Application of recombinant protein in treatment of muscular atrophy

Abstract

The invention discloses an application of a recombinant protein in treatment of muscular atrophy. Researches show that the mouse Serpina3n recombinant protein and the human Serpina3 recombinant protein can play a role in treating the muscular atrophy by improving myotubular cells and skeletal muscle atrophy, and a new direction is provided for research and development of muscular atrophy treatment drugs. The Serpina3n and Serpina3 recombinant protein is expected to become a safe and effective drug for treating muscular atrophy, and has wider application in the field of muscular atrophy treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology. Specifically, the present invention relates to the application of a recombinant protein in the treatment of muscle atrophy. More specifically, the present invention relates to the application of Serpina3n recombinant protein in the prevention and / or treatment of muscle atrophy. Background Art

[0002] Muscle atrophy is a clinical manifestation of muscle loss after the imbalance between catabolism and anabolism of muscle tissue. Patients show characteristics such as reduced skeletal muscle volume, decreased muscle strength, and reduced limb and trunk motor ability, which lead to a decline in their quality of life and an increase in the incidence and mortality of various diseases. Muscle atrophy often occurs in the elderly and is also secondary to various diseases such as cancer, long-term starvation, diabetes, Cushing's syndrome (caused by excessive glucocorticoid secretion or external intake in the body). With the aggravation of population aging, muscle atrophy is gradually becoming a social health problem.

[0003] Currently, there is no effective treatment for muscle atrophy. Related research such as gene therapy, stem cell therapy, and anti-inflammatory drug therapy has become the main direction of the treatment of this disease. In recent years, research on the treatment of muscle atrophy has continuously made progress, and new treatment strategies and methods have emerged in an endless stream. Previously promising treatment strategies have also been re-verified in subsequent research. However, at present, there is no conclusion on the treatment strategies for muscle atrophy, and further research is still needed. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a new treatment strategy for muscle atrophy. Specifically, it provides the application of Serpina3n recombinant protein in the prevention and / or treatment of muscle atrophy.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides the application of a recombinant protein.

[0007] Furthermore, the recombinant protein includes murine Serpina3n recombinant protein and human Serpina3 recombinant protein.

[0008] Furthermore, the application includes:

[0009] 1) Application in the preparation of products for preventing and / or treating muscle atrophy;

[0010] 2) Application in the preparation of reagents for alleviating and / or inhibiting myotube atrophy in vitro;

[0011] 3) Application in the preparation of reagents for alleviating and / or inhibiting skeletal muscle atrophy in vitro;

[0012] 4) Use in screening products for preventing and / or treating muscle atrophy.

[0013] Furthermore, the muscle atrophy includes neurogenic muscular atrophy, myogenic muscular atrophy, disuse muscular atrophy, and senile muscular atrophy.

[0014] Furthermore, the products include drugs, foods for special medical purposes, and health products.

[0015] Furthermore, the dosage forms of the drugs include granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions.

[0016] In some embodiments, the Serpina3n recombinant protein is an important plasma protein, and its main function is to inhibit the activities of digestive enzymes such as proteases and maintain the balance of protein digestion in the intestine. It is closely related to the development of inflammatory diseases and cancers and has the potential to become a treatment strategy or drug candidate for related diseases. It has now been found in the amyloid plaques of the hippocampus in the Alzheimer's disease brain and is related to liver diseases, Parkinson's disease, and chronic obstructive pulmonary disease. In the present invention, the inventors have demonstrated through experiments that murine Serpina3n recombinant protein and human Serpina3 recombinant protein have a therapeutic effect on muscle atrophy.

[0017] In the present invention, the sources of the murine Serpina3n recombinant protein and the human Serpina3 recombinant protein are not limited, and Serpina3n recombinant protein or Serpina3 recombinant protein from any source can achieve the technical effects described in the present invention. For example, the recombinant proteins described in the present invention can be obtained by purchasing through existing channels or can be prepared by oneself according to the sequences of wild-type Serpina3n protein and wild-type Serpina3 protein. In a specific embodiment of the present invention, the Serpina3n recombinant protein is prepared by the applicant himself, and its amino acid sequence has deleted 24 amino acid sequences at the N-terminus on the basis of the wild-type Serpina3n protein. The wild-type Serpina3n protein sequence can be queried from the NCBI website, with the number NP_033278.2; the deleted 24 amino acid sequences at the N-terminus are AFIAALGLLMAGICPAVLCFPDGT.

