CN111647051B - Short peptide based on sodium channel structure and application thereof - Google Patents

Short peptide based on sodium channel structure and application thereof Download PDF

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CN111647051B
CN111647051B CN201910160234.5A CN201910160234A CN111647051B CN 111647051 B CN111647051 B CN 111647051B CN 201910160234 A CN201910160234 A CN 201910160234A CN 111647051 B CN111647051 B CN 111647051B
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short peptide
sodium channel
epilepsy
channel structure
application
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CN111647051A (en
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郭凤
赵伟东
郝丽英
胡慧媛
王韵
徐晓雪
封瑞
孙雪菲
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China Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention relates to the field of epilepsy treatment, in particular to a sodium channel-based short peptide and application thereof in epilepsy treatment, and particularly relates to the application of the sodium channel-based short peptide in treatment of epilepsy, in particular to the application of the sodium channel-based short peptide in treatment of protecting neurons, wherein the sodium channel-based short peptide is used for reducing channel abnormal discharge through amino acid site mutation and binding site competition. The invention provides a short peptide based on a sodium channel, the amino acid sequence of which is shown as SEQ ID No.1, the short peptide can be obtained by artificial synthesis and purification, and can be prepared into an antiepileptic drug. The short peptide can play the pharmacological action of resisting epilepsy by improving the survival rate of nerve cells, improving the discharge of neurons and reducing the apoptosis.

