABA B1 and It can be classified as GABA B2 . Among them, GABA B1 receptor is a structure linked to G-protein, which activates K + channel, which is an ion channel of cell membrane, and is known to have non-indirect inhibition. However, GABA B1 receptor role in epilepsy, especially its expression in the fetus and There is no known distribution. In particular, it is not known that diagnosis of epilepsy may be possible from analysis by the hippocampal neuron region of the fetus. In addition, it has not been reported that the diagnosis and self-screening of the disease may be possible by analyzing the expression of GABA B1 receptor and expression of cytochrome-c, caspase-3 and PKA together.
Accordingly, the present inventors confirmed the expression change of GABA, especially GABA B1 receptor, PKA, cytochrome-c or caspase-3, in the epileptic model induced by PTZ or ethanol, and the change in the expression sensitization as the candidate material was treated. By confirming that the screening for the treatment of neurological disorders, including epilepsy, can be screened, and the expression of the protein as described above by confirming the presence or absence of neurological disorders of the fetus can be seen and confirmed The invention was completed.
It is an object of the present invention to administer a candidate substance to an epileptic model induced by administration of pentylenetetrazol (PTZ) or ethanol to a subject, while the GABA B1 receptor, PKA, cytochrome-c or caspase-3 in hippocampal neurons of the subject. By identifying the expression pattern, it provides a method for screening a therapeutic agent for neurological disorders.
Another object of the present invention is to provide a method for providing information on neurodegenerative disorders of fetuses by identifying the GABA B1 receptor, PKA, cytochrome-c or caspase-3 expression patterns in hippocampal neurons of suspected epileptic subjects and normal fetuses. To provide.
The present invention relates to a method for screening the fetal nervous system disorder therapeutics due to epileptic seizures during pregnancy, particularly, (a) GABA B1 receptors in hippocampal neurons of the object (GABA B1 receptor) and PKA In some embodiments confirming the expression of (protein kinase A); (b) administering pentylene tetrazol (PTZ) or ethanol to the subject; (c) selecting an individual whose expression level of GABA B1 receptor and PKA in said individual is reduced compared to step (a); (d) administering the candidate agent to the selected subject; And (e) relates to the screening method of the therapeutic agent for neurological dysfunction comprising the step of confirming the increase in the expression level of GABA B1 receptor and PKA.
In still another aspect, the present invention provides a method of preparing a cytochrome cell comprising (a) confirming the expression of cytochrome-c and caspase-3 in hippocampal neurons of an individual; (b) administering PTZ or ethanol to the subject; (c) selecting the individual whose expression level of cytochrome-c and caspase-3 in said individual is increased compared to step (a); (d) administering the candidate agent to the selected subject; And (e) relates to the screening method of the therapeutic agent neurological disorders comprising the step of identifying the decrease in the expression level of cytochrome-c and caspase-3.
The present invention is directed to screening therapeutic agents by confirming the expression of GABA B1 receptor, PKA, cytochrome-c or caspase-3 in hippocampal neurons of an individual, and confirming the change in the expression of the proteins before and after treatment with the candidate. Can be. Preferably the present invention can screen for therapeutic agents by confirming the expression of any one or more proteins selected from the group consisting of GABA B1 receptor, PKA, cytochrome-c and caspase-3, wherein any two or more of the four types of proteins By identifying three, more, or all of the expression patterns. In addition, these expression patterns can be performed sequentially or simultaneously, and can be identified at the mRNA level rather than the protein level as needed.
In the present invention, GABA, a neurotransmitter responsible for the inhibitory function, means a molecule that binds to the GABA receptor present in the membrane protein of synapse neurons. GABA receptors that bind to these GABA is largely composed of GABA-A and GABA-B and GABA-B can be divided into GABA B1 and GABA B2 . The term "GABA B1 receptor" in the present invention, mediates a slow response through the G-protein, which mainly suppresses the excretion of glutamic acid as a Glutamatergic presynaptic receptor (glutamic acid) to suppress the excitatory function (excitatory function) Suppress
The term "PKA (protein kinase A)" in the present invention is a protein that plays a role in cell growth regulation. When a hormone or neurotransmitter binds to a receptor present in the cell membrane for survival and growth of cells, a signal is generated. This signal is transmitted by a secondary messenger that is responsible for delivering it inside the cell. In particular, the signal by cAMP is known to be transferred to a phosphatase called PKA to cause the action by phosphorylating other proteins. Thus, PKA is a major mediator of synaptic transmission that is involved in the process of epileptogenesis.
On the other hand, the term "cytochrome-c" in the present invention refers to the caspase as the release of cytochrome-c into the cytoplasm due to increased calcium concentration in the mitochondria after epileptic seizures in the early cell death phase. Acts as an important key to activate -3.
In addition, the term "caspases-3" in the present invention is activated during neuronal cell death in epileptic seizures due to nerve cell damage, and activated caspase-3 is widely used in cells. It breaks down proteins, attacks the cytoskeleton, and inactivates DNA repair enzymes in the nucleus to break down DNA. Eventually, caspase-3 is activated, causing intracellular proteolysis and DNA damage in the nucleus, which breaks down into several apoptotic bodies consisting of some small nucleus fragments and membrane parts of the cell.
The present invention can screen novel therapeutic agents for neurological dysfunction disorders, including epilepsy, in hippocampal neurons of the subject through confirmation of increased or decreased expression of GABA B1 receptor, PKA, cytochrome-c or caspase-3.
The relationship between GABA receptors and epilepsy has been known, but how maternal epileptic seizures affect pregnancy's hippocampal neurons and, in addition, maternal epilepsy causes changes in the expression of related proteins in fetal hippocampal neurons. It is not known whether or not epilepsy induces epilepsy, but changes in GABA B1 receptor, PKA, cytochrome-c, or caspase-3 expression patterns in fetal hippocampal neurons of pregnant mothers, especially those who caused epileptic seizures by PTZ. The present invention is the first study on whether the fetus epilepsy according to.
In addition, the term "PTZ (pentylenetetrazol)" in the present invention is known as a substance causing convulsant cognitive deficits in rodents (Corda and Biggio, 1986; Becker et al., 1994), GABA antagonists ( Experimental results have shown that GABA antagonist is effective in improving learning ability and memory by administering pentylenetetrazol (PTZ, Metrazol), which is a central nerve stimulant, to animals. In addition, the drug is also used as a circulatory system and respiratory stimulator, and it has been used as a shock therapy because it causes cramps when excessively ingested. However, it is also a drug that was rejected by the FDA in 1982 because of the risk of causing seizures when prescribed as a drug for treating mental disorders.
The term "ethanol" in the present invention is a kind of alcohol in which one hydrogen atom is substituted with a hydroxy group (-OH) in two-carbon ethanol, also referred to as ethyl alcohol. In particular, in relation to the nervous system, ingestion of ethanol becomes an excited state due to the suppression of cerebral function, and then the effect of central nervous suppression appears. These ethanols are one of the main causes of preventable cause of birth defects, mental retardation and neurorodevelopmental disorders, and ethanol intake in pregnant women is long-term cognitive. , Neurorobehavioral disruption, growth deficits, and epilepsy (Berman and Hannigan, 2000; Mattson, 2001, Kelly et al., 2000, Sampson et al., 2000; Autti-Ramoet al , 2000), causing damage to the development of the central nervous system (Zhou et al. 2000; McGoey et al., 2003; Guerri, 2002).
In the present invention, in order to determine how maternal epilepsy affects the fetus, the epileptic seizures were induced by administering the PTZ or ethanol to pregnant rats. Injections were first identified to affect GABA B1 receptor, PKA, cytochrome-c or caspase-3 expression in fetal hippocampal neurons. As a result, it was confirmed that the expression of GABA B1 receptor in mRNA and protein levels in hippocampal neurons of fetuses in the PTZ-induced epileptic seizure model was significantly reduced in the PTZ-induced seizure model compared to the control group (FIG. 5). In addition, in order to elucidate the regulatory effects of maternal epilepsy on the expression of PKA protein, PKA levels were measured by RT-PCR and Western blot in epileptic seizure models, indicating that the expression of the protein was reduced in the PTZ-induced seizure model. (FIG. 6C). In addition, it was confirmed that the expression of cytochrome-c or caspase-3 was increased in the PTZ-induced seizure model in the expression of cytochrome-c or caspase-3 (FIGS. 2C, 3A and 3B).
By measuring the change in the expression of the protein as described above, it is possible to confirm whether or not the invention of the neurological dysfunction disorder, including epilepsy in the mother's fetus during pregnancy, and through the change in the expression of the protein to treat a novel neurological dysfunction disease Can be excavated.
In addition, preferably, the present invention induced epileptic seizures by administering PTZ or ethanol to pregnant rats, and in particular, cytochrome-in the fetal hippocampal neurons by intraperitoneal injection of PTZ, widely used as an epileptic inducer. It was first identified how it affects the expression of c or caspase-3. As a result, cytochrome-c secretion into the cytoplasm from the increased expression of cytochrome-c in the PTZ-induced seizure model compared to the group not treated with PTZ in cultured hippocampal neurons (nerve). apoptosome) formation (Fig. 2c). In addition, the present inventors examined the expression change of caspase-3 in neurons of the hippocampus during the early developmental stage by Western blot analysis whether it is due to the caspase-3 activity in the PTZ-induced seizure model, compared to the control group. It was confirmed that the expression of cleaved caspase-3 was increased by the caspase-3 activity in neurons of the prenatal rat hippocampus (FIGS. 3A and 3B).
On the other hand, the subject used in the present invention means a whole mammal, including dogs, cattle, horses, rabbits, mice, rats, chickens or humans, but the mammal of the present invention is not limited by the above examples. Preferably, rats were used to determine whether neuronal dysfunction was associated with PTZ or ethanol exposure in pregnant rats.
In the present invention, the administration of the PTZ or ethanol in the method, oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, transdermal, etc. can be used a variety of methods, the dosage is the age, sex of the individual In the present invention, PTZ is intraperitoneally injected at a dose of 40 mg / kg (Example 1).
In addition, preferably the expression level of the GABA B1 receptor and PKA, or cytochrome-c and caspase-3 of the present invention can be used without limitation the conventional expression confirmation method used in the art. In addition, confirmation of the expression level can be determined from the mRNA level or the protein level encoded by them according to the selection or need of those skilled in the art, for example, in the case of mRNA can be confirmed through a primer or probe sequence complementary to the sequence, the protein In this case, it can be confirmed by using an antibody which binds to a protein or a fragment thereof. Preferably, the present invention may use RT-PCR to confirm the expression pattern of GABA B1 receptor and PKA at the mRNA level. In addition, Western blot can be used to confirm the expression pattern at the protein level of GABA B1 receptor and PKA, or cytochrome-c and caspase-3.
In the present invention, the term 'mRNA expression level confirmation' is a process of identifying the expression level of GABA B1 receptor and PKA, or cytochrome-c and caspase-3 and mRNA in a subject to diagnose a neurological disorder-related disease. Measure the amount of. Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting, DNA chips, etc., but are not limited to these. In the present invention, the term 'protein expression level confirmation' refers to the presence of a protein expressed from GABA B1 receptor and PKA or cytochrome-c and caspase-3 mRNA in an individual for diagnosing a neurological disorder-related disease. In the process of confirming whether the expression and the degree of expression, preferably, the amount of the protein can be confirmed using an antibody that specifically binds to the protein of the gene. In addition, the expression of the protein can be confirmed by immunoassay using specific antibodies against GABA B1 receptor and PKA, or cytochrome-c and caspase-3 proteins, respectively, and analytical methods therefor. Western blots, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, oukterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining , Immunoprecipitation assays, complement fixation assays, FACS and protein chips, but are not limited to these. In addition, when the antibody is used as a method for determining the amount of the protein, a second antibody linked to a specific and detectable label may be added to the target protein, and detection may also be performed after the addition of the second antibody. A third antibody having a binding affinity and linked to a detectable label can also be added. As a label that can be detected by the second or third antibody, an appropriate chromogenic substrate and an enzyme that displays color in culture can be used. The detectable moiety may comprise a composition detectable by spectroscopic, enzymatic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means, for example fluorescent markers and dyes, magnetic labels , Linked enzymes, mass spectroscopy tags, spin labels, electron transfer donors and acceptors, etc., but are not limited to these examples.
As used herein, the term 'neuro dysfunction disorder' refers to any disease that occurs in association with the GABA B1 receptor and PKA or cytochrome-c and caspase-3 in an individual, and epilepsy as an example of a neurological disorder. Schizophrenia, panic disorder, depression, mental retardation, Huntington's disease, spasticity, drug addiction or neurodegenerative disease degenaration), but the neurological dysfunction diseases of the present invention are not limited by the above examples.
On the other hand, the individuals with reduced expression of GABA B1 receptor or PKA or those with increased expression of cytochrome-c or caspase-3 are selected, and candidates are administered to them to confirm the increase or decrease in their expression levels. The substance may be determined as a therapeutic agent for a neurological disorder-related disease, thereby screening for a therapeutic agent for the disease.
As used herein, the term "candidate substance" means any substance that is thought to be capable of preventing or treating neurological dysfunction-related diseases such as epileptic seizures in rats during pregnancy, particularly in hippocampal neurons of the fetus, and GABA in the brain. Refers to a subject measuring the ability to treat the disease by directly or indirectly altering the B1 receptor and PKA, or changes in cytochrome-c and caspase-3 expression levels. Such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and a wide variety of compounds. Such candidates also include both natural and synthetic materials.
