A polypeptide and use thereof in the preparation of a medicament for ischemic stroke

Abstract

The application relates to the field of biological medicine, and particularly discloses a polypeptide and application thereof in preparation of a medicine for ischemic cerebral stroke. The amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1. By establishing a mouse tMCAO / R model, it is confirmed that the polypeptide can significantly improve nerve function defect, reduce cerebral infarction volume, reduce expression of brain tissue inflammatory factors and whole-body oxidative stress level. In an in-vitro experiment, the polypeptide not only has a protective effect on injury of mouse hippocampal neuron HT22 cells and human brain microvascular endothelial cells hCMEC / D3 induced by sugar-oxygen deprivation, but also can significantly resist PC12 cell injury induced by CORT and MPP + , effectively reduce intracellular active oxygen accumulation caused by CORT. The polypeptide has the potential to play a stroke treatment role through multiple mechanisms such as nerve protection, blood vessel protection and antioxidation, and provides a solid experimental basis for development of the polypeptide into a novel multi-mechanism treatment medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide and its application in the preparation of drugs for ischemic stroke. Background Technology

[0002] Ischemic stroke (IS) is an acute cerebrovascular disease caused by a sudden blockage of blood vessels in the brain, leading to interruption of blood flow to local brain tissue and subsequent neuronal damage and neurological deficits. It is characterized by high incidence, high disability rate, high recurrence rate, and high mortality rate, and is one of the leading causes of death and long-term disability among adults worldwide. With the increasing aging population and the rising prevalence of underlying diseases such as hypertension, diabetes, and atherosclerosis, the burden of ischemic stroke continues to increase, becoming a major public health problem.

[0003] The pathological mechanisms of cerebral ischemia-reperfusion injury are extremely complex, involving a cascade of reactions and interactions among multiple biological processes. During the ischemic phase, hypoxia leads to mitochondrial energy metabolism disorders, causing cellular ATP depletion and ion imbalance. Simultaneously, damaged cells release damage-related molecules, activating the nuclear factor κB signaling pathway via Toll-like receptor 4, initiating an inflammatory response. Upon reperfusion, the restoration of blood oxygenation is accompanied by a surge in reactive oxygen species (ROS), further exacerbating mitochondrial dysfunction and activating matrix metalloproteinases, leading to increased blood-brain barrier permeability. Crucially, these pathological processes mutually reinforce each other: mitochondrial damage promotes ROS generation, ROS activate inflammasome-mediated pyroptosis, and lipid peroxidation can induce neuronal death via ferroptosis. This multi-target pathological network makes it difficult for single intervention strategies to achieve ideal neuroprotective effects.

[0004] In recent years, peptide drugs have shown unique application prospects in the treatment of ischemic stroke due to their high selectivity, low immunogenicity, and good biocompatibility. However, most of the peptide neuroprotective agents reported so far still have significant shortcomings. They mostly act on single targets in the pathological network of ischemia-reperfusion injury, and cannot comprehensively address the multi-factor, multi-stage cascade characteristics of this injury, resulting in limited clinical application efficacy and failing to meet the actual needs of clinical treatment.

[0005] Therefore, there is an urgent need to develop a polypeptide drug that can intervene in the pathological process of cerebral ischemia-reperfusion injury at multiple targets and stages, and has both high efficacy and high safety, so as to provide better treatment options for patients with ischemic stroke. Summary of the Invention

[0006] To address the aforementioned deficiencies in existing technologies, this invention provides a polypeptide (sequence ESVPEFPLSPPK) and its applications. This polypeptide not only exhibits significant neuroprotective effects in classic ischemic stroke models but also demonstrates broad-spectrum cytoprotective activity in various neurological injury-related models, showing outstanding drug development potential and broad application prospects.

[0007] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: In a first aspect, the present invention provides a polypeptide (denoted as ES-PK) with the amino acid sequence: ESVEFPLSPPK (SEQ ID NO. 1).

[0008] This invention, through the establishment of multiple in vivo and in vitro models of nerve injury, has for the first time demonstrated that this polypeptide exhibits excellent protective effects in the prevention and / or treatment of nerve injury-related diseases, especially in the intervention of ischemic stroke. Moreover, its mechanism of action covers multiple pathways, including anti-inflammatory, antioxidant, and direct neurovascular protection, and has significant clinical application value.

[0009] It should be noted that the polypeptide ES-PK described in this invention is prepared by chemical synthesis using conventional solid-phase synthesis techniques (such as Fmoc solid-phase synthesis). In one specific embodiment of this invention, the polypeptide ES-PK was chemically synthesized by Nanjing Genscript Biotech Co., Ltd. The synthesized polypeptide was identified as having sufficient purity and correct sequence, meeting the requirements for subsequent experiments and applications.

[0010] Secondly, as a specific embodiment of the present invention, the nerve injury-related disease is ischemic cerebrovascular disease.

[0011] Furthermore, the ischemic cerebrovascular disease is ischemic stroke; in a more specific embodiment, the ischemic stroke is induced by transient middle cerebral artery occlusion or reperfusion injury.

[0012] In in vivo pharmacodynamic experiments, this invention utilized a mouse transient middle cerebral artery occlusion / reperfusion (tMCAO / R) model highly correlated with clinicopathology to demonstrate that the peptide ES-PK significantly improved neurological deficit scores and enhanced motor coordination in the model mice, indicating its positive promoting effect on neurological function recovery after cerebral ischemia-reperfusion injury. Simultaneously, TTC staining revealed that the peptide significantly reduced cerebral infarction volume, confirming its substantial protective effect on the ischemic core area and penumbra. Furthermore, this invention also examined the expression levels of inflammatory factors (such as TNF-α, IL-1β, and IL-6) in brain tissue and systemic oxidative stress indicators. The results showed that the peptide effectively reduced the excessive release of inflammatory factors and inhibited oxidative stress levels, comprehensively demonstrating its clear neuroprotective effect at the whole animal level.

[0013] At the in vitro cellular level, by constructing various injury models closely related to the pathological processes of ischemic stroke, the ES-PK peptide was systematically validated to possess broad and multi-mechanistic cellular protective activities, as detailed below: 1. Targeting ischemic-hypoxic core injury: This peptide significantly improved the survival rate of mouse hippocampal HT22 neurons treated with 10 hours of oxygen-glucose deprivation (OGD) and human brain microvascular endothelial cells hCMEC / D3 treated with 6 hours of OGD. This result directly confirms its dual potential for direct neuroprotection and vascular endothelial protection, which helps to simultaneously protect the integrity of neural units and the blood-brain barrier.

[0014] 2. Targeting neurotoxic stress injury: This peptide is effective against 4 mM MPP. + Induced PC12 cell damage has a significant protective effect and can improve cell viability, suggesting that it may exert its effects by antagonizing pathways such as mitochondrial dysfunction or oxidative stress, thus expanding its application prospects in combating neurotoxic damage.

[0015] 3. Targeting hormone stress damage: Through reactive oxygen species (ROS) staining combined with live cell imaging and absolute quantitative analysis using an enzyme-linked immunosorbent assay (ELISA), it was directly confirmed that it can significantly reduce the excessive accumulation of ROS in PC12 neurons, clearly revealing the key molecular mechanism by which this peptide alleviates oxidative stress.

[0016] In summary, the ES-PK peptide provided by this invention, validated through multiple in vitro and in vivo models, not only demonstrated definite neuroprotective, anti-inflammatory, and antioxidant effects in animal models simulating clinical ischemic stroke, but also confirmed its broad application potential in various cell models covering neurons, endothelial cells, and different injury mechanisms. These findings indicate that this peptide can exert neuroprotective effects through multiple targets and pathways, and holds promise for development into an innovative drug for treating ischemic cerebrovascular diseases and a wider range of neurological injury-related diseases.

[0017] Thirdly, the present invention provides a polypeptide derivative, which is a fusion polypeptide obtained by attaching a protein purification tag or detection tag that retains polypeptide activity to the amino or carboxyl terminus of the polypeptide ES-PK. Any such fusion polypeptide capable of retaining the core therapeutic activity of the polypeptide ES-PK should be included within the scope of equivalent embodiments of the present invention.

[0018] Specifically, the protein purification tag or detection tag can be selected from the His tag or the FLAG tag.

[0019] On the other hand, this invention also includes active derivatives obtained by subjecting the amino acid sequence shown in SEQ ID NO: 1 to one or more conventional chemical modifications. These conventional chemical modifications include, but are not limited to, amination, amidation, acetylation, phosphorylation, glycosylation, or biotinylation. Such chemical modifications generally do not alter the biological function of the peptide ES-PK; rather, they may improve its in vitro stability, in vivo pharmacokinetic properties, or reduce its potential immunogenicity. All of the above derivatives, as long as they retain neuroprotective activity substantially consistent with that of the peptide ES-PK shown in SEQ ID NO: 1, fall within the scope of protection of this invention.

[0020] Fifthly, the present invention provides the use of the above-mentioned polypeptide derivatives in the preparation of medicaments for the prevention and / or treatment of neurological injury-related diseases.

[0021] In one specific embodiment of the present invention, the nerve injury-related disease is ischemic cerebrovascular disease.

[0022] Furthermore, the ischemic cerebrovascular disease is ischemic stroke.

[0023] In a sixth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective dose of the aforementioned polypeptide ES-PK or a polypeptide derivative thereof, and a pharmaceutically acceptable carrier.

[0024] The carriers include, but are not limited to: diluents, disintegrants, binders, lubricants, flow aids, flavoring agents, colorants, preservatives, excipients, surfactants, and other functional excipients required for the preparation of specific dosage forms.

[0025] Furthermore, the dosage form of the pharmaceutical composition may be selected from injection, lyophilized powder for injection or nasal spray.

[0026] The pharmaceutical composition provided by the present invention can be prepared into various dosage forms, such as injections, lyophilized powder injections, or nasal sprays, etc., and can be prepared according to conventional dosage form preparation methods in the art. The present invention does not impose any special limitations on this.

[0027] The pharmaceutical composition of the polypeptide ES-PK or its derivatives described in this invention can be administered to subjects via suitable routes of administration known in the art, including but not limited to oral, intravenous, intramuscular, subcutaneous, and mucosal administration.

[0028] The pharmaceutical composition effectively protects nerve cells, cerebral vascular endothelial cells, and neuroendocrine cells against cell damage induced by various damaging factors. These damaging factors include, but are not limited to: ischemic hypoxia injury (e.g., glucose-oxygen deprivation), oxidative stress injury (e.g., reactive oxygen species accumulation), inflammatory damage, and neurotoxins (such as 1-methyl-4-phenylpyridinium ion, MPP). +Damage induced by stress hormones (such as corticosterone, CORT).

[0029] In summary, this invention provides a polypeptide ES-PK with the amino acid sequence shown in SEQ ID NO: 1 and its active derivatives. Experimental studies have confirmed that this polypeptide exhibits significant protective efficacy in various in vitro and in vivo neurological injury models: in a constructed mouse transient middle cerebral artery occlusion / reperfusion (tMCAO / R) model, this polypeptide can effectively improve neurological deficit symptoms, reduce cerebral infarction volume, and significantly downregulate the expression levels of inflammatory factors in brain tissue and systemic oxidative stress response; in in vitro cell experiments, this polypeptide not only has a significant protective effect against glucose-oxygen deprivation (OGD)-induced damage to mouse hippocampal neurons HT22 cells and human brain microvascular endothelial cells hCMEC / D3, but also effectively resists corticosterone (CORT) and 1-methyl-4-phenylpyridinium ion (MPP). + The invention can reduce PC12 cell damage induced by CORT and significantly decrease intracellular reactive oxygen species (ROS) accumulation. These experimental results demonstrate that the polypeptide ES-PK of this invention exerts a neuroprotective effect through multiple mechanisms, including anti-inflammatory, antioxidant, and direct neurovascular protection, exhibiting broad-spectrum neuroprotective activity. Furthermore, this invention also covers active derivatives of the polypeptide, applications of the polypeptide derivatives (fifth aspect), and pharmaceutically acceptable pharmaceutical compositions thereof (sixth aspect), providing a complete and feasible technical implementation scheme for the application of this polypeptide in the preparation of drugs for the prevention or treatment of neurological injury-related diseases (especially ischemic stroke). Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 Example 2 of this invention describes how the peptide ES-PK improved the body weight, cerebral infarction area, and behavior of mice with ischemic stroke in the tMCAO / R model, where (A) changes in body weight; (B) cerebral infarction area; and (C) neurological deficit behavioral scores. Figure 2 Example 2 of this invention uses peptide ES-PK to reduce the levels of brain inflammatory factors and oxidative stress in rats with ischemic stroke under tMCAO / R modeling, wherein (A) brain IL-1β level; (B) brain IL-6 level; (C) brain TNF-α level; (D) brain iNOS level; (E) serum MDA level; Figure 3The protective effects of the polypeptide ES-PK in Example 3 of this invention in various cell injury models closely related to the pathological process of ischemic stroke are as follows: (A) Effect on cell survival rate of mouse hippocampal neuron HT22 cells damaged by 10h glucose-oxygen deprivation (OGD); (B) Effect on cell survival rate of human brain microvascular endothelial cells hCMEC / D3 cells damaged by 6h glucose-oxygen deprivation (OGD). Figure 4 The protective effects of peptide ES-PK in Example 4 of this invention in various cell injury models closely related to the pathological process of ischemic stroke include: (A) its effect on cell survival in PC12 cell injury induced by 250 μM corticosterone (CORT); and (B) its effect on the cell survival of 4 mM 1-methyl-4-phenylpyridinium ion (MPP). + The effect of induced neurotoxicity on cell survival in PC12 cells; Figure 5 The regulatory effect of peptide ES-PK on ROS in damaged neuronal cells in Example 5 of this invention: (A) Representative results of live-cell imaging of intracellular reactive oxygen species (ROS) levels after PC12 cell injury induced by 250 μM CORT; (B) Absolute quantitative analysis results of intracellular reactive oxygen species (ROS) levels after PC12 cell injury induced by 250 μM CORT using an enzyme-linked immunosorbent assay (ELISA) reader. Figure 6 To illustrate the effects of the polypeptide EP-DL in Comparative Example 1 of this invention on the body weight, behavior, infarct area, serum MDA level, brain inflammatory factor level, and HT22 cells and hCEMEC / D3 cells under protective OGD / R conditions in mice with ischemic stroke, the following were observed: (A) Changes in body weight; (B) Neurological deficit behavioral score; (C) Infarct area; (D) Serum MDA level; (E) Brain TNF-α level; (F) Brain IL-6 level; (G) Brain IL-1β level; (H) Effect on cell survival of mouse hippocampal HT22 cells damaged by 10 h of glucose-oxygen deprivation (OGD); (I) Effect on cell survival of human brain microvascular endothelial hCMEC / D3 cells damaged by 6 h of glucose-oxygen deprivation (OGD). Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] To better illustrate the present invention, further examples are provided below.

[0034] Example 1 This invention provides a polypeptide ES-PK, whose amino acid sequence is: ESVPEFPLSPPK (SEQ ID NO.1).

[0035] The methods for obtaining and preparing the above-mentioned polypeptide ES-PK are as follows: 1. Sources of sequence construction The amino acid sequence of the polypeptide ES-PK (SEQ ID NO: 1) was identified from the target protein sample brain protein hydrolysate I (Hebei Zhitong Biopharmaceutical Co., Ltd.) using high-resolution mass spectrometry.

[0036] Mass spectrometry analysis was performed on a Thermo Scientific instrument equipped with a nanoliter spray ionization source. TM OrbitrapFusion TM Lumos TM Tribrid TM The analysis was performed on a mass spectrometer. The raw mass spectrometry data were analyzed using PEAKS® Studio bioinformatics software for database searching and de novo sequencing. Key parameters were set as follows: precursor ion mass tolerance ±10 ppm, fragment ion mass tolerance ±0.02 Da. Through the above analysis, a polypeptide sequence with potential neuroprotective activity was finally identified from brain protein hydrolysate I with a confidence level higher than 95%. This sequence was named ES-PK, and its amino acid sequence corresponds to SEQ ID NO: 1, ESVPEFFPLSPPK (three-letter code: Glu-Ser-Val-Pro-Glu-Phe-Pro-Leu-Ser-Pro-Pro-Lys).

[0037] 2. Chemical Synthesis and Sample Acquisition The polypeptide ES-PK described in this invention is chemically prepared using the standard Fmoc solid-phase polypeptide synthesis method. The L-type amino acid raw materials (E, S, V, P, F, L, K) and other synthetic reagents (such as resins, condensing agents, deprotecting agents, etc.) used in the synthesis are all commercially available analytical grade or pharmaceutical grade (≥99%), and can be purchased from conventional reagent suppliers such as Sinopharm Chemical Reagent Co., Ltd. and Shanghai Aladdin Biochemical Technology Co., Ltd.

[0038] To obtain high-purity samples that meet pharmaceutical research standards, the polypeptide ES-PK used in this invention was custom-produced by Nanjing Genscript Biotech Co., Ltd., a professional polypeptide synthesis company, based on the aforementioned Fmoc solid-phase synthesis principle. The production process complies with relevant GMP regulations. The synthesized crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and the final product was identified using liquid chromatography-mass spectrometry (LC-MS). The results showed that the measured molecular weight was consistent with the theoretical value; analytical RP-HPLC analysis confirmed a chemical purity of ≥98%.

[0039] Using the above method, the present invention successfully obtained sufficient quantity and high purity of polypeptide ES-PK, which can be used for subsequent pharmacodynamic experimental studies.

[0040] Example 2 Therapeutic effect of peptide ES-PK on tMCAO / R-induced ischemic stroke in mice Step 1: Experimental Animals, Grouping, and Dosing Regimen Male C57BL / 6J mice aged 6-8 weeks and weighing 20-25g (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., Animal License No.: SYXK(Su)2021-0011) were selected and acclimatized for one week in a standard SPF environment (temperature 22-25℃, humidity 50%-60%, 12h light / 12h dark cycle, free access to food and water). They were then randomly divided into the following 3 groups using a random number table: i) Sham group: 6 mice, only the neck vessels were separated, no suture embolism was inserted, and no cerebral ischemia-reperfusion treatment was performed. After the mice were housed separately, 0.2 mL of physiological saline was injected intraperitoneally for the first time 1 day before modeling. On the day of modeling and after modeling, intraperitoneal injection was continued once a day until the end of the experiment (72 h after modeling).

[0041] ii) Model group (tMCAO / R same as I / R): 12 mice were used to establish a tMCAO / R (middle cerebral artery occlusion / reperfusion) model (abbreviated as I / R). The drug administration regimen was the same as the sham-operated group, that is, after the mice were housed separately, 0.2 mL of physiological saline was injected intraperitoneally for the first time one day before modeling; intraperitoneal injection was continued once a day on the day of modeling and after modeling until the end of the experiment (72 hours after modeling).

[0042] iii) Peptide treatment group (I / R+ES-PK): 12 mice were used to establish the tMCAO / R model. The peptide ES-PK was prepared fresh and used immediately, with a stock solution concentration of 10 mg / mL (solvent was physiological saline). Before use, it was diluted with physiological saline to 1 mg / mL. After the mice were housed separately, the first administration was started one day before the model was established. The above-mentioned diluted peptide ES-PK solution was injected intraperitoneally daily (the dosage was 10 mg / kg, and the volume of administration was calculated based on the weight of the mouse to ensure accurate dosage). On the day of model establishment and after model establishment, the administration was continued once a day until the end of the experiment (72 hours after model establishment).

[0043] Explanation of the number of experimental animals: During the construction of the tMCAO / R model, some mice died postoperatively due to factors such as surgical trauma, cerebral ischemia-reperfusion stress, and postoperative infection. After screening, the number of valid animals included in the final data statistical analysis was: sham surgery group n=6, model group n=10, and peptide treatment group n=5. All statistical analyses were completed based on valid animal data, and the feeding and handling of experimental animals complied with the requirements of the "Guidelines for Ethical Review of Laboratory Animals" (GB / T 35892-2018).

[0044] Step 2: Establish a mouse model of ischemic stroke induced by middle cerebral artery occlusion / reperfusion (tMCAO / R). A mouse tMCAO / R model was established using the suture occlusion method, with ischemia time strictly controlled to 1 hour. After reperfusion, the mice were fed for 72 hours (3 days). The specific procedures are as follows: i) Anesthesia and Fixation: Mice were placed in an anesthesia induction box and anesthesia was induced using a gas mixture containing 3%–4% isoflurane (oxygen flow rate: 0.8–1.0 L / min). After the mice lost their righting reflex (a marker of adequate anesthesia depth), they were moved to the operating table, and anesthesia was maintained by continuously administering a gas mixture containing 1.5%–2.0% isoflurane through a nasal cone. The mice were fixed in a supine position on the operating table, and their core body temperature was monitored and maintained at 37.0 ± 0.5 °C in real time using a rectal temperature probe combined with a feedback heating pad (body temperature stability is a key prerequisite for the successful construction of the tMCAO / R model, and can avoid the impact of body temperature fluctuations on experimental results).

[0045] ii) Neck incision and blood vessel separation: After preparing the anterior neck area, disinfect the area three times with 75% medical alcohol (with a 30-second interval between each disinfection). Make a longitudinal incision in the midline of the neck (approximately 1.5-2.0 cm in length). Under a surgical microscope (10-20x magnification), bluntly dissect the subcutaneous connective tissue, salivary glands, and sternocleidomastoid muscle of the neck to fully expose the left common carotid artery, external carotid artery, and internal carotid artery. The dissection process should be gentle to avoid damaging the blood vessel walls and surrounding nerves and to prevent bleeding from affecting the stability of the model.

[0046] iii) Vascular pretreatment: Carefully dissect the external carotid artery with micro-forceps to avoid traction on the blood vessel and causing vasospasm, and permanently ligate its distal end with 5-0 medical silk suture; temporarily clamp the proximal end of the common carotid artery with a miniature arterial clamp to block blood flow, providing conditions for subsequent suture insertion and avoiding blood flow interference.

[0047] iv) Insertion of the suture and ischemia: Approximately 2 mm proximal to the ligation point of the external carotid artery, make a small 45° oblique incision using microvascular scissors (the incision size should be just large enough to allow for smooth insertion of the suture without significant bleeding). Gently insert a nylon suture (0.22 mm in diameter, approximately 18-20 mm in length, with a silicone coating thickness of 0.02-0.03 mm) with its tip coated with silicone into the internal carotid artery through this incision. Slowly advance the suture along the internal carotid artery into the cranial cavity to a depth of approximately 9-11 mm (adjust slightly according to the mouse's weight: 9-10 mm for mice weighing 20-22 g, and 10-11 mm for mice weighing 23-25 ​​g), until slight resistance is felt, indicating that the tip of the suture has reached and blocked the origin of the middle cerebral artery. Gently ligate the stump of the external carotid artery with another 5-0 medical suture to fix the suture and prevent displacement. Immediately begin recording the ischemia time, which should be strictly controlled to 1 hour (with an error not exceeding ±5 minutes).

[0048] v) Reperfusion: After 1 hour of ischemia, gently clamp the suture thrombus at the stump of the external carotid artery with micro-forceps, and slowly pull out the nylon suture thrombus into the stump of the external carotid artery (the pulling speed should be controlled at 1~2 mm / s to avoid excessive traction and damage to the internal carotid artery wall). Visually observe the recovery of blood flow in the internal carotid artery (criteria for judging the recovery of blood flow: the vessel is full and continuous blood flow pulsation is visible). After confirming the recovery of blood flow, reperfusion is performed. Then, slowly release the micro-arterial clamp on the common carotid artery to restore normal blood flow in the neck vessels and avoid blood flow impact that could cause vascular damage.

[0049] vi) Wound Closure and Postoperative Recovery: After carefully checking the surgical area for active bleeding, suture the neck muscles and skin incision layer by layer with 4-0 medical sutures. After suturing, disinfect the incision again with 75% medical alcohol to prevent postoperative infection. Turn off the isoflurane supply and maintain a continuous oxygen supply (oxygen flow rate adjusted to 0.5~0.8L / min). Transfer the mice to a preheated recovery cage (the recovery cage temperature is maintained at 30~32℃ to simulate the normal body temperature environment of mice and promote recovery). Closely observe the mice's respiration and activity until they are fully recovered. Keep the mice in individual cages after surgery. Within 24 hours, administer a subcutaneous injection of 0.5mL of preheated saline solution at 37℃ to replenish body fluids and reduce postoperative mortality. Continuously monitor the mice's mental state, food and water intake, activity, and other vital signs, and promptly remove any mice with abnormal conditions.

[0050] Step 3: Assessment of body weight, infarct volume, and neurological deficits in mice with ischemic stroke. i) Weight monitoring: The weight of mice was measured before daily administration, the weight change was recorded, and a time-series graph of weight change was plotted. The results are shown in Figure 1.

[0051] ii) Infarct volume determination (TTC staining method): 72 h after reperfusion, mice were deeply anesthetized (anesthesia protocol as in step ii). After perfusion via the aorta with pre-cooled saline to 4°C (perfusion rate 5 mL / min, until the mouse liver turned pale white, indicating adequate perfusion), the brain was quickly harvested, and the meninges and blood vessels on the surface of the brain tissue were removed. The intact brain tissue was flash-frozen at -20°C for approximately 15 min to facilitate subsequent sectioning. Using a brain sectioning mold, the brain tissue was continuously cut along the coronal plane into slices approximately 1 mm thick. The brain slices were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution and incubated at 37°C in the dark for 15–20 min, gently shaking the incubation container every 5 min to ensure uniform staining. After staining, normal brain tissue appeared brick red, while the infarcted area appeared pale white. Brain slice images were acquired using a digital camera under the same lighting conditions. The infarct area of ​​each brain slice was analyzed using ImageJ software, and then the percentage of total brain infarct volume was calculated (percentage of infarct volume = volume of infarcted area / total brain volume × 100%).

[0052] iii) Neurological deficit behavioral assessment: Neurological behavioral assessment was conducted at 24h, 48h, and 72h post-reperfusion using the Longa and Bederson scoring methods. Higher scores indicated more severe neurological deficits in the mice. The assessments were performed blinded by two trained researchers (to avoid subjective bias). Specific scoring criteria are as follows: 0 points: No neurological deficits, normal activity and eating; 1 point: When placed on a table, the paralyzed side's forepaw cannot be fully extended. When the tail is lifted, the paralyzed side's forepaw retracts and tucks under the abdomen. 2 points: When walking, the person turns in circles towards the paralyzed side, and the resistance to pushing objects towards the paralyzed side is significantly lower than that on the normal side; 3 points: When walking, the person leans to the paralyzed side and is unable to maintain a normal standing posture; 4 points: Unable to walk automatically, exhibiting loss of consciousness, and showing no obvious response to external stimuli.

[0053] After the experiment, statistical analysis was performed on the changes in mouse body weight, cerebral infarction volume, and neurological deficit scores using SPSS statistical software. Data are expressed as mean ± standard deviation. "±s" indicates that the difference was statistically significant using one-way ANOVA combined with LSD post-hoc test, and P<0.05 was considered statistically significant. This study clarified the therapeutic effect of peptide ES-PK on tMCAO / R model mice.

[0054] The results are as follows Figure 1 As shown in Tables 1 to 3.

[0055] Table 1. Effects of peptide ES-PK on body weight in I / R mice

[0056] Table 2. Effects of peptide ES-PK on the proportion of cerebral ischemic infarct area in I / R mice.

[0057] Table 3. Effects of peptide ES-PK on neurological deficit scores in mice 3 days after modeling.

[0058] Experimental results showed that the body weight of mice with ischemic stroke increased after administration ( Figure 1 .A), the volume of cerebral infarction in mice was significantly reduced ( Figure 1 .B), decreased neurological deficit behavioral score ( Figure 1 (C), demonstrating that the peptide ES-PK can increase the body weight of mice with ischemic stroke, reduce neurological deficits, and improve motor coordination.

