Application of a recombinant protein in the treatment of muscle atrophy

Abstract

This invention discloses the application of a recombinant protein in the treatment of muscle atrophy. The research of this invention shows that murine Serpina3n recombinant protein and human Serpina3 recombinant protein can exert a therapeutic effect on muscle atrophy by improving myotube cell and skeletal muscle atrophy. This provides a new direction for the research and development of drugs for the treatment of muscle atrophy. Serpina3n and Serpina3 recombinant proteins are expected to become safe and effective drugs for the treatment of muscle atrophy, with wider applications in the field.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to the application of a recombinant protein in the treatment of muscle atrophy, and more specifically, this invention relates to the application of Serpina3n recombinant protein in the prevention and / or treatment of muscle atrophy. Background Technology

[0002] Muscle atrophy is the clinical manifestation of muscle loss resulting from an imbalance between catabolism and anabolism in muscle tissue. Patients exhibit reduced skeletal muscle volume, decreased muscle strength, and reduced motor function of the limbs and trunk, leading to a decline in quality of life and an increase in the incidence and mortality of various diseases. Muscle atrophy commonly occurs in the elderly and is secondary to various diseases such as cancer, prolonged starvation, diabetes, and Cushing's syndrome (caused by excessive intake of glucocorticoids, either endogenously or exogenously). With the increasing aging of the population, muscle atrophy is gradually becoming a social health problem.

[0003] Currently, there is no effective treatment for muscular dystrophy. Research on gene therapy, stem cell therapy, and anti-inflammatory drugs has become the main focus of treatment. In recent years, research on the treatment of muscular dystrophy has made continuous progress, with new treatment strategies and methods emerging one after another. Previously promising treatment strategies have also been re-evaluated in subsequent studies. However, a definitive treatment strategy for muscular dystrophy is still pending and further research is needed. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide a novel treatment strategy for muscle atrophy, specifically, to provide the application of recombinant Serpina3n protein in the prevention and / or treatment of muscle atrophy.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides an application of a recombinant protein.

[0006] Furthermore, the recombinant protein includes mouse Serpina3n recombinant protein and human Serpina3 recombinant protein.

[0007] Furthermore, the applications include: 1) Use in the preparation of products for the prevention and / or treatment of muscle atrophy; 2) Application in the preparation of reagents for relieving and / or inhibiting myotube cell 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 the prevention and / or treatment of muscle atrophy.

[0008] Furthermore, the muscle atrophy includes neurogenic muscle atrophy, myogenic muscle atrophy, disuse muscle atrophy, and senile muscle atrophy.

[0009] Furthermore, the product includes pharmaceuticals.

[0010] Furthermore, the dosage form of the drug includes granules, powder, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions, or solutions.

[0011] In some embodiments, the Serpina3n recombinant protein is an important plasma protein whose main function is to inhibit the activity of digestive enzymes such as proteases, maintaining the balance of protein digestion in the intestine. It is closely related to the development of inflammatory diseases and cancer, and has the potential to be a therapeutic strategy or drug candidate for these diseases. It has been found in hippocampal amyloid plaques in the brains of Alzheimer's disease patients and is associated with liver disease, Parkinson's disease, and chronic obstructive pulmonary disease. In this invention, the inventors experimentally demonstrated that murine Serpina3n recombinant protein and human Serpina3 recombinant protein have therapeutic effects on muscle atrophy.

[0012] In this invention, the sources of the mouse-derived Serpina3n recombinant protein and the human-derived Serpina3 recombinant protein are not limited; any source of Serpina3n recombinant protein or Serpina3 recombinant protein can achieve the technical effects described in this invention. For example, the recombinant protein described in this invention can be purchased through existing channels or prepared by the applicant based on the sequences of wild-type Serpina3n protein and wild-type Serpina3 protein. In a specific embodiment of this invention, the Serpina3n recombinant protein is prepared by the applicant, and its amino acid sequence is based on the wild-type Serpina3n protein with 24 amino acids deleted from the N-terminus. The wild-type Serpina3n protein sequence can be found on the NCBI website, with the number NP_033278.2; the deleted N-terminal 24 amino acid sequence is AFIAALGLLMAGICPAVLCFPDGT.

[0013] In this invention, muscle atrophy refers to a reduction in the normal volume of striated muscles due to various causes, with muscle fibers becoming thinner or even disappearing. Clinical manifestations mainly include muscle weakness, hypotonia or rigidity, muscle atrophy or hypertrophy, and decreased or absent tendon reflexes, without sensory disturbances or fasciculations.

[0014] In this invention, senile muscular atrophy refers to the progressive reduction in skeletal muscle mass, muscle strength, and motor function that occurs along with the aging process.

