Use of plasmin inhibitor NKI10 in preparation of drugs for preventing and treating cerebral ischemia-reperfusion injury

Abstract

The application discloses application of a plasmin inhibitor NKI10 in preparation of a medicine for preventing and treating cerebral ischemia-reperfusion injury, and belongs to the technical field of biological medicine. The application discloses application of the plasmin inhibitor NKI10 in preparation of the medicine for preventing and treating cerebral ischemia-reperfusion injury, and the amino acid sequence of the plasmin inhibitor NKI10 is shown in any one of SEQ ID NO. 1-10. The NKI10 can significantly improve the nerve function of a cerebral ischemia-reperfusion mouse, reduce the cerebral infarction volume and nerve cell necrosis of the mouse, namely, has a significant protective effect on cerebral ischemia-reperfusion injury. The NKI10 can be used for preventing and treating cerebral ischemia-reperfusion injury.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and more particularly to application of a plasmin inhibitor NKI10 in preparation of a medicine for preventing and treating cerebral ischemia-reperfusion injury. BACKGROUND

[0002] Stroke, commonly known as apoplexy, is one of the major diseases threatening people's health. Stroke has the characteristics of high incidence, high recurrence rate, high disability rate, high mortality rate and heavy economic burden. Because of its rapid onset and rapid progression, it can cause limb paralysis, dysphagia, cognitive impairment and other symptoms, which seriously threatens the life and health of residents and the quality of life. Ischemic stroke (IS), also known as cerebral infarction, refers to the stenosis or occlusion of the cerebral blood vessels, leading to ischemia and necrosis of the brain tissue, and loss of corresponding brain function. According to the data of the China Guidelines for Diagnosis and Treatment of Acute Ischemic Stroke 2023, acute ischemic stroke accounts for about 69.6%-72.8% of all stroke cases. Recanalization of blood vessels is the preferred method for treating ischemic stroke. The best strategy for treating IS in the clinic is to use drug thrombolysis and interventional thrombectomy to restore blood flow. However, blood flow restoration can further aggravate brain injury and worsen clinical symptoms, which is known as cerebral ischemia-reperfusion injury (CIRI). The pathophysiological mechanism of CIRI is very complex, and some drugs for improving brain injury are currently used to alleviate the symptoms of IS patients, including excitatory amino acid modulating drugs, free radical scavengers, neurotrophic factors, calcium ion antagonists, nitric oxide synthase inhibitors, and anti-apoptotic drugs. There is a lack of specific drugs for preventing and treating CIRI, so it is urgent to develop new drugs for preventing and treating CIRI.

[0003] As mentioned above, recanalization of blood vessels is the main method for treating IS, and the use of alteplase (t-PA) for thrombolysis to recanalize blood vessels is the main treatment for IS. Alteplase can directly activate plasminogen to convert it into plasmin, thereby dissolving thrombus, and is a commonly used fibrinolytic drug in the clinic. NKI10 discovered in the previous study of the application is a highly efficient and specific plasmin inhibitor, which is expected to be used as an antifibrinolytic drug. The antifibrinolytic effect of NKI10 is obviously different from the function of fibrinolytic drugs used in the treatment of IS: t-PA is a fibrinolytic drug, while NKI10 is an antifibrinolytic drug. However, we were surprised to find that after ischemia-reperfusion in a mouse model of transient middle cerebral artery occlusion (tMCAO), administration of NKI10 showed a significant protective effect on CIRI, which could significantly improve brain tissue injury in cerebral ischemia-reperfusion in mice. The research results of the application show that NKI10 can be applied to prevent and treat CIRI.

[0004] Therefore, the application of the plasmin inhibitor NKI10 in the preparation of a drug for preventing and treating cerebral ischemia-reperfusion injury is an urgent problem for those skilled in the art to solve. SUMMARY

[0005] Therefore, the application of the plasmin inhibitor NKI10 in the preparation of a drug for preventing and treating cerebral ischemia-reperfusion injury is an urgent problem for those skilled in the art to solve.

[0006] The present application finds that the plasmin inhibitor NKI10 has a significant protective effect on cerebral ischemia-reperfusion injury (CIRI) in a mouse model of transient middle cerebral artery occlusion (tMCAO), indicating that NKI10 can be used as a drug for preventing and treating cerebral ischemia-reperfusion injury in clinical applications.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] The application of the plasmin inhibitor NKI10 in the preparation of a drug for preventing and treating cerebral ischemia-reperfusion injury, wherein the amino acid sequence of the plasmin inhibitor NKI10 is shown in any one of SEQ ID NO. 1-10.

[0009] Further, a drug preparation for preventing and treating cerebral ischemia-reperfusion injury, wherein the plasmin inhibitor NKI10 is directly used as an active ingredient or is formulated with a pharmaceutically acceptable carrier; and the amino acid sequence of the plasmin inhibitor NKI10 is shown in any one of SEQ ID NO. 1-10.

[0010] SEQ ID NO. 1 amino acid sequence (NKI10-1):

[0011] GHLCNGDLLPGPCRAKMERWGLDKESGKCKKFIYGGCGGNRNNFES EEEKCKKRCKVD; SEQ ID NO. 1.

[0012] Gene sequence encoding SEQ ID NO. 1 (NKI10-1):

[0013] ggtcatctatgcaatggggatttactaccaggaccttgcagagctaaaatggaaagatggggattggataagga atcaggaaagtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaa gaaacgttgcaaagtggattga (containing stop codon tga); SEQ ID NO. 11.

[0014] SEQ ID NO. 2 amino acid sequence (NKI10-2):

[0015] MGMKGSGHLCNGDLLPGPCRAKMERWGLDKESGKCKKFIYGGCGG NRNNFESEEKCKKRCKVD; SEQ ID NO. 2.

[0016] Gene sequence encoding SEQ ID NO. 2 (NKI10-2):

[0017] atgggaatgaagggaagtggtcatctatgcaatggggatttactaccaggaccttgcagagctaaaatggaaa gatggggattggataaggaatcaggaaagtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttg aaagtgaagagaaatgcaagaaacgttgcaaagtggattga (with stop codon tga); SEQ ID NO. 12.

[0018] SEQ ID NO. 3 amino acid sequence (NKI10-3):

[0019] CNGDLLPGPCRAKMERWGLDKESGKCKKFIYGGCGGNRNNFESEEK CKKRC; SEQ ID NO. 3.

[0020] Gene sequence encoding SEQ ID NO. 3 (NKI10-3):

[0021] tgcaatggggatttactaccaggaccttgcagagctaaaatggaaagatggggattggataaggaatcaggaa agtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaagaaacgtt gc; SEQ ID NO. 13.

[0022] SEQ ID NO. 4 amino acid sequence (NKI10-4):

[0023] GHLCNGDLLPGPCKAKMERWGLDKESGKCKKFIYGGCGGNRNNFES EEKCKKRCKVD; SEQ ID NO. 4.

[0024] Gene sequence encoding SEQ ID NO. 4 (NKI10-4):

[0025] ggtcatctatgcaatggggatttactaccaggaccttgcaaagctaaaatggaaagatggggattggataagga atcaggaaagtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaa gaaacgttgcaaagtggattga (with stop codon tga); SEQ ID NO. 14.

[0026] Amino acid sequence of SEQ ID NO. 5 (NKI10-5):

[0027] GHLCNGDLLPGPCKARMERWGLDKESGKCKKFIYGGCGGNRNNFES EEKCKKRCKVD; SEQ ID NO. 5.

[0028] Gene sequence encoding SEQ ID NO. 5 (NKI10-5):

[0029] ggtcatctatgcaatggggatttactaccaggaccttgcaaagctagaatggaaagatggggattggataagga atcaggaaagtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaa gaaacgttgcaaagtggattga (with stop codon tga); SEQ ID NO. 15.

[0030] Amino acid sequence of SEQ ID NO. 6 (NKI10-6):

[0031] GHLCNGDLLPGPCRARMERWGLDKESGKCKKFIYGGCGGNRNNFES EEKCKKRCKVD; SEQ ID NO. 6.

[0032] Gene sequence encoding SEQ ID NO. 6 (NKI10-6):

[0033] Ggtcatctatgcaatggggatttactaccaggaccttgcagagctagaatggaaagatggggattggataaggaatcaggaaagtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaagaaacgttgcaaagtggattga (with stop codon tga); SEQ ID NO. 16.

[0034] SEQ ID NO. 7 amino acid sequence (NKI10-7):

[0035] CNGDLLPGPCKAKMERWGLDKESGKCKKFIYGGCGGNRNNFESEEK CKKRC; SEQ ID NO. 7.

[0036] Gene sequence encoding SEQ ID NO. 7 (NKI10-7):

[0037] Ggtcatctatgcaatggggatttactaccaggaccttgcagagctagaatggaaagatggggattggataaggaatcaggaaagtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaagaaacgttgcaaagtggattga (with stop codon tga); SEQ ID NO. 16.

[0038] SEQ ID NO. 8 amino acid sequence (NKI10-8):

[0039] CNGDLLPGPCKARMERWGLDKESGKCKKFIYGGCGGNRNNFESEEK CKKRC; SEQ ID NO. 8.

[0040] Gene sequence encoding SEQ ID NO. 8 (NKI10-8):

[0041] Ggtcatctatgcaatggggatttactaccaggaccttgcagagctagaatggaaagatggggattggataaggaatcaggaaagtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaagaaacgttgcaaagtggattga (with stop codon tga); SEQ ID NO. 16.

[0042] SEQ ID NO. 9 amino acid sequence (NKI10-9):

[0043] CNGDLLPGPCRARMERWGLDKESGKCKKFIYGGCGGNRNNFESEEK CKKRC; SEQ ID NO. 9.

[0044] Gene sequence encoding SEQ ID NO. 9 (NKI10-9):

[0045] tgcaatggggatttactaccaggaccttgcagagctagaatggaaagatggggattggataaggaatcaggaa agtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaagaaacgtt gc; SEQ ID NO. 19.

[0046] SEQ ID NO. 10 amino acid sequence (NKI10-10):

[0047] GHLCNGDLLPGPCRVKMERWGLDKESGKCKKFIYGGCGGNRNNFES EEKCKKRCKVD; SEQ ID NO. 10.

[0048] Gene sequence encoding SEQ ID NO. 10 (NKI10-10):

[0049] ggtcatctatgcaatggggatttactaccaggaccttgcagagttaaaatggaaagatggggattggataagga atcaggaaagtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaa gaaacgttgcaaagtggattga (containing stop codon tga); SEQ ID NO. 20.

[0050] According to the technical solution, compared with the prior art, the application provides the application of the plasmin inhibitor NKI10 in the preparation of a medicine for preventing and treating cerebral ischemia-reperfusion injury. The tMCAO mouse model is used, and NKI10 can significantly improve the neurological function of a cerebral ischemia-reperfusion mouse, reduce the cerebral infarction volume and nerve cell necrosis of the mouse, that is, has a significant protective effect on cerebral ischemia-reperfusion injury. BRIEF DESCRIPTION OF DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings required by the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only are the embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative effort based on the provided drawings.

[0052] Figure 1 The brain coronal section (TTC staining) of rNKI10 to tMCAO mice , n≥6) results; A: brain coronal section of mice, B: cerebral infarction volume (%);

