CN115057917A - Neuroprotective active polypeptide and application thereof - Google Patents

Abstract

The invention discloses a neuroprotective active polypeptide and application thereof. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. The application of the polypeptide is the application in preparing the medicine for treating brain nerve injury and sequelae thereof. The invention obtains a polypeptide OL-FS13 with neuroprotective activity from skin secretion of rana grahami, the polypeptide has simple structure and shows stronger nerve injury repair promoting activity under low dosage (10 mug/kg in vivo and 100 pM in vitro) in vivo and in vitro. Compared with the neuroprotective peptide OM-LV20, the mature peptide sequence of the polypeptide OL-FS13 provided by the invention is shorter, so that the synthesis cost is lower. In addition, different from OM-LV20, OM-LV20 alleviates nerve injury of cerebral ischemia reperfusion rats through preventive administration, OL-FS13 plays a role in repairing after reperfusion injury by administration, has higher clinical application value, and also provides a new direction for development of neuroprotective drugs.

Description

Neuroprotective active polypeptide and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a neuroprotective active polypeptide and application thereof.

Background

Stroke (stroke) is an acute cerebrovascular disease that is one of the causes of death and permanent disability in many humans. In recent years, stroke mortality has decreased dramatically with advances in clinical diagnosis and treatment. However, according to the world health organization data, one third of stroke patients may be permanently disabled. This high disability rate not only reduces the quality of life for patients, but also increases their family and social burden.

Most stroke cases are classified as ischemic stroke (about 80%). Treatment of this acute disease relies primarily on early restoration of cerebral blood flow (reperfusion) to preserve hypoperfused brain tissue and limit neurological dysfunction. Therefore, reperfusion is considered to be the most effective method for treating ischemic stroke. Currently, only two stroke therapies, thrombolytics (tissue plasminogen activators) to dissolve occlusive thrombi and direct cerebrovascular thrombus clearance, are approved by the U.S. food and drug administration. However, both of these approaches have strict indications and only a few patients can benefit from early treatment. Therefore, ischemia/reperfusion remains a considerable challenge in most ischemic stroke patients and can lead to blood brain barrier damage, brain edema, neurological deficits, and even devastating cerebral hemorrhage.

In recent years, amphibians have attracted considerable attention in the biomedical field as a potential natural drug resource. Researchers have obtained a variety of amphibian derived peptide molecules with novel structures and unique functions, including peptides with wound healing, hypoglycemic, antibacterial, analgesic, and antioxidant properties and activities. However, only a few neuroprotective peptides obtained from amphibian skin have been reported.

The invention aims to provide a neuroprotective active polypeptide which has a simple structure and can promote the repair of nerve injury in vivo and in vitro.

Disclosure of Invention

The first object of the present invention is to provide a polypeptide, the second object of the present invention is to provide a nucleic acid molecule encoding said polypeptide, and the third object of the present invention is to provide the use of said polypeptide.

The first object of the present invention is achieved by a polypeptide having an amino acid sequence shown in SEQ ID NO. 1.

The second object of the present invention is achieved by a nucleic acid molecule which is a DNA molecule or an RNA molecule; the DNA molecule is cDNA, genome DNA or recombinant DNA; the RNA molecule is mRNA or hnRNA.

The third purpose of the invention is realized by the application of the polypeptide in preparing medicines for treating brain nerve injury and sequelae thereof.

The invention has the beneficial effects that:

the polypeptide OL-FS13 with neuroprotective activity is obtained from skin secretion of rana grahami, has simple structure, shows stronger nerve injury repair activity in vivo and in vitro under low dose, has higher clinical application value, and provides a new direction for the development of neuroprotective drugs.

Drawings

FIG. 1 is a sequence diagram of a cDNA encoding the damage-resistant polypeptide OA-FS13 of the present invention;

FIG. 2 is a graph showing neuroprotection of the neuroprotective peptide OL-FS13 of the present invention in an oxygen deprivation/reoxygenation cell model

An activity profile;

FIG. 3 is a graph showing the anti-cerebral ischemia reperfusion injury activity of the neuroprotective peptide OL-FS13 in animal model: A. TTC dyeing; B. quantifying the cerebral infarction area; C. neurobehavioral scores for each group of rats.

