Neuroprotective small peptide taking proline as pharmacological active component and application thereof

Abstract

The invention relates to a neuroprotective small peptide with proline as a pharmacological active ingredient and application of the neuroprotective small peptide, the neuroprotective small peptide is a synthesized small peptide with the molecular weight smaller than 500Da, and the amino acid sequence of the neuroprotective small peptide comprises at least one unmodified proline which can be hydrolyzed into single free proline, at least one of peptide bonds connected with the proline is a protease hydrolysis point, so that the proline is hydrolyzed into free single amino acid to play a pharmacological role in protecting neurons; and at least one peptide terminal is connected with L-Phe or D-Phe, so that the small peptide can penetrate through a cell membrane and / or a blood brain barrier. Experiments prove that the small peptide can penetrate through the blood brain barrier to enter the brain and be hydrolyzed into single free proline in the brain, so that the proline can play a role in protecting neurons and brain tissues, and the small peptide can be used for preparing neuroprotective drugs for treating nervous system diseases, especially brain injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically to a neuroprotective small peptide with proline as a pharmacologically active ingredient and application thereof. Background Art

[0002] Ischemic brain injury refers to a series of brain cell and tissue damage caused by insufficient blood supply to the brain (ischemia). Taking stroke as an example, the ischemic area can be subdivided into two independent pathophysiological zones: the rapidly apoptotic necrotic core zone and the salvageable penumbra zone. The neurons in the ischemic core zone die within minutes to hours, while the penumbra neurons can repolarize after ischemia, but the energy required is large, and the body usually has difficulty supporting this polarization. There is currently no more effective treatment. Due to its high morbidity, high disability rate and high mortality rate, it has become the leading cause of death in my country and the most important cause of disability in adults.

[0003] Stroke includes ischemic stroke and hemorrhagic stroke. Ischemic stroke is mainly treated by intravenous thrombolysis, which is intravascular thrombolysis to enhance vascular recanalization, such as intravenous injection of thrombolytic drug rt-PA (recombinant tissue plasminogen activator). However, this drug needs to be treated within 3 hours after the onset of symptoms. Its treatment time window is short, and the patient may have missed the treatment time after admission. The use of neuroprotective peptides and neurorepair agents to ensure that the ischemic penumbra is protected from ischemic and hypoxic damage is an emerging treatment method. These drugs mainly exert neuroprotective effects by targeting one or several aspects of the potential pathophysiology of stroke. Although some drugs targeting stroke pathophysiology have achieved preclinical success, most of these drugs are difficult to replicate their significant preclinical performance in human stroke volunteers, mainly because these existing drugs are difficult to penetrate the blood-brain barrier (BBB) ​​and cannot reach therapeutic concentrations in the brain parenchyma.

[0004] Recent studies on single amino acids have shown that in addition to synthesizing proteins to provide nutrition to the body, single amino acids can also be used to treat diseases, such as proline. Proline is a non-essential amino acid. Although it has been shown to have neuroprotective properties in the central nervous system, the low permeability of proline to the blood-brain barrier (BBB) ​​limits its clinical application. Our team's previous studies have found that proline-loaded chitosan nanoparticles prepared by ionic crosslinking can effectively penetrate the blood-brain barrier and deliver proline to the damaged brain, thereby providing neuroprotection ("Proline-Loaded Chitosan Nanoparticles Penetrate the Blood-Brain Barrier to Confer Neuroprotection in Mice Cerebral Ischemia Injury"), but the biodegradability and potential toxicity of chitosan nanoparticles need further evaluation. Therefore, how to more safely and effectively promote proline to penetrate the blood-brain barrier and specifically target the site of brain injury is the main challenge currently faced by its application in the treatment of brain injury. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a neuroprotective small peptide with proline as a pharmacologically active ingredient and its application, the purpose of which is to find that D-phenylalanine can be used as a transport carrier to synthesize a small peptide with a molecular weight of less than 500 Da with proline and / or glycine, which can carry proline to penetrate the blood-brain barrier and enter neurons, and be hydrolyzed into free proline at the site of brain damage to exert its neuroprotective effect, thereby solving the technical problem that proline alone is difficult to penetrate the blood-brain barrier.

[0006] To achieve the above object, according to one aspect of the present invention, a neuroprotective small peptide with proline as a pharmacologically active ingredient is provided, which is a synthetic small peptide with a molecular weight of less than 500 Da, and its amino acid sequence includes at least one unmodified proline, and at least one of the peptide bonds connected to the proline is a protease hydrolysis point, so that it is hydrolyzed into free single amino acids to play a pharmacological effect of protecting neurons;

[0007] At least one peptide end is connected with L-Phe or D-Phe, so that the small peptide can penetrate the cell membrane or the blood-brain barrier.

[0008] Preferably, the neuroprotective small peptide is a small peptide containing 2 to 4 amino acids, and its amino acid sequence is selected as follows:

[0009] X1-X2-X3-F, where phenylalanine F is attached to the C-terminus of the small peptide;

[0010] In the sequence, one or two of X1, X2, and X3 are proline, P.

[0011] Preferably, the amino acid sequence of the neuroprotective peptide is selected as follows:

[0012] X1-X2-X3-F, where phenylalanine F is attached to the C-terminus of the small peptide;

[0013] In the sequence, one or two of X1, X2, and X3 are proline, P.

[0014] Preferably, in the amino acid sequence of the neuroprotective peptide, if X3 is P and X2 is G, then X1 is 0 or any amino acid;

[0015] If X2 is P, then X1 and X3 are G or F;

[0016] If X1 is P and X2 is 0, then X3 is 0, G or F;

[0017] Or when X1 is P and X2 is G or F, then X3 is 0 or any amino acid.

[0018] Preferably, the amino acid sequence of the neuroprotective peptide is selected as follows:

[0019] PF, PGF, GPF, GPGF, CGPF or PGPF.

[0020] Preferably, the amino acid sequence of the neuroprotective peptide is PF, PGF, or GPF.

[0021] According to another aspect of the present invention, there is also provided a use of the neuroprotective peptide according to the present invention in the preparation of drugs for treating nervous system diseases, wherein the nervous system diseases include neurodegenerative diseases and brain damage.