[0015] In this invention, the term "treatment" refers to the process of intervening in or changing a particular health condition, including eliminating the cause, symptomatic treatment, or supportive treatment.

[0016] In this invention, the term "prevention" includes preclinical prevention and clinical prevention. Preclinical prevention refers to preventing changes in the disease in its preclinical or early clinical stages through early detection, early diagnosis, and appropriate treatment. This allows the disease to be detected and treated at an early stage, avoiding or reducing complications, sequelae, and disabilities, or shortening the time to disability. Clinical prevention refers to using various clinical treatment methods to treat patients with diseases related to muscle atrophy in a timely manner, preventing deterioration, promoting early recovery, reducing adverse effects of the disease, and preventing complications and disabilities.

[0017] In some embodiments, the medicament described in this invention can be manufactured using methods well-known in the art, such as conventional granulation, mixing, dissolving, encapsulation, lyophilization, or emulsification. The medicament can be formulated 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.

[0018] A second aspect of the present invention provides a pharmaceutical composition for the prevention and / or treatment of muscle atrophy.

[0019] Furthermore, the pharmaceutical composition includes mouse-derived Serpina3n recombinant protein or human-derived Serpina3 recombinant protein.

[0020] In this invention, the term "pharmaceutical composition" refers to a composition containing at least one bioactive compound. The pharmaceutical compositions of this invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted drug delivery device. In some embodiments, oral administration is preferred. The pharmaceutical compositions of this invention may contain any commonly used non-toxic pharmaceutically acceptable carrier, excipient, or excipient. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can be administered to the receptor via any route, provided the target tissue can be reached.

[0021] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0022] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: diluent, binder, surfactant, humectant, adsorbent, lubricant, filler, and disintegrant.

[0023] In this invention, the term "pharmaceutically acceptable carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other materials known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of the carrier will depend on the route of administration for the specific application. As used herein, the term "pharmaceuticalally acceptable carrier" refers to a nontoxic material that does not interfere with the efficacy or bioactivity of the pharmaceutical composition according to the invention. Formulation of pharmaceutical active ingredients using pharmaceutically acceptable carriers is known in the art, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005), and any subsequent editions). Non-limiting examples of pharmaceutically acceptable carriers include: salts (e.g., acidic / anionic salts, basic / cationic salts), excipients, buffers, diluents, solubilizers, tension modifiers, surfactants, preservatives, isotonic agents, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers may be used to formulate the pharmaceutical compositions of the invention.

[0024] In some implementations, pharmaceutically acceptable carriers include acid salts / anionic salts. Non-limiting examples of acid salts / anionic salts include, but are not limited to, acetates, benzenesulfonates, benzoates, bicarbonates, tartrates, bromides, calcium edetate, camphorsulfonates, carbonates, chlorides, citrates, dihydrochlorides, edetate, ethanedisulfonate, etolate, ethanesulfonate, fumarate, gluconate, gluconate, glutamate, p-hydroxyacetaminophenarsine, hexylresorcinol, hyaluronic acid, hydrobromide, hydrochloride, hydroxynaphthyl salt, iodides, hydroxyethylsulfonate, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, methyl bromide, methyl nitrate, methyl sulfate, mucilage, naphthalenesulfonate, nitrate, dihydroxynaphthyl salt, pantothenate, phosphates / bisphosphates, polygalacturonic acid salts, salicylates, stearates, hypoacetates, succinates, sulfates, tannates, tartrates, theochlorophosphate, toluenesulfonate, and triethyl iodide.

[0025] In some embodiments, pharmaceutically acceptable carriers include alkali salts / cationic salts. Non-limiting examples of alkali salts / cationic salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, aminobutanetriol, or “TRIS”), ammonia, benzathine penicillin, tert-butylamine, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucosamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.

[0026] In some embodiments, pharmaceutically acceptable carriers include buffers. Non-limiting examples of buffers include, but are not limited to, arginine, aspartic acid, dihydroxyethylglycine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, diglycine peptide, 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.

[0027] In some embodiments, pharmaceutically acceptable carriers include preservatives. Non-limiting examples of preservatives include, but are not limited to, benzyl chloride, benzoic acid, benzyl alcohol, bromonitropropylene glycol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorophenylglycerol ether, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidureus, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thimerosal, and mixtures thereof.

[0028] In some embodiments, pharmaceutically acceptable carriers include isotonic agents. Non-limiting examples of isotonic 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 isotonic agents includes sugars. Non-limiting examples of sugars can be monosaccharides, disaccharides, or polysaccharides, or water-soluble dextran, including, for example, fructose, glucose, mannose, sorbitol, xylose, and maltose.

[0029] Furthermore, the pharmaceutical composition also includes other substances for the prevention and / or treatment of muscle atrophy.

[0030] Furthermore, the other substances mentioned for preventing and / or treