Application of ASS1 in preparation of medicine for treating ischemic myocardial injury

Abstract

The invention discloses application of ASS1 as a drug target in development of myocardial ischemia injury treatment drugs and application of ASS1 as a biomarker in diagnosis of myocardial ischemia injury. The invention provides a new way for diagnosis and treatment of myocardial ischemia injury.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the application of ASS1 (argininosuccinate synthase 1) in the development of drugs for treating myocardial ischemia injury and / or myocardial infarction, and the use of ASS1 or its gene in the preparation of diagnostic reagents for myocardial ischemia injury and / or myocardial infarction. Background Art

[0002] Argininosuccinate Synthase 1 (ASS1) is a key rate-limiting enzyme in the urea cycle, responsible for catalyzing the formation of argininosuccinate from citrulline and aspartic acid, thereby synthesizing arginine. Arginine is an important semi-essential amino acid in the body, which not only serves as a basic raw material for protein synthesis, but also participates in regulating a variety of important physiological processes, including nitric oxide (NO) production, immune response, cell proliferation, and oxidative stress response. As the core enzyme controlling arginine synthesis, the absence or abnormality of ASS1 function can lead to metabolic disorders and affect the body's homeostasis.

[0003] The expression and function of ASS1 have attracted much attention in tumor metabolism research. In various cancers, such as liver cancer, lung adenocarcinoma, glioma, and mesothelioma, the expression of ASS1 is often down-regulated, and this change increases the dependence of tumor cells on exogenous arginine. This feature is called "arginine auxotrophy", which provides a new direction for the metabolic targeted therapy of tumors.

[0004] The function of ASS1 is also involved in gene expression regulation and signal pathway regulation. Research shows that ASS1 can affect the processes of cell proliferation, differentiation, and apoptosis by participating in the arginine-nitric oxide (NO) metabolic pathway. In the research of non-tumor diseases, the regulatory role of ASS1 in vascular endothelial cell function has been reported. ASS1 supports the production of nitric oxide (NO) by regulating arginine synthesis, thereby maintaining the stability of vascular endothelial function. In the atherosclerosis model, the abnormal expression of ASS1 can lead to a decrease in NO production and exacerbate vascular inflammatory responses. However, the research on the role of ASS1 in endothelial cells is relatively limited, and there is still a gap compared with its comprehensiveness in tumor research. Summary of the Invention

[0005] Although ASS1 shows important functional regulatory roles in various cell types and pathological processes, there is currently no direct research report on the role of ASS1 in cardiomyocytes. This indicates that the specific mechanism of ASS1 in the pathophysiological process of the heart still needs to be further explored, and its possible functions and potential diagnostic and therapeutic values are worthy of in-depth study.

[0006] In the study of the mechanism of myocardial death in acute myocardial infarction, by detecting the changes in the expression levels of numerous proteins in the serum samples of patients with myocardial infarction (MI), it was unexpectedly found that the expression of ASS1 was significantly decreased in MI patients. Further, by knocking down ASS1 in neonatal rat cardiomyocytes, it was found that its ability to tolerate hypoxia was significantly decreased, and the apoptosis level increased. Since myocardial infarction (MI) is mainly an acute disease caused by myocardial ischemia and necrosis, the research results indicate that ASS1 can be used as a potential diagnostic marker and therapeutic target for myocardial ischemia injury and / or myocardial infarction, providing a new research direction for the diagnosis and treatment of myocardial ischemia injury and / or myocardial infarction. Based on the above research findings, the present invention includes the following technical solutions.

[0007] The first aspect of the present invention provides the use of ASS1, namely argininosuccinate synthase 1 (Argininosuccinate Synthase 1), in the development of drugs for treating myocardial ischemia injury and / or drugs for treating myocardial infarction (MI), wherein ASS1 or its expression gene is used as a drug target.

[0008] Preferably, the above-mentioned ASS1 is human ASS1, with the NCBI accession number NP_000041.2 (the NCBI mRNA accession number corresponding to the encoding gene is NM_000050.4), the amino acid sequence is as shown in SEQ ID NO:1, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO:2.

[0009] The second aspect of the present invention provides a drug for treating myocardial ischemia injury, wherein the active pharmaceutical ingredient (API) is a substance that promotes the expression of ASS1, a substance that upregulates the expression level of ASS1, or a substance that enables an individual to express ASS1.

[0010] Preferably, the above-mentioned active pharmaceutical ingredient has the function of reducing the apoptosis level of cardiomyocytes after myocardial infarction.

[0011] Optionally, the above-mentioned active pharmaceutical ingredient can be a small molecule agonist of the ASS1 gene, the ASS1 gene, the ASS1 gene mRNA, an expression plasmid containing the ASS1 gene, or an expression plasmid containing the coding region of the ASS1 gene (SEQ ID NO:2).

[0012] The vector of the above-mentioned expression plasmid can be any eukaryotic expression vector as long as it can mediate the expression of the target gene.

