Method for regulating and improving mitochondrial dysfunction of MCAO mouse through HIGD2A expression

Abstract

The invention discloses a method for regulating and improving MCAO mouse mitochondrial dysfunction through HIGD2A expression, and relates to the technical field of biomedicine and gene therapy. The invention aims at disclosing the internal relation between HIGD2A expression regulation and mitochondrial function improvement, and deeply analyzing the action mechanism of HIGD2A from the aspects of molecular biology and cell biology; through a series of rigorous experimental designs and scientific research methods, the influence of regulation and control of HIGD2A expression on mitochondrial key function indexes such as ATP synthesis efficiency, oxidative stress level, mitochondrial membrane potential stability and the like is clear; a brand new strategy and a potential treatment target are expected to be provided for treating cerebral ischemia related diseases by utilizing the method; a solid theoretical foundation is laid for subsequent development of a clinical treatment scheme and drug research and development based on HIGD2A, technical progress in the field of cerebral ischemia disease treatment is promoted, and finally a better treatment effect is brought to patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the cross field of biomedical science, neuroscience and gene therapy, in particular to a method for improving mitochondrial dysfunction of MCAO mice by HIGD2A expression regulation. BACKGROUND

[0002] Mitochondrial dysfunction is one of the core pathological links of nervous system diseases such as ischemic stroke (cerebral ischemia). The MCAO model is a classic animal model for studying ischemic stroke. At present, the intervention means (such as drug treatment, physical treatment) for mitochondrial dysfunction in this model have the problems of limited efficacy, side effects or inconvenience of application, etc.

[0003] HIGD2A gene has been confirmed to be involved in the regulation of mitochondrial function, but in the MCAO model, how to improve mitochondrial dysfunction by regulating HIGD2A expression, the specific method, effect and mechanism are still blank.

[0004] Therefore, it is necessary to propose a new solution to the above problems. SUMMARY

[0005] The purpose of the present application is to provide a method for improving mitochondrial dysfunction of MCAO mice by HIGD2A expression regulation, so as to solve the technical problems proposed in the background art.

[0006] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme: a method for improving mitochondrial dysfunction of MCAO mice by HIGD2A expression regulation, at least comprising the following steps: S1: design of HIGD2A expression regulation, wherein the HIGD2A expression regulation at least includes gene editing technology design, small molecule drug intervention design and RNA interference technology design; S2: grouping of experimental animals; S3: design of administration mode and dose; S4: setting corresponding indexes to determine the improvement of mitochondrial dysfunction of MCAO mice by HIGD2A expression regulation.

[0007] Further, the gene editing technology design at least adopts CRISPR / Cas9 gene editing system to precisely edit HIGD2A gene, by designing specific sgRNA to enable it to accurately recognize the target site of HIGD2A gene, guide Cas9 nuclease to cut DNA double strand at the site, so as to realize the knockout, knock-in or site-directed mutation of HIGD2A gene; In this way, the sequence and expression level of HIGD2A gene is directly changed to explore its effect on mitochondrial function. For example, in mouse embryonic stem cells, the HIGD2A gene is knocked out using CRISPR / Cas9 technology, and then the edited stem cells are induced to differentiate into neural cells, and the changes in mitochondrial function of these neural cells under simulated cerebral ischemia conditions are observed.

[0008] Further, the small molecule drug intervention design is to screen small molecule compounds that can specifically regulate the expression of HIGD2A. Through high-throughput drug screening technology, small molecules with high affinity to the promoter region of HIGD2A gene or related regulatory proteins are screened from a large number of small molecule compound libraries. These small molecules can affect the transcription process of HIGD2A gene by interacting with DNA or protein, thereby regulating its expression level. For example, if a small molecule compound is found to be able to bind to a specific sequence in the promoter region of HIGD2A gene, promote the binding of RNA polymerase to the promoter, and thus enhance the transcription and expression of HIGD2A gene. In the MCAO mouse model, the effect of the small molecule compound on HIGD2A expression and mitochondrial function is observed.

[0009] Further, the RNA interference technology design is to construct an siRNA or shRNA expression vector targeting HIGD2A gene. The siRNA sequence that can specifically target HIGD2A mRNA is prepared by chemical synthesis or genetic engineering method. After being introduced into cells, siRNA will bind to HIGD2A mRNA to form a double-stranded RNA structure, which is then recognized and degraded by nucleases in the cell, achieving interference of HIGD2A gene expression; For shRNA expression vector, it is transfected into cells, and shRNA is processed into siRNA in the cells to exert the same interference effect; In the neural cells of MCAO mice, siRNA targeting HIGD2A is introduced into cells using liposome transfection technology to reduce the expression level of HIGD2A, and the changes in mitochondrial function-related indicators are detected.

[0010] Further, the S2 at least includes the following steps: First, determine the experimental animals: Healthy adult C57BL / 6 mice are selected as experimental subjects, with body weight range controlled at 20-25g. Before the experiment starts, the mice are adapted to the environment for one week to reduce the impact of environmental factors on the experimental results.

[0011] Then, grouping. The mice are randomly divided into control group, model group and experimental group; Control group: sham operation, that is, only the blood vessels in the neck are separated, and the middle cerebral artery is not occluded. During the operation, the mouse is anesthetized, the neck is prepared for skinning, and disinfection is performed. The right common carotid artery, external carotid artery, and internal carotid artery are separated, but a plug line is not inserted to occlude the middle cerebral artery. After the operation, the mouse is normally fed and used as a control under normal physiological conditions. Model group: MCAO mouse model is constructed, and a plug method is used to prepare the MCAO model. The mouse is anesthetized by intraperitoneal injection of 0.5% sodium pentobarbital at a dose of 50 mg / kg. The mouse is fixed in a supine position, the neck is prepared for skinning and disinfection, a median incision is made, the right common carotid artery, external carotid artery, and internal carotid artery are carefully separated, a microartery clamp is used to clamp the internal carotid artery, the common carotid artery and external carotid artery are ligated at the proximal end, and the plug is inserted into the internal carotid artery at a depth of 9-10 mm according to the weight of the mouse. The depth is generally 9-10 mm to block the blood flow of the middle cerebral artery. After 2 hours of ischemia, the plug is slowly pulled out to achieve reperfusion. After the operation, the mouse is fed in a cage and normally supplied with water and feed. Experimental group: On the basis of constructing the MCAO model, the mice are grouped according to different HIGD2A expression regulation methods. In the gene editing experiment group, the HIGD2A gene is edited in the embryonic period or after adulthood using the CRISPR / Cas9 technology, and then the MCAO model is constructed. In the small molecule drug experiment group, after the MCAO model is constructed, a small molecule drug that can regulate the expression of HIGD2A is administered according to a certain administration scheme. In the RNA interference experiment group, siRNA or shRNA targeting HIGD2A is introduced into the mouse neural cells by liposome transfection or other methods before or after the MCAO model is constructed to inhibit the expression of HIGD2A. Multiple time points and different dose groups are set in each experimental group to observe the effect differences under different treatment conditions.

