A polypeptide and uses thereof

Abstract

The application discloses a polypeptide and application thereof, and belongs to the technical field of biochemistry and pharmacy. The polypeptide sequence disclosed by the application is RKKRRQRRRAPSDPRLR, as shown in SEQ ID NO. 1, and is composed of 17 amino acids. At present, no related function has been reported. The polypeptide has an improvement effect on astrocyte glucose oxygen deprivation (OGD) / reperfusion (R) injury, and can improve the activity of damaged astrocytes. Meanwhile, the polypeptide has a good application prospect in preparation of a drug or a pharmaceutical composition for treating human brain astrocyte injury caused by OGD / R, and is expected to provide a new possibility for treatment of focal cerebral ischemia.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemical pharmaceutical technology, in particular to a polypeptide and its application, and specifically to a polypeptide RKKRRQRRRAPSDPRLR and its application in the preparation of a drug for treating OGD / R-induced damage to human brain astrocytes and a pharmaceutical composition. BACKGROUND

[0002] Astrocytes are a type of glial cells, and their number is 5 times that of neurons, and they are one of the main components of the central nervous system (CNS), playing an important role in biological processes such as structural support, regulation of brain 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, and playing an indispensable connecting role between cells. The oxygen-glucose deprivation and reperfusion (OGD / R) model is a model used to study the response of cells to reperfusion after ischemia after the restoration of glucose and oxygen supply after hypoxia and glucose deprivation. This model is often used to study the pathogenesis, drug efficacy and treatment strategies of brain ischemia and stroke and other diseases.

[0003] When cerebral ischemia occurs, the glucose and oxygen supply in the ischemic core area is completely stopped, causing irreversible damage to all neural 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 longer period of time. Studies using the oxygen-glucose deprivation (OGD) model have shown that astrocytes are more resistant to OGD than neurons; further studies have shown that astrocytes can generate ATP for energy using glycolysis during OGD, thereby exerting a neuroprotective effect. However, during ischemic stroke, due to persistent and severe hypoxia and glucose deprivation, i.e., hypoxia / ischemia exceeds the body's tolerance level, the regulatory function of astrocytes is impaired, inflammatory cytokines are produced, and the damage to the brain is increased, making it difficult to repair the damage even after oxygen is restored.

[0004] Therefore, finding an effective drug to help treat OGD / R-induced astrocyte damage has important theoretical significance and practical application value for hypoxic / ischemic brain injury, but in combination with clinical practice, the current drug for treating hypoxic damage to astrocytes does not have obvious efficacy.

[0005] Polypeptide is a compound formed by alpha-amino acids connected together by peptide bonds, which has the characteristics of both small molecule drugs and biological agents such as macromolecular proteins. Polypeptide exists in the form of enzymes, hormones, neurotransmitters, ion channel ligands, immune response mediators, etc. in the body, and can mimic various physiological metabolic pathways to regulate homeostasis when endogenous secretion is lacking. Polypeptide is the natural ligand of many receptors and ion channels, which makes its binding to the target or receptor specific, so it has higher selectivity, which can greatly avoid the side effects caused by off-target and trigger the immune response, and has lower immunogenicity. The half-life of polypeptide is short, and its metabolites will not accumulate in tissues, which also avoids the risk of metabolic toxicity.

[0006] Studies have shown that polypeptide drugs have outstanding performance in antibacterial, prevention and treatment of thrombosis, hypertension and hyperlipidemia, can delay aging, improve the body's anti-tumor ability, and have outstanding performance in diagnostic reagents. Therefore, it is feasible to use polypeptide drugs to study the regulation of astrocyte damage caused by oxygen-glucose deprivation and reperfusion, but there are few related reports in the field, and the corresponding polypeptide drug research and development has great development space and huge market blank. SUMMARY

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

[0008] In order to achieve the above technical purpose, the present application is realized by the following technical scheme: a polypeptide, the polypeptide sequence is RKKRRQRRRAPSDPRLR, and the amino acid sequence is shown as SEQ ID NO. 1.