[0013] In one embodiment, the above-mentioned drug can be a pharmaceutical composition, which in addition to containing a therapeutically effective amount of the active pharmaceutical ingredient, also contains a pharmaceutically acceptable carrier.

[0014] In another embodiment, the above-mentioned drug can be a pharmaceutical composition, which, in addition to containing a therapeutically effective amount of the above-mentioned drug active ingredient, further contains one or more other drug ingredients for treating myocardial ischemia injury.

[0015] The dosage form of the above-mentioned drug can be an injection dosage form or an oral preparation.

[0016] The third aspect of the present invention provides the use of ASS1 or its gene in the preparation of a diagnostic reagent for myocardial ischemia injury and / or myocardial infarction, wherein ASS1 or its expressed gene is used as a biomarker for judging myocardial ischemia injury.

[0017] Specifically, when the level (concentration / content) of ASS1 or its gene in the biological sample of the subject to be tested is more than 21%, preferably more than 30%, more preferably more than 31% lower than the average level of ASS1 or its gene in healthy individuals, it indicates that the subject to be tested has myocardial ischemia injury.

[0018] Optionally, the above-mentioned biological sample is selected from the following group: whole blood, plasma, serum, saliva, oral mucosa, nasopharyngeal secretion, tissue fluid, body fluid, urine, and more preferably serum.

[0019] Furthermore, when the subject to be tested is a human and the biological sample is serum, if the content of ASS1 protein in the serum sample is not higher than 6.0 ng / ml, for example not higher than 5.7 ng / ml, it indicates that the subject to be tested has myocardial ischemia injury.

[0020] The fourth aspect of the present invention provides a diagnostic kit for myocardial ischemia injury and / or myocardial infarction, which is used for implementing the above-mentioned use, and its detection target is ASS1 in a biological sample.

[0021] Specifically, the detection method of the above-mentioned diagnostic kit for myocardial ischemia injury and / or myocardial infarction is to measure the content of ASS1 protein or gene level in a biological sample.

[0022] For example, the content of antigen ASS1 in a biological sample can be measured by using the principle of antibody-antigen affinity binding, including:

[0023] (1) An antibody capable of binding ASS1; and

[0024] (2) A standard sample composed of a solution containing a known amount of ASS1, wherein ASS1 is ASS1 protein extracted from a human or a mammal or recombinant ASS1 protein produced by fermentation of a genetically engineered bacterium.

[0025] In one embodiment, the above-mentioned myocardial ischemia injury and / or myocardial infarction diagnostic kit can be a latex immunoturbidimetry kit, a magnetic particle chemiluminescence kit, a radioimmunoassay kit, or an immunochromatographic test strip.

[0026] Furthermore, the above-mentioned myocardial ischemia injury and / or myocardial infarction diagnostic kit further includes: (3) a labeled antibody that can bind to ASS1 when ASS1 binds to the antibody defined in (1).

[0027] For example, the above-mentioned diagnostic kit is a sandwich immunoassay kit, selected from the following methods:

[0028] A. Enzyme-linked immunosorbent assay kit;

[0029] B. Magnetic particle chemiluminescence kit;

[0030] C. Immunochromatographic test strip.

[0031] Preferably, an instruction manual is also included in any of the above-mentioned kits.

[0032] The above-mentioned instruction manual can be written on bottles, test tubes and the like, plates, or on a separate piece of paper, or on the outside or inside of the container. For example, it is a paper piece with a download window for an operation demonstration video APP such as a QR code. The instruction manual can also be in a multimedia form, such as a CD, a USB flash drive, a network disk, etc.

[0033] In another embodiment, the detection object of the above-mentioned myocardial ischemia injury and / or myocardial infarction diagnostic kit further includes other myocardial ischemia injury biomarkers in biological samples, that is, the kit simultaneously detects or jointly detects ASS1 and other types of myocardial ischemia injury and / or myocardial infarction biomarkers. The other myocardial ischemia injury and / or myocardial infarction biomarkers include, but are not limited to, myocardial enzymes and myocardial proteins, etc. Among them, the myocardial enzymes are selected from serum aspartate aminotransferase, serum lactate dehydrogenase and its isoenzymes, serum creatine kinase and its isoenzymes; the myocardial proteins are selected from troponin, myoglobin, etc.

[0034] The present invention first discovers that ASS1 is closely related to myocardial ischemia injury and / or myocardial infarction and can be used as a biomarker for this disease. At the same time, based on this discovery, ASS1 has the potential to be used as a drug target for developing drugs for the treatment of myocardial ischemia injury and / or myocardial infarction, providing a new approach for the diagnosis and treatment of myocardial ischemia injury and / or myocardial infarction. Description of the Drawings

[0035] Figure 1The western blot detection of ASS1 protein in serological samples of myocardial infarction patients and healthy subjects is shown. Among them, A is the SDS electrophoresis photo of ASS1 protein detection; B is the statistical result of ASS1 level; *P<0.05, **P<0.01.