Description

Short peptide based on sodium channel structure and application thereof
Technical Field
The invention relates to the field of epilepsy treatment, in particular to a sodium channel-based short peptide and application thereof in epilepsy treatment, and particularly relates to the application of the sodium channel-based short peptide in treatment of epilepsy, in particular to the application of the sodium channel-based short peptide in treatment of protecting neurons, wherein the sodium channel-based short peptide is used for reducing channel abnormal discharge through amino acid site mutation and binding site competition.
Background
Epilepsy is a chronic disease in which there is a sudden abnormal firing of cerebral neurons, resulting in transient cerebral dysfunction. According to the latest Chinese epidemiological data, the total prevalence rate of domestic epilepsy is 7.0 per thousand, the annual incidence rate is 28.8/10 ten thousand, and the prevalence rate of active epilepsy with attacks within 1 year is 4.6 per thousand. Therefore, about 900 million epilepsy patients are estimated in China, 500-600 million of the epilepsy patients are active epilepsy patients, about 40 million epilepsy patients are newly added every year, and epilepsy has become the second most common disease of the neurology department, second to headache in China. Studies have shown that voltage-gated sodium channel expression and dysfunction in neuronal cell membranes in the central nervous system are important causes of epileptogenesis. In recent years, with the continuous disclosure of molecular mechanisms of epileptogenesis, the attention on epileptic specific target therapy is increasing, and the acquisition of a suitable target is a key link of epileptic therapy. With the rapid development of molecular biology methods, methods for inducing a large amount of purified polypeptides are continuously updated, and a new link is opened for targeted therapy of epilepsy.
Studies have shown that increased sodium channel persistent sodium current, which is closely associated with the regulation of calmodulin binding, leads to the development of epilepsy. Calmodulin is capable of specifically binding to the C-terminal IQ-specific domain of the sodium channel to increase sodium current. Therefore, the application of the sodium channel short peptide to realize targeted epilepsy treatment also becomes a new idea of the current antiepileptic drugs. Since the nature of epileptogenesis is that neurons die due to abnormal firing of the neurons and thus cause brain damage, protection of neurons from damage due to abnormal firing is an important scientific problem that has yet to be solved.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide a short peptide based on a sodium channel structure and application thereof, wherein the short peptide is characterized by having similarity with the sodium channel structure and being capable of competitively binding with a binding site of calmodulin and a sodium channel, so that the short peptide plays a role in suppressing discharge neuroprotection and lays a theoretical foundation for the research and development of targeted antiepileptic drugs.
In order to achieve the above object, the present invention adopts the following technical solutions.
A short peptide based on a sodium channel structure is characterized in that the amino acid sequence of the short peptide is shown as SEQ ID NO. 1:
YGRKKRRQRRRVSAVIQRAYRRHLLKRAVK, wherein YGRKKRRQRRR is a tool-penetrating peptide.
Further, the short peptide based on the sodium channel structure comprises a part of the original sequence of the sodium channel, and can competitively bind to calmodulin; in addition, the short peptide has a mutation site which is a mutation phosphorylation site and can inhibit the phosphorylation of a channel, and the final effects of the two principles are that the short peptide reduces the discharge of neurons and protects nerve cells.
Further, the short peptide based on the sodium channel structure is a mutant peptide derived from the amino acid sequence composition by replacing an amino acid.
Further, a short peptide based on a sodium channel structure is obtained by artificial synthesis and purification.
A short peptide based on a sodium channel structure is applied to anti-epileptic treatment and is prepared into a targeted anti-epileptic medicament.
Compared with the prior art, the invention has the following beneficial effects.
1) The short peptide is based on the structure of a voltage-gated sodium channel, but has a mutation site, and has original innovation in amino acid sequence.
2) The short peptide is obtained by artificial synthesis and purification, and the preparation method is simple and has controllable purity.
3) The short peptide can play the pharmacological action of resisting epilepsy by improving the survival rate of nerve cells, improving the discharge of neurons and reducing the apoptosis through experimental verification.
Drawings
FIG. 1 shows Cell Counting Kit-8 tests the effect of the short peptide of the present invention on the survival rate of neuro-2a nerve cells in the magnesium-free epilepsy model.
FIG. 2 is a multichannel microelectrode array system for detecting the effect of the short peptide of the invention on the abnormal discharge of primary culture neurons of a magnesium-free epilepsy model rat.
FIG. 3 shows Western blot for detecting the effect of the short peptide of the present invention on neuro-2a nerve cell apoptosis after administration of magnesium-free epilepsy model cells.
FIG. 4 is a morphological diagram of neuro-2a nerve cells after 24 hours of administration of the short peptide of the present invention to a magnesium-free epilepsy model.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments. The following examples will help to understand the present invention, but they are only for illustrative purposes and the present invention is not limited to these contents. The methods of operation in the examples are conventional in the art.
The embodiment relates to a method for establishing a magnesium-free epileptic cell model, experimental grouping and administration.
First, cell culture is performed. Culturing primary neurons: taking newborn rat brain, separating bilateral hippocampus under a microscope, and placing the hippocampus in D-Hanks liquid; adding 0.125% trypsin, and digesting for 15-30 min in an incubator at 37 ℃. The digestion was stopped by the plating medium (DMEM/F12 + 15% serum) and the cell density was adjusted to 2X 105/cm2Then planted on a 2.0 cm × 2.0 cm cover glass. Changing the feeding culture solution by half 3-4 days (2% B27+ Neurobasal)TM-a-Medium). When the cell density of Neuro-2a reaches 80% -90%, removing the culture medium, and washing twice with 10mL of PBS. 3mL of trypsin containing 0.25% EDTA was added and placed in a cell incubator for 3 min. Pancreatin digestion was stopped by adding 1 mL of DMEM complete medium and transferred to a 15mL centrifuge tube. The cells were washed by adding 10mL of PBS and transferred to a 15mL centrifuge tube, centrifuged at 2000rpm for 2min, and the supernatant was discarded. Then 10mL of PBS was added, blown uniformly, 10. mu.L of PBS was aspirated and counted at 1X 106Plate inoculation in 5% CO2The cell culture box continues to culture. Magnesium-free liquid (mmol. L)-1）：NaCl 145，KCl 2.5，CaCl 22, HEPES 10, glucose 10, glycine 0.001, adjusting the pH to 7.4 by NaOH; extracellular fluid (mmol. L)-1）：NaCl 135，KCl 5.4，MgCl21.0，NaH2PO40.33, HEPES 10, glucose 5.5, pH adjusted to 7.4 with NaOH; after 10 days of culture, the neuron or Neuro-2a cell is put into magnesium-free extracellular fluid for treatment for 3 hours, and then put into magnesium-containing normal extracellular fluid again for culture. The nerve cells are the magnesium-free epileptic cell model. The following experiment was performed 24 hours after treatment with different concentrations of short peptides administered while recovering normal extracellular fluid. The experiment is divided into a normal group, a magnesium-free epileptic cell model group, a magnesium-free epileptic cell group and a short peptide group.
