A polypeptide analogue and its application in medicine

Abstract

The present invention discloses a polypeptide analog and its application in medicine. The polypeptide analog has an amino acid sequence as shown in SEQ ID NO.3, including an amino acid fragment in the transcriptional transactivator protein Tat and an amino acid fragment with recognition function. The amino acid fragment in the transcriptional transactivator protein Tat drives the polypeptide analog to penetrate and enter the cell, and the amino acid fragment in the cold-induced protein CIRP can recognize GluR1 and bind to GluR1, and inhibit the lower membrane of the synaptic membrane protein GluR1, thereby achieving the purpose of stabilizing the synaptic membrane protein GluR1 under high altitude exposure. The polypeptide analog provided by the present invention can play an effective reference role in preventing / treating learning and memory decline induced by high altitude exposure, and can further play an effective drug role.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a polypeptide analog and its application in medicine. Background Art

[0002] GluR1 is a protein with three transmembrane domains expressed on the postsynaptic membrane of the central nervous system. It is closely associated with the pathogenesis of learning and memory impairment and related functional disorders, and is a major component of the AMPA receptor. Under the influence of the unique physiological and environmental factors of high altitude, GluR1 is abnormally expressed and aggregated. Through a series of biochemical reactions, including phosphorylation and dephosphorylation, it interacts with the upper and lower membranes of the AMPA receptor, thereby participating in the pathogenesis of learning and memory impairment. GluR1 has an extracellular amino terminus and an intracellular carboxyl terminus, and aggregates with other AMPA receptor subunits to form an amino acid-gated, ion-permeable channel. GluR1 differs from other subunits in that its carboxyl terminus can interact with different specific PDZ scaffold proteins, forming a large protein complex to regulate signal transduction and stabilize the role of AMPA receptors in learning and memory. Studies have found that high altitude exposure significantly inhibits the onset of long-term depression, which is associated with the dissociation of GluR1 from synaptosomes. Therefore, identifying drugs that inhibit the lower membrane of the synaptic GluR1 protein under altitude exposure is highly effective in inhibiting learning and memory impairment induced by altitude exposure.

[0003] During their long-term research, the Nantong University research team discovered that there is currently a lack of effective drugs for treating learning and memory impairment induced by high altitude exposure. Therefore, developing a drug to prevent or treat learning and memory impairment induced by high altitude exposure is particularly important. Summary of the Invention

[0004] In response to the deficiencies of the prior art, the present invention provides a polypeptide analog and its use in medicine, which can play an effective reference role in preventing / treating learning and memory decline induced by plateau exposure, and can further play an effective pharmaceutical role.

[0005] The present invention is achieved through the following technical solutions:

[0006] A polypeptide analogue has an amino acid sequence as shown in SEQ ID NO. 3, comprising an amino acid fragment in the transcriptional transactivator protein Tat and an amino acid fragment with recognition function.

[0007] Preferably, the amino acid fragment in the transcriptional transactivator protein Tat is located at the N-terminus of the polypeptide analog, and the amino acid fragment with recognition function is located at the C-terminus of the polypeptide analog.

[0008] Preferably, the amino acid fragment in the transcriptional transactivator protein Tat is the 47-57 amino acid fragment in the transcriptional transactivator protein Tat, having the amino acid sequence shown in SEQ ID NO.1; the amino acid fragment with recognition function is the 141-152 amino acid fragment in the cold-induced protein CIRP, having the amino acid sequence shown in SEQ ID NO.2.

[0009] Use of the above-mentioned polypeptide analogs in the preparation of drugs for preventing / treating learning and memory decline induced by plateau exposure.

[0010] A pharmaceutical composition for preventing / treating learning and memory decline induced by plateau exposure, comprising the above-mentioned polypeptide analogue.

[0011] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0012] The beneficial effects of the present invention are as follows:

