Targeting peptide compound for specifically degrading NDRG2 and application thereof

Abstract

The invention discloses a specific structure of a targeting peptide compound capable of specifically degrading NDRG2, the structure of the targeting peptide compound is disclosed for the first time, the chemical formula of the targeting peptide compound is clear to be C293H474N96O68, the molecular weight of the targeting peptide compound is 6429.62, the compound improves the structural domain of targeting peptide, and the targeting peptide compound can be used for degrading NDRG2. The method comprises the design and synthesis of three structural domains: (1) synthesizing a transmembrane structural domain (CMPD) by utilizing a known membrane penetrating peptide fragment TAT; (2) specifically binding an NDRG2 structural domain (PBD); and (3) serially connecting a degradation structural domain (CTM / gene). The main effect of the targeting peptide compound is to alleviate inflammatory response and oxidative stress injury, so that the targeting peptide compound is applied to the new application of drugs for treating cerebral ischemia injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of targeted peptide drug applications, and specifically relates to a targeted peptide compound for specifically degrading NDRG2 and an application thereof, and particularly relates to an application thereof in alleviating inflammatory response and oxidative stress damage. Background Art

[0002] The targeted peptides reported in the latest research can cross the blood-brain barrier and degrade the target protein specifically, rapidly and reversibly through the lysosomal pathway. They have high potency, good safety and a wide range of applications. They have been preliminarily proven to improve damage caused by neurological diseases and are expected to become a new strategy for preventing and treating neurological diseases.

[0003] NDRG2 (N-myc downstream regulated gene 2) is a tumor suppressor gene that plays a key role in a variety of physiological and pathological processes. The protein encoded by this gene belongs to the NDRG (N-myc downstream-regulated gene) family, and members of this family have multiple functions, including cell differentiation, cell cycle regulation, cell stress response, and tumor suppression. NDRG2 plays a wide and important role in a variety of diseases, including cancer and stroke, as well as neurodegenerative diseases (such as Alzheimer's disease), cardiovascular diseases (such as myocardial infarction), metabolic diseases (such as diabetes), kidney diseases (such as chronic kidney disease), liver diseases (such as liver fibrosis and cirrhosis), inflammatory diseases (such as inflammatory bowel disease), and autoimmune diseases (such as systemic lupus erythematosus). In these diseases, NDRG2 affects the occurrence and progression of the disease through multiple mechanisms. For example, in cancer, NDRG2 inhibits tumor growth by inhibiting cell cycle progression, inducing apoptosis and autophagy; in stroke, it may reduce neuronal cell death in ischemic injury by regulating the excitotoxicity of glutamate. In addition, NDRG2 may play a role in neurodegenerative diseases by regulating neuroinflammation and apoptosis, and is associated with tissue damage and fibrosis in cardiovascular and liver diseases. In metabolic and renal diseases, NDRG2 may affect disease progression by regulating metabolic pathways and inflammatory responses. Due to its multiple biological functions in these wide-ranging diseases, NDRG2 shows great potential for clinical research and treatment, becoming an important target in future treatment strategies.

[0004] In recent years, with the in-depth study of NDRG2, scientists have begun to explore the possibility of using NDRG2 as a target for disease treatment. Due to the small molecular weight and good biocompatibility of peptides, they can penetrate the cell membrane and interact with NDRG2 in the cell to achieve efficient targeted therapy. NDRG2 targeting peptide is a specific peptide segment specifically targeting NDRG2. It can interfere with its normal function or trigger specific biological effects by binding to NDRG2, showing therapeutic potential in various types of diseases. In cancer treatment, NDRG2 targeting peptide can inhibit the proliferation and migration of tumor cells, induce cell apoptosis or enhance the sensitivity of chemotherapeutic drugs by enhancing the expression or function of NDRG2. However, the current research on NDRG2 targeting peptide is still in its early stages, and its exact mechanism of action, optimal peptide sequence and administration method still need to be further explored and optimized. Although peptide drugs have many potential advantages, they also face challenges such as poor stability, easy degradation and high production cost. Therefore, in-depth research is needed in the design, synthesis and clinical application of peptide drugs in the future to promote their application in the treatment of various diseases.

[0005] A Chinese patent application with publication number CN 112142856 A discloses a specific degradation NDRG2 targeting peptide for improving excitotoxic damage in stroke, which mainly works by reducing the excitotoxicity of glutamate. However, the targeting peptide has certain structural defects, such as poor stability and easy enzymatic degradation in the body, resulting in low bioavailability. In addition, the targeting and specificity of the targeting peptide may be insufficient, which may affect its therapeutic effect. Therefore, although it shows certain potential in reducing excitotoxicity in stroke, there is still a lack of relevant reports on its role in reducing inflammatory response and oxidative stress damage. In view of these structural defects and functional limitations, it is necessary to further optimize and improve the targeting peptide to enhance its stability and specificity, and expand its scope of application in disease treatment, aiming to improve its effectiveness and specificity in clinical applications. Summary of the invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a targeted peptide compound and its application for specifically degrading NDRG2. The targeted peptide with improved structural domain can effectively enhance the degradation effect on NDRG2, and at the same time provide pharmaceutical applications for reducing inflammatory response and oxidative stress damage.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The invention discloses a targeting peptide compound for specifically degrading NDRG2, wherein the chemical formula of the targeting peptide compound is C 293 H 474 N96 O 68 , molecular weight is 6429.62, and the structural formula is as follows: .

