Application of CKB K11 lactic acid modification as cerebral apoplexy I / R injury diagnosis or neuroprotection drug target

Abstract

The invention discloses application of CKB K11 lactic acid modification as a cerebral apoplexy I / R injury diagnosis or neuroprotection drug target, and belongs to the technical field of biological medicine. The CKB K11 lactic acid modification provided by the invention can obviously improve the cerebral apoplexy ischemia reperfusion injury and motor function, and the CKB K11 lactic acid modification is closely related to the cerebral apoplexy ischemia reperfusion injury and can be used as a specific diagnosis index of the cerebral apoplexy ischemia reperfusion injury. By detecting the CKB K11 lactic acid modification level in a biological sample, the brain tissue damage degree and the postoperative recovery condition of a detected object can be rapidly and accurately evaluated, and a new effective means is provided for clinical diagnosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of CKB K11 lactation modification as a target for diagnosis of stroke I / R (i.e., ischemia-reperfusion) injury or neuroprotective drugs. Background Technology

[0002] Ischemic stroke is a leading cause of long-term neurological dysfunction and death worldwide, characterized by sudden occlusion of cerebral blood vessels, leading to localized cerebral ischemia, metabolic failure, and neuronal loss. Ischemic stroke accounts for approximately 87% of all stroke cases, with its incidence continuing to rise, particularly among the elderly. The primary treatment for ischemic stroke is reperfusion therapy via thrombolysis or mechanical thrombectomy to restore cerebral blood flow, but this can also lead to ischemia-reperfusion (I / R) injury. I / R pathological injury results from a cascade of reactions triggered by oxidative stress, mitochondrial dysfunction, calcium overload, lipid peroxidation, and neuroinflammation following thrombolysis or mechanical thrombectomy. Neurons, with their high energy demands and limited energy reserves, are highly susceptible to the effects of blood flow obstruction and energy supply deficits. Therefore, maintaining neuronal metabolic homeostasis during I / R is a crucial issue that needs to be addressed in stroke treatment.

[0003] Lactic acid, a byproduct of glycolysis, serves as both an energy substrate and a metabolic signaling agent. In the central nervous system, lactate is actively produced by astrocytes and transported to neurons as an alternative fuel under energy stress. However, its role in stroke remains controversial. On the one hand, exogenous lactate regulation has been shown to improve hypoxic-ischemic injury; on the other hand, excessive intracellular lactate accumulation during ischemia may exacerbate acidosis and neuronal damage. Recent studies have shown that lactate also participates in epigenetic and post-translational regulation through a newly discovered lysine lactation modification. In this modification, a lactyl group is covalently attached to a lysine residue, which has been shown to regulate histone assembly, gene transcription activity, and protein function. Under ischemic conditions, lactation is associated with the regulation of neuronal apoptosis, glycolysis, inflammation, and mitochondrial translocation, highlighting its multifaceted role in brain injury.

[0004] The common pathological mechanism of ischemia-reperfusion injury in stroke is nerve damage. However, there are currently no neuroprotective agents with significant clinical efficacy, and there has been no breakthrough in the treatment of ischemia-reperfusion injury in stroke.

[0005] Creatine kinase (CK) is widely distributed in various organs and exhibits tissue and cell specificity. There are four isoenzyme forms of creatine kinase: muscle creatine kinase (CKM), brain creatine kinase (CKB), mitochondrial creatine kinase 1 (CKMT1), and mitochondrial creatine kinase 2 (CKMT2). ​​CKB is highly expressed in the central nervous system and is a key component of the creatine-phosphocreatine (PCr) shuttle system, which buffers intracellular ATP levels. CKB rapidly rephosphorylates ADP to ATP via PCr, thereby stabilizing energy supply during acute stress. CKB is present in the cytoplasm and mitochondria; only a very small amount of CKB crosses the cell membrane. Therefore, serum creatine kinase (sCK) levels are low and relatively stable under normal physiological conditions. Elevated serum CKB levels often indicate encephalitis or brain injury, but current detection methods can only measure the relative level of serum CKB and do not detect the kinase activity of serum CKB. Summary of the Invention

[0006] Based on lactation-modified proteomics sequencing data, this application found that CKB K11 was significantly upregulated in the brain tissue of a mouse model of stroke ischemia-reperfusion, accompanied by an increase in CKB kinase activity. Further investigation showed that CKBK11 lactation modification increased CKB kinase activity, reduced oxidative stress, reactive oxygen species and lipid peroxidation levels in neurons, reduced oxidative damage and apoptosis in neurons in brain tissue, and improved motor function in mice with stroke ischemia-reperfusion.

[0007] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide an application of CKB K11 lactation modification as a diagnostic tool for ischemia-reperfusion injury in stroke or as a target for neuroprotective drugs, thereby solving the technical problem of the lack of effective diagnostic indicators and neuroprotective drugs with significant therapeutic effects for ischemia-reperfusion injury in the prior art.

[0008] To achieve the above-mentioned technical objectives, the present invention provides an application of CKB K11 lactation modification as a diagnostic tool for ischemia-reperfusion injury in stroke or as a target for neuroprotective drugs.

[0009] This invention also proposes the application of genes or proteins with enhanced CKB K11 lactation modification in the preparation of neuroprotective drugs.

[0010] In any embodiment, the neuroprotective drug is a neuroprotective drug for stroke ischemia-reperfusion injury.

[0011] This invention also proposes the application of CKB K11 lactation modification in the preparation of diagnostic reagents for stroke ischemia-reperfusion injury or in the screening of neuroprotective drugs.

[0012] In any embodiment, the diagnostic reagent assesses the degree of ischemia-reperfusion injury or postoperative recovery of the test subject by detecting the level of CKB K11 lactation modification in a biological sample; the biological sample includes cerebrospinal fluid, serum, or brain tissue samples; the diagnostic reagent contains antibodies, probes, or primers for specifically detecting CKB K11 lactation modification; and / or, the screening method includes the following steps:

[0013] S1. Construct an OGD / R cell model or an MCAO / R biomodel;

[0014] S2, administer candidate drugs to the model;

[0015] S3. Detect at least one of the following indicators in the detection model: CKB K11 lactation modification level, CKB kinase activity, neuronal cell viability, oxidative stress level, mitochondrial function, cerebral infarction area, neuronal apoptosis level, or motor function; if the candidate drug can improve the CKB K11 lactation modification level and / or CKB kinase activity, and improve at least one of the above indicators, it is judged as a potential neuroprotective drug.

[0016] In addition, the present invention also proposes a neuroprotective drug, the active ingredient of which is a substance capable of increasing the lactation modification level of CKB K11; said substance includes a recombinant vector encoding wild-type CKB protein, a recombinant virus, or a CKB K11 lactation modification activator.

[0017] In any embodiment, the backbone of the recombinant vector encoding the CKB wild-type protein is pcNDA3.1-N-3xFlag, and the inserted target gene is a nucleotide sequence encoding the CKB wild-type protein; the vector of the recombinant virus encoding the CKB wild-type protein is pAAV-hSyn-EGFP-P2A-3xFlag-WPRE.

[0018] Furthermore, this invention also proposes the application of creatine kinase B as a screening target for neuroprotective drugs against ischemia-reperfusion injury in stroke.

[0019] Furthermore, this invention also proposes the application of reagents that enhance creatine kinase B activity or increase creatine kinase B protein expression in the preparation of neuroprotective drugs for stroke ischemia-reperfusion injury.

[0020] Furthermore, this invention also proposes the application of reagents for detecting creatine kinase B activity or reagents for detecting creatine kinase B K11 lactation modification in the preparation of diagnostic kits for stroke ischemia-reperfusion injury.

[0021] Compared with existing technologies, the beneficial effects of this invention include: This invention is the first to discover that CKB K11 lactation modification is closely related to ischemia-reperfusion injury in stroke, and its modification level is highly consistent with CKB kinase activity, thus serving as a specific diagnostic indicator for ischemia-reperfusion injury in stroke. By detecting the level of CKB K11 lactation modification in biological samples, the degree of brain tissue damage and postoperative recovery in the tested subjects can be rapidly and accurately assessed, providing a new and effective means for clinical diagnosis.

[0022] This invention also discovered that CKB K11 lactation modification has neuroprotective effects. It can increase CKB kinase activity, reduce oxidative stress, reactive oxygen species, and lipid peroxidation levels in neurons, decrease neuronal apoptosis, improve mitochondrial function and motor function in mice, and reduce the area of ​​cerebral infarction. Screening neuroprotective drugs targeting this could provide a new direction for drug development in the treatment of ischemia-reperfusion injury after stroke, solving the current clinical problem of a lack of effective neuroprotective agents.

[0023] Based on the neuroprotective effect of CKB K11 lactation modification, the neuroprotective drug provided by this invention, which contains a recombinant vector encoding wild-type CKB protein, a recombinant virus, or a CKB K11 lactation modification activator, has the characteristics of strong targeting and significant efficacy. It can effectively improve ischemia-reperfusion injury in stroke and has important clinical application value. Attached Figure Description

[0024] Figure 1 This is Example 1 of the present invention, showing the sequencing process of cortical brain tissue from mice in the MCAO / R model group and sham-operated group, and the identification of CKBK11 lactation modification sites; A: Schematic diagram of lactation modification proteomics experimental process; B: Volcano plot of differentially modified protein sites in lactation modification proteomics; C: Mass spectrum of CKB K11 lactation modification identification (n = 3).

[0025] Figure 2Example 2 of this invention verifies CKB K11 lactation modification and explores its cellular function in a cell model; A: Sequencing identification maps of Flag-CKB and Flag-CKB K11R plasmids; B: Flag-IP detection of OGD / R-induced CKB K11 lactation modification; C: Cell viability assay kit to detect the effect of CKB K11 lactation modification on OGD / R-induced cell viability; D: Creatine kinase assay kit to detect the effect of CKB K11 lactation modification on OGD / R-induced creatine kinase activity; E: Creatine content assay kit to detect the effect of CKB K11 lactation modification on OGD / R-induced intracellular creatine content (n = 3).

[0026] Figure 3 Example 3 of this invention investigates the effects of CKB K11 lactation modification on oxidative stress and mitochondrial function in a cell model; A: DCFH-DA staining to detect changes in intracellular reactive oxygen species induced by OGD / R induced by CKB K11 lactation modification; B: Statistical results of reactive oxygen species; C: TMRE staining to detect changes in mitochondrial membrane potential induced by OGD / R induced by CKB K11 lactation modification; D: Statistical results of mitochondrial membrane potential; E: Seahorse Mitochondrial Stress Kit to detect changes in mitochondrial energy metabolism induced by OGD / R induced by CKB K11 lactation modification; F: ATP content assay kit to detect changes in intracellular ATP induced by OGD / R induced by CKB K11 lactation modification (n = 3).

[0027] Figure 4 Example 4 of this invention demonstrates that CKB K11 lactation modification can improve brain injury caused by ischemia-reperfusion in an animal model; A: Experimental timeline; B: Changes in CKB protein expression in the cortical tissue of mice in the MCAO / R group and sham-operated group 3 weeks after tail vein injection of AAV-GFP-CKB / CKB K11R adenovirus; C: TTC staining of brain tissue sections; D: TTC staining and counting results of brain tissue sections (n ​​= 3); E: Changes in CKB K11 lactation modification in the cortical tissue of mice in the MCAO / R group and sham-operated group detected by Flag-IP; F: Nissl staining of brain tissue sections.

[0028] Figure 5Example 5 of this invention investigates the effects of CKB K11 lactation modification on creatine kinase activity, oxidative stress, and apoptosis in an animal model; A: Changes in CK activity in the cortical tissue of mice in the MCAO / R group and sham-operated group; B: Changes in creatine content in the cortical tissue of mice in the MCAO / R group and sham-operated group; C: Changes in MDA content in the cortical tissue of mice in the MCAO / R group and sham-operated group (n = 3); D: Changes in Active-Caspase 3 expression in the cortical tissue of mice in the MCAO / R group and sham-operated group; E: Changes in Active-Caspase 3 expression in immunofluorescence staining of brain tissue sections from mice in the MCAO / R group and sham-operated group.

[0029] Figure 6 Example 6 of this invention investigates the effect of CKB K11 lactation modification on motor function in mice in an animal model; A: Experimental timeline for histochemical experiments and behavioral analysis; BD: Behavioral evaluation of motor function in mice after stroke ischemia-reperfusion injury. Detailed Implementation

[0030] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0031] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0032] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0033] This specific embodiment provides an application of CKB K11 lactation modification as a diagnostic tool for ischemia-reperfusion injury in stroke or as a target for neuroprotective drugs.

[0034] This specific embodiment also proposes the application of genes or proteins that enhance CKB K11 lactation modification in the preparation of neuroprotective drugs, wherein the neuroprotective drugs are neuroprotective drugs for ischemia-reperfusion injury in stroke.

[0035] Furthermore, this specific embodiment also proposes the application of CKB K11 lactation modification in the preparation of diagnostic reagents for stroke ischemia-reperfusion injury or in the screening of neuroprotective drugs. The diagnostic reagent assesses the degree of stroke ischemia-reperfusion injury or postoperative recovery in the tested subject by detecting the level of CKB K11 lactation modification in biological samples. The biological samples include cerebrospinal fluid, serum, or brain tissue samples. The diagnostic reagent contains antibodies, probes, or primers for specifically detecting CKB K11 lactation modification. And / or, the screening method includes the following steps:

[0036] S1. Construct an OGD / R cell model or an MCAO / R biomodel;

[0037] S2, administer candidate drugs to the model;

[0038] S3. Detect at least one of the following indicators in the detection model: CKB K11 lactation modification level, CKB kinase activity, neuronal cell viability, oxidative stress level, mitochondrial function, cerebral infarction area, neuronal apoptosis level, or motor function; if the candidate drug can improve the CKB K11 lactation modification level and / or CKB kinase activity, and improve at least one of the above indicators, it is judged as a potential neuroprotective drug.

[0039] Furthermore, this specific embodiment also proposes a neuroprotective drug, the active ingredient of which is a substance capable of increasing the lactation modification level of CKBK11; the substance includes a recombinant vector encoding wild-type CKB protein, a recombinant virus, or a CKB K11 lactation modification activator.

[0040] In some embodiments, the backbone of the recombinant vector encoding the CKB wild-type protein is pcNDA3.1-N-3xFlag, and the inserted target gene is a nucleotide sequence encoding the CKB wild-type protein; the vector of the recombinant virus encoding the CKB wild-type protein is pAAV-hSyn-EGFP-P2A-3xFlag-WPRE.

[0041] Furthermore, this specific embodiment also proposes the application of creatine kinase B as a screening target for neuroprotective drugs against ischemia-reperfusion injury in stroke.

[0042] Furthermore, this specific embodiment also proposes the application of reagents that increase creatine kinase B activity or increase creatine kinase B protein expression in the preparation of neuroprotective drugs for stroke ischemia-reperfusion injury.

[0043] Furthermore, this specific embodiment also proposes the application of reagents for detecting creatine kinase B activity or reagents for detecting creatine kinase B K11 lactation modification in the preparation of diagnostic kits for ischemia-reperfusion injury in stroke.

[0044] This invention reveals that creatine kinase B kinase activity is highly correlated with the level of lactation modification of CKB K11. Assessing creatine kinase B kinase activity by detecting changes in CKB K11 lactation modification is a stable and reliable method.