[0036] Figure 2 The statistical results of the ELISA detection of the abundance of ASS1 protein in serological samples of myocardial infarction patients and healthy subjects are shown. *P<0.05, **P<0.01.

[0037] Figure 3 The detection of the content of ASS1 protein and the cardiomyocyte apoptosis protein cleaved-caspase3 (CC3) in neonatal rat cardiomyocytes transfected with small interfering siRNA molecules siASS1 and negative control siNC under normal condition (Ctrl) induction and hypoxia induction is shown, with β-actin as the internal reference. Among them, A is the SDS electrophoresis photo of western blot detection; B is the statistical result of CC3 protein level; C is the statistical result of ASS1 level, *P<0.05, **P<0.01.

[0038] Figure 4 The detection of the Tunel staining of neonatal rat cardiomyocytes transfected with small interfering siRNA molecules siASS1 and negative control siNC under hypoxia induction is shown. Among them, A is the Tunel staining photo; B is the comparison result of Tunel staining, *P<0.05, **P<0.01. Detailed implementation

[0039] In our work on studying the types of proteins closely related to the occurrence and development of myocardial infarction (MI), it was detected that the expression level of ASS1 in the serum samples of MI patients was significantly decreased. It is suggested that ASS1 has the potential for a new use as a diagnostic marker for myocardial ischemia injury, and thus may be used as a new tool for the clinical diagnosis of myocardial ischemia injury and / or myocardial infarction. In further animal experimental studies, by knocking down the expression of ASS1 in neonatal rat cardiomyocytes with siRNA, it was found that the tolerance of cardiomyocytes to hypoxia was significantly decreased, while the apoptosis level increased. It is predicted that ASS1 has the potential for a new use as a therapeutic target for myocardial ischemia injury, providing a new drug screening target for the treatment of myocardial ischemia injury and / or myocardial infarction, and having important clinical transformation value.

[0040] It should be understood that in phrases such as "A and / or B" described herein, the terms "and / or", "and / or" are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, in phrases such as "A, B and / or C", the term "and / or" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0041] In view of the use of ASS1 as a biomarker and / or drug target for myocardial ischemia injury and / or myocardial infarction (MI), it is obvious to those skilled in the art that various types of reagent products such as kits can be developed as auxiliary diagnostic tools for myocardial ischemia injury and / or myocardial infarction (MI). For example, the forms of kits that can be developed include but are not limited to: latex immunoturbidimetry kits containing only one antibody (monoclonal antibody or polyclonal antibody, preferably monoclonal antibody), chemiluminescence kits such as magnetic particle chemiluminescence kits, radioimmunoassay kits; enzyme-linked immunosorbent assay (ELISA) kits, magnetic particle chemiluminescence kits, immunochromatographic test strips, etc. containing two different antibodies (monoclonal antibody and / or polyclonal antibody).

[0042] What can be used in the preparation of reagents for detecting myocardial ischemia injury and / or myocardial infarction (MI) can be natural proteins extracted from humans or mammals, or recombinant human proteins produced by fermentation of genetically engineered bacteria. Preferably, the amino acid sequence of the protein is identical to that of the human protein and retains the same antigen recognition epitope / antigen determinant.

[0043] Monoclonal antibodies and polyclonal antibodies can be commercially available antibodies purchased through commercial channels, or can be prepared by conventional technical means, for example, prepared using, preferably human source, as an antigen. Typically, monoclonal antibodies can be prepared by immunizing animals with proteins. The steps include: after fully mixing the protein as an immunogen with an equal volume of Freund's complete adjuvant, immunizing animals such as mice by subcutaneous multi-point injection; measuring the serum titer several weeks later, selecting mice with good immunoreactivity and boosting the immunization multiple times, and then fusing splenocytes with myeloma cells by PEG-mediated conventional method and selecting and culturing with HAT conditioned medium; after fusion, placing it in CO 2After culturing in an incubator at 37°C for 9 - 11 days, larger cell clones appeared in the wells; screening was started on the 11th day using indirect ELISA; the wells with positive primary screening were subjected to 4 rounds of cloning culture using the limiting dilution method to allow the screened cells to divide and multiply in large numbers, and then the cells were amplified, cryopreserved, and ascites was prepared; the mice were treated with pristane, and after one week, hybridoma cells were inoculated intraperitoneally, and ascites was collected 10 days later. The titer of the monoclonal antibody was determined by the indirect ELISA method to obtain a monoclonal antibody with a titer meeting the requirements; after ammonium sulfate precipitation, it was further purified by Protein G affinity purification, and the monoclonal antibody was isolated and purified, and after aliquoting, it was freeze-dried and stored at low temperature.

