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

Abstract

This invention relates to the field of biomedical technology, specifically disclosing a polypeptide and its application in the preparation of drugs for ischemic stroke. The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1. This invention utilizes an in vivo tMCAO / R model and in vitro studies covering ischemic hypoxia (OGD) and neurotoxicity (MPP). + Multiple cell models of NL-PT, including those for cellular stress and hormonal stress (CORT), were used to reveal the application potential of the peptide NL-PT from multiple dimensions and levels. These results collectively demonstrate that NL-PT not only has a significant protective effect against the core pathological processes of ischemic stroke, but also exhibits broad-spectrum biological activity against various related injuries. In particular, its well-defined mechanism of anti-oxidative stress through scavenging reactive oxygen species (ROS) further highlights its multi-target intervention characteristics, providing solid and reliable experimental evidence for its development into a novel, multi-target therapeutic drug for ischemic stroke.

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 a clinical syndrome caused by impaired blood supply to the brain, leading to localized ischemia, hypoxic necrosis, and consequently, corresponding neurological deficits. Its incidence, mortality, and disability rates remain high, making it a major disease threatening human health. Currently, clinical treatment for ischemic stroke primarily focuses on early vascular recanalization, such as thrombolytic therapy using tissue plasminogen activator or mechanical thrombectomy, to restore blood flow to the ischemic penumbra. However, the time window for these treatments is very strict, usually limited to within a few hours of onset, and thrombolytic therapy may also increase the risk of intracranial hemorrhage transformation, leading to secondary brain injury.

[0003] In-depth research has revealed that the pathophysiological mechanisms of ischemic stroke are extremely complex, involving multiple interrelated biological processes such as excitatory amino acid toxicity, oxidative stress, inflammatory response, and apoptosis. In the ischemic core, irreversible neuronal necrosis occurs due to complete interruption of blood supply; while in the surrounding ischemic penumbra, although blood flow is significantly reduced, cells still retain basic metabolic functions, making it a key target for clinical treatment. However, even if blood flow is restored through thrombolysis and thrombectomy, subsequent ischemia-reperfusion injury can further aggravate neuronal damage and death through pathways such as the generation of large amounts of free radicals, activation of inflammatory cascades, and mitochondrial dysfunction, thereby weakening the overall efficacy of vascular recanalization. This complex pathological cascade reaction involving multiple links and multiple targets means that traditional neuroprotective agents targeting single links (such as excitatory amino acid antagonists and antioxidants) often fail to meet treatment needs in clinical translation due to limited efficacy or significant adverse reactions.

[0004] Therefore, there is an urgent need to develop new drugs that can prolong the treatment time window, reduce ischemia-reperfusion injury, and promote the recovery of nerve function. Summary of the Invention

[0005] To address the technical shortcomings in the existing treatment of ischemic stroke, this invention innovatively provides a polypeptide (with the amino acid sequence NLDIERPT) and its application in the treatment of neurological injury-related diseases. This NL-PT polypeptide not only exhibits significant neuroprotective efficacy in classic ischemic stroke animal models, but also displays broad-spectrum cytoprotective activity in various neurological injury-related cell models, demonstrating outstanding drug development potential and broad prospects for clinical application.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a polypeptide (denoted as NL-PT) with the amino acid sequence: NLDIERPT (SEQ ID NO: 1).

[0007] This invention, through in-depth exploration and systematic research into the pathogenesis of ischemic stroke, has identified a novel polypeptide, NL-PT, with a novel amino acid sequence structure. Multidimensional in vitro and in vivo experiments have verified that this polypeptide effectively improves neurological deficits following cerebral ischemia through multiple pathways, including inhibiting neuronal apoptosis, reducing oxidative stress damage, and regulating inflammatory responses. It exhibits significant protective efficacy in both cellular and animal-level ischemic injury models. The NL-PT polypeptide provided by this invention offers a novel and highly effective drug candidate strategy for the clinical treatment of ischemic stroke, possessing significant social value and market application potential.

[0008] It should be noted that the polypeptide NL-PT described in this invention is chemically synthesized using conventional solid-phase synthesis methods (such as Fmoc solid-phase synthesis). In one specific embodiment of this invention, the polypeptide NL-PT is custom-produced by Nanjing Genscript Biotech Co., Ltd., a professional polypeptide synthesis company, based on the standard Fmoc solid-phase synthesis principle. The synthesis process strictly adheres to relevant GMP specifications. Verification has shown that the synthesized polypeptide sequence is completely identical to the sequence shown in SEQ ID NO: 1, and its purity meets the standards, stably meeting the requirements for subsequent in vivo and in vitro pharmacodynamic experiments and clinical applications.