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0046] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0047] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0048] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0049] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0050] Terminology Explanation:

[0051] The MCAO / R model, or middle cerebral artery occlusion / reperfusion model, is an animal model used to study ischemia-reperfusion injury after stroke. This model simulates the brain tissue injury process caused by ischemia-hypoxia followed by reperfusion by temporarily occluding the middle cerebral artery and then restoring blood flow to it. The MCAO / R model is an internationally recognized standard animal model of focal cerebral ischemia, and its pathogenesis is similar to the symptoms of cerebral ischemia in humans.

[0052] OGD / R model: Oxygen-glucose deprivation / reperfusion model, is a cell model used to study ischemic stroke. This model simulates the damage process of cells in vivo after ischemia-hypoxia and reperfusion by depriving cells of nutrients and oxygen for a short period of time and then restoring glucose and oxygen.

[0053] Lactylation is a post-translational modification of proteins. Lactic acid and coenzyme A (CoA) combine under the action of a specific enzyme to form lactyl-CoA, which serves as the donor for lactylation. A specific lactate transferase then transfers the lactate group to a lysine residue in the protein, resulting in lactylation. This process alters the protein's charge state, structure, and function, thereby affecting its activity, localization, and interactions with other molecules, further regulating a wide range of cellular physiological processes and pathological states. CKB K11 represents the 11th lysine residue (K11) of brain-type creatine kinase (CKB). CKB K11 lactylation modification indicates that the 11th lysine residue (K11) of brain-type creatine kinase (CKB) undergoes lactylation.

[0054] Lactic acid modification proteomics sequencing: After whole protein extraction, the protein sample is enzymatically digested into small molecule peptides. Then, high-quality lactic acid modification antibodies and biological materials are used to enrich the lactic acid modification peptides. Finally, LC-MS / MS is used for on-machine detection, analysis and quantification. This sequencing technology is used to search libraries to quantify lactic acid modified proteins and modification sites.

[0055] Experimental materials

[0056] Plasmid list:

[0057] All plasmids used in the following examples were purchased from Wuhan Aoke Dingsheng Biotechnology Co., Ltd., as shown in Table 1.

[0058] Table 1

[0059] plasmid name plasmid backbone resistance pcNDA3.1-3xFlag-GFP pcNDA3.1-N-3xFlag Amp pcNDA3.1-3xFlag-CKB pcNDA3.1-N-3xFlag Amp pcNDA3.1-3xFlag-CKB K11R pcNDA3.1-N-3xFlag Amp

[0060] Virus list:

[0061] All viruses used in the following examples were purchased from Shanghai Heyuan Biotechnology Co., Ltd., as shown in Table 2.

[0062] Table 2

[0063] Virus Name pAAV-hSyn-EGFP-P2A-3xFlag-WPRE pAAV-hSyn-EGFP-P2A-CKB-3xFlag-WPRE pAAV-hSyn-EGFP-P2A-CKB K11R-3xFlag-WPRE

[0064] The antibody list is shown in Table 3.

[0065] Table 3

[0066] Antibody name brand Item number Pan Kla antibody PTMab PTM-1401RM HA antibody ABclonal AE105 DYKDDDDK antibody Proteintech 20543-1-AP β-actin proteintech 66009-1-Ig Active-Caspase3 BD 559665 NeuN Proteintech 66836-1-Ig

[0067] Cell culture and plasmid transfection

[0068] We cultured Neuro-2a cells (Chinese Academy of Sciences Cell Bank) in DMEM high-glucose medium mixed with 10% fetal bovine serum (Hyclone, China). All plasmids were transfected into Neuro-2a cells according to the instructions of PEI transfection reagent (Thermo Fisher Scientific, USA). The medium was changed 6 hours after transfection. The medium was removed at specific time points for further analysis.

[0069] Laboratory animal husbandry and treatment

[0070] Male C57BL / 6 mice, aged 7-8 weeks, were purchased from the Animal Experiment Center of Huazhong Agricultural University. Animal management and use during the experiment followed the *Guide for the Care and Use of Laboratory Animals* (2011) published by the National Academy Press, the *Regulations on the Management of Laboratory Animals* revised by the State Science and Technology Commission in 2017, and the *Regulations on the Management of Laboratory Animals in Hubei Province* promulgated by the Hubei Provincial Department of Science and Technology in 2005. The animal management, use, and related procedures involved in the following examples were approved by the Animal Care and Use Committee of Agricultural Institutions of Huazhong University, with ethics number HZAURAB-2023-0027. Male mice weighing 21 ± 0.5 g were selected and anesthetized daily by intraperitoneal injection of 1.5% isoflurane. The mice were then injected intravenously with 3*10¹² vg / ml of pAAV-hSyn-EGFP-P2A-CKB-3xFlag-WPRE virus (Heyuan Biotechnology, China) at 100 μl. The mice were then maintained in this manner. Control mice were injected with the same dose of pAAV-hSyn-EGFP-P2A-3xFlag-WPRE virus. Subsequent experiments were conducted 21 days after the mice were neutered.

[0071] MCAO / R modeling

[0072] Healthy 7-8 week old male C57BL / 6 mice weighing 22-24 g were selected for MCAO / R modeling surgery. Mice were anesthetized with 2% isoflurane and continuously anesthetized with 1.5% isoflurane. The mice were fixed supine on the operating table. Hair removal cream was used to remove the skin from the mouse's neck to prevent bacterial infection from hair. An incision was made slightly to the left of the middle of the neck. The muscles and glands were separated, and the pulsating common carotid artery was located. The vagus nerve was separated, and the common carotid artery was clamped with an arterial clamp. The external carotid artery and internal carotid artery were separated upwards from the distal end of the common carotid artery. The external carotid artery was ligated with 5-0 suture. A small incision was made below the ligated external carotid artery, and a suture plug was inserted to the hemostatic clamp. The suture plug was then inserted into the internal carotid artery, and continued to be pushed into the middle cerebral artery about 8-9 mm, stopping when slight resistance was encountered. The wound was sutured, and the mouse was placed in an incubator at a constant temperature of 28 degrees Celsius for warmth. Two hours after the ischemia, the suture plug was removed, reperfusion was performed, the opening was ligated with sutures, and the wound was sutured. After the mouse regained consciousness, it was returned to its rearing environment.

[0073] Lactic acidification modification proteomics analysis

[0074] Protein enzymatic hydrolysis and peptide preparation

[0075] The samples were ground in liquid nitrogen and lysed using a lysis buffer containing 8 M urea and 1% protease inhibitor. The lysis buffer was then subjected to sonication, reduction with dithiothreitol, alkylation with iodoacetamide, and finally digested with trypsin.

[0076] Post-translational modification enrichment based on pan-antibody

[0077] To enrich the modified peptides, the enzyme-digested peptides were dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) and incubated overnight at 4°C with pre-washed antibody beads (catalog number PTM1404, PTM Bio) using gentle shaking. The beads were then washed four times with NETN buffer and twice with deionized water. The bound peptides were eluted with 0.1% trifluoroacetic acid, and the eluates were collected, combined, and vacuum-dried. The resulting peptides were desalted using C18 ZipTips (Millipore) according to the manufacturer's instructions for subsequent LC-MS / MS analysis.

[0078] LC-MS / MS analysis of lactation-modified proteomics

[0079] Peptides enriched with pan-antibody were dissolved in solvent A (0.1% formic acid, 2% acetonitrile aqueous solution) and directly loaded onto a self-assembled reversed-phase analytical column (25 cm long, 100 μm inner diameter). The mobile phase consisted of solvent A and solvent B (0.1% formic acid and acetonitrile solution), with the following gradient elution program: 0–42 min, 6%–22% B; 42–52 min, 22%–30% B; 52–56 min, 30%–80% B; 56–60 min, 80% B, at a constant flow rate of 450 nl / min, using a NanoElute ultra-high performance liquid chromatography system (Bruker Daltonics). Peptides were introduced into a timsTOF Pro2 mass spectrometer via a capillary ion source at an electrospray voltage of 1.50 kV. Precursor ions and fragment ions were analyzed in a TOF detector, with the MS / MS scan range set to 100–1700. Mass spectrometry was performed in parallel cumulative continuous fragmentation (PASEF) mode, selecting precursor ions with charge states of 0–5 for fragmentation. Ten PASEF-MS / MS spectra were acquired in each cycle, and the dynamic exclusion time was set to 24 seconds.

[0080] Database search for lactation modification proteomics

[0081] The obtained MS / MS data were processed using the MaxQuant search engine (v.1.6.15.0). The tandem mass spectra were searched using the Mus_musculus_10090_SP_20230103.fasta database (17132 sequences) and its reverse pseudo-database. Enzyme digestion was set to Trypsin / P, allowing a maximum of 4 missed cleavage sites. The initial and main search mass error for precursor ions was set to 20 ppm; the fragment ion mass error was also set to 20 ppm. The fixed modification was carbamoyl methylation of cysteine; variable modifications included N-terminal acetylation, methionine oxidation, and lysine lactation. The false positive rate (FDR) threshold was set to < 1%.

[0082] TTC staining

[0083] Mice were anesthetized with 10% chloral hydrate at a dose of 400 mg / kg body weight. After complete anesthesia, the mice were decapitated, and their brains were removed on ice and placed in a brain mold. The brains were then evenly sliced ​​into five sections at 2 mm each. Staining was performed using 1% TTC solution at 37°C. During staining, a coverslip was placed over each brain slice, and the slices were incubated in the dark for 20 minutes. After complete staining, the TTC solution was replaced with 4% paraformaldehyde, and the slices were fixed overnight at 4°C. Images were then captured of the stained brain slices.

[0084] Nissl stain

[0085] Mouse brain sections were stained using a Nissl staining kit. The procedure involved PSB washing, Nissl staining at 65°C for 10 min, Nissl differentiation solution for 3 min, Nissl blue staining for 5 min, gradient dehydration with alcohol for 1 min each, xylene clearing for 2 min, and mounting with neutral resin. The slides were then photographed under an optical microscope. Local brain regions were photographed under a 10x objective lens, and the approximate cellular morphology was photographed at 25x to analyze neuronal damage and recovery.

[0086] Behavioral testing

[0087] Balance beam experiment

[0088] The spontaneous movement of test mice was analyzed by placing them on a 1 m long and 4 cm wide square wooden beam. The test time varied from 1 to 5 minutes depending on the animal's activity level. During the test, two experimenters stood on either side of the balance beam to record the upper limb movements of the animals when they faced away from the experimenters. All scores were blind-scored by the experimenters. The mice were placed in a dark room for 2 hours before the test. Three days before the test, the experimenters trained the test animals to grasp them so that they were familiar with the experimenters' grasping methods. There was no need to fast or reward the animals with food during the experiment. The number of times the animal's limbs slipped off the balance beam was recorded during the test: the mouse was placed on one side of the 1 m long and 2 cm wide square wooden beam, and the mouse spontaneously walked to the other side. The number of times the mouse's limbs fell was recorded. If the mouse did not fall at all, it was scored as 10 points. One point was deducted for each slip. The test stopped when the mouse crossed the balance beam and entered the dark room. The test score was the full score (10 points) minus the number of slips. If the mouse cannot pass or falls off midway, the lowest score (0 points) is recorded. The experiment is performed three times consecutively, and the average of the three tests is calculated. After the test, the animal's motor behavior ability is assessed by calculating the number of slips on the right side (the injured side).

[0089] Rotating bar experiment

[0090] This test was used to assess the overall motor coordination ability of mice. During the procedure, mice were placed on a rotating rotundus at 40 rpm, and a timer was started simultaneously. The fall latency and the rotational speed of the rotundus at the moment of fall were recorded when the mouse fell from the rotundus into its corresponding lane. Insulators separated the lanes to prevent interference between mice in adjacent lanes. If a mouse survived for 5 minutes without falling, the latency was recorded as 300 s. The experiment was repeated three times, with a 30-minute interval between each test. To ensure the stability of the test, 2-3 training sessions were conducted before each test. The test recorded the time it took for the mouse to fall from the rotundus. The mouse was placed on a rotating rotundus at 40 rpm, and a timer was started simultaneously. The fall latency and the rotational speed of the rotundus at the moment of fall were recorded when the mouse fell from the rotundus into its corresponding lane. If the mouse survived for 5 minutes without falling, the latency was recorded as 300 s. The experiment was conducted three times consecutively, with a 30-minute interval between each time.

[0091] Horizontal ladder test

[0092] The spontaneous movement of mice was analyzed by placing them on a horizontal ladder measuring 70 x 15 cm with 5 cm intervals between the two side panels. The ladder side panels had 121 circular holes, each 2 mm in diameter and spaced 5 mm apart. Stainless steel crossbars, 8 cm long and 1 mm in diameter, were inserted at varying irregular intervals into the ladder structure. The test time ranged from 3 to 10 minutes depending on the animal's activity level. During the test, two experimenters stood on either side of the ladder to record and analyze the movement of the animal's limbs simultaneously. The mouse started from the beginning and reached the target point along the ladder, and the time taken to traverse the ladder was recorded, excluding pauses. If the mouse could not walk within 300 seconds, the test was stopped, and the time was recorded as 300 seconds. After training, mice that could traverse the entire ladder within 180 seconds were selected for subsequent experiments. All scores were blind-reviewed by the experimenters. Mice were placed in a dark room for 2 hours before the experiment. The test recorded the number of times the animal's limbs slipped while walking on a horizontal ladder: The mouse was placed on one side of the ladder and spontaneously walked to the other side. The number of times the mouse's limbs fell was recorded. If the mouse did not fall at all, it was scored as 10 points; one point was deducted for each slip. The test stopped when the mouse passed the ladder. The test score was the full score (10 points) minus the number of slips. If the mouse could not pass, the lowest score (0 points) was recorded.

[0093] OGD / R modeling

[0094] Neuro-2a cells in the logarithmic growth phase were selected, and the original medium was replaced with preheated Dulbecco modified medium. The cell culture plates were then quickly placed in a hypoxic chamber at 37°C, 5% CO2 + 94% N2 + 1% O2 for oxygen and glucose deprivation for 4 h. Immediately afterwards, the Dulbecco modified medium was replaced with normal medium, and the cell culture plates were reoxygenated for 24 h at 37°C, 5% CO2. Cell morphological changes were observed after reoxygenation, and relevant stress indicators were measured.

[0095] CCK-8 cell viability assay

[0096] The CCK-8 assay kit (GlpBio) was used to detect cell proliferation. Logarithmic growth phase neurons were harvested and their concentration adjusted to 1×10⁻⁶ cells / mL using DMEM medium containing 10% FBS. 5Cells / mL were seeded into 96-well plates, with 100 μL of cell suspension added to each well. The plates were incubated for 48 h. After OGD / R treatment, CCK-8 assays were performed at 24 h and 48 h. The supernatant was quickly discarded, and 10 μL of CCK-8 solution was added to each well. The plates were incubated at 37 ℃ for 2 h, and the absorbance at 450 nm was measured using a microplate reader. Six parallel wells were set up for each group, and the average value was taken.

[0097] Reactive oxygen species (ROS) detection

[0098] Intracellular ROS levels were detected using the DCFH-DA fluorescence method. DCFH-DA was diluted 1:1000 with serum-free culture medium to a final concentration of 10 μM / mL. The cell culture medium was removed, and 1 mL of DCFH-DA staining working solution was added; the cells were incubated at 37°C for 30 min. Cells were washed three times with serum-free cell culture medium to thoroughly remove any uninfiltrated DCFH-DA. 1 mL of DAPI staining working solution was added, and the cells were incubated at room temperature for 10 min. After incubation, the supernatant was aspirated, and the cells were washed three times with serum-free cell culture medium; the cells were fixed in 4% paraformaldehyde solution at room temperature for 15 min. Images were captured using a confocal microscope at 20x objective lens, with dual-channel acquisition: DAPI (Blue) and DCFH-DA (Green).

[0099] Mitochondrial membrane potential detection

[0100] TMRE staining was used to detect intracellular mitochondrial membrane potential levels. TMRE was diluted 1:1000 with detection buffer to a final concentration of 1x TMRE staining working solution. Cell culture medium was removed, and cells were washed once with PBS buffer. 1 ml of TMRE staining working solution was added, and the cells were incubated at 37ºC for 30 min. After incubation, the supernatant was aspirated, and the cells were washed twice with pre-warmed cell culture medium. 1 ml of Hoechst staining working solution was added, and the cells were incubated at room temperature for 10 min. After incubation, the supernatant was aspirated, and the cells were washed twice with pre-warmed cell culture medium. 500 μL of pre-warmed cell culture medium was added, and images were captured using a confocal microscope at 20x objective lens, with dual-channel acquisition: Hoechst (Cyan) and DCF (Red).

[0101] Immunoprecipitation

[0102] Preparation of cell lysates

[0103] After removing the culture medium, wash the cells once with ice-cold PBS; remove the PBS, add 0.5 ml of ice-cold 1x IP lysis buffer to each 10 cm plate, and incubate the plates on ice for 5 min; scrape the cells off the plate, and transfer the extract to a microcentrifuge tube and place it on ice; sonicate the sample three times on ice for 5 s each time; microcentrifuge at 14000 xg at 4°C for 10 min, and then transfer the supernatant to a new test tube.

[0104] Immunoprecipitation

[0105] Transfer 20 μl of Flag-beads to a clean centrifuge tube. Place the tube on a magnetic separator for 10–15 s. Once the solution becomes clear, carefully remove the buffer. Add 500 μl of 1x cell lysis buffer to the magnetic bead precipitate, vortex slightly to wash the beads, repeat twice; add 200 μl of cell lysis to 20 μl of pre-washed Flag / HA-beads; incubate by rotation at room temperature for 60 min; precipitate the magnetic beads using a magnetic separator; wash the precipitate five times with 500 µl of 1x cell lysis buffer. During the washing interval, keep the sample on ice; resuspend the precipitate in 80–100 μl of 2x SDS sample buffer, vortex slightly to mix, then centrifuge briefly to allow the sample to precipitate; heat the sample to 95–100°C for 10 min; precipitate the magnetic beads using a magnetic separator. Transfer the supernatant to a new tube; the supernatant is the sample.

[0106] Protein extraction and immunoblotting analysis

[0107] Cell protein extraction: Remove the culture medium and add 1 ml of cell lysis buffer containing protease inhibitors. Mix thoroughly, incubate on ice for 20 min, centrifuge at 4°C, 12,000 r / min for 5 min, and collect the supernatant.

[0108] Protein concentration determination: The protein sample was removed and diluted 1:20 with RIPA lysis buffer containing PMSF. The sample concentration was determined using a BCA protein concentration assay kit (enhanced version). The OD562 value of the cell sample was measured using a microplate reader, and the protein concentration was calculated based on the standard curve.

[0109] Protein sample preparation: Add 5× protein loading buffer to adjust the concentration of cell protein sample to 2 μg / μL, boil for 10 min, and let stand on ice for 10 min.

[0110] Electrophoresis: First set the electrophoresis to 80 V for about 30 minutes. Once the bands have passed the stacking gel, adjust the V to 100 V.

[0111] Transfer: After SDS-PAGE electrophoresis, the gel is not stained. Proteins are directly transferred to a PVDF membrane using a wet transfer apparatus at 100 V for 60 min. Remove the gel from the electrophoresis apparatus, cut off the portion containing the desired band, and discard the rest. Immerse the membrane in distilled water. Cut six 1 mm thick filter papers and one PVDF membrane, ensuring the membrane size is the same as or slightly smaller than the gel. Gently immerse the PVDF membrane in methanol, then soak it in transfer buffer for 3 min. Simultaneously, immerse the six filter papers, the clamps, and the sponge in the transfer buffer. Assemble the clamps in the following order (cathode-three-layer filter paper-gel-membrane-three-layer filter paper-anode), adjusting the gel orientation so that the marker is on the left after transfer, from top to bottom and from largest to smallest. After completion, roll a clean glass rod back and forth on the filter paper a few times to remove air bubbles. Clamp the clamps, place the membrane in the transfer tank, and add 1 L of buffer to the tank. Finally, add ice cubes to the transfer tank, turn on the power, and transfer at 4℃.

[0112] Blocking: After the transfer is complete, remove the PVDF membrane with tweezers and cut off a corner for marking. Place the membrane in 10 mL of TBST and rinse three times, 5 min each time, aspirating all residual liquid on the last rinse. Add 10-15 mL of blocking buffer (TBST containing 5% BSA) and block on a shaker at room temperature for 2 h.

[0113] Primary antibody incubation: After removing the membrane from the blocking solution, rinse it three times with 10 mL TBST for 5 min each time, and aspirate any residual solution. Place the rinsed PVDF membrane in a suitable amount of primary antibody diluted with TBST containing 5% BSA and incubate overnight at 4°C on a shaker.

[0114] Secondary antibody incubation: Remove the PVDF membrane and wash it three times with 10 mL of TBST for 10 min each time, aspirating all residual solution on the last wash. Then, place the PVDF membrane in a solution of secondary antibody appropriately diluted (1:10000) with TBST containing 5% BSA and incubate on a shaker at room temperature for 1 h. Afterward, wash the membrane three times with PBST for 10 min each time, aspirating all residual solution.

[0115] Development: Prepare fresh developer (A and B solutions in a 1:1 ratio). Remove the membrane from the TBST and drain. Evenly add the developer and develop in the dark for 30-60 seconds. Then blot off any remaining substrate solution on the membrane and expose it using an imaging system to take a picture.

[0116] Software was used to perform strip grayscale analysis on the image.

[0117] ATP detection

[0118] Weigh approximately 0.1 g of tissue or 10 g of other tissue. 6Cells were added to 1 ml of lysis buffer and homogenized using a glass homogenizer. After thorough homogenization, the cells were centrifuged at 12000g for 5 minutes at 4°C, and the supernatant was collected for subsequent assays.

[0119] Thaw the reagents on ice. Dilute the ATP standard solution with ATP detection lysis buffer to the following concentration gradients: 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM. Prepare the ATP detection working solution.

[0120] Determine the RLU values ​​of the standard curve sample and the test sample; plot the standard curve based on the results of the standard curve sample and calculate the ATP content of the test sample.

[0121] MDA detection

[0122] Sample preparation: Weigh approximately 0.1 g of tissue or 10 g of sample. 6 Cells were lysed in an ice bath with 1 mL of extract.

[0123] Centrifuge at 8000 g, 4°C for 10 min, collect the supernatant and place it on ice.

[0124] Prepare the system according to the steps in Table 4:

[0125] Table 4 MDA Detection Reaction System

[0126] Reagent Name (μL) Measurement tube Blank tube MDA detection working solution 600 600 distilled water - 200 sample 200 - Reagent 3 200 200

[0127] After incubating the mixture in a 100°C water bath for 60 min, it was cooled in an ice bath and centrifuged at 10000 g at room temperature for 10 min. 200 μL of the supernatant was transferred to a 96-well plate, and the OD values ​​of each sample were measured at 450 nm, 532 nm, and 600 nm. The following values ​​were calculated: ΔOD 450 = OD 450 measurement - OD 450 blank; ΔOD 532 = OD 532 measurement - OD 532 blank; ΔOD 600 = OD 600 measurement - OD 600 blank.