[0008] Preferably, the targeting peptide compound comprises a domain that can specifically target the NDRG2 protein, and the transmembrane peptide in the targeting peptide is a transmembrane domain transformed from TAT, YGR-Orn-Orn-KRRQRRR, and the amino acid sequence of the transmembrane peptide is shown in SEQ.ID.NO.1.

[0009] Further preferably, the targeting peptide compound is composed of a transmembrane peptide, a domain that can specifically target the NDRG2 protein, and a lysosome plus proteasome-induced degradation peptide segment; The amino acid sequence of the domain that can specifically target the NDRG2 protein is shown in SEQ.ID.NO.2; the amino acid sequence of the lysosome plus proteasome induced degradation peptide segment is shown in SEQ.ID.NO.3.

[0010] The present invention also discloses the use of the above-mentioned targeted peptide compound for specifically degrading NDRG2 in preparing a medicine for alleviating oxidative stress damage.

[0011] Preferably, the drug is a drug that uses NDRG2 as an intervention target to significantly reduce oxygen-glucose deprivation damage in astrocytes.

[0012] Further preferably, the drug can inhibit the activation of type A1 astrocytes and promote the activation of type A2 astrocytes.

[0013] The invention also discloses the use of the above-mentioned targeted peptide compound for specifically degrading NDRG2 in preparing a medicine for alleviating cerebral ischemic damage.

[0014] Preferably, the drug is a drug that inhibits overexpression of NDRG2 after cerebral ischemia, thereby alleviating cerebral ischemic damage.

[0015] The present invention also discloses the use of the above-mentioned targeted peptide compound for specifically degrading NDRG2 in preparing a drug for alleviating inflammatory response.

[0016] Preferably, the drug is a drug that reduces the levels of IL-1β, TNF-α, and IL-6 The important role of the modified targeting peptide compound in this application is its pharmaceutical use for cerebral ischemic injury, while the targeting peptide disclosed in the prior art is aimed at the therapeutic effect of ischemic stroke. There are significant differences between cerebral ischemic injury and ischemic stroke in terms of concepts, clinical manifestations, pathogenesis and treatment methods. Specifically, it is reflected in: Cerebral ischemic injury: usually refers to brain tissue damage caused by insufficient blood supply to the brain. This injury can be acute or chronic, and its degree and scope depend on the duration, severity and brain region of the ischemia. Cerebral ischemic injury may be caused by a variety of reasons, such as atherosclerosis, thrombosis, vasospasm, etc. The clinical manifestations vary, depending on the location, degree and duration of ischemia. Mild cases may only show symptoms such as short-term dizziness, headache, blurred vision, etc., while severe cases may have serious neurological dysfunction such as hemiplegia, aphasia, coma, etc.

