CN109553685B

CN109553685B - Small molecular polypeptide and application thereof in preparation of medicine for preventing and treating cerebral arterial thrombosis

Info

Publication number: CN109553685B
Application number: CN201710875498.XA
Authority: CN
Inventors: 朱铃强; 周雅帆; 刘丹; 汪晶; 邓曼菲
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2017-09-25
Filing date: 2017-09-25
Publication date: 2021-07-27
Anticipated expiration: 2037-09-25
Also published as: CN109553685A

Abstract

The invention discloses a small molecular polypeptide TAT-CDK5-CTM and application thereof in preparing a medicine for preventing or treating cerebral arterial thrombosis. By synthesizing a TAT protein transduction domain, a protein polypeptide capable of combining and inhibiting CDK5 and a fusion protein TAT-CDK5-CTM of an autophagy original CTM, TAT is utilized to carry CDK5 protein polypeptide, so that the protein polypeptide can permeate blood brain barrier through blood transportation and is taken up by neurons. The polypeptide is applied to in vitro and in vivo ischemic stroke models, can effectively play a role in blocking the combination of NR2B subunit of N-methyl aspartate receptor (NMDAR) and cyclin dependent protein kinase 5(CDK5), and polypeptide molecules combined with CDK5 protein are mediated by CTM, enter lysosome for degradation, inhibit neuronal apoptosis and necrosis caused by CDK5 downstream, reduce brain injury after ischemic stroke, and provide a molecular target for developing drugs for treating ischemic stroke.

Description

Small molecular polypeptide and application thereof in preparation of medicine for preventing and treating cerebral arterial thrombosis

Technical Field

The invention belongs to the field of medicines, relates to small molecular polypeptides and medical application thereof, and particularly relates to artificially synthesized small molecular polypeptide TAT-CDK5-CTM and application of the small molecular polypeptide TAT-CDK5-CTM in preparation of medicines for preventing or treating ischemic stroke.

Background

Ischemic stroke is a serious neurological disease such as cerebral ischemia caused by thrombosis or embolism due to interruption of cerebral blood flow, and paralysis, language disorder, visual loss and even death of patients can occur. Due to its high morbidity, mortality and disability rate, it is considered a common intractable disease that seriously harms human health and life safety. According to 2016 cerebral apoplexy epidemiological reports, 7000 million patients with cerebral apoplexy, 200 new cerebral apoplexy and 165 million dead people with cerebral apoplexy occur in every 12 seconds, one person dies from cerebral apoplexy in every 21 seconds, the number of dead people due to cerebral apoplexy accounts for 22.45% of all dead people in every year, and the data still gradually rises along with the aging of the population. Stroke is not only high in mortality rate, but also has poor prognosis, 70% of patients have different degrees of impaired working ability, and 30% of patients cannot take care of themselves, which causes serious economic burden and mental stress to families and the whole society of patients. However, the current therapeutic approaches for ischemic stroke are very limited, and the only effective therapy is thrombolysis therapy using tissue plasminogen activator (tPA), but the therapeutic time window is very narrow (4.5 hours), so most patients can only be treated symptomatically. To date, over 1000 small molecule compounds have been developed globally for ischemic stroke and about 200 clinical trials have been conducted, but all have failed. Therefore, it is very important to explore new therapeutic approaches to combat brain damage caused by ischemic stroke and reduce neuronal death.

Molecular mechanisms of cell death caused by stroke include excitotoxicity, oxygen/nitrogen stress, mitochondrial dysfunction, calcium overload, and the like. In the case of ischemia, glucose deprivation hypoxia causes excessive accumulation of glutamate at the synapse, activating postsynaptic NMDA and AMPA receptors, the NMDA receptor playing a critical role. Large amount of Ca²⁺Influx into cells via NMDA receptor channels, causing Ca²⁺And (4) performing super-treatment. The intracellular segment of the NMDA receptor interacts with intracellular proteins, activating a series of proteases, ribozymes and esterases to initiate a lethal downstream response, resulting in neuronal death. Since glutamate receptors play an important physiological role in normal neuronal activity, direct blockade of glutamate receptors is not suitable for the treatment of ischemic stroke. Inhibition of neuronal damage by blocking the interaction of NMDA receptors with downstream neuronal death-causing proteinsThe normal physiological activity of the neuron is not influenced, and the method is a safer and more effective treatment strategy.

TAT is cell-penetrating peptides (cell-penetrating peptides) and is a novel efficient transportation vector capable of penetrating cell membranes and nuclear membranes. TAT can carry polypeptides, proteins, DNA molecules and the like to enter cytoplasm and nucleus through active transport of receptors, so that the carried molecules exert corresponding biological effects. At present, in vitro and in vivo experiments show that HIV-TAT can pass through all tissue cells including nerve cells, and has no obvious toxic or side effect. TAT has high transport efficiency, can be transported through blood, can penetrate through a blood brain barrier to enter neurons and glial cells, and carried proteins or polypeptides can keep the original biological activity and play corresponding biological effects.

CTM is a molecular Chaperone mediated autophagy Targeted Motif (Lys-Phe-Glu-Arg-Gln (KFERQ)) and the core element thereof can mediate the protein combined with the Lys-Phe-Glu-Arg-Gln (KFERQ) to enter the lysosome to be degraded.

Based on the recent research of the applicant and TAT technology, the applicant synthesizes a membrane penetration small molecule polypeptide TAT-CDK5-CTM consisting of an amino acid sequence (Arg-Arg-Pro-Pro-Arg-Ser-Pro-Asp-His-Lys-Arg-Tyr-Phe-Arg-Asp-Lys-Glu, RRPPRSPDHKRYFRDKE) of NR2B and a CTM sequence (Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln-Arg-Phe-Glu, KFERQKILDQRFFE) and TAT membrane penetrating peptide (Tyr-Gly-Arg-Lys-Arg-Arg, YGKRRQRRR), and applies the membrane penetration small molecule polypeptide TAT-CDK5-CTM to an in vitro and carrier ischemic stroke model to effectively play the role of blocking the NR2B subunit of an N-methyl aspartate receptor (NMDAR) and a cyclin dependent protein The kinase 5(CDK5) is combined and leads CDK5 to lysosome for degradation, so that neuronal apoptosis and necrosis caused by CDK5 downstream are inhibited, brain damage after ischemic stroke is reduced, and a molecular target is provided for further developing a medicament for clinically treating the ischemic stroke.

Disclosure of Invention

The invention aims to provide a polypeptide and application of the polypeptide in preparing a medicine for preventing or treating cerebral arterial thrombosis.

The technical scheme for realizing the invention is as follows:

the polypeptide provided by the invention is a small molecular polypeptide TAT-CDK5-CTM, and the amino acid sequence of the polypeptide is shown as a sequence 1(SEQ ID NO.1) in a sequence table.

The small molecular polypeptide TAT-CDK5-CTM provided by the invention can be used for preparing a medicine for preventing or treating ischemic stroke, a medicine for reducing neuronal necrosis after ischemia and a medicine for reducing neuronal apoptosis after ischemia.

The TAT cell-penetrating peptide (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, YGRKKRRQRRR) is connected with a segment of amino acid sequence (Arg-Arg-Pro-Pro-Arg-Ser-Pro-Asp-His-Lys-Arg-Tyr-Phe-Arg-Asp-Lys-Glu, RRPPRSPDHKRYFRDKE) and a CTM sequence (Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln-Arg-Phe-Phe-Glu, KFERQKILDQRFFE) of NR2B to obtain the TAT-5-CTM small molecular polypeptide with biological activity. TAT is used for carrying CDK5 polypeptide, and the polypeptide can permeate blood brain barrier through blood transportation and be taken by cerebral neurons so as to play the biological function of inducing CDK5 degradation.

The invention provides application of a small molecular polypeptide TAT-CDK5-CTM in preparing a medicine for preventing or treating cerebral arterial thrombosis, and the small molecular polypeptide TAT-CDK5-CTM is co-incubated with neuronal cells or injected into tail veins of mice, so that the small molecular polypeptide TAT-CDK5-CTM can be found to be capable of effectively reducing the cerebral infarction area after cerebral arterial thrombosis, inhibiting the apoptosis or necrosis of the neurons, and improving the nervous system symptoms and corresponding behavioral phenotypes.

The inventors of the present application found that after ischemic stroke, the NR2B subunit of the N-methylaspartate receptor (NMDAR) interacts with cyclin-dependent protein kinase 5(CDK5) and mediates downstream neuronal death (necrosis and apoptosis). The interaction of the NR2B subunit of the N-methyl aspartate receptor (NMDAR) and the cyclin dependent protein kinase 5(CDK5) is blocked, and the neuronal death after ischemic stroke can be effectively reduced. In response to this finding, the applicants linked TAT-penetrating peptides (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, YGRKKRRQRRR) with a stretch of amino acid sequence of NR2B (Arg-Arg-Pro-Pro-Arg-Ser-Pro-Asp-His-Lys-Arg-Tyr-Phe-Arg-Asp-Lys-Glu, RRPPRSPDHKRYFRDKE) and CTM sequence (Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln-Arg-Phe-Phe-Glu, KFERQKILDQRFFE) to obtain biologically active TAT-5-CTM. TAT-CDK5-CTM is incubated with neurons cultured in vitro, and TAT-CDK5-CTM polypeptide can be directly taken up by the neurons. Through intravenous injection on the tail of a mouse, TAT-CDK5-CTM polypeptide enters blood and penetrates through the blood brain barrier to be taken up by cerebral neurons, so that the biological effect of the TAT-CDK5-CTM polypeptide is exerted.

The sequence of the small molecular polypeptide TAT-CDK5-CTM is as follows:

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-Arg-Pro-Pro-Arg-Ser-Pro-Asp-His-Lys-Arg-Tyr-Phe-Arg-Asp-Lys-Glu-Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln-Arg-Phe-Phe-Glu(YGRKKRRQRRR-RRPPRSPDHKRYFRDKE-KFERQKILDQRFFE)

the TAT-CDK5-CTM control is TAT-scramble-CDK5(TAT-s-CDK5), the sequence of which is as follows:

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-His-Pro-Arg-Ser-Arg-Pro-Arg-Lys-Glu-Asp-Asp-Lys-Arg-Tyr-Phe-Arg-Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln-Arg-Phe-Phe-Glu(YGRKKRRQRRR-PHPRSRPRKEDDKRYFR-KFERQKILDQRFFE)

TAT-CDK5-CTM and its control TAT-s-CDK5 were synthesized by commercial companies.

The application of the small molecular polypeptide TAT-CDK5-CTM in preparing the medicine for preventing or treating cerebral arterial thrombosis comprises the following application processes:

(1) application of TAT-CDK5-CTM in ischemic stroke cell model

Oxygen Glucose Deprivation (OGD) is an in vitro cellular model of ischemic stroke. Experiment on neurons primary cultured for 10 days in vitro were OGD treated for 60 min and 90 min, with PI marking 44% of the cells necrotic and TUNNEL marking 20% of the cells apoptotic at 60 min of OGD treatment. In the case of OGD90 min treatment, PI marked 86% cell necrosis and TUNNEL marked 18% apoptosis. It can be seen that most neurons die under OGD treatment. After the primary neurons cultured in vitro were incubated with 5um TAT-CDK5-CTM, the number of cells positive for PI and TUNNEL was significantly reduced in the primary neurons treated with OGD, indicating that TAT-CDK5-CTM treatment could reduce neuronal death after ischemia at the cellular level.

(2) Application of TAT-CDK5-CTM in animal model of cerebral arterial thrombosis

The Middle Cerebral Artery Occlusion (MCAO) model is a well-established animal model of cerebral ischemia. The insertion of a wire plug through the mouse carotid artery, blocking the middle cerebral artery, can cause cortical striatal ischemia innervated by the middle cerebral artery. Ischemia reperfusion injury can be caused by blood flow reperfusion after the wire plug is pulled out. So as to simulate the symptoms of clinical cerebral embolism or cerebral infarction. After the model is established, the volume of ischemic infarction is detected by TTC staining, the death of neurons is detected by FJ and TUNNEL staining, the balance coordination ability of mice is reflected by Rotarod test of fatigue, and the functions of the nervous system after ischemia are detected by nervous system Score (N.S.). TAT-CDK5-CTM solution of 1mg/ml is injected into tail vein at 3 hours and 6 hours after ischemia-reperfusion of model mice respectively, TTC staining and nuclear magnetic resonance detection are carried out on 3 days of animal ischemia-reperfusion, FJ staining and TUNNEL staining are carried out on seven days, and animal behavior experiments are carried out within corresponding time intervals. TTC staining results showed that the cerebral ischemic infarct volume of mice given TAT-CDK5-CTM was significantly lower than that of TAT-s-CDK5 or normal saline group of the control group. FJ and TUNNEL staining results showed that the number of neuronal deaths following ischemia in mice given TAT-CDK5-CTM was significantly lower than in the control group TAT-s-CDK5 or saline Vehicle. In addition, the results of the fatigue rotarod and water maze experiments show that the balance coordination ability and the learning and memory ability of mice injected with TAT-CDK5-CTM are obviously improved compared with those of a control group TAT-s-CDK5 or normal saline Vehicle. The results all prove that the TAT-CDK5-CTM has exact therapeutic effect on cerebral ischemic stroke.

Compared with the prior art, the invention has the following characteristics: the small molecular polypeptide TAT-CDK5-CTM designed by the invention has high synthesis purity, good solubility, no toxic or side effect, is suitable for intravenous injection, and has advantages in conversion production and clinical application.

The invention uses the artificially synthesized TAT protein transduction structural domain and the fusion protein polypeptide combined with CDK5, and aims to treat ischemic stroke by intravenous injection, reduce nerve cell death and brain injury caused by ischemic stroke, and improve nervous system symptoms after ischemic stroke. In the TAT-CDK5-CTM recombinant protein polypeptide disclosed by the invention, TAT is used as a high-efficiency transduction protein, can carry polypeptide to be transported by blood and penetrate through a blood brain barrier to be absorbed by neurons, can be converted and applied to nervous system diseases such as cerebral arterial thrombosis and the like, and has feasibility of practical operation.

Detailed Description

The method is further described with reference to the accompanying drawings and specific embodiments. All procedures involved in the embodiments of cell culture, apoptosis and necrosis staining, animal ischemia models, tail vein injection, and behavioral testing are well known to those skilled in the art. For material and method parts not described in detail in the present invention, reference is made to the literature (Tu W, Xu X, Peng L, Zhong X, Zhang W, Soundarapandian MM, Balel C, Wang W, Jia N, Zhang W, Lew F, Chan SL, Chen Y, Lu Y (2010) DAPK1interaction with NMDA receptor NR2B subbunits media library in column. cell 140: 222-234).

Example 1:

artificial synthesis of TAT-CDK5-CTM

The sequence of TAT-CDK5-CTM is shown in SEQ ID NO.1, and the TAT-CDK5-CTM is artificially synthesized by Jiangsu Qiangyao Biotechnology Co., Ltd, the synthesis report is shown in the following, and the chromatogram is shown in FIG. 1.

Artificial synthesis of HPLC report by TAT-CDK5-CTM

TABLE 1

	Wavelength of light	Time	Peak area	Percentage of Peak area (%)
					1	220nm	7.310	17438	0.2265
2	220nm	9.067	55117	0.716
					3	220nm	9.360	7404161	96.18
4	220nm	9.933	118775	1.543
					5	220nm	11.207	38910	0.5055
6	220nm	12.239	63670	0.8271

The synthesized TAT-CDK5-CTM polypeptide has the purity of 96.18 percent, each TAT-CDK5-CTM polypeptide is 1mg, is white powder, is completely soluble in water, and is stored at-20 ℃ in a sealed and dark manner. Before use, the preparation is diluted by normal saline for injection according to a specified concentration and is used as it is.

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Pro-His-Pro-Arg-Ser-Arg-Pro-Arg-Lys-Glu-Asp-Asp-Lys-Arg-Tyr-Phe-Arg-Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln-Arg-Phe-Phe-Glu (YGRKKRRQRRR-PHPRSRPRKEDDKRYFR-KFERQKILDQRFFE), also synthesized by this company.

Example 2:

TAT-CDK5-CTM blocks the combination of neuron NR2B receptors and CDK5 under the condition of ischemia and hypoxia, and inhibits the neuron apoptosis and necrosis caused by the downstream of CDK 5.

Primary neurons from day 10 were cultured in vitro and treated with sugar oxygen deprivation (OGD) to mimic the cellular model of ischemic stroke. After 90 minutes of OGD treatment for 2 hours of normal culture, 5uM of TAT-CDK5-CTM polypeptide or control TAT-s-CDK5 polypeptide or vehicle was administered for incubation. Cellular proteins were extracted after 2 hours. The interaction of NR2B with CDK5 was demonstrated by black blots on NC membranes, which were detected by first precipitating the cellular proteins with anti-NR 2B antibody and then detecting the precipitated proteins with anti-CDK 5 antibody. The results show that: antibodies against CDK5 barely detected CDK5 protein on NC membranes after administration of TAT-CDK5-CTM, suggesting that TAT-CDK5-CTM blocks the interaction of NR2B with CDK 5. Whereas the control group administered with TAT-s-CDK5 clearly detected CDK5 protein, indicating that TAT-s-CDK5 failed to block the interaction of NR2B with CDK 5.

Example 3:

application of TAT-CDK5-CTM in ischemic stroke cell model

(1) Construction of primary neuron culture and glucose-oxygen deprivation simulated ischemic stroke cell model

Fetal mice at embryonic stage day 18.5 were removed from the uterus of pregnant mice, and after decapitation, the prefrontal cortex of the brain was removed from the dissecting fluid (Hank's Balance Solution), and the cells were isolated by 0.125% trypsinization. The cells were seeded on 20ng/ul polylysine and laminin coated coverslips, which were placed in 12-well plates at a cell seeding density of 100-. Immunofluorescent labeling of the cultured cells with the neuronal marker β -tubulin iii (Tuj1) demonstrated that more than 90% of the cultured cells were neurons.

To establish a cellular model of ischemic stroke, primary neurons cultured in 12-well plates were given a sugar oxygen deprivation (OGD) treatment on day 10, discarding the original cell culture medium, rinsing 3 times with oxygen and sugar free bicarbonate buffer, adding 500ul of this buffer, and culturing the cells in 37 degree anaerobic incubator for 60 minutes and 90 minutes, respectively, to simulate ischemic stroke. Then replacing the anaerobic sugarless bicarbonate buffer with normal medium containing oxygen and sugar at 37 deg.C/5% CO₂/10％H₂/85％O₂Maintained in a humidified incubator for 72 hours, simulating reperfusion injury. Therefore, the establishment of the cell model of the cerebral arterial thrombosis is completed, and the neuron can be correspondingly detected.

(2) Application of TAT-CDK5-CTM in ischemic stroke cell model

In order to investigate the optimal action concentration of TAT-CDK5-CTM, the primary cultured neurons were incubated with 10ul of 1uM/3uM/5uM TAT-CDK5-CTM in 500ul culture medium per well of 12-well plate at 2 hours after 90 minutes of OGD treatment and returned to normal culture conditions, and the effect of inhibiting the interaction of NR2B with CDK5 was observed. The best concentration of TAT-CDK5-CTM was selected as the group with the strongest inhibitory effect and was used in subsequent experiments.

Optimum application concentrations of TAT-CDK 5-CTM: co-immunoprecipitation experiments were performed by incubating OGD-treated neuronal cells with different concentrations of TAT-CDK5-CTM and extracting the corresponding cellular proteins. Precipitation of cellular proteins with antibodies against NR2B followed by detection of the precipitated proteins with antibodies against CDK5 revealed a lighter black blot on NC membranes, indicating a weaker interaction between NR2B and CDK 5. At a concentration of 5uM TAT-CDK5-CTM administered, the black bands on NC membranes were almost disappeared, indicating that at this concentration TAT-CDK5-CTM polypeptides completely inhibited the interaction of NR2B with CDK5, and also indicating that 5uM TAT-CDK5-CTM was the optimal dosing concentration.

Performing OGD treatment on primary cultured neurons on the tenth day of culture, adding 10ul 5uM TAT-CDK5-CTM polypeptide into 500ul culture medium per well of a 12-well plate for incubation after restoring normal culture conditions for 2 hours, and continuously performing incubation at 37 ℃/5% CO₂/10％H₂/85％O₂Maintained in a humidified incubator for 70 hours, after which the cells were subjected to PI and TUNNEL staining, which equates to the number of necrotic and apoptotic cells. (see FIG. 3).

(3) Evaluation of Effect of TAT-CDK5-CTM application

Propidium Iodide (PI) is a nucleic acid dye. It cannot penetrate the cell membrane of normal cells, but can penetrate the cell membrane of necrotic cells and stain the nucleus of necrotic cells red. PI staining may therefore reflect the number of necrotic cells. DAPI is also a nucleic acid dye that labels both live and dead cells, so DAPI staining reflects the total number of cells. The specific experimental steps are as follows: 10uM PI solution was prepared in PBS and added to the cell culture medium at 1/10 volumes (50ul/500 ul). After incubation for 15 minutes at 37 degrees, the cells were washed twice with PBS buffer. Then 500ul of nuclear dye DAPI (1/1000PBS dilution) was added to each well of the 12-well plate, and after 5 minutes of incubation, washed twice with PBS buffer. Then directly observing and photographing under a fluorescence microscope, wherein the red marked cells are necrotic cells, and the blue marked cells are total cells. The percentage of necrotic cells was calculated.

In situ terminal transferase labelling (tunel) of apoptotic cells may be used to label apoptotic cells. During apoptosis, intracellular endonucleases are activated, resulting in the cleavage of the DNA double strand. The fragmented DNA fragments expose 3' -OH, and are combined with fluorescein (TRITC) labeled nucleotides under the catalysis of Terminal Deoxynucleotidyl Transferase (TdT) and DNA polymerase, so that the apoptotic cells are specifically labeled, and therefore, the DNA fragments can be used for reflecting the apoptosis degree of the cells. The specific experimental steps are as follows: adherent cells were washed 2 times with PBS buffer, fixed on 4% paraformaldehyde ice for 10 minutes, and then washed twice with PBS buffer. Followed by incubation on ice for 2 minutes with sodium citrate buffer containing 0.1% Trition-X-100. 30ul of freshly prepared TUNNEL reaction solution (enzyme solution: labeling solution, mixed at 1: 9) was added to the cell slide, incubated for one hour at 37 ℃ in the absence of light, then incubated for 5 minutes with DAPI (1/1000PBS dilution), and washed twice with PBS buffer. Directly observing and taking a picture under a fluorescence microscope under the irradiation of 562nm exciting light, wherein cells marked as red are apoptotic cells, and observing and taking a picture under the irradiation of 660nm exciting light, and cells marked as blue are total cells. The percentage of apoptotic cells was calculated therefrom.

We performed OGD treatment on 10-day in vitro cultured neurons for 60 min and 90 min, respectively. With OGD treatment for 60 min, PI marked 44% cell necrosis and TUNNEL marked 20% apoptosis. In the case of OGD90 min treatment, PI marked 86% cell necrosis and TUNNEL marked 18% apoptosis. It can be seen that most neurons die under OGD treatment. After the primary neurons cultured in vitro were incubated with 5um TAT-CDK5-CTM, the number of PI and TUNNEL positive cells was significantly reduced after OGD treatment for 90 minutes (FIG. 4B), and the apoptosis and necrosis rates were 3% and 11%, respectively, indicating that TAT-CDK5-CTM treatment can reduce neuronal death after ischemia at the cellular level, indicating that TAT-CDK5-CTM has the effect of treating ischemic stroke.

Example 4:

application of TAT-CDK5-CTM in mouse model with cerebral arterial thrombosis

(1) Establishment of ischemic stroke mouse model

The Middle Cerebral Artery Occlusion (MCAO) model is a recognized cerebral ischemia animal model, and the specific operation is as follows: preoperative anesthesia (0.1ml/10g body weight, intraperitoneal injection) is carried out by 6% chloral hydrate, supine fixation is carried out, neck skin preparation is carried out, 1-2 cm of median incision is made on the neck after iodophor disinfection, the right common carotid artery is separated, the proximal end of the common carotid artery is ligated by a loose knot, the external carotid artery and the internal carotid artery are exposed to be branched, ligation is carried out at the position 1.5 cm far from the external carotid artery, a nylon thread plug coated by silica gel with the diameter of about 0.22mm is inserted into the internal carotid artery from the external carotid artery, the insertion depth is about 0.9-1.0 cm, and the distance reaches the beginning of the middle cerebral artery. After 1 hour the plug was removed and the common carotid artery ligation was released, its perfusion restored and the wound closed. Sham (sham) animals were identical to MCAO group model mice except that no wire plugs were inserted. In the whole operation process, bleeding is reduced as much as possible, aseptic operation is carried out, and the body temperature of the animal is maintained by a heating pad.