[0128] Lactic acid content detection

[0129] Sample preparation: Weigh approximately 0.1 g of tissue or 10 g of sample. 6 Cells were lysed in an ice bath with 1 mL of extract.

[0130] Centrifuge at 10000 g for 10 min at 4°C, collect the supernatant and place it on ice.

[0131] Take 75 μL of supernatant from each sample and mix it with 75 μL of Lactate assay buffer. Set aside for later use.

[0132] Prepare the system according to the steps in Table 5:

[0133] Table 5 Lactate detection reaction system / reaction

[0134] Reagent Name (μL) reaction mixture Blank tube Lactate Assay Buffer 46 48 Probe 2 2 Enzyme 2 -

[0135] Take 50 μL of sample mixture and 50 μL of reaction mixture, mix gently, and incubate at room temperature in the dark for 30 min. Measure the OD value of each sample at 570 nm, and calculate the lactic acid content of each sample according to the standard curve.

[0136] Relative lactic acid content (μg / 10) 4 cell count = lactic acid content / number of cells

[0137] Creatine kinase activity assay

[0138] The reaction solution preparations for creatine kinase detection are shown in Table 6.

[0139] Table 6

[0140] Reagent Name volume ratio Reagent 1 70 Reagent 2 4 Reagent 3 7 Reagent 4 10 Reagent 5 90

[0141] Weigh approximately 0.1 g of tissue or 10 g of other tissue. 6 Add 1 ml of extraction solution to the cells and sonicate on ice for 3 min to disrupt the cells; centrifuge at 10000 g for 10 min at 4°C and collect the supernatant.

[0142] Set up the blank group and experimental group according to the proportions shown in Table 7 below:

[0143] Table 7 Creatine kinase detection reaction system / reaction

[0144] experimental group Blank group Sample supernatant 40 - working fluid 90 90 water 70 110

[0145] Add the above reagents to the 96-well UV plate, mix thoroughly, and measure the absorbance value A1 at 340 nm for 10 s. Then, quickly place the plate in a 37°C incubator for 3 min and take it out to measure the absorbance value A2 at 190 s. ▲A = (A2 measurement - A1 measurement) - (A2 blank - A1 blank). Calculate the CK activity based on ▲A.

[0146] Creatine content detection

[0147] Weigh approximately 0.1 g of tissue or 10 g of other tissue. 6 Cells were subjected to 1 ml of extraction buffer and sonicated on ice for 3 min. The cells were then centrifuged at 12000 g for 10 min at 4°C. 0.8 mL of the supernatant was taken and 0.15 mL of extraction buffer II was added. The cells were gently pipetted until no more bubbles were generated. The cells were then centrifuged at 12000 g for 10 min at 4°C. The supernatant was collected for analysis.

[0148] Set up the blank group and experimental group according to the proportions shown in Table 8 below:

[0149] Table 8 Reaction system / reaction for creatine content detection

[0150] experimental group Blank group Sample supernatant 20 20 working fluid 40 40 Reagent 2 20 - distilled water - 20

[0151] After mixing all the above components, the mixture was kept at room temperature in the dark for 10 minutes. Then, 120 μL of distilled water was added and mixed thoroughly. The absorbance was measured at 530 nm, and the creatine content was calculated based on the standard curve.

[0152] Immunofluorescence

[0153] Brain tissue was embedded in OCT and then frozen sectioned to a thickness of 15 µm. The sections were placed in a slide cassette and immersed in PBS solution. After the OCT embedding agent was washed away, 0.5% Triton-X 100 solution was added for permeation for 15 min. Then, the tissue to be stained was circled using an immunohistochemical pen, and an appropriate amount of immunofluorescence blocking solution was added, with blocking for 60 min. The immunofluorescence blocking solution was discarded, and appropriate amounts of Anti-NeuN antibody (1:500) and Anti-Active Caspase 3 antibody (1:250) were added and incubated overnight at 4°C. The secondary antibody was recovered, washed three times with PBS, and then incubated with the corresponding species' fluorescent secondary antibody. After washing three times with PBS, the slides were stained with Hoechst staining solution, washed three times with PBS, and then mounted. The slides were placed under a fluorescence microscope for imaging under a 40x objective lens. Four channels: Hoechst (Blue), GFP (Green), NeuN (Purple), and Active Caspase 3 (Red). Imaging parameters were within the following ranges: Blue (0.1 s exposure), Green (0.3 s exposure), Purple (0.3 s exposure), and Red (0.5 s exposure). The exposure time for each animal's slide remained constant across all four channels. After imaging, NeuN and Active Caspase 3 positive cell labeling and analysis were performed using ImageJ.

[0154] Example 1: Sequencing procedure of cortical brain tissue from MCAO / R model group and sham-operated group mice and identification of CKB K11 lactation modification sites

[0155] I. Experimental Methods

[0156] A transient focal cerebral ischemia model was established in male C57BL / 6 mice (7-8 weeks old, 22-24 g) using the MCAO / R method. Mice were anesthetized with 2% isoflurane (RWD, China), and a midline cervical incision was made to expose the left common carotid artery, external carotid artery, and internal carotid artery. The distal end of the common carotid artery was ligated, and ligatures were placed at the distal end of the external carotid artery and at its bifurcation with the internal carotid artery. Subsequently, a silicone-coated suture was inserted into the internal carotid artery through a small incision in the external carotid artery to occlude the middle cerebral artery. The suture was removed 2 hours after occlusion to achieve reperfusion. The rectal temperature was maintained at 37°C throughout the procedure. The sham surgery group underwent the same procedure as the sham surgery group, except that no suture was inserted.

[0157] Cerebral cortex tissue samples from the above model were ground with liquid nitrogen, followed by lysis with lysis buffer and trypsin digestion. The digested peptides were incubated overnight at 4°C with antibody magnetic beads (batch number PTM1404, PTM Bio) by gentle shaking. The bound peptides were then eluted with 0.1% trifluoroacetic acid. The eluates were combined and vacuum dried. The resulting peptides were desalted using C18 ZipTips according to the manufacturer's instructions and then analyzed by LC-MS / MS.

[0158] The differentially modified proteins and modification sites identified above were plotted, clustered, and identified by mass spectrometry.

[0159] II. Experimental Results

[0160] The experimental procedure is illustrated in Figure 1A. Brain cortex tissues from three mice in both the sham-operated group and the MCAO / R group were collected, fragmented, enzymatically digested, enriched with Pan Kla antibody, and sequenced for identification. Lactation modification proteomics data showed that 128 differentially modified proteins, including CKBK11, underwent lysine lactation modification. Of these, 115 proteins showed upregulation of lactation modification in the MCAO / R model brain cortex tissue, while 13 proteins showed downregulation. A volcano plot illustrates the distribution of lactation sites in the proteins identified by lactation modification proteomics. Figure 1 B). Furthermore, focusing on the important regulatory pathways of energy metabolism and conversion in ischemia-reperfusion injury during stroke, we identified the CKB K11 site elevated by lactation modification and identified the characteristic spectral lines of the modified site using mass spectrometry. Figure 1 C).

[0161] Example 2: Effects of simulated CKB K11 delactation modification on cell function in an OGD / R cell model

[0162] I. Experimental Methods

[0163] To further investigate the role of CKB K11 lactation, we constructed wild-type CKB (pcDNA3.1-C-Flag-CKB) and mutant CKB K11R (pcDNA3.1-C-Flag-CKB K11R) plasmids, transfected Neuro-2a cells, and performed OGD / R (oxygen-glucose deprivation-reperfusion) modeling. We detected changes in CKB protein lactation modification using Flag-IP, and combined this with cell viability data, CK activity, and creatine content to infer the role of CKB K11 lactation modification.

