Membrane-penetrating peptide for inhibiting MD2 function and application thereof

Abstract

The invention discloses a fusion oligopeptide. The fusion oligopeptide is formed by fusing a cell-penetrating peptide and an oligopeptide for inhibiting combination of MD2 and CIRP. The invention also discloses an application of the fusion oligopeptide in preparation of a medicine for treating central nervous cell diseases mediated by an MD2-TLR4 signal channel. The fusion oligopeptide provided by the invention only has 15 amino acids, not only reduces immunological rejection of an organism, but also is relatively low in synthesis cost, and is used as a bioactive molecule to effectively block central nervous cell disease injury mediated by an MD2-TLR4 signal channel, relieve post-stroke nerve injury caused by nerve excitation toxicity and reduce infarct area, so that the fusion oligopeptide can be used as a biologically active molecule to effectively inhibit apoplexy. The nerve function is effectively recovered.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a short peptide that inhibits the function of MD2 protein and an application thereof. Background Art

[0002] Surveys show that stroke has become the leading cause of death in my country and the leading cause of disability among Chinese adults. Stroke is characterized by high morbidity, high mortality and high disability rate. Stroke is divided into ischemic and hemorrhagic stroke, of which ischemic stroke accounts for more than 80% of all types. Currently, tPA (tissue plasminogen activator) is the only treatment drug approved by the US FDA. However, due to its short treatment time window (within 4.5 hours after ischemia) and increased bleeding tendency, only 4% to 7% of patients can currently receive tPA treatment. Therefore, how to treat stroke is still a difficult problem that needs to be solved urgently.

[0003] In animal experiments, a large number of drugs and interventions have been shown to significantly reduce the neurological damage caused by stroke. However, hundreds of completed clinical trials have ended in failure. Combining clinical and animal research, in-depth analysis found that one of the main reasons for failure is that there are certain differences in the physiological structure of cells and rodents and humans. Therefore, researchers need to focus on verifying rodent research in primates and ultimately conducting clinical transformation.

[0004] Nerve injury involves a variety of pathological processes, including excitotoxic injury and neuroimmune injury, and these two pathological processes can promote each other and form a vicious cycle. Eventually, it leads to cell necrosis, apoptosis and necroptosis. Since necrosis is a non-programmed death and cannot be regulated; therefore, in the past few decades of research, researchers have focused on apoptosis. However, in recent years, researchers have found that if we simply inhibit apoptosis, cells will transform into another regulatable cell death mode-necroptosis. This transformation not only fails to reduce the damage, but aggravates the degree of damage. This largely explains why a large number of drugs that inhibit apoptosis signals cannot play a protective role in clinical practice. Therefore, in-depth understanding of the common signaling pathways of different pathological processes and different death modes can develop effective therapeutic drugs. A large number of studies have shown that immune inflammatory responses play an important role in multiple stages such as acute injury and later repair after stroke.

[0005] Myeloid differentiation protein-2 (MD-2) is a secretory protein with a molecular weight of 20 to 30 kD. It binds to the extracellular region of Toll-like receptor 4 (TLR4) and can form a complex with TLR4 (TLR4-MD2). The MD2-mediated signaling pathway is an important component of the immune-inflammatory response and is also involved in the pathological process of ischemic and hemorrhagic stroke. More importantly, the TLR4-MD2-mediated signaling pathway is a common signaling pathway for multiple pathological processes such as inflammation, apoptosis, and necroptosis. However, current reports on MD2 mainly focus on its involvement in the immune-inflammatory response of peripheral tissues and organs. However, whether MD2 mediates central nervous system damage, stroke damage, and therapeutic measures with MD2 as an intervention target have not been reported.