[0018] In the present invention, the muscle atrophy refers to the reduction in the volume of striated muscle caused by various reasons, with the muscle fibers becoming thinner or even disappearing. The clinical manifestations are mainly muscle weakness, reduced or increased muscle tone, muscle atrophy or hypertrophy, and decreased or absent tendon reflexes, without sensory disturbances and fasciculations.

[0019] In the present invention, the senile muscular atrophy refers to the progressive reduction in skeletal muscle mass, as well as the weakening of muscle strength and motor function, which occur along with the aging process.

[0020] In the present invention, the term "treatment" refers to the process of intervening or changing a specific health state, including eliminating the cause, symptomatic treatment, or supportive treatment.

[0021] In the present invention, the term "prevention" includes pre-clinical prevention and clinical prevention. Pre-clinical prevention refers to preventing the changes in the pre-clinical or initial clinical stages of a disease through early detection, early diagnosis, and appropriate treatment, enabling the disease to be detected and treated at an early stage, avoiding or reducing the occurrence of complications, sequelae, and disabilities, or shortening the time of disability. Clinical prevention refers to using various clinical treatment methods to promptly treat patients with diseases related to muscle atrophy, preventing deterioration, enabling the disease to recover at an early date, reducing the adverse effects of the disease, and preventing complications and disabilities.

[0022] In the present invention, the special medical food refers to a food for special medical purposes, which is a formulated food specifically processed and prepared to meet the special nutritional or dietary needs of people with limited intake, impaired digestion and absorption, metabolic disorders, or specific disease states. Such foods must be used under the guidance of a doctor or clinical dietitian and can be used alone or in combination with ordinary foods or other special dietary foods.

[0023] In some embodiments, the drug of the present invention can be manufactured by methods well known in the art, such as conventional granulation, mixing, dissolution, encapsulation, lyophilization, or emulsification methods. The drug can be made into various forms, including granules, precipitates, or microparticles, powders (including lyophilized powders, rotary dried powders, or spray dried powders, amorphous powders), tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions.

[0024] The second aspect of the present invention provides a pharmaceutical composition for preventing and / or treating muscle atrophy.

[0025] Furthermore, the pharmaceutical composition includes a murine Serpina3n recombinant protein or a human Serpina3 recombinant protein.

[0026] In the present invention, the term "pharmaceutical composition" refers to a composition comprising at least one bioactive compound. The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by an implanted reservoir device. In some embodiments, oral administration is preferred. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carriers, excipients, or diluents. In certain cases, pharmaceutically acceptable acids, bases, or buffers may be used to adjust the pH of the formulation to enhance the stability of the formulated compound or its dosage form. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention can be administered to a recipient by any route as long as the target tissue can be reached.

[0027] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0028] Furthermore, the pharmaceutically acceptable carrier includes one or more of a diluent, a binder, a surfactant, a humectant, an adsorbent carrier, a lubricant, a filler, and a disintegrant.

[0029] In the present invention, the term "pharmaceutically acceptable carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicles, microspheres, liposome encapsulation, or other materials known in the art for pharmaceutical formulations. It should be understood that the characteristics of the carrier will depend on the route of administration of the specific application. As used in the present invention, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effect of the pharmaceutical composition according to the present invention or the biological activity of the pharmaceutical composition according to the present invention. Formulating a pharmaceutically active ingredient with a pharmaceutically acceptable carrier is known in the art, for example, Remington: The Science and Practice of Pharmacy (such as the 21st edition (2005), and any subsequent editions). Non-limiting examples of pharmaceutically acceptable carriers include: salts (such as acid salts / anion salts, base salts / cation salts), excipients, buffers, diluents, solubilizers, tonicity regulators, surfactants, preservatives, isotonic agents, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used to formulate the pharmaceutical compositions of the present invention.

[0030] In some embodiments, pharmaceutically acceptable carriers include acid salts / anionic salts. Non-limiting examples of acid salts / anionic salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, ethanedisulfonate, etidronate, esylate, fumarate, glucoheptonate, gluconate, glutamate, p-acetamidobenzenearsonate, hexylresorcinol, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, naphthalenesulfonate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate, toluenesulfonate, and triethyl iodide.

[0031] In some embodiments, pharmaceutically acceptable carriers include base salts / cationic salts. Non-limiting examples of base salts / cationic salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethamine or "TRIS"), ammonia, benzathine penicillin, tert-butylamine, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.