[0013] The drug provided by the present invention for preventing / treating learning and memory impairment induced by plateau exposure includes a polypeptide analogue, which includes: an amino acid fragment in the transcriptional transactivator protein Tat and an amino acid fragment in the cold-induced protein CIRP. Among them, the amino acid fragment in the transcriptional transactivator protein Tat drives the above-mentioned polypeptide analogue to penetrate and enter the cell, and the amino acid fragment in the cold-induced protein CIRP can recognize GluR1 and bind to GluR1, and inhibit the lower membrane of the synaptic membrane protein GluR1, thereby achieving the purpose of stabilizing the synaptic membrane protein GluR1 under plateau exposure. That is, the polypeptide analogue provided by the present invention can play an effective reference role in preventing / treating learning and memory impairment induced by plateau exposure, and can further play an effective drug role. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the results of the Morris water maze test for the three experimental groups in Example 2: A is the acquisition training curve of the escape latency of mice on days 2-6 of the Morris water maze test; B is the motion trajectory of mice in the spatial memory exploration in the Morris water maze on day 6; C is a statistical graph of the total swimming distance of mice in the target quadrant on day 6 of the Morris water maze test; D is a statistical graph of the time spent by mice passing the platform position for the first time on day 6 of the Morris water maze test; E is a statistical graph of the total number of times mice passed the platform position on day 6 of the Morris water maze test; F is a statistical graph of the total time spent by mice in the target quadrant on day 6 of the Morris water maze test; G is a statistical graph of the swimming speed of mice on day 6 of the Morris water maze test;

[0015] Figure 2Schematic diagram of the results of the Golgi staining experiment for the three experimental groups in Example 2: A is a graph showing the results of Golgi staining of mouse brain tissue (scale 100 μm); B is a statistical graph showing the number of Golgi-stained nerve fibers in the cortical region (CTX) of mouse brain tissue; C is a statistical graph showing the density of dendritic spines of neurons in the cortical region (CTX) of mouse brain tissue that are Golgi-stained; D is a statistical graph showing the number of Golgi-stained nerve fibers in the CA1 region of the hippocampus of mouse brain tissue; E is a statistical graph showing the density of dendritic spines of neurons in the CA1 region of the hippocampus of mouse brain tissue that are Golgi-stained; F is a statistical graph showing the number of Golgi-stained nerve fibers in the DG region of the hippocampus of mouse brain tissue; G is a statistical graph showing the density of dendritic spines of neurons in the DG region of the hippocampus of mouse brain tissue that are Golgi-stained. DETAILED DESCRIPTION

[0016] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the accompanying drawings and specific embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0017] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The experimental materials used in the following examples, unless otherwise specified, are all commercially available at conventional biochemical reagent stores. The quantitative experiments in the following examples were performed in triplicate, and all results are expressed as mean ± standard error (SEM). Data were analyzed using two-way ANOVA; post hoc analysis was performed using the Turkey test. P < 0.05 was considered significant.

[0018] Example 1

[0019] A polypeptide analogue Tat-CIRP comprises an amino acid fragment in the transcription transactivator protein Tat and an amino acid fragment in the cold-induced protein CIRP with recognition function.

[0020] The transcriptional transactivator protein Tat can cross the cell membrane and enter the cell interior, showing a strong internalization effect. The Tat protein is generally composed of 86-102 amino acid residues. In this example, the amino acid fragment 47-57 of the Tat protein was selected, having the amino acid sequence shown in SEQ ID NO.1, abbreviated as: YGRKKRRQRRR, specifically:

[0021] Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg.

[0022] Of course, in other embodiments, other longer fragments of the Tat protein including the above-mentioned amino acids 47-57 may also be selected.

[0023] In this example, the amino acid fragment at positions 141-152 of the cold-inducible protein CIRP was selected. The amino acid fragment at positions 141-152 can effectively recognize and bind to the synaptic membrane protein GluR1. The amino acid fragment at positions 141-152 has the amino acid sequence shown in SEQ ID NO. 2, abbreviated as: YYASRSQGGGSYG, specifically:

[0024] Tyr-Tyr-Ala-Ser-Arg-Ser-Gln-Gly-Gly-Gly-Ser-Tyr-Gly.

[0025] Of course, in other embodiments, other longer fragments of the CIRP protein comprising the aforementioned amino acids 141-152 may also be selected.

[0026] In this example, the polypeptide analog is a fusion protein formed by amino acid fragments 47-57 of the transcriptional transactivator protein Tat and amino acid fragments 141-152 of the cold protein CIRP. The fusion protein in this example is formed by genetic engineering to connect the two amino acid fragments and express the corresponding protein fusion product.

[0027] In the fusion protein of this example, the amino acid fragment 47-57 of the transcriptional transactivator protein Tat is located at the N-terminus of the fusion protein, and the amino acid fragment 141-152 of the cold-inducible protein CIRP is located at the C-terminus of the fusion protein. That is, in this example, the sequence of the polypeptide analog Tat-CIRP is shown in SEQ ID NO. 3, abbreviated as: YGRKKRRQRRRYYASRSQGGGSYG, specifically:

[0028] Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Tyr-Tyr-Ala-Ser-Arg-Ser-Gln-Gly-Gly-Gly-Ser-Tyr-Gly.