[0053] The brain of the sham operation group mice did not appear infarction; the brain of the model group mice appeared a large range of obvious infarction; the cerebral infarction range of the rNKI10-1 low dose group (1.00 mg / kg, rNKI10-1-L group), the rNKI10-1 middle dose group (2.00 mg / kg, rNKI10-1-M group), the rNKI10-1 high dose group (4.00 mg / kg, rNKI10-1-H group), the rNKI10-2 group (4.00 mg / kg, rNKI10-2-H group), the rNKI10-3 group (4.00 mg / kg, rNKI10-3-H group), the rNKI10-4 group (4.00 mg / kg, rNKI10-4-H group), the rNKI10-5 group (4.00 mg / kg, rNKI10-5-H group), the rNKI10-6 group (4.00 mg / kg, rNKI10-6-H group), the rNKI10-7 group (4.00 mg / kg, rNKI10-7-H group), the rNKI10-8 group (4.00 mg / kg, rNKI10-8-H group), the rNKI10-9 group (4.00 mg / kg, rNKI10-9-H group) and the rNKI10-10 group (4.00 mg / kg, rNKI10-10-H group) appeared different degrees of reduction (P<0.001), thereby indicating that rNKI10 can effectively reduce the cerebral infarction rate; within a certain range, with the increase of the dose, the cerebral infarction rate decreases ** P<0.01, compared with the sham operation group; ## P<0.01, compared with the model group; && P<0.01, compared with the NKI10-1-L group; ^^ P<0.01, compared with the NKI10-1-M group; $$ P<0.01, compared with the NKI10-1-H group); the white part in the figure is the cerebral infarction part; tranexamic acid (100.00 mg / kg, TXA);

[0054] Figure 2Pathological observation of NKI10's effect on the hippocampal structure in mice (Hippocampal region HE staining, observation of the whole hippocampus under a microscope: 40×; dentate gyrus, CA1 region, CA2 region, CA3 region: 200×);

[0055] In the sham-operated group, hippocampal neurons in mice were neatly arranged, regularly shaped, with intact cell membranes and clearly visible nuclei. In the model group, numerous neuronal condensation, vacuolization, and necrosis were observed in the hippocampus (dentate gyrus, CA1, CA2, and CA3 regions). Hippocampal neurons in mice treated with low (1.00 mg / kg, NKI10-1-L), medium (2.00 mg / kg, NKI10-1-M), and high (4.00 mg / kg, NKI10-1-H) NKI10-1 showed varying degrees of improvement, with significantly reduced neuronal necrosis, more neat arrangement, and more regular morphology. Detailed Implementation

[0056] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0057] Example 1: Protective effect of NKI10 on CIRI in mice

[0058] 1) Main reagents

[0059] Tranexamic acid (TXA) is a product of Sigma-Aldrich.

[0060] Preparation of recombinant NKI10 (rNKI10): The method for preparing the crude product refers to Chinese Patent Application No. 201810505110.1, followed by purification. ① Preparation of crude product: Taking the expression of the amino acid sequence shown in SEQ ID NO.1 (NKI10-1) as an example, in short: design upstream primer NI10-1e and downstream primer NI10-2e for recombinant expression encoding, using the gene sequence encoding the amino acid sequence of SEQ ID NO.1 (SEQ ID NO.11) as a template, to amplify the gene sequence encoding the amino acid sequence shown in SEQ ID NO.1. Amplification system: ddH2O 38μL, dNTP (2mmol / L) 5.0μL, 10×PCR buffer (containing Mg) 2+)5.0 μL, 0.6 μL of each of the upstream and downstream primers (20 μmol / L), 0.5 μL of the template, and 0.3 μL of Pfu DNA polymerase (5 U / μL). The reaction conditions were 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, for a total of 30 cycles. The obtained gene sequence was ligated into a prokaryotic expression vector pET32a-sumo, and the correct recombinant plasmid was transformed into E. coli BL21 (DE3). The host bacteria were induced to express by IPTG, and the host bacteria were separated and ultrasonically broken. The expression product was purified by nickel affinity chromatography to obtain the fusion protein. The fusion partner was cleaved by SUMO protease, and the fusion partner was further removed by affinity chromatography to obtain the crude product of recombinant NKI10-1.

[0061] The amino acid sequences shown in SEQ ID NO. 2 (NKI10-2), SEQ ID NO. 3 (NKI10-3), SEQ ID NO. 4 (NKI10-4), SEQ ID NO. 5 (NKI10-5), SEQ ID NO. 6 (NKI10-6), SEQ ID NO. 7 (NKI10-7), SEQ ID NO. 8 (NKI10-8), SEQ ID NO. 9 (NKI10-9), and SEQ ID NO. 10 (NKI10-10) were prepared according to the above method. The corresponding coding sequences (such as those shown in SEQ ID NO. 12-20) were used as templates, and the primers shown in Table 1 were used to amplify the coding genes.

