Polypeptide and application thereof

Abstract

The invention discloses a polypeptide and application thereof, and belongs to the technical field of biochemical pharmacy. The sequence of the polypeptide disclosed by the invention is RKKRRQRRRRAPSDPRLR, the sequence is shown as SEQ ID NO.1, the polypeptide is composed of 17 amino acids, and no related function report exists at present. The polypeptide has an improvement effect on the sugar-oxygen deprivation reperfusion injury of the astrocytes, and can improve the activity of the damaged astrocytes; meanwhile, the polypeptide has a good application prospect in preparation of drugs or pharmaceutical compositions for treating OGD / R-induced human brain astrocyte injury, and is expected to provide new possibility for treatment of focal cerebral ischemia.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical pharmacy, in particular to a polypeptide and an application thereof, specifically to a polypeptide RKKRRQRRRAPSDPRLR and an application thereof in preparing a medicine and a pharmaceutical composition for treating OGD / R-induced human brain astrocyte damage. Background Art

[0002] Astrocytes are a type of glial cells, with a number five times that of neurons. They are one of the main components of the entire central nervous system (CNS). They play an important role in biological processes such as structural support, regulation of cerebral blood flow, stabilization of intercellular communication, neuronal metabolism, neurotransmitter synthesis, maintenance of the extracellular environment, formation of the blood-brain barrier, and resistance to oxidative stress. They play an indispensable role in connecting cells. The oxygen-glucose deprivation-reperfusion (OGD / R) model is a model used to study the recovery of cells after hypoxia and glucose deprivation to study the response of cells to reperfusion after ischemia. This model is often used to study the pathogenesis, drug efficacy, and treatment strategies of diseases such as cerebral ischemia and stroke.

[0003] During cerebral ischemia, the glucose and oxygen supply in the ischemic core area is completely terminated, resulting in irreversible damage to all nerve cells, including neurons and astrocytes. However, the glucose and oxygen supply in the ischemic penumbra is still partially maintained, allowing astrocytes to survive for a long time. Studies using the oxygen-glucose deprivation (OGD) model have shown that astrocytes tolerate OGD better than neurons; further studies have shown that astrocytes can use glycolysis to produce ATP energy during OGD, thereby exerting a neuroprotective effect. However, during ischemic stroke, due to persistent and severe hypoxia and glucose deprivation, that is, hypoxia / ischemia exceeds the body's tolerance level, the regulatory function of astrocytes is impaired, inflammatory cytokines are produced, and damage to the brain is increased. Even if oxygen supply is restored, it is difficult to make up for the damage.

[0004] Therefore, finding effective drugs to help treat astrocyte damage caused by OGD / R has important theoretical significance and practical application value for hypoxic / ischemic brain damage. However, from a clinical perspective, the efficacy of current drugs for treating hypoxic astrocyte damage is not obvious.

[0005] Peptides are compounds formed by α-amino acids linked together by peptide bonds. Peptides have the characteristics of both small molecule drugs and biological agents such as large molecule proteins. Peptides exist in the body in the form of enzymes, hormones, neurotransmitters, ion channel ligands, immune response mediators, etc. When endogenous secretion is lacking, they can mimic various physiological metabolic pathways and regulate homeostasis. Peptides are natural ligands for many receptors and ion channels, which makes their binding to targets or receptors specific, so they have higher selectivity, which can greatly avoid side effects caused by off-target, and can also avoid the triggering of immune responses, and have lower immunogenicity. Peptides have a short half-life, and their metabolites will not accumulate in tissues, which also avoids the risks of metabolic toxicity.

[0006] Studies have shown that peptide drugs not only have outstanding performance in antibacterial, prevention and treatment of thrombosis, hypertension and hyperlipidemia, but also can delay aging and improve the body's anti-tumor ability. In addition, peptides also have outstanding performance in diagnostic reagents. Therefore, it is feasible to use peptide drugs to study the regulation of astrocyte damage caused by oxygen-glucose deprivation reperfusion, but there are few related reports in this field, and there is still a lot of room for development and a huge market gap in the research and development of corresponding peptide drugs. Summary of the invention

[0007] The purpose of the present invention is to solve the problems existing in the prior art, provide a polypeptide RKKRRQRRRAPSDPRLR, and disclose that the polypeptide can increase the activity of astrocytes damaged after oxygen glucose deprivation reperfusion. The polypeptide has broad application prospects in the preparation of drugs and pharmaceutical compositions for treating OGD / R-induced human brain astrocyte damage.