[0059] Step 4: Assessment of the effects of peptide ES-PK on intracranial inflammatory factors and oxidative stress levels: i) Detection of mRNA expression levels of inflammatory factors in brain tissue (RT-qPCR): 72 h after reperfusion, approximately 50 mg of cerebral cortex tissue from the ischemic side (left side) of mice in each group was collected. Total RNA was extracted using Trizol reagent (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: R411-01) strictly following the reagent instructions. After extraction, RNA purity was measured (A260 / A280 ratio controlled between 1.8 and 2.0). Subsequently, a reverse transcription kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: R323-01) was used to reverse transcribe the qualified total RNA into cDNA. The reverse transcription conditions were: incubation at 37°C for 15 min, heating at 85°C for 5 s, and storage at 4°C. Using a Roche LightCycler 480II real-time quantitative PCR instrument (model: Roche LightCycler 480II), with β-actin as an internal reference gene, and the SYBR Green real-time quantitative PCR kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: Q711-02), the relative mRNA expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) were detected. The PCR reaction system (20 μL) consisted of: 10 μL SYBR Green Mix, 0.8 μL each of forward and reverse primers, 2 μL cDNA template, and 6.4 μL RNase-free ddH2O; cycling conditions: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, 40 cycles; melting curve analysis: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s, to verify primer specificity. The relative expression levels of mRNAs of each inflammatory factor were calculated using the 2^(-ΔΔCt) method. The sham-operated group (Sham) was used as a control to evaluate the regulatory effect of the peptide ES-PK on the expression of inflammatory factors in the brain.

[0060] ii) Serum malondialdehyde (MDA) detection (assessment of oxidative stress level): 72 h after reperfusion and before mouse brain tissue collection, blood was collected via the orbital venous plexus (0.3–0.5 mL per mouse). Whole blood was collected in anticoagulant-free centrifuge tubes, incubated at room temperature for 30 min, and then centrifuged at 8000 rpm for 5 min at 4°C. The supernatant serum was carefully aspirated and stored at -80°C for later use. The MDA detection kit (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number: BC0025) was strictly followed according to the instructions. An ELISA reader (model: BioTek Synergy HTX multi-functional ELISA reader) was used to measure the absorbance (OD value) of each well at 532 nm. The serum MDA content was calculated based on the standard curve to quantitatively assess the effect of peptide ES-PK on oxidative stress levels in mice.

[0061] Statistical analysis was performed on the levels of pro-inflammatory factors in the mouse cortex and serum MDA levels using SPSS statistical software. Data are expressed as mean ± standard deviation. "±s" indicates that the difference was statistically significant using one-way ANOVA combined with LSD post-hoc test, and P<0.05 was considered statistically significant. This study clarified the therapeutic effect of peptide ES-PK on tMCAO / R model mice.

[0062] The results are as follows Figure 2 As shown in Tables 4 to 8.

[0063] Table 4. Effects of peptide ES-PK on IL-1β mRNA expression in the cerebral cortex of I / R mice

[0064] Table 5. Effects of peptide ES-PK on IL-6 mRNA expression in the cerebral cortex of I / R mice.

[0065] Table 6. Effects of peptide ES-PK on TNF-α mRNA expression in the cerebral cortex of I / R mice.

[0066] Table 7. Effects of peptide ES-PK on iNOS mRNA expression in the cerebral cortex of I / R mice.

[0067] Table 8. Effects of peptide ES-PK on serum MDA levels in I / R mice

[0068] Experimental results showed that tMCAO / R modeling significantly upregulated the levels of pro-inflammatory factors in the mouse brain, and administration of ES-PK increased the levels of the pro-inflammatory factor IL-1β in the brains of ischemic stroke mice. Figure 2 .A), IL-6 ( Figure 2 .B) TNF-α level ( Figure 2 .C) and iNOS level ( Figure 2 The significant reduction in MDA (diethyltoluamide) levels indicates that peptide ES-PK can reduce the levels of inflammatory factors in the brains of mice with ischemic stroke. Furthermore, peptide ES-PK can significantly reduce serum MDA (diethyltoluamide) levels in mice with ischemic stroke. Figure 2 .E) level.

[0069] Example 3: Protective effect of peptide ES-PK on oxygen-glucose deprivation (OGD)-induced damage to HT22 neurons and hCMEC / D3 endothelial cells. Experimental materials: HT22 mouse hippocampal neurons and hCMEC / D3 human brain microvascular endothelial cells were purchased from Wuhan Pronosei Biotechnology Co., Ltd. (cell line numbers: HT22:CVCL_0321 and hCMEC / D3:CVCL_U985, respectively); high-glucose DMEM (catalog number: 11965084) and glucose-free DMEM (catalog number: 11966025) culture media were purchased from Gibco; fetal bovine serum (FBS) was purchased from Suzhou Ecosei Biotechnology Co., Ltd. (catalog number: FSP500); CCK-8 reagent was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (catalog number: C0039).

[0070] Step 1: Protective effect of peptide ES-PK on OGD-induced hippocampal neuronal damage in HT22 mice 1. Cell seeding and grouping HT22 cells in logarithmic growth phase were digested with 0.25% trypsin, resuspended in DMEM high-glucose medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) with 10% fetal bovine serum, and then seeded at 1.2 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μL per well in 96-well plates. The plates were incubated at 37°C with 5% CO2 for 12 h. When cell confluence reached 70%–80%, the cells were randomly divided into three groups, with four biological replicates per group. A blank control well (containing only culture medium, without cells, used to subtract background absorbance) was also included. Control group: The original culture medium was discarded and replaced with serum-free high-glucose DMEM medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin), and cultured under normal culture conditions (37℃, 5% CO2, saturated humidity) for 10 h. OGD model group: Discard the original culture medium and replace it with serum-free and glucose-free DMEM culture medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin). Transfer the culture plate to a three-gas incubator (94% N2, 5% CO2, 1% O2) and incubate at 37℃ and saturated humidity for 10 h to establish a glucose-oxygen deprivation cell damage model. Peptide treatment group (ES-PK group): The original culture medium was discarded and replaced with serum-free and sugar-free DMEM culture medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) containing different final concentrations of peptide ES-PK (0.1 μg / mL, 0.5 μg / mL, 1.5 μg / mL). The culture was placed under the above OGD conditions for 10 h. Peptide ES-PK was prepared and used immediately. The stock solution concentration was 10 mg / mL (solvent was serum-free and sugar-free DMEM culture medium). Before use, it was diluted with serum-free and sugar-free DMEM culture medium to the corresponding final concentration to ensure accurate concentration.

