Fusion polypeptide based on GluD1 receptor and application thereof

Abstract

The invention discloses a fusion polypeptide based on a GluD1 receptor and application of the fusion polypeptide in cerebral ischemia treatment. The transduction structural domain of the TAT protein and the motif 3 sequence of the GluD1 receptor form the fusion polypeptide, and the polypeptide can obviously interfere the positive regulation effect of the GluD1 receptor on the NMDA receptor, so that the excessive activation of the NMDA receptor after cerebral ischemia is inhibited, and the fusion polypeptide shows a good anti-cerebral ischemia effect on both in-vivo and in-vitro cerebral ischemia models.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptides, and in particular relates to a polypeptide and its application in the treatment of cerebral ischemia. Background Art

[0002] After cerebral ischemia, excitotoxicity caused by excessive activation of N-methyl-D-aspartate (NMDA) receptors is one of the main causes of neuronal damage. This receptor is an important anti-cerebral ischemia target and has a broad application prospect in the treatment of cerebral ischemia. Although a variety of NMDA receptor antagonists have been developed, they have not been clinically applied due to limited efficacy or serious adverse reactions. Therefore, it is urgent to develop new NMDA receptor modulators to promote the clinical application of therapeutic strategies targeting NMDA receptors.

[0003] GluD1 receptor is an ionotropic glutamate receptor. Studies have shown that the minimal GluD1 construct, which is connected by a transmembrane region and contains only the N-terminal cerebelin binding region and the C-terminal intracellular domain, can selectively regulate the activity of the NMDA receptor, among which the motif 3 sequence of the GluD1 receptor is a key regulatory sequence, and the motif 3 mutant of the GluD1 receptor can reduce the activity level of the NMDA receptor by about 40%. It can be seen that the GluD1 receptor has a positive regulatory effect on the NMDA receptor. In view of the positive regulatory effect of the GluD1 receptor on the NMDA receptor and the key role played by the motif 3 sequence of the GluD1 receptor, the present invention designs a fusion polypeptide composed of the transmembrane peptide Tat and the motif 3 sequence of the GluD1 receptor, verifies the anti-cerebral ischemia effect of the polypeptide, and indicates its potential clinical use. Summary of the invention

[0004] The purpose of the present invention is to provide a novel polypeptide based on GluD1 receptor and having anti-cerebral ischemia effect and its application, characterized in that the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1.

[0005] The polypeptide provided by the invention is formed by fusion of two parts, which are respectively a Tat peptide fragment and a motif 3 sequence of a GluD1 receptor.

[0006] The polypeptide provided by the present invention has good pharmacokinetic properties. After administration, it can significantly inhibit the excitotoxic damage of cortical neurons and acute hippocampal slices induced by NMDA, and can also significantly reduce the cerebral infarction volume, neurological deficit score and crawling wrong step rate in the grid experiment of MCAO rats, significantly improve the total movement distance, central area movement distance, central area retention time and number of times entering the central area in the open field experiment, and significantly improve the target quadrant distance ratio, residence time and number of times crossing the platform in the water maze experiment. In short, the polypeptide shows a definite anti-cerebral ischemia effect in vivo and in vitro, and is expected to be used to treat ischemic stroke. The mechanism of action of the polypeptide is to interfere with the signal transduction of GluD1 receptors, thereby reducing the function of NMDA receptors to counteract neuronal damage caused by excessive activation of NMDA receptors after cerebral ischemia.

[0007] The polypeptide provided by the present invention has a high blood-brain barrier permeability and can be prepared into a variety of preparations for administration according to conventional pharmacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 :Effects of fusion peptide (Tat-D1M3) on neuronal NMDA excitotoxic injury (survival rate, n=8).

[0009] Note: **Compared with the Control group, P<0.01; ##Compared with the Vehicle group, P<0.01; nsCompared with the Vehicle group, P>0.05.

[0010] Figure 2 :Effect of fusion peptide (Tat-D1M3) on neuronal NMDA excitotoxic injury (Calcein AM / PI double staining).

[0011] Note: Scale bar=50 μm.

[0012] Figure 3 :Statistical results of the percentage of live cells after Calcein AM / PI double staining (n=6).

[0013] Note: **Compared with the Control group, P<0.01; #Compared with the Vehicle group, P<0.05; nsCompared with the Vehicle group, P>0.05.

[0014] Figure 4 : Effects of fusion peptide (Tat-D1M3) on NMDA excitotoxic injury in hippocampal slices (LDH release, n=6).

[0015] Note: **Compared with the Control group, P<0.01; ##Compared with the Vehicle group, P<0.01; nsCompared with the Vehicle group, P>0.05.

[0016] Figure 5 :Effects of fusion peptide (Tat-D1M3) on NMDA excitotoxic injury in hippocampal slices (PI staining).

[0017] Note: ae represents Control group, Vehicle group, Tat-D1M3 group, Tat-D1M3△AA group and ifenprodil group respectively.

[0018] Scale bar=500 μm.

[0019] Figure 6 : Effect of fusion peptide (Tat-D1M3) on cerebral infarction volume in MCAO rats (TTC staining).

[0020] Figure 7 : Effect of fusion peptide (Tat-D1M3) on cerebral infarction volume in MCAO rats (TTC staining statistical results, n=10).

[0021] Note: **Compared with Sham group, P<0.01; ##Compared with Vehicle group, P<0.01; #Compared with Vehicle group, P<0.05; nsCompared with Vehicle group, P>0.05.

[0022] Figure 8 :Effect of fusion peptide (Tat-D1M3) on the misstep rate of forelimb on the affected side of MCAO rats (n=15).