[0044] Similarly, polyclonal antibodies can be prepared by immunizing animals with proteins, and the steps include: selecting rabbits as immunized animals; in the primary immunization, the protein is used as an immunogen and mixed with an equal volume of Freund's complete adjuvant - after thorough emulsification, it is subcutaneously injected at multiple points on the back of the rabbit; the booster immunization is carried out once every 4 weeks, and after thorough emulsification of the antigen with incomplete Freund's adjuvant, it is subcutaneously injected at multiple points on the back; on the 10th day after the last booster immunization, blood is taken from the carotid artery and the serum is separated; the titer of the prepared polyclonal antibody is measured by the indirect ELISA method to obtain a polyclonal antibody with a titer meeting the requirements, blood is taken by carotid artery intubation and the serum is separated; after ammonium sulfate precipitation, it is further purified by Protein G affinity purification, and the monoclonal antibody is isolated and purified, and after aliquoting, it is freeze-dried and stored at low temperature.

[0045] Detecting antigen proteins in biological samples using monoclonal antibodies and polyclonal antibodies is based on immunological techniques. There are various detection means for immunological techniques well-known in the field of clinical medicine. Generally speaking, the level (content) in biological samples can be measured using methods selected from the following group: Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunoassay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; immunofluorescence assay; mass spectrometry; fluorescence-activated cell sorting (FACS); immunoelectrophoresis assay. In some embodiments, the antibody or its antigen-binding portion is detectably labeled or generates a detectable signal.

[0046] In some embodiments, the antibody reagent can be detectably labeled. In some embodiments, the antibody reagent can be attached to a solid support (e.g., bound to a solid support). In some embodiments, the solid support can comprise particles (including but not limited to agarose or latex beads or particles or magnetic particles), beads, nanoparticles, polymers, substrates, slides, coverslips, plates, culture dishes, wells, membranes, and / or gratings. The solid support can include many different materials, including but not limited to polymers, plastics, resins, polysaccharides, silicon- or silica-based materials, carbon, metals, inorganic glasses, and membranes.

[0047] In one embodiment, the assays, methods, and / or systems described herein may include ELISA. In an exemplary embodiment, a first antibody reagent may be immobilized on a solid support (commonly a polystyrene microtiter plate). The solid support may be contacted with a sample obtained from a subject, and the antibody reagent will bind (“capture”) its specific antigen. The solid support may then be contacted with a second labeled antibody reagent (e.g., a detection antibody reagent). The detection antibody reagent may, for example, comprise a detectable signal, be covalently linked to an enzyme, or itself may be linked to an enzyme via bioconjugation for detection by a secondary antibody. The presence of a signal indicates that both the first antibody reagent immobilized on the support and the second “detection” antibody reagent have bound to the antigen, i.e., the presence of a signal indicates the presence of the molecule. Between each step, the plate is typically washed with a mild detergent solution to remove any non-specifically bound proteins or antibodies. After the final wash step, the plate is developed by adding an enzyme substrate to produce a visible signal that indicates the content in the sample. Older ELISAs used chromogenic substrates, but newer assays use fluorescent substrates with higher sensitivity. There are also other different forms of ELISA, which are well known to those skilled in the art.

[0048] On the other hand, the close relationship between ASS1 and myocardial ischemia injury and / or myocardial infarction (MI) makes ASS1 or its gene suitable as a drug target for developing drugs for treating myocardial ischemia injury and / or myocardial infarction (MI), preferably a drug that can increase the expression level of ASS1 in a patient.

[0049] The terms “increase” or “elevate” or “raise” of (ASS1 expression level) are used herein to mean a statistically significant amount of increase. In some embodiments, “increase” or “elevate” or “raise” generally means an increase of at least 10% compared to a reference level, and may include, for example, an increase of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more.

[0050] The drugs for treating myocardial ischemia injury and / or myocardial infarction (MI) developed by the present invention are substances that promote ASS1 expression, upregulate the ASS1 expression level, or enable an individual to express ASS1. This substance has the function of reducing the level of myocardial cell apoptosis after myocardial infarction.

[0051] The drug can be either a chemical drug or a genetically engineered drug, including small molecule agonists of the ASS1 gene, the ASS1 gene, ASS1 gene mRNA, an expression plasmid containing the ASS1 gene, or an overexpression plasmid containing the ASS1 coding gene (SEQ ID NO:2).

[0052] The drug can be a single-component drug containing a therapeutically effective amount of a drug active ingredient, or a pharmaceutical composition further containing other components such as a pharmaceutically acceptable carrier.

[0053] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of humans and animals within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic reaction or other problems or complications, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers are well known in the art, and these carriers include liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient, including, for example, aqueous solutions (such as water or physiological buffer saline) or other solvents or vehicles (such as glycols, glycerol, oils (such as olive oil) or injectable organic esters). Excipients can be selected, for example, to achieve delayed release of the medicament or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in unit dosage form, such as tablets, capsules (including dispersed capsules and gelatin capsules), granules, powders, solutions, syrups, suppositories, injections, etc.

[0054] As used herein, the term "effective amount" refers to the therapeutically effective amount required to alleviate at least one or more symptoms of a disease or condition and relates to an adequate amount of a drug that provides the desired effect. Thus, the term "therapeutically effective amount" refers to the amount of a treatment that, when administered to a typical subject, is sufficient to cause a specific effect. In various contexts, the effective amount as used herein also includes an amount sufficient to delay the development of a disease condition, alter the course of a disease condition (e.g., but not limited to, slowing the progression of a disease condition), or reverse a disease condition. It should be understood that there are many ways known in the art to determine the effective amount for a given application. For example, pharmacological methods for dose determination can be used in a therapeutic context. In the context of therapeutic or prophylactic applications, the amount of the composition administered to a subject will depend on the type and severity of the disease as well as the characteristics of the individual, such as general health status, age, sex, weight, and tolerance to the drug. It also depends on the extent, severity, and type of the disease. Those skilled in the art will be able to determine the appropriate dose based on these and other factors. For example, the therapeutically effective amount of a small molecule agonist and a plasmid can be determined with reference to the safe usage amount when they are currently administered to patients with myocardial ischemia injury and / or myocardial infarction (MI) for the treatment of myocardial ischemia injury and / or myocardial infarction (MI), and through clinical investigations. The appropriate effective dosage also needs to consider therapeutic factors such as the drug dosage form, the physical constitution of the administered individual, weight, age, disease progression, and administration site.

[0055] For chemical drugs such as small molecule agonists for the treatment of myocardial ischemia injury and / or myocardial infarction (MI), the drug dosage form may contain, in addition to the main component, a pharmaceutically acceptable carrier. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars (such as lactose, glucose, and sucrose); (2) starches (such as corn starch and potato starch); (3) cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate); (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients (such as cocoa butter and suppository wax); (9) oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil); (10) diols (such as propylene glycol); (11) polyols (such as glycerol, sorbitol, mannitol, and polyethylene glycol); (12) esters (such as ethyl oleate and ethyl laurate); (13) agar; (14) buffering agents (such as magnesium hydroxide and aluminum hydroxide); (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0056] A pharmaceutical preparation can be administered to a subject by any one of many routes of administration, including, for example, injections such as lyophilized powder injections, oral administration (e.g., as a drench in an aqueous or non-aqueous solution or suspension, tablets, capsules (including dispersed capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment, or spray applied to the skin). Chemical drugs can also be formulated for inhalation. In certain embodiments, a chemical drug can simply be dissolved or suspended in a sterile solvent.

[0057] For a genetically engineered drug such as a plasmid for treating myocardial ischemia injury and / or myocardial infarction (MI), the pharmaceutical dosage form can be in various forms as long as it is suitable for administration of the corresponding disease and appropriately maintains the activity of the DNA molecule. For example, for an injectable drug delivery system, the dosage form can be a lyophilized powder. Optionally, any pharmaceutically acceptable carrier and adjuvant can be included in the above pharmaceutical dosage form as long as it is suitable for the corresponding drug delivery system and appropriately maintains the activity of the DNA molecule.

[0058] The preparation of a pharmacological composition containing an active ingredient dissolved or dispersed therein is well known in the art and does not require limitation based on the formulation. Generally, such compositions are prepared as injectable liquid solutions or suspensions. However, solid forms suitable for solutions or suspensions to be placed in a liquid before use can also be prepared. The preparation can also be emulsified or exist in the form of a liposome composition. The active ingredient can be mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient and in an amount suitable for the treatment methods described herein. Suitable excipients include, for example, water, saline, glucose, glycerol, ethanol, etc. and combinations thereof. In addition, if necessary, the composition can contain a small amount of auxiliary substances, such as wetting agents or emulsifying agents, pH buffering agents, etc., which enhance or maintain the effectiveness of the active ingredient. An exemplary liquid carrier is a sterile aqueous solution that contains nothing other than the active ingredient and water, or contains a buffer such as sodium phosphate at physiological pH, saline, or both, such as phosphate buffered saline. Further, the aqueous carrier can contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol, and other solutes. In addition to water, the liquid composition can contain a liquid phase. Examples of such other liquid phases are glycerol, vegetable oils (e.g., cottonseed oil), and water-oil emulsions. The amount of the active ingredient used effectively to treat myocardial ischemia injury and / or myocardial infarction (MI) in the present invention will depend on the nature of the disease or disorder and can be determined by standard clinical techniques.

[0059] The above-mentioned medicament can be administered by subcutaneous injection, intramuscular injection, intravenous injection or intravenous infusion gradually over time. Appropriate formulations for a given route, for example, the medicament that can be used in the methods and compositions described herein can be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, and if necessary, can be delivered by a peristaltic pump, via a venous port, or by other means known to those skilled in the art. Intravenous or intramuscular administration is preferred for treating patients with myocardial ischemic injury and / or myocardial infarction (MI).

[0060] The individual or subject receiving this treatment for myocardial ischemic injury and / or myocardial infarction (MI) is any animal in need, including primates (especially humans) and other mammals (such as horses, cows, pigs, sheep, cats and dogs).

[0061] To make the present invention more clearly understandable, preferred embodiments are described in detail below in conjunction with the accompanying drawings. Those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and are not intended to limit the present invention.

[0062] Example

[0063] The siRNA and PCR primers in the examples were synthesized by Biomics Biotechnologies Co., Ltd.

[0064] In the embodiments of the present invention, if no specific description is made for the experimental operation temperature, the temperature generally refers to room temperature (10-30 °C).

[0065] In this article, the addition amounts, contents and concentrations of various substances are involved. Among them, the percentage content, unless otherwise specified, all refers to the mass percentage content.

[0066] Statistical analysis: All data in the examples are expressed as mean ± standard deviation (SD), and statistical analysis is performed using GraphPad Prism 8.0. Student's t-test is used to compare the differences between two groups, and one-way analysis of variance (ANOVA) and Tukey's multiple comparison test are used to compare the differences between three or more groups after that. A P value less than 0.05 between groups is considered statistically significant.

[0067] Example 1: Detection method of argininosuccinate synthetase (ASS1)

[0068] Serum samples from myocardial infarction patients and healthy subjects collected from Zhongshan Hospital Affiliated to Fudan University were used to detect the protein of argininosuccinate synthetase 1 (ASS1) by western blot. Subsequently, a large number of serum samples from healthy subjects (n = 80) and myocardial infarction patients were detected by ELISA to determine the abundance of ASS1 protein, including the following steps.

[0069] 1. Western Blot Experiment

[0070] (1) Preparation of SDS-Polyacrylamide Gel: After preparing the glass plates and checking their tightness, prepare a 10% separating gel, pour it into the glass plates, cover the liquid surface with anhydrous ethanol to ensure flatness, and wait for complete polymerization at room temperature. Then pour out the covering liquid, rinse the surface with a small amount of deionized water, and then prepare a 5% stacking gel. After thoroughly mixing it, add it on top of the separating gel, insert the comb and remove the air bubbles, and let it stand until the gel solidifies.

[0071] (2) Sample Loading: Gently remove the solidified comb, and slowly add the processed protein samples to each sample well using a micropipette, taking care to avoid overflow or contamination.

[0072] (3) Electrophoresis: Set the constant voltage mode. The running conditions for the stacking gel are 60 V, and for the separating gel are 90 V until the bromophenol blue dye reaches the bottom of the gel, at which point the electrophoresis is complete.

[0073] (4) Protein Transfer: Cut the separating gel and soak it in the wet transfer buffer for about 10 minutes. Prepare the PVDF membrane, activate it with methanol for 10 seconds first, then rinse it with distilled water, and then place it in the wet transfer buffer for pre-equilibration. Place the sponge pads, filter papers, gel, PVDF membrane, filter papers, and another sponge pad in the wet transfer cassette in sequence, ensuring no air bubbles between the membrane and the gel during assembly. Insert the wet transfer cassette into the transfer tank and start the electrotransfer instrument.

[0074] (5) Blocking: After the transfer is complete, place the PVDF membrane in a blocking solution containing 5% non-fat milk powder and incubate it on a shaker at room temperature for 1 hour to reduce non-specific binding.

[0075] (6) Primary Antibody Incubation: Dilute the primary antibody according to the instructions (usually 1:1000), immerse the PVDF membrane in the antibody dilution solution, and incubate it on a shaker at 4°C overnight to ensure sufficient antibody binding.

[0076] (7) Membrane Washing: After the primary antibody incubation, wash the PVDF membrane with TBST buffer for 7 - 10 minutes each time, for a total of three times to remove the unbound primary antibody.

[0077] (8) Secondary Antibody Incubation: Add the diluted HRP-labeled secondary antibody (usually 1:5000) to the washed membrane, incubate it at room temperature for 1 hour, and then wash it three times with TBST, 3 - 5 minutes each time.

[0078] (9) Protein development: After removing the liquid on the surface of the PVDF membrane with absorbent paper, add the developing solution. Place the membrane in the imaging system and capture and analyze the signal according to the development intensity of the target protein.

[0079] 2. ELISA detection

[0080] (1) In this experiment, the Human ASS1 (Argininosuccinate synthase 1) ELISA Kit (Catalogue No.: EH2494) from Fine Test was used for detection. ELISA detection is based on the sandwich enzyme-linked immunosorbent assay technique. First, the anti-ASS1 antibody was pre-coated on a 96-well plate. After adding the sample or standard, it binds to the antibody to form a complex. The biotin-labeled antibody was used as the detection antibody. Through the catalytic action of HRP-streptavidin, the TMB substrate was converted into a blue reaction product, and then turned yellow after adding the stop solution. The OD value was read at 450 nm, and the OD value was positively correlated with the concentration of ASS1 in the sample.