[0008] In order to achieve the above technical objectives, the present invention is implemented through the following technical scheme: a polypeptide, the polypeptide sequence is RKKRRQRRRAPSDPRLR, and its amino acid sequence is shown in SEQ ID NO.1.

[0009] The present application provides a type of biomaterial, including any one or more of 1) to 5):

[0010] 1) A polypeptide or a polypeptide composition comprising the polypeptide RKKRRQRRRAPSDPRLR;

[0011] 2) a nucleic acid molecule encoding the polypeptide RKKRRQRRRAPSDPRLR;

[0012] 3) a vector comprising a nucleic acid sequence encoding the polypeptide RKKRRQRRRAPSDPRLR;

[0013] 4) a cell culture medium containing the polypeptide or polypeptide combination as described in 1);

[0014] 5) The product obtained by conventional modification on the amino acid side chain groups of the polypeptide RKKRRQRRRAPSDPRLR, at the amino terminus or carboxyl terminus of the polypeptide RKKRRQRRRAPSDPRLR.

[0015] Furthermore, the conventional modification is amination, hydroxylation, carboxylation, amidation, carbonylation, alkylation, acetylation, esterification, glycosylation, cyclization or biotinylation.

[0016] The present application provides a pharmaceutical preparation, which contains the above-mentioned biological material, and the pharmaceutical preparation is in a pharmaceutically acceptable form, including but not limited to one or more of tablets, pills, granules, creams, patches, capsules, suppositories, oral liquids or injections. Drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0017] The above polypeptide, biological material or pharmaceutical preparation can be used to prepare drugs and pharmaceutical compositions for treating OGD / R-induced damage to human brain astrocytes.

[0018] The present application discloses a pharmaceutical composition for treating OGD / R-induced damage to human brain astrocytes. The pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or a pharmaceutically active substance and is prepared into any pharmaceutically acceptable dosage form. The pharmaceutically active substance comprises a pharmaceutically effective dose of the polypeptide RKKRRQRRRAPSDPRLR.

[0019] Furthermore, the pharmaceutically acceptable carrier is an excipient, a filler, a diluent, a binder, a disintegrant, an absorption enhancer, a surfactant or a lubricant.

[0020] The beneficial effects of the present invention are:

[0021] 1. The present application discloses a novel active polypeptide RKKRRQRRRAPSDPRLR, which has an improving effect on astrocyte oxygen-glucose deprivation reperfusion injury and can increase the activity of damaged astrocytes after oxygen-glucose deprivation reperfusion;

[0022] 2. Since the polypeptide disclosed in the present application has the effect of improving glucose-oxygen deprivation-reperfusion injury of astrocytes, it can be used to prepare drugs or pharmaceutical compositions for treating OGD / R-induced damage to human brain astrocytes, promote the development of the polypeptide RKKRRQRRRAPSDPRLR, and is expected to provide new possibilities for the treatment of focal cerebral ischemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1To determine whether the modeling is successful, the activity of astrocytes before and after modeling was changed; Panel A shows the effect of oxygen-glucose deprivation and reperfusion on the activity of astrocytes, Panel B shows the effect of oxygen-glucose deprivation and reperfusion on the activation of astrocytes, Note: CON refers to the normal untreated rat primary cortical astrocyte group, O / R refers to the rat primary cortical astrocyte group after oxygen-glucose deprivation and reperfusion; Panel C shows the GFAP immunofluorescence staining of cells in the CON group; Panel D shows the GFAP immunofluorescence staining of cells in the O / R group;

[0024] Figure 2 The live-dead double staining results of cells in the normal control group (CON group), model control group (OGD / R group, marked as O / R in the figure) and peptide intervention group (OGD / R+peptide, peptide concentration is 20μM) and the schematic diagram of cell morphological changes under bright field conditions; Panel A is a statistical chart of the live-dead ratio of cells; Panel B is a live-dead double staining picture of primary cortical astrocytes; Panel C is a picture of morphological analysis of primary cortical astrocytes;