[0032] In some embodiments, pharmaceutically acceptable carriers include buffers. Non-limiting examples of buffers include, but are not limited to, arginine, aspartic acid, bis(2-hydroxyethyl)glycine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, triazine, and tris(hydroxymethyl)aminomethane and mixtures thereof.

[0033] In some embodiments, pharmaceutically acceptable carriers include preservatives. Non-limiting examples of preservatives include, but are not limited to, benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butylparaben, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethylparaben, imidurea, methylparaben, phenol, 2-phenoxyethanol, 2-phenylethanol, propylparaben, sodium dehydroacetate, thimerosal, and mixtures thereof.

[0034] In some embodiments, the pharmaceutically acceptable carrier includes an isosmotic agent. Non-limiting examples of isosmotic agents include, but are not limited to, amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), sugar alcohols (such as glycerol, 1,2-propanediol, propylene glycol, 1,3-propanediol, and 1,3-butanediol), polyethylene glycol (e.g., PEG400), and mixtures thereof. Another example of an isosmotic agent includes sugars. Non-limiting examples of sugars can be monosaccharides, disaccharides, or polysaccharides, or water-soluble dextrans, including, for example, fructose, glucose, mannose, sorbose, xylose, and maltose.

[0035] Further, the pharmaceutical composition further includes other substances for preventing and / or treating muscle atrophy.

[0036] Further, the other substances for preventing and / or treating muscle atrophy include glucocorticoids, vitamin drugs, and immunosuppressive drugs.

[0037] Further, the vitamin drugs include vitamin E and vitamin B1.

[0038] Further, the immunosuppressive drugs include nucleic acid drugs.

[0039] Further, the muscle atrophy includes neurogenic muscular atrophy, myogenic muscular atrophy, disuse muscular atrophy, and senile muscular atrophy.

[0040] The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating, preventing, alleviating, and / or relieving muscle atrophy. Other compounds for treating, preventing, alleviating, and / or relieving muscle atrophy can be administered simultaneously with the main active ingredient (e.g., Serpina3n recombinant protein), or even in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from that of the main active ingredient.

[0041] In the present invention, the pharmaceutical composition can be formulated for oral administration, parenteral administration, or rectal administration. In addition, the pharmaceutical composition of the present invention can be made in solid form (including but not limited to capsules, tablets, pills, granules, powders, or suppositories), or in liquid form (including but not limited to solutions, suspensions, or emulsions).

[0042] The effective amount of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, those skilled in the art can easily determine the optimal dose to be administered, and the optimal dose will vary depending on the specific drug used, the mode of administration, the strength of the formulation, and the progression of the disease condition. Additionally, factors related to the specific subject being treated, including the subject's age, weight, diet, and time of administration, will result in the need to adjust the dose to an appropriate therapeutic level.

[0043] According to specific embodiments, an effective amount or effective dose refers to a therapeutically effective amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder, or condition to be treated or the symptoms associated therewith; (ii) reducing the duration of the treated disease, disorder, or condition or the symptoms associated therewith; (iii) preventing the development of the treated disease, disorder, or condition or the symptoms associated therewith; (iv) causing the regression of the treated disease, disorder, or condition or the symptoms associated therewith; (v) preventing the development or onset of the treated disease, disorder, or condition or the symptoms associated therewith; (vi) preventing the recurrence of the treated disease, disorder, or condition or the symptoms associated therewith; (vii) reducing hospitalization of a subject having the treated disease, disorder, or condition or the symptoms associated therewith; (viii) reducing the length of hospitalization of a subject having the treated disease, disorder, or condition or the symptoms associated therewith; (ix) increasing the survival of a subject having the treated disease, disorder, or condition or the symptoms associated therewith; (xi) inhibiting or reducing the treated disease, disorder, or condition or the symptoms associated therewith in a subject; and / or (xii) enhancing or improving the prophylactic or therapeutic effect of another therapy.

[0044] The dosage form of the pharmaceutical composition of the present invention is a dosage form favorable for administration prepared by conventional methods, including but not limited to: parenteral dosage forms, enteral dosage forms, and specific examples include but are not limited to: aqueous injection solutions, powder for injection, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder inhalations, sustained-release agents, and controlled-release agents, etc.