[0023] Note: **Compared between the Vehicle group and the Sham group, P<0.01; ##Compared between the Tat-D1M3 group and the Vehicle group, P<0.01; #Compared between the Tat-D1M3 group and the Vehicle group, P<0.05; ++Compared between the ifenprodil group and the Vehicle group, P<0.05.

[0024] Fig. 9 :Statistical chart of mNSS neurological function score results (n=15).

[0025] Note: **Compared between the Vehicle group and the Sham group, P<0.01; ##Compared between the Tat-D1M3 group and the Vehicle group, P<0.01; #Compared between the Tat-D1M3 group and the Vehicle group, P<0.05; ++Compared between the ifenprodil group and the Vehicle group, P<0.01; +Compared between the ifenprodil group and the Vehicle group, P<0.05.

[0026] Fig.10: Statistical graph of open field test results on the 14th day after drug administration (n=15).

[0027] Note: **Compared with the Sham group, P<0.01; *Compared with the Vehicle group, P<0.05; ##Compared with the Vehicle group, P<0.01; #Compared with the Vehicle group, P<0.05; nsCompared with the Vehicle group, P>0.05.

[0028] Fig.11 : Statistical graph of open field test results on the 21st day after drug administration (n=15).

[0029] Note: **Compared with the Sham group, P<0.01; *Compared with the Vehicle group, P<0.05; ##Compared with the Vehicle group, P<0.01; #Compared with the Vehicle group, P<0.05; nsCompared with the Vehicle group, P>0.05.

[0030] Fig.12 : Statistical graph of open field test results on the 28th day after drug administration (n=15).

[0031] Note: **Compared with the Sham group, P<0.01; *Compared with the Vehicle group, P<0.05; ##Compared with the Vehicle group, P<0.01; #Compared with the Vehicle group, P<0.05; nsCompared with the Vehicle group, P>0.05.

[0032] Fig.13 : Statistical chart of water maze positioning navigation results (n=15).

[0033] Note: **Compared between the Vehicle group and the Sham group, P<0.01; ##Compared between the Tat-D1M3 group and the Vehicle group, P<0.01; ++Compared between the ifenprodil group and the Vehicle group, P<0.01.

[0034] Fig.14 : Statistical graph of water maze space exploration results (n=15).

[0035] Note: **Compared with the Sham group, P<0.01; ##Compared with the Vehicle group, P<0.01; nsCompared with the Vehicle group, P>0.05. DETAILED DESCRIPTION

[0036] The above scheme is further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0037] Example 1. Effects of fusion polypeptide (Tat-D1M3) on neuronal NMDA excitotoxic injury.

[0038] The cortical neurons were divided into blank control (Ctrl) group, solvent control (Vehicle) group, ifenprodil group, Tat-D1M3 group and negative control peptide Tat-D1M3ΔAA group. The NMDA excitotoxicity model was established by incubating NMDA (50 μM) and glycine (10 μM) for 20 min. The dosages of ifenprodil, Tat-D1M3 and Tat-D1M3ΔAA were 10, 20 and 20 μM, respectively. The drugs were given at the same time as NMDA incubation. After the incubation, all drugs were withdrawn. After continuing to culture for 24 h, the cell survival rate was determined by CCK-8 method and Calcein AM / propidium iodide (PI) double staining method.

[0039] Example 2. Effects of fusion polypeptide (Tat-D1M3) on NMDA excitotoxic injury in hippocampal slices.

[0040] The hippocampal slices were divided into Ctrl group, Vehicle group, ifenprodil group, Tat-D1M3 group and Tat-D1M3ΔAA group. The NMDA excitotoxicity model was established by incubating NMDA (100 μM) and glycine (10 μM) for 1 h. Drugs were given during NMDA incubation. After the incubation, all drugs were withdrawn, and the slices were washed 3 times and placed in an incubator to recover for 3 h. After 3 h, LDH release assay and PI staining were used to detect brain slice damage.

[0041] Example 3. Effects of fusion polypeptide (Tat-D1M3) on cerebral ischemic injury in MCAO rats.

[0042] The middle cerebral artery occlusion (MCAO) model of cerebral ischemia was established by internal carotid artery suture method. After 1.5 h of ischemia, blood supply was restored for reperfusion. The animals were divided into sham group, vehicle group, ifenprodil group, Tat-D1M3 group and Tat-D1M3ΔAA group. After the end of cerebral ischemia, the drugs were injected into the lateral ventricle at a dose of 6, 9 and 9 μg / mouse, respectively. Each group of animals was divided into 4 subgroups for index detection: (1) Evaluation of the degree of ischemic injury: On the first day after modeling, the cerebral infarction area and water content were detected; (2) Evaluation of neurobehavioral function: Neurological function score, cylinder test and open field test were performed on the day before modeling and on the 1st, 7th, 14th and 21st days after modeling; (3) Evaluation of spatial learning and memory ability: On the 22nd-27th day after modeling, Morris water maze test was performed. The above indicators reflect the severity of cerebral ischemic injury and the degree of recovery in rats.

[0043] The above examples are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A polypeptide based on GluD1 receptor, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. Use of the polypeptide as claimed in claim 1 in treating cerebral ischemic injury.

3. Use of the polypeptide according to claim 1 in the preparation of a drug for preventing cerebral ischemic injury.

4. A drug for treating cerebral ischemic injury, characterized in that: The active ingredient of the drug is the fusion polypeptide according to claim 1.

5. The drug according to claim 4, characterized in that: The drug further contains a combination of one or more pharmaceutically acceptable excipients and / or carriers.

6. The drug according to claim 4, characterized in that: The dosage form of the medicine is injection, powder injection, solution, suspension, tablet or capsule.

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