[0022] Preferably, in the application, the neurodegenerative disease is Alzheimer's disease or Parkinson's disease, and the brain injury is ischemic brain injury.

[0023] Preferably, the application is used for preparing a drug for treating ischemic brain injury.

[0024] Preferably, in the application, the neuroprotective peptide is used as an active ingredient of a drug, and its amino acid sequence is PF, PGF, GPF, PGPF, or GPGF.

[0025] Preferably, in the application, the amino acid sequence of the neuroprotective peptide is PF, PGF, or GPF.

[0026] In general, the above technical scheme conceived by the present invention is compared with the prior art. Since the present invention finds that proline has a neuroprotective effect, and also finds that dextrorotatory phenylalanine can penetrate the blood-brain barrier, it can be used as a transmembrane carrier for synthesizing a series of small peptides containing proline and L-Phe or D-Phe, and can achieve the following beneficial effects:

[0027] The neuroprotective small peptide provided by the present invention is a synthetic small peptide with a molecular weight of less than 500 Da, and its amino acid sequence includes at least one proline that can be hydrolyzed into a single unmodified proline, and at least one of the peptide bonds connected to the proline is a protease hydrolysis point, so that it is hydrolyzed into a free single amino acid to play a pharmacological effect of protecting neurons; and at least one peptide end is connected to L-Phe or D-Phe, so that the small peptide can penetrate the cell membrane or penetrate the blood-brain barrier to enter the brain, and be hydrolyzed into a single free proline in the brain, thereby playing the protective effect of proline on nerves and brain tissues, and can be used to prepare drugs for preventing or treating nervous system diseases or brain injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the change of Pro content after neuronal damage;

[0029] Figure 2 It is the change of Pro content in brain tissue after ischemia / reperfusion brain injury;

[0030] Figure 3 is the change of Pro content in brain tissue of infarcted area in I / R model rats;

[0031] Figure 4 The effect of different doses of proline injection on the infarct volume in rats with I / R model;

[0032] Figure 5 This is the purity test result of the dipeptide PF synthesized in Example 3;

[0033] Figure 6 is the effect of different doses of PF on the activity of neuronal cells after OGD / R injury;

[0034] Figure 7 is the effect of injection of different doses of PF on the infarct volume in I / R rats;

[0035] Figure 8 is a schematic diagram of the co-localization of drug PF and nerves;

[0036] In A, the green is FITC-PF, the red is NeuN, and B is the quantitative analysis result;

[0037] Fig. 9 This is the result of TTC staining analysis of brain sections of I / R rats after one or two injections of PF;

[0038] Fig.10 is the effect of PF, AC-PF, and Phe on cell survival after neuronal OGD / R injury;

[0039] Fig.11 is the effect of PF and AC-PF on Pro content in neurons after OGD / R;

[0040] Fig.12 is the result of TTC staining analysis of brain sections of I / R rats after injection of PF, AC-PF, and Phe;

[0041] Fig.13 The purity test results of PGPF and GPF synthesized in Example 5 are shown in FIG. 5 , wherein the left side shows the purity analysis result of PGPF and the right side shows the purity analysis result of GPF;

[0042] Fig.14 It is a schematic diagram of the fluorescence distribution of FITC, FITC-PGPF, and FITC-GPF in the brain tissue of I / R rats; Fig.14 A and B in the middle are both left-sided infarctions, in which A in the left picture is injected with FITC, B is injected with FITC-PGPF, and in the right picture A is injected with FITC, B is injected with FITC-GPF;

[0043] Fig.15 is the comparison of fluorescence intensity between the healthy and affected sides in the brain tissue of I / R rats; Fig.15 A in the middle is the fluorescence image of the healthy side, and B is the fluorescence image of the affected side, where the upper image is the injection of FITC-PGPF, and the lower image is the injection of FITC-GPF;

[0044] Fig.16 It is the fluorescence image of the affected side in the brain tissue of I / R rats after injection of FITC, FITC-PGPF, and FITC-GPF;

[0045] Fig.17 is the effect of different concentrations of PGPF on neuronal cell survival after OGD / R;

[0046] Fig.18 is the effect of different concentrations of PGPF on the infarct volume in I / R rats;

[0047] Fig.19 is the effect of different concentrations of GPF on neuronal cell survival after OGD / R;

[0048] Fig. 20 The effect of different concentrations of GPF on the cerebral infarction volume in I / R rats. DETAILED DESCRIPTION

[0049] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0050] The amino acid abbreviations are as follows:

[0051] Glycine: Gly, G; Phenylalanine: Phe, F, including L-Phe for L-phenylalanine and D-Phe for D-phenylalanine; Proline: Pro, P; Arginine: Arg, R.

[0052] Proline (Pro) is a non-essential amino acid. Unlike the amino acids that generally make up proteins, Pro has no free α-amino group and is an α-imino acid. Pro plays a key role in maintaining osmotic pressure, regulating carbon and nitrogen metabolism, regulating cell signaling pathways and promoting cell survival. The synthetic peptide in patent CN 106220709 B (a synthetic peptide that easily penetrates the blood-brain barrier) uses L-Phe as a transport carrier to carry CG through the blood-brain barrier, enter the brain and hydrolyze to produce free Gly and L-Cys to exert neuroprotective effects, among which L-Pro promotes the penetration of synthetic peptides through the cell membrane. The preliminary experimental results of this team show that proline (Pro, P) has a neuroprotective effect and can be used as a pharmacologically active ingredient of neuroprotective small peptides, but single free proline has poor penetration of the blood-brain barrier, making it difficult to exert a good pharmacodynamic effect in the brain and cannot be directly used clinically.

[0053] The present invention has found that D-phenylalanine can also be used as a carrier to penetrate the cell membrane. In order to study how to make proline safely and effectively pass through the blood-brain barrier and play a protective role at the site of brain injury, we synthesized a series of small peptides with a molecular weight of less than 500 Da, whose amino acid sequence includes at least one unmodified proline, and at least one of the peptide bonds connected to the proline is a protease hydrolysis point, so that it is hydrolyzed into a free single amino acid to play a pharmacological role in protecting neurons; and at least one peptide end is connected to L-Phe or D-Phe; preferably, its amino acid sequence also includes glycine, so that it can be used as one of the pharmacologically active ingredients after hydrolysis. Experimental verification shows that this series of small peptides can penetrate the cell membrane and the blood-brain barrier, and can be hydrolyzed into a single free proline in the brain to play its role in protecting nerves. It is a safe and effective neuroprotective small peptide that can be used to prepare relevant drugs for the treatment of nervous system diseases.

[0054] The neuroprotective small peptide can reduce the volume of cerebral infarction and has a significant effect in treating ischemic brain injury such as ischemic stroke, especially polypeptides with a molecular weight of less than 400 Da such as PF and PGF.

[0055] Based on this, the present invention provides a neuroprotective small peptide with proline as a pharmacologically active ingredient. The neuroprotective small peptide is a synthetic small peptide with a molecular weight of less than 500 Da, and its amino acid sequence includes at least one unmodified proline (Pro, P), and at least one of the peptide bonds connected to the proline is a protease hydrolysis site, which can hydrolyze the small peptide to generate a single free proline; and at least one peptide end is connected to L-Phe or D-Phe that can carry proline to penetrate the blood-brain barrier. As a transport carrier, it can enable the small peptide to smoothly penetrate the blood-brain barrier and enter the brain tissue, and concentrate on neurons in the brain damage site, hydrolyze to generate at least one free natural proline, and then play its role in protecting nerves and reducing brain damage.

[0056] Preferably, the neuroprotective small peptide is a synthetic small peptide containing 2 to 4 amino acids, and its amino acid sequence is selected as follows:

[0057] X1-X2-X3-F, where phenylalanine F is attached to the C-terminus of the small peptide;

[0058] One or two of X1, X2, and X3 in the sequence are proline, P, which can be hydrolyzed into a single free proline.

[0059] Wherein, if X3 is P and X2 is G, then X1 is 0 or any amino acid, preferably an amino acid with neuroprotective effect. In this type of small peptide, the peptide bond between F and P at the C-terminus and the peptide bond between P and G can be hydrolyzed by peptidases to release a free single proline (X3).

[0060] If X2 is P, then X1 and X3 are G or F. This type of small peptide can release a free single proline (X2) after hydrolysis;

[0061] If X1 is P and X2 is 0, then X3 is 0, G or F; or if X1 is P and X2 is G or F, then X3 is 0 or any amino acid, preferably an amino acid with neuroprotective effect. Such small peptides can release a free single proline (X1) after hydrolysis.

[0062] In some embodiments, the amino acid sequence of the neuroprotective peptide includes the following:

[0063] PF, PGF, GPF, GPGF, or X1-GPF, wherein X1 is any amino acid, preferably an amino acid with neuroprotective properties or an amino acid capable of penetrating the blood-brain barrier, such as P, G, C, R, or F; more preferably, X1 is P or C, and the polypeptide can be hydrolyzed to produce more neuroprotective proline or cysteine.

[0064] In some embodiments, the neuroprotective small peptide is a synthetic dipeptide with an amino acid sequence of PF; this type of synthetic peptide contains a proteolytic enzyme cleavage site, i.e., the connection point between Phe at one end of the peptide and any amino acid, and free natural proline is generated after proteolytic enzyme cleavage.

[0065] In some embodiments, the neuroprotective small peptide is a synthetic tripeptide whose amino acid sequence is PGF and GPF; there are two proteolytic enzyme cleavage sites in this type of synthetic peptide, one is the connection point between Phe at one end of the peptide and any amino acid, and the other is the connection point between Pro and Gly. After proteolytic enzyme cleavage, free proline and glycine are produced, wherein the proline is natural proline.

[0066] In some embodiments, the neuroprotective small peptide is a synthetic tetrapeptide whose amino acid sequence is GPGF or X1-GPF, wherein X1 is P or C. There are two proteolytic enzyme cleavage sites in this type of synthetic peptide, one is the connection point between Phe at one end of the peptide and any amino acid, and the other is the connection point between Pro and Gly. Free natural proline is produced after proteolytic enzyme cleavage.

[0067] The neuroprotective small peptide of the present invention is preferably a small peptide with a molecular weight of ≤400 Da, which is more conducive to successfully penetrating the blood-brain barrier.

[0068] In addition, the present invention also provides a use of the neuroprotective small peptide according to the present invention in the preparation of drugs for preventing or treating nervous system diseases.

[0069] The nervous system diseases include neurodegenerative diseases and brain damage. Neurodegenerative diseases include Alzheimer's disease and Parkinson's disease; brain damage includes ischemic brain damage such as ischemic stroke. It is preferably used in the preparation of drugs for treating ischemic brain damage.

[0070] The neuroprotective peptide is used as an active ingredient of the drug, and its amino acid sequence is PF, PGF, GPF, PGPF, or GPGF; preferably, its amino acid sequence is PF, PGF, or GPF.

[0071] In some embodiments, the neuroprotective peptide directly acts on damaged neurons, preferably the concentration of dipeptide PF is 200 μmol / L, the concentration of tripeptide GPF is 200-250 μmol / L, or the concentration of tetrapeptide PGPF is 100-120 μmol / L.

[0072] In some embodiments, the neuroprotective peptide acts on damaged brain tissue. Taking the injection concentration in mice as an example, the preferred injection concentration of dipeptide PF is 50 mg / kg, the injection concentration of tripeptide GPF is 50 mg / kg, or the injection concentration of tetrapeptide PGPF is 30-40 mg / kg, and the more preferred injection concentration of tetrapeptide PGPF is 3 mg / kg.

[0073] In the present invention, the conversion can be performed according to the conversion relationship between existing cell experiments, animal experiments and human experiments, so as to obtain the appropriate injection concentration and injection dose corresponding to the human body.