[0045] In some embodiments, the injectable dosage forms include but are not limited to: intravenous injection solutions, intramuscular injection solutions, subcutaneous injection solutions, intradermal injection solutions, and intracavitary injection solutions, etc.

[0046] In some embodiments, the cavity dosage forms include but are not limited to: suppositories, aerosols, effervescent tablets, drops, dripping pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.

[0047] In some embodiments, the mucosal dosage forms include but are not limited to: eye drops, nasal drops, ophthalmic ointments, gargles, sublingual tablets, adhesive tablets, film dressings, etc.

[0048] In some embodiments, the skin dosage forms include but are not limited to: topical solution, lotion, liniment, ointment, plaster, paste, patch, etc.

[0049] The third aspect of the present invention provides a method for evaluating whether a substance to be screened has a prophylactic and / or therapeutic effect on muscle atrophy.

[0050] Further, the method includes using murine Serpina3n recombinant protein or human Serpina3 recombinant protein as a positive drug to evaluate the effect of the drug to be screened.

[0051] Further, the drug to be screened includes, but is not limited to: substances, small molecule compounds that promote the expression or activity of murine Serpina3n recombinant protein or human Serpina3 recombinant protein.

[0052] In some embodiments, the drug to be screened further includes substances having an amino acid sequence with 80% identity to murine Serpina3n recombinant protein or human Serpina3 recombinant protein.

[0053] In some embodiments, the substance that promotes the expression or activity of murine Serpina3n recombinant protein or human Serpina3 recombinant protein refers to any substance that can increase the activity of Serpina3n protein, improve the stability of Serpina3n protein, up-regulate the expression of Serpina3n protein, and increase the effective action time of Serpina3n protein. These substances can all be used in the present invention as substances useful for up-regulating Serpina3n, and thus can be used for preventing or treating muscular atrophy.

[0054] The fourth aspect of the present invention provides a method for alleviating and / or inhibiting myotube atrophy for non-therapeutic purposes in vitro.

[0055] Further, the method includes the following steps: treating myotubes with an effective amount of murine Serpina3n recombinant protein or human Serpina3 recombinant protein or a pharmaceutical composition containing an effective amount of murine Serpina3n recombinant protein or human Serpina3 recombinant protein.

[0056] The fifth aspect of the present invention provides a system for alleviating and / or inhibiting myotube atrophy or alleviating and / or inhibiting skeletal muscle.

[0057] Further, the system includes a treatment unit for treating myotubes or skeletal muscle with a therapeutically effective amount of murine Serpina3n recombinant protein or human Serpina3 recombinant protein.

[0058] In the present invention, the term "therapeutically effective amount" refers to an amount sufficient to achieve the desired result when administered to a subject (including mammals, such as humans). The effective amount of the Serpina3n recombinant protein described herein can vary depending on factors such as the disease state, age, gender, and weight of the subject. As understood by those skilled in the art, the dosage or treatment regimen can be adjusted to provide an optimal therapeutic response.

[0059] In the present invention, the subject to be treated by the drug includes any member of the animal kingdom, usually a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic animals and farm animals, as well as zoo, sports or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. As a preferred embodiment, the subject to be treated is a human.

[0060] In certain specific embodiments, the system provided by the present invention includes a computer-readable storage medium for storing a computer-readable program or instruction, which, when executed by a processor, can implement the functions of the treatment unit in the system of the present invention.

[0061] In some embodiments, the system may be the user's electronic device or a computer system remotely located relative to the electronic device.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0063] The term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the element. The indefinite articles "a" and "an" before an element or component of the present invention do not limit the number requirement (i.e., the number of occurrences) of the element or component. Therefore, "a" or "an" should be construed as including one or at least one, and the singular form of an element or component also includes the plural form unless the quantity clearly refers only to the singular form. The meaning of "a plurality" is at least two, such as two, three, etc., unless otherwise specifically defined.

