Polypeptide for treating ischemic stroke and application thereof

Abstract

The application discloses a polypeptide for treating ischemic stroke. The application also discloses application of the polypeptide in preparation of a medicine for treating ischemic stroke. The polypeptide provided by the application shows a protective effect on ischemic stroke disease models of cells and animals, and provides a new direction for future treatment of the disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptides, in particular to a polypeptide for treating ischemic stroke and application thereof. BACKGROUND

[0002] TRPM2, as a member of the TRP family, has structural similarities with other TRP channels. It has an intracellular N-terminal and C-terminal, 6 transmembrane domains, among which S5 and S6 form the channel pore region, which can permeate sodium ions, potassium ions and calcium ions. Previous studies have shown that ADPR, as a classic endogenous agonist of TRPM2, binds to the NUDT9H domain of the C-terminal, and unlike other TRP channels, TRPM2 requires dual ligand activation, i.e. ADPR and calcium ions. At the same time, TRPM2 can also be activated by cADPR, hydrogen peroxide, etc.

[0003] With the development of cryo-EM technology and further analysis of TRPM2 structure, it is found that the structure of hsTRPM2 (human TRPM2) can be divided into three layers, and there are three important interaction interfaces, namely interaction interfaces I and II formed within the subunit and interaction interface III formed between the subunits. As the first TRPM2 structure to be resolved, nvTRPM2 (sea anemone TRPM2) is found to be activated without the involvement of the C-terminal NUDT9H domain, which means that the interaction interface III found in hsTRPM2 does not play a role in the gating process of nvTRPM2. In the structure of drTRPM2 (zebrafish TRPM2), which is more advanced, it is found that apo-state TRPM2 exists within the subunit interaction, but after ADPR binds to the channel, the interaction between the subunits appears, which may help zebrafish TRPM2 to maintain in the activated state. Finally, looking at the gating process of hsTRPM2, it is found that after ADPR binds, the interaction interface III is broken, followed by further rotation of MHR and conformational change of TRPH1, which ultimately leads to the opening of the channel. These data show that from sea anemone to zebrafish and finally to human, the gating process of TRPM2 becomes more and more sophisticated and complex, and the importance of the interaction interface also becomes greater and greater.

[0004] Many studies have shown that TRPM2 is involved in oxidative stress-related diseases such as stroke and Alzheimer's disease.

[0005] Ischemic stroke is the most common type of stroke, accounting for about 85% of all strokes. It is caused by the blockage or reduction of blood flow to a certain part of the brain, leading to hypoxia and ischemia in that area, resulting in brain cell damage or death. Common causes include thrombosis caused by atherosclerosis and embolism caused by heart disease, etc.

[0006] In ischemic stroke, due to blood flow interruption, brain tissue is hypoxic, leading to the production of a large amount of reactive oxygen species (ROS). These ROS can activate the TRPM2 channel, leading to channel opening. After TRPM2 is activated, a large amount of calcium ions enter the cell through the channel, causing intracellular calcium overload. Calcium overload can trigger a series of fatal cellular responses, including mitochondrial damage, excessive activation of enzymes, and apoptosis or necrosis. The activation of TRPM2 is also associated with inflammatory response. Through increasing calcium ion concentration, TRPM2 can activate inflammatory signaling pathways, inducing the release of inflammatory factors such as TNF-α and IL-1β, which can exacerbate brain tissue damage. Therefore, TRPM2 plays a promoting role in the pathological process of ischemic stroke, exacerbating brain damage through calcium overload and inflammatory response. Therefore, inhibiting the function of TRPM2 may be an effective treatment strategy for reducing brain damage caused by ischemic stroke and improving patient prognosis.

[0007] The present application screens a polypeptide targeting TRPM2 interaction interface III. The inventors have carried out research on the protective activity of the polypeptide in ischemic stroke disease at the cell and animal levels. The results show that the polypeptide has protective activity, which will have a significant role in the future development of drugs for ischemic stroke. SUMMARY

[0008] The purpose of the present application is to provide a polypeptide for treating ischemic stroke and its application, in order to solve the problems in the prior art.

[0009] The purpose of the present application is achieved by the following technical solutions:

[0010] The present application provides a polypeptide for treating ischemic stroke, whose amino acid sequence is YILREDGLGERTCLD.

[0011] The present application provides the use of the above-mentioned polypeptide in the preparation of a drug for treating ischemic stroke.

[0012] The present application has the following beneficial effects:

[0013] The present application provides a polypeptide C0224 (YILREDGLGERTCLD), which shows protective effect in ischemic stroke disease models of cells and animals, providing a new direction for the treatment of this disease in the future. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1The results of IC50 detection of polypeptide C0224 and calcium imaging experiment; A, B, C, D, E, F are the results obtained by using whole cell patch clamp technology, wherein A, B, C, D, E, F are current maps after 10 nM, 100 nM, 300 nM, 500 nM, 1 uM and 100 uM C0224 are given in the electrode internal solution, G is the IC50 obtained according to the current maps of A, B, C, D, E, F; H, I are the real-time fluorescence change curve and the fluorescence statistical diagram of calcium imaging respectively; ACA is an inhibitor of TRPM2, and ADPR is an agonist of TRPM2.

[0015] Figure 2 The selective detection of polypeptide C0224; A, B are the current change maps of TRPM8 without giving and giving 100 uM C0224; C, D are the current change maps of TRPV2 without giving and giving 100 uM C0224; E, F are the current change maps of TRPV3 without giving and giving 100 uM C0224; G is Figure 1 F and Figure 2 A-D comparison result map. Menthol is menthol, 2-APB is 2-aminoethyl diphenyl borate, and wash is elution.

[0016] Figure 3 The results of cell OGD / R are shown in the figure; A is a schematic diagram of the OGD / R experiment process; B is a statistical diagram of the cck-8 experiment results under different OGD / R treatment conditions.

[0017] Figure 4 The effect of polypeptide c0224 on the infarction volume of the ischemic side of the brain of a mouse with cerebral ischemia-reperfusion injury; Figure 4 A is a representative image of TTC staining of the coronal section of the brain tissue of a mouse after 24 h of cerebral ischemia; Figure 4 B is a statistical diagram of the infarction volume. DETAILED DESCRIPTION

[0018] The present application will be further explained in conjunction with the examples and the accompanying drawings. The following examples are only used to illustrate the present application, but not to limit the scope of the present application.

[0019] Example 1

[0020] The polypeptide C0224 is screened according to the NC interaction interface of TRPM2, that is, the interaction interface III, and the amino acid sequence of the polypeptide C0224 is YILREDGLGERTCLD. In subsequent cell and animal experiments of ischemic stroke (calcium imaging in Example 2, Example 4, Example 5), a transmembrane sequence (YGRKKRRQRRR) is added to C0224 to form a transmembrane peptide C0224 (tat-C0224), and the amino acid sequence of tat-C0224 is YGRKKRRQRRRYILREDGLGERTCLD.

[0021] C0224 and tat-C0224 are synthesized by GenScript (Shanghai) Co., Ltd., and the purity is 95%. Before the experiment, the stock solution of C0224 and tat-C0224 is prepared with ddH2O to an appropriate concentration.

[0022] Example 2

[0023] Whole-cell patch clamp and calcium imaging are used to detect the activity of the polypeptide C0224

[0024] The whole-cell patch clamp experiment is as follows:

[0025] 1. Prepare experimental materials: HEK293T cells are cultured in a suitable culture medium (Gibco DMEM medium, item number: 8123194), and the cells are transfected with invitrogen TM Lipofectamine TM 3000 reagent 24 hours in advance, and the next day, the cells are plated on a glass slide according to the appropriate cell density (cell density 70%-80%).

[0026] 2. Prepare a glass microelectrode: draw a glass microelectrode, and then fill it with an electrode internal solution (the electrode internal solution is composed of 150 mM KCl, 0.05 mM EGTA, 10 mM HEPES, and 100 um ADPR, pH 7.3. According to the experimental requirements, different concentrations of C0224 are added to the electrode internal solution, including 10 nM, 100 nM, 300 nM, 500 nM, 1 uM and 100 uM). The tip of the electrode should be very smooth to ensure a good seal.

[0027] 3. Contact the cell membrane: under a microscope, use a manipulator to move the glass microelectrode to a single cell, and make the tip of the electrode contact the cell membrane.

[0028] 4. Form a seal: Apply slight negative pressure to form a high resistance seal (Giga-Ohm level) between the electrode tip and the cell membrane. At this point, enter the "cell-attached" mode.