[0081] (2) Standard preparation: According to the instructions, the lyophilized standard was reconstituted with the sample diluent and serially diluted. The standard concentration range was from 10 ng / mL to 0.156 ng / mL. The sample diluent was added to the blank wells as a control.

[0082] (3) Sample treatment: After collecting the blood sample, it was left overnight at 4°C to allow the blood to clot fully. Centrifuge at 1000×g for 20 minutes and take the supernatant for detection.

[0083] (4) Sample addition and incubation: Add 100 μL of the standard, sample, or blank control to each well. Gently tap the plate to ensure even distribution of the liquid and incubate at 37°C for 90 minutes.

[0084] (5) Plate washing: After incubation, wash the plate with the pre-diluted washing solution. Add 350 μL of the washing solution each time, let it stand for 30 seconds and then pour out the liquid. Wash twice in total.

[0085] (6) Detection antibody incubation: Add 100 μL of the biotin-labeled detection antibody working solution and incubate at 37°C for 60 minutes. Repeat the plate washing step 3 times after incubation.

[0086] (7) Enzyme-labeled reaction: Add 100 μL of the HRP-streptavidin working solution to each well and incubate at 37°C for 30 minutes. Then wash the plate 5 times with the washing solution to ensure removal of unbound substances.

[0087] (8) Color reaction: Add 90 μL of TMB substrate solution to each well, incubate at 37 °C in the dark for 10 - 20 minutes, and observe the degree of color development. When the gradient color development reaches the expected effect, add 50 μL of stop solution, and the color changes from blue to yellow.

[0088] (9) Data determination: Use an enzyme-linked immunosorbent assay (ELISA) reader to read the absorbance value (OD value) of each well at 450 nm. If necessary, perform background correction using 630 nm or 570 nm. Calculate the concentration of ASS1 in the sample according to the standard curve.

[0089] Experimental results: First, it was found by Western blot that the protein expression of argininosuccinate synthetase 1 (ASS1) decreased in patients with acute myocardial infarction. The ASS1 level in the serum samples of patients with acute myocardial infarction was 35.4% lower than the average ASS1 level of healthy individuals, as Figure 1 shown.

[0090] By performing ELISA to detect the protein abundance of ASS1 in the sera of a large number of healthy subjects (n = 80) and myocardial infarction patients, it was found that the content of ASS1 in the sera of myocardial infarction patients was significantly lower than that of healthy subjects, as Figure 2 shown. The content of ASS1 protein in the serum samples of myocardial infarction patients was no higher than 12.41 ng / ml, and the average content of ASS1 protein was 6.182 ng / ml; while the average content of ASS1 protein in the serum samples of healthy individuals was 7.919 ng / ml.

[0091] This result indicates that ASS1 is a secreted protein, may be involved in the pathophysiological process of myocardial infarction in clinical patients, and may play a certain role in predicting the prognosis of patients.

[0092] Example 2: Experiment on knocking out the ASS1 gene in neonatal rat cardiomyocytes

[0093] In this study, the effect of ASS1 on the growth state of cardiomyocytes was investigated by knocking out the ASS1 gene in cardiomyocytes.

[0094] 1. Design siRNA targeting the ASS1 gene

[0095] A small interfering siRNA molecule was designed against the conserved region of the ASS1 coding gene. The siRNA was designed according to the ASS1 mRNA sequence in the NCBI database (accession number NP_000041.2) and synthesized by Shanghai Qianmo Biotechnology Co., Ltd. A siRNA sequence that is non-homologous to the human gene was used as a negative control (siRNA negative control, siNC). The siASS1 sequence is as follows:

[0096] siASS1 sense strand: 5′-CUCCACUUUCACUCUACAA dTdT-3′ (SEQ ID NO: 3);

[0097] siASS1 antisense strand: 5′-GCAAGGAAUUUGUGGAAGA dTdT-3′ (SEQ ID NO: 4).

[0098] siNC sense strand: 5′-UUCUCCGAACGUGUCACGUdTdT-3′ (SEQ ID NO: 5);

[0099] siNC antisense strand: 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (SEQ ID NO: 6).

[0100] According to the kit instructions, siRNA was transfected into cells using the ThermoFisher 3000 (ThermoFisher Scientific, USA).

[0101] 2. Extraction and Culture of Neonatal Rat Cardiomyocytes

[0102] (1) Preparation of experimental materials: Select healthy lactating rats aged 1-3 days (purchased from Shanghai Shengchang Biotechnology Co., Ltd., male, 1-3 days old), prepare calcium- and magnesium-free PBS buffer, 0.25% trypsin solution, and DMEM medium containing 10% fetal bovine serum in advance. Use sterile operating tools, including surgical scissors, forceps, culture dishes, centrifuge tubes, etc.

[0103] (2) Collection of cardiac tissue: After the rats were killed by cervical dislocation, the chest and abdomen were disinfected with 75% alcohol. The chest cavity was cut open along the midline of the sternum, and after the heart was exposed, the heart was quickly removed with sterile scissors and placed in a culture dish filled with ice-cold calcium- and magnesium-free PBS buffer.