[0071] 2. Cell viability assay After OGD treatment, carefully discard the culture medium from each well. Add 10 μL of CCK-8 reagent and 90 μL of serum-free high-glucose DMEM medium (without penicillin or streptomycin to avoid interfering with the detection results) to each well. Gently shake the culture plate for 1–2 min to mix thoroughly (shaking speed 50 rpm). Place the culture plate back in a 37°C, 5% CO2, saturated humidity incubator and incubate in the dark for 0.5–1 h (incubation time determined based on preliminary experimental optimization). Use a microplate reader (Thermo Scientific Multiskan FC) to measure the absorbance (OD) of each well at 450 nm. 450 Before measurement, let the culture plate stand for 10 minutes to ensure the liquid in the wells is uniform, and the absorbance value of the blank control wells should be <0.1. Using the control group cell viability as 100%, calculate the relative cell viability of each group using the following formula: Relative cell survival rate (%) = (Experimental group OD) 450 - Blank control hole OD 450 ) / (control group OD 450 - Blank control hole OD 450 (mean × 100%) Step 2: Protective effect of peptide ES-PK on OGD-induced hCMEC / D3 human brain microvascular endothelial cell injury. 1. Cell seeding and grouping Logarithmically growing hCMEC / D3 human brain microvascular endothelial cells were digested with 0.25% trypsin, resuspended in DMEM high-glucose medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) with 10% fetal bovine serum, and then seeded at 8 × 10⁻⁶ cells per well. 3 Cells were seeded at a density of 100 μL per well in 96-well plates. The plates were incubated at 37°C with 5% CO2 for 12 hours. When cell adhesion and confluence reached 60%–70%, the cells were randomly divided into three groups, with four biological replicates in each group. A blank control well (containing only DMEM high-glucose medium, without cells, used to subtract background absorbance) was also included. Control group: The original culture medium was discarded and replaced with serum-free DMEM high glucose medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin), and cultured under normal culture conditions (37℃, 5% CO2, saturated humidity) for 6 h; OGD model group: Discard the original culture medium and replace it with serum-free and glucose-free DMEM culture medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin). Transfer the culture plate to a three-gas incubator (94% N2, 5% CO2, 1% O2) and incubate at 37℃ and saturated humidity for 6 h to establish a glucose-oxygen deprivation cell damage model. Peptide treatment group (ES-PK group): The original culture medium was discarded and replaced with serum-free and sugar-free DMEM culture medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) containing different final concentrations of peptide ES-PK (0.1 μg / mL, 0.5 μg / mL, 1.5 μg / mL). The culture was placed under the above OGD conditions for 6 h. Peptide ES-PK was prepared and used immediately. The stock solution concentration was 10 mg / mL (solvent was serum-free and sugar-free DMEM culture medium). Before use, it was diluted with serum-free and sugar-free DMEM culture medium to the corresponding final concentration to ensure accurate concentration.

[0072] 2. Cell viability assay After OGD treatment for 6 hours, CCK-8 assay was performed according to the method in step 1.2 to determine OD. 450 The values ​​were then calculated to determine the relative cell viability.

[0073] 3. Explanation of the scientific validity of the experimental design The replication settings were based on the following criteria: both the HT22 cell experiment and the hCMEC / D3 cell experiment used 4 biological replicates to meet the requirements of statistical analysis, which can effectively reduce experimental errors and ensure the reliability and reproducibility of experimental results.

[0074] Data statistics principles: Experimental data are expressed as mean ± standard deviation. "±s") indicates that SPSS statistical software was used, and one-way ANOVA combined with LSD post-hoc test was used for inter-group comparisons. P<0.05 was considered statistically significant.

[0075] The results are as follows Figure 3 As shown in Tables 9 and 10.

[0076] Table 9. Effect of peptide ES-PK on the survival rate of HT22 cells damaged by OGD for 10 h.

[0077] Table 10. Effect of peptide ES-PK on the survival rate of OGD-damaged hCMEC / D3 cells 6 h ago.

[0078] The above experimental results show that, compared with the control group, the survival rates of HT22 cells and hCMEC / D3 cells in the OGD model group were significantly decreased (P<0.0001). This demonstrates that peptide ES-PK treatment can increase the survival rate of both cell types after OGD injury in a concentration-dependent manner (P<0.001). Figure 3 A、 Figure 3 (B) This study demonstrates that it has a direct protective effect on neurons and cerebral vascular endothelial cells.

[0079] Example 4: Protective effect of peptide ES-PK against chemically induced PC12 cell damage. Experimental materials: PC12 rat adrenal medullary pheochromocytoma cells were purchased from the Cell Bank of the Chinese Academy of Sciences (cell line number: CSTR:19375.09.3101RATSCSP517); high-glucose DMEM (catalog number: 11965084) and glucose-free DMEM (catalog number: 11966025) culture media were purchased from Gibco; fetal bovine serum (FBS, catalog number: FSP500) was purchased from Suzhou Ekosei Biotechnology Co., Ltd.; corticosterone (CORT, purity ≥98%, catalog number: C119329) and 1-methyl-4-phenylpyridinium ion (MPP) were also used. + The reagent (purity ≥99%, catalog number: N137206) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the CCK-8 reagent was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; all reagents were sterile and met the standards for cell experiments.

[0080] Step 1: Protective effect of peptide ES-PK against corticosterone (CORT)-induced PC12 cell damage 1. Cell seeding and grouping PC12 cells in logarithmic growth phase were digested with 0.25% trypsin and then resuspended in high-glucose DMEM medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and 100 μg / mL streptomycin) to adjust the cell density to 1.0 × 10⁶ cells / mL. 4 Cells were seeded per well in a 96-well plate, with a final volume of 100 μL per well. The plates were incubated at 37°C with 5% CO2 for 12 h. When cell adhesion and confluence reached 70%–80%, the cells were randomly divided into four groups, with four biological replicates (n=4) in each group. A blank control well (containing only serum-free, high-glucose DMEM medium, without cells, used to subtract background absorbance) was also included. Control group: The original culture medium was discarded and replaced with fresh serum-free high-glucose DMEM culture medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin), without the addition of any damaging agents or peptides; Corticosterone model group (CORT group): The original culture medium was discarded and replaced with serum-free high-glucose DMEM culture medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) with a final concentration of 250 μM corticosterone (CORT) to establish a PC12 cell chemical damage model. Peptide treatment group (ES-PK group): The original culture medium was discarded and replaced with serum-free high-glucose DMEM culture medium (containing 100U / mL penicillin and 100μg / mL streptomycin) containing a final concentration of 250μM CORT and different concentrations of peptide ES-PK (0.5μg / mL, 2μg / mL, 10μg / mL). Peptide ES-PK was prepared and used immediately, with a stock solution concentration of 10mg / mL (solvent being serum-free high-glucose DMEM culture medium). Before use, it was diluted with serum-free high-glucose DMEM culture medium to the corresponding final concentration to ensure accurate concentration.