[0006] CN108864311A discloses a short peptide Tat-CIRP (amino acid sequence: YGRKKRRQRRR---GRGFSRGGGDRGYGG) that can cross the blood-brain barrier and inhibit the binding of MD2 to CIRP. However, the current drug treatment for ischemic stroke is mainly tPA for thrombolysis, and plasmin is the most active protease in plasma, but its specificity is poor. tPA can hydrolyze the sites where each lysine-arginine (KR) on the peptide chain is connected. Tat-CIRP contains 26 amino acids and contains the KR amino acid sequence, which is easily degraded by plasmin and tPA in the blood, affecting the therapeutic effect. Therefore, how to optimize the amino acid sequence of the short peptide, reduce the difficulty of synthesis, and enhance the therapeutic effect of the short peptide has become an important part of the clinical transformation of the short peptide. Summary of the invention

[0007] The purpose of the present invention is to provide a short peptide for inhibiting the function of MD2 protein and application thereof.

[0008] Based on the above purpose, the present invention first provides a fusion short peptide, which is formed by fusion of a membrane-penetrating peptide and a short peptide that inhibits the binding of MD2 and CIRP. The membrane-penetrating peptide is named 5R (RRRRR) in the present invention. The 5R can be used as a membrane-penetrating guide short peptide to effectively penetrate the blood-brain barrier. Its amino acid sequence is shown in SEQ ID NO. 1. The short peptide that inhibits the binding of MD2 and CIRP is obtained by truncating the binding region of MD2 protein and CIRP protein and replacing the amino acid sequence. It is named CIRP-R in the present invention, and its amino acid sequence is shown in SEQ ID NO. 2. CIRP-R is used to inhibit the binding of MD2 and CIRP, thereby blocking NMDA-mediated neuronal excitotoxicity.

[0009] In the fusion peptide of the present invention, the 5R can be located at the N-terminus of the fusion peptide or at the C-terminus of the fusion peptide. In a preferred embodiment, 5R is located at the N-terminus of the fusion peptide, and the amino acid sequence of the fusion peptide is shown in SEQID NO.3.

[0010] Secondly, the present invention provides the use of the above-mentioned fusion peptide in the preparation of a therapeutic drug for central nervous system diseases caused by elevated MD2. The fusion peptide provided by the present invention plays a therapeutic role in central nervous system diseases caused by elevated MD2 by specifically inhibiting the TLR4-MD2 mediated signaling pathway.

[0011] In a preferred embodiment, the therapeutic drug is a therapeutic drug for central nervous system excitotoxicity diseases, which refers to a series of diseases caused by increased central nervous system excitotoxicity mediated by the MD2-TLR4 signaling pathway. The fusion peptide of the present invention can reduce central nervous system excitotoxicity by inhibiting the binding of MD2 and TLR4.

[0012] In a more preferred embodiment, the therapeutic drug is a drug that reduces N-methyl-D-aspartate (NMDA)-mediated neuronal cell death. Overactivation of NMDA receptors is an important pathological process that causes nerve damage after ischemic and hemorrhagic stroke. The fusion peptide described in the present invention can reduce neuronal cell death, including apoptosis, necroptosis, etc., by inhibiting overactivation of NMDA receptors.

[0013] In another preferred embodiment, the therapeutic drug is an ischemic stroke therapeutic drug. The fusion peptide of the present invention can reduce various neural damages caused by ischemic stroke by inhibiting the binding of MD2 and TLR4.

[0014] More preferably, the therapeutic drug is a drug for reducing the infarct area after ischemic stroke. In a specific application of the present invention, the fusion peptide can effectively reduce neurons after stroke and reduce the area of ​​cerebral infarction.

[0015] In another preferred embodiment, the therapeutic drug is a drug for improving neurological function. In a specific application of the present invention, the fusion peptide can improve the motor function and limb strength of the mouse focal cerebral ischemia model (MCAO) by inhibiting the binding of MD2 to TLR4.

[0016] In another preferred embodiment, the therapeutic drug is a drug for improving neuroimmune damage. The fusion peptide of the present invention can reduce inflammatory damage caused by the immune response of the nervous system by inhibiting the binding of MD2 and TLR4.

[0017] In another preferred embodiment, the therapeutic drug is a drug for treating hemorrhagic stroke. The fusion peptide of the present invention can reduce the neurological damage caused by hemorrhagic stroke by inhibiting the binding of MD2 and TLR4, thereby reducing various neurological damages caused by ischemic stroke.