On the other hand, after selecting the individuals in which the expression of GABA B1 receptor and PKA is reduced in the administered subject compared to the subject not administered PTZ or ethanol by the above method, the expression of GABA B1 receptor and PKA in the subject is increased or By administering candidates that are expected to be reduced and then checking for changes in their expression patterns, it is determined whether the treated candidates restore the expression of GABA B1 receptors and PKA to the state prior to exposure to PTZ or ethanol. Can be. Specifically, when increasing the expression of the GABA B1 receptor and PKA from the selected individuals, the substance can be determined as a treatment for a reproductive dysfunction-related disease, on the contrary, candidate substances that reduce the expression of the GABA B1 receptor and PKA May be regarded as an inhibitor for the treatment of neurological dysfunction-related diseases. In addition, in the same manner as described above, compared to the subjects who did not receive PTZ or ethanol, the subjects with increased cytochrome-c and caspase-3 were selected, and then the subjects were treated with cytochrome-c and caspase-. By administering candidates expected to increase or decrease 3, and confirming changes in their expression patterns, the treated candidates before exposure of the cytochrome-c and caspase-3 expression to PTZ or ethanol You can check whether you are recovering to a state. Specifically, when the expression of cytochrome-c and caspase-3 is reduced from the selected individuals, the substance may be determined as a treatment for diseases related to reproductive dysfunction, and conversely, cytochrome-c and caspase- Candidates that increase the expression of 3 can be judged as inhibitors for the treatment of neurological dysfunction related diseases.
As such, the method for measuring GABA B1 receptor and PKA, or cytochrome-c and caspase-3 expression increase and decrease in the present invention can be used without limitation the methods used in the art as described above, but preferably the present invention RT-PCR and Western blot were used and the candidates were treated by neuronal dysfunction by confirming the increase in GABA B1 receptor and PKA, or cytochrome-c and caspase-3 expression levels after administration of the candidates to the subject. Can be screened as a therapeutic.
In another aspect, the present invention provides a method for diagnosing neuropathy disorders of a fetus by measuring changes in GABA B1 receptor and PKA expression in fetuses of suspected epilepsy and normal subjects. Specifically, (a) comparing GABA B1 receptor and PKA expression in hippocampal neurons of suspected epileptic and normal fetuses; (b) selecting the subjects whose GABA B1 receptor or PKA expression in suspected epileptic subjects is reduced than in normal subjects; And (c) identifying a fetus selected in step (b) as an individual having a neuropathy disorder.
In addition, the present invention comprises the steps of (a) comparing cytochrome-c and caspase-3 expression in hippocampal neurons of suspected epileptic subjects and normal subject fetuses; (b) selecting individuals whose cytochrome-c and caspase-3 expressions in suspected epileptics have increased than those in normal individuals; And (c) identifying a fetus selected in step (b) as an individual having a neuropathy disorder.
In order to achieve the above object, the present invention can identify the expression pattern of any one or more proteins selected from the group consisting of GABA B1 receptor, PKA, cytochrome-c and caspase-3 as mentioned above, in this case also Expression of any one or more or all of the proteins can be confirmed, simultaneous or sequential identification is possible, as well as identification at the mRNA level as well as protein. This can be confirmed by comparing the suspected epilepsy with the normal subject (control), the expression of GABA B1 receptor and PKA decreases, and in the case of cytochrome-c and caspase-3 neurons An individual with a dysfunctional disorder can be determined.
The present invention, after confirming the GABA B1 receptor, PKA, cytochrome-c or caspase-3 expression increase and decrease in hippocampal neurons of suspected epilepsy and normal fetus, the expression of GABA B1 receptor or PKA is increased or cytochrome- By selecting the c or caspase-3 reduced expression of the individual may provide information that can determine that the individual has a neurological disorder of the fetus.
Preferably, the expression pattern identification method of the present invention can be used without limitation the method of confirming the GABA B1 receptor, PKA, cytochrome-c or caspase-3 expression pattern used in the screening method for the treatment of neurological disorders of the fetus. Preferably, in the case of GABA B1 receptor and PKA, by confirming the decrease in the expression of mRNA and protein of the protein, it can be determined that the subject has a neurological dysfunction disorder of the fetus. In addition, in the case of cytochrome-c and caspase-3, it can be seen that the subject is an individual with neurological dysfunction by confirming the increased expression of the protein.
The present invention can determine whether the maternal epilepsy of the fetus is neuronal dysfunction by confirming the increase or decrease of expression of GABA B1 receptor, PKA, cytochrome-c or caspase-3 in the hippocampal neurons of the fetus. In addition, after administering a candidate substance to the epilepsy induction model, it is useful to develop a novel therapeutic agent related to neurological disorders of the fetus by checking whether the protein expression is increased or decreased before and after administration.
Hereinafter, preferred examples are provided to help understanding of the present invention. The following examples are provided only to more easily understand the present invention, but the contents of the present invention are not limited by the examples.
Example 1 Animal Treatment
Female (n = 48) Sprague-Dawley rats (250 g, Gyeongsang National University, Neurobiology Laboratory, Jinju, South Korea) were fed to light at 06: 00-20: 00 h Breeding in a controlled environment. Gestational days (GD) After 17.5 days, intravenous injection of pentobarbital sodium (3 mg / 100 g bw) followed by pregnancy Sprague-Dawley rats were killed and killed.