[0017] Ischemic stroke: It is a type of stroke, specifically referring to necrosis and softening caused by ischemia and hypoxia of brain tissue due to sudden blockage of cerebral blood vessels. Ischemic stroke is usually related to causes such as atherosclerotic stenosis and cardiogenic embolism. It is the main type of stroke, accounting for 70% to 80% of the total number of strokes. The main clinical manifestations are sudden symptoms of neurological deficits, such as hemiplegia, hemisensory disturbances, aphasia, ataxia, etc. These symptoms usually reach a peak in a short period of time and may persist or gradually worsen. The clinical manifestations of ischemic stroke are closely related to the location, range and severity of ischemia.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time a specific structure of a targeted peptide compound for specific degradation of NDRG2, the chemical formula of which is C 293 H 474 N 96 O 68 , with a molecular weight of 6429.62. This compound improves the domain of the targeting peptide, which includes the design and synthesis of three domains: ① using the known membrane-penetrating peptide TAT to synthesize the membrane-penetrating domain (CMPD) ② specifically binding to the NDRG2 domain (PBD) ③ tandem degradation domain (CTM / degron). Among them, the amino acid sequence of the functional region that binds to the NDRG2 protein is shown in SEQ.ID.NO.1, the amino acid sequence of the lysosome plus proteasome-induced degradation peptide is shown in SEQ.ID.NO.2, and the amino acid sequence of the membrane-penetrating peptide TAT is shown in SEQ.ID.NO.3. The main function of this targeting peptide compound is to reduce inflammatory response and oxidative stress damage, so it can be used in new drug uses for cerebral ischemic injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The structural formula of the targeted peptide compound for specifically degrading NDRG2 provided by the present invention; Figure 2 The HPLC spectrum of the targeted peptide compound for specific degradation of NDRG2 provided by the present invention; Figure 3A mass spectrum of the targeted peptide compound for specific degradation of NDRG2 provided by the invention; Figure 4 The targeting peptide with improved structural domain can effectively enhance the degradation of NDRG2; Figure 5 The results of the targeted peptide compound that specifically degrades NDRG2 reversibly degraded the expression of NDRG2 in mouse brain and primary astrocytes; A is a schematic diagram of the penetration of the blood-brain barrier by the NDRG2 targeted peptide compound; B is a Western blot showing the expression of NDRG2 protein in the mouse brain after treatment with the NDRG2 targeted peptide; C is the neurological deficit score assessed 3 days after treatment with the NDRG2 targeted peptide; D is a representative immunofluorescence staining showing the blood-brain barrier permeability of the NDRG2 targeted peptide in the brain; E is a Western blot showing the permeability of the NDRG2 targeted peptide in the brain. Blot shows the expression of NDRG2 protein in primary astrocytes after treatment with NDRG2 targeting peptide at different physiological concentrations; F is the CCK-8 experiment testing the cell viability of primary astrocytes after treatment with NDRG2 targeting peptide at different doses; G is the LDH release experiment testing the cytotoxicity of primary astrocytes after treatment with different doses of NDRG2 targeting peptide; H is immunofluorescence staining showing the co-localization of NDRG2 targeting peptide dye and GFAP in primary astrocytes; I is the expression of NDRG2 protein in primary astrocytes at different treatment times of NDRG2 targeting peptide; J is the expression of NDRG2 protein at different time points after treatment with NDRG2 targeting peptide for 6 hours.

[0020] Figure 6 The experimental results of the targeted peptide compound for specific degradation of NDRG2 inhibiting the overexpression of NDRG2 after cerebral ischemia and alleviating cerebral ischemic injury; A is Western blot showing the expression of NDRG2 in the ischemic penumbra after cerebral ischemic injury; B is Western blot showing the expression of NDRG2 in the ischemic penumbra after treatment with NDRG2 targeted peptide; C and D are the cerebral infarction volumes of each group after cerebral ischemic injury; E is the neurological function injury score of each group after cerebral ischemic injury; F and G are the pathological changes of each group after cerebral ischemic injury, Nissl staining shows the morphological changes of neurons, NeuN staining shows the survival of neurons, and TUNEL staining shows neuronal apoptosis.

[0021] Figure 7The experimental results of the targeted peptide compound for specific degradation of NDRG2 inhibiting the activation of type A1 astrocytes and promoting the activation of type A2 astrocytes; A is the expression of GFAP, S100A10 and C3d proteins shown by Western blot; B is the percentage of C3d-positive cells to GFAP-positive cells (type A1 astrocytes) in the ischemic penumbra; C is the percentage of S100A10-positive cells to GFAP-positive cells (type A2 astrocytes) in the ischemic penumbra.

[0022] Figure 8 The results of targeted peptide compounds that specifically degrade NDRG2 in alleviating oxidative stress and inflammatory response; wherein, A is Western blot showing the expression of p38-MAPK, C / EBP and SOCS1 in the ischemic penumbra after treatment with NDRG2 targeted peptide; B is Western blot showing the expression of NF-κB, p-Stat3 / Stat3 and BDNF in the ischemic penumbra after treatment with NDRG2 targeted peptide; C is Western blot showing the expression of Nrf2 and HO-1 in the ischemic penumbra after treatment with NDRG2 targeted peptide; D is the percentage of BDNF-positive cells in the ischemic penumbra after treatment with NDRG2 targeted peptide; E is the levels of IL-1β, IL-6 and TNF-α in the ischemic penumbra after treatment with NDRG2 targeted peptide; F is the level of MDA in the ischemic penumbra after treatment with NDRG2 targeted peptide; G is the level of SOD in the ischemic penumbra after treatment with NDRG2 targeted peptide. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings: The present invention discloses a targeting peptide for specific degradation of NDRG2. The targeting peptide for specific rapid and reversible degradation of NDRG2 is constructed by using a proteomics method, which includes the design and synthesis of three domains: ① using the known membrane penetrating peptide TAT to synthesize the transmembrane domain (CMPD) ② specific binding domain of NDRG2 (PBD) ③ tandem degradation domain (CTM / degron). Among them, the amino acid sequence of the functional region binding to the NDRG2 protein is shown in SEQ.ID.NO.1, the amino acid sequence of the lysosome plus proteasome induced degradation peptide is shown in SEQ.ID.NO.2, and the amino acid sequence of the transmembrane peptide TAT is shown in SEQ.ID.NO.3.