[0018] Compared with the prior art, the present invention has the following beneficial technical effects: The present invention optimizes and restructures the original short peptide Tat-CIRP (amino acid sequence: YGRKKRRQRRR---GRGFSRGGGDRGYGG) that has been reported. On the one hand, the Tat amino acid sequence (11 amino acids, including KR) is optimized to 5R (RRRRR), which reduces the molecular weight of the target peptide, inhibits the degradation of the short peptide by plasmin and tPA, and increases the membrane penetration efficiency of the membrane-penetrating peptide to ensure that it can pass through the blood-brain barrier and enter the cell. On the other hand, the present invention truncates and restructures the short peptide CIRP (16 amino acids, amino acid sequence: GRGFSRGGGDRGYGG) in the functional region, and names it CIRP-R (10 amino acids, amino acid sequence GRGFSRGRRR). After experimental verification, the short peptide constructed by the present invention can effectively block NMDA-mediated neuronal cell death, reduce nerve damage after ischemic stroke, and reduce the infarct area.

[0019] At the same time, the short peptide synthesized by the present invention has only 15 amino acids, which not only reduces the body's immune rejection reaction, but also has a low synthesis cost. It is used as a biologically active molecule to prepare stroke drugs, which has a very broad prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect and dose-effect diagram of 5R-CIRP-R treatment on primary cultured neurons after NMDA-induced neurotoxic injury; Figure 2 This is an immunofluorescence image showing that the short peptide 5R-CIRP-R inhibits NMDA and significantly inhibits neuronal apoptosis; Figure 3 This is a comparison of the efficacy of 5R-CIRP-R and TAT-CIRP in inhibiting neurons from NMDA-induced neurotoxic injury in an in vitro experiment; Figure 4 This is a comparison of the effects of tail vein injection of TAT-CIRP and 5R-CIRP-R on reducing cerebral infarction area after ischemic stroke; Figure 5 This is a comparison of the effects of tail vein injection of TAT-CIRP and 5R-CIRP-R on improving neurological function after ischemic stroke. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0022] Preparation Example, Design and Preparation of Chemically Synthesized Tat-CIRP and 5R-CIRP-R Short Peptides Design objectives and requirements: First, Tat-CIRP was synthesized according to the reported method.

[0023] In order to reduce the molecular weight of the target peptide and increase the penetrating efficiency of the membrane-penetrating peptide, RRRRR was first synthesized to replace Tat as a membrane-penetrating peptide that guides the passage through the blood-brain barrier. Secondly, the amino acids were truncated and reconstructed from the functional region of the original CIRP. The truncated amino acid sequence must also be able to maintain a high ability to bind to MD2, and it is required to be a pseudosubstrate that prevents CIRP from binding to MD2, thereby inhibiting the activity of MD2, and then inhibiting the inflammatory response, apoptosis and necroptosis mediated by the MD2-TLR4 signaling pathway.

[0024] The cell-penetrating peptide 5R used in the present invention has only 5 amino acids, which can effectively introduce biological macromolecules such as proteins and polypeptides into cells in a non-receptor-mediated and non-energy-consuming manner, and will not cause cell damage within a certain concentration range and is not easily degraded by tPA.

[0025] The present invention also prepares the Tat-CIRP disclosed in CN108864311A as a control. The short peptide Tat-CIRP and 5R-CIRP-R are synthesized by Shanghai Jier Biotechnology Co., Ltd., wherein the amino acid sequence of Tat-CIRP is YGRKKRRQRRR---GRGFSRGGGDRGYGG (SEQ ID NO.4), the amino acid sequence of the optimized CIRP-R functional region is: GRGFSRGRRR (SEQ ID NO.2), the amino acid sequence of 5R is: RRRRR (SEQ ID NO.1), and the full-length amino acid sequence of 5R-CIRP-R is RRRRR-GRGFSRGRRR (SEQ ID NO.3).

[0026] Application Example 1. Construction of NMDA Stimulated Primary Neuron Model and Evaluation of 5R-CIRP-R Therapeutic Effect A large amount of research evidence shows that excessive release of excitatory amino acids activates NMDA receptors, leading to swelling of depolarized neurons, Ca+ influx and even complex processes such as second messengers, which are important pathological processes causing nerve damage after ischemic and hemorrhagic stroke. Therefore, the present invention uses a model of NMDA stimulating primary neurons to simulate a stroke model for in vitro exploration.