(2) TAT-CDK5-CTM intravenous injection

After 3 hours and 6 hours of mouse ischemia reperfusion, 1mg/kg of the polypeptide TAT-CDK5-CTM and the control polypeptide TAT-s-CDK5 or saline for injection (vehicle) were injected via the femoral vein word of mice. The polypeptide concentration is 1mg/ml, and the polypeptide is dissolved by normal saline for injection. After intravenous injection, after 3 days of surgical treatment, brain tissue of a mouse is taken to carry out TTC staining and nuclear magnetic resonance to detect infarct volume, after 7 days, TUNNEL and FJ staining is carried out to observe apoptosis and necrosis, water maze experiments are carried out from 7 days to 14 days to detect the learning and memory ability of the mouse, a fatigue rod turning experiment is carried out during 28 days of ischemia reperfusion to detect the balance coordination ability of the mouse, and a nervous system score (N.S.) is carried out on 28 days to evaluate the nervous system function of the mouse, so that the experimental result proves the treatment effect of TAT-CDK5-CTM on ischemic stroke.

(3) Assessment of the efficacy of TAT-CDK5-CTM treatment

TTC dyeing: TTC staining was used to detect ischemic infarct volume. TTC (2, 3, 5-triphenyltetrazolium chloride) is a fat-soluble photosensitive compound. Can react with succinate dehydrogenase in living cell mitochondria to generate red formazan. The activity of succinate dehydrogenase in mitochondria of cells in ischemic tissues is reduced, and the reaction cannot be carried out, so that the tissues are pale. The specific operation steps are as follows: the MCAO model mouse is anesthetized and killed after 3 days of ischemia reperfusion, the whole brain tissue is rapidly taken out, rapidly frozen at the temperature of minus 20 ℃ for 20 minutes, prepared with 1 percent TTC-PBS solution and stored in a 37 ℃ incubator in a dark place. After brain tissue was removed at-20 ℃, placed on ice, serial coronal sections of approximately 1 mm thickness were rapidly prepared from front to back, the sections were immersed in 1% TTC-PBS solution, incubated at 37 ℃ in the dark for 15 minutes, and the sections were inverted and incubated again for 15 minutes. The staining result was visible as red in the normal brain tissue area and pale in the ischemic area. After staining, the sections were fixed in 4% paraformaldehyde PBS for 15 minutes, and finally the fixed brain slices were photographed for analysis. Continuous 6 slices of 0.3cm area before and after bregma were selected and analyzed for ischemic volume (infarcation, mm3) by Image analysis software Image J.

Nuclear magnetic resonance: magnetic Resonance (MRI) was used to detect the area of ischemia. MRI is one of tomographic imaging, a biomagnetic spin imaging technique. It uses the characteristic of nuclear spin movement, under the condition of external magnetic field, it can produce signal by means of radio-frequency pulse excitation, and after the signal is detected by detector, it can be inputted into computer, and after the signal is processed and converted by computer, it can display image. MRI can detect the ischemic area of the mouse without killing the mouse, and the result is more accurate. The method comprises the following specific steps: after weighing, the mice were anesthetized with 6% chloral hydrate before surgery (0.1ml/10g body weight, i.e. intraperitoneal injection), after anesthesia the metal ear tags were carefully removed, the mice were fixed on a nuclear magnetic resonance apparatus, the shape of the mice was adjusted to ensure scanning of the heads, and the parameters were set to start scanning.

Fluoro-Jade C (FJ-C) and TUNNEL staining: MCAO model mice were anesthetized 7 days after ischemia reperfusion and perfused through the heart aorta. Blood was rapidly perfused and flushed from the mice with pre-cooled 0.9% normal saline until the right atrial effluent became colorless, after which it was perfused with 4% pre-cooled paraformaldehyde solution for 10 minutes at 4 ℃. Completely taking out brain tissue, soaking in the same paraformaldehyde solution for 12-24 hours, dewatering in 30% sucrose solution, freezing and slicing the brain tissue after the brain tissue sinks to the bottom, wherein the thickness of the slice is 30 micrometers, soaking the slice in PBS buffer solution, and storing at 4 degrees. FJ-C staining: the FJ-C dye solution is an anionic ligand histochemical dye with fluorescence, can be combined with denatured neuron, and can emit green fluorescence under the excitation of 488nm excitation light of a microscope, and normal neuron can not be combined with the FJ-C dye solution, so that the fluorescence is not emitted. The specific operation is as follows: the slices were mounted on gelatin coated slides and air dried, and placed in 100% ethanol, 70% ethanol and double distilled water for 1 min. And then incubating for 30 minutes in a mixed solution containing 0.01 percent of Fluoro-Jade and 0.1 percent of acetic acid (1:10) at room temperature in a dark place, rinsing for 3 times by double-distilled water, quickly clearing xylene, directly observing under a microscope after a DPX mounting agent is mounted, and observing green marked cells under 488nm exciting light, namely denatured and dead cells. TUNNEL dyeing: the principles of TUNNEL staining are as described above. The specific operation is as follows: sections were mounted on gelatin coated slides and air dried, rinsed with PBS and incubated on ice for 2 minutes with 0.1% Trition-X-100 in sodium citrate buffer. 50ul of freshly prepared TUNNEL reaction solution (enzyme solution: labeled solution, mixed at 1: 9) was added to each brain plate, incubated at 37 ℃ for one hour in the dark, and directly photographed under a fluorescence microscope under 562nm excitation light irradiation, and the cells labeled red were apoptotic cells.

