Transmembrane polypeptide with neuroprotection function and application thereof

Abstract

The invention relates to a transmembrane polypeptide with neuroprotection and application thereof, the transmembrane polypeptide comprises 2-4 amino acids, and the amino acid sequence comprises at least one unmodified amino acid with neuroprotection and leucine connected with at least one peptide terminal as a transmembrane carrier; wherein at least one of the peptide bonds connected with the unmodified amino acid is a protease hydrolysis site, so that the unmodified amino acid can be hydrolyzed into free single amino acid. Experiments prove that the leucine can be used as a carrier for penetrating the blood brain barrier, carries amino acid which is difficult to penetrate through BBB and has neuroprotection, penetrates through the blood brain barrier to enter the brain, is hydrolyzed into corresponding specific amino acid in the brain, plays a role in protecting the brain, can be applied to preparation of medicines for treating nervous system diseases, and has broad application prospects. The compound is especially used for preparing a neuroprotective drug for treating brain injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically to a neuroprotective membrane-penetrating polypeptide and applications thereof. Background Art

[0002] Neurodegenerative diseases usually involve gradual damage and loss of function of neurons and the nervous system; and after brain damage, a large number of free radicals are produced, leading to oxidative stress and damaging neurons, such as stroke, which is caused by rupture or embolism of blood vessels in the brain, damage to brain tissue due to ischemia, or further brain damage caused by cerebral ischemia-reperfusion. Their common feature is mainly neuronal damage. Slowing down or preventing neuronal damage and loss of function in neurodegenerative diseases is conducive to improving the quality of life and prognosis of patients; in the context of brain damage, reducing neuronal death and damage may limit or reverse the consequences of brain damage. Therefore, preventing neuronal damage or promoting neuronal survival is conducive to protecting the nervous system from damage or promoting recovery after damage.

[0003] Currently, neuroprotective drugs are mainly used to protect neurons, but the most difficult problem with neuroprotective drugs is that the blood-brain barrier (BBB) ​​has low permeability. If the drug can pass through the blood-brain barrier smoothly and can play a brain-protective role, it will have great therapeutic prospects. The BBB is a dynamic structure organized by endothelial cells through interactions with astrocytes, neurons, and microglia. Under normal circumstances, the BBB relies on factors such as solute carriers, ion transporters, and transcytosis to regulate inflow and outflow transport; although the permeability of the BBB will change under pathological conditions, it is still strictly selective for substances entering and leaving the BBB.

[0004] In the development of neuroprotective drugs, it is necessary to ensure that the drug itself has a neuroprotective effect and secondly, that it can smoothly pass through the BBB and enter the neuronal cell site. It is reported that a variety of single amino acids have been shown to play a neuroprotective role after stroke through different molecular mechanisms, thereby promoting stroke recovery, such as glycine (Reference 1), glutamic acid (Reference 2), arginine (Reference 3), proline (Reference 4), etc. However, the blood-brain barrier (BBB) ​​has a strict selective absorption of amino acids, among which proline, alanine, glycine, cysteine, glutamic acid and aspartic acid are difficult to enter the brain to play their role. Although L-phenylalanine can penetrate the BBB and enter neurons, high concentrations of phenylalanine can inhibit the formation of neuronal synapses and may aggravate damage after cerebral ischemia-reperfusion. Therefore, more amino acids that can penetrate the BBB and enter neurons are found and used as transmembrane carriers, which is conducive to the synthesis of peptides that have neuroprotective properties and can penetrate the blood-brain barrier, so that they can play the role of specific amino acids in neuroprotection after hydrolysis in the brain.

[0005] Prior art documents:

[0006] Document 1: LINNIK M D, ZOBRIST R H, HATFIELD M D. Evidence supporting a rolefor programmed cell death in focal cerebral ischemia in rats[J]. Stroke, 1993, 24(12):2002-2008; discussion 2008-2009;

[0007] Document 2: KAUR H, PRAKASH A, MEDHI B. Drug therapy in stroke:frompreclinical to clinical studies[J]. PharMCAOlogy, 2013, 92(5-6):324-334;

[0008] Document 3: BROWN C S, MANUEL F C, MATTSON A E, et al. Implementation ofTenecteplase for Acute Ischemic Stroke Treatment[J]. Journal of emergencynursing, 2023;

[0009] Document 4: Jingchen Gao, Xiyuran Wang, Xiangyi Kong, et al. Proline-LoadedChitosan Nanoparticles Penetrate the Blood-Brain Barrier to ConferNeuroprotection in Mice Cerebral Ischemia Injury[J]. ACSAppl.Polym.Mater. 2023, 5, 10234-10244。 Summary of the invention

[0010] In view of the above defects or improvement needs of the prior art, the present invention provides a neuroprotective transmembrane polypeptide and its application, the purpose of which is to find that leucine can be used as a transmembrane carrier to carry a synthetic polypeptide containing a specific neuroprotective amino acid to penetrate the blood-brain barrier and enter neurons, thereby exerting the neuroprotective effect of the specific amino acid in the brain, thereby solving the technical problem that the existing amino acids with neuroprotective effects have low penetration of the blood-brain barrier, which limits their clinical application.