[0027] 1. Cultivation of primary neurons Add 30 μg / ml L-polylysine to 12-well and 24-well cell culture plates (0.5 ml), wrap with sterile tin foil, place in a clean bench, and incubate overnight at room temperature (coating). The next day, use a vacuum suction pump to remove the polylysine, and rinse with high-temperature and high-pressure sterilized double distilled water three times for primary neuron culture.

[0028] After deep anesthesia of 16-day-old C57BL / 6 mice, the pregnant mice were placed supine on a sterile tray. The abdomen of the pregnant mice was disinfected with alcohol and iodine. The abdominal skin was cut open, and the abdominal muscles and peritoneum were cut open with new sterile scissors. The embryo was cut out with scissors and placed in pre-cooled D-Hanks solution. New sterile ophthalmic scissors were used to cut the head of the fetal mouse and place it in new D-Hanks solution. Under a microscope, the meninges and blood vessels were carefully separated with microtweezers. The cortical brain tissue was removed, placed in new D-Hanks solution, and fully minced. 2 ml of 0.25% trypsin was added to the minced tissue and digested in an incubator (37°C, 5% CO2) for 10 minutes. The trypsin digestion was terminated by adding serum-containing culture medium. The tissue solution after the termination of digestion was transferred to a 15 ml centrifuge tube with a pipette and centrifuged at 1000 rpm for 10 minutes. Carefully aspirate the supernatant with a pipette and add culture medium (containing 20% ​​fetal bovine serum) to the centrifuge tube. Use a dropper to fully disperse the cells to obtain a single cell suspension. Aspirate 10 μl of the cell suspension, dilute it 100 times, and count it using a glass counting plate. Add culture medium according to the total number of cells, mix thoroughly, and inoculate into 12-well plates and 24-well plates. The inoculation density for 12-well plates is 3.0×10 5 , 24-well plate: 2.0×10 5 . Culture in a 37°C, 5% CO2 incubator. After 3 days, change half of the medium, and then change 1 / 3 of the medium every 3 days. Before cell treatment and administration, observe neuronal morphology under a microscope to determine neuronal purity and status.

[0029] 2. Release rate of lactate dehydrogenase in primary neurons after NMDA stimulation In order to ensure the effectiveness of NDMA stimulation, we first detected the release rate of lactate dehydrogenase (LDH) after NMDA stimulation (LDH release can reflect the overall cell death, including necrosis, apoptosis and necroptosis). The results showed that after 24 hours of NMDA (50μM; 1 h) stimulation of primary neurons, the release of LDH by neurons increased significantly. After treatment with 5R-CIRP-R, cell death was significantly blocked; and the treatment dose ranged from 1 micromolar to 20 micromolar. Figure 1 The relative LDH were: control group 1, NMDA group 2.26±0.16, 1μmol group 1.46±0.09; 5μmol group 1.11±0.11; 10μmol group 1.2±0.85; 20μmol group 1.29±0.10. Data are expressed in Mean+SEM). Immunofluorescence results are shown in Figure 2 As shown, MAP-2 positive cells indicate that the cells under study are neurons, and Tunnel is an apoptotic neuron. In normal neurons, Tunnel is expressed in small amounts; after NMDA stimulation, apoptotic neurons increased significantly, and after 5R-CIRP-R treatment, neuronal apoptosis was significantly inhibited.

[0030] After comparing the efficacy, no obvious cell death was found after treatment with Tat-CIRP and 5R-CIRP-R, which completely blocked the NMDA pathway, indicating a ceiling effect for the short peptide treatment in the in vitro model ( Figure 3 , the relative LDH were: control group 1, NMDA group 2.17±0.10, Tat-CIRP group 1.10±0.07; 5R-CRIP-R group 1.17±0.13. Data are expressed in Mean+SEM). Therefore, there was no significant difference in the efficacy between the two groups.