[0081] 2. Cell viability assay After incubating each group of cells at 37°C and 5% CO2 for 24 hours, cell viability was detected using the CCK-8 assay as described in step one of Example 3. The specific procedure was as follows: Carefully discard the culture medium from each well. Add 10 μL of CCK-8 reagent and 90 μL of serum-free, high-glucose DMEM culture medium (without penicillin or streptomycin to avoid interference with the results) to each well. Gently shake the culture plate for 1-2 minutes to mix (shaking speed 50 rpm). Return the culture plate to a 37°C, 5% CO2, saturated humidity incubator and incubate in the dark for 0.5-1 hour (incubation time determined based on preliminary experiments). Measure the absorbance (OD) at 450 nm using a microplate reader (Thermo Scientific Multiskan FC). 450 Before measurement, let the culture plate stand for 10 minutes to ensure the liquid in the wells is uniform, and the absorbance value of the blank control wells should be <0.1; with the cell viability of the control group as 100%, calculate the relative cell viability of each group according to the following formula: Relative cell survival rate (%) = (Experimental group OD) 450 - Blank control hole OD 450 ) / (Control group OD 450 - Blank control hole OD 450 (mean × 100%) Step 2: Peptide ES-PK against MPP + Protective effect against induced neurotoxicity in PC12 cells 1. Cell seeding and grouping PC12 cells in logarithmic growth phase were digested with 0.25% trypsin and resuspended in high-glucose DMEM medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) with 10% fetal bovine serum, and the cell density was adjusted to 8 × 10⁶ cells / mL. 3 Cells were seeded per well in a 96-well plate, with a final volume of 100 μL per well. The plates were incubated at 37°C with 5% CO2 for 24 h. When cell adhesion and confluence reached 70%–80%, the cells were randomly divided into four groups, with four biological replicates (n=4) in each group. A blank control well (containing only serum-free, high-glucose DMEM medium, without cells, used to subtract background absorbance) was also included. Control group: The original culture medium was discarded and replaced with fresh serum-free high-glucose DMEM culture medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin), without the addition of any damaging agents or peptides; MPP + Model Group (MPP) + Group 1): Discard the original culture medium and replace it with a medium containing 4 mM 1-methyl-4-phenylpyridinium ions (MPP). + The PC12 cell neurotoxicity injury model was established by using serum-free, high-glucose DMEM medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) and avoiding light throughout the above process. Peptide treatment group (ES-PK group): The original culture medium was discarded and replaced with a medium containing a final concentration of 4 mM MPP. + Serum-free high-glucose DMEM medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) with different concentrations of peptide ES-PK (0.2 μg / mL, 0.5 μg / mL, 2.5 μg / mL) was prepared and used immediately. The stock solution concentration was 10 mg / mL (solvent was serum-free high-glucose DMEM medium). Before use, it was diluted with serum-free high-glucose DMEM medium to the corresponding final concentration to ensure accurate concentration. All the above processes were carried out in the dark.

[0082] 2. Cell viability assay After incubating each group of cells at 37°C and 5% CO2 for 24 hours, cell viability was detected using the CCK-8 assay as described in step one of Example 3, and OD was measured. 450 The relative cell viability was calculated, and the procedure was consistent with step one to ensure experimental reproducibility.

[0083] 3. Explanation of the scientific validity of the experimental design The replication factor is based on the following: In this embodiment, each group of PC12 cells is configured with 4 biological replicates (n=4). PC12 cells are neuroendocrine cells, which are resistant to chemical damage (CORT, MPP).+ The sensitivity of the sample is high, and four biological replicates can effectively reduce random errors in the experiment and further ensure the accuracy of the experimental results.

[0084] Concentration gradient design basis: for two different damage models (CORT and MPP) + ), and appropriate peptide ES-PK concentration gradients were set. The CORT model used a wide range of gradients of 0.5 μg / mL, 2 μg / mL, and 10 μg / mL to cover low, medium, and high concentrations for preliminary assessment of protective effects; MPP + The model uses relatively low concentration gradients of 0.2 μg / mL, 0.5 μg / mL, and 2.5 μg / mL to explore the effective dose range more precisely, avoid the potential toxicity of high-concentration peptides to cells, and thus accurately determine the optimal concentration range for ES-PK to exert its neuroprotective effect.

[0085] Data statistics principles: Experimental data are expressed as mean ± standard deviation. "±s") indicates that SPSS statistical software was used, and one-way ANOVA combined with LSD post-hoc test was used for inter-group comparisons. P<0.05 was considered statistically significant.

[0086] The results are as follows Figure 4 As shown in Tables 11 and 12.

[0087] Table 11 Effect of peptide ES-PK on the survival rate of PC12 cells damaged by 250 μM CORT

[0088] Table 12 Effects of peptide ES-PK on 4mM MPP + Effect of damage on PC12 cell survival

[0089] Experimental results show that treatment with 250 μM CORT for 24 h or 4 mM MPP... + Treatment for 24 hours significantly reduced PC12 cell viability (P<0.0001). Co-treatment with peptide ES-PK significantly antagonized CORT and MPP. + The decrease in cell viability was caused in a concentration-dependent manner (P<0.001). Figure 4 A, Figure 4 B) This suggests that the polypeptide has a protective effect against both hormone-induced stress damage and neurotoxic damage.

[0090] Example 5: Effect of peptide ES-PK on CORT-induced intracellular reactive oxygen species (ROS) levels in PC12 cells Experimental materials: PC12 rat adrenal medullary pheochromocytoma cells were purchased from the Cell Bank of the Chinese Academy of Sciences (cell line number: CSTR:19375.09.3101RATSCSP517); high-glucose DMEM (catalog number: 11965084) and glucose-free DMEM (catalog number: 11966025) culture media were purchased from Gibco; fetal bovine serum (FBS, catalog number: FSP500) was purchased from Suzhou Ekosei Biotechnology Co., Ltd.; corticosterone (CORT, purity ≥98%, catalog number: C119329) and 1-methyl-4-phenylpyridinium ion (MPP) were also used. + The reagent (purity ≥99%, catalog number: N137206) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the CCK-8 reagent was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; all reagents were sterile and met the standards for cell experiments.

[0091] The reactive oxygen species (ROS) detection kit (DCFH-DA fluorescent probe method) was purchased from Beyotime Biotechnology Co., Ltd.; the live cell imaging system was purchased from Agilent Technologies (product number: Lionheart FX). Both the live cell imaging system and the BioTek Synergy HTX multi-functional microplate reader were calibrated to ensure accurate and reliable detection data.

[0092] Experimental steps: i) Cell treatment PC12 cells were seeded in 24-well culture plates. The seeding density, culture conditions, and grouping scheme were performed according to the method described in step one of Example 4. The cell seeding density was adjusted to 1.0 × 10⁶ cells / well. 4 Each well contains 500 μL of samples and is randomly divided into 3 groups, with 3 biological replicates (n=3) in each group: control group, 250 μM CORT model group, and CORT+ES-PK peptide group (the final peptide concentration was selected as 10 μg / mL, which showed the best protective effect in Example 4, to ensure the relevance and continuity of the experiment). Each group strictly followed the corresponding protocol to complete the drug treatment and the culture conditions were kept consistent.

[0093] ii) ROS detection After 24 hours of CORT treatment, carefully discard the culture medium from each well (avoid scratching adherent cells to prevent affecting fluorescence detection results). Strictly follow the ROS detection kit instructions: Use serum-free, high-glucose DMEM medium pre-warmed to 37°C. Dilute the DCFH-DA fluorescent probe at a ratio of 1:1000 to prepare the probe working solution. Add 500 μL of the diluted working solution to each well, ensuring complete coverage of the cells. Incubate the 24-well plate in a 37°C, 5% CO2 incubator in the dark for 30 minutes, avoiding shaking the plate during incubation to prevent cell detachment. After incubation, gently wash the cells three times with pre-warmed serum-free, high-glucose DMEM medium, allowing them to stand for 2 minutes after each wash to thoroughly remove any undeclared free probes and eliminate background interference.