[0164] II. Experimental Results

[0165] Sequencing results showed that the coding sequence of the K11 site in pcDNA3.1-C-Flag-CKB K11R was mutated from AAG to AGG compared to the pcDNA3.1-C-Flag-CKB plasmid, and its amino acid sequence was mutated from lysine (Lys, K) to arginine (Arg, R). Figure 2 A). The aim was to prevent OGD / R-induced lactation modification by mutating the lysine residues in the original sequence. Furthermore, our Flag-IP experiments revealed that, compared to the control group expressing Flag-CKB Neuro-2a cells, the Pan Kla signal was significantly increased in the OGD / R group expressing Flag-CKB Neuro-2a cells, indicating that OGD / R treatment can significantly induce lactation modification of the CKB protein. On the other hand, the Pan Kla signal in the OGD / R group expressing Flag-CKB K11R Neuro-2a cells was significantly lower than that in cells expressing Flag-CKB Neuro-2a cells, indicating that the CKB K11R mutation can effectively mimic the delactation state of CKB K11. Figure 2 B).

[0166] Cell viability data showed that, compared with the control group, the cell viability of all groups treated with OGD / R was significantly lower than that of the control group, indicating that OGD / R treatment significantly reduced cell viability. However, compared with Neuro-2a cells expressing Vector, the viability of Neuro-2a cells expressing Flag-CKB was partially salvaged, while the viability of Neuro-2a cells expressing Flag-CKB K11R remained almost unchanged. Figure 2C). Next, we examined intracellular creatine kinase activity and creatine content. The results showed that CK activity in Neuro-2a cells expressing Flag-CKB and Flag-CKB K11R was increased to varying degrees in both the control and OGD / R groups. However, the increase in CK activity was greater in the OGD / R group of Neuro-2a cells expressing both Flag-CKB and Flag-CKB K11R. Furthermore, the CK activity in Neuro-2a cells expressing Flag-CKB K11R was significantly lower in the OGD / R group compared to those expressing Flag-CKB, indicating that CKB K11 lactation modification can significantly increase CK activity. Figure 2 D). Creatine, as a substrate for CK kinase activity, can reflect changes in CK activity to some extent. The results showed ( Figure 2 E) Compared with the control group, the creatine content of cells in each group treated with OGD / R was significantly lower than that in the control group, indicating that OGD / R treatment significantly reduced intracellular creatine content; moreover, the creatine content of Neuro-2a cells expressing Flag-CKB in the OGD / R group was lower than that of Neuro-2a cells expressing Flag-CKBK11R, indicating that Neuro-2a cells expressing Flag-CKB in the OGD / R group consumed more creatine, which may be due to the increased CK activity caused by CKB K11 lactation. Figure 2 (D) Based on the fact that cells with increased CK activity after OGD / R treatment have stronger cell viability, we infer that OGD / R-induced CKB K11 lactation exerts a cytoprotective effect by upregulating CK activity.

[0167] Example 3: Investigating the effects of CKB K11 lactation modification on oxidative stress and mitochondrial function in an OGD / R cell model.

[0168] I. Experimental Methods

[0169] Neuro-2a cells were transfected with pcDNA3.1-C-Flag-CKB and pcDNA3.1-C-Flag-CKB K11R for OGD / R modeling. Changes in intracellular reactive oxygen species and mitochondrial membrane potential were detected by DCFH-DA staining and TMRE staining. Combined with the oxygen consumption rate under mitochondrial stress and intracellular ATP content, the role of CKB K11 lactation modification was inferred.

[0170] II. Experimental Results

[0171] ROS results showed that, compared with the control group, the ROS levels in all OGD / R treated groups were significantly higher than those in the control group, indicating a sharp increase in intracellular ROS content after OGD / R treatment. However, in the OGD / R group, compared with Neuro-2a cells expressing Vector, the ROS content in Neuro-2a cells expressing Flag-CKB was slightly reduced, while the ROS content in Neuro-2a cells expressing Flag-CKB K11R remained almost unchanged. Figure 3 AB). Mitochondrial membrane potential results showed that, compared with the control group, the mitochondrial membrane potential levels in all OGD / R treated groups were significantly lower than those in the control group, indicating that intracellular mitochondrial function was impaired and mitochondrial membrane potential was reduced after OGD / R treatment. Further analysis revealed that, in the OGD / R group, compared with Neuro-2a cells expressing Vector, both Flag-CKB and Flag-CKB K11R-expressing Neuro-2a cells showed a slight increase in mitochondrial membrane potential, but the increase was not as large in Neuro-2a cells expressing Flag-CKB K11R as in those expressing Flag-CKB. Figure 3 CD).

[0172] To further determine how CKB K11 lactation modification affects mitochondrial function, we used the Seahorse high-throughput mitochondrial metabolism analyzer to detect mitochondrial oxygen consumption rate. The results showed that, compared with the control group, the oxidative respiration levels in all OGD / R-treated groups were significantly lower than those in the control group, indicating that intracellular mitochondrial function was reduced after OGD / R treatment. Within the OGD / R group, the reserve respiration value in Neuro-2a cells expressing Flag-CKB was slightly higher than that in cells expressing Vector and Flag-CKB K11R. Figure 3 E). Subsequent intracellular ATP content data showed that, compared with the control group, the ATP content of cells in all groups treated with OGD / R decreased, but there was no statistically significant difference between the groups. Figure 3 F). This suggests that CKBK11 lactation modification may exert a cytoprotective effect by upregulating CK activity and enhancing intracellular mitochondrial reserve respiration.

[0173] Example 4: CKB K11 lactation modification in the MCAO / R animal model can improve brain injury caused by ischemia-reperfusion.

[0174] I. Experimental Methods

[0175] To specifically mutate the lactation modification site of CKB K11 in mouse brain neurons, two vectors using adeno-associated virus (BrainVTA Technology Co., Ltd.) were constructed to highly express the target gene AAV-hSyn-EGFP-3xFlag-CKB and AAV-hSyn-EGFP-3xFlag-CKB K11R, respectively, into neurons. AAV-hSyn-EGFP-3xFlag was used as a control.

[0176] Tail vein injection of AAV-hSyn virus can target and enrich it in brain tissue, while an AAV-hSyn-EGFP virus is constructed as a control. This method achieves specific expression in neurons within brain tissue without any extraneuronal effects.

[0177] Therefore, AAV-hSyn-EGFP-3xFlag, AAV-hSyn-EGFP-3xFlag-CKB, and AAV-hSyn-EGFP-3xFlag-CKB K11R were expressed in 5-week-old C57 mice via tail vein. After 21 days of viral expression, MCAO / R modeling was performed on the mice to detect CKB protein expression in brain tissue, brain infarct area, changes in CKB protein lactation modification, and Nissl bodies in neurons.

[0178] II. Experimental Results

[0179] The experimental procedure is shown in Figure 4A. Mice were modeled using MCAO / R 21 days after viral expression. Twenty-four hours after reperfusion, tissue samples were collected for protein immunoblotting, TTC staining, Flag-IP, and Nissl staining. The protein immunoblotting results showed that, compared to AAV-hSyn-EGFP-3xFlag, CKB protein expression was significantly increased in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB and AAV-hSyn-EGFP-3xFlag-CKB K11R, indicating successful CKB protein expression. Figure 4 B).

[0180] Next, TTC staining results showed that, compared with the sham-operated group expressing AAV-hSyn-EGFP-3xFlag, the infarct area in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag in the MCAO / R group increased dramatically, indicating successful modeling. In the MCAO / R group, the infarct area in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB was significantly reduced, while the infarct area in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB K11R was only slightly reduced. Figure 4 CD). Flag-IP results showed that, compared with the sham-operated group, CKB lactation modification was increased in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB and AAV-hSyn-EGFP-3xFlag-CKB K11R in the MCAO / R group, and the increase in CKB lactation modification was more significant in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB. Figure 4 E).