[0062] Table 1 Primers used for amplifying the NKI10 coding sequence

[0063]

[0064]

[0065] wherein the amplification of the coding sequence of SEQ ID NO. 2 (NKI10-2) uses NI10-3e paired with NI10-2e; the amplification of the coding sequence of SEQ ID NO. 4 (NKI10-4), SEQ ID NO. 5 (NKI10-5), SEQ ID NO. 6 (NKI10-6) and SEQ ID NO. 10 (NKI10-10) uses NI10-1e paired with NI10-2e; the amplification of the coding sequence of SEQ ID NO. 3 (NKI10-3), SEQ ID NO. 7 (NKI10-7), SEQ ID NO. 8 (NKI10-8), SEQ ID NO. 9 (NKI10-9) uses NI10-5e paired with NI10-6e.

[0066] The concentration of NKI10 prepared above is all > 2.0 mg / mL. Before use, configure on ice to the concentration of administration.

[0067] 2) Animals

[0068] A total of 114 SPF level 8-week-old C57BL / 6J male wild-type mice weighing 18-22 g were selected, purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd., and the qualified certificate was SCXK (Yue) 2022-0063. Before the experiment, the mice were fed in the SPF level environment of the Experimental Animal Center of Guangdong Medical University for 2 weeks to adapt to the environment. The experimental scheme was approved by the Animal Experiment Ethics Committee of Guangdong Medical University, and the approval number was GDMU-2024-000008.

[0069] 3) Modeling

[0070] The mouse body temperature was maintained at 36.5-37.5℃, and a middle cerebral artery transient ischemia model (tMCAO) was formed by intravascular insertion of a thread plug. After anesthesia with 1.5% isoflurane, the mouse was fixed on the operating table in a supine position, the neck fur was cut off, the neck skin was disinfected with 75% alcohol and iodophor disinfectant in turn, about 0.5 cm of the left common carotid artery was exposed, the external jugular vein was separated and ligated, a slipknot was made on the upper end of the common carotid artery, and a dead knot was made on the lower end. Under a body microscope, a small opening was cut on the common carotid artery near the dead knot end with microscissors, and a thread plug was inserted into the small opening. The slipknot was opened, and the middle cerebral artery was entered through the internal jugular vein. The neck skin was sutured, the wound was disinfected with iodophor disinfectant, and ischemia was performed for 30 min. The sham operation group did not insert the thread plug, and the other steps were the same as above. During the experiment, the mice were given normal diet, and the conditions of the mice in each group were observed. All mice and drug preparation were randomly assigned to the operator by an independent person who did not participate in animal experiments, data collection and analysis. The following conditions of tMCAO mice were not included in the test drug group and model group (exclusion criteria): ① The mouse died within 24 h after tMCAO; ② Subarachnoid hemorrhage (SAH) or intracerebral hemorrhage (ICH) (such as macroscopic evaluation when brain sampling); ③ Postoperative neurological behavior score score = 0 or 4 (30 min after tMCAO).