[0009] Secondly, the present invention provides the use of the above-mentioned polypeptide NL-PT in the preparation of medicaments for the prevention and / or treatment of neurological injury-related diseases.

[0010] In one 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 that highly matches clinicopathological characteristics to verify that the peptide NL-PT significantly improved the neurological deficit scores of the model mice and significantly enhanced their motor coordination and limb movement abilities, indicating that it has a positive promoting effect on the recovery of neurological function after cerebral ischemia-reperfusion injury. Simultaneously, professional detection methods such as TTC staining confirmed that the peptide can significantly reduce the volume of cerebral infarction, clearly demonstrating its substantial protective efficacy against the ischemic core area and penumbra brain tissue. Furthermore, this invention systematically detected the expression levels of inflammatory factors (such as TNF-α, IL-1β, IL-6, etc.) in brain tissue and systemic oxidative stress indicators using relevant detection technologies. The results showed that the peptide can effectively inhibit the excessive release of inflammatory factors and significantly downregulate systemic oxidative stress levels, comprehensively confirming its clear and stable neuroprotective effect at the whole animal level.

[0013] At the in vitro cellular level, this invention systematically verified that the peptide NL-PT possesses broad and multi-mechanistic cellular protective activity by constructing various cell injury models closely related to the core pathological processes of ischemic stroke, as detailed below: 1. Targeting ischemic-hypoxic core injury: The peptide NL-PT significantly improved the survival rate of mouse hippocampal HT22 cells treated with 10 hours of oxygen-glucose deprivation (OGD) and human brain microvascular endothelial hCMEC / D3 cells treated with 6 hours of OGD. This result directly confirms that the peptide possesses both direct neuroprotective and vascular endothelial protective potential, helping to simultaneously maintain the structural and functional integrity of neural units and the blood-brain barrier, providing important cellular-level experimental support for its application in the treatment of ischemic stroke.

[0014] 2. Targeting neurotoxic stress injury: Peptide NL-PT against 4 mM MPP + Induced PC12 cell damage has a significant protective effect and can effectively improve cell viability, suggesting that it may exert neuroprotective effects by antagonizing key pathways such as mitochondrial dysfunction or regulating oxidative stress, further expanding its application prospects in the field of combating neurotoxic damage.

[0015] 3. Targeting Hormonal Stress Injury: The peptide NL-PT significantly alleviated PC12 cell damage induced by 250 μM corticosterone (CORT). CCK-8 assay results clearly demonstrated that this peptide effectively improved cell viability and proliferation. Furthermore, reactive oxygen species (ROS) fluorescence staining combined with live-cell imaging and ELISA quantitative analysis further confirmed its ability to significantly reduce CORT-induced intracellular ROS accumulation and decrease oxidative stress-mediated cell damage. These results clearly suggest that reducing oxidative stress levels and clearing excess intracellular ROS are among the important molecular mechanisms by which this peptide exerts its neuroprotective effect.

[0016] In summary, this invention utilizes an in vivo mouse transient middle cerebral artery occlusion / reperfusion (tMCAO / R) model, combined with in vitro studies encompassing ischemia-hypoxia (OGD) and neurotoxicity (MPP). + Multiple cell models, including those for cellular stress and hormonal stress (CORT), were used to systematically reveal the application value and development potential of the peptide NL-PT from multiple dimensions and levels. The experimental results fully confirm that this peptide NL-PT not only has significant protective efficacy against the core pathological processes of ischemic stroke, but also exhibits broad-spectrum biological activity against various related injuries. In particular, its well-defined mechanism of anti-oxidative stress through specific scavenging of reactive oxygen species (ROS) further highlights its unique advantage of multi-target synergistic intervention, providing solid and reliable experimental support for its development into a novel, multi-target regulated treatment for ischemic stroke.

[0017] Thirdly, this invention provides a polypeptide derivative, which is a fusion polypeptide formed by coupling a protein purification tag or detection tag that retains the original activity of the polypeptide NL-PT to the amino or carboxyl terminus. Any such fusion polypeptide capable of retaining the core therapeutic activity of the polypeptide NL-PT should be included within the scope of equivalent embodiments of this invention.