[0074] The following are examples

[0075] The following examples use adult male Sprague-Dawley (SD) rats and pregnant rats, with male rats weighing 250g and pregnant rats with a gestation period of 17 to 20 days, all purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. 2 to 3 rats are kept in each cage, with a room temperature of 23 to 25°C. Except for experimental requirements, there is sufficient water and bedding is changed regularly. The experimental mice were acclimatized in the animal room for one week before the experiment and were randomly grouped during the experiment. Animal ethics number: QDU-AEC-2023423. The preparation of some of the main solutions is as follows:

[0076] (1) Electrophoresis buffer: Use an electronic balance to weigh 144 g of glycine, 30.3 g of Tris-Base, and 10 g of SDS, dilute to 1 L with double distilled water, add magnetic beads, and mix thoroughly with a magnetic stirrer to prepare 10× electrophoresis buffer. Keep it at room temperature for use. When using, dilute 100 mL of 10× electrophoresis buffer to 1 L with double distilled water for western blot experiments.

[0077] (2) Transfer solution: Weigh 144 g of glycine and 30.3 g of Tris-Base on a balance, pour into a reagent bottle, dilute to 1 L with double distilled water, add magnetic beads, mix thoroughly with a magnetic stirrer, prepare 10× transfer solution, and keep it at room temperature for use. When using, add 200 mL of methanol to every 100 mL of 10× electrophoresis solution, dilute to 1 L with double distilled water, cool in ice, and then perform western blot experiments.

[0078] (3) TBST buffer (1×): Use an electronic balance to weigh 16 g of NaCl, 0.4 g of KCl, 6 g of Tris-Base, and 2 mL of Tween 20. Add deionized water to make up to 2 L. Adjust the pH to 7.4 with hydrochloric acid and keep at room temperature for later use.

[0079] (4) 5% skim milk powder: Weigh 5% skim milk powder by mass according to the required amount on a balance and pour it into a centrifuge tube. Add TBST buffer (1×) to the volume and vortex the tube. Mix thoroughly and store at 4°C.

[0080] (5) Neuron culture medium: Neurobasal Medium: B-27 additive: glutaMax additive: penicillin-streptomycin = 96:2:1:1, mixed evenly to prepare neuron culture medium. It is recommended to prepare it in bottles for distribution and use.

[0081] (6) Sugar-free extracellular solution (ECS-): Weigh 9.01 g NaCl, 0.40 g KCl, 0.203 g MgCl2·6H2O, 5.96 g Tris-Base, and 0.222 g CaCl2 on a balance, pour into a reagent bottle, make up to 1 L with double distilled water, mix thoroughly, and adjust the pH to 7.30-7.40.

[0082] (7) Extracellular solution with glucose (ECS+): Weigh 9.01 g NaCl, 0.40 g KCl, 0.203 g MgCl2·6H2O, 5.96 g Tris-Base, 0.222 g CaCl2, and 6.54 g glucose on a balance, pour into a reagent bottle, make up to 1 L with double distilled water, mix thoroughly, and adjust the pH to 7.30-7.40.

[0083] (8) 2,3,5-triphenyl tetrazoliumchloride (TTC) dye solution: Weigh 1 g of TTC powder on a balance, add PBS buffer to 50 mL, shake well to mix, and prepare a TTC dye solution with a mass fraction of 2%. Cut the blood-free fresh tissue block with ophthalmic scissors, add TTC dye solution, and place it in a 37°C incubator for activation. After 6 hours, it will be successfully activated and stored at 4°C for use. It needs to be placed in a 37°C incubator for 30 minutes before each use.

[0084] (9) 30% sucrose solution: Weigh 30% sucrose by mass on a balance and pour into a reagent bottle. Make up to volume with double distilled water, add magnetic beads and place on a magnetic stirrer to mix thoroughly. Store at 4°C until used.

[0085] (10) Acetate buffer: High performance liquid chromatography (HPLC) requires the preparation of 500 mL of 1 mol / L acetic acid solution. Based on the density of glacial acetic acid being 1.05 mol / L, the mass percent concentration being 99%, and the molar concentration being 17.5 mol / L, the required volume of glacial acetic acid can be calculated as "1×0.5×60÷1.05=28.6 mL" (glacial acetic acid is liquid above 16.7°C). Dissolve 8.2 g of sodium acetate in water, add the prepared 1 mL of acetic acid solution, filter, and dilute to 1 L with pure water.

[0086] (11) Physiological saline: Weigh 9 g of NaCl using an electronic balance, dilute to 1 L with deionized water, mix thoroughly and keep at room temperature for later use.

[0087] (12) Tissue lysis buffer: RIPA: PMSF: protein phosphatase inhibitor = 100:1:1.

[0088] (3) Synthesis of polypeptides: Referring to the solid phase synthesis method in patent CN 106188235 B, the polypeptides required in the following examples were synthesized and purified and separated by HPLC, wherein the phenylalanine was dextrorotatory phenylalanine (D-Phe).

[0089] Example 1 Pro in neurons decreases after ischemia / reperfusion brain injury

[0090] In order to study the relationship between Pro and ischemic brain injury, this example observed the changes in Pro content in rat cortical neurons after ischemia / reperfusion injury through an in vitro cell model, as follows:

[0091] The OGD / R model is an in vitro model used to study cerebral ischemia and neuronal injury. In this model, neurons or neural cell cultures are deprived of oxygen and glucose to simulate cerebral ischemia, and then the oxygen and glucose supply is restored to simulate the reperfusion process. The details are as follows:

[0092] ①Build OGD / R model

[0093] Primary neuron culture: Pregnant mice were killed by dislocation of the neck and disinfected with alcohol. The abdominal skin and tissue were cut open to remove the whole bunch of embryos wrapped in the uterus. The amniotic membranes that wrapped the embryos were cut open one by one, the embryo heads were cut off from the neck, the skull was cut open along the middle of the skull to expose the brain tissue, and the complete brain tissue of the fetal mice was removed. The left and right brains were separated and the cerebral cortex was taken. It was placed in a 15mL centrifuge tube filled with DMEM and centrifuged (10000r, 15min). The upper separation solution was discarded, 0.05% trypsin was added and slowly blown 3 to 5 times, and the mixture was placed in an incubator for digestion for 15 to 20 minutes. Two times the volume of DMEM containing serum was added and slowly blown 2 to 3 times to terminate the digestion. The above mixture was sieved using a cell sieve to remove cell tissues other than cortical neurons.