[0094] iii) Imaging and Quantitative Analysis ① Live cell imaging: After washing the cells, the culture plate is immediately placed under the live cell imaging system. Three cells are evenly distributed in each well and the field of view is selected for imaging to obtain fluorescence images. The intracellular ROS level is qualitatively assessed by fluorescence intensity (the higher the fluorescence intensity, the more ROS is generated).

[0095] ② Absolute quantification using an ELISA reader: After imaging observation, a multi-functional ELISA reader is used to detect the fluorescence intensity value of each well under the conditions of excitation wavelength 488nm and emission wavelength 525nm. The fluorescence intensity value reflects the relative content of ROS in the cell, and the absolute quantitative analysis is completed.

[0096] After the test, the fluorescence intensity data of each group were statistically analyzed using SPSS statistical software. Data are expressed as mean ± standard deviation. "±s" indicates that one-way ANOVA combined with LSD post-hoc test was used, and P<0.05 was considered statistically significant, clarifying the regulatory role of peptide ES-PK on CORT-induced intracellular ROS levels in PC12 cells.

[0097] For detailed qualitative imaging results and quantitative data, please refer to [link to relevant documentation]. Figure 5 And Table 13.

[0098] Table 13 Effects of peptide ES-PK on CORT-induced ROS levels in PC12 cells

[0099] Live-cell imaging showed that the green fluorescence intensity in PC12 cells of the CORT model group was significantly stronger than that of the control group, indicating a large accumulation of ROS; while compared with the CORT model group, the fluorescence intensity in cells of the peptide co-treatment group was significantly weakened. Microplate reader quantification results were consistent with the imaging results: compared with the control group, the ROS level in the CORT model group was significantly increased (P<0.001); while compared with the CORT model group, the peptide ES-PK significantly reduced the ROS level (P<0.001). Figure 5 A, Figure 5 (B). This result confirms that the peptide ES-PK can effectively alleviate CORT-induced oxidative stress in PC12 cells.

[0100] Comparative Example 1 Design and synthesis of control peptide EP-DL: To verify the specific efficacy of the polypeptide ES-PK (SEQ ID NO: 1) of this invention in treating ischemic stroke and to eliminate non-specific interference that may be caused by arbitrary amino acid sequences, this invention designed and prepared a control peptide with no significant pharmacological activity, named EP-DL. The amino acid sequence of this control peptide is Glu-Pro-Pro-Thr-Val-Val-Pro-Gly-Gly-Asp-Leu (single-letter code: EPPTVVPGGDL), which is the sequence shown in SEQ ID NO: 2.

[0101] To ensure the accuracy and reliability of the comparative experiment, the control peptide EP-DL was prepared using the same method and quality control standards as the peptide ES-PK of this invention.

[0102] Specifically, the control peptide EP-DL was chemically prepared using the Fmoc solid-phase peptide synthesis method, which is well-known in the art. The L-type amino acid raw materials (E, P, T, V, G, D, L) and other reagents used in the synthesis were all commercially available analytical grade or pharmaceutical grade (≥99%), and could be purchased from conventional reagent suppliers such as Sinopharm Chemical Reagent Co., Ltd. and Shanghai Aladdin Biochemical Technology Co., Ltd.

[0103] The control peptide EP-DL used in this comparative example was custom-produced by Nanjing Genscript Biotech Co., Ltd., a professional peptide synthesis company, based on the aforementioned Fmoc solid-phase synthesis principle. The production process complied with relevant GMP requirements. The synthesized crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and the final product was identified using liquid chromatography-mass spectrometry (LC-MS). The results showed that the measured molecular weight was consistent with the theoretical value; analytical RP-HPLC analysis showed that its chemical purity was ≥98%.

[0104] The control peptide EP-DL was subjected to the same assays as the peptide ES-PK to measure body weight, behavioral parameters, cerebral infarction area, levels of cerebral inflammatory factors, and HT22 cells and hCMEC / D3 under OGD protection conditions. The results are as follows: Figure 6 As shown in Tables 14 to 22.

[0105] Table 14 Effects of EP-DL peptides on body weight in I / R mice

[0106] Table 15 Effects of EP-DL peptides on neurological deficit scores in I / R mice

[0107] Table 16 Effects of EP-DL peptides on cerebral infarction volume in I / R mice

[0108] Table 17 Effect of control peptide EP-DL on serum MDA levels in I / R mice

[0109] Table 18 Effects of EP-DL peptides on TNF-α levels in the cerebral cortex of I / R mice

[0110] Table 19 Effects of EP-DL peptides on IL-6 levels in the cerebral cortex of I / R mice

[0111] Table 20 Effects of EP-DL peptides on IL-1β levels in the cerebral cortex of I / R mice

[0112] Table 21 Effect of EP-DL peptide on the survival rate of HT22 cells damaged by OGD for 10 h (%)

[0113] Table 22 Effect of EP-DL peptide on the survival rate of hCMEC / D3 cells damaged by OGD for 6 h (%)

[0114] Experimental results showed that there were no significant differences in body weight, cerebral infarction volume, and neurological deficit scores between the EP-DL control group and the model group (P>0.05). Specifically, the body weight continued to decrease, consistent with the trend of body weight change in the model group. Figure 6 .A); No significant decrease in behavioral scores for neurological deficits ( Figure 6 .B); The volume of the cerebral infarction did not decrease significantly ( Figure 6 .C); Serum MDA levels did not decrease significantly ( Figure 6 .D).

[0115] The levels of inflammatory factors in the cerebral cortex of mice in the EP-DL control group were not significantly different from those in the model group (P>0.05). Specifically, the levels of inflammatory factors TNF-α, IL-6, and IL-1β showed a significantly increased trend consistent with those in the model group. Figure 6 .E, Figure 6 .F, Figure 6 .G). The cell viability of the EP-DL peptide-treated group was not significantly improved compared to the OGD group. Figure 6 .H and Figure 6 The results indicate that EP-DL peptides have no significant therapeutic effect on ischemic stroke, further confirming that the significant therapeutic effect of ES-PK peptides is specific and not a non-specific effect of random peptides.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of a polypeptide preparation in a medicament for the prevention and / or treatment of ischemic stroke, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The polypeptide was prepared by solid-phase synthesis using amino acid raw materials, and its purity was >98.0% as determined by HPLC-MS.

3. The use of a polypeptide derivative in the preparation of a medicament for the prevention and / or treatment of ischemic stroke, characterized in that, The polypeptide derivative is a fusion polypeptide obtained by attaching a protein purification tag or detection tag that can retain polypeptide activity to the amino or carboxyl terminus of the polypeptide described in claim 1.

4. The application according to claim 3, characterized in that, The protein purification tag or detection tag is a His tag or a FLAG tag.

Images