[0018] Specifically, the protein purification tag or detection tag may be a His tag or a FLAG tag.

[0019] On the other hand, this invention also includes active derivatives obtained by performing one or more conventional chemical modifications on the amino acid sequence shown in SEQ ID NO: 1. 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 NL-PT peptide; on the contrary, they can significantly improve its in vitro stability, in vivo pharmacokinetic properties, or reduce its potential immunogenicity, further enhancing its drug potential. All the above derivatives, as long as they retain neuroprotective activity substantially consistent with the NL-PT peptide shown in SEQ ID NO: 1, fall within the scope of protection of this invention.

[0020] Fourthly, 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 specifically refers to ischemic stroke.

[0023] Fifthly, the present invention provides a pharmaceutical composition comprising a therapeutically effective dose of the aforementioned polypeptide NL-PT 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 NL-PT 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, and the administration method can be flexibly selected according to actual clinical treatment needs.

[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, the polypeptide NL-PT, with the amino acid sequence shown in SEQ ID NO: 1, provided by this invention exhibits significant neuroprotective effects in both in vivo and in vitro experiments. This invention, through the construction of a mouse transient middle cerebral artery occlusion / reperfusion (tMCAO / R) model, confirmed that this polypeptide can significantly improve neurological deficit symptoms in model mice, reduce cerebral infarction volume, and effectively reduce the expression levels of inflammatory factors in brain tissue and systemic oxidative stress levels, indicating that it has a good comprehensive effect of anti-inflammation, anti-oxidation, and promoting neurological function recovery in vivo. In in vitro cell experiments, this polypeptide not only has a direct and 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 ions (MPP). + The peptide NL-PT induced PC12 cell damage and significantly reduced CORT-induced intracellular reactive oxygen species (ROS) accumulation. These results further confirm that the peptide NL-PT possesses clear multi-target cytoprotective activity, synergistically exerting therapeutic effects on ischemic stroke from multiple dimensions, including reducing infarct volume, inhibiting inflammatory oxidative stress, and protecting neurons and vascular endothelial cells, demonstrating great potential for development into a novel and highly effective neuroprotective agent. Furthermore, this invention also covers active derivatives of the peptide and pharmaceutical compositions containing the peptide or its derivatives, providing a complete and feasible technical implementation scheme for the application of this peptide 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 To illustrate how the polypeptide NL-PT reduced the cerebral infarction area and behavioral score in mice with ischemic stroke in the tMCAO / R model in Example 2 of this invention, the following images are included: (A) TTC staining image; (B) percentage of cerebral infarction area; and (C) behavioral score of neurological deficit. Figure 2 Example 2 of this invention uses polypeptide NL-PT 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 NL-PT 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 polypeptide NL-PT 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 rate 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 NL-PT 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 in the tMCAO / R model, 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 rate of mouse hippocampal HT22 cells damaged by 10 h of glucose-oxygen deprivation (OGD); and (I) effect on cell survival rate of human brain microvascular endothelial cells hCMEC / D3 cells damaged by 6 h of glucose-oxygen deprivation (OGD). Detailed Implementation

[0032] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following detailed description of the polypeptide NL-PT, its preparation method, and applications, in conjunction with specific embodiments, is provided. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the scope of protection of this invention.

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

[0034] Example 1 This invention provides a polypeptide NL-PT with the amino acid sequence NLDIERPT (SEQ ID NO: 1). The purpose of this embodiment is to disclose the sequence source, chemical synthesis method, and quality testing standards of this polypeptide, ensuring the acquisition of sufficient, high-purity polypeptide samples to provide reliable support for subsequent pharmacodynamic experiments.

[0035] The method for obtaining and preparing the above-mentioned polypeptide NL-PT is as follows: 1. Sources of sequence construction The amino acid sequence of peptide NL-PT (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. The specific identification process is as follows: Mass spectrometry analysis was performed using a Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer equipped with a nanoliter spray ionization source. The acquired raw mass spectrometry data were analyzed using PEAKS® Studio bioinformatics software, combining database searching and de novo sequencing to ensure the accuracy of the identified sequence. Key detection parameters were set as follows: precursor ion mass tolerance ±10 ppm, fragment ion mass tolerance ±0.02 Da. Through the above systematic analysis, a peptide sequence with potential neuroprotective activity was finally identified from brain protein hydrolysate I with a confidence level higher than 95%. This invention names it NL-PT, and its amino acid sequence corresponds to NLDIERPT (three-letter code: Asn-Leu-Asp-Ile-Glu-Arg-Pro-Thr) shown in SEQ ID NO: 1.