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

[0010] 1) polypeptide or polypeptide composition, containing polypeptide RKKRRQRRRAPSDPRLR;

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

[0012] 3) vector, including a nucleic acid sequence encoding polypeptide RKKRRQRRRAPSDPRLR;

[0013] 4) cell culture solution, containing polypeptide or polypeptide composition as described in 1);

[0014] 5) products obtained by subjecting the amino acid side chain groups of the polypeptide RKKRRQRRRAPSDPRLR, the amino terminal or carboxyl terminal of the polypeptide RKKRRQRRRAPSDPRLR to conventional modifications.

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

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

[0017] The above-mentioned polypeptide, biomaterial or pharmaceutical preparation can be used for preparing a medicament and a pharmaceutical composition 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, which comprises a pharmaceutically acceptable carrier and / or a pharmaceutically active substance, and is prepared into any pharmaceutically acceptable dosage form, wherein the pharmaceutically active substance comprises a pharmaceutically effective dose of the polypeptide RKKRRQRRRAPSDPRLR.

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

[0020] The present application has the following beneficial effects:

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

[0022] 2. Since the polypeptide disclosed in the present application has an improving effect on astrocyte glucose-oxygen deprivation reperfusion injury, it can be used for preparing a medicament or a pharmaceutical composition for treating OGD / R-induced damage to human brain astrocytes, and promotes the development of the polypeptide RKKRRQRRRAPSDPRLR, which is expected to provide a new possibility for the treatment of focal cerebral ischemia. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1Figure 2 shows the vitality of astrocytes before and after modeling, and the effect of oxygen-glucose deprivation and reperfusion on the vitality of astrocytes. Subfigure A shows the vitality of astrocytes before and after modeling, and subfigure B shows the effect of oxygen-glucose deprivation and reperfusion on the vitality of astrocytes. CON refers to the normal untreated rat primary cortical astrocyte group, and O / R refers to the oxygen-glucose deprivation and reperfusion rat primary cortical astrocyte group. Subfigure C shows the GFAP immunofluorescence staining of the cells in the CON group, and subfigure D shows the GFAP immunofluorescence staining of the cells in the O / R group.

[0024] Figure 2 Figure 3 shows the results of live and dead double staining 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, polypeptide concentration is 20 μM), and the morphological changes of the cells under bright field conditions. Subfigure A is a statistical diagram of the live and dead ratio of the cells. Subfigure B is a live and dead double staining image of the primary cortical astrocytes. Subfigure C is a morphological analysis image of the primary cortical astrocytes.

[0025] Figure 3 Figure 4 shows the results of GFAP activation and CCK8 test 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). Subfigure A shows the vitality of astrocytes in different experimental groups. The 0.1 μM, 1 μM, 20 μM, and 100 μM marked on the horizontal axis represent the use of polypeptide drugs of corresponding concentrations to intervene in rat primary cortical astrocytes. Subfigure B shows the activation degree of astrocytes in different experimental groups. The 20 μM marked on the horizontal axis represents the use of 20 μM polypeptide drugs to intervene in rat primary cortical astrocytes. Subfigure C is a staining image of GFAP immunofluorescence staining of cells in different experimental groups. DETAILED DESCRIPTION

[0026] The following examples further illustrate the present application, but should not be construed as limiting the present application. Modifications and substitutions of the method, steps or conditions of the present application, without departing from the essence of the present application, all belong to the scope of the present application.