[0064] Advantages of the present invention:

[0065] The present invention has for the first time discovered and confirmed the new use of murine Serpina3n recombinant protein or human Serpina3 recombinant protein as a therapeutic drug for muscle atrophy, providing a new direction for the research and development of therapeutic drugs for muscle atrophy, and is expected to become a safe and effective drug for the treatment of muscle atrophy, having broad application prospects in the field of muscle atrophy treatment. Description of the Drawings

[0066] Figure 1 It is a diagram showing the results of the effect of Serpina3n recombinant protein on senescent myotube cells; among them Figure 1 A is the Myh staining diagram of myotube cells; Figure 1 B and C are the quantitative diagrams of the length and diameter of myotubes respectively; Figure 1 D is the diagram showing the expression of the muscle atrophy marker gene Atrogen1 detected by Western Blot;

[0067] Figure 2 It is a diagram showing the results of the effect of Serpina3n recombinant protein on dexamethasone-induced myotube atrophy; among them Figure 2 A is the Myh staining, length and diameter quantitative diagram of myotube cells; Figure 2 B is the diagram showing the expression of the muscle atrophy marker genes Atrogen1 and Murf1 detected by Western Blot;

[0068] Figure 3 It is a diagram showing the results of the effect of Serpina3n recombinant protein on senescence-induced skeletal muscle atrophy; among them, Figure 3 A is the diagram of the weights of four types of muscle fibers in mice injected with PBS and mice injected with Serpina3n recombinant protein; Figure 3 B and C are the diagrams of the grasping force and endurance of mice injected with PBS and mice injected with Serpina3n recombinant protein respectively;

[0069] Figure 4 It is a diagram showing the results of the effect of Serpina3n recombinant protein on disuse skeletal muscle atrophy in mice; among them, Figure 4 A is the flowchart for establishing the mouse models of each group; Figure 4 B is the diagram of the weights of four types of muscle fibers in mice of different groups; Figure 4 C is the diagram of the grasping force and endurance of mice in each group; Figure 4 D is the diagram showing the expression of the muscle atrophy marker genes Atrogen1 and Murf1 detected by Western Blot in mice of each group.

[0070] Figure 5 It is a diagram showing the results of the effect of human Serpina3 recombinant protein on dexamethasone-induced myotube atrophy; among them Figure 5 A is the diagram showing the expression of the muscle atrophy marker genes Atrogen1 and Murf1 detected by RT-PCR in atrophied myotubes; Figure 5 B is the diagram showing the expression of the muscle atrophy marker genes Atrogen1 and Murf1 detected by Western Blotting in atrophied myotubes. Detailed Implementation Modes

[0071] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0072] Accordingly, it is intended that the present invention cover such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present invention are disclosed in or are obvious from the following detailed description. Those of ordinary skill in the art should understand that this discussion is only a description of exemplary embodiments and is not intended to limit the broader aspects of the present invention.

[0073] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. In the following examples, all quantitative tests are set up with three repeated experiments, and the results are averaged.

[0074] Example 1 Serpina3n recombinant protein can alleviate atrophy of senescent myotubes

[0075] 1. Experimental method

[0076] Purification of murine Serpina3n recombinant protein: The target sequence of Serpina3n (24 amino acid sequences at the N-terminus were deleted based on the wild-type Serpina3n protein, and the wild-type Serpina3n protein sequence can be queried on the NCBI website with the accession number NP_033278.2, and the deleted 24 amino acid sequences at the N-terminus are AFIAALGLLMAGICPAVLCFPDGT) was constructed into the pET-28a(+) expression vector containing a His tag. The Serpina3n containing the His tag was expressed in Transetta(DE3) (Transgene, CD801-02), cultured in LB medium at 37 °C, and at OD 600When it is about 0.6, add IPTG with a final concentration of 1 mM to induce protein expression, and continue culturing at 37 °C for 12 - 16 hours; after protein expression, use lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole) to lyse the bacteria, and perform sonication. The lysate is loaded onto Ni NTA Beads 6FF (Tiandi Renhe, SA005005) equilibrated with equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole) for affinity chromatography. Then, use wash buffer (50 mM Tris-HCl, 150 mM NaCl, 150 mM imidazole) to load onto the nickel column to elute the protein. The collected protein is further purified and separated on a Superdex TM 75 Increase chromatography column (Cytiva, 29148721). The purified Serpina3n recombinant protein is dialyzed to remove imidazole, and the purified Serpina3n recombinant protein is stored at -80 °C.

[0077] Culture and induced differentiation of mouse myoblast cell line C2C12:

[0078] (1) Culture of C2C12 cells in the proliferation stage: In the proliferation stage, culture C2C12 cells with complete medium DMEM containing 10% fetal bovine serum and 2% penicillin / streptomycin mixture. During the culture, the cell density should not be too high. When the cell density reaches 50% - 60%, cell passage is required.