[0036] 2. Chemical Synthesis and Sample Acquisition The polypeptide NL-PT described in this invention is chemically prepared using the standard Fmoc solid-phase polypeptide synthesis method. The L-type amino acid raw materials (N, L, D, I, E, R, P, T) and other synthetic reagents (such as resins, condensing agents, deprotecting reagents, 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. To obtain high-purity samples that meet pharmaceutical research standards, the polypeptide NL-PT used in this embodiment was custom-produced by Nanjing Genscript Biotech Co., Ltd., a professional polypeptide synthesis company, based on the aforementioned Fmoc solid-phase synthesis principle. Its production process complies with relevant GMP specifications. 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%.

[0037] Through the above-mentioned sequence identification, chemical synthesis and purification identification steps, this invention successfully obtained a sufficient amount of high-purity polypeptide NL-PT (purity ≥98%, sequence completely identical to SEQ ID NO: 1). The sample is stable in quality and reliable in activity, and can be directly used for subsequent in vivo and in vitro pharmacodynamic experiments and related studies, providing a solid sample foundation for subsequent verification of the neuroprotective efficacy of the polypeptide and development of related drugs.

[0038] Example 2: Therapeutic effect of peptide NL-PT 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.) were selected and acclimatized for one week in a standard SPF environment (temperature 22-25℃, humidity 50%-60%, 12h light / 12h dark cycle). They were then randomly divided into three groups using a random number table to ensure no statistically significant differences in weight or age among the groups (P>0.05). i) Sham group: 6 mice, only the neck blood vessels were separated, and no suture plug was inserted. After the mice were housed separately, 0.2 mL of physiological saline was injected intraperitoneally for the first time 1 day before modeling. The drug was administered once a day on the day of modeling and after modeling until the end of the experiment (72 hours after modeling).

[0039] ii) Model group (I / R): 12 animals, tMCAO / R model was established, and the drug administration regimen was the same as that of the sham operation group (0.2 mL of physiological saline was injected intraperitoneally every day, and the drug administration time and cycle were the same as those of the sham operation group).

[0040] iii) Peptide treatment group (I / R+NL-PT): 12 mice were used to establish the tMCAO / R model. Peptide NL-PT was prepared fresh (stored at 4℃ protected from light after preparation and used within 2 hours), with a stock solution concentration of 10 mg / mL, which was diluted with physiological saline to 1 mg / mL before use. After the mice were housed separately, the first administration was started 1 day before modeling. Peptide NL-PT solution was injected intraperitoneally daily (dose 10 mg / kg, volume calculated based on mouse body weight, i.e., 10 μL / g). The administration was continued once daily on the day of modeling and after modeling until the experimental endpoint (72 hours after modeling).

[0041] 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 and cerebral ischemia-reperfusion stress. After screening (screening criteria: no severe postoperative bleeding, no infection, and able to eat and drink normally), the final number of valid animals included in the statistical analysis was: sham-operated group n=6, model group n=10, and peptide treatment group n=11. All statistical analyses were performed based on valid animal data to ensure the reliability of the experimental results.

[0042] Step 2: Establish a mouse model of ischemic stroke induced by middle cerebral artery occlusion / reperfusion (tMCAO / R). Anesthesia and Fixation: Mice were first placed in an anesthesia induction box and anesthesia was induced using a mixed gas containing 3%–4% isoflurane (oxygen flow rate 0.8–1.0 L / min). After the mice lost their righting reflex, they were quickly moved to the operating table, and anesthesia was maintained by continuously administering a mixed gas containing 1.5%–2.0% isoflurane through a nasal cone. The mice were fixed in a supine position on the operating table, and a rectal temperature probe combined with a feedback heating pad was used to monitor and maintain the mice's core body temperature at 37.0 ± 0.5℃ in real time to avoid the influence of body temperature fluctuations on the experimental results.

[0043] ii) Neck incision and blood vessel separation: After preparing the anterior neck area of ​​the mouse, the skin was disinfected with 75% ethanol, and a midline neck incision (approximately 1.5 cm in length) was made. Under a stereomicroscope (model: Olympus SZ61), the subcutaneous tissue, salivary glands and neck muscles were bluntly separated to fully expose the left common carotid artery, external carotid artery and internal carotid artery. During the separation process, damage to blood vessels and surrounding nerves was avoided.