[0026] The targeting peptide of the present invention was commissioned to be synthesized by Hefei Guopi Biotechnology Co., Ltd., with a TAT amino acid sequence of YGR(Orn)(Orn)KRRQRRR, a specific binding domain of NDRG2: YPYYGKLLQPKYLQPLLAVQ, and a lysosome plus proteasome induced degradation peptide segment: KFERQKILDQRFFERRRG.

[0027] In particular, the Orn domain refers to a specific protein domain that is usually closely related to the function of arginine decarboxylase and is highly conserved during evolution. Orn stands for ornithine. Ornithine is a basic amino acid. Although it is not one of the 20 amino acids that make up proteins, it is present in some antimicrobial peptides such as tyrosinase and tyrosinase S. Ornithine is mainly involved in the urea cycle, plays an important role in the excretion of ammonia nitrogen in the body, and is commonly used in medicine. In this modified TAT sequence, the introduction of ornithine may be to increase certain properties of the membrane-penetrating peptide, such as enhancing its interaction with the cell membrane, improving its membrane-penetrating efficiency, or changing its metabolic pathway in the body. The basic properties of ornithine may help the membrane-penetrating peptide interact with the negative charge on the cell membrane, thereby promoting its penetration of the cell membrane. In addition, the introduction of ornithine may also affect the secondary structure and conformation of the membrane-penetrating peptide, which may further affect its biological activity and penetration ability. It is worth noting that the modification of the membrane-penetrating peptide needs to take into account many factors, including its stability, toxicity, specificity, and in vitro and in vivo behavior.

[0028] The structural formula of the targeted peptide compound for specific degradation of NDRG2 of the present invention is as follows Figure 1 Its liquid phase spectrum and mass spectrum are shown in Figure 2 and Figure 3 As shown. The compound of the present invention has restructured and innovated the existing targeting peptide structure. The domain of the old targeting peptide is: TAT: YGRKKRRQRRR; the domain of the new targeting peptide is: TAT: YGR (Orn) (Orn) KRRQRRR). The new structure has new structural characteristics and functional activity.

[0029] 1. Experimental methods to verify the neuroprotective effect of targeted peptide compounds that specifically degrade NDRG2 1.1 Experimental animals SPF grade C57BL / 6 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 First Medical Center of the General Hospital of the People's Liberation Army. The animal culture environment is SPF grade, 12-hour day / night rhythm regulation, free diet. The experimental process strictly complies with the Animal Experiment Guide of the First Medical Center of the General Hospital of the People's Liberation Army.

[0030] 1.2. Design and synthesis of targeting peptides The targeting peptide designed according to the following amino acid sequence was commissioned to Hefei Guopi Biotechnology Co., Ltd. for synthesis.

[0031] TAT:YGR(Orn)(Orn)KRRQRRR Specific binding to NDRG2 domain: YPYYGKLLQPKYLQPLLAVQ Lysosome plus proteasome induced degradation peptide: KFERQKILDQRFFERRRG 1.3 Culture of primary astrocytes Add 30 μg / ml L-polylysine to the cell culture bottle, wrap it with sterile tin foil, and place it in an incubator (37°C, 5% CO2) for overnight incubation (encapsulation). The next day, use a vacuum suction pump to remove the polylysine, and rinse it three times with high-temperature and high-pressure sterilized double-distilled water.

[0032] Place the newborn 1-3 days old C57BL / 6 mice in 75% alcohol for disinfection for 5-10 minutes, cut off the head of the mice with sterile ophthalmic scissors, and place them in pre-cooled D-Hanks solution. Under a microscope, carefully separate the scalp, meninges, and blood vessels with microtweezers. Remove the cortical brain tissue, place it in new D-Hanks solution, and cut it into pieces. Add 2 ml of 0.25% trypsin to the cut tissue and digest it in an incubator (37°C, 5% CO2) for 10 minutes. Add serum-containing culture medium to terminate trypsin digestion. Use a pipette to transfer the tissue fluid after the termination of digestion to a 15 ml centrifuge tube and centrifuge it at 1000 rpm for 10 minutes. Use a pipette to carefully aspirate the supernatant and add culture medium (containing 10% fetal bovine serum) to the centrifuge tube. Use a dropper to blow the cells thoroughly to obtain a single cell suspension. Add the single cell suspension to a cleaned sterile culture bottle and place it in an incubator at 37°C and 5% CO2 for culture. After 3 days, change half of the medium, and then change 1 / 3 of the medium every 3 days. After 7 days, observe the cells under the microscope and seal the mouth of the culture bottle with a sealing film. Shake it on a 37℃ shaker at a speed of 190 for 20 hours. After 20 hours, take out the culture bottle, remove the sealing film in a sterile environment, and wipe the bottle mouth with 75% alcohol for sterilization. Discard the supernatant in the culture bottle, add 3 ml of 0.25% trypsin to digest for 10 minutes, add serum-containing culture medium after digestion to terminate trypsin digestion, use a sterile pipette to suck out the cell homogenate and transfer it to a 15 ml centrifuge tube, and centrifuge it at 1000 rpm for 10 minutes. Discard the supernatant and add culture medium (containing 10% fetal bovine serum) to the centrifuge tube. Use a dropper to blow the cells fully to obtain a single cell suspension, which is the primary astrocyte. Then transfer the single cell suspension to a 6 cm culture dish for culture.

[0033] Astrocyte injury was induced by oxygen glucose deprivation (OGD), and cell death was determined by detecting lactate dehydrogenase (LDH) released into the culture medium.

[0034] 1.4 Construction of mouse focal cerebral ischemia model (MCAO) After inducing anesthesia in mice with 2.5% isoflurane, the anesthesia was maintained with 1.5% isoflurane, and the animals were able to breathe spontaneously during the operation. The mice were fixed and placed supine on a thermostatic plate (the temperature of the thermostatic plate was 37°C). The rectal temperature was monitored to maintain the body temperature at 37.0±0.5°C. The blood flow in the surface projection area of ​​the middle cerebral artery was monitored by laser Doppler blood flowmeter before, during and after the operation. After disinfecting the neck of the mouse with iodine, a small incision was made in the middle neck skin with a scalpel to fully expose and carefully separate the right common carotid artery, external carotid artery and internal carotid artery. A slipknot was tied at the proximal end of the common carotid artery with a No. 3 mousse thread to temporarily block the blood flow of the common carotid artery; the distal end of the right external carotid artery was ligated, and a small incision was made in the external carotid artery with a syringe. A 15 mm long thread plug (Shenzhen Ruiwode Company) was inserted along the external carotid artery. Use microtweezers to push the thread plug forward, advancing 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 thread plug has reached the starting point of the middle cerebral artery, and the insertion is stopped. According to the results of the cerebral blood flow detector, after the thread plug is blocked, the blood flow value of the middle cerebral artery is reduced to less than 30% of the level before the blockage, which is considered to be effective in modeling, otherwise it is excluded. The incision was sutured and the mice were placed in a constant temperature box to maintain their body temperature between 37.0±0.5℃. After blocking the blood flow for 1 hour, the mice were re-anesthetized with isoflurane, the incision was reopened, the thread plug was slowly pulled out, and the perfusion was restored. Within 10 minutes, the blood flow recovered to more than 70% of the basal value, which was considered a successful model, otherwise it was excluded. The skin was sutured, the mice were placed in an incubator, and they were waited for the mice to wake up naturally. The mice in the sham operation group only underwent surgical operations without thread plugs. NDRG2 targeting peptide (20 mg / kg) and the control group were given saline through the tail vein 60 minutes after surgery.

[0035] 1.5 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 and referred to the Garcia Test scoring method.

[0036] 1.6 Determination of cerebral infarction volume by TTC staining After the mouse was deeply anesthetized, the head of the mouse was quickly removed with scissors, the skull was quickly peeled off, the brain was taken out, and placed in a 4°C refrigerator to cool for 10 minutes. Then the brain was placed in the brain trough and the brain was evenly cut into 1 mm thick (coronal section) with a blade. The 1 mm thick brain slices were transferred to a preheated (37°C) 2% TTC solution. After 10 minutes, the TTC dye solution was recovered and the stained brain slices were fixed with 4% paraformaldehyde for 24 h. A digital camera was used to take pictures, and the brain slice images were imported into the computer, and the cerebral infarction volume was analyzed using Adobe Photoshop cs 3.0 software. In order to reduce the effect of edema of the ischemic side of the brain tissue on the infarction volume, this experiment used the percentage of infarction volume to express the degree of infarction: Infarct volume percentage = (contralateral cerebral hemisphere volume - infarct side normal brain tissue volume) / contralateral cerebral hemisphere volume * 100%.

[0037] 1.7 Western Blot and immunofluorescence were performed according to conventional methods.

[0038] 1.8 Statistical analysis SPSS19.0 statistical software was used to analyze the data in this study. Quantitative data such as percentage of infarct area and relative protein expression were expressed as mean ± standard deviation (mean ± SD). The Student's t test was used for comparison between the two groups of quantitative data; one-way ANOVA was used for comparison between multiple groups and between groups, and Dunnett's test or Bonferroni test was used according to experimental requirements. Neurobehavioral scores were expressed as medians. Nonparametric tests (Mann-Whitney U) were used for comparison between the two groups, and Kruskal-Wallis H test was used for comparison between multiple groups, and Bonferroni method was used for correction. The difference between the groups was considered statistically significant if the P value was less than 0.05.

[0039] 2. Experimental results 2.1 The targeting peptide with improved domain structure can effectively enhance the degradation of NDRG2 The improved targeting peptide of the present invention is optimized in design for its original defects, thereby significantly enhancing the degradation effect on NDRG2. NDRG2 targeting peptide 10 mg / kg was injected into the mouse tail vein once a day for 3 consecutive days, and the effect of the NDRG2 targeting peptide before improvement (specific degradation NDRG2 targeting peptide disclosed in Chinese patent publication number CN 112142856 A) and the improved NDRG2 targeting peptide of the present invention on NDRG2 expression was detected by Western Blot. The results are shown in Figure 4 , it can be found that the targeting peptides with improved domains can effectively enhance the degradation of NDRG2. This is because, by introducing more stable amino acid sequences or chemical modifications, the improved targeting peptides show stronger resistance to enzymatic hydrolysis, greatly improving their stability and bioavailability in the body. In addition, the improved targeting peptides have enhanced their specific binding ability with NDRG2 in terms of domain design, ensuring that they can more effectively recognize and bind to the NDRG2 protein, further promoting its degradation. Such optimization not only enhances the efficacy of the targeting peptide, but also reduces potential nonspecific effects, thereby reducing the risk of side effects.

[0040] 2.2 NDRG2-targeted peptide compounds reversibly degrade NDRG2 expression in mouse brain and primary astrocytes In order to explore the role of NDRG2 targeting peptide compounds in ischemic stroke, it is necessary to test their ability to pass the blood-brain barrier (BBB) ​​and their safety. The NDRG2 targeting peptide is composed of a penetrating peptide (TAT sequence), a functional domain sequence that specifically binds to the NDRG2 protein, and a proteasome that degrades the peptide. The latter is a targeting peptide that specifically degrades NDRG2 ( Figure 5 (A) NDRG2 targeting peptide 10 mg / kg was injected through the tail vein once a day for different durations (1, 2, 3 days) to explore the effective dose for downregulating NDRG2 expression. Figure 5 As shown in Figure B, we found that after 2 or 3 consecutive days of NDRG2 targeting peptide administration, NDRG2 expression in the brain decreased rapidly. Three days after treatment, mice showed no behavioral evidence of toxicity ( Figure 5 C). FITC-Tat-NDRG2 dye was injected into the tail vein once a day for 3 consecutive days, and the ability of NDRG2 targeting peptide to cross the BBB in the brain was verified by IF staining ( Figure 5 (middle D).

[0041] In the in vitro experiment, primary astrocytes were exposed to different physiological concentrations (1.0, 2.5, 5.0 μM) of NDRG2 targeting peptide for 6 h. Compared with the Con group, the NDRG2 expression in these three groups was significantly decreased ( Figure 5 In addition, we used CCK-8 method to detect the effect of NDRG2 targeting peptide on cell viability; LDH release method was used to detect the effect of NDRG2 targeting peptide on cytotoxicity. The cell viability of astrocytes did not decrease after treatment ( Figure 5 There was no significant difference in cytotoxicity between the Con group and the group treated with 2.5 μM NDRG2 targeting peptide for 6 h, but the cytotoxicity of the group treated with 5.0 μM NDRG2 targeting peptide increased ( Figure 5 Then, astrocytes were exposed to 2.5 μM FITC-NDRG2 targeting peptide dye for 6 h, and IF staining showed that the NDRG2 targeting peptide dye co-localized with GFAP, indicating that the NDRG2 targeting peptide compound synthesized by the present invention can penetrate the cell membrane of astrocytes and enter the cytoplasm ( Figure 5 H). Figure 5As shown in Figure I, primary astrocytes were exposed to 2.5 μM NDRG2 targeting peptide at different time points (3, 6, and 12 h), and the NDRG2 expression in these three groups was significantly reduced compared with the Con group. Therefore, we selected 2.5 μM NDRG2 targeting peptide for 6 hours as the effective dose and duration for in vitro experiments. Subsequently, we detected the NDRG2 expression of astrocytes at different time points (0, 8, 16, 24, and 48 h) after exposure to 2.5 μM NDRG2 targeting peptide for 6 h. Figure 5 J), we found that NDRG2 expression decreased after astrocytes were exposed to 2.5 μM NDRG2 targeting peptide for 6 h, while NDRG2 expression was restored within 8, 16, 24, and 48 h after astrocytes were treated with NDRG2 targeting peptide for 6 h.