Example two CCK8 method to determine the effect of short peptides on the survival of Neuro-2a cell line.
The Cell Counting Kit-8 (CCK-8 for short) reagent can be used for simple and accurate Cell proliferation and toxicity analysis. The basic principle is as follows: the reagent contains WST-8 [ chemical name: 2- (2-Methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt ] which is reduced to a highly water-soluble yellow Formazan product (Formazan dye) by a dehydrogenase in cells under the action of the electron carrier 1-Methoxy-5-methylphenazinium dimethylsulfate (1-Methoxy PMS). The amount of formazan produced was proportional to the number of living cells. Therefore, the cell proliferation and toxicity analysis can be directly carried out by utilizing the characteristic.
1) Cell suspensions were prepared in 96-well plates. The plate was placed in an incubator for 24 hours.
2) Polypeptide drugs were added to the cultured cells at various concentrations and allowed to act for 24 hours.
3) To each well was added 10 μ LCCK8 solution.
4) The plates were placed in an incubator and incubated for 3 hours.
5) Absorbance at 450nm was measured with a microplate reader.
The Cell Counting Kit-8 test result is shown in FIG. 1, and the short peptide concentrations given to the neuro-2a nerve Cell without magnesium treatment are 100 nM, 1. mu.M and 10. mu.M, wherein the effect of 1. mu.M short peptide on improving the survival rate of magnesium-free epileptic cells is the best.
Example three multichannel microelectrode array detection systems (MEA) detect the effect of short peptides on neuronal discharge in primary cultured rats of the magnesium-free epileptic cell model.
MEA is a novel electrophysiological technique that allows extracellular recording from multiple electrodes embedded in the bottom of the MEA channels without infiltration into the tissue. Electrical activity can be measured at multiple points where the embedded electrodes are in contact with the tissue. The stimulating and recording electrodes may be selected from any of the electrodes depending on the location of the cells. Therefore, the probability of generating false negatives may be lower than that of conventional extracellular recording techniques. Furthermore, compared to conventional microelectrode recording techniques, MEA techniques allow long-term analysis of the spatiotemporal distribution of network-level electrical activity and stable recordings that are less sensitive to factors related to microelectrode insertion into tissue (e.g., mechanical vibrations). MEA fusion fineThe method for detecting the discharge of a cluster of neurons on a 96-hole MEA special plate has the advantages of an extracellular microelectrode array and an intracellular microelectrode. The MEA 96-well plate was pretreated with polylysine at 50. mu.L per well in advance by 48 hours, and polylysine in the 96-well plate was aspirated and washed three times with distilled water. Rat hippocampal neurons were added to 96-well plates at 2X 10 per well5/cm2Plating was performed, and medium change culture was performed half a day. Cell discharge detection was performed 24 hours after the polypeptide was treated with the cells. Activity was recorded using MEA-1060-INV-BC single channel and MC software from a multichannel system. Each single-channel MEA contained 59 recording electrodes (diameter 30 μm, electrode spacing 200 μm) and one internal ground electrode. All MEA recordings were performed in 37 ℃ media without perfusion. The signal from the amplifier is digitized at a rate of 25 kHz and high-pass filtered (cut-off frequency of 100 Hz). The threshold for peak detection is 3 times the noise standard deviation. The effect of the drug on the spike rate is expressed as a percentage, i.e., quantified relative to baseline. As shown in fig. 2, the magnesium-free epileptic cell model showed strong cell discharge compared to normal primary cultured neurons, but 1 μ M short peptide administration significantly improved epileptic neuronal discharge.
Example four Western Blot method to detect apoptosis.
RIPA lysate lyses adherent cells. And (4) centrifuging at low temperature after homogenizing and ultrasonic treatment, and taking supernatant BCA protein concentration determination kit to determine the protein concentration. Adding sample buffer solution, boiling for denaturation, performing SDS-PAGE on 50 μ g/well, 5% (mass concentration) concentrated gel and 12% (mass concentration) separation gel at a gel voltage of 80V 50min and a gel voltage of 120V 70 min. Transfer to PVDF membrane (200 mA, 2 h). 5% (mass concentration) BSA normal temperature blocking 1h add anti rabbit anti mouse polyclonal antibody Caspase-3 and Bcl-2, according to the instructions to give the respective optimal concentration, room temperature shaking table slowly to incubate overnight. PBS was washed 3 times. Adding a secondary goat anti-rabbit antibody at a ratio of 1:3000, and incubating for 2 hours at room temperature on a side-shaking table with slow shaking. ECL chemiluminescence is carried out after TBST membrane washing, the position of a target band is determined according to the relative molecular mass of protein on a primary antibody specification, the gray value is determined, and the ratio of the gray value of the target band to the gray value of GAPDH is taken as the relative expression quantity of the target protein. The Western blot method reveals that the short peptide can improve the apoptosis result of magnesium-free epilepsy model cells, and the result is shown in figure 3, compared with normal neuro-2a nerve cells, the magnesium-free epilepsy model cells show that the apoptosis indexes Caspase-3 and Bcl protein are up-regulated, but the expression of magnesium-free epilepsy cell proteins Caspase-3 and Bcl can be obviously reduced after 1 mu M short peptide is administrated.
Example five Neuro-2a cell cultures and light microscopy.
Neuro-2a as 1X 106Plate inoculation in 5% CO2The cell culture box of (3). After 24 hours of administration of the short peptide, cell morphology was examined by light microscopy, as shown in FIG. 4 (scale: 10 μm), and 3 hours after administration of the magnesium-free treatment to Neuro-2a cells, cell morphology was damaged, and cells were swollen and necrotic. After 24h administration of the short peptide, the magnesium-free epileptic cell morphology partially recovered.
Sequence listing
<110> university of Chinese medical science
<120> short peptide based on sodium channel structure and application
<141> 2021-12-27
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> PRT
<213> human (Homo sapiens)
<400> 1
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Val Ser Ala Val Ile
1 5 10 15
Gln Arg Ala Tyr Arg Arg His Leu Leu Lys Arg Ala Val Lys
20 25 30