[0071] 4) Grouping and administration

[0072] The experimental mice were randomly divided into 15 groups, namely: sham operation group (10), model group (10), tranexamic acid control group (100.00 mg / kg, TXA, 10), rNKI10-1-L group (1.00 mg / kg, 10), rNKI10-1-M group (2.00 mg / kg, 10), rNKI10-1-H group (4.00 mg / kg, 10), rNKI10-2-H group (4.00 mg / kg, 6), rNKI10-3-H group (4.00 mg / kg, 6), rNKI10-4-H group (4.00 mg / kg, 6), rNKI10-5-H group (4.00 mg / kg, 6), rNKI10-6-H group (4.00 mg / kg, 6), rNKI10-7-H group (4.00 mg / kg, 6), rNKI10-8-H group (4.00 mg / kg, 6), rNKI10-9-H group (4.00 mg / kg, 6), and rNKI10-10-H group (4.00 mg / kg, 6). Double-blind drug administration was used. Sham operation group: ligate the common carotid artery without inserting the thread plug. After 30 min of local cerebral ischemia, physiological saline (0.9% NaCl) was injected through the tail vein at a dose of 10 mL / kg. Model group: insert the thread plug from the common carotid artery and enter the middle cerebral artery through the internal carotid artery, remove the thread plug to restore blood flow after 30 min of local cerebral ischemia, then inject an equal amount of physiological saline (0.9% NaCl) through the tail vein at a dose of 10 mL / kg, and reperfuse for 23.5 h. TXA group: insert the thread plug from the common carotid artery and enter the middle cerebral artery through the internal carotid artery, remove the thread plug to restore blood flow after 30 min of local cerebral ischemia, then inject an equal volume of TXA (100.00 mg / kg) through the tail vein, and reperfuse for 23.5 h. Protein rNKI10 administration group: insert the thread plug from the common carotid artery and enter the middle cerebral artery through the internal carotid artery, remove the thread plug to restore blood flow after 30 min of local cerebral ischemia, then inject the corresponding dose of drug through the tail vein, and reperfuse for 23.5 h. After the operation, the mice were closely observed for postoperative conditions, and their hair, mental state, respiratory rate, and body movement were recorded.

[0073] 5) Sampling

[0074] After 24 h of mouse modeling, the behavioral changes were evaluated according to the 5-point method of Zea-Longa, and then the mice were anesthetized with isoflurane, the eyeballs were removed, and the blood was collected. The collected blood samples were placed in 1.5 mL anticoagulant EP tubes and rested in a 4°C refrigerator for 2 h. After the upper plasma was separated, the 1.5 mL anticoagulant tube was placed in a 4°C low-temperature centrifuge, centrifuged at 3500 r / min for 15 min, and the plasma was separated. After cervical dislocation, the brain was removed, and 3 mice from each group were taken for TTC staining to observe the cerebral infarction.

[0075] 6) Animal neurobehavioral evaluation

[0076] Neurological function of each group of animals was evaluated using the Zea-Longa scoring system (Table 2). Neurological function scores were assessed for each group of animals before sacrifice.

[0077] Table 2. Zea-Longa scoring criteria

[0078]

[0079] Note: Levels 0 and IV indicate model failure, while Levels I, II, and III indicate model success.

[0080] 7) Calculation of infarction rate [percentage of infarct volume; infarct volume (%)]:

[0081] Mice were euthanized by cervical dislocation, and their brains were harvested. The olfactory bulb, cerebellum, and lower brainstem were removed. The remaining brain tissue was coronally sliced ​​into five sections, each 2 mm thick, and completely immersed in 0.5% TTC staining solution. The sections were stained at 37°C in the dark for 30 minutes. After staining, the brain slices were fixed in 10% formaldehyde solution for 3 hours and then scanned to obtain images, which were then saved. Normal tissue appeared red after staining, while infarcted tissue appeared white. The infarct volume was calculated using Image Tool 3.0. The infarct rate was calculated using the following formula:

[0082] Infarction rate = infarct volume / total volume x 100%.