[0029] Of course, the polypeptide analog provided in this embodiment can also be a polypeptide analog derived from the sequence shown in SEQ ID NO. 3 by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 3.

[0030] Example 2

[0031] This example uses specific data to illustrate that the peptide analog Tat-CIRP provides an effective reference for preventing / treating learning and memory decline induced by plateau exposure.

[0032] In this example, male C57BL / 6 (B6) mice were provided by the Animal Experiment Center of Nantong University and weighed 20-25 g. The peptide analog Tat-CIRP was artificially synthesized, and its amino acid sequence is shown in SEQ ID NO.3.

[0033] 1. First, 30 B6 mice were trained in the Morris water maze experiment to acquire memory and then randomly divided into groups. The hypobaric hypoxia treatment group and the hypobaric hypoxia peptide administration group were then placed in a hypobaric oxygen chamber to simulate plateau exposure. The specific principles and methods are as follows:

[0034] When the body is exposed to the low-oxygen environment of the plateau for a long time, the body cannot adapt well to the plateau environment through long-term natural selection and self-regulation. The body's internal environmental homeostasis is broken, and a series of functional disorders or pathological morphological changes that occur are called altitude depression.

[0035] The experiment used a Tawa Technology animal hypobaric oxygen chamber to simulate plateau altitude (oxygen partial pressure at 6000 meters) for 14 days (the ascent to 6000 meters took 20 minutes, with a ventilation rate of 22 times / min). The mice were trained in the Morris water maze before modeling. The specific experimental steps are as follows:

[0036] (1) Place the mouse head-down toward the wall of the Morris water maze into one of four random starting positions: east, west, south, or north. Record the time it takes the animal to find the underwater platform (s). During the first few training sessions, if this time exceeds 60 s, guide the animal to the platform. Allow the animal to remain on the platform for 10 s.

[0037] (2) Remove the animal and dry it. If necessary, place the animal under a 150W incandescent lamp for 5 minutes and return it to its cage. Each animal should be trained 4 times a day, with a 15-20 minute interval between training sessions, for 5 consecutive days.

[0038] (3) On the day after the last acquisition training session, the platform was removed and a 60-second exploration test was initiated. The animal was placed in the water from the quadrant opposite the original platform. The time the animal spent in the target quadrant (the quadrant where the platform was originally placed) and the number of times it entered that quadrant were recorded as indicators of spatial memory.

[0039] 2. After Morris water maze training, the mice were divided into three experimental groups for feeding as follows:

[0040] (1) Experimental Group 1 (Control + Saline Group): Ten mice were randomly selected and fed normally. Saline (0.9%) was injected into the tail vein starting on the 8th day. The injection dose was 100 μL, once every other day for 1 week, and the experiment ended on the 14th day.

[0041] (2) Experimental Group 2 (Hypobaric and Hypoxic Treatment Group + Saline Group): 10 mice were randomly selected and placed in a hypobaric oxygen chamber (Tawang Technology) simulating an altitude of 6000 m for continuous exposure to hypobaric and hypoxic conditions. Starting from the 8th day, the mice were taken out of the chamber every day and injected with saline (0.9%, 100 μL) through the tail vein within 30 minutes. After that, they were placed in the hypobaric oxygen chamber for exposure. The mice were injected once every other day and continued to be exposed to the hypobaric oxygen chamber for 1 week. The experiment ended on the 14th day.

[0042] (3) Experimental group 3 (low-pressure hypoxia treatment group + Tat-CIRP group): 10 mice were randomly selected and placed in a hypobaric oxygen chamber (Tawang Technology) simulating an altitude of 6000 m for low-pressure hypoxia exposure. On the 8th day, the peptide analog Tat-CIRP (20 mg / kg, 100 μL) was injected into the tail vein and then placed in the hypobaric oxygen chamber for exposure. The mice were injected once every other day and continued to be exposed to the hypobaric oxygen chamber for 1 week. The experiment ended on the 14th day.