[0094] Centrifuge the sieved mixture at 10,000r for 15min. Discard the supernatant, add 10mL of the prepared neuron culture medium, and blow slowly to disperse the neuron cells. Pipette 10μL from the evenly blown culture medium into the cell counting plate, count using a cell counter under a microscope, repeat three times and take the average value. Calculate the number of wells according to the counting results, add the corresponding neuron culture medium according to the number of wells, put it into the neuron incubator, and culture it for 12 days for use. Observe the cell state under a microscope before the experiment to ensure that the neurons selected for each group of experiments are in good condition and have similar density.

[0095] ②Construct an OGD / R model to detect the Pro content after neuronal injury: aspirate the culture medium in the well plate of neurons that require OGD, wash with sterile PBS buffer 1 to 3 times, add ECS-, and then place the well plate in an anaerobic workstation (temperature: 37°C, gas parameters: 1% O2+94% N2+5% CO2) for 2 to 3 hours of glucose and oxygen deprivation, resulting in 50%-60% cell damage. Perform resugar and reoxygenation to simulate reperfusion injury, construct an OGD / R model, and detect the Pro content in the normal control group (Non-OGD / R), 3h, and 6h after OGD / R. In the normal control group (Non-OGD / R), aspirate the culture medium in the well plate of neurons in this group, replace it with ECS+ and place it in an incubator. The test results of each group are as follows Figure 1 As shown, the results showed that Pro content was significantly reduced after neuronal injury.

[0096] Example 2 Pro in brain tissue decreases after ischemia / reperfusion brain injury

[0097] Ischemia / reperfusion (I / R) animal model is an important tool for studying myocardial ischemia / reperfusion injury, cerebral ischemia / reperfusion injury, ischemic stroke and other diseases. Ischemia / reperfusion is a disease caused by stenosis or occlusion of the cerebral blood supply artery, resulting in insufficient blood supply to the brain, which in turn causes brain tissue necrosis. The I / R model simulates the pathological and physiological process caused by temporary interruption of blood flow (ischemia) and subsequent restoration of blood flow (reperfusion) in the human body. This embodiment uses the suture method to construct a rat ischemia / reperfusion (I / R) animal model, and its path is common carotid artery-internal carotid artery-middle cerebral artery. The specific surgical operation is briefly described as follows:

[0098] (1) Animal grouping and preoperative preparation

[0099] The rats required for the experiment were randomly divided into sham group, ischemia / reperfusion (I / R) group, control group (Vehicle), drug treatment group (Pro), etc. SD rats were weighed and fasted one night in advance. 75% alcohol, iodine, cotton balls, cotton swabs, restraint bandages, 6-0 type sterile silk thread, 3-0 type sterile silk thread, electrocoagulation pen, 3600 thread plug, animal isoflurane, etc., which were needed for the surgery, were prepared. The surgical instruments were sterilized one night in advance for standby use.

[0100] The rat was placed in the induction box of the gas anesthesia machine and anesthesia was quickly induced using 3% isoflurane. The rat was taken out after the tail pinching reflex disappeared, placed in supine position on the constant temperature pad on the operating table and inhaled 1.5-2% isoflurane using an anesthesia mask, and fixed with a restraint bandage. Before the operation, the rat was observed to see whether the tail pinching reflex disappeared, the breathing was stable, and the color of the gingival mucosa was normal. The rat's tongue was pulled out to ensure smooth breathing. After all the above were confirmed, the surgical operation began.

[0101] (2) Construction of ischemia / reperfusion (I / R) animal model

[0102] ① Neck surgery: After the rats were anesthetized, the neck was shaved and disinfected, the skin was incised along the midline of the neck, and the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were separated.

[0103] ② Ligation of the proximal end of the common carotid artery: To protect the vagus nerve, use 6-0 sterile thread to tie a slipknot at the proximal end of the common carotid artery to block blood flow, continue to peel upward along the external carotid artery to expose the external carotid artery, pass through the double strands of 6-0 sterile silk thread and cut it into a single strand with scissors, tie a knot with the upper sterile thread (tighten the knot as far away from the bifurcation of the common carotid artery as possible), and tie a slipknot with the lower sterile thread, leaving a certain distance between the two threads for plugging the opening. Continue to peel the internal carotid artery, thread the thread away from the bifurcation of the blood vessel, tie a slipknot and tighten it to block blood flow, so that the blood vessel forms a closed loop.

[0104] ③ Insert the thread plug: Open the slipknot of the internal carotid artery, make sure there is no blood flowing out, and cut the blood vessel above the opening of the external carotid artery. Fold the thread plug toward the internal carotid artery and insert it into the internal carotid artery until you see the black dot of the thread plug and there is slight resistance. Stop inserting the thread plug to block the blood flow of the MCA and cause focal cerebral ischemia.

[0105] ④Fix the thread plug: After the thread plug enters the middle cerebral artery, tie the sterile thread at the opening and immediately record the time. Next, open the slipknot at the common carotid artery, put all the sterile threads and thread plugs into the neck opening, and then suture the wound with 3-0 sterile silk thread and disinfect it.

[0106] ⑤ Reperfusion: 90 minutes after the embolization, the rat was anesthetized and placed on the operating table using the same method as above, the suture was cut, the slipknot at the common carotid artery was tied, and the embolization was gently pulled out. The common carotid artery was opened, the blood supply to the middle cerebral artery was restored, and reperfusion was performed. The untied sterile thread was removed, the skin was sutured, iodine was used for disinfection, and a label was hung to mark it. At the same time, attention was paid to intraoperative and postoperative care.

[0107] To verify whether the rat I / R model was successfully constructed, this example used a laser Doppler blood flowmeter to observe the changes in local blood flow in the left and right hemispheres of the rats after modeling. The results are as follows: Figure 2 As shown in the figure, the local blood flow on the ischemic side was significantly lower than that on the contralateral side, indicating that the rat I / R model was successfully established. In addition, the changes in Pro content in rat brain tissue after ischemia / reperfusion injury were observed. The Pro content in the brain tissue of the infarct site was measured 3h and 6h after I / R. The results are shown in the figure. Figure 3 As shown in Figure 3, Pro was also significantly decreased in rat brain tissue after ischemia / reperfusion injury.