[0079] (2) Induce C2C12 cells to differentiate into myotubes: When the C2C12 cell density reaches 90% - 100%, induce differentiation with complete medium DMEM containing 2% horse serum and 2% penicillin / streptomycin mixture (referred to as differentiation medium). During this period, replace the fresh differentiation medium every two days until it differentiates into mature myotubes (generally, it differentiates into mature myotubes in 7 days).

[0080] Perform Myh staining on mature myotube cells that have differentiated for 7 days. Add 10 ng and 50 ng of Serpina3n (SPN) recombinant protein to the medium of senescent myotube cells that have differentiated for 11 days, and then perform Myh staining. Add Vehicle empty control liquid PBS to the control group medium; then, perform quantification of the myotube length and diameter on the stained myotube cells; and use Western Blotting to detect the expression of the muscle atrophy marker gene Atrogen1 in senescent myotubes.

[0081] Western Blotting: Cell samples were lysed in lysis buffer (50 mM tris, pH 7.5, 150 mM NaCl, 0.5% NP-40) supplemented with protease inhibitor for 1 hour, followed by SDS-PAGE and then transferred to a membrane using the wet transfer method. After the transfer was completed, the membrane was blocked with 5% non-fat milk for 1 hour, then incubated with the primary antibody overnight at 4°C. After incubation, the membrane was washed 3 times for 10 minutes each with the washing solution, then incubated with the corresponding secondary antibody, and washed 3 times for 10 minutes each with the washing solution again. Finally, chemiluminescence reaction was carried out using horseradish peroxidase.

[0082] 2. Experimental Results

[0083] Myh staining was performed on mature myotubes differentiated for 7 days and senescent myotubes differentiated for 11 days, and the results are as Figure 1 shown in A. Quantitative analysis of the length and diameter of the stained myotubes was carried out, and the results are as Figure 1 shown in B and C respectively. The myotube length on Day 11 became shorter and the diameter became thinner, indicating that senescent myotubes underwent atrophy. Treatment with two different concentrations of Serpina3n recombinant protein (SPN) could extend the myotube length and increase the diameter width, indicating that the phenomenon of myotube atrophy was alleviated. Meanwhile, the expression of Atrogen1, a marker gene for muscle atrophy in senescent myotubes, was also detected by Western Blotting and showed a decreasing trend ( Figure 1 D), and this result supported that SPN could alleviate the atrophy of senescent myotube cells.

[0084] Example 2 Serpina3n Recombinant Protein Can Alleviate Dexamethasone-Induced Myotube Atrophy

[0085] 1. Experimental Method

[0086] Long-term use of the anti-inflammatory drug dexamethasone (DEX) can cause the side effect of muscle atrophy. Mature myotubes were pretreated with 80 uM dexamethasone for 24 hours to create a drug-induced atrophy phenotype with reduced myotube length and thinner diameter. In this case, the myotubes were treated with two concentrations of 10 ng and 50 ng of SPN recombinant protein respectively, and the control group was added with Vehicle (PBS as the blank control), and then Myh staining and Western Blotting were performed for detection.

[0087] 2. Experimental Results

[0088] The results of the length and diameter of the myotubes after treatment with SPN are as Figure 2As shown in Figure A, after treatment with two concentrations of SPN, both the length and diameter of myotubes were alleviated. Meanwhile, the expression of Atrogen1 and Murf1, the marker genes of muscle atrophy in senescent myotubes, was also detected by Western Blotting, showing a decreasing trend. Figure 2 B), which supports that SPN can alleviate dexamethasone-induced myotube atrophy.

[0089] Example 3 Serpina3n recombinant protein can alleviate skeletal muscle atrophy caused by aging

[0090] 1. Experimental method

[0091] Mouse feeding and drug administration: 20-month-old C57BL / 6 mice were purchased from Beijing Speywood Co., Ltd., and 8-week-old C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experimental operation procedures were approved by the Animal Ethics Committee of Peking Union Medical College. All mice used in the experiment were housed and bred in a SPF-level animal room, with the indoor temperature strictly controlled at 21-25°C, humidity 50%-60%, 12h light, 12h dark. The feed, drinking water, and bedding used by the mice were all strictly sterilized by high temperature. The mouse feed and drinking water were supplemented or replaced daily, and the mouse bedding was replaced twice a week. The growth and development of the mice were closely monitored during the breeding process.