The experimental animals were randomly divided into two groups as follows:
(1) Group treated with PTZ: Female rats intraperitoneally injected with PTZ (40mg / kg) for 2-16 days after fertilization
(2) Control group: intraperitoneal injection of 0.9% saline
Example 2. Seizure Confirmation Process and EEG Recording
For at least 17 days, experimental animals received intraperitoneally (i.p) in saline at a semiconvulsive dose for 24 hours in saline and the control group received only saline. After administration of each reagent, seizure behavior was observed for 30 minutes, and the resulting seizures were scored as follows:
Administration of 10 (mg / kg) PTZ to experimental animals was thought to stimulate the seizure of stage 4 or
Seizure behavior and electroencephalograms (EEGs) during seizures revealed a minimum seizure equivalent to score 3-4 after 15 days of chronic administration at a subconvulsive dose of 40 mg / kg during pregnancy. It became. In addition, barrel rotation and tonic seizures have been identified in some animals: from 65% of animals administered 10 th (10th) to 85% administered 15 th (15th). Seizures induced by PTZ in pregnant rats typically begin with hind limb kicks, followed by stiffness and intermittent spasms of the extremities.
The exact observations after PTZ administration are summarized in Table 1.
To confirm the seizure, electroencephalography (EEG) started at 5 for 30 minutes and was confirmed at 15 minutes after PTZ administration. Five rats of the control group and 15 rats treated with PTZ were observed. EEG evaluation results are shown in FIG. 1.
Example 3. Initial Cell Culture and Reagent Treatment
Pregnant rats were ip injected with PTZ (40 mg / kg) daily for 2-16 days of gestational days. Cultures were prepared from hippocampal neurons of fetal rats on day 1 G of gestation from pregnant rats. Hippocampal tissues were treated with 0.25% trypsin EDTA for 20 minutes and dissociated by mechanical grinding in cold calcium- and magnesium-free Hank's balanced salt solution (pH 7.4). After pelleting by centrifugation, cells were seeded on cell culture plates and chamber slides pre-coated with poly-lysine (0.02 g / l) (1 × 10 6 cells / ml). Medium was 10% heat-inactivated fetal bovine serum, 1 mM pyruvate, 4.2 mM sodium bicarbonate, 20 mM HEPES, 0.3 g / l bovine serum albumin, 50 U / Consisted of ml Penicillin, and DMEM (Dulbecco's modified Eagle medium) with 50 mg / l streptomycin. The culture was maintained at 37 ° C. in 5% CO 2 and 95% humid air. Neuroglial cells were inhibited with medium containing 100 μM Ara-C (Cytosine β-D-ArabinoFuranoside (Sigma)) for 12 hours. After 3 days, hippocampal neuron cells were treated with medium containing 100 mM ethanol, PTZ 10 mM, 50 μM baclofen and 100 μM paclofen combined into different groups. All groups treated with reagents were incubated for 20 minutes in vitro.
Example 4. Reverse transcriptase-polymerase chain reaction (RT-PCR)
RT-PCR analysis was performed with cDNA from the reagent treated group. Total RNA was isolated by Trizol Reagent (Life Technologies, Rockville, MD). The first strand cDNA was transcribed from 2 μg of RNA using oligo (dt) 15, M-mlV reverse transcriptase (Promega), according to the method provided by the manufacturer. Total 4 μl cDNA was used for PCR amplification in the presence of 1 μl Taq DNA polymerase. Thermal cycling was performed under the following conditions: 5 minutes at 94 ° C., 1 minute at 94 ° C., 1 minute at 68 ° C., 1 cycle at 72 ° C., 1 minute at 72 ° C., and then at 72 ° C. for final extension. 5 minutes.
GAPDH (58 ° C., 25 cycles) was used as a negative control. PCR products were run on a 1% agarose gel containing ethidium bromide (EtBr) and visualized under UV light.
Primers used were as follows.
Early cultured cells, baclofen, and paclofen treated cells cultured in controls to elucidate the effects on fetal hippocampal neuronal cells with respect to changes in GABA B1 receptor and PTZ-induced expression in vivo And GABA B1 receptor mRNA levels were examined by RT-PCR. As a result, GABA B1 receptor expression was significantly decreased in the mRNA level of fetal hippocampal neurons of the PTZ-induced seizure model compared to the control (Figs. 5A and 5B).
In contrast, PKA expression was not significantly reduced in ethanol, KA, ethanol and PTZ, baclofen and paclofen treatment in hippocampal neurons (FIG. 6A).
Example 5. Western blotting
Initially cultured hippocampal cells were homogenized with a cell lysis buffer (Cell signaling # 9803) containing 100 mM PMSF, a protease inhibitor. Samples were left on ice for 20 minutes prior to sonication for 4 minutes (operate 15 sec, pause 10sec). After ultracentrifugation (12,000 rpm, 10 min x 2), the protein including the supernatant was separated. Protein content was measured spectrophotometrically at 295 nm using a Bio-Rad Protein Assay, and 30 μl protein was applied to each line. Soluble fraction (30 μg) was duplicated in 12% SDS-polyacrylamide gels (30% Acrylamide, 1% Bis, 1 M Tris, 10% SDS, 10% APS, TEMED) Separated. One gel was stained with Coassie Blue, and the protein in the other gel was nitrocellulose membrane (90 V for 1 h in a 48 mM Tris, 39 mM glycine, 20% MeOH and 0.037%). SDS transfer buffer). Nitrocellulose as a blocking solution (Tris-buffered saline (TBS)) containing 0.1% (v / v) Tween 20 and 6% (w / v) non-fat dry milk to reduce nonspecific binding The membrane was treated. Immune responses to rabbit polyclonal IgG GABA B1 receptor and PKA-α antibodies (1: 1000, 24 h, 4 ° C., Santa Cruz, Cell signaling) or rabbit-derived anti-rat GABA B1 receptor antibodies (1: 1000, Abcam Limited, UK). After washing, goat anti-mouse attached horseradish peroxidase (HRP) and goat anti-rabbit IgG-HRP (1: 10000, Bio-Rad) were added at room temperature. Incubate for minutes. Protein was measured by chemiluminescence using ECL-detecting reagent (Amersham Pharmacia Biotech, Western blotting detection reagents) according to the manufacturer's test method. Western blots were analyzed by densitometry using a computer-based Sigma Gel (SPSS Inc. Chicago, USA) system.
To investigate the effects of PTZ-induced seizures of cytochrome-c during pregnancy, ethanol, PTZ, kainic acid (KA 1 μM) and baclofen (50 μM) were applied to hippocampal neurons in fetal rats. And paclofen (100 μM) were treated in different combinations for 20 minutes to secrete cytochrome-c by Western blot analysis. As a result, increased expression of cytochrome-c was observed in PTZ-induced seizures, indicating that cytochrome-c secretion into the cytoplasm induces apoptosome formation. In addition, the expression change of caspase-3 was investigated using Western blot analysis whether the PTZ-induced seizure was due to the activity of caspase-3. When caspase-3 is activated, it is cleaved into two small subunits of 17 and 12 kDa, respectively. As a result, the expression of cleaved caspase-3 in neurons of fetal rat hippocampus increased in vitro compared to the control (FIG. 3).
We have significantly reduced expression at the protein level of GABA B1 receptor in fetal hippocampal neurons of the PTZ-induced seizure model (FIGS. 5C and 5D), while treating paclofen (GABA B1 receptor antagonist). One GABA B1 receptor expression was found to be more reduced than the former. Thus, the results indicate that expression of GABA B1 receptors in the PTZ-induced seizure model during pregnancy can be regulated at the protein level rather than at the mRNA level. On the other hand, the effect of the PTZ-induced seizure with respect to PKA was confirmed that the expression pattern is significantly reduced at the protein level (Figs. 6c and 6d)
Example 6 Visualization of Mitochondrial Cytochrome-c Secretion and Nuclear Morphology
In situ analysis of cytochrome-c was performed by immunofluorescence technique. Briefly, initial cultures of hippocampal neuron cells (1 × 10 6 cells in culture plates) were treated with ethanol, PTZ, ethanol and PTZ fixed with 4% paraformaldehyde and washed with PBS under cooling. Cytochrome-c is a mouse anti-cytochrome-c antibody and a rabbit anti-mouse FITC-labeled antibody (1: 250 and 1: 200, respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, chromatin was stained (developed) with PI (1 mg / ml in PBS) in the dark for 20 minutes and the slides were attached with reagent (Molecular Probes, Eugene, OR, USA). Cytochrome-c (green) and chromatin (red) staining patterns were obtained using a confocal laser scanning microscope (Fluoview FV 1000, Olympus, Japan).
As a result, by treating PTZ, ethanol and PTZ and ethanol, caspase-3 (TRITC-label, red) means the secretion of cytochrome-c from mitochondria (cytochrome-c, green FITC-label), and cytochrome Confocal microscopy revealed cytochrome-c staining in early hippocampal neuronal cells by translocation of -c. The fused image (yellow) shows that the expression of cytochrome-c and caspase-3 is secreted into the cytoplasm in hippocampal neuronal cell culture as a result of PTZ and ethanol treatment.