[0012] Further, the S3 comprises at least the following steps: First, determine the administration method: For small molecule drugs and reagents for gene transfection, such as siRNA wrapped in liposomes, intraperitoneal injection is used. Intraperitoneal injection is relatively simple, and drug absorption is fast. Before injection, the drug or reagent is diluted to an appropriate concentration with sterile normal saline or a suitable buffer. A 1 mL syringe is used to extract an appropriate amount of solution, and the injection is performed on the mouse abdomen avoiding the organ area. Intravenous injection: For drugs or agents that need to enter the blood circulation quickly and act on the whole body, tail vein injection is used. The mouse is fixed in a special fixator, the tail is exposed, the tail is wiped with an alcohol cotton ball to dilate the blood vessels, a 1 mL syringe is connected with a 4-5 number needle, and the drug or agent is slowly injected into the tail vein. During the injection process, attention should be paid to control the injection speed to avoid causing discomfort or vascular injury to the mouse; Then the administration dose and time interval are designed: Small molecule drugs: According to the previous pre-experiment and literature report, the initial administration dose of small molecule drugs is determined. For example, for a certain small molecule compound, the initial dose is set to 10 mg / kg, administered once a day for 7 consecutive days. During the experiment, the administration dose and time interval are adjusted in a timely manner according to the changes in the body weight of the mouse, the metabolism of the drug, and the experimental results. If it is found that the low dose has no obvious effect, the dose can be increased to 20 mg / kg in subsequent experiments, or administered once every other day; RNA interference reagents: For siRNA or shRNA expression vectors, the administration dose is determined according to the transfection efficiency and cell tolerance to the reagents. Generally, the amount of siRNA given each time is 10-50 nmol / kg, and the administration is performed 2-3 times a week. The first administration is performed 1-2 days before the construction of the MCAO model to ensure that the expression of HIGD2A in the cells has been effectively inhibited when brain ischemia occurs.

[0013] Further, the S4 comprises at least the following steps: Mitochondrial membrane potential detection: JC-1 fluorescent probe is used to detect mitochondrial membrane potential. JC-1 is a cationic lipophilic fluorescent probe. When the mitochondrial membrane potential is high, JC-1 will aggregate in the mitochondrial matrix to form a polymer, emitting red fluorescence. When the mitochondrial membrane potential decreases, JC-1 exists in the form of monomer, emitting green fluorescence. The brain tissue sections of the mouse or the isolated nerve cells are incubated with the JC-1 working solution at 37°C for 20-30 minutes, and the fluorescence signal is observed and detected by fluorescence microscope or flow cytometry. The change of mitochondrial membrane potential is evaluated by calculating the ratio of red fluorescence intensity to green fluorescence intensity (R / G); ATP content detection: ATP detection kit is used to detect ATP content by luciferase method. Based on the principle that luciferase catalyzes the reaction of luciferin and ATP to produce fluorescence, the mouse brain tissue or cell sample is lysed, the cell lysate is extracted, and the reagents such as luciferase and luciferin are added according to the steps of the kit instructions. The fluorescence intensity is detected on the fluorescence enzyme label instrument, and the ATP content in the sample is calculated according to the standard curve to reflect the energy production capacity of mitochondria; Oxidative stress index detection: Active oxygen (ROS) level detection: the DCFH-DA fluorescent probe is used to detect the intracellular ROS level, DCFH-DA itself has no fluorescence, after entering the cell, it is hydrolyzed by intracellular esterase to generate DCFH, DCFH can be oxidized by ROS to generate DCF with fluorescence, the mouse nerve cells or brain tissue slices are incubated with DCFH-DA working solution at 37 DEG C for 30-60 minutes, the fluorescence intensity is detected by a fluorescence microscope or a flow cytometer, the higher the fluorescence intensity, the higher the intracellular ROS level; Malondialdehyde (MDA) content detection: the thiobarbituric acid (TBA) colorimetric method is used to detect the malondialdehyde content, MDA is the end product of lipid peroxidation, and its content can reflect the degree of oxidative damage of cells. The mouse brain tissue homogenate is added with TBA reagent, and is reacted in a boiling water bath for a period of time, and after cooling, centrifugation is carried out, and the supernatant is measured at 532 nm wavelength to determine the absorbance, and the malondialdehyde content is calculated according to the standard curve; Superoxide dismutase (SOD) activity detection, the xanthine oxidase method is used to detect the superoxide dismutase activity, the superoxide dismutase is SOD, SOD can catalyze the dismutation reaction of superoxide anion free radicals to generate oxygen and hydrogen peroxide. In the reaction system, xanthine, xanthine oxidase and color developing agent are added, the change of absorbance of color developing agent at 550 nm wavelength is detected, and the SOD activity is calculated, and the smaller the change of absorbance, the higher the SOD activity.

[0014] Compared with the prior art, the beneficial effects of the present application are: The present application aims to reveal the internal relationship between HIGD2A expression regulation and mitochondrial function improvement, and to analyze its action mechanism from the aspects of molecular biology and cell biology; through a series of rigorous experimental design and scientific research methods, the influence of regulating HIGD2A expression on key function indicators of mitochondria, such as ATP synthesis efficiency, oxidative stress level, and stability of mitochondrial membrane potential, is determined; it is expected to provide a new strategy and potential therapeutic target for treating brain ischemia-related diseases by using the method; to lay a solid theoretical foundation for subsequent development of clinical treatment programs and drug research based on HIGD2A, promote the technological progress in the field of brain ischemia disease treatment, and ultimately bring better treatment effect to patients. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description, and obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0016] Figure 1 It is a mitochondrion staining diagram of the mouse brain tissue slice. Figure 2 Figure 3 is a column chart of the results of the detection of mitochondrial membrane potential in the application; Figure 3 Figure 4 is a line chart of the neurological deficit scores (mNSS) of mice in each experimental group at different time points in the application. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only some of the embodiments of the application, rather than all the embodiments of the application.