[0092] Drug administration plan for 20-month-old C57BL / 6 mice: Serpina3 recombinant protein (50 ng) was injected into the right hind leg muscle of the mice in situ three times a week for 3 consecutive weeks. After three weeks, the mice were sacrificed, and four types of muscle fibers of the injected hind limb skeletal muscle were isolated: tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (Gas), and soleus (Sol). The weights of various muscle fibers of the mice in the PBS control group and the SPN group were compared, and the grip strength and endurance tests were performed on the two groups of mice.

[0093] 2. Experimental results

[0094] The weight results of various muscle fibers of the mice in the PBS control group and the SPN group are shown in Figure 3 Figure A. The weights of the extensor digitorum longus (EDL) and soleus (Sol) of the injected mice increased significantly, indicating an increase in muscle mass and improvement in atrophy. The grip strength Figure 3 Figure B) and endurance Figure 3 Figure C) tests of the two groups of mice showed that the grip strength and endurance of the SPN injection group mice increased after injection, while there was no change in the PBS control group, indicating an enhancement of the muscle function of the SPN injection group mice.

[0095] Example 4 Serpina3n recombinant protein can alleviate disuse skeletal muscle atrophy in mice

[0096] 1. Experimental methods

[0097] For 8-week-old C57BL / 6 mice induced with muscle atrophy and the drug administration protocol: Three groups of mice were designed for the experiment. One group was the modeling control (Control) group, and the mice in this group were not treated with anything. The other two groups used bandages to tie the right hind legs of the mice for 8 weeks to establish a model of disuse skeletal muscle atrophy. After 10 days of tying, injections of 50 ng of SPN or the vehicle control liquid PBS (Vehicle) were started three times a week for 3 consecutive weeks. Three weeks later, the three groups of mice were sacrificed, and four types of muscle fibers of the injected hind limb skeletal muscle were isolated: tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (Gas), and soleus (Sol), and the weights of various muscle fibers were compared. Before sacrifice, endurance and grip strength tests were performed on the three groups of mice, and the expression of the muscle atrophy marker genes Atrogen1 and Murf1 in the skeletal muscle of the three groups of mice was detected by Western Blotting.

[0098] 2. Experimental results

[0099] The schematic diagram of the modeling is as shown in Figure 4 A. The weight results of various muscle fibers are as shown in Figure 4 B. Compared with the modeling control (Control) group, the four types of muscle fibers in the two SPN groups or the Vehicle group after modeling decreased, indicating that the muscle atrophy modeling was successful. At the same time, compared with the PBS injection group, the fiber quality of the extensor digitorum longus (EDL), gastrocnemius (Gas), and soleus (Sol) in the SPN injection group increased significantly, indicating that the muscle atrophy was alleviated. Grip strength and endurance tests were performed on the mice, and the results are as shown in Figure 4 C. The grip strength and endurance of the mice in the SPN injection group were stronger than those of the mice in the Vehicle injection group, indicating that SPN injection enhanced the function of skeletal muscle. The expression of the muscle atrophy marker genes Atrogen1 and Murf in the skeletal muscle of the three groups of mice was detected by Western Blotting, which also showed that SPN injection improved disuse skeletal muscle atrophy ( Figure 4 D).

[0100] Example 5: Human SerpinA3 recombinant protein can reduce dexamethasone-induced atrophy of human muscle fibers

[0101] 1. Experimental methods

[0102] Purification of human SerpinA3 recombinant protein: The target sequence of human SerpinA3 (25 amino acid sequences at the N-terminus were deleted based on the wild-type SerpinA3 gene. The wild-type SerpinA3 protein sequence can be queried on the NCBI website with the accession number NP_001076.2. The deleted 25 amino acid sequences at the N-terminus are MERMLPLLALGLLAAGFCPAVLCHP) was constructed into the pET-28a(+) expression vector containing a His tag. SerpinA3 with a His tag was expressed in Transetta(DE3) (Transgene, CD801-02), cultured in LB medium at 37 °C, and IPTG with a final concentration of 1 mM was added when the OD 600 was approximately 0.6 to induce protein expression, and the culture was continued at 37 °C for 12 - 16 hours; after protein expression, the bacterial cells were lysed using a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole), and sonicated. The lysate was loaded onto Ni NTA Beads 6FF (Tiandi Renhe, SA005005) equilibrated with an equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole) for affinity chromatography. Then, the protein was eluted by loading the washing buffer (50 mM Tris-HCl, 150 mM NaCl, 150 mM imidazole) onto the nickel column. The collected protein was further purified and separated on a Superdex TM 75 Increase chromatography column (Cytiva, 29148721). The purified SerpinA3 recombinant protein was dialyzed to remove imidazole, and the purified SerpinA3 recombinant protein was stored at -80 °C. Human muscle stem cells were purchased from the immortalized human skeletal muscle stem cells (YB7086HPC) of Shanghai Yubo Biotechnology Co., Ltd. After being induced to differentiate for 5 days with 2% horse serum and matured, the pre-treated and differentiated myotube cells were treated with 80 μM dexamethasone for 24 hours to create a drug-induced atrophy phenotype with reduced myotube length and thinner diameter.