[0029] 5. Break into whole-cell mode: Apply a rapid negative pressure or break the patch under the cell membrane by a transient electrical pulse to directly connect the microelectrode with the inside of the cell, enter the whole-cell mode, at this time the current change of the whole cell can be recorded.

[0030] 6. Record membrane current: Record the ion channel current on the cell membrane by voltage clamp mode (20uM ACA is given after the current rises and stabilizes to inhibit).

[0031] The steps of calcium imaging experiment are as follows:

[0032] 1. Cell culture and dye loading:

[0033] (1) The stable hsTRPM2 cells are cultured in appropriate culture medium (Gibco DMEM medium, item number: 8123194).

[0034] (2) The cells are stained with calcium indicator (such as Fura-8 AM).

[0035] (3) Wash to remove the dye (calcium indicator) that is not absorbed by the cells.

[0036] The stable hsTRPM2 cells are referred to Zhao S, Zhang H, Jin H, Cai X, Zhang R, Jin Z, Yang W, Yu P, Zhang L, Liu Z. Design, synthesis and biological activities of benzo[d]imidazo[1,2-a]imidazole derivatives as TRPM2-specfic inhibitors. Eur J Med Chem. 2021 Dec 5;225:113750. doi: 10.1016 / j.ejmech.2021.113750. Epub 2021 Aug 14. PMID: 34416664.

[0037] 2. Stimulate the cells:

[0038] Cells were stimulated with H2O2 to induce changes in calcium ion concentration. The experiment was divided into three groups: the H2O2 group, which was only given 500 uM H2O2, the H2O2 + tat-C0224 group, which was given 500 uM H2O2 + 100 uM tat-C0224, and the control group, which was not given any treatment. H2O2 and tat-C0224 were prepared with ddH2O.

[0039] 3. Real-time monitoring of calcium signals:

[0040] During the stimulation process, the dynamic changes of intracellular calcium ions were monitored in real time, and the concentration of calcium ions was indirectly measured by changes in fluorescence intensity.

[0041] The results, as shown in Figure 1 Whole-cell patch clamp experiments showed that the IC50 of C0224 was 508.7 nM; calcium imaging experiments showed that the fluorescence intensity of the group given 500 uM H2O2 + 100 uM tat-C0224 decreased significantly compared to the group given only 500 uM H2O2.

[0042] Example 3

[0043] Whole-cell patch clamp detection of the selectivity of polypeptide C0224

[0044] 1. Prepare experimental materials: HEK293T cells were cultured in a suitable culture medium (Gibco DMEM medium, item number: 8123194) and transfected with invitrogen TM Lipofectamine TM 3000 transfection reagent 24h in advance. The next day, the corresponding plasmids (TRPM8 plasmid, TRPV2 plasmid, TRPV3 plasmid) were transfected, and the cells were then plated on glass slides according to the appropriate cell density (cell density 70%-80%).

[0045] TRPM8 plasmid reference Xu L, Han Y, Chen X, Aierken A, Wen H, Zheng W, Wang H, Lu X, Zhao Z, Ma C, Liang P, Yang W, Yang S, Yang F. Molecular mechanisms underlying menthol binding and activation of TRPM8 ion channel. Nat Commun. 2020 Jul 29;11(1):3790. doi: 10.1038 / s41467-020-17582-x. PMID: 32728032; PMCID: PMC7391767.

[0046] TRPV2 plasmid reference Su N, Zhen W, Zhang H, Xu L, Jin Y, Chen X, Zhao C, Wang Q, Wang X, Li S, Wen H, Yang W, Guo J, Yang F. Structural mechanisms of TRPV2 modulation by endogenous and exogenous ligands. Nat Chem Biol. 2023 Jan;19(l):72-80. doi: 10.1038 / s41589-022-01139-8. Epub 2022 Sep 26. PMID: 36163384.

[0047] TRPV3 plasmid reference Cao X, Yang F, Zheng J, Wang K. Intracellular proton-mediated activation of TRPV3 channels accounts for the exfoliation effect of a-hydroxyl acids on keratinocytes. J Biol Chem. 2012 Jul 27;287(31):25905-16. doi: 10.1074 / jbc.M112.364869. Epub 2012 Jun 7. PMID: 22679014; PMCID: PMC3406675.

[0048] 2. Prepare glass microelectrode: Pull a glass microelectrode and then fill it with internal solution (internal solution formula: 150 mM KCl, 0.05 mM EGTA, 10 mM HEPES, pH 7.3, according to experimental requirements, add 100 uM C0224 to the internal solution). The electrode tip should be very smooth to ensure good sealing.

[0049] 3. Contact cell membrane: Under the microscope, use the manipulator to move the glass microelectrode to a single cell and make the electrode tip contact the cell membrane.

[0050] 4. Form a seal: Apply a slight negative pressure to form a high resistance seal (GΩ level) between the electrode tip and the cell membrane, at which point the "cell-attached" mode is entered.

[0051] 5. Break-in whole-cell mode: Apply a rapid negative pressure or break the patch under the cell membrane by a transient electrical pulse to connect the microelectrode directly with the inside of the cell, and enter the whole-cell mode. At this time, the current change of the whole cell can be recorded.

[0052] 6. Record membrane current: Record the ion channel current on the cell membrane by voltage clamp mode.

[0053] The results are shown in Figure 2 Fig. 4. C0224 has no obvious inhibitory effect on TRPM8, TRPV2 and TRPV3, indicating that C0224 has good selectivity.

[0054] Example 4

[0055] This example is divided into the following groups according to different treatment conditions: Control group: no oxygen-glucose deprivation and reperfusion treatment; OGD / R group: only oxygen-glucose deprivation and reperfusion treatment; 30uM S-tatC0224+OGD / R group: oxygen-glucose deprivation and reperfusion treatment and then 30uM S-tatC0224 (S-tatC0224 is tatC0224 with the C0224 sequence scrambled, and its amino acid sequence is YGRKKRRQRRRILYERDLGGRTEDCL) treatment; 1uM tatC0224+OGD / R group: oxygen-glucose deprivation and reperfusion treatment and then 1uM tatC0224 treatment; 10uM tatC0224+OGD / R group: oxygen-glucose deprivation and reperfusion treatment and then 10uM tatC0224 treatment; 30uM tatC0224+OGD / R group: oxygen-glucose deprivation and reperfusion treatment and then 30uM tatC0224 treatment.

[0056] 1. Establishment of SH-SY5Y cell oxygen-glucose deprivation model

[0057] (1) Cell preparation: SH-SY5Y neuroblastoma cells were seeded in culture dishes or flasks and cultured with Dulbecco's Modified Eagle Medium / Ham's F-12 (DMEM / F-12) medium containing 10v / v% fetal bovine serum (FBS).

[0058] (2) Cell state: When the cells were cultured to 70-80% confluence, the experiment was prepared.

[0059] (3) Change of culture medium (sugar-free environment): The cell culture medium was replaced with DMEM medium without sugar and serum to ensure that there was no glucose in the medium.

[0060] (4) Anaerobic environment: Placed in a hypoxic tube filled with mixed gas (95v / v% N2+ 5v / v% CO2) for 10 min to ensure that there is no oxygen in the culture medium.

[0061] (5) Treatment time: Cells were exposed to OGD conditions (OGD, oxygen-glucose deprivation) for 8 h.

[0062] 2. Reperfusion treatment

[0063] (1) Normal culture recovery: After OGD treatment, the anaerobic environment was removed, and the culture medium was quickly replaced with DMEM / F-12 medium containing 10v / v% FBS, and 30uM S-tatC0224+OGD / R group, 1uM tatC0224+OGD / R group, 10uM tatC0224+OGD / R group, 30uM tatC0224+OGD / R group were added with corresponding concentrations of S-tatC0224 or tatC0224.

[0064] (2) Reperfusion time: Cells were placed back in the regular incubator (37℃, 5v / v% CO2) for reperfusion (restored to the original sugar and oxygen environment) treatment, and the time was set to 12 h.

[0065] 3. Cell viability detection: Cell viability was detected by CCK-8 to evaluate cell survival rate.

[0066] The results are shown in Figure 3 , compared with the OGD / R group, the cell viability of the 10uM tatC0224+OGD / R group, the 30uM tatC0224+OGD / R group and the OGD / R group was significantly improved, indicating that the polypeptide C0224 had a protective effect on relieving the damage caused by OGD / R at 10uM and 30uM.