[0031] Application Example 2. Construction and evaluation of a mouse focal cerebral ischemia model (MCAO) 1. Experimental Animals SPF grade C57BL / 6 mice, MD2 knockout mice, male, 8-10 weeks old, weighing 20-25 g, healthy female C57 pregnant mice, 16 days of gestation. Purchased from the Experimental Animal Center of the Fourth Military Medical University. The animal culture environment was SPF grade, 12-hour day / night rhythm regulation, free diet. The experimental process strictly followed the Fourth Military Medical University Animal Experiment Guide.

[0032] 2. Construction of mouse focal cerebral ischemia model (MCAO) Since NMDA (N-methyl-D-aspartate)-induced excitotoxic injury can only partially simulate the pathological process of ischemic stroke, a focal transient ischemia model was constructed in vivo to simulate clinical ischemic stroke. The specific construction process is as follows: 1) After inducing anesthesia in mice with 2.5% isoflurane, anesthesia was maintained with 1.5% isoflurane and the animals were allowed to breathe spontaneously during the surgery.

[0033] 2) Fix the mouse and place it supine on a thermostatic plate (the temperature of the thermostatic plate is 37°C). Monitor the rectal temperature to maintain the body temperature at 37.0±0.5°C. Use a laser Doppler blood flowmeter to monitor the blood flow in the surface projection area of ​​the middle cerebral artery before, during, and after the operation.

[0034] 3) After disinfecting the mouse neck with iodine, use a scalpel to make a small incision in the middle of the neck skin to fully expose and carefully separate the right common carotid artery, external carotid artery and internal carotid artery. Be careful to prevent vagus nerve damage during the operation.

[0035] 4) Use No. 3 mousse thread to tie a slipknot at the proximal end of the common carotid artery to temporarily block the blood flow of the common carotid artery; ligate the distal end of the right external carotid artery, and make a small incision in the external carotid artery with a syringe. Insert a 15 mm long thread plug (Shenzhen Ruiwode Company) along the external carotid artery.

[0036] 5) Use micro forceps to push the suture plug forward about 8 to 9 mm (from the bifurcation of the internal and external carotid arteries). When there is a slight resistance, it indicates that the suture plug has reached the starting point of the middle cerebral artery and the insertion should be stopped.

[0037] 6) According to the results of the cerebral blood flow detector, after the occlusion of the suture, the blood flow value of the middle cerebral artery is reduced to 30% lower than the level before occlusion, the modeling is considered effective, otherwise it is excluded.

[0038] 7) Suture the incision and place the mouse in an incubator to maintain its body temperature at 37.0±0.5℃.

[0039] 8) After blocking the blood flow for 1 hour, the mouse was re-anesthetized with isoflurane, the incision was reopened, the suture was slowly removed, and the perfusion was restored. If the blood flow recovered to more than 70% of the basal value within 10 minutes, the model was considered successful, otherwise it was excluded.

[0040] 9) Suture the skin, place the mouse in an incubator, and wait for the mouse to wake up naturally.

[0041] 10) The mice in the sham operation group only underwent surgery without suture insertion. The mice in the treatment group were given Tat-CIRP and 5R-CIRP-R (20 mg / kg) through the tail vein at 0h and 6h after surgery, respectively. The control group was given normal saline.

[0042] 3. Neurological Functional Assessment of Cerebral Ischemia After the stroke model was successfully established, scores were performed at corresponding time points according to the experimental plan. The scores were performed by two researchers who were unaware of the experimental groups, using the Grip strength and Rotarod test methods to evaluate the motor function and limb strength of the mice.

[0043] 4. Determination of infarct volume by TTC staining Use scissors to quickly remove the mouse head, quickly peel off the skull, take out the brain, and put it in a 4°C refrigerator to cool for 10 minutes. Then put the brain into the brain trough and use a blade to evenly cut the brain into 1 mm thick (coronal section). Transfer the 1 mm thick brain slices to a preheated (37°C) 2% TTC solution. After 10 minutes, recover the TTC dye solution and fix the stained brain slices with 4% paraformaldehyde for 24 hours. Use a digital camera to take pictures, import the brain slice images into the computer, and use Adobe Photoshop cs 3.0 software to analyze the cerebral infarction volume. In order to reduce the effect of edema of the ischemic side of the brain tissue on the infarction volume, this embodiment uses the percentage of infarction volume to express the degree of infarction: The percentage of infarct volume = (the volume of the contralateral cerebral hemisphere - the volume of normal brain tissue on the infarcted side) / the volume of the contralateral cerebral hemisphere × 100%.