[0103] 2. Experimental results

[0104] Human SerpinaA3 is the homologous protein of murine Serpina3n in humans. In the dexamethasone-induced atrophy model, adding 50 ng of human SerpinA3 recombinant protein (SPN) and treating for 12 hours achieved the effect of alleviating atrophy ( Figure 5 ). At the same time, RT-PCR ( Figure 5 A) and Western Blotting ( Figure 5The expression of Atrogen1 and Murf1, markers of muscle atrophy, in atrophied myotubes was detected by the method of (B). The results showed a decreasing trend in these two markers of muscle atrophy, which supported that human SPN could alleviate dexamethasone-induced atrophy of human myotube cells.

[0105] The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Use of a recombinant protein, characterized in that, The recombinant protein includes murine Serpina3n recombinant protein and human Serpina3 recombinant protein.

2. The application according to claim 1, characterized in that, The applications include: 1) Application in the preparation of products for preventing and / or treating muscle atrophy; 2) Application in the preparation of reagents for relieving and / or inhibiting myotube atrophy in vitro; 3) Application in the preparation of reagents for relieving and / or inhibiting skeletal muscle atrophy in vitro; 4) Application in screening products for preventing and / or treating muscle atrophy.

3. The application according to claim 1, characterized in that, The muscle atrophy includes neurogenic muscular atrophy, myogenic muscular atrophy, disuse muscular atrophy, and senile muscular atrophy; The products include drugs, special medical foods, and health products; Preferably, the dosage forms of the drugs include granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions or solutions.

4. A pharmaceutical composition for preventing and / or treating muscle atrophy, characterized in that, The pharmaceutical composition includes murine Serpina3n recombinant protein or human Serpina3 recombinant protein.

5. The pharmaceutical composition according to claim 4, wherein The pharmaceutical composition further includes a pharmaceutically acceptable carrier; Preferably, the pharmaceutically acceptable carrier includes one or several of diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, disintegrants; Preferably, the pharmaceutical composition further includes other substances for preventing and / or treating muscle atrophy; Preferably, the other substances for preventing and / or treating muscle atrophy include glucocorticoids, vitamin drugs, immunosuppressive drugs; Preferably, the vitamin drugs include vitamin E and vitamin B1; Preferably, the immunosuppressive drugs include nucleic acid drugs.

6. The pharmaceutical composition according to claim 4, characterized in that, The muscle atrophy includes neurogenic muscular atrophy, myogenic muscular atrophy, disuse muscular atrophy, and senile muscular atrophy.

7. A method for evaluating whether a substance to be screened has the effect of preventing and / or treating muscle atrophy, characterized in that, The method includes using murine Serpina3n recombinant protein or human Serpina3 recombinant protein as a positive drug to evaluate the effect of the drug to be screened.

8. The method according to claim 7, characterized in that, The drugs to be screened include, but are not limited to, substances that promote the expression or activity of murine Serpina3n recombinant protein or human Serpina3 recombinant protein, and small molecule compounds.

9. A method for relieving and / or inhibiting myotube atrophy for non-therapeutic purposes in vitro, characterized in that, The method includes the following steps: treating myotube cells with an effective amount of murine Serpina3n recombinant protein or human Serpina3 recombinant protein, or a pharmaceutical composition containing an effective amount of murine Serpina3n recombinant protein or human Serpina3 recombinant protein.

10. A system for alleviating and / or inhibiting myotube atrophy or alleviating and / or inhibiting skeletal muscle, characterized in that, The system includes a treatment unit for treating myotube cells or skeletal muscle with a therapeutically effective amount of murine Serpina3n recombinant protein or human Serpina3 recombinant protein.

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