[0042] 2.2 NDRG2-targeted peptide compounds inhibit NDRG2 overexpression after cerebral ischemia and reduce cerebral ischemic damage Next, the present invention also detected the expression of NDRG2 in MCAO-R injured mice. Western blot analysis showed that compared with the corresponding areas of Con group mice, the expression of NDRG2 in the ischemic penumbra increased in a time-dependent manner at different reperfusion durations (1, 2, and 3 days) after MCAO 1 h. Figure 6 Then, immediately after reperfusion after stroke, we injected NDRG2-targeting peptide compounds into the tail vein to regulate the expression of NDRG2, and we found that 3 days after reperfusion, the expression of NDRG2 in the ischemic penumbra was significantly increased ( Figure 6 (B), while the NDRG2 targeting peptide compound reduced the expression of NDRG2 compared with the MCAO group.

[0043] The present invention also uses TTC staining and neurological function scoring to evaluate the brain damage of the three groups of mice. Figure 6 In CD, after cerebral ischemic injury, the NDRG2-targeted peptide compound significantly reduced the infarct volume compared with the MCAO group. In addition, the neurological function score in the MCAO group was significantly reduced compared with the Con group, indicating a worse neurological condition. Interestingly, the neurological function score of mice in the NDRG2-targeted peptide compound group was significantly increased compared with the MCAO group ( Figure 6 Middle E).

[0044] Nissl staining, NeuN staining, and TUNEL staining were used to evaluate neuronal damage and survival in the ischemic penumbra after MCAO-R injury. Figure 6F and G). Nissl-positive cells (Nissl+) represent intact neurons with cell bodies, while damaged neurons have atrophy of cell bodies, atrophy and pyknosis of nuclei. Compared with the MCAO group, the proportion of Nissl-positive cells in the NDRG2 targeted peptide group increased significantly. NeuN-positive (NeuN+) cells represent neuronal survival. Compared with the MCAO group, the NDRG2 targeted peptide group significantly increased the proportion of NeuN+ cells. TUNEL-positive cells (TUNEL+) represent apoptotic neurons. Compared with the MCAO group, the NDRG2 targeted peptide group significantly reduced the proportion of TUNEL+ cells.

[0045] 2.3 Specific degradation of NDRG2-targeted peptide compounds inhibits the activation of type A1 astrocytes and promotes the activation of type A2 astrocytes Next, the present invention also studies the effect of NDRG2 targeting peptide compounds on the activation of A1 / A2 astrocytes. Figure 7 In (a) and (b) of Figure A, western blot analysis showed that the GFAP expression in the MCAO group was significantly higher than that in the Con group, while the NDRG2-targeted peptide compound group significantly reduced the GFAP expression compared with the MCAO group. Compared with the Con group, the protein level of S100A10, a marker of A2-type astrocytes, in the MCAO group was significantly reduced, while the NDRG2-targeted peptide compound was able to reverse the reduction in S100A10 levels. Compared with the Con group, the protein level of C3d, a marker of A1-type astrocytes, was significantly increased in the MCAO group, and this effect was reversed by the NDRG2-targeted peptide compound.

[0046] Subsequently, the present invention evaluated the effect of NDRG2 targeting peptide compound treatment on the number of C3d / GFAP positive cells and S100A10 / GFAP positive cells around the infarct by immunofluorescence staining. Figure 7 As shown in Figures B and C, compared with the Con group, the C3d / GFAP positive cells in the MCAO group increased significantly, and the S100A10 / GFAP positive cells decreased significantly. Compared with the MCAO group, the NDRG2 targeted peptide compound group significantly reduced the number of C3d / GFAP positive cells and increased the number of S100A10 / GFAP positive cells. This indicates that the specific degradation of NDRG2 targeted peptide compounds inhibits the activation of A1 type astrocytes and promotes the activation of A2 type astrocytes.

[0047] 2.3 Specific degradation of NDRG2-targeted peptide compounds to reduce oxidative stress and inflammatory response To investigate the regulatory effect of NDRG2 targeting peptide on the upstream signaling pathways of A1 / A2 astrocyte activation, we used Western blot to analyze the protein expressions of p38-MAPK, C / EBP, and SOCS1 in the ischemic penumbra ( Figure 8 The results showed that compared with the control group, the levels of these three proteins were significantly increased in the MCAO model mice, while the levels of these proteins were significantly reduced in the NDRG2 targeted peptide compound treatment group. In addition, NF-κB and p-Stat3 are key signals for the activation of A1 and A2 astrocytes. NF-κB was significantly increased and p-Stat3 / Stat3 was significantly decreased in the MCAO group, while NF-κB decreased and p-Stat3 / Stat3 increased after treatment with NDRG2 targeted peptide compounds ( Figure 8 Middle B). BDNF is secreted in A2 astrocytes. The BDNF level in the MCAO group was significantly decreased, while the BDNF level in the NDRG2 targeting peptide group was significantly increased, and the number of BDNF / GFAP positive cells also increased ( Figure 8 Nrf2 / HO-1 signaling pathway is important in anti-oxidative stress. Nrf2 and HO-1 levels were significantly decreased in the MCAO group, while they were significantly increased after NDRG2 targeting peptide treatment ( Figure 8 Middle C).