[0011] To achieve the above object, according to one aspect of the present invention, a neuroprotective membrane-penetrating polypeptide is provided, wherein the membrane-penetrating polypeptide comprises 2 to 4 amino acids, and its amino acid sequence comprises at least one neuroprotective unmodified amino acid, wherein at least one of the peptide bonds connected to the unmodified amino acid is a protease hydrolysis site, so that it can be hydrolyzed into free single amino acids;

[0012] At least one peptide end is connected to leucine as a transmembrane carrier.

[0013] Preferably, the amino acid sequence of the membrane-penetrating polypeptide comprises the following:

[0014] X 1 -X 2 -X 3 -L, where X 3 is unmodified proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K, X 1 , X 2 is 0 or any amino acid;

[0015] or LX 2 -X 3 -X 4 , where X 2 is unmodified proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K, X 3 , X 4 is 0 or any amino acid.

[0016] Preferably, the amino acid sequence of the membrane-penetrating polypeptide comprises the following:

[0017] X 1 -X 2 -X 3 -L,X 3 is proline P, glycine G, cysteine ​​C, or arginine R;

[0018] Where X 3 P, X 2 0, G, F or L, X 1 is 0 or any amino acid;

[0019] X 3 G, X 2 0, P, C, F or L, X 1 is 0 or any amino acid;

[0020] X 3 C, X 2 0, G, F or L, X 1 is 0 or any amino acid;

[0021] X 3 R, X 2 0, G, P, or A, X 1 is 0 or any amino acid.

[0022] Preferably, the cell-penetrating polypeptide is a dipeptide having an amino acid sequence selected from the following:

[0023] PL, GL, CL or RL.

[0024] Preferably, the amino acid sequence of the membrane-penetrating polypeptide contains an amino acid with neuroprotective effect, and the amino acid sequence is selected from the following:

[0025] LPL, LGL, LCL, LRL, FPL, FGL, FCL, or FRL;

[0026] Or its amino acid sequence contains two amino acids with neuroprotective effects, and the amino acid sequence is selected from the following:

[0027] GPL, GCL, GRL, PGL, PCL, PRL, CGL, CPL, CRL, RGL, RPL, or RCL.

[0028] Preferably, the cell-penetrating polypeptide is a tetrapeptide whose amino acid sequence is selected from the following:

[0029] X 1 GPL, X 1 GCL, X 1 GRL, X 1 PGL, X 1 PCL, X 1 PRL, X 1 CGL, X 1 CPL, X 1 CRL, X 1 RGL, X 1 RPL, or X 1 RCL,X 1 is any amino acid.

[0030] Preferably, the cell-penetrating polypeptide, wherein X 1 is P, G, C, R, L, A, or K.

[0031] According to another aspect of the present invention, there is also provided a use of the membrane-penetrating polypeptide according to the present invention in the preparation of a drug for preventing or treating a nervous system disease.

[0032] Preferably, in the application, the nervous system diseases include neurodegenerative diseases and brain injuries.

[0033] Preferably, in the application, the neurodegenerative diseases include Alzheimer's disease and Parkinson's disease, and the brain injury includes ischemic brain injury and traumatic brain injury.

[0034] Preferably, in the application, the ischemic brain injury is ischemic stroke.

[0035] In general, compared with the prior art, the above technical solution conceived by the present invention can achieve the following beneficial effects because the present invention finds that leucine can be used as a transmembrane carrier to carry amino acids with neuroprotection that are difficult to penetrate the blood-brain barrier into the brain:

[0036] The membrane-penetrating polypeptide provided by the present invention has an amino acid sequence including 2 to 4 amino acids, the amino acid sequence including at least one unmodified amino acid with neuroprotection, and at least one peptide end connected to leucine as a membrane-penetrating carrier; wherein at least one of the peptide bonds connected to the unmodified amino acid is a protease hydrolysis site, so that it can be hydrolyzed into a free single amino acid. Since the N-terminus and / or C-terminus of the polypeptide are connected to leucine as a membrane-penetrating carrier, it can carry specific amino acids with neuroprotection that are difficult to penetrate the BBB through the blood-brain barrier into the brain, and hydrolyze into corresponding specific amino acids in the brain, thereby playing the role of the specific amino acid in protecting the brain. Compared with the existing polypeptides with L-phenylalanine as a membrane-penetrating carrier, the membrane-penetrating polypeptide provided by the present invention can not only penetrate the blood-brain barrier, but also avoid the side effects of phenylalanine as a membrane-penetrating carrier. In addition, leucine as a membrane-penetrating carrier in the present invention also has a certain neuroprotective effect, and its hydrolysis to form a free single amino acid will also play a certain neuroprotective effect, which can further enhance the protective effect of the membrane-penetrating polypeptide on the brain. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a comparison of the protective effects of different treatment groups on neurons after OGD / R injury;

[0038] Figure 2 is the change of Pro and Leu content in neurons added to PL after OGD / R;

[0039] Figure 3 The effects of different transmembrane peptides on cerebral infarction in I / R model rats;

[0040] Figure 4 is the changes in Pro and Leu content in the brain of rats supplemented with PL after MCAO;

[0041] Figure 5 The localization of FITC-PL and FITC in the brains of rats in the Sham group and MCAO group was observed under a 4x microscope;

[0042] Figure 6 The colocalization of FITC-PL and FITC with nerves in the Sham group and MCAO group was observed under a 20x microscope; DETAILED DESCRIPTION

[0043] 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.

[0044] the term:

[0045] Standard amino acid abbreviations: proline (Pro, P); glycine (Gly, G); cysteine ​​(Cys, C); arginine (Arg, R); alanine (Ala, A); lysine (Lys, K); leucine (Leu, L).

[0046] Acidic amino acids are amino acids with negative charges on their side chains, which are acidic under physiological pH conditions. Among the 20 standard amino acids in proteins, there are two acidic amino acids: aspartic acid (Asp, D) and glutamic acid (Glu, E).

[0047] Polar amino acids are amino acids with polar groups on their side chains. These groups can be hydrophilic or hydrophobic, but all have an electric charge or the ability to form hydrogen bonds.

[0048] Among the 20 standard amino acids in protein, the following are included:

[0049] Serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N), glutamine (Gln, Q).

[0050] Our team's previous research found that by using L-phenylalanine, which can penetrate the blood-brain barrier, as a transmembrane carrier, a peptide of a certain size can successfully penetrate the blood-brain barrier and enter the brain, and hydrolyze in the brain to produce specific amino acids with neuroprotection, such as glycine. However, L-phenylalanine as a transmembrane carrier may cause high concentrations of phenylalanine in the brain to inhibit the formation of neuronal synapses, thereby exacerbating damage after cerebral ischemia-reperfusion. We screened existing amino acids to find amino acids that can replace L-phenylalanine as new transmembrane carriers and verify their ability to penetrate the blood-brain barrier. The results showed that leucine can be used as a transmembrane carrier to penetrate the blood-brain barrier. Based on this, we synthesized a series of transmembrane peptides that can penetrate the blood-brain barrier, as follows:

[0051] The amino acid sequence of the membrane-penetrating polypeptide includes 2 to 4 amino acids, and the amino acid sequence includes at least one unmodified amino acid with neuroprotective properties, wherein at least one of the peptide bonds connected to the unmodified amino acid is a protease hydrolysis site, so that it can be hydrolyzed into free single amino acids to exert a neuroprotective effect; and at least one peptide end is connected to leucine as a membrane-penetrating carrier, so that the polypeptide can penetrate the blood-brain barrier.

[0052] The unmodified amino acids with neuroprotective properties include unmodified proline (Pro, P), glycine (Gly, G), cysteine ​​(Cys, C), arginine (Arg, R), alanine (Ala, A), or lysine (Lys, K); wherein at least one of the peptide bonds connected to the unmodified amino acid is a protease hydrolysis site, so that it can be hydrolyzed into free single amino acids, thereby exerting a neuroprotective effect.

[0053] Furthermore, the amino acid sequence of the membrane-penetrating polypeptide includes the following:

[0054] X 1 -X 2 -X 3 -L, where X 3 is unmodified proline (Pro, P), glycine (Gly, G), cysteine ​​(Cys, C), arginine (Arg, R), alanine (Ala, A), or lysine (Lys, K), X 1 , X 2 is 0 or any amino acid;

[0055] or LX 2 -X 3 -X 4 , where X 2is unmodified proline (Pro, P), glycine (Gly, G), cysteine ​​(Cys, C), arginine (Arg, R), alanine (Ala, A), or lysine (Lys, K), X 3 , X 4 It is 0 or any amino acid; and can be hydrolyzed into free single neuroprotective amino acids in the brain, thereby exerting its neuroprotective effect.

[0056] Preferably, the amino acid sequence of the polypeptide is 1 -X 2 -X 3 -L, where X 3 is an unmodified proline (Pro, P), glycine (Gly, G), cysteine ​​(Cys, C), or arginine (Arg, R) having a neuroprotective effect, X 1 , X 2 It is 0 or any amino acid, where "0" means no amino acid.

[0057] In some embodiments, the X 3 P, X 2 0, G, F or L, X 1 is 0 or any amino acid; if X 2 If X is 0, 1 Preferably 0 or selected from X 3 The peptide bonds formed are easily hydrolyzed by amino acids.

[0058] In some embodiments, the X 3 G, X 2 0, P, C, F or L, X 1 is 0 or any amino acid; if X 2 If X is 0, 1 Preferably 0 or selected from X 3 The peptide bonds formed are easily hydrolyzed by amino acids.

[0059] In some embodiments, the X 3 C, X 2 0, G, F or L, X 1 is 0 or any amino acid; if X 2 If X is 0, 1 Preferably 0 or selected from X 3 The peptide bonds formed are easily hydrolyzed by amino acids.

[0060] In some embodiments, the X 3 R, X 2 is O, G, P, A, acidic or polar amino acid, X 1 is 0 or any amino acid; if X 2 If X is 0,1 Preferably 0 or selected from X 3 The peptide bonds formed are easily hydrolyzed by amino acids.

[0061] In the present invention, acidic amino acids refer to amino acids with negative charges on their side chains, which are acidic under physiological pH conditions. 2 Acidic amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E).

[0062] In the present invention, polar amino acids refer to amino acids with polar groups on the side chains. These groups can be hydrophilic or hydrophobic, but all have charges or the ability to form hydrogen bonds. 2 is a polar amino acid, including serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N), glutamine (Gln, Q); preferably X 2 It is cysteine ​​(Cys, C).

[0063] In some embodiments, the membrane-penetrating polypeptide is a dipeptide, and its amino acid sequence is selected from the following:

[0064] PL, GL, CL, or RL; there is a protease hydrolysis site in this type of dipeptide, that is, the connection point between leucine (Leu, L) at one end of the peptide and any amino acid, which can be hydrolyzed into free proline, glycine, cysteine ​​or arginine in the brain, thereby playing its role in protecting nerves and reducing brain damage. PL, GL, and CL are preferred, and the peptide bonds in these dipeptides are easily hydrolyzed in the brain.

[0065] In some embodiments, the membrane-penetrating polypeptide is a tripeptide, the amino acid sequence of which contains an unmodified amino acid with neuroprotective effect, and the amino acid sequence thereof includes the following:

[0066] LPL, LGL, LCL, LRL, FPL, FGL, FCL, or FRL; there is a protease hydrolysis site in this type of tripeptide, that is, the connection point between leucine (Leu, L) at one end of the peptide and any amino acid, which can be hydrolyzed into free proline, glycine, cysteine ​​or arginine in the brain, thereby exerting its nerve-protecting effect and reducing brain damage.

[0067] The tripeptide, whose amino acid sequence contains two unmodified amino acids with neuroprotective effects, includes the following:

[0068] GPL, GCL, GRL, PGL, PCL, PRL, CGL, CPL, CRL, RGL, RPL, or RCL. There are two protease hydrolysis sites in this type of tripeptide, one is the connection point between leucine (Leu, L) at one end of the peptide and any amino acid, and the other is the connection point between glycine (Gly, G) and cysteine ​​(Cys, C) or proline (Pro, P), or the connection point between arginine (Arg, R) and P, G, or C. They can be hydrolyzed into free proline, glycine, cysteine ​​or arginine in the brain, thereby playing its role in protecting nerves and reducing brain damage.

[0069] In some embodiments, the membrane-penetrating polypeptide is a tetrapeptide, and its amino acid sequence includes the following:

[0070] X 1 GPL, X 1 GCL, X 1 GRL, X 1 PGL, X 1 PCL, X 1 PRL, X 1 CGL, X 1 CPL, X 1 CRL, X 1 RGL, X 1 RPL, or X 1 RCL,X 1 is any amino acid; preferably X 1 Amino acids with neuroprotective effects or amino acids that can penetrate the blood-brain barrier, such as P, G, C, R, L, A, or K.

[0071] In addition, the present invention also provides a use of the membrane-penetrating polypeptide according to the present invention in the preparation of a drug for preventing or treating a nervous system disease.

[0072] The nervous system diseases include neurodegenerative diseases and brain damage.

[0073] The neurodegenerative diseases include Alzheimer's disease and Parkinson's disease; the brain injury includes ischemic brain injury and traumatic brain injury, among which ischemic brain injury is preferably ischemic stroke.

[0074] The following are examples

[0075] The following examples use adult male Sprague-Dawley (SD) rats and pregnant rats, male rats weighing 250g; 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, the room temperature is 23 to 25 ° C, and there is sufficient water and bedding is changed regularly except for experimental requirements. The experimental rats were acclimatized in the animal room for one week before the experiment and randomly grouped during the experiment. Animal ethics number: QDU-AEC-2023423.

[0076] In the following examples, the following polypeptides were synthesized by referring to the solid phase synthesis method in patent CN 106188235 B and purified and separated by HPLC. The amino acid sequences are shown below:

[0077] GPL, PL, PGPL, GL, CL; these polypeptides were purified and separated by HPLC, and the purity of each polypeptide was measured to be above 98%.

[0078] Among them, leucine (Leu, L): hydrophobic parameter 3.8, molecular weight 121.20; cysteine ​​(Cys, C): hydrophobic parameter 2.5, molecular weight 121.18; glycine (Gly, G): hydrophobic parameter -0.4, molecular weight 75.08; proline (Pro, P): hydrophobic parameter -1.6, molecular weight 115.15.

[0079] Example 1 Protective effect of unmodified PL on ischemia-reperfusion neurons

[0080] In this example, dipeptides PL and AC-PL were synthesized, wherein the dipeptide PL was formed by connecting unmodified proline and leucine, abbreviated as PL, and AC-PL was formed by connecting acetylated proline and leucine, abbreviated as AC-PL.

[0081] Construction of OGD / R model: Fetal rat primary neurons were extracted and cultured for about 12 days. The cell state was observed under a microscope, and the neuron culture medium with good neuron state and similar density was selected. The old neuron culture medium was aspirated and washed three times with PBS. The control group (Non-OGD group) was added with an appropriate volume of extracellular solution with sugar (ECS+) and placed in a 37°C cell culture incubator; the model group (OGD / R group) and the drug treatment group (OGD / R+drug group) were added with an appropriate amount of extracellular solution without sugar (ECS-) and placed in an anaerobic workstation (temperature: 37°C, gas parameters: 1% O 2 +94% N 2 +5% CO 2) for two hours to form a neuronal OGD model. Cell damage was 50%-60%, and re-glucose and re-oxygenation were performed to simulate reperfusion injury. The original culture medium of neurons in all groups was discarded and an appropriate amount of normal neuronal culture medium was added, which was OGD / R. After the model was successfully constructed, the specific drug treatments were as follows:

[0082] Drug group: After OGD, the neuron culture plate was taken out, the ECS-solution was replaced with neuron culture medium, and drug treatment was performed (Pro: 200μmol / L; Leu: 200μmol / L; PL: 200μmol / L; AC-PL: 200μmol / 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 was also aspirated from the Non-OGD / R group and an equal volume of sterile PBS buffer was added.

[0083] After 24 hours, the CCK-8 method was used to detect the effects of supplementation of Pro, Leu, PL, and AC-PL on neuronal survival rate after OGD / R injury. Figure 1 shown.

[0084] Depend on Figure 1 The results showed that compared with the OGD group, in addition to supplementation of AC-PL, supplementation of amino acids Pro, Leu and dipeptide PL could improve the survival rate of neurons, indicating that amino acids Pro, Leu and dipeptide PL all have the effect of protecting neurons, and there is no significant difference in the protective effect of single amino acids Pro and Leu on neurons; PL treatment can enhance the activity of ischemia-reperfusion neuronal cells, while acetylated proline-leucine has no neuroprotective effect.

[0085] To further investigate whether the effect of supplementation with dipeptide PL on neuronal survival is due to the dipeptide itself or the free amino acids formed after dipeptide hydrolysis. This example further uses HPLC to detect the changes in Pro and Leu content in neurons after OGD / R and PL is added, and compares the proline and leucine content in neurons after GOD group and dipeptide PL supplementation. The results are as follows: Figure 2 shown.

[0086] The high performance liquid chromatography (HPLC) method was as follows:

[0087] Sample extraction: neurons in different groups were taken out from the cell room, the culture medium was sucked out, the water around the culture dish was absorbed with absorbent paper, and the dish was weighed on an electronic balance, and the mass was recorded as N1 mg; cell lysis solution was evenly added to the neurons for lysis, and after 5 minutes, it was scraped off with a cell scraper and sucked into a 1.5 mL centrifuge tube; the liquid around the culture dish was sucked out again, and the dish was weighed on an electronic balance, and the mass was recorded as N2 mg; the centrifuge tube was placed in a pre-cooled 4°C centrifuge for centrifugation at 12,000 r for 10 min; after centrifugation, the supernatant was taken out and added to a new 5 mL centrifuge tube, and 300 mL of acetonitrile was added to the centrifuge tube for centrifugation again at 12,000 r for 10 min; the centrifuge tube was taken out after centrifugation, and 1 mL of supernatant was aspirated for later use.