[0043] On day 8, the three experimental groups were injected with 0.9% saline or a peptide analog as follows: First, the animal was placed in a restrainer and the tail was manually straightened. The tail was rubbed with a cotton ball containing 70% to 75% alcohol / hot water and then dried with a dry cotton ball. The right thumb and index finger were used to squeeze the vein at the base of the tail and push toward the tip. Simultaneously, the tail was rotated approximately 90 degrees to the left or right, causing one tail vein to swell and point upward. With the left hand facing upward, the operator placed the index and middle fingers beneath the tail and the thumb above it. The operator grasped the tail and used the thumb and index finger to control the angle of the tail so that the tip of the tail pointed downward. The left index and middle fingers rested against the edge of the experimental table, keeping the tail parallel to the surface. The operator held a 1 mL syringe (gauge 4 needle) in the right hand, keeping the needle parallel to the vein (<15°), and inserted the needle into the lower third of the tail. Insert the needle parallel to the bend of the mouse's tail (pointing downward) about 5 mm into the already filled tail vein. If blood returns after a slight withdrawal, it means that the needle has been accurately inserted into the vein. The operator releases the syringe, pinches the syringe jacket with the index and middle fingers, pushes the syringe button with the thumb, and pushes the liquid medicine into the vein at a certain speed while releasing the squeezed blood vessel. After insertion, slowly inject a small amount of liquid medicine. If there is no resistance, it means that the needle has entered the vein and you can continue to inject. If there is a lot of resistance during injection, it means that the needle has not penetrated the blood vessel and the injection should be moved back to the base of the tail.

[0044] 3. After the injection of peptide analogs and normal saline (0.9%), and after 14 days of hypobaric hypoxia modeling, the mice were tested for memory loss using the Morris water maze. The test method is as follows:

[0045] Remove the platform from the training pool and place the mouse in the water on the opposite side of the platform, ensuring that the mouse's back is facing the pool boundary. Start timing when the mouse enters the water, and use ANY-maze software to record the number of times the mouse crosses the original platform within 60 seconds, the time it first reaches the original platform, the distance and time it stays in the quadrant where the original platform is located, and the swimming speed.

[0046] The results are as follows Figure 1 As shown, mice in each group acquired memory before hypobaric hypoxia exposure ( Figure 1 A) The movement trajectory of mice in the Morris water maze spatial memory exploration on day 6. Figure 1 As shown in B. Based on this, different treatments are carried out in different groups. Figure 1 It can be seen that compared with experimental group 1, the total swimming distance of mice in experimental group 2 in the quadrant where the original platform was located ( Figure 1 C) The time it takes to reach the original platform for the first time ( Figure 1 D) increased significantly; while the number of times passing the original platform ( Figure 1 E) and the total swimming time in the quadrant where the platform is located ( Figure 1 F) were significantly reduced; while the changes of the above indicators were significantly reversed in the experimental group 3 mice, and the differences were statistically significant (P < 0.05). There was no difference in swimming speed among the mice in the above groups ( Figure 1 G). The above behavioral results suggest that the changes in these indicators are not caused by changes in the mice's motor ability.

[0047] 4. After the injection of peptide analogs and normal saline (0.9%), and after 14 days of hypobaric hypoxia modeling, the mice were dissected and the brain tissue was removed for Golgi staining using the HitoBiotec Golgi staining kit (PreKit). The experimental method is as follows:

[0048] (1) Slicing steps:

[0049] ① Use appropriate anesthetics to deeply anesthetize experimental animals.

[0050] ② Do not use buffer or fixative for perfusion. Remove the specimen quickly according to the experimental routine. Be careful to avoid damaging the tissue. Large specimens should be cut into tissue blocks about 1 cm thick using a sharp blade.

[0051] ③ Rinse the tissue block with double-distilled water for 2-3 seconds to remove residual blood on the surface.

[0052] ④ Transfer the tissue block to at least five times the volume of the pre-prepared immersion solution and store it at room temperature in the dark.

[0053] ⑤ The next day (12-24 hours later), replace the soaking solution with an equal amount and store at room temperature (20-25°C) in the dark for 14 days. To avoid nonspecific staining, do not extend the soaking time.

[0054] ⑥ After 14 days, the tissue will turn yellow-brown, become soft and loose, and easily damaged. Carefully transfer the tissue to at least five times the volume of Solution 3 (provided in the kit, the same below) and store at 4°C in the dark. After 12 hours, replace the solution with an equal amount and continue to store at 4°C in the dark for 24 to 72 hours.