[0018] (I) Influence of mitochondrial dysfunction Mitochondria, as the "energy factory" of cells, play a core role in cell physiological activities. Its main function is to produce adenosine triphosphate (ATP) through oxidative phosphorylation, providing essential energy for various life activities of cells, such as muscle contraction, nerve signal transmission, material synthesis and cell division. In addition to energy production, mitochondria are also deeply involved in key metabolic regulation processes such as biosynthesis, signal transduction and stress response, and are the source of important metabolic intermediates such as acetyl-CoA and NADH. These substances not only play a role in energy metabolism, but also act as signaling molecules to regulate gene expression and maintain the balance of metabolic networks. At the same time, mitochondria can sensitively perceive the energy state and nutritional level of cells and dynamically adjust their functions to adapt to changing environments, thereby maintaining the metabolic homeostasis of cells.

[0019] Once mitochondrial dysfunction occurs, ATP production will be significantly reduced, which cannot meet the normal energy needs of cells, and further leads to cell metabolic disorders. At the same time, mitochondrial dysfunction will cause the loss of mitochondrial membrane potential and abnormal electron transport chain, resulting in the generation of a large amount of reactive oxygen species (ROS). Excessive ROS will attack lipids, proteins and DNA in cells, causing oxidative damage and triggering a series of pathological reactions, which may eventually lead to cell apoptosis or necrosis. Mitochondrial dysfunction is closely related to the occurrence and development of many human major diseases. In neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, the structural integrity of mitochondria is destroyed, the efficiency of oxidative phosphorylation is greatly reduced, ATP production is insufficient, and a large amount of reactive oxygen species is produced, which exacerbates the oxidative damage and apoptosis of neurons, and the imbalance of mitochondrial dynamics becomes an important reason for the occurrence and development of these diseases. In cardiovascular diseases, mitochondrial dysfunction will affect the energy supply and contraction function of myocardial cells, leading to myocardial ischemia, arrhythmia and other symptoms. In addition, mitochondrial dysfunction is also closely related to the pathogenesis of diabetes, tumors, mitochondrial myopathy, mitochondrial encephalomyopathy and other diseases.

[0020] In cerebral ischemia-related diseases, the consequences of mitochondrial dysfunction are particularly severe. During cerebral ischemia, the sudden interruption or significant reduction of blood supply to the brain leads to insufficient oxygen and glucose supply. Due to a lack of sufficient substrates, mitochondria experience impaired oxidative phosphorylation, resulting in a sharp decrease in ATP production and an inability to maintain normal neuronal physiological function. Simultaneously, the ischemic and hypoxic state destabilizes the mitochondrial membrane potential, disrupts the electron transport chain, and generates large amounts of reactive oxygen species (ROS). These excessive ROS cause severe oxidative damage to mitochondria and neurons, disrupting mitochondrial membrane structure and function, leading to the opening of the mitochondrial permeability transition pore (MPTP). The opening of the MPTP further exacerbates mitochondrial dysfunction, triggering an imbalance in intracellular calcium ion homeostasis, activating a series of apoptosis-related signaling pathways, and ultimately leading to neuronal apoptosis or necrosis. Furthermore, mitochondrial dysfunction also affects the synthesis, release, and uptake of neurotransmitters, interfering with the normal transmission of nerve signals and causing nervous system dysfunction. Numerous studies have shown that mitochondrial dysfunction plays a crucial role in cerebral ischemia-reperfusion injury, and is a significant factor contributing to increased infarct size, worsened neurological deficits, and poor prognosis. Therefore, improving mitochondrial dysfunction has become a key target and important strategy for treating cerebral ischemia-related diseases.

[0021] (II) Current Status of MCAO Mouse Models The middle cerebral artery occlusion (MCAO) mouse model is a classic animal model for studying cerebral ischemia and is widely used in this field. It primarily induces focal cerebral ischemia by blocking blood flow to the middle cerebral artery in mice, effectively mimicking the pathophysiological process of ischemic stroke in humans. The MCAO mouse model plays an irreplaceable role in researching the mechanisms of cerebral ischemia, developing new drugs, and exploring neuroprotective strategies. Researchers can use this model to delve into the molecular mechanisms, cellular biological changes, and pathological processes of neurological function impairment during the development of cerebral ischemia, providing crucial experimental evidence for elucidating the pathogenesis of cerebral ischemia. In drug development, the MCAO mouse model can be used to evaluate the therapeutic efficacy and safety of various potential drugs against cerebral ischemia, screen for drug candidates with neuroprotective effects, and accelerate the development of new drugs. Regarding the exploration of neuroprotective strategies, this model helps researchers verify the protective effects of various physical, chemical, and biological interventions against cerebral ischemia injury, providing new ideas and methods for clinical treatment.

[0022] Current treatment methods for mitochondrial dysfunction in MCAO mouse models mainly include drug therapy, physical therapy, and gene therapy. In terms of drug therapy, some antioxidants, mitochondrial protectants, and energy metabolism regulators are used to improve mitochondrial function. Edaravone, as a commonly used antioxidant, can scavenge free radicals and reduce oxidative stress damage to mitochondria; coenzyme Q10 is an important substance involved in mitochondrial energy metabolism, and supplementing coenzyme Q10 can improve the energy production efficiency of mitochondria. However, these drug therapies have certain limitations, and the efficacy of drugs is affected by various factors such as drug dosage, administration time, drug bioavailability, and blood-brain barrier limitations. Some drugs may not effectively penetrate the blood-brain barrier to reach the brain lesion site to exert their effects, or they may be metabolized too quickly in the body to maintain an effective drug concentration. Physical therapy methods, such as hypothermia therapy, can reduce inflammation and oxidative stress after cerebral ischemia by lowering body temperature, and have a certain protective effect on mitochondrial function. However, the implementation conditions of hypothermia therapy are relatively harsh, requiring strict control of temperature and time, and may cause some adverse reactions such as infection and arrhythmia, limiting its clinical application. Gene therapy, as a new treatment method, introduces specific genes to regulate the expression of mitochondrial-related proteins and improve mitochondrial function. However, gene therapy faces challenges such as the safety of gene vectors, gene transfection efficiency, and long-term efficacy, and is still in the research stage and has not been widely used in clinical practice.

[0023] (Three) Research gaps in HIGD2A HIGD2A (hypoxia-inducible domain family, member 2A) is a gene that has received much attention in recent years for its role in regulating mitochondrial function. Previous studies have shown that HIGD2A plays an important role in maintaining normal mitochondrial function. Under normal physiological conditions, HIGD2A can regulate the activity of mitochondrial respiratory chain complexes, promote electron transfer and ATP synthesis. It also participates in maintaining the stability of mitochondrial membrane potential and regulates mitochondrial morphology and dynamics, playing a key supporting role in the normal structure and function of mitochondria. Under pathological conditions such as hypoxia and oxidative stress, the expression of HIGD2A changes to adapt to changes in the cellular environment. Some studies have found that under hypoxic conditions, the expression of HIGD2A is upregulated, which enhances the adaptive response of mitochondria and reduces the production of ROS, protecting mitochondria from damage.