longa behavioral disorder scoring and fatigue rotarod test: fifth-score scoring of Longa: 0 point, no nerve damage symptoms; 1 minute, when the tail is lifted, the injury adducts and flexes the contralateral forelimb, and the contralateral forelimb cannot be fully extended; 2 min, turning to the opposite side during crawling; 3 min, and pouring towards the opposite side when standing; 4 points, spontaneous walking or loss of consciousness is not possible. Higher scores indicate more severe animal behavior disorders. Fatigue rod turning experiment: the exercise coordination ability of the animals was tested by performing a fatigue rotarod test before the operation of the mice (day 0), on the 7 th, 14 th, 21 th and 28 th days after the operation, respectively. The specific operation is as follows: the time interval from the placement of the mice to the fall of the mice at 4 rpm was recorded as retention time, reflecting the limb strength and motor coordination of the animals. Mice were trained two days 3 times a day before surgery and their average residence time was taken as a reference baseline. After the experiment, the test was carried out 3 times a day with 28 days intervals. The longer the residence time of the mouse, the stronger the motor ability of the mouse.

Morris water maze experiment: the Morris water maze experiment is established in 1981 by Morris, is a classic experiment for detecting spatial learning and memory of experimental animals, and is specifically operated as follows: firstly, a positioning navigation experiment detects the space learning ability of a mouse: a circular pool (diameter 120cm, height 60cm) is filled with water, and a circular platform with the diameter of 15cm is placed at the position of a designated quadrant in the center of the pool and is below 1cm of the water surface. The experimenter holds the mouse and puts the mouse into water gently facing the pool wall, and if the mouse can find the platform within 60 seconds, the time for finding the platform is the incubation period of the mouse in the experiment. If the platform is not found successfully within 90s, the platform is guided to the platform and taken out after 30 s. The training is carried out for 7 days, and the training is carried out four times every day, wherein each time the training is carried out by different terms. The incubation period and swimming distance of each group of mice are calculated every day, and the smaller the incubation period is, the stronger the learning ability is. Secondly, detecting the spatial memory ability of the mouse by a spatial exploration experiment: after training for one week, the platform is removed after one day of rest, the mouse is vertically placed into water from the opposite side quadrant of the platform, swims for 60 seconds, and the percentage of the movement time of the mouse in the target quadrant is counted to reflect the spatial memory capacity of the mouse.

Mice were ischemia-reperfused for 3 hours and 6 hours, and injected with 1mg/kg TAT-CDK5-CTM solution or TAT-s-CDK5 solution of control group, or vehicle group of physiological saline via tail vein. TTC results showed that the ischemic infarct volume of mice was 10.1. + -. 2.2mm after TAT-CDK5-CTM administration³Is obviously lower than that of a TAT-s-CDK5 control group with the thickness of 19.1 +/-2.3 mm³And physiological saline vehicle group 19.6 +/-2.0 mm³(FIG. 7A). Therefore, TAT-CDK5-CTM can reduce the cerebral ischemic infarction volume of mice with ischemic stroke after being injected. The MRI results showed that the ischemic infarct size of mice was 0.18. + -. 0.1cm after TAT-CDK5-CTM administration²Is obviously lower than 0.75 +/-0.3 cm of TAT-s-CDK5 control group²And physiological saline vehicle group 0.81 plus or minus 0.3cm²(FIG. 7B), it is shown that TAT-CDK5-CTM can reduce the size of cerebral ischemic infarction lesion of mice with ischemic stroke after being injected. FJ-C and TUNNEL staining showed that the number of neuronal necrosis and apoptosis in mice given TAT-CDK5-CTM was 30. + -.2 and 6. + -.1, significantly lower than 139. + -.27 and 38. + -.12 of TAT-s-CDK5 in the TAT-s-CDK5 control group or 147. + -. 25 and 49. + -.12 in the saline vehicle group, and close to the sham group level (16. + -.2 and 6. + -.1) (FIG. 7A/FIG. 7B). Say thatInjection of TAT-CDK5-CTM can improve necrosis and apoptosis of neurons after ischemic stroke of mice. And the results of the mouse fatigue bar-rotating experiment show that the residence time of TAT-CDK5-CTM group mice is 72 +/-5 seconds at the seventh day after ischemia, is significantly higher than that of TAT-s-CDK5 control group mice 42 +/-3 seconds and that of normal saline vehicle group mice 39 +/-4 seconds, and the trend continues until day 28 (FIG. 8A). Therefore, TAT-CDK5-CTM injection can improve the motor coordination ability of mice after cerebral arterial thrombosis. The neurological score at day 28 after ischemia (N.S.) results showed that mice in the TAT-CDK5-CTM group scored 1.3 ± 0.2 points, significantly lower than 3.3 ± 0.5 points in the TAT-s-CDK5 control group and 3.5 ± 0.5 points in the normal saline vehicle group. (FIG. 8B). Therefore, TAT-CDK5-CTM injection can improve the nervous system function of mice after cerebral arterial thrombosis. The results of the water maze behavioural experiments showed that mice in the TAT-CDK5-CTM group found a platform on day seven with a latency of 17. + -.5 seconds, significantly lower than 33. + -.5 seconds in the TAT-s-CDK5 control group and 35. + -.6 seconds in the saline vehicle group, and close to 15. + -.5 seconds in the sham group. (FIG. 9A). The time required for TAT-CDK5-CTM group mice to find the platform is shorter, which indicates that TAT-CDK5-CTM can improve the learning ability of ischemic stroke mice. In the ninth day of space exploration experiments, the percentage of time that mice in the TAT-CDK5-CTM group stayed at the target threshold was 62. + -.5%, significantly higher than 29. + -.2% in the TAT-s-CDK5 control group and 31. + -. 3% in the normal saline vehicle group, and close to 67. + -.5% in the sham group. (FIG. 9B). The result shows that TAT-CDK5-CTM can significantly improve the memory capacity of mice with ischemic stroke after being injected.