[0067] Example 5

[0068] Protective effect of polypeptide C0224 on ischemic stroke in mice

[0069] 1. Animal selection: Male C57BL / 6 mice weighing 22-25g were selected to ensure that all animals were in good health before the experiment began.

[0070] 2. Grouping:

[0071] (1) Sham group: After isolating the common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA), no transient middle cerebral artery occlusion (tMCAO) surgery was performed as a control.

[0072] (2) Model group (tMCAO group): After isolating CCA, ICA and ECA, tMCAO operation was performed. After pulling out the plug, only the same volume of normal saline (tail vein injection) as tatC0224 or S-tatCO224 was given, without any drug treatment.

[0073] (3) Treatment groups: After isolating CCA, ICA and ECA, tMCAO operation was performed. After pulling out the plug, 3 mg / kg mouse tatC0224 or S-tatCO224 treatment was given (tail vein injection, tatC0224 and S-tatCO224 were prepared with normal saline, 100 uL tatC0224 or S-tatCO224 was injected for each mouse).

[0074] 3. Operation steps:

[0075] (1) The mouse was weighed and induced anesthesia with 3 m / v% isoflurane in 30 v / v% O2 / 67 v / v% N2 mixed gas. The mouse was pinched between the toes of the foot to test the depth of anesthesia, and then maintained with 1.5 m / v% isoflurane in 30 v / v% O2 / 68.5 v / v% N2 mixed gas.

[0076] (2) After the mouse was anesthetized, it was placed in a supine position on the surface of the operation table, and the neck was exposed. After disinfecting the skin of the neck, a midline incision was made. Under the stereomicroscope, the right CCA, ECA and ICA were isolated, and the occipital artery and superior thyroid artery were coagulated and cut off.

[0077] (3) The ECA and CCA were ligated with 6-0 suture, and the ICA was clamped with an artery clamp. After cutting off the ECA, a wedge-shaped small opening was cut between the ECA and CCA bifurcation.

[0078] (4) The MCAO thread plug was inserted from the ECA incision, the artery clamp was loosened, and the thread plug was inserted through the CCA bifurcation and along the ICA direction to block the middle cerebral artery (MCA), and the thread plug was fixed.

[0079] (5) After 90 min of ischemia, the thread plug was pulled out for blood reperfusion, the ECA was ligated, the CCA suture was loosened, the neck incision was sutured, and the brain ischemia-reperfusion injury mouse was formed. After suturing, the model group was injected with normal saline through the tail vein, and the treatment group was injected with tatC0224 or S-tatCO224 through the tail vein.

[0080] 4. Brain infarction volume measurement: The brain was removed at 24 h after operation, sectioned and stained with 2, 3, 5-Triphenyltetrazolium Chloride (TTC) to evaluate the brain infarction volume. The specific operation method is as follows: After the mice were sacrificed by cervical dislocation, the brain was removed. The mouse brain tissue was placed in a -20℃ refrigerator for 15-20 min, quickly taken out and cut into 2 mm thick brain slices on ice in coronal position. The cut brain slices were immersed in 2 m / v% TTC solution, incubated at 37℃ for 30 min in the dark. Then the TTC solution was replaced with 4 m / v% paraformaldehyde (PFA) solution, and fixed at 4℃ overnight. The next day, the brain slices were scanned with a scanner, and the percentage of brain infarction volume was calculated by Image·J software. Because of brain edema after cerebral infarction, the following formula was used to calculate the brain infarction volume (%):

[0081]

[0082] The results are shown in Figure 4 , P is less than 0.0001, indicating that the modeling is successful, and after modeling, the brain infarction volume is significantly increased. Compared with the model group, the C0224 treatment group significantly reduced the brain infarction volume (P less than 0.01), indicating that CO224 has a protective effect on improving the brain infarction volume of mice with cerebral ischemia-reperfusion injury at 3 mg / kg. Compared with the model group, the brain infarction volume of the S-C0224 treatment group was not significantly different, indicating that S-C0224 has no protective effect on improving the brain infarction volume of mice with cerebral ischemia-reperfusion injury, indicating that the amino acids of CO224 must be arranged in a certain order to play a role, and not any random order.

Claims

1. A polypeptide for treating ischemic stroke, characterized in that, YILREDGLGERTCLD.

2. Use of the polypeptide of claim 1 in the preparation of a medicament for treating ischemic stroke.

Images