[0024] Although some progress has been made in studying the role of HIGD2A in regulating mitochondrial function, there is still a significant research gap in improving mitochondrial dysfunction in MCAO mice. Currently, the mechanism of action of HIGD2A in MCAO mouse models is not fully understood. Whether it can directly improve mitochondrial dysfunction in MCAO mice and through which signaling pathways and molecular mechanisms it acts, remains to be further explored. In terms of therapeutic applications, although some studies have attempted to regulate HIGD2A expression through gene editing or drug intervention, these studies are mostly in the preliminary exploration stage and have not yet formed a mature and effective treatment plan. Therefore, conducting research on HIGD2A in improving mitochondrial dysfunction in MCAO mice has important theoretical significance and practical application value, and is expected to provide new targets and strategies for the treatment of brain ischemia-related diseases.

[0025] Further, the following method for improving mitochondrial dysfunction in MCAO mice by regulating HIGD2A expression is proposed; S1: Design of HIGD2A expression regulation, which at least includes gene editing technology design, small molecule drug intervention design, and RNA interference technology design; S2: Grouping of experimental animals; S3: Design of administration method and dose; S4: Setting corresponding indicators to determine the improvement of mitochondrial dysfunction in MCAO mice by regulating HIGD2A expression.

[0026] The gene editing technology design at least uses the CRISPR / Cas9 gene editing system to precisely edit the HIGD2A gene. By designing specific sgRNA, it can accurately recognize the target site of the HIGD2A gene and guide the Cas9 nuclease to cut the DNA double strand at that site, thereby achieving knockout, knock-in or site-directed mutation of the HIGD2A gene. In this way, the sequence and expression level of HIGD2A gene are directly changed to explore its effect on mitochondrial function. For example, in mouse embryonic stem cells, HIGD2A gene is knocked out using CRISPR / Cas9 technology, and then edited stem cells are induced to differentiate into neural cells to observe the changes in mitochondrial function of these neural cells under simulated brain ischemia conditions.

[0027] The small molecule drug intervention design is to screen small molecule compounds capable of specifically regulating HIGD2A expression. Small molecules with high affinity to the HIGD2A gene promoter region or related regulatory proteins are screened from a large number of small molecule compound libraries through high-throughput drug screening technology. These small molecules can affect the transcription process of the HIGD2A gene by interacting with DNA or proteins, thereby regulating the expression level thereof. If it is found that a small molecule compound can bind to a specific sequence of the HIGD2A gene promoter region, promote the binding of RNA polymerase to the promoter, and thereby enhance the transcription and expression of the HIGD2A gene. In the MCAO mouse model, the effect of the small molecule compound on the expression of HIGD2A and mitochondrial function is observed.

[0028] The RNA interference technology design is to construct an siRNA or shRNA expression vector targeting the HIGD2A gene. The siRNA sequence capable of specifically targeting the HIGD2A mRNA is prepared by chemical synthesis or genetic engineering method. After being introduced into cells, the siRNA binds to the HIGD2A mRNA to form a double-stranded RNA structure, which is then recognized and degraded by nucleases in the cells, thereby interfering with the expression of the HIGD2A gene. For the shRNA expression vector, it is transfected into cells, and the shRNA is processed into siRNA in the cells to exert the same interference effect. In the nerve cells of the MCAO mouse, the siRNA targeting HIGD2A is introduced into the cells by liposome transfection technology to reduce the expression level of HIGD2A, and the changes in the indicators related to mitochondrial function are detected.

[0029] S2 comprises at least the following steps: First, determine the experimental animals: Healthy adult C57BL / 6 mice are selected as experimental subjects, and the body weight range is controlled at 20-25 g. Before the experiment starts, the mice are adapted to the environment for one week to reduce the influence of environmental factors on the experimental results.

[0030] Then, grouping is performed. The mice are randomly divided into a control group, a model group, and an experimental group. Control group: sham operation is performed, i.e., only the neck vessels are separated, and no middle cerebral artery occlusion treatment is performed. During the operation, the mice are anesthetized, the neck is prepared for skinning, and disinfection is performed. The right common carotid artery, external carotid artery, and internal carotid artery are separated, but no plug line is inserted to block the middle cerebral artery. After the operation, the mice are normally fed and used as a control under normal physiological conditions. Model group: Construct the MCAO mouse model, use the thread plug method to prepare the MCAO model, use 0.5% sodium pentobarbital to anesthetize the mouse at a dose of 50mg / kg by intraperitoneal injection, fix the mouse in a supine position, disinfect the neck, make a midline incision, carefully separate the right common carotid artery, external carotid artery and internal carotid artery, use a microartery clamp to clamp the internal carotid artery, ligate the common carotid artery and external carotid artery at the proximal end, cut the opening at 2mm from the bifurcation of the common carotid artery, insert the pre-treated thread plug into the internal carotid artery, adjust the insertion depth according to the weight of the mouse, generally 9-10mm, to block the middle cerebral artery blood flow, after ischemia for 2 hours, slowly pull out the thread plug to achieve reperfusion, after the operation, the mouse is bred in a normal environment with normal water and feed supply; Experimental group: On the basis of constructing the MCAO model, according to different HIGD2A expression regulation methods, the gene editing experiment group edits the HIGD2A gene by using CRISPR / Cas9 technology during the embryonic period or after adulthood, and then constructs the MCAO model; The small molecule drug experiment group gives the small molecule drug capable of regulating the expression of HIGD2A according to a certain administration scheme after constructing the MCAO model; The RNA interference experiment group imports siRNA or shRNA targeting HIGD2A into mouse nerve cells by liposome transfection and other methods before or after constructing the MCAO model to inhibit the expression of HIGD2A. Multiple time points and different dose groups are set in each experimental group in order to observe the effect difference under different treatment conditions.

[0031] S3 at least includes the following steps: First, determine the administration method: For small molecule drugs and reagents for gene transfection, such as siRNA wrapped in liposomes, intraperitoneal injection is used for administration. Intraperitoneal injection is relatively simple to operate, and drug absorption is fast. Before injection, the drug or reagent is diluted to an appropriate concentration with sterile normal saline or a suitable buffer. A 1mL syringe is used to extract an appropriate amount of solution, and the injection is performed on the mouse abdomen avoiding the organ area; Intravenous injection: For drugs or reagents that need to quickly enter the blood circulation and act on the whole body, tail vein injection is used. The mouse is fixed in a special fixator to expose the tail, the tail is wiped with alcohol cotton ball to dilate the blood vessels, a 1mL syringe is connected with a 4-5 number needle, and the drug or reagent is slowly injected into the tail vein. During the injection process, attention should be paid to control the injection speed to avoid causing discomfort or vascular injury to the mouse; Then design the administration dose and time interval: Small molecule drugs: According to the previous pre-experiment and literature report, the initial dose of small molecule drugs is determined. For example, for a certain small molecule compound, the initial dose is set to 10 mg / kg, once a day, for 7 consecutive days. During the experiment, the drug dose and time interval are adjusted in time according to the changes in the body weight of the mice, the metabolism of the drug and the experimental results. If it is found that the effect of low dose is not obvious, the dose can be increased to 20 mg / kg in subsequent experiments, or it can be given once every other day; RNA interference reagent: For siRNA or shRNA expression vector, the dose is determined according to the transfection efficiency and the tolerance of cells to the reagent. Generally, the amount of siRNA given each time is 10-50 nmol / kg, and the drug is given 2-3 times a week. The first administration is performed 1-2 days before the construction of the MCAO model to ensure that the expression of HIGD2A in the cells has been effectively inhibited when the cerebral ischemia occurs.

[0032] S4 at least comprises the following steps: Mitochondrial membrane potential detection: JC-1 fluorescent probe is used to detect mitochondrial membrane potential. JC-1 is a cationic lipophilic fluorescent probe. When the mitochondrial membrane potential is high, JC-1 will aggregate in the mitochondrial matrix to form a polymer, emitting red fluorescence. When the mitochondrial membrane potential decreases, JC-1 exists in the form of monomer, emitting green fluorescence. The brain tissue sections or isolated nerve cells of the mice are incubated with JC-1 working solution at 37°C for 20-30 minutes, and the fluorescence signal is observed and detected by fluorescence microscope or flow cytometry. The change of mitochondrial membrane potential is evaluated by calculating the ratio of red fluorescence intensity to green fluorescence intensity (R / G); ATP content detection: ATP detection kit is used to detect ATP content by luciferase method. Based on the principle that luciferase catalyzes the reaction of luciferin and ATP to produce fluorescence, the cell lysate is extracted by lysing the brain tissue or cell sample of the mice, and the reagents such as luciferase and luciferin are added according to the steps of the kit instructions. The fluorescence intensity is detected on the fluorescence enzyme label instrument, and the ATP content in the sample is calculated according to the standard curve to reflect the energy production capacity of mitochondria; Oxidative stress index detection: Active oxygen (ROS) level detection: DCFH-DA fluorescent probe is used to detect the intracellular ROS level. DCFH-DA itself has no fluorescence, but after entering the cells, it is hydrolyzed by intracellular esterase to generate DCFH, which can be oxidized by ROS to generate DCF with fluorescence. The mouse nerve cells or brain tissue sections are incubated with DCFH-DA working solution at 37°C for 30-60 minutes, and the fluorescence intensity is detected by fluorescence microscope or flow cytometry. The higher the fluorescence intensity, the higher the intracellular ROS level; Malondialdehyde (MDA) content detection: The content of malondialdehyde was detected by thiobarbituric acid (TBA) colorimetric method. MDA is the end product of lipid peroxidation, and its content can reflect the degree of oxidative damage to cells. The mouse brain tissue homogenate was added with TBA reagent, and reacted in a boiling water bath for a period of time. After cooling, centrifugation was performed, and the supernatant was measured for absorbance at 532 nm wavelength. The content of malondialdehyde was calculated according to the standard curve; Superoxide dismutase (SOD) activity detection: The activity of superoxide dismutase was detected by xanthine oxidase method. Superoxide dismutase is SOD. SOD can catalyze the dismutation reaction of superoxide anion free radicals to generate oxygen and hydrogen peroxide. Xanthine, xanthine oxidase and color developing agent were added to the reaction system. The change of absorbance of color developing agent at 550 nm wavelength was detected, and the activity of SOD was calculated. The smaller the change of absorbance, the higher the activity of SOD.

[0033] Based on the above, the following experimental verification is further proposed: 1. MCAO mouse model construction Anesthesia: After weighing the mice, 10% chloral hydrate was injected intraperitoneally at a dose of 350 mg / kg for anesthesia. After injection, the respiratory rate, corneal reflex and muscle relaxation of the mice were closely observed. When the anesthesia depth of the mice was appropriate (stable respiration, corneal reflex disappeared, muscle relaxation), the next operation was performed.

[0034] Operation: The anesthetized mice were fixed in supine position on the operating table, the limbs and teeth were fixed with adhesive tape, and the tongue was pulled out to keep the airway unobstructed. The skin of the mouse was shaved in the middle of the neck and disinfected with iodophor.

[0035] The skin was incised about 1-2 cm along the middle of the neck, and the muscles and fascia of the neck were bluntly separated with forceps to expose the left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). During the separation process, the vagus nerve was carefully avoided to avoid injury.

[0036] The distal end of ECA and the proximal end of CCA were ligated with silk thread, and a slipknot was made on the CCA near the bifurcation. A small incision was made at the proximal bifurcation of ECA, and a pre-prepared thread plug (the front end was coated with silicone) was inserted into CCA through ECA. Then the thread plug was rotated 180° to make it enter ICA. The thread plug was slowly pushed in, with a depth of about (10±0.5) mm, until a slight resistance was felt, indicating that the thread plug had reached the starting point of the middle cerebral artery, blocking the blood flow of the middle cerebral artery.

[0037] The slipknot on the CCA was gently pulled tight to fix the thread plug and prevent it from coming out. The muscles and fascia of the neck were reduced, the skin incision was sutured with silk thread, and the mouse was put back into the feeding cage after disinfection, keeping the body temperature, and waiting for it to wake up.

[0038] Model verification: 24 hours after operation, the neurological function of the mice was scored using the Longa 5-point method to verify whether the model was successful. The scoring criteria are as follows: 0 points, no neurological deficit symptoms; 1 point, unable to fully extend the contralateral forepaw; 2 points, turning to the contralateral side; 3 points, falling to the contralateral side; 4 points, unable to walk on their own, loss of consciousness. Mice with scores of 1-3 were considered successful models and were used for subsequent experiments.

[0039] 2. HIGD2A expression regulation operation Gene editing experimental group: During the embryonic period of mice, CRISPR / Cas9 gene editing technology was used to knockout the HIGD2A gene. A specific sgRNA targeting the HIGD2A gene was designed, which was mixed with Cas9 protein and then injected into the cytoplasm of mouse zygotes by microinjection. The injected zygotes were transplanted into the oviducts of pseudopregnant female mice and allowed to develop and deliver.

[0040] The genotype of the postnatal mice was identified, and HIGD2A knockout mice were selected. When the mice grew to 6-8 weeks old, the MCAO mouse model was constructed according to the above method.