[0044] iii) Vascular pretreatment: Carefully dissect the external carotid artery with microforceps and permanently ligate its distal end with 5-0 silk suture; temporarily clamp the proximal end of the common carotid artery with miniature arterial clamps to block blood flow and prepare for subsequent suture insertion.

[0045] iv) Insertion of the suture and ischemia: Approximately 2 mm proximal to the ligation point of the external carotid artery, make a 45° oblique incision using microvascular scissors. Gently insert a nylon suture (0.22 mm in diameter and 18 mm in length) with its tip coated with silicone (approximately 0.02 mm thick to increase friction and prevent suture dislodgement) through this incision into the internal carotid artery. Slowly advance the suture along the internal carotid artery into the cranium to a depth of approximately 9-11 mm 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 silk suture to secure the suture. Begin recording the ischemia time, which is strictly controlled to be 1 hour.

[0046] v) Reperfusion: After 1 hour of ischemia, slowly pull out the nylon suture into the stump of the external carotid artery and observe the recovery of blood flow in the internal carotid artery by visual inspection (the criteria for blood flow recovery are: vascular filling and normal pulsation). After confirming that reperfusion has been achieved, release the mini arterial clamp on the common carotid artery to restore normal blood flow in the neck vessels.

[0047] vi) Wound closure and postoperative recovery: After carefully checking the surgical area for active bleeding, the neck muscles and skin incision were sutured layer by layer using 4-0 silk sutures. The isoflurane supply was turned off, while oxygen supply was maintained continuously for 5-10 minutes. The mice were then transferred to a preheated (37°C) recovery cage and closely observed until they were fully recovered. After surgery, the mice were housed individually. Within 24 hours, all mice were subcutaneously injected with preheated saline (0.5 mL) to replenish fluids and reduce postoperative mortality. Simultaneously, the mice's mental state, food and water intake, and vital signs were continuously monitored, and any abnormal mice were promptly removed.

[0048] Step 3: Assessment of infarct volume and neurological deficits in mice with ischemic stroke i) Infarct volume determination (TTC staining method): 72 h after reperfusion, mice were deeply anesthetized with an excess of isoflurane (5%). After the mice stopped breathing, they were rapidly perfused with 4°C pre-cooled physiological saline (perfusion rate 5 mL / min) through the heart until the liver turned pale. The brain was then quickly harvested (within 5 min). The intact brain tissue was flash-frozen at -20°C for 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 (6-7 slices per mouse). All brain slices were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution (prepared with 0.1 mol / L PBS buffer, pH=7.4) and incubated at 37°C in the dark for 15-20 min, gently shaking the incubation solution every 5 min to ensure uniform staining. After staining, normal brain tissue appeared brick red, while infarcted areas appeared pale white. Brain slice images were acquired using a digital camera, and the infarct area of ​​each brain slice was analyzed using ImageJ software. The percentage of total cerebral infarction volume was calculated using the following formula: Percentage of total cerebral infarction volume = (Sum of infarct areas of each brain slice ÷ Sum of total area of ​​each brain slice) × 100%. The effect of peptide NL-PT on cerebral infarction volume was quantitatively assessed.

[0049] ii) Neurological deficit behavioral assessment: At 24h, 48h, and 72h after reperfusion, the Longa and Bederson scoring methods were used to assess the neurological deficit behavior of mice in each group (the scoring was done in a blinded manner to avoid subjective bias). The scoring criteria are as follows (the higher the score, the more severe the neurological deficit in the mouse): 0 points: No neurological deficits, normal activity; 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 on its own, showing signs of loss of consciousness, near death or death.

[0050] The experimental results of the above-mentioned cerebral infarction volume measurement and neurological deficit scoring are as follows: Figure 1 As shown in Tables 1 and 2, the experimental data are expressed as mean ± standard deviation (x ± s). Statistical analysis was performed using SPSS software, and P < 0.05 was considered statistically significant.

[0051] The results are as follows Figure 1 As shown in Tables 1 and 2.

[0052] Table 1. Effect of peptide NL-PT on the percentage of cerebral ischemic infarct volume in I / R mice.

[0053] Table 2. Effects of peptide NL-PT on neurological deficit scores in mice 3 days after tMCAO / R model.