Claims (4)

1. A short peptide based on a sodium channel structure is characterized in that the amino acid sequence of the short peptide is shown as SEQ ID NO. 1: YGRKKRRQRRRVSAVIQRAYRRHLLKRAVK, wherein YGRKKRRQRRR is a tool-penetrating peptide.
2. A short peptide based on the sodium channel structure of claim 1, wherein said short peptide is capable of competitively binding to calmodulin; in addition, the short peptide has a mutation site which is a mutation phosphorylation site and can inhibit the phosphorylation of a channel, and the final effects of the two principles are that the short peptide reduces the discharge of neurons and protects nerve cells.
3. The short peptide based on a sodium channel structure as claimed in claim 1, wherein the short peptide is obtained by artificial synthesis and purification.
4. Use of the short peptide based on a sodium channel structure according to claim 1 in the preparation of a medicament for targeting antiepileptic.
CN201910160234.5A 2019-03-04 2019-03-04 Short peptide based on sodium channel structure and application thereof Active CN111647051B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106220713A (en) * 2016-08-08 2016-12-14 大连医科大学 A kind of heat-resisting synthetic peptide of scorpion venom and application thereof
CN107213465A (en) * 2017-06-23 2017-09-29 武汉大学 Inhibitors of dipeptidyl IV is preparing the application in preventing and treating the medicine of epilepsy

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106220713A (en) * 2016-08-08 2016-12-14 大连医科大学 A kind of heat-resisting synthetic peptide of scorpion venom and application thereof
CN107213465A (en) * 2017-06-23 2017-09-29 武汉大学 Inhibitors of dipeptidyl IV is preparing the application in preventing and treating the medicine of epilepsy

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