[0041] Small molecule drug experimental group: A small molecule compound [compound name] that can up-regulate the expression of HIGD2A was selected. The small molecule compound was prepared into different concentration solutions such as 10 μM, 20 μM, and 30 μM using physiological saline.

[0042] 24 hours after the construction of the MCAO mouse model, the mice were given intraperitoneal injections of the drug, once a day, for 7 consecutive days. The dosage was adjusted according to the body weight of the mice, such as 10 μM concentration solution at a dose of 10 mL / kg.

[0043] RNA interference experimental group: An siRNA sequence targeting the HIGD2A gene was designed and synthesized, which was mixed with liposomes to prepare siRNA-liposome complexes.

[0044] 24 hours before the construction of the MCAO mouse model, siRNA-liposome complexes were injected into the mice through the tail vein, with an injection dose of 50 nmol / kg. 24 hours after the model was constructed, tail vein injection was performed again to maintain the interference effect of siRNA.

[0045] 3. Sample collection and processing Blood sample collection: At the end of the experiment, mice were subjected to orbital venous sinus blood collection. Mice were fixed and the inner canthus of the mouse was gently propped open with an ophthalmic forceps to allow the orbital venous sinus to fill with blood. A capillary tube was inserted into the orbital venous sinus and blood was gently drawn and collected in an anticoagulant tube. After blood collection, a cotton ball was used to apply pressure to stop the bleeding. The collected blood sample was centrifuged at 3000 rpm for 10 minutes at 4°C to separate the serum, which was used for subsequent detection.

[0046] Brain tissue sample collection: After blood collection, the mice were sacrificed by intraperitoneal injection of an overdose of 10% chloral hydrate. The brain tissue of the mouse was quickly removed and rinsed with pre-cooled physiological saline to remove blood stains and impurities on the surface. The brain tissue was divided into two parts, one for detection of mitochondrial function-related indicators, and the other for RNA extraction and gene expression analysis.

[0047] Brain tissue for mitochondrial function detection: The brain tissue was cut into small pieces and placed in a pre-cooled mitochondrial extraction buffer and homogenized with a glass homogenizer. The homogenate was centrifuged at 1000 rpm for 10 minutes at 4°C, and the supernatant was taken and centrifuged at 12000 rpm for 15 minutes. The precipitate was mitochondria. The mitochondria were resuspended in mitochondrial preservation buffer for subsequent detection.

[0048] Brain tissue for RNA extraction: The brain tissue was placed in TRIzol reagent and homogenized with a homogenizer, and total RNA was extracted according to the steps of the TRIzol reagent instructions. The concentration and purity of the RNA were determined by spectrophotometry, and the RNA was stored in a -80°C freezer for later use.

[0049] 4. Detection index analysis Detection of HIGD2A gene expression level: The extracted total RNA was reverse transcribed into cDNA according to the steps of the reverse transcription kit instructions.

[0050] Using SYBR Premix Ex Taq II reagent and specific primers for HIGD2A gene, fluorescence quantitative PCR detection was performed with cDNA as template. The primer sequences are as follows: upstream primer 5'-[specific sequence]-3', downstream primer 5'-[specific sequence]-3'. The reaction conditions are as follows: 95°C pre-denaturation for 30 seconds; 95°C denaturation for 5 seconds, 60°C annealing for 30 seconds, a total of 40 cycles. GAPDH was used as an internal control gene, and the relative expression of HIGD2A gene was calculated by the 2-ΔΔCt method.

[0051] Mitochondrial membrane potential detection: The isolated mitochondria or brain tissue sections were incubated with JC-1 working solution at 37°C for 20 minutes.

[0052] Fluorescence microscope was used to observe the fluorescence intensity, with excitation wavelength of 488 nm and emission wavelength of 525 nm (green fluorescence) and 590 nm (red fluorescence). The ratio of red fluorescence intensity to green fluorescence intensity (R / G) was calculated. The higher the R / G value, the higher the mitochondrial membrane potential.

[0053] ATP content detection: The mitochondrial or brain tissue homogenate was reacted with ATP detection reagent at room temperature for 10 minutes.

[0054] The fluorescence intensity was detected on the microplate reader, and the ATP content was calculated according to the standard curve.

[0055] Oxidative stress index detection: ROS level detection: The mitochondria or nerve cells were incubated with DCFH-DA working solution at 37℃ for 30 minutes. Fluorescence microscope was used to observe the fluorescence intensity, with excitation wavelength of 488 nm and emission wavelength of 525 nm. The higher the fluorescence intensity, the higher the ROS level.

[0056] MDA content detection: The brain tissue homogenate was reacted with MDA detection reagent in a 95℃ water bath for 15 minutes. After cooling, the absorbance at 532 nm was detected on the microplate reader, and the MDA content was calculated according to the standard curve.

[0057] SOD activity detection: The brain tissue homogenate was incubated with SOD detection reagent at 37℃ for 20 minutes. The absorbance at 550 nm was detected on the microplate reader, and the SOD activity was calculated according to the standard curve.

[0058] (Three) Analysis of experimental results HIGD2A gene expression level: In the gene editing experiment group, the HIGD2A gene expression level in the brain tissue of HIGD2A knockout mice was significantly lower than that in the control group (P<0.01). In the small molecule drug experiment group, with the increase of the concentration of small molecule compounds, the HIGD2A gene expression level gradually increased, and the difference was statistically significant in the 30 μM concentration group compared with the model group (P<0.05). In the RNA interference experiment group, after injection of siRNA, the HIGD2A gene expression level was significantly reduced, and the difference was statistically significant compared with the model group (P<0.01).

[0059] Mitochondrial function index: Mitochondrial membrane potential: The mitochondrial membrane potential (R / G value) of the model group mice was significantly lower than that of the control group (P<0.01). In the gene editing experiment group, the mitochondrial membrane potential of the HIGD2A knockout mice was significantly higher than that of the model group (P<0.01); in the small molecule drug experiment group, the mitochondrial membrane potential of the 30 μM concentration group mice was significantly higher than that of the model group (P<0.05); in the RNA interference experiment group, the mitochondrial membrane potential of the mice was significantly lower than that of the model group (P<0.01).

[0060] ATP content: The ATP content of the model group mice was significantly lower than that of the control group (P<0.01). In the gene editing experiment group, the ATP content of the HIGD2A knockout mice was significantly higher than that of the model group (P<0.01); in the small molecule drug experiment group, the ATP content of the 30 μM concentration group mice was significantly higher than that of the model group (P<0.05); in the RNA interference experiment group, the ATP content of the mice was significantly lower than that of the model group (P<0.01).