[0054] Experimental results showed that the infarct volume in mice with ischemic stroke was significantly reduced after administration. Figure 1 A、 Figure 1 .B), decreased neurological deficit behavioral score ( Figure 1 (C), demonstrating that the peptide NL-PT can significantly reduce neurological deficits and improve motor coordination in mice with ischemic stroke.

[0055] Step 4: Assessment of the effects of peptide NL-PT on brain 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 each inflammatory factor mRNA 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 NL-PT on the expression of inflammatory factors in the brain.

[0056] 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, allowed to stand at room temperature for 30 min, and then centrifuged at 4°C and 8000 rpm for 5 min. 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 NL-PT on oxidative stress levels in mice.

[0057] The results of the above-mentioned inflammatory factor mRNA expression detection and serum MDA detection are expressed as mean ± standard deviation (x ± s). Statistical analysis was performed using SPSS software. One-way ANOVA was used for inter-group comparisons, and P < 0.05 was considered statistically significant.

[0058] The results are as follows Figure 2 As shown in Tables 3 to 7.

[0059] Table 3. Effects of peptide NL-PT on IL-1β mRNA expression in the cerebral cortex of I / R mice.

[0060] Table 4. Effects of peptide NL-PT on IL-6 mRNA expression in the cerebral cortex of I / R mice.

[0061] Table 5. Effects of peptide NL-PT on TNF-α mRNA expression in the cerebral cortex of I / R mice.

[0062] Table 6. Effects of peptide NL-PT on iNOS mRNA expression in the cerebral cortex of I / R mice.

[0063] Table 7. Effects of peptide NL-PT on serum MDA levels in I / R mice

[0064] Experimental results showed that tMCAO / R modeling significantly upregulated the levels of pro-inflammatory factors in the mouse brain, and administration of peptide NL-PT increased the levels of pro-inflammatory factor IL-1β in the brains of ischemic stroke mice. Figure 2 .A), IL-6 ( Figure 2 .B), TNF-α ( Figure 2 .C), iNOS ( Figure 2 .D) and serum MDA ( Figure 2 The levels of inflammatory factors (E) were significantly reduced. This indicates that the peptide NL-PT can significantly reduce the levels of inflammatory factors and oxidative stress in the brains of mice with ischemic stroke, and enhance antioxidant capacity.

[0065] Example 3: Protective effect of peptide NL-PT on oxygen-glucose deprivation (OGD)-induced damage to HT22 neurons and hCMEC / D3 endothelial cells. HT22 mouse hippocampal neurons and hCMEC / D3 human brain microvascular endothelial cells were purchased from Wuhan Pronosai 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 Ecosai Biotechnology Co., Ltd. (catalog number: FSP500); and CCK-8 reagent was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (catalog number: C0039).

[0066] Step 1: Protective effect of peptide NL-PT on OGD-induced hippocampal neuronal cell 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. Polypeptide treatment group (NL-PT 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 polypeptide NL-PT (0.1 μg / mL, 0.5 μg / mL, 1.5 μg / mL). The medium was then cultured under the above OGD conditions for 10 h. Polypeptide NL-PT was prepared fresh 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.

[0067] 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 NL-PT on OGD-induced damage to human brain microvascular endothelial hCMEC / D3 cells. 1. Cell seeding and grouping Human brain microvascular endothelial cells (hCMEC / D3) in the 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 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. Polypeptide treatment group (NL-PT 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 polypeptide NL-PT (0.1 μg / mL, 0.5 μg / mL, 1.5 μg / mL). The medium was then cultured under the above OGD conditions for 6 h. Polypeptide NL-PT 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] The results are as follows Figure 3 As shown in Tables 8 and 9.

[0072] Table 8. Effect of peptide NL-PT on the survival rate of HT22 cells damaged by OGD for 10 h.

[0073] Table 9. Effect of peptide NL-PT on the survival rate of hCMEC / D3 cells damaged by OGD 6h

[0074] 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 NL-PT treatment can increase the survival rate of both cell types after OGD injury in a concentration-dependent manner (P<0.05, P<0.01, or P<0.001). Figure 3 A、 Figure 3 (B), demonstrating that it has a direct protective effect on neurons and cerebral vascular endothelial cells.

[0075] Example 4: Protective effect of peptide NL-PT 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.

[0076] Step 1: Protective effect of peptide NL-PT 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 (NL-PT 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 NL-PT (0.5μg / mL, 2μg / mL, 10μg / mL). Peptide NL-PT 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.