Detailed Description

The invention is further described in detail below with reference to the drawings and examples, but the invention is not limited in any way, and any changes or modifications made based on the teachings of the invention fall within the scope of the invention.

The invention provides a polypeptide, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

The invention further provides a nucleic acid molecule encoding said polypeptide.

The nucleic acid molecule is a DNA molecule or an RNA molecule; the DNA molecule is cDNA, genome DNA or recombinant DNA; the RNA molecule is mRNA or hnRNA.

The invention further comprises a recombinant vector, a recombinant bacterium, an expression cassette or a transgenic cell line of the nucleic acid molecule.

The invention also provides application of the polypeptide in preparing a medicament for treating brain nerve injury and sequelae thereof.

The invention further provides a pharmaceutical composition comprising said polypeptide as an active agent and a pharmaceutically effective carrier.

EXAMPLE 1 preparation of neuroprotective peptide OL-FS13

The first step is as follows: adult large odorous frog comes from Yunnan province of China. The large Rana grahami is raised in a 21 cm × 21 cm × 15 cm jar for 7 days to adapt to the environment, and water is periodically changed to feed the Rana grahami with Tenebrio molitor as feed. The samples were from the skin of bullfrog. After the skin of the rana grahami was washed with deionized water, the skin was quickly sacrificed and harvested by smashing the brain marrow.

The second step is that: the large rana grahami cDNA library was constructed as follows. To screen for cDNA encoding mature OL-FS13, Polymerase Chain Reaction (PCR) templates were derived from cDNA synthesized by SMART technology and 5'PCR primers (5' -CCAAA (G/C) ATGTTCACC (T/A) TGAAGAA-3 ') and 3' PCR primers (5 'ATTCAGGCCGAGGCC GACA TG-3') synthesized by BGI, Inc. (China). The obtained PCR product was cloned into E.coli DH5a active cells and DNA sequencing was carried out using an Applied Biosystems DNA sequencer (ABI 3730XL, Foster, Calif.) as shown in FIG. 1, the nucleotide sequence of the cDNA is shown in SEQ ID NO.2, the full length of the cDNA sequence is 311bd (containing 311 bases) encoding 58 amino acid residues, and the mature peptide sequence is "FSLLLTWWRRRVC" (underlined in FIG. 1). This peptide was searched and named OL-FS13 as a novel peptide.

The third step: the OL-FS13 is prepared by conventional chemical synthesis or gene expression.

EXAMPLE 2 Activity assay of the polypeptide OL-FS13

The activity of the polypeptide OL-FS13 prepared in example 1 was assayed by an in vitro oxygen deprivation/reoxygenation assay and an in vivo ischemia-reperfusion assay.

In vitro neuroprotective Activity detection

The test method comprises the following steps: in vitro cell oxygen sugar deprivation/reoxygenation experiments

PC12 cells in the presence of 5% CO ₂ And culturing in a constant temperature incubator at 37 ℃. DMEM/F12 medium supplemented with 1% antibiotic (100U/mL penicillin, 100U/mL streptomycin) and 10% fetal bovine serum was selected for culture. PC12 cells (5000 per well) were cultured in 96-well plates. After the cells were attached, PC12 cells were washed twice with PBS and the original medium was replaced with sugar-free DMEM. The cells were divided into two groups, negative control group was cultured in the original medium under the general environment, and the remaining groups were placed in 95% N ₂ And 5% CO ₂ In the environment of (2). After 4 h, the cells were reaerated and the sugar-free DMEM medium of the model group (PBS) and the administered group (OL-FS 13) PC12 cells was replaced with the original complete medium.

After cell reaeration, model and dosing groups were intervened with PBS and various concentrations of OL-FS13, respectively, and incubation continued for 24 h. According to the manufacturer's instructions, to each well CellTiter 96A AQueous One Solution Assay Assay was added and the absorbance values at 490 nm were determined. The viability of the cells in each group was calculated by taking the negative control group as 100%.