[0055] ⑦ Use low-melting-point agarose gel to embed the tissue, trim it with a sharp blade, and fix the sample on the sample holder of the vibration microtome. Set the slice thickness to 100μm and start slicing. Collect the tissue slices in distilled water and use the included brush to stick the slices floating in the distilled water onto the gelatin-coated slide.

[0056] ⑧ After absorbing excess distilled water with a filter paper strip, use a dropper to add a small amount of solution 3 to the tissue section. Wait for 1 to 2 minutes and then absorb the excess solution 3 with a filter paper strip. After confirming that the section no longer floats or slides, place the slide at an angle in the dark at room temperature to dry overnight.

[0057] ⑨ The dried sections should be stained and sealed as soon as possible. If conditions do not permit, they can be stored in the dark at room temperature for 2 to 3 days. However, they must not be stored for a long time, otherwise a large amount of black crystalline background will be generated inside the tissue.

[0058] (2) Staining steps:

[0059] ① Soak the slide with the tissue section in distilled water twice, 3 minutes each time.

[0060] ② Mix 2 mL of solution 4 (provided by the kit), 2 mL of solution 5 (provided by the kit), and 6 mL of double-distilled water in the 12 mL staining jar included in the kit, place the slide with the tissue section, cover tightly, and wait for 10 minutes (this mixture can only be used once).

[0061] ③ After the reaction is completed, soak the slices in distilled water twice, each time for 4 minutes.

[0062] ④ As an optional step, if Nissl-stained cell nuclei counterstaining is required, it can be performed at this time.

[0063] ⑤ Place the slices treated in distilled water in 50%, 70% and 95% alcohol for 5 minutes each for gradient dehydration.

[0064] ⑥ Dehydrate the slices in anhydrous alcohol three times, 5 minutes each time.

[0065] ⑦ Place the slices in xylene for transparent treatment twice, 5 minutes each time, and then use coverslips and undiluted high-concentration neutral gum mounting medium to seal the slices.

[0066] ⑧After the xylene evaporates and dries, it can be observed under a microscope.

[0067] The results are as follows Figure 2 The results of Golgi staining of mouse brain tissue are shown in Figure 2 As shown in A. Figure 2 It can be seen that compared with experimental group 1, the experimental group 2 mice had Figure 2 B) Hippocampal CA1 region ( Figure 2 D) and dentate gyrus DG area ( Figure 2 F) The number of neuronal fibers was significantly reduced; compared with experimental group 1, the number of neurons in the cerebral cortex ( Figure 2 C), hippocampal CA1 area ( Figure 2 E) and dentate gyrus DG area ( Figure 2 G) Dendritic spine density also decreased significantly. Experimental group 3 can effectively inhibit the reduction of neuronal fiber number and dendritic spine density in various regions of mouse brain tissue, and the above differences are statistically significant (P < 0.05).

[0068] The above experimental results show that injection of the peptide analog Tat-CIRP into the tail vein of mice can significantly inhibit the reduction in the number of dendritic spines in the brain caused by hypobaric and hypoxic exposure, and inhibit the learning and memory impairment caused by plateau exposure.

[0069] Based on Example 1 and Example 2, their applications can be further expanded. For example, the present invention also provides a drug for preventing / treating learning and memory decline induced by plateau exposure, including the polypeptide analog Tat-CIRP.

[0070] In addition, in the present invention, the drug for preventing / treating learning and memory impairment induced by plateau exposure may further include a pharmaceutically acceptable carrier, such as an inert diluent, a filler, water, etc. If necessary, the drug may further include additional ingredients, such as flavoring agents, binders, etc.

[0071] In the present invention, the effective dose of the drug depends on the patient's species, sex, weight, age, medical condition, route of administration and severity of the condition being treated. A skilled physician can easily determine and prescribe the effective dose of the drug for preventing / treating a disease.

[0072] The above description is only an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the description and drawings of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A polypeptide analog, characterized in that: The amino acid sequence of the polypeptide analog is shown in SEQ ID NO. 3, which includes an amino acid fragment in the transcriptional transactivator protein Tat and an amino acid fragment with recognition function.

2. Use of the polypeptide analogue according to claim 1 in the preparation of a medicament for preventing / treating learning and memory impairment induced by plateau exposure.

3. A pharmaceutical composition for preventing / treating learning and memory decline induced by plateau exposure, characterized in that: Including the polypeptide analogue as claimed in claim 1.

4. The pharmaceutical composition according to claim 3, characterized in that Also included are pharmaceutically acceptable carriers.

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