[0048] Next, the present invention determined the effects of NDRG2-targeted peptide compounds on inflammation and oxidative stress after cerebral ischemia. The expression of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in the ischemic penumbra was measured 3 days after reperfusion. Figure 8 In Figure E, the IL-1β level in the control group was 20.9 pg / ml ± 6.6 pg / ml, while the IL-1β level in the MCAO group was 27.9 pg / ml ± 2.8 pg / ml, but NDRG2 targeted peptide treatment significantly reduced the IL-1β level to 13.4 pg / ml ± 1.6 pg / ml. Similarly, the IL-6 level in the control group was 93.8 pg / ml ± 7.4 pg / ml, and the IL-6 level in the ischemic penumbra of the MCAO group after cerebral ischemia increased to 760.3 pg / ml ± 44.1 pg / ml, but NDRG2 targeted peptide compound treatment reversed the IL-6 level to 419.6 pg / ml ± 18.0 pg / ml. As expected, the TNF-α level in the control group was 19.0 pg / ml ± 0.4 pg / ml, while the TNF-α level in the MCAO group increased significantly to 32.1 pg / ml ± 1.0 pg / ml, and the NDRG2-targeted peptide compound treatment reduced the TNF-α level to 23.7 pg / ml ± 3.4 pg / ml.

[0049] In addition, we measured the levels of MDA (a byproduct of lipid peroxidation) and the activity of SOD (a major antioxidant enzyme). Figure 8In Figure F, the MDA level in the control group was 2.7 ± 0.1 nmol / ml, and that in the MCAO group was 2.8 ± 0.2 nmol / ml, while the MDA level in the NDRG2 targeting peptide compound group was significantly reduced to 1.7 ± 0.3 nmol / ml. Figure 8 Middle G shows the SOD levels of the three groups, which were 2.4 ± 0.4 ng / ml in the control group, 1.2 ± 0.1 ng / ml in the MCAO group, and the SOD level in the NDRG2 targeting peptide compound group increased significantly to 1.6 ± 0.1 ng / ml.

[0050] Based on the above results, the present invention confirms for the first time that the specific degradation NDRG2 targeted peptide compound provided by the present invention can significantly reduce oxidative stress and inflammatory response, inhibit the activation of type A1 astrocytes, promote the activation of type A2 astrocytes, reduce astrocyte oxygen-glucose deprivation damage, and improve the prognosis of cerebral ischemia-reperfusion injury by taking NDRG2 as the intervention target.

[0051] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A targeted peptide compound for specific degradation of NDRG2, characterized in that: The chemical formula of the targeting peptide compound is C 293 H 474 N 96 O 68 , molecular weight is 6429.62, and the structural formula is as follows: 。 2. The targeting peptide compound for specific degradation of NDRG2 according to claim 1, characterized in that: The targeting peptide compound comprises a domain that can specifically target the NDRG2 protein, and the transmembrane peptide in the targeting peptide is a transmembrane domain transformed from TAT, YGR-Orn-Orn-KRRQRRR, and the amino acid sequence of the transmembrane peptide is shown in SEQ.ID.NO.

1.

3. The targeting peptide compound for specific degradation of NDRG2 according to claim 2, characterized in that: The targeting peptide compound is composed of a membrane-penetrating peptide, a domain that can specifically target the NDRG2 protein, and a lysosome plus proteasome-induced degradation peptide segment; The amino acid sequence of the domain that can specifically target the NDRG2 protein is shown in SEQ.ID.NO.2; the amino acid sequence of the lysosome plus proteasome induced degradation peptide segment is shown in SEQ.ID.NO.

3.

4. Use of the targeted peptide compound for specifically degrading NDRG2 according to any one of claims 1 to 3 in the preparation of a drug for alleviating oxidative stress damage.

5. The use according to claim 4, characterized in that The drug is a drug that uses NDRG2 as an intervention target to significantly reduce oxygen-glucose deprivation damage in astrocytes.

6. The use according to claim 5, characterized in that The drug can inhibit the activation of type A1 astrocytes and promote the activation of type A2 astrocytes.

7. Use of the targeted peptide compound for specifically degrading NDRG2 according to any one of claims 1 to 3 in the preparation of a drug for alleviating cerebral ischemic injury.

8. The use according to claim 7, characterized in that The drug is a drug that inhibits the overexpression of NDRG2 after cerebral ischemia and thus reduces cerebral ischemic damage.

9. Use of the targeted peptide compound for specifically degrading NDRG2 according to any one of claims 1 to 3 in the preparation of a drug for alleviating inflammatory response.

10. The use according to claim 9, characterized in that The drug is a drug for reducing the levels of IL-1β, TNF-α, and IL-6.

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