[0088] Amino acid derivatization: Prepare solution A: PITC + acetonitrile (1: 4), solution B: triethylamine + acetonitrile (1: 4); use 5mL centrifuge tubes to prepare 1mL of the amino acid standard to be tested, with concentrations of: 0.05mg / mL, 0.1mg / mL, and 0.2mg / mL; place the above tissue extracts and the amino acid standards to be tested on a centrifuge tube rack, add 0.4mL of amino acid derivatization solution A and 0.4mL of amino acid derivatization solution B to each tube, shake evenly, and place in a 37°C water bath in a dark bath for 30min; after the water bath, add 0.4mL of n-hexane solution and shake for 5min; after shaking, keep it still in a dark place for 20min, and wait for the amino acid derivatization solution to be stratified; after standing, use a 1mL syringe to take the bottom layer of solution, and use an organic filter to filter the bottom layer of solution into an HPLC injection bottle for later use.

[0089] Chromatographic conditions: Mobile phase D is 0.05 mol / L sodium acetate buffer (pH=6.5), B is methanol, gradient elution program: mobile phase B is increased from 14% at the beginning to 30% after 14 minutes, and to 100% after 30 minutes. Flow rate is 1 mL / min, detection wavelength is 254 nm, column temperature is 43°C, and 5 μL is injected for analysis.

[0090] Depend on Figure 2 It can be seen that compared with the OGD group, the content of amino acids Pro and Leu in neurons increased significantly after supplementation with dipeptide PL, among which Pro increased by about 5 times and leucine increased by about 50%. It can be seen that after supplementation with dipeptide PL, it is mainly the free proline formed after its hydrolysis that exerts its neuroprotective effect, and the leucine formed by its hydrolysis also plays a certain neuroprotective effect.

[0091] Example 2 Protective effect of unmodified PL on ischemia-reperfusion brain tissue

[0092] In this example, dipeptides PL and AC-PL were synthesized, wherein the dipeptide PL was formed by connecting unmodified proline and leucine, abbreviated as PL, and AC-PL was formed by connecting acetylated proline and leucine, abbreviated as AC-PL.

[0093] The MCAO model, or middle cerebral artery occlusion model, simulates the occurrence of ischemic stroke by blocking the middle cerebral artery (MCA). In this model, the MCA is blocked, causing ischemia in the area it supplies (usually the cerebral cortex and some subcutaneous structures). This model can be used to study the pathophysiology of cerebral ischemia, the mechanisms of neuronal damage, and to evaluate potential neuroprotective treatments. The MCAO model can be permanent or temporary. The latter, during the experiment, restores blood flow to simulate the reperfusion process in clinical treatment, forming an I / R model, i.e., an ischemia / reperfusion model.

[0094] An ischemia-reperfusion (I / R) animal model was constructed. After the preoperative work was prepared, the rats were fixed on the operating table in a supine position during the operation. The skin was prepared and disinfected on the neck, and the skin was neatly cut with tissue scissors. The subcutaneous tissue was separated with curved micro-tweezers to expose the superficial fascia and platysma. Under a low-power microscope, micro-tweezers were used to carefully and bluntly separate the muscles, blood vessels, nerves and other tissues of the neck, and the common carotid artery was exposed in the deep layer. The common carotid artery was separated upward to reveal the "Y"-shaped branches, which were the external carotid artery and the internal carotid artery.

[0095] Ligate the proximal end of the separated common carotid artery and tie a slipknot to reserve space for embolization. Tie a dead knot at the distal end of the external carotid artery and a slipknot that can be inserted at the proximal end. Tie a tight slipknot at the internal carotid artery or clamp it with a vascular clamp. After the ligation is completed, use ophthalmic scissors to make a "V"-shaped incision of appropriate size between the two ligature points of the external carotid artery, and insert the thread plug from the external carotid artery into the common carotid artery through the incision. After the thread plug enters the common carotid artery, tighten the slipknot at the proximal end of the external carotid artery.

[0096] Then, the blood vessel was cut at the distal end of the incision of the external carotid artery, and the internal carotid artery was entered from the common carotid artery. After the suture entered the internal carotid artery, the loose knot of the internal carotid artery was tied again, and the suture was inserted along the blood vessel to the distal end until the small black dot on the suture entered the incision of the external carotid artery. At this time, the suture entered the middle cerebral artery, which is the rat middle cerebral artery occlusion (MCAO) model.

[0097] 90 minutes after the embolization, the rats were anesthetized again with the same steps, the neck incision was opened, the embolism was slowly withdrawn, and the slipknot of the embolism at the proximal end of the external carotid artery was tied. The slipknot of the common carotid artery was opened, the blood supply was restored, and suture was performed. After the embolism was withdrawn, the blood supply of the middle cerebral artery was restored, and this was the rat ischemia reperfusion (I / R) model.

[0098] After the I / R model was successfully established, the drug treatment group was immediately injected with GPL (50 mg / kg, 100 mg / kg), PL (50 mg / kg, 100 mg / kg), and Leu (25 mg / kg, 50 mg / kg, 75 mg / kg, and 100 mg / kg) through the tail vein. NA-1 (3 nmol), (Edaravone, 3 mg / kg), NBP stock solution, and NBP Nacl injection were used as positive controls, and normal saline (NS, pH: 3-5) was used as a blank control.

[0099] Among them, NA-1 (Nerinetide) is a neuroprotective agent, mainly used to treat acute ischemic stroke (AIS); Edaravone is a brain protective agent, mainly used to treat acute cerebral infarction; NBP concentrate refers to butylphthalide (3-Butylphthalide), also known as Enbipu, which is a drug used to treat mild and moderate acute ischemic stroke.

[0100] After 24 hours, the rat brain TTC staining sections were analyzed. The results of some sections are as follows: Figure 3 As shown in the middle left, the volume of cerebral infarction in each group was counted, and the results were as follows Figure 3 As shown in the middle right, Figure 3 The numbers marked on the middle bars indicate the sample size.

[0101] Depend on Figure 3 The results show that compared with the I / R group, the cerebral infarction volume of rats can be reduced in the polypeptide drug treatment group, wherein the cerebral infarction volume of rats in each group injected with the polypeptide provided by the present invention is equivalent to that of the positive control group, and as the concentration of the polypeptide increases, the cerebral infarction volume of rats shows a downward trend overall. It shows that the therapeutic effect of the polypeptide provided by the present invention on stroke is equivalent to that of existing drugs, and can be used to prepare potential drugs for treating stroke, especially for preparing potential drugs for treating ischemic stroke.

[0102] Furthermore, this example also uses HPLC to detect the changes in the amino acid content in the brain after supplementation with the above polypeptides after MCAO, and takes the changes in the Pro and Leu content in the brain after supplementation with PL as an example for illustration. The detection results are as follows: Figure 4 As shown. Wherein high performance liquid chromatography (HPLC) detection:

[0103] Sample extraction: After the brain tissue is taken out, a certain mass of rat infarction cortical brain tissue is weighed. Note that the weight of rat brain tissue in different groups is the same. The brain tissue is placed in a 1.5 mL centrifuge tube, marked and placed on ice; steel balls and 9 volumes of acetonitrile: water (1:1) solution are added to the centrifuge tube for grinding at 70 Hz, 10 seconds each time, and a total of 10 times; after grinding, the centrifuge tube is placed in a pre-cooled 4 ° C centrifuge for centrifugation at 12,000 r for 10 min; after centrifugation, the supernatant is taken and aspirated into a marked 1.5 mL centrifuge tube, and 300 mL of acetonitrile is added; the above mixture is centrifuged again at 12,000 r for 10 min; after centrifugation, 1 mL of the supernatant is aspirated and placed in a 5 mL centrifuge tube for use for amino acid derivatization.

[0104] Depend on Figure 4 It can be seen that compared with the MCAO model group, the proline and leucine contents in the brain of rats supplemented with dipeptide PL after MCAO increased significantly. It is speculated that the supplemented dipeptide PL can penetrate the blood-brain barrier and be hydrolyzed in the brain to form single proline and leucine, thereby exerting the protective effect of proline and leucine on the brain.

[0105] Example 3 Verification of the ability of membrane-penetrating polypeptides to penetrate the blood-brain barrier

[0106] It can be seen from Examples 1 and 2 that the above-mentioned synthesized polypeptide has a neuroprotective effect. It is speculated that it can be hydrolyzed in the brain to form a single corresponding amino acid with neuroprotective effect, thereby exerting the protective effect of the amino acid on neurons or the brain. In order to further verify that leucine in the above-mentioned polypeptide can serve as a transmembrane carrier, carry the corresponding polypeptide to penetrate the blood-brain barrier, and be hydrolyzed in the brain to form a single corresponding amino acid with neuroprotective effect, thereby exerting a protective effect on the brain.

[0107] In order to make the results more intuitive, this example further uses FITC-PL for verification, which shows that PL can penetrate the BBB as a membrane-penetrating polypeptide in the rat I / R model, as follows:

[0108] After the MCAO model was successfully established, the drug group was immediately injected with FITC-PL (0.13mmol / g) through the tail vein, and the injection of FITC (fluorescein isothiocyanate, which can emit green fluorescence under ultraviolet excitation, 0.13mmol / g) was used as a blank control; the sham group was also injected with equal amounts of FITC-PL and FITC. The co-localization of nerves and drugs in each group was detected, and the results are shown in Figure 5 and Figure 6 As shown, FITC-PF (green), NeuN (red), Figure 5 For 4x observation results, Figure 6 20x observation results.

[0109] Depend on Figure 5 and Figure 6 The results showed that after intravenous injection of FITC-PL, FITC-PL could enter the ischemic brain tissue in large quantities and co-localize with nerves, indicating that PL could not only penetrate the blood-brain barrier, but also enter neurons, and could be hydrolyzed into single free proline and leucine in brain tissue, exerting the neuroprotective effect of proline, which was beneficial to reducing the volume of cerebral infarction in I / R rats. It can be seen that leucine not only has a certain neuroprotective effect, but also can be used as a transmembrane carrier to carry neuroprotective amino acids such as proline that are difficult to penetrate the blood-brain barrier, penetrate the BBB and enter neurons, and exert the neuroprotective effect of specific amino acids after hydrolysis in the brain, which is beneficial to reducing the volume of cerebral infarction.

[0110] 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 membrane-penetrating polypeptide having neuroprotective properties, characterized in that: It is a synthetic polypeptide comprising 2 to 4 amino acids, the amino acid sequence of which includes at least one unmodified amino acid with neuroprotective properties, and at least one peptide end is connected to a leucine that serves as a transmembrane carrier; At least one of the peptide bonds connecting the unmodified amino acid is a protease hydrolysis site, so that it can be hydrolyzed into free single amino acids.

2. The membrane-penetrating polypeptide according to claim 1, characterized in that Its amino acid sequence includes the following: X1-X2-X3-L, wherein X3 is unmodified proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K, and X1 and X2 are 0 or any amino acid; Or L-X2-X3-X4, wherein X2 is unmodified proline P, glycine G, cysteine ​​C, arginine R, alanine A, or lysine K, and X3 and X4 are 0 or any amino acid.

3. The membrane-penetrating polypeptide according to claim 2, characterized in that Its amino acid sequence is X1-X2-X3-L, X3 is proline P, glycine G, cysteine ​​C, or arginine R; Wherein X3 is P, X2 is O, G, F or L, and X1 is O or any amino acid; X3 is G, X2 is O, P, C, F or L, and X1 is O or any amino acid; X3 is C, X2 is O, G, F or L, and X1 is O or any amino acid; X3 is R, X2 is O, G, P, or A, and X1 is O or any amino acid.

4. The membrane-penetrating polypeptide according to claim 3, characterized in that The membrane-penetrating polypeptide is a dipeptide, and its amino acid sequence is selected from the following: PL, GL, CL, or RL.

5. The membrane-penetrating polypeptide according to claim 3, characterized in that The membrane-penetrating polypeptide is a tripeptide, and its amino acid sequence contains an amino acid with neuroprotective effect, and the amino acid sequence is selected from the following: LPL, LGL, LCL, LRL, FPL, FGL, FCL, or FRL; Or its amino acid sequence contains two amino acids with neuroprotective effects, and the amino acid sequence is selected from the following: GPL, GCL, GRL, PGL, PCL, PRL, CGL, CPL, CRL, RGL, RPL, or RCL.

6. The membrane-penetrating polypeptide according to claim 3, characterized in that The membrane-penetrating polypeptide is a tetrapeptide, and its amino acid sequence is selected from the following: X1GPL, X1GCL, X1GRL, X1PGL, X1PCL, X1PRL, X1CGL, X1CPL, X1CRL, X1RGL, X1RPL, or X1RCL, X1 is any amino acid.

7. The membrane-penetrating polypeptide according to claim 6, characterized in that The X1 is P, G, C, R, L, A, or K.

8. Use of the membrane-penetrating polypeptide according to any one of claims 1 to 7 in the preparation of a drug for treating a nervous system disease.

9. The use according to claim 8, characterized in that The nervous system diseases include neurodegenerative diseases and brain damage.

10. The use according to claim 9, characterized in that The neurodegenerative diseases include Alzheimer's disease and Parkinson's disease; the brain injuries include ischemic brain injury and traumatic brain injury.

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