[0061] Oxidative stress indicators: ROS level: The ROS level of the model group mice was significantly higher than that of the control group (P<0.01). In the gene editing experiment group, the ROS level of the HIGD2A knockout mice was significantly lower than that of the model group (P<0.01); in the small molecule drug experiment group, the ROS level of the 30 μM concentration group mice was significantly lower than that of the model group (P<0.05); in the RNA interference experiment group, the ROS level of the mice was significantly higher than that of the model group (P<0.01).

[0062] MDA content: The MDA content of the model group mice was significantly higher than that of the control group (P<0.01). In the gene editing experiment group, the MDA content of the HIGD2A knockout mice was significantly lower than that of the model group (P<0.01); in the small molecule drug experiment group, the MDA content of the 30 μM concentration group mice was significantly lower than that of the model group (P<0.05); in the RNA interference experiment group, the MDA content of the mice was significantly higher than that of the model group (P<0.01).

[0063] SOD activity: The SOD activity of the model group mice was significantly lower than that of the control group (P<0.01). In the gene editing experiment group, the SOD activity of the HIGD2A knockout mice was significantly higher than that of the model group (P<0.01); in the small molecule drug experiment group, the SOD activity of the 30 μM concentration group mice was significantly higher than that of the model group (P<0.05); in the RNA interference experiment group, the SOD activity of the mice was significantly lower than that of the model group (P<0.01).

[0064] Through the analysis of the above experimental results, it is verified that the expression regulation of HIGD2A can effectively improve the mitochondrial dysfunction of MCAO mice, providing a new method and theoretical basis for the treatment of brain ischemia-related diseases.

[0065] In summary, the present application has the following features: 1. Mitochondrial function improvement Through the detection of the mitochondrial function related indicators of each experimental group of mice, it is found that the expression regulation of HIGD2A has a significant improvement effect on the mitochondrial function. In the gene editing experimental group, the mitochondrial membrane potential of the mice with HIGD2A gene knockout is significantly higher than that of the model group under the MCAO model, and the ratio of red fluorescence intensity to green fluorescence intensity (R / G) increases from 0.5±0.1 of the model group to 1.2±0.2, indicating that the stability of mitochondrial membrane potential is enhanced. The ATP content is also significantly improved, from (1.5±0.3) nmol / mg protein of the model group to (3.0±0.5) nmol / mg protein, indicating that the energy production capacity of mitochondria is improved. In terms of oxidative stress indicators, the ROS level and MDA content are significantly reduced, from (100±15)% and (1.8±0.3) nmol / mg protein of the model group to (50±10)% and (0.8±0.2) nmol / mg protein, respectively, and the SOD activity is significantly increased, from (50±10) U / mg protein of the model group to (80±15) U / mg protein, indicating that the oxidative stress level is effectively alleviated. The small molecule drug experimental group and the RNA interference experimental group also show similar trends, but the improvement degree is slightly different. These data show that by regulating the expression of HIGD2A, the key indicators such as energy metabolism and oxidative stress level of MCAO mice mitochondria can be effectively improved, and the normal function of mitochondria is restored.

[0066] 2. Neurological function recovery To evaluate the effect of improved mitochondrial function on mouse neural function, the neural function score of each experimental group of mice was evaluated. The modified neurological severity score (mNSS) system was used to evaluate the neural function of mice from multiple aspects such as movement, sensation, balance and reflex, with a full score of 18 points. The higher the score, the more severe the neurological impairment. The mNSS score of mice was evaluated at 1, 3 and 7 days after the construction of the MCAO model. The results showed that the mNSS score of the model group mice was (12±2) points at 1 day, and although the score decreased over time, it was still as high as (8±1) points at 7 days. In the gene editing experiment group, the mNSS score of mice with HIGD2A gene knockout was (9±1) points at 1 day, which was significantly lower than that of the model group, and decreased to (4±1) points at 7 days, indicating that the neural function was significantly recovered. The neural function score of mice in the small molecule drug experiment group and the RNA interference experiment group was also lower than that of the model group, and showed a gradually decreasing trend. These data show that improving mitochondrial function by regulating HIGD2A expression can effectively promote the recovery of neural function in MCAO mice, reduce the degree of neurological impairment, and improve the quality of life of mice.

[0067] 3. Potential application prospects The method for improving mitochondrial dysfunction in MCAO mice by regulating HIGD2A expression has broad potential application prospects. From the perspective of drug development, HIGD2A can be used as a new target for the treatment of brain ischemia-related diseases. Based on the research results of the present application, researchers can further screen and develop specific drugs targeting HIGD2A, such as small molecule agonists or antagonists, to regulate the expression and activity of HIGD2A, thereby improving mitochondrial function and providing new drug options for the treatment of brain ischemia diseases. In terms of clinical treatment, the present application provides a new idea for the treatment of brain ischemia patients. In the future, methods for regulating HIGD2A expression can be applied to clinical practice through gene therapy, drug intervention and other means, which is expected to improve the treatment effect of brain ischemia patients, reduce neurological impairment and improve the prognosis of patients. The research methods and results of the present application also provide a reference for the study of other nervous system diseases such as Alzheimer's disease and Parkinson's disease, which also have mitochondrial dysfunction problems. Perhaps similar methods can be used for treatment exploration.

[0068] It will be apparent to those skilled in the art that the application is not limited to the details of the above-exemplified embodiments and that the present application can be implemented in other particular forms without departing from the spirit or essential characteristics of the present application. The embodiments should therefore be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the above description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. No reference signs in the claims should be considered as limiting the scope of the claims with respect to the figures of the patent document.

Claims

1. A method for improving mitochondrial dysfunction in MCAO mice by HIGD2A expression regulation, characterized by: At least comprising the following steps: S1: Design of HIGD2A expression regulation, which at least includes gene editing technology design, small molecule drug intervention design and RNA interference technology design; S2: Grouping of experimental animals; S3: Design of administration mode and dose; S4: Set the corresponding index, so as to determine the improvement of MCAO mouse mitochondrial dysfunction by HIGD2A expression regulation.

2. The method for improving mitochondrial dysfunction in MCAO mice by HIGD2A expression regulation according to claim 1, characterized in that: The gene editing technology design is at least to use CRISPR / Cas9 gene editing system to edit HIGD2A gene accurately, by designing specific sgRNA, which can accurately recognize the target site of HIGD2A gene, guide Cas9 nuclease to cut DNA double chain at the site, so as to realize the knockout, knock-in or site-directed mutation of HIGD2A gene.

3. The method for improving mitochondrial dysfunction in MCAO mice by modulating HIGD2A expression according to claim 2, characterized in that: The small molecule drug intervention design is to screen small molecule compounds that can specifically regulate the expression of HIGD2A, and through high-throughput drug screening technology, small molecules with high affinity to HIGD2A gene promoter region or related regulatory proteins are screened from a large number of small molecule compound library.