[0027] To identify therapeutic peptides that can improve oxygen-glucose deprivation / reperfusion injury in astrocytes, a bioinformatics approach using peptideomics analysis was used to establish a rat MCAO model. By analyzing differentially expressed peptides between the sham-operated and MCAO model groups, several peptides with potential bioactivity were identified. These peptides were then validated for bioactivity in an astrocyte oxygen-glucose deprivation / reperfusion model. The peptide RKKRRQRRRAPSDPRLR met the criteria. RKKRRQRRR is a penetrating peptide sequence that facilitates the entry of the peptide into cells, while APSDPRLR is the target sequence that primarily exerts its effect upon entry into the body. APSDPRLR has a molecular weight of 911.03, an isoelectric point of 9.64, and a mass-to-charge ratio of 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 rat cortical astrocytes 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 model is successful. The judgment 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 Panel A shows that cell viability decreased to approximately 50% after 5 hours of oxygen-glucose deprivation followed by 2 hours of reperfusion. Furthermore, GFAP immunofluorescence staining revealed a significant increase in the mean fluorescence intensity of cells after oxygen-glucose deprivation and reperfusion (Panel B), confirming the successful establishment of the oxygen-glucose deprivation and reperfusion model for astrocytes. Therefore, the group undergoing 5 hours of oxygen-glucose deprivation followed by 2 hours of oxygen-glucose reperfusion was designated 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 objects. Primary cultured rat cortical astrocytes without any treatment were used as the normal control group (CON group). The cells were treated with glucose and oxygen deprivation for 5 hours and then restored to glucose and oxygen perfusion for 2 hours and 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 glucose and oxygen deprivation and reperfusion, serving as the peptide intervention group (OGD / R+peptide).

[0035] Each experimental group received intervention according to the following scheme:

[0036] After the cells in the CON group were rinsed twice with PBS, they were added with normal DMEM high-glucose medium and continued to be cultured in a 37°C, 5% CO2 incubator. After the cells in the OGD / R group were rinsed twice with PBS, they were added with sugar-free DMEM medium and cultured in a hypoxic incubator at 37°C, 5% CO2, 0.1% O2, and 94.9% N2 for 5 h. Then, the sugar-free DMEM medium was replaced with DMEM high-glucose medium and cultured in a 37°C, 5% CO2 incubator for 2 h. For the peptide intervention group (OGD / R+peptide), the cells were rinsed twice with PBS, added with sugar-free DMEM medium and cultured in a hypoxic incubator at 37°C, 5% CO2, 0.1% O2, and 94.9% N2 for 5 h. Then, the sugar-free DMEM medium was replaced with DMEM high-glucose medium. When the sugar and oxygen supply was restored, the peptide drug was added and diluted with DMEM high-glucose medium. Then, the peptide drug was 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 panels), GFAP expression increased ( Figure 3 (Small Figure 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 20μM peptide drugs, cell viability was significantly improved (p<0.001). Figure 3 (Small image in center A).

[0040] Compared with the model control group, the GFAP activation of the astrocytes was reduced after the sugar and oxygen supply was restored and 20 μM polypeptide drugs were given ( Figure 3 Fig. 2B), and the CCK8 results showed that the cell activity was obviously improved ( Figure 3 Fig. 2A). Compared with the model control group, the survival proportion of the astrocytes was increased after the sugar and oxygen supply was restored and 20 μM polypeptide drugs were given ( Figure 2 Fig. 2A and B).

[0041] Therefore, the polypeptide RKKRRQRRRAPSDPRLR provided in the application has the effect of improving the astrocyte damage caused by OGD / R, and can be further used for preparing a drug or reagent for treating the corresponding damage diseases.

[0042] The above shows and describes the basic principles, main features and advantages of the present application. However, the above description is only a specific embodiment of the present application, and the technical features of the present application are not limited to this. Any other implementation derived by those skilled in the art without departing from the technical solution of the present application should be covered in the patent range of the present application.

Claims

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

1.

2. Biomaterial, characterized in that Including any one or more of 1) to 4): 1) A polypeptide or 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 described in 1).

3. A pharmaceutical preparation, characterized in that The pharmaceutical preparation contains the biomaterial according to claim 2 and is in a pharmaceutically acceptable form.

4. The pharmaceutical preparation according to claim 3, wherein The dosage form of the pharmaceutical preparation is one or more of tablets, pills, granules, creams, patches, capsules, suppositories, oral solutions or injections.

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