[0083] 8) Pathological examination of the hippocampus

[0084] ① Fixation: Brain tissue soaked in 10% formaldehyde solution for more than 24 hours is removed. The olfactory bulb, cerebellum, and lower brainstem are removed. The remaining tissue is coronally cut into 5 sections using a scalpel. The hippocampus is selected and placed in an embedding cassette, and marked. ② Dehydration: Brain tissue is sequentially soaked in double-distilled water for 2 hours, 70% alcohol for 2 hours, 95% alcohol for 3 hours, and 100% alcohol for 1 hour. ③ Clearing: The tissue is soaked in xylene for 1 minute. ④ Paraffin Infiltration: The tissue is placed in a paraffin bath at 60℃ for 2-3 hours. ⑤ Embedding: The paraffin-infiltrated tissue is placed in an embedding cassette containing paraffin solution, and the paraffin solution is injected to form a paraffin block. ⑥ Sectioning: After the paraffin solidifies, continuous sections (5μm thick) are made. The sections are naturally flattened in 41℃ warm water, separated, and transferred to glass slides. They are then dried in a constant temperature oven at 60℃ for 30 minutes and cooled for later use. ⑦ Dewaxing: Immerse paraffin sections sequentially in xylene I for 10 min, xylene II for 10 min, 100% ethanol for 5 min, 75% ethanol for 5 min, and tap water for 5 min. ⑧ HE staining: Stain with hematoxylin for 5 min, rinse repeatedly with tap water 3-4 times, stain with eosin for 10 s, and rinse with tap water. ⑨ Mounting: After air-drying at room temperature, add neutral resin, then cover with a coverslip for microscopic observation.

[0085] 9) Statistical methods

[0086] Data were expressed as mean ± SD and analyzed by SPSS 17.0 software. The LSD least significant difference method of one-way ANOVA was used for difference test among multiple groups. P < 0.05 was considered statistically significant.

[0087] 10) Results

[0088] The Zea-Longa score results of mice after 24 hours of cerebral ischemia reperfusion are shown in Table 3.

[0089] Table 3 Neurological function score of mice in each group n≥6

[0090]

[0091] Note: *** P < 0.001 compared with the sham operation group; ## P < 0.01, ### P < 0.001 compared with the model group. The sham operation group and the model group were injected with the same volume of 0.9% NaCl.

[0092] Table 3 shows that compared with the sham operation group, the neurological function score of mice in the model group increased (P < 0.001), indicating that the tMCAO model was successfully established. Compared with the model group, the neurological function score of mice in the TXA group and the rNKI10 administration group decreased (P < 0.01), indicating that rNKI10 significantly improved the neurological state and behavior of mice after local cerebral ischemia reperfusion.

[0093] The TTC staining results of brain slices of mice in each group after 24 hours of modeling are shown in Figure 1 A: The brain of the mouse in the sham operation group did not appear infarction; the brain of the mouse in the model group appeared a large range of obvious infarction. The brain infarction volume percentage was calculated using Image Tool 3.0. Figure 1 B: The statistical results of brain infarction volume (%) of mice in each group. Compared with the sham operation group (blank control group), the brain infarction volume of mice in the model group increased significantly (P < 0.001). Compared with the model group, the brain infarction volume of mice in the rNKI10 administration group decreased significantly (P < 0.001). Compared with the rNKI10-1-L group, the brain infarction volume (%) of mice in the rNKI10-1-M group and the rNKI10-1-H group decreased significantly (P < 0.001). Compared with the rNKI10-1-M group and the rNKI10-H group respectively, the brain infarction volume (%) of mice in the TXA group increased significantly (P < 0.001).

[0094] HE staining results of the hippocampus structure are as follows Figure 2 As shown: In the sham-operated group, the hippocampal neurons of the mice were neatly arranged, regularly shaped, with intact cell membranes and clearly visible nuclei. In the model group, numerous neuronal condensation, vacuolization, and necrosis were observed in the hippocampus (dentate gyrus, CA1 region, CA2 region, and CA3 region) of the mice. The hippocampus of mice in the low, medium, and high rNKI10-1 administration groups showed varying degrees of improvement, with significantly reduced neuronal necrosis, more neat arrangement, and more regular morphology.

[0095] 12) Conclusion

[0096] NKI10 can significantly improve the neurological function of mice with cerebral ischemia-reperfusion injury, reduce the volume of cerebral infarction and neuronal necrosis, and has a significant protective effect against CIRI.

[0097] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of the plasmin inhibitor NKI 10 for the preparation of a medicament for the prevention and treatment of cerebral ischemia-reperfusion injury, characterized in that, The amino acid sequence of the plasmin inhibitor NKI10 is shown in any one of SEQ ID NO. 1~10. The amino acid sequence of the plasmin inhibitor NKI10 is shown in any one of SEQ ID NO. 1~10.

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