Example 7. Histological Analysis and Measurement of Cell Death
Propidium iodide (PI) and Fluoro-Jade-B staining were performed according to the previously disclosed methods. After 17.5 days of gestation, PTZ (intraperitoneally administered 40 mg / kg for 15 days) was exposed to the mother, and experimental animals were anesthetized with sodium pentobarbital (50 mg / g. I.p). The fetus was removed, fixed in cold 4% NBP for 48 hours, and then frozen in antifreeze by precipitation in 20% sucrose phosphate buffer at 4 ° C. for 48 hours. Whole fetuses were frozen in O.C.T compound (A.O. USA) and then frozen 14 μm sections were prepared with coronal planes (Leica cryostat CM 3050C, Germany). Sections were thawed on a slide with a positively charged probe (Fisher). The slides were gently shaken, then immersed in a 1 μg / ml PI solution in PBS for 20 minutes at room temperature and washed twice with PBS for 10 minutes. Glass cover slips were placed on glass slides with mounting medium. PI filtration was used to identify PI staining (Red color) and FITC filtration was used to identify Fluoro-Jade-B (Green color). For imaging we used a Zeiss fluorescent microscope (Zeiss, Germany) and a confocal microscope (Olympus, Japan). The picture was taken with a soft imaging systems video camer.
PI and Fluoro-Jade-B (FJB) staining were performed to determine whether PTZ-induced seizures during pregnancy induced in vivo neuronal cell death in the hippocampus. PIs typically stain nucleic acids by contrast staining in multicolor fluorescence techniques. In the tissue section, the PI is used as a nuclear marker and sometimes used to identify nuclei that show deadly changes. Confocal microscopic analysis confirmed that the entire hippocampal region was reliably stained in the prenatal rat brain from a PTZ-induced seizure model compared to the control group. In the CA1 subfields of the hippocampus, pyramidal neurons were scattered and shrunk (shrunken), and nuclei were clearly accumulated (Figure 4, A-F). PI and FJB staining appeared in neurons with normal morphological features appearing around the nucleus in the control group, but nuclei accumulated in many groups withdrawn and treated groups. Fluoro-JadeB and PI staining, as a potent neuronal damage marker, showed neurodegeneration throughout the fetal rat hippocampal region from a PTZ-induced seizure model (FIG. 4). In addition, co-treatment with Fluoro-JadeB and PI staining and high magnification confirmed that significantly increased Fluoro-JadeB and PI in the hippocampal region of prenatal rats from the PTZ-induced seizure model compared to the saline treated group. Seemed.
Example 8. Data Analysis and Statistics
Objective bands obtained from RT-PCR and Western blot were scanned and analyzed with a densitometer using a Sigma Gel System (SPSS Inc., Chicago, IL) using a basal computer. Density values are expressed as mean ± SEM. As a method according to the TukeyKramer multiple-comparisons test, the ANOVA analysis measured significant differences between the relevant treatment groups. In all cases, the acceptable ranges for statistical significance were * P < and ** P <0.01 .
1 shows representative EEG tracing in pregnant rats after 15 th administration. Representative 30s EEG samples recorded at 5 and 15 minutes after 15 th administration of PTZ (40 mg / kg) during pregnancy at 16 th . A represents normal EEG in the saline group. B is EEG at 5 minutes after 15 th PTZ administration C represents EEG at 15 minutes after 15 th PTZ administration.
FIG. 2 shows that seizure-induced PTZ increases cytochrome-c secretion from fetal rat hippocampal neurons.
2A shows Western blot analysis of cytochrome-c in hippocampal neuron cells initially cultured at GD 17.5 from a PTZ-induced seizure model during pregnancy. Cells were treated as control (C) normal medium, medium containing 100 mM ethanol (E), medium containing 10 mM PTZ (pentylenetetrazol), medium containing 1 μM Kainic acid (KA), PTZ and ethanol (E + PTZ). Treated with ethanol containing medium, medium containing baclofen and paclofen (E + B + P), medium containing 50 μM baclofen (B), and 100 μM paclofen (P) for 20 minutes, respectively. It was. β-actin was used as loading control in each case.
2a: Immunoblot of cytochrome-c of hippocampal neuron cells under different treatment conditions. Immuno blots were labeled with anti cytochrome-c antibodies. 2b: the density value is The mean ± SEM of the protein corresponding to cytochrome-c (n = 4, average 4 per group). * P <0.05 and ** P <0.01 for the control. 2C shows visualization of mitochondrial cytochrome-c secretion and caspase-3 expression in the same neuron. Effect of PTZ and Ethanol on Secretion of Mitochondrial Cytochrome-c and Caspase-3 in Hippocampal Neuronal Cell Cultures. Initial neuronal cultures of fetal rats exposed to PTZ and ethanol for 20 min. Detailed procedures are disclosed in the Examples. Immunofluorescence of hippocampal cells double stained for cytochrome-c (FITC-labeled cytochrome-c antibody, green) and caspase-3 (TRITC-labeled, red). Yellow color (green + red; fused image) shows the secretion of cytochrome-c from the mitochondria to the cytoplasm and caspase-3 secretion from the same neurons measured by confocal microscopy. Arrows indicate dead cells with secretion of cytochrome-c and caspase-3 expression. Magnification 40 X, Scale bar for AC = 20.