[0181] Finally, we assessed the changes in Nissl bodies in neuronal cells. The results showed that, compared with the sham-operated group, the number and density of Nissl bodies in the brain tissue of mice in all MCAO / R groups were reduced to varying degrees, indicating neuronal damage. The loss of Nissl bodies was most severe in neurons expressed by mice with AAV-hSyn-EGFP-3xFlag, while the loss of Nissl bodies was protected to varying degrees in neurons expressed by AAV-hSyn-EGFP-3xFlag-CKB and AAV-hSyn-EGFP-3xFlag-CKB K11R. Figure 4 F).

[0182] Example 5: Investigating the effects of CKB K11 lactation modification on creatine kinase activity, oxidative stress, and apoptosis in an MCAO / R animal model.

[0183] I. Experimental Methods

[0184] Five-week-old C57 mice were administered AAV-hSyn-EGFP-3xFlag, AAV-hSyn-EGFP-3xFlag-CKB, and AAV-hSyn-EGFP-3xFlag-CKB K11R via tail vein. Twenty-one days after viral expression, MCAO / R modeling was performed on the mice, and CK activity, creatine content, MDA content, and neuronal apoptosis levels in brain tissue were detected.

[0185] II. Experimental Results

[0186] Twenty-one days after viral expression, mice were modeled using the MCAO / R method. Twenty-four hours after reperfusion, tissue samples were collected for biochemical analysis, protein immunoblotting, and immunofluorescence staining. CK activity results showed that, compared to the sham-operated group, CK activity in the brain tissue of mice in all MCAO / R groups was increased to varying degrees. In the MCAO / R groups, the CK activity in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB and AAV-hSyn-EGFP-3xFlag-CKB K11R was significantly higher than that in the AAV-hSyn-EGFP group, with the highest CK activity observed in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB. Figure 5 A). Creatine content results showed that the creatine content in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB and AAV-hSyn-EGFP-3xFlag-CKB K11R in the MCAO / R group was significantly lower than that in the AAV-hSyn-EGFP group. Among them, the creatine content in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB was the lowest, and a negative correlation between creatine content and CK activity was found. Figure 5 B).

[0187] Subsequent lipid peroxidation (MDA) results showed that, compared with the sham-operated group, the MDA content in the brain tissue of mice in all MCAO / R groups was increased to varying degrees; among them, the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB had the lowest MDA content. Figure 5 (C) This result is completely consistent with the TTC staining results, indicating that MCAO / R-induced CKB K11 lactation plays an important protective role.

[0188] Finally, we detected the expression changes of Active-Caspase 3 in the brain tissue of mice in each group using Western blotting and immunofluorescence assays. The results showed that compared with the sham-operated group, the expression of Active-Caspase 3 in the brain tissue of mice in each MCAO / R group was increased to varying degrees. However, in the MCAO / R group, the expression of Active-Caspase 3 in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB and AAV-hSyn-EGFP-3xFlag-CKB K11R was significantly lower than that in the AAV-hSyn-EGFP group, with the lowest expression of Active-Caspase 3 in the brain tissue of mice expressing AAV-hSyn-EGFP-3xFlag-CKB. Figure 5 DE).

[0189] Example 6: Investigating the effects of CKB K11 lactation modification on motor function in a mouse MCAO / R animal model.

[0190] I. Experimental Methods

[0191] Five-week-old C57 mice were administered AAV-hSyn-EGFP-3xFlag, AAV-hSyn-EGFP-3xFlag-CKB, and AAV-hSyn-EGFP-3xFlag-CKB K11R via tail vein. MCAO / R modeling was performed on the mice 21 days after viral expression. Motor function of the mice was then assessed on days 1, 3, 7, 14, 21, and 28 after modeling.

[0192] II. Experimental Results

[0193] The experimental procedure is shown in Figure 6A. Mice were modeled using the MCAO / R method 21 days after viral expression. Motor function was assessed on days 1, 3, 7, 14, 21, and 28 post-modeling using balance beam, horizontal ladder, and rotarod tests. Results showed that compared to the sham-operated group, motor function impairment was significant on day 1 post-modeling. With the restoration of reperfusion blood flow, the damage continued to progress in mice expressing AAV-hSyn-EGFP in the MCAO / R group, reaching its most severe level on day 7. In the same period, mice expressing AAV-hSyn-EGFP-3xFlag-CKB and AAV-hSyn-EGFP-3xFlag-CKB K11R showed the most severe damage around day 3, with varying degrees of recovery by day 7. Mice expressing AAV-hSyn-EGFP-3xFlag-CKB showed the least motor function impairment and the earliest recovery. Furthermore, motor function gradually recovered in all groups over time. Figure 6 BD).

[0194] In summary, CKB K11 lactation can significantly improve ischemia-reperfusion injury and motor function in stroke patients. Therefore, using CKB K11 lactation levels as a neuroprotective target for stroke can effectively address the key issue that neurological drugs cannot slow down or repair nerve damage or improve post-injury effects. Furthermore, detecting CKB K11 lactation and CK activity can serve as indicators for diagnosing neurological injury in stroke patients. In clinical applications, samples can be quickly and easily collected via cerebrospinal fluid, and the degree of brain tissue damage and postoperative recovery can be assessed by detecting CKB K11 lactation and CK activity.

[0195] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of CKB K11 lactation modification as a diagnostic tool for stroke I / R injury or as a target for neuroprotective drugs.

2. Enhance the application of CKB K11 lactation-modified genes or proteins in the preparation of neuroprotective drugs.

3. The application according to claim 1 or 2, characterized in that, The neuroprotective drug is a neuroprotective drug for ischemia-reperfusion injury caused by stroke.

4. Application of CKB K11 lactation modification in the preparation of diagnostic reagents for ischemia-reperfusion injury in stroke or in the screening of neuroprotective drugs.

5. The application according to claim 4, characterized in that, The diagnostic reagent assesses the degree of ischemia-reperfusion injury or postoperative recovery of the test subject by detecting the level of CKBK11 lactation modification in biological samples; the biological samples include cerebrospinal fluid, serum or brain tissue samples; the diagnostic reagent contains antibodies, probes or primers for specifically detecting CKB K11 lactation modification. And / or, the screening method includes the following steps: S1. Construct an OGD / R cell model or an MCAO / R biomodel; S2, administer candidate drugs to the model; S3. Detect at least one of the following indicators in the detection model: CKB K11 lactation modification level, CKB kinase activity, neuronal cell viability, oxidative stress level, mitochondrial function, cerebral infarction area, neuronal apoptosis level, or motor function; if the candidate drug can improve the CKB K11 lactation modification level and / or CKB kinase activity, and improve at least one of the above indicators, it is judged as a potential neuroprotective drug.

6. A neuroprotective drug, characterized in that, Its active ingredient is a substance that can increase the level of CKB K11 lactation modification; the substance includes a recombinant vector encoding wild-type CKB protein, a recombinant virus, or a CKB K11 lactation modification activator.

7. The neuroprotective drug according to claim 6, characterized in that, The backbone of the recombinant vector encoding the wild-type CKB protein is pcNDA3.1-N-3xFlag, and the inserted target gene is a nucleotide sequence encoding the wild-type CKB protein; the vector of the recombinant virus encoding the wild-type CKB protein is pAAV-hSyn-EGFP-P2A-3xFlag-WPRE.

8. Application of creatine kinase B as a screening target for neuroprotective drugs against ischemia-reperfusion injury in stroke.

9. Application of reagents that increase creatine kinase B activity or increase creatine kinase B protein expression in the preparation of neuroprotective drugs for stroke ischemia-reperfusion injury.

10. Application of reagents for detecting creatine kinase B activity or reagents for detecting creatine kinase B K11 lactation modification in the preparation of diagnostic kits for ischemia-reperfusion injury in stroke.

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