[0104] (3) Separation of myocardial tissue: Use forceps to remove blood vessels and excess tissue on the surface of the heart, and cut the ventricle into 1-2 mm 3 The pieces were transferred to calcium- and magnesium-free PBS buffer and washed three times to remove blood and debris.

[0105] (4) Tissue digestion: Place the myocardial tissue block in a centrifuge tube containing 0.25% trypsin solution and gently shake it in a 37°C water bath for 5 minutes. Then collect the digestion solution and add DMEM medium containing 10% fetal bovine serum to terminate the digestion. Repeat the above digestion steps 5-7 times until the tissue block is basically digested.

[0106] (5) Cell collection: After combining all the digestive fluids, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet with an appropriate amount of DMEM medium containing 10% fetal bovine serum.

[0107] (6) Cell culture: Inoculate the resuspended cardiomyocyte suspension into a sterile culture flask and culture it in an incubator at 37°C and 5% CO 2 for 1.5 hours. During this period, fibroblasts will adhere to the wall preferentially, and the non-adherent cardiomyocytes will remain in the culture medium. Carefully collect the suspension and transfer it to a new culture flask for subsequent culture.

[0108] (7) Purify cells: To improve the purity of cardiomyocytes, a certain concentration of BrdU (bromodeoxyuridine, 0.1 mmol / L) can be added to the medium to inhibit the proliferation of fibroblasts.

[0109] (8) Transfect cardiomyocytes with small interfering siRNA molecules siASS1 (knockout group) and siNC (negative control) respectively.

[0110] (9) Cell observation and subculture: After culturing for 48 hours, observe the adhesion and beating status of cardiomyocytes. When the cell density reaches 80%-90%, routine subculture or experimental treatment can be carried out.

[0111] Experimental results: Compared with the group transfected with the control small interfering RNA (siNC), the apoptotic protein cleaved-caspase3 (CC3) of cardiomyocytes induced by 6-hour hypoxia in the siASS1 transfection group decreased, as Figure 3 shown; in addition, Tunel staining showed that siASS1 could reduce the apoptosis of cardiomyocytes induced by hypoxia (Hypoxia), as Figure 4 shown.

[0112] The above experimental results indicate that the expression of ASS1 decreases after myocardial infarction, and down-regulating the expression of ASS1 can exacerbate the apoptotic level of cardiomyocytes after myocardial infarction. It is suggested that ASS1 is closely related to myocardial ischemia injury and myocardial infarction and can be used as a biomarker for this disease.

[0113] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of ASS1, i.e. arginine succinate synthetase 1, in the development of a drug for treating myocardial ischemic injury and / or a drug for treating myocardial infarction, characterized in that: ASS1 or its expressed genes as drug targets.

2. A drug for treating myocardial ischemia, characterized in that: The active ingredient of the drug is a substance that promotes the expression of ASS1, a substance that upregulates the expression level of ASS1, or a substance that causes an individual to express ASS1.

3. The drug according to claim 2, characterized in that The active ingredient of the medicine has the function of reducing the apoptosis level of myocardial cells after myocardial infarction.

4. The drug according to claim 2, characterized in that The active pharmaceutical ingredient is an ASS1 gene agonist, an ASS1 gene, an ASS1 gene mRNA, an expression plasmid containing the ASS1 gene, or an expression plasmid containing the ASS1 gene coding region.

5. The drug according to claim 3, characterized in that The medicine is a pharmaceutical composition, which contains not only a therapeutically effective amount of active pharmaceutical ingredients but also a pharmaceutically acceptable carrier.

6. The drug according to claim 3, characterized in that The medicine is a pharmaceutical composition, which, in addition to containing a therapeutically effective amount of the above-mentioned active pharmaceutical ingredients, also contains one or more other pharmaceutical ingredients for treating myocardial ischemic injury.

7. Use of ASS1 or its gene in the preparation of a diagnostic reagent for myocardial ischemia and / or myocardial infarction, characterized in that: ASS1 or its expressed gene is used as a biomarker for judging myocardial ischemic damage and / or myocardial infarction.

8. The use according to claim 7, characterized in that When the level (concentration / content) of ASS1 or its gene in the biological sample of the test subject is lower than the average ASS1 or its gene level of healthy individuals by more than 21%, preferably more than 30%, it indicates that the test subject suffers from myocardial ischemia.

9. The use according to claim 8, characterized in that When the subject is a human, and the biological sample is serum, if the ASS1 protein content in the serum sample is not higher than 6.0 ng / ml, for example, not higher than 5.7 ng / ml, it indicates that the subject suffers from myocardial ischemia.

10. A diagnostic kit for myocardial ischemic injury and / or myocardial infarction, for implementing the use as claimed in claim 7, characterized in that: The detection target is ASS1 in biological samples.

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