Figure 3 shows increased PTZ induced seizures with expression of cleaved caspase-3 in fetal rat hippocampal neurons.
Western blot of caspase-3 in hippocampal neuron cells initially cultured at day 17.5 of pregnancy in a PTZ-induced seizure model during pregnancy. Cells were exposed to other reagents for 20 minutes as previously described. Detailed procedures are disclosed in the Examples. β-actin was used as loading control, respectively. 3a: Immuno blot of caspase-3 in hippocampal and cortical neuronal cells under different treatment conditions. Immuno blots are labeled with anti caspase-3 antibody. 3b: Density values are expressed as mean ± SEM (n = 4, 4 averages per group) corresponding to caspase-3 protein. * P <0.05 and ** P <0.01 for the control.
4 shows a PTZ-induced seizure inducing acute death of neurons.
4a shows a P.I (Propidium iodide) and Fluoro-jade B staining analysis of neurodegeneration in the hippocampus of prenatal rats in a PTZ-induced seizure model. The detailed procedure is shown in the examples. The histological sections are GD 17.5 day old fetal rats in vehicle-treated mothers. Maternal exposure of PTZ during pregnancy induces neurodegeneration in the hippocampal region of the fetal rat brain (A-F). Interruptions, apparent neurodegeneration and changes in cell morphology in CA1 lamination were identified in the prenatal period of pregnant rats treated with PTZ. Photographs show that the PTZ-induced seizure model stimulates a strong neurodegenerative response throughout the entire area of the fetal rat hippocampus, whereas saline is associated with deadly degeneration due to normal physiological cell death in the developing brain. It indicates that a lean brain remains. Panels (B, E = X100 and C, F = X40) are magnified images from panels (A, D = X20) and show PI and FJB staining in the hippocampus.
4b shows co-treatment with FJB and PI staining in the hippocampal region of the brain. Zoom 100 X, Scale bar = 10
5 shows that PTZ-induced seizures during pregnancy reduce mRNA and protein levels of GABA B1 receptors in fetal rat hippocampal neurons. 5a: In PTZ-Induced Seizure Models During Pregnancy RT-PCR analysis (5a) and protein level analysis of GABA B1 receptor (5c) in mRNA in early cultured hippocampal neuron cells with GD 17.5. The cells were exposed to other reagents for 20 minutes as previously described. Detailed procedures are shown in the Examples. GAPDH and β-actin were used as controls. Western blots were labeled with anti-GABA B1 receptor antibody.
5b and 5d: Density values are mean corresponding to mRNA and protein of GABA B1 receptor ± SEM (n = 4, average 4 mice per group). * P <0.05 and ** P <0.01 for the control.
FIG. 6 shows that PTZ-induced seizures during pregnancy reduced mRNA expression levels of PKA in prenatal rat hippocampal neurons.
6a and 6c: In PTZ-Induced Seizure Models During Pregnancy RT-PCR analysis on mRNA in hippocampal neuron cells initially cultured at GD 17.5 shows protein levels of PKA. The cells were exposed to other reagents for 20 minutes as previously described. Detailed procedures are shown in the Examples. GAPDH and β-actin were used as controls.
Western blots were labeled with anti PKA antibodies. 6b and 6d: Density values are expressed as mean ± SEM (n = 4, average 4 per group) corresponding to mRNA and protein of GABA B1 receptor and PKA. * P <0.05 and ** P <0.01 for the control.
<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> METHOD FOR SCREENING OF AGENT FOR TREATING AND DIAGNOSING PRENATAL NEURONAL DISEASE IN MATERNAL EPILEPSY <130> PA090197 / KR <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GABA B1 receptor <400> 1 aattgaattc cgctaccatc caacagacca 30 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GABA B1 receptor <400> 2 aattaagctt tcctgtgacg tcatgttgga a 31 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PKA <400> 3 gtggcaagga gtttactgag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PKA <400> 4 ccagtatctg actttcctgc 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 5 gccatcaatg accccttcat t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GAPDH <400> 6 cgcctgcttc accaccttct t 21
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| KR20190054902A (en) | 2017-11-13 | 2019-05-22 | 단국대학교 천안캠퍼스 산학협력단 | Screening and evaluation method of pain suppressing drug |
| WO2022220403A1 (en) * | 2021-04-16 | 2022-10-20 | 가톨릭대학교 산학협력단 | Use of neogenin as marker for diagnosing brain damage induced by epilepsy |
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| KR20190054902A (en) | 2017-11-13 | 2019-05-22 | 단국대학교 천안캠퍼스 산학협력단 | Screening and evaluation method of pain suppressing drug |
| WO2022220403A1 (en) * | 2021-04-16 | 2022-10-20 | 가톨릭대학교 산학협력단 | Use of neogenin as marker for diagnosing brain damage induced by epilepsy |
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