[0025] Figure 3 It is a schematic diagram of the GFAP activation results and CCK8 test results of the normal control group (CON group), model control group (OGD / R group, marked as O / R in the figure) and polypeptide intervention group (OGD / R+polypeptide); Panel A shows the viability of astrocytes in different experimental groups, where 0.1μM, 1μM, 20μM and 100μM marked on the horizontal axis indicate the use of corresponding concentrations of polypeptide drugs to intervene in primary cortical astrocytes of rats; Panel B shows the degree of astrocyte activation in different experimental groups, and 20μM marked on the horizontal axis indicates the use of 20μM polypeptide drugs to intervene in primary cortical astrocytes of rats; Panel C is a staining diagram of GFAP immunofluorescence staining of cells in different experimental groups. DETAILED DESCRIPTION

[0026] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0027] In order to screen out pharmaceutical peptides that can be used to improve oxygen-glucose deprivation-reperfusion injury in astrocytes, the bioinformatics method of peptideomics analysis was used to construct a MCAO model in rats. By analyzing the differential peptides between the sham operation group and the MCAO model group, several peptides with possible biological activity were obtained and verified for biological activity in the oxygen-glucose deprivation-reperfusion model of astrocytes. It was found that the peptide RKKRRQRRRAPSDPRLR met the requirements. Among them, RKKRRQRRR is a penetrating peptide sequence that promotes the entry of peptides into cells, and APSDPRLR is the target sequence that plays a major role after entering the body. The molecular weight of APSDPRLR is 911.03, the isoelectric point is 9.64, and the mass-to-charge ratio is 456.2559.

[0028] The amino acid sequence of the polypeptide is Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Ser AspPro Arg Leu Arg, as shown in SEQ ID NO.1. Currently, there is no report on its relevant function.

[0029] 1) Modeling

[0030] Primary cortical astrocytes of rats were extracted and cultured, and then randomly divided into two groups: a group without any treatment and a group deprived of glucose and oxygen for 5 hours and then restored to glucose and oxygen perfusion for 2 hours.

[0031] First, determine whether the modeling is successful. The determination method is: after the perfusion is completed, use CCK8 reagent to test the changes in cell viability, and GFAP immunofluorescence staining to observe the degree of astrocyte activation.

[0032] The results are as follows Figure 1 As shown, from Figure 1 As can be seen from the middle panel A, the cell viability decreased to about 50% after 5h of oxygen-glucose deprivation and 2h of reperfusion; and the results of GFAP immunofluorescence staining showed that the average fluorescence intensity of the cells increased significantly after oxygen-glucose deprivation and reperfusion (panel B), which confirmed that the oxygen-glucose deprivation and reperfusion model of astrocytes was successfully established. Therefore, the group with 5h of oxygen-glucose deprivation and then 2h of oxygen-glucose perfusion was defined as the model group for subsequent studies.

[0033] 2) Effects of the peptide RKKRRQRRRAPSDPRLR on astrocytes in the model group after administration

[0034] Primary cultured rat cortical astrocytes were used as the research object. Primary cultured rat cortical astrocytes without any treatment were used as the normal control group (CON group). The cells treated with glucose and oxygen deprivation for 5 hours and then restored to glucose and oxygen perfusion for 2 hours were divided into two groups: one group was the model control group treated with glucose and oxygen deprivation and reperfusion only (OGD / R group), and the other group was intervened with the peptide RKKRRQRRRAPSDPRLR during reperfusion after glucose and oxygen deprivation, as the peptide intervention group (OGD / R+peptide).

[0035] Each experimental group was intervened according to the following scheme:

[0036] After the cells in the CON group were rinsed twice with PBS, normal DMEM high-glucose medium was added and the cells were continued to be cultured in a 37°C, 5% CO2 incubator; after the cells in the OGD / R group were rinsed twice with PBS, sugar-free DMEM medium was added and the cells were cultured in a hypoxic incubator at 37°C, 5% CO2, 0.1% O2, and 94.9% N2 for 5 h, and then the sugar-free DMEM medium was replaced with DMEM high-glucose medium and the cells were continued to be cultured in a 37°C, 5% CO2 incubator for 2 h; for the polypeptide intervention group (OGD / R+polypeptide), the cells were rinsed twice with PBS, sugar-free DMEM medium was added and the cells were cultured in a hypoxic incubator at 37°C, 5% CO2, 0.1% O2, and 94.9% N2 for 5 h, and then the sugar-free DMEM medium was replaced with DMEM high-glucose medium, and when the sugar and oxygen supply was restored, the polypeptide drug was added and diluted with DMEM high-glucose medium, and then the cells were continued to be cultured in a 37°C, 5% CO2 incubator for 2 h.