[0077] 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 NL-PT on 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. Polypeptide treatment group (NL-PT 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 NL-PT (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.

[0078] 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.

[0079] 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.

[0080] Concentration gradient design basis: for two different damage models (CORT and MPP) + ), and appropriate peptide NL-PT 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 NL-PT to exert its neuroprotective effect.

[0081] 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.

[0082] The entire experimental procedure was performed in strict accordance with aseptic techniques to avoid contamination that could affect the experimental results. The results are as follows: Figure 4 As shown in Tables 10 and 11.

[0083] Table 10 Effect of peptide NL-PT on the survival rate of PC12 cells damaged by 250 μM CORT

[0084] Table 11. Effects of peptide NL-PT on 4mM MPP + Effect of damage on PC12 cell survival

[0085] 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 NL-PT significantly antagonized CORT and MPP. + The decrease in cell viability was caused in a concentration-dependent manner (P<0.01 or P<0.001). Figure 4 A, Figure 4 (B), suggesting that this polypeptide has a protective effect against both hormone stress injury and neurotoxic injury.

[0086] Example 5 Effect of peptide NL-PT 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.

[0087] 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.

[0088] 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+NL-PT 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.

[0089] ii) ROS detection After 24 hours of CORT treatment, carefully discard the culture medium from each well (avoiding scratching of 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 to 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.

[0090] 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).

[0091] 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.

[0092] 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 NL-PT on CORT-induced intracellular ROS levels in PC12 cells.

[0093] Experimental results are as follows Figure 5 As shown in Table 12.

[0094] Table 12 Effect of peptide NL-PT on CORT-induced ROS levels in PC12 cells

[0095] 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. Quantitative results from the microplate reader were consistent with the imaging results: compared with the control group, the ROS level in the CORT model group was significantly increased (P<0.0001); while compared with the CORT model group, the peptide NL-PT significantly reduced the ROS level (P<0.01). Figure 5 A, Figure 5 (B). This result confirms that the peptide NL-PT can effectively alleviate CORT-induced oxidative stress in PC12 cells.

[0096] Comparative Example 1 Design and synthesis of control peptide EP-DL: To verify the specific efficacy of the polypeptide NL-PT (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.

[0097] 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 NL-PT of this invention.

[0098] 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.

[0099] 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%.

[0100] The control peptide EP-DL was subjected to behavioral, infarct area, brain inflammatory factor levels, and protective OGD-conditioning HT22 cell and hCMEC / D3 assays using the same methods as peptide NL-PT. The results are as follows: Figure 6 As shown in Tables 13 to 21.

[0101] Table 13 Effects of EP-DL peptides on body weight in I / R mice

[0102] Table 14 Effects of EP-DL peptides on neurological deficit scores in I / R mice

[0103] Table 15 Effects of EP-DL peptide on cerebral infarction volume in tMCAO / R mice

[0104] Table 16 Effect of control peptide EP-DL on serum MDA levels in I / R mice

[0105] Table 17 Effects of EP-DL peptides on TNF-α levels in the cerebral cortex of I / R mice

[0106] Table 18 Effects of EP-DL peptides on IL-6 levels in the cerebral cortex of I / R mice

[0107] Table 19 Effects of EP-DL peptides on IL-1β levels in the cerebral cortex of I / R mice

[0108] Table 20 Effect of EP-DL peptide on the survival rate of HT22 cells damaged by OGD for 10 h (%)

[0109] Table 21 Effect of EP-DL peptide on the survival rate of hCMEC / D3 cells damaged by OGD for 6 h (%)

[0110] 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).

[0111] 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 NL-PT peptides is specific and not a non-specific effect of random peptides.

[0112] 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. A polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The polypeptide 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. A polypeptide derivative, 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 polypeptide derivative according to claim 3, characterized in that, The protein purification tag or detection tag is a His tag or a FLAG tag.

5. The use of the polypeptide of claim 1 or the polypeptide derivative of claim 3 in the preparation of a medicament for the prevention and / or treatment of neurological injury-related diseases.

6. The application according to claim 5, characterized in that, The neurological injury-related disease is ischemic cerebrovascular disease.

7. The application according to claim 6, characterized in that, The ischemic cerebrovascular disease mentioned is ischemic stroke.

8. A pharmaceutical composition, characterized in that, The product comprises a therapeutically effective amount of the polypeptide of claim 1 or the polypeptide derivative of claim 3, and a pharmaceutically acceptable carrier.

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