As a result: as shown in FIG. 2, the neuroprotective peptide OL-FS13 has strong activity of promoting nerve injury repair in vitro. Oxygen sugar deprivation/reoxygenation reduced cell viability in the model group by 70.12% ± 3.08% (compared to the negative control group) compared to the control group. Finally, treatment with OL-FS13 (1, 10 and 100 pM) reduced hypoxic ischemic damage to PC12 cells. In addition, OL-FS13 treatment demonstrated in vitro neuroprotective activity in a concentration-dependent manner at 46.07% ± 4.80%, 70.20% ± 8.20% and 79.55% ± 5.23%, respectively (100% with respect to the negative control).

Second, in vivo neuroprotective Activity detection

The test method comprises the following steps: cerebral ischemia reperfusion experiment of rat

SD rats were randomly divided into sham, model, and drug administration groups (post-modeling given OL-FS13 intervention). Rats were anesthetized with sodium pentobarbital (50 mg/kg) by intraperitoneal injection. A0.36 mm artificial wire plug is inserted into the internal carotid artery of the rat and advanced until it meets resistance (18-22 mm) to block the middle cerebral artery. After 2 h, the pegs were gently removed to create reperfusion and the wound was closed. OL-FS13 intervenes after suturing the wound, and sham rats used the same procedure but without the insertion of a peg.

To assess the extent of brain injury in rats, assessment of cerebral infarction volume and neurological deficit was performed.

Infarct volume was calculated by staining with 2,3, 5-triphenyltetrazolium chloride (TTC). First, excess sodium pentobarbital was used to euthanize rats. Then, the brain was frozen at-20 ℃ for about 15 min and uniformly cut into 6 pieces (coronal sections). Finally, brain sections were incubated in 1% TTC solution at 37 ℃ until infarction clearly appeared and removed for observation. Infarct area was calculated for each section using Image J software, with the rat cerebral infarct volume = 100% x infarct area divided by section area. Neurological deficit scoring in rats was performed 24 h after cerebral ischemia-reperfusion. The improved nerve severity score was used to assess the degree of nerve damage in rats.

As a result: as shown in figure 3, the neuroprotective peptide OL-FS13 can reduce cerebral infarction area of cerebral ischemia-reperfusion rats and reduce neurological deficit, and has strong activity of promoting nerve injury repair. TTC staining is commonly used to assess cerebral ischemic injury, and red indicates non-infarcted brain tissue. As shown in fig. 3A, ischemia reperfusion of the left brain of the rat resulted in severe cerebral infarction. Under the intervention of OL-FS13 (10. mu.g/kg), the cerebral infarction volume was significantly reduced. Similarly, treatment with OL-FS13 (10 μ g/kg) improved the neurological deficit score in cerebral ischemia-reperfused rats (FIG. 3C).

As a neuroprotective peptide, the OL-FS13 provided by the invention has the following advantages: first, OL-FS13 showed significant neuroprotective capacity at low doses (10. mu.g/kg in vivo, 100 pM in vitro). Secondly, the mature peptide sequence of OL-FS13 is shorter (13: 20) and thus the synthesis cost is lower than that of the known neuroprotective peptide OM-LV20 (OM-LV 20 is a neuroprotective peptide extracted from Rana grahami skin). Finally, although OM-LV20 can reduce nerve damage in cerebral ischemia-reperfused rats at lower doses (20 ng/kg), it works by prophylactic administration, whereas OL-FS13 is administered after reperfusion injury, which improves its clinical utility.

SEQUENCE LISTING

<110> university of Kunming medical science

<120> neuroprotective active polypeptide and application thereof

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<170> PatentIn version 3.3

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<213> Odorrana graminea

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Phe Ser Leu Leu Leu Thr Trp Trp Arg Arg Arg Val Cys

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ctctgtcagg aagagagaaa tgacgaagaa gaaagaagag aagaagaggt tgctaaagac 120

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Claims (5)

1. A polypeptide, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

2. A nucleic acid molecule encoding the polypeptide of claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule; the DNA molecule is cDNA, genome DNA or recombinant DNA; the RNA molecule is mRNA or hnRNA.

4. The use of the polypeptide of claim 1 in the preparation of a medicament for the treatment of brain nerve injury and its sequelae.

5. A pharmaceutical composition comprising the polypeptide of claim 1 as an active agent and a pharmaceutically effective carrier.

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