[0108] From the results of Examples 1 and 2, it can be seen that the Pro content in rat cortical neurons and brain tissues was significantly reduced after ischemia / reperfusion injury. It is speculated that the reduction in Pro content may be involved in the process of ischemia / reperfusion brain injury, and supplementation of Pro may reduce the damage of ischemia / reperfusion to neurons and brain tissues.

[0109] Furthermore, to establish a rat I / R model, 200 mg / kg, 400 mg / kg, 600 mg / kg, and 800 mg / kg of proline were injected into the tail vein immediately after thrombus removal. The brain was perfused and harvested 1 hour after tail vein injection. TTC-stained sections of the rat brain were sliced ​​and analyzed for cerebral infarction volume 24 hours later. The results are as follows: Figure 4 .

[0110] The results showed that compared with the I / R+Vehicle group, the drug treatment group was intravenously injected with 200-800 mg / kg Pro, and only the concentrations of 600 mg / kg and 800 mg / kg groups showed a significant increase in Pro content in ischemic brain tissue and a significant decrease in cerebral infarction volume. However, in the group injected with Pro concentrations below 400 mg / kg, there was no significant change in Pro content in ischemic brain tissue and cerebral infarction volume compared with the control group. It can be seen that although Pro has a neuroprotective effect, it needs to be injected at a high concentration to enter the brain tissue to exert a neuroprotective effect, thereby reducing the volume of cerebral infarction, indicating that Pro has low penetration of the blood-brain barrier.

[0111] Example 3 Effect of PF treatment on Pro content in ischemic / reperfused neurons and brain tissue

[0112] In patent CN 106220709 B, L-Phe is used as a transport carrier to carry GC through the blood-brain barrier and enter the brain tissue. In order to study whether D-Phe can also be used as a transport carrier, this example dehydrates unmodified Pro and D-Phe to synthesize a dipeptide, abbreviated as PF, with a molecular weight of 262.31 Da. HPLC separation and purification are used, and the mass report of PF is as follows: Figure 5 .

[0113] Depend on Figure 5 It can be seen that the purity of PF calculated from the peak area by HPLC purity analysis of PF is above 98%.

[0114] The method for constructing the OGD / R model is the same as in Example 1. After the model is successfully constructed, the groups are treated with drugs. Among them, the drug group: after OGD, the neuron culture plate is taken out, and the ECS-solution is replaced with neuron culture medium, and drug treatment is performed (PF: 50μmol / L, 100μmol / L, 150μmol / L, 200μmol / L, 250μmol / L), and placed in an incubator for reoxygenation for a specified time, with OGD / R as a blank control. At this time, the culture medium of the Non-OGD / R group is also aspirated and an equal volume of sterile PBS buffer is added to the drug. After 24 hours, the CCK-8 method is used to detect the cell activity of neurons after OGD / R injury. The results are as follows Figure 6 .

[0115] Depend on Figure 6 Compared with the OGD / R group, the survival rate of cells added with PF after OGD / R injury increased, indicating that PF has a neuroprotective effect, and the protective effect gradually increases within a certain range. The optimal addition concentration is 200 μmol / L.

[0116] Immediately after I / R, rats in each group were injected with 30 mg / kg, 40 mg / kg, and 50 mg / kg of PF via tail vein. After 24 hours, the brains were harvested and the whole brain infarction volume was determined by TTC staining. Figure 7 .

[0117] Depend on Figure 7 Compared with the I / R+Vehicle group, the whole brain infarction volume of rats injected with PF after I / R decreased significantly, and the infarction volume showed a downward trend with the increase of PF concentration. Proline is difficult to penetrate the blood-brain barrier, so it is speculated that D-Phe can penetrate the blood-brain barrier, prompting the dipeptide PF to penetrate the blood-brain barrier, and then carry proline into the brain to play its role in protecting nerves and brain.

[0118] In order to make the results more intuitive, this example further uses NeuN and FITC-PF for co-staining to verify that PF can penetrate the BBB in the rat I / R model; NeuN (neuronal nuclear antigen) is a protein specifically expressed in neurons, mainly localized in the neuronal nuclei and the cytoplasm around the nuclei of the central nervous system of mammals, as follows:

[0119] After the I / R model was successfully established, the drug group was immediately injected with FITC-PF (0.13mmol / g) via the tail vein, and the injection of FITC (fluorescein isothiocyanate, which can emit green fluorescence when excited by ultraviolet light, 0.13mmol / g) was used as a blank control; the sham group was also injected with equal amounts of FITC-PF and FITC. The co-localization of nerves and drugs in each group was detected, and the results are shown in Figure 2. Figure 8 As shown in A, FITC-PF (green), NeuN (red), scale bar = 50 μm; Figure 8 The quantitative analysis results of A are as follows Figure 8 As shown in B.

[0120] The results showed that intravenous injection could allow FITC-PF to enter the ischemic brain tissue in large quantities and co-localize with nerves, indicating that PF could not only penetrate the blood-brain barrier, but also enter neurons and be hydrolyzed into single free proline in brain tissue, exerting its neuroprotective effect and reducing the volume of cerebral infarction in I / R rats.

[0121] Further mechanistic studies found that proline exerts a neuroprotective effect by regulating the PTEN / EMSY signaling pathway in neurons after oxygen-glucose deprivation / reoxygenation (OGD / R), inhibiting neuronal ferroptosis.

[0122] Example 4 The therapeutic effect of two injections of PF on ischemic stroke

[0123] This example simulates multiple administrations in clinical practice, as follows:

[0124] After the rat I / R model was successfully established, 50 mg / kg PF was immediately injected into the tail vein of the drug-treated group 1, and the brain was harvested by perfusion 24 hours later, while the blank control group was injected with the same dose of normal saline;

[0125] The drug-treated group 2 was immediately injected with 50 mg / kg PF in the tail vein, and 5 hours later, a second injection of PF of the same dose was given. After 24 hours, the brain was perfused and removed. After 24 hours, TTC staining sections of the rat brains of each group were analyzed. The results are shown in the figure. Fig. 9 shown.

[0126] The experimental results show that compared with drug treatment group 1 (one injection of PF into the tail vein immediately after I / R), the whole brain infarction volume of rats in drug treatment group 2 (one injection of PF into the tail vein immediately after I / R, and the second injection 5 hours later) was significantly reduced, indicating that PF can still play a neuroprotective effect and reduce the volume of cerebral infarction when administered within 5 hours after I / R.

[0127] rt-PA (recombinant tissue plasminogen activator) is a drug used to dissolve blood clots. It belongs to the glycoprotein class of drugs, which directly activates plasminogen to convert into plasmin, thereby degrading fibrin and dissolving blood clots. Currently, rt-PA is mainly used to treat acute myocardial infarction, acute massive pulmonary embolism, and ischemic stroke, but rt-PA treatment must be administered within 3 hours after the onset of symptoms. It can be seen that compared with the existing rt-PA, PF has a longer administration time, which is beneficial for the treatment of acute myocardial infarction, acute massive pulmonary embolism, and ischemic stroke.

[0128] Comparative Example 1 Acetylated polypeptide (Ac-PF) has no neuroprotective effect

[0129] In this example, another acetylated dipeptide was synthesized, which was formed by linking acetylated proline and phenylalanine, and was abbreviated as AC-PF.

[0130] The OGD / R model construction method is the same as in Example 1. After the model is successfully constructed, the groups are treated with drugs. In the drug group: after OGD, the neuron culture plate is taken out, the ECS-solution is replaced with neuron culture medium, and the drug treatment is performed (PF: 200μmol / L; AC-PF: 200μmol / L; Phe: 200μmol / L), and the plate is placed in an incubator for reoxygenation for a specified time, with OGD / R as a blank control. At this time, the culture medium of the Non-OGD / R group is also aspirated and an equal volume of sterile PBS buffer is added.

[0131] After 24 hours, the CCK-8 method was used to detect the effects of supplementation of PF, AC-PF, and Phe on neuronal survival rate after OGD / R injury. Fig.10 HPLC was used to detect the changes in Pro content in PF and AC-PF neurons after OGD / R. The results are shown in Fig.11 shown.

[0132] Depend on Fig.10 and Fig.11 The results showed that among the supplementation of PF, AC-PF and Phe, only PF could improve the survival rate of neurons and significantly increase the Pro content in neurons. It can be seen that PF can not only penetrate the cell membrane, but also be hydrolyzed into free natural proline to exert the neuroprotective effect of Pro, indicating that D-phenylalanine can also serve as a transport carrier, while acetylated proline has no neuroprotective effect.

[0133] The method for constructing the ischemia / reperfusion (I / R) model was the same as in Example 2. After the model was successfully established, the drug treatment group was immediately injected with 50 mg / kg PF, 50 mg / kg AC-PF, and 50 mg / kg Phe through the tail vein. After 24 hours, the rat brain TTC staining sections were analyzed. The results are shown in Fig.12 shown.

[0134] Fig.12 The results showed that compared with the I / R group, only the PF-injected group could reduce the infarct volume and brain damage in rats among the three drug-treated groups, indicating that PF can penetrate the BBB into the ischemic side and hydrolyze into a single free Pro in the brain tissue to exert a neuroprotective effect. It can be used as a neuroprotective small peptide for the preparation of drugs for the treatment of nervous system diseases, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, ischemic brain damage such as ischemic stroke, etc. However, acetylated proline has no neuroprotective effect.

[0135] Example 5 PGPF and GPF can penetrate the blood-brain barrier and enter neurons

[0136] Previous experiments by our team have shown that glycine (Gly, G) has a neuroprotective effect. In this example, two neuroprotective peptides were synthesized, one of which has a tetrapeptide amino acid sequence of PGPF. The polypeptide is abbreviated as PGPF, and its molecular weight is 416.47 Da. The specific molecular structure is as follows:

[0137]

[0138] The purity analysis of the finished tetrapeptide is as follows Fig.13 Center left, by Fig.13 As can be seen from the middle left, the HPLC purity analysis of PGPF shows that the purity of PGPF is above 98% based on the peak area calculation.

[0139] The other polypeptide is a tripeptide, whose amino acid sequence is GPF. The polypeptide is abbreviated as GPF, and its molecular weight is 319.35Da. The specific structural formula is as follows:

[0140]

[0141] The purity analysis of the tripeptide product is as follows Fig.13 Shown in the middle right. Fig.13 As can be seen in the middle right, the HPLC purity analysis of GPF shows that the purity of GPF is above 98% based on the peak area calculation.

[0142] Using 50mg / kg FITC as the standard, the corresponding dose of FITC-PGPF is 118mg / kg and the corresponding dose of FITC-GPF is 106mg / kg according to the molecular weight. SD rats were injected with corresponding doses of FITC, FITC-PGPF, and FITC-GPF immediately after I / R. The brains were taken out by perfusion in the tail vein 1 hour later. Frozen sections were made after about one week of sedimentation. The fluorescence distribution in the brain tissue was photographed using a confocal microscope, and the parameters used for photographing different brain slices remained unchanged. The BBB permeability of neuroprotective peptides can be obtained by comparing and analyzing the fluorescence intensity. The fluorescence analysis results are shown in Figure 2. Fig.14 and Fig.15 To observe whether the neuroprotective peptides PGPF and GPF can enter neurons, FITC-PGPF and FITC-GPF brain slices obtained from the tail vein injection were perfused and co-stained with NeuN 1 hour later, and the fluorescence co-localization was observed under a 40x microscope. The results are shown in Fig.16 .

[0143] Fig.14 The scale bar in the middle is 2000 μm. A and B are both left-sided infarctions. Fig.14 A in the middle left is for injection of FITC, and B is for injection of FITC-PGPF. Fig.14 A in the middle right is injected with FITC, and B is injected with FITC-GPF; comparison Fig.14 From the fluorescence intensity of A and B, it can be seen that FITC alone is difficult to penetrate the blood-brain barrier, while FITC-PGPF and FITC-GPF can easily penetrate the blood-brain barrier, indicating that PGPF and GPF can penetrate the blood-brain barrier.

[0144] Fig.15 The scale bar is 200 μm. Fig.15 A in the middle upper center is the fluorescence image of the healthy side after injection of FITC-PGPF, and B is the fluorescence image of the affected side after injection of FITC-PGPF. Fig.15 A in the middle and lower middle is the fluorescence image of the healthy side after injection of FITC-GPF, and B is the fluorescence image of the affected side after injection of FITC-GPF. Fig.15From the fluorescence intensity of A and B, we can see that after injection of FITC-PGPF and FITC-GPF, the fluorescence intensity of the affected side is stronger than that of the healthy side, indicating that significantly more PGPF and GPF reach the affected side than the healthy side.

[0145] Fig.16 The scale bar in the middle is 50 μm. The figure shows the fluorescence image of the affected side after tail vein injection of FITC, FITC-PGPF, and FITC-GPF. Fig.16 It can be seen that after injection of FITC-PGPF or FITC-GPF, high FITC fluorescence intensity can be seen on the affected side and co-stained with NeuN, indicating that PGPF and GPF can not only penetrate the blood-brain barrier but also enter neurons. However, after injection of FITC, low fluorescence intensity on the affected side and no co-stained with NeuN can be seen, indicating that FITC has difficulty penetrating the blood-brain barrier and cannot enter neurons.

[0146] Example 6 PGPF can reduce brain damage

[0147] Primary neurons of fetal rats were extracted and cultured. OGD / R was performed on the 12th day when the cells were in good condition. During re-glucose and re-oxygenation, PGPF, a neuroprotective peptide, was added to the culture medium at concentrations of 24 μmol / L, 48 μmol / L, 72 μmol / L, 120 μmol / L, and 168 μmol / L, respectively. After 24 hours, CCK8 was added to determine the neuronal survival rate. The results are shown in the figure. Fig.17 shown.

[0148] Depend on Fig.17 It can be seen that compared with the blank control group OGD / R, the neuronal survival rate was significantly improved after the addition of PGPF and gradually increased within a certain concentration range, indicating that PGPF has a neuroprotective effect, and its preferred addition concentration is 100-120 μmol / L.

[0149] After I / R, rats were divided into groups by tail vein injection. Rats in different groups were injected with normal saline (control group, Vehicle), 20 mg / kg, 30 mg / kg, and 50 mg / kg of the small peptide PGPF. After 24 hours, the brains were harvested by perfusion and TTC staining was used to measure the whole brain infarction volume of the rats. Fig.18 .

[0150] Depend on Fig.18 It can be seen that compared with the control group, tail vein injection of the small peptide PGPF after I / R can significantly reduce the volume of cerebral infarction, indicating that PGPF has the effect of treating ischemic stroke, and the preferred addition concentration is 30-40 mg / kg.

[0151] Example 7 GPF can reduce brain damage

[0152] Primary neurons were cultured for about 12 days and then OGD / R was performed in good condition. During glucose rehydration and reoxygenation, 100 μmol / L, 150 μmol / L, 200 μmol / L, 250 μmol / L, and 300 μmol / L of the neuroprotective peptide GPF were added to the culture medium, respectively. After 24 hours, CCK8 was added to determine the neuronal survival rate. The results are shown in the figure. Fig.19 .

[0153] Depend on Fig.19 It can be seen that compared with the OGD / R group, the addition of GPF significantly increased the viability of neuronal cells, indicating that GPF has a neuroprotective effect, and the preferred addition is 200-250 μmol / L.

[0154] Immediately after I / R, rats in each group were injected with saline, 30 mg / kg, 50 mg / kg, and 100 mg / kg of GPF through the tail vein. Brains were harvested 24 hours later and the whole brain infarction volume was determined by TTC staining. Fig. 20 .

[0155] Depend on Fig. 20 It can be seen that compared with the I / R+Vehicle group, the whole brain infarction volume of rats injected with GPF was significantly reduced, indicating that GPF can significantly reduce the volume of cerebral infarction and can be used to treat ischemic stroke, preferably injected at 50 mg / kg.

[0156] In the above Examples 1-7, phenylalanine may also be selected from L-phenylalanine (L-Phe).

[0157] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A neuroprotective peptide with proline as a pharmacologically active ingredient, characterized in that: The neuroprotective small peptide is a synthetic small peptide with a molecular weight of less than 500 Da, and its amino acid sequence includes at least one unmodified proline, and at least one of the peptide bonds connected to the proline is a protease hydrolysis point, so that it is hydrolyzed into free single amino acids to play a pharmacological role in protecting neurons; And at least one peptide end is connected with L-Phe or D-Phe, so that the small peptide can penetrate the cell membrane or the blood-brain barrier.

2. The neuroprotective peptide according to claim 1, characterized in that It is a small peptide containing 2 to 4 amino acids, and its amino acid sequence is selected as follows: X1-X2-X3-F, where phenylalanine F is attached to the C-terminus of the small peptide; In the sequence, one or two of X1, X2, and X3 are proline, P.

3. The neuroprotective peptide according to claim 2, characterized in that: In its amino acid sequence, if X3 is P and X2 is G, then X1 is 0 or any amino acid; If X2 is P, then X1 and X3 are G or F; If X1 is P and X2 is 0, then X3 is 0, G or F; Or when X1 is P and X2 is G or F, then X3 is 0 or any amino acid.

4. The neuroprotective peptide according to claim 3, characterized in that: The amino acid sequence is selected as follows: PF, PGF, GPF, GPGF, CGPF or PGPF.

5. The neuroprotective peptide according to claim 4, characterized in that: Its amino acid sequence is PF, PGF, or GPF.

6. Use of the neuroprotective peptide according to any one of claims 1 to 5 in the preparation of a drug for treating nervous system diseases, characterized in that: The nervous system diseases include neurodegenerative diseases and brain damage.

7. The use according to claim 6, characterized in that The neurodegenerative disease is Alzheimer's disease or Parkinson's disease, and the brain injury is ischemic brain injury.

8. The use according to claim 7, characterized in that It is used to prepare drugs for treating ischemic brain damage.

9. The use according to claim 8, characterized in that The neuroprotective peptide is used as an active ingredient of the drug, and its amino acid sequence is PF, PGF, GPF, PGPF, or GPGF.

10. The use according to claim 9, characterized in that The amino acid sequence of the neuroprotective peptide is PF, PGF, or GPF.

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