[0037] After the intervention, CCK8 cell viability test was performed on the astrocytes of each experimental group, and the morphological changes of the cells were observed under bright field conditions. GFAP immunofluorescence staining and live-dead double staining were used to observe the activation of astrocytes and the survival of the cells.

[0038] from Figure 2 and Figure 3 It can be seen that the cell viability of the model control group was significantly decreased compared with the normal control group ( Figure 3 A), the proportion of surviving cells decreased ( Figure 2 A and B in the middle), GFAP expression increased ( Figure 3 (B in the middle), its trend is similar to Figure 1 The displayed results remain consistent.

[0039] Compared with the model control group, after administration of 20 μM peptide drugs, cell viability was significantly increased (p < 0.001). Figure 3 (A in the middle).

[0040] Compared with the model control group, after restoring the glucose and oxygen supply and administering 20 μM peptide drugs, the GFAP activation of astrocytes decreased ( Figure 3 The results of CCK8 showed that the cell activity was significantly improved ( Figure 3 Compared with the model control group, after restoring the glucose and oxygen supply and administering 20 μM peptide drugs, the survival rate of astrocytes increased ( Figure 2 (A and B in the middle).

[0041] It can be seen that the polypeptide RKKRRQRRRAPSDPRLR provided in the present application has the effect of improving astrocyte damage caused by OGD / R, and can be further used to prepare drugs or reagents for treating corresponding injury diseases.

[0042] The above shows and describes the basic principles, main features and advantages of the present invention. However, the above is only a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation methods derived by any technician in the field without departing from the technical solution of the present invention should be included in the patent scope of the present invention.

Claims

1. A polypeptide, characterized in that The polypeptide sequence is RKKRRQRRRAPSDPRLR, as shown in SEQ ID NO.

1.

2. A biomaterial, characterized in that Including any one or more of 1) to 5): 1) A polypeptide or a polypeptide composition comprising the polypeptide according to claim 1; 2) a nucleic acid molecule encoding the polypeptide of claim 1; 3) A vector comprising a nucleic acid sequence encoding the polypeptide of claim 1; 4) a cell culture medium containing the polypeptide or polypeptide composition as described in 1); 5) Products obtained by conventionally modifying the amino acid side chain groups of the polypeptide of claim 1, or the amino terminus or carboxyl terminus of the polypeptide of claim 1.

3. The biomaterial according to claim 2, characterized in that The conventional modifications are amination, hydroxylation, carboxylation, amidation, carbonylation, alkylation, acetylation, esterification, glycosylation, cyclization or biotinylation.

4. A pharmaceutical preparation, characterized in that The pharmaceutical preparation contains the biomaterial according to claim 2, and the pharmaceutical preparation is in a pharmaceutically acceptable form, including but not limited to one or more of tablets, pills, granules, creams, patches, capsules, suppositories, oral solutions or injections.

5. Use of the polypeptide, biomaterial or pharmaceutical preparation according to any one of claims 1 to 4 in the preparation of a drug or pharmaceutical composition for treating OGD / R-induced damage to human brain astrocytes.

6. A pharmaceutical composition for treating OGD / R-induced damage to human brain astrocytes, characterized in that: The pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or a pharmaceutically active substance, and is prepared into any pharmaceutically acceptable dosage form. The pharmaceutically active substance comprises a pharmaceutically effective dose of the polypeptide according to claim 1.

7. A pharmaceutical composition for treating OGD / R-induced human brain astrocyte damage according to claim 6, characterized in that: The pharmaceutically acceptable carrier is an excipient, a filler, a diluent, a binder, a disintegrant, an absorption enhancer, a surfactant or a lubricant.

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