[0044] 5. Statistical analysis SPSS19.0 statistical software was used to analyze the data. Quantitative data such as percentage of infarct area and relative protein expression were expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used for comparison between multiple groups and between groups, and Dunnett test or Bonferroni test was used according to experimental requirements. Neurobehavioral scores were expressed as medians. P values ​​less than 0.05 were considered statistically significant between groups.

[0045] 6. Experimental results In order to clarify whether the therapeutic effect of the modified short peptide is better than Tat-CIRP, the present invention intravenously administered normal saline, Tat-CIRP and 5R-CIRP-R to mice immediately after ischemia-reperfusion injury (20 mg / kg, iv). According to the time course of MD2 changes in ischemic stroke, an additional treatment was given 6 hours after reperfusion, and the neurological function was evaluated 72 hours later and the infarct volume was calculated by TTC staining. Figure 4 As shown in the figure, the infarct area in the 5R-CIRP-R group was significantly smaller than that in the Tat-CIRP group (the relative infarct area of ​​each group was: 0.59±0.03 in the control group, 0.43±0.01 in the Tat-CIRP group, and 0.34±0.02 in the 5R-CIRP-R group. The data were expressed as Mean+SEM). Figure 5As shown, 5R-CIRP-R is significantly better than Tat-CIRP in improving the neurological function of mice (the Grip strength scores of each group are: 45.30±2.64 for the control group, 56.80±2.44 for the Tat-CIRP group, and 64.90±2.97 for the 5R-CIRP-R group. The rotarod test scores are 141.80±10.60 for the control group, 200.13±5.90 for the Tat-CIRP group, and 245.63±10.61 for the 5R-CIRP-R group. The data are expressed in the form of Mean+SEM). The above results show that 5R-CIRP-R is significantly better than Tat-CIRP in alleviating ischemic nerve damage.

[0046] Based on the above results, the present invention confirms for the first time that 5R-CIRP-R can be used as an inhibitor of the NMDA signaling pathway to block NMDA-induced excitotoxicity. The present invention uses MD2 as the intervention target and optimizes and modifies Tat-CIRP to synthesize a short peptide 5R-CIRP-R that can significantly reduce excitotoxic damage and improve the prognosis of ischemic and hemorrhagic stroke.

Claims

1. A fusion peptide, wherein the fusion peptide is formed by fusion of a membrane-penetrating peptide and a short peptide that inhibits the binding of MD2 and CIRP, characterized in that: The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.1, and the amino acid sequence of the short peptide that inhibits the binding of MD2 and CIRP is shown in SEQ ID NO.

2.

2. The fusion peptide according to claim 1, characterized in that: The amino acid sequence of the fusion short peptide is shown in SEQ ID NO.

3.

3. Use of the fusion peptide according to claim 1 or 2 in the preparation of a drug for treating central nervous system diseases caused by elevated MD2.

4. The use according to claim 3, characterized in that: The therapeutic drug is a therapeutic drug for central nervous system excitotoxicity diseases.

5. The use according to claim 3, characterized in that: The therapeutic drug is a drug that reduces N-methyl-D-aspartate-mediated neuronal cell death.

6. The use according to claim 3, characterized in that: The therapeutic drug is an ischemic stroke therapeutic drug.

7. The use according to claim 6, characterized in that: The therapeutic drug is a drug for reducing the infarct area after ischemic stroke.

8. The use according to claim 3, characterized in that: The therapeutic drug is a drug that improves nerve function.

9. The use according to claim 3, characterized in that: The therapeutic drug is a drug for treating hemorrhagic stroke.

10. The use according to claim 3, characterized in that: The therapeutic drug is a drug that improves neuroimmune damage.

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