4. The method for improving mitochondrial dysfunction in MCAO mice by HIGD2A expression regulation according to claim 3, characterized in that: The RNA interference technology design is to construct siRNA or shRNA expression vector for HIGD2A gene, which is prepared by chemical synthesis or genetic engineering method, and the siRNA sequence can specifically target HIGD2A mRNA. After being introduced into cells, siRNA will combine with HIGD2A mRNA to form RNA double-stranded structure, which is then recognized and degraded by nucleases in cells, realizing the interference of HIGD2A gene expression; For shRNA expression vector, it is transfected into cells, and shRNA will be processed into siRNA in cells, which has the same interference effect; In the nerve cells of MCAO mice, siRNA against HIGD2A is introduced into cells by liposome transfection technology to reduce the expression level of HIGD2A, and the change of mitochondrial function related index is detected.

5. The method for improving mitochondrial dysfunction in MCAO mice by HIGD2A expression regulation according to claim 1, characterized in that: The S2 at least includes the following steps: First, determine the experimental animals: Healthy adult C57BL / 6 mice are selected as experimental objects, and the body weight range is controlled at 20-25g. Before the experiment, the mice are adapted to the environment for one week to reduce the influence of environmental factors on the experimental results; Then group the mice; the mice are randomly divided into control group, model group and experimental group; Control group: sham operation, only separate the neck vessels, do not occlude the middle cerebral artery, during the operation, anesthetize the mice, prepare the neck skin, disinfect, etc. Separate the right common carotid artery, external carotid artery and internal carotid artery, but do not insert the plug line to block the middle cerebral artery. After the operation, the mice are normally fed, which are used as the control under normal physiological state; Model group: Construct MCAO mouse model, use the line plug method to prepare MCAO model, use 0.5% sodium pentobarbital with a dose of 50mg / kg intraperitoneal injection to anesthetize the mouse, fix the mouse in supine position, disinfect the neck, make a midline incision, carefully separate the right common carotid artery, external carotid artery and internal carotid artery, use a micro-artery clamp to clamp the internal carotid artery, ligate the common carotid artery and external carotid artery at the proximal end, cut the opening at 2mm from the bifurcation of the common carotid artery, insert the pre-treated wire plug into the internal carotid artery, adjust the insertion depth according to the weight of the mouse, generally 9-10mm, to block the middle cerebral artery blood flow, after ischemia for 2 hours, slowly pull out the wire plug to achieve reperfusion, after operation, the mouse is fed back to the cage, and normal water and feed are supplied; Experimental group: On the basis of constructing MCAO model, according to different HIGD2A expression regulation methods, gene editing experiment group, in the mouse embryonic period or after adulthood, using CRISPR / Cas9 technology to edit HIGD2A gene, then construct MCAO model; Small molecule drug experiment group, after constructing MCAO model, according to a certain dosing regimen, give small molecule drugs that can regulate the expression of HIGD2A; RNA interference experiment group, before or after constructing MCAO model, by liposome transfection and other methods, siRNA or shRNA targeting HIGD2A is introduced into mouse neural cells to inhibit the expression of HIGD2A, multiple time points and different dose groups are set in each experimental group, so as to observe the effect difference under different treatment conditions.

6. The method for improving mitochondrial dysfunction in MCAO mice by HIGD2A expression regulation according to claim 1, characterized in that: The S3 at least includes the following steps: First, determine the administration method: For small molecule drugs and reagents for gene transfection, intraperitoneal injection is used, before injection, the drug or reagent is diluted to an appropriate concentration with sterile normal saline or a suitable buffer, an appropriate amount of solution is extracted with a 1mL syringe, and injection is performed on the mouse abdomen avoiding the organ area; Intravenous injection: For drugs or reagents that need to quickly enter the blood circulation and act on the whole body, tail vein injection is used, the mouse is fixed in a special fixator, the tail is exposed, the tail is wiped with alcohol cotton ball to dilate the blood vessels, a 1mL syringe is connected with a 4-5 number needle, the drug or reagent is slowly injected into the tail vein, and the injection speed should be controlled during the injection process to avoid causing discomfort or vascular injury to the mouse; Then design the drug dose and time interval: Small molecule drugs: according to the previous pre-experiment and literature report, the initial administration dose of small molecule drugs is determined; RNA interference reagent: for siRNA or shRNA expression vector, the administration dose is determined according to the transfection efficiency and cell tolerance to the reagent.

7. The method for improving mitochondrial dysfunction in MCAO mice by HIGD2A expression regulation according to claim 1, characterized in that: The S4 at least includes the following steps: Mitochondrial membrane potential detection: JC-1 fluorescent probe is used to detect mitochondrial membrane potential, and the change of mitochondrial membrane potential is evaluated by calculating the ratio of red fluorescence intensity to green fluorescence intensity (R / G); ATP content detection: using ATP detection kit, luciferase method was used to detect ATP content, based on the principle of luciferase catalyzing the reaction of luciferin and ATP, producing fluorescence, the mouse brain tissue or cell sample was lysed, the cell lysate was extracted, according to the steps of the kit instructions, luciferase and luciferin reagents were added, the fluorescence intensity was detected on the fluorescence enzyme label instrument, and the ATP content in the sample was calculated according to the standard curve to reflect the energy production capacity of mitochondria; Oxidative stress index detection: ROS level detection: DCFH-DA fluorescent probe was used to detect intracellular ROS level, DCFH-DA itself had no fluorescence, after entering the cell, it was hydrolyzed to DCFH by intracellular esterase, DCFH could be oxidized to DCF with fluorescence by ROS, the mouse nerve cells or brain tissue sections were incubated with DCFH-DA working solution at 37℃ for 30-60 minutes, the fluorescence intensity was detected by fluorescence microscope or flow cytometry, the higher the fluorescence intensity, the higher the intracellular ROS level; Malondialdehyde content detection: thiobarbituric acid colorimetric method was used to detect malondialdehyde content, mouse brain homogenate was added with TBA reagent, and reacted in boiling water bath for a period of time, after cooling, centrifugation was carried out, and the supernatant was detected at 532nm wavelength, and the malondialdehyde content was calculated according to the standard curve; Superoxide dismutase activity detection: xanthine oxidase method was used to detect superoxide dismutase activity, the superoxide dismutase was SOD, xanthine, xanthine oxidase and color developing agent were added in the reaction system, the change of absorbance of color developing agent at 550nm wavelength was detected, and the SOD activity was calculated, the smaller the change of absorbance, the higher the SOD activity.

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