The experimental results of the histology and the ethology show that after TAT-CDK5-CTM polypeptide is injected, the infarct volume of a cerebral arterial thrombosis mouse can be obviously reduced, the necrosis and the apoptosis of neurons are reduced, the motor balance capability and the space learning and memory capability of the mouse are improved, the nervous system symptoms after ischemia are relieved, and the TAT-CDK5-CTM polypeptide is a potential target for developing a clinical therapeutic drug for cerebral arterial thrombosis.

Drawings

FIG. 1 is an artificial chromatogram of a small molecule polypeptide TAT-CDK 5-CTM.

FIG. 2 is a graph showing the results of TAT-CDK5-CTM interfering with binding of neuronal NR2B receptor to CDK 5. The result of western blotting is shown in FIG. A, and the result is shown in FIG. B.

FIG. 3 is a flow chart of TAT-CDK5-CTM on primary neurons for treatment of ischemic stroke.

FIG. 4 is a graph showing the results of screening for optimal polypeptide treatment concentrations. The result of western blotting is shown in FIG. A, and the result is shown in FIG. B.

FIG. 5 is a statistical plot of the results of TAT-CDK5-CTM on primary neurons reducing neuronal apoptosis and necrosis following OGD treatment. Panel A is a histogram of TUNNEL staining results and panel B is a histogram of PI staining results.

FIG. 6 is a flow chart of TAT-CDK5-CTM in animal models for treating ischemic stroke.

FIG. 7 is an MRI test result and a statistical chart of the reduction of ischemic infarct size after ischemic stroke by TAT-CDK5-CTM in animal models. Panel A is a control group and panel B is a saline-injected group. Panel C shows the injection of disordered polypeptide (TAT-s-CTM) and panel D shows the injection of normal polypeptide (TAT-CDK 5-CTM). Fig. E is a statistical chart of results.

FIG. 8 is a statistical plot of the results of TAT-CDK5-CTM in animal models to reduce ischemic infarct volume (A), neuronal necrosis (B) and apoptosis (C) following ischemic stroke.

FIG. 9 is a statistical chart of the results of TAT-CDK5-CTM in animal models to improve motor function (A) and neurological symptoms (B) after ischemic stroke.

FIG. 10 is a statistical chart of the results of TAT-CDK5-CTM improving learning memory function after ischemic stroke in animal models. Graph A is a latency statistic and graph B is a target term retention percentage statistic.

The following is an amino acid sequence table of the polypeptide related to the patent application, wherein a sequence 1 is a small molecule polypeptide TAT-CDK 5-CTM; the sequence 2 is TAT-scramble-CDK5(TAT-s-CDK 5).

Sequence listing

SEQUENCE LISTING

<110> university of science and technology in Huazhong

<120> small molecular polypeptide and application thereof in preparing medicine for preventing and treating cerebral arterial thrombosis

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 42

<212> PRT

<213> Artificial sequence

<400> 1

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Arg Arg Pro Pro Arg

1 5 10 15

Ser Pro Asp His Lys Arg Tyr Phe Arg Asp Lys Glu Lys Phe Glu Arg

20 25 30

Gln Lys Ile Leu Asp Gln Arg Phe Phe Glu

35 40

<210> 2

<211> 42

<212> PRT

<213> Artificial sequence

<400> 2

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro His Pro Arg Ser

1 5 10 15

Arg Pro Arg Lys Glu Asp Asp Lys Arg Tyr Phe Arg Lys Phe Glu Arg

20 25 30

Gln Lys Ile Leu Asp Gln Arg Phe Phe Glu

35 40

Claims

1. An amino acid sequence of the artificially synthesized small molecular polypeptide is shown in SEQ ID NO. 1.

2. The use of the polypeptide of claim 1in the preparation of a medicament for the treatment of cerebral arterial thrombosis.

3. The use of the polypeptide of claim 1 for the manufacture of a medicament for reducing neuronal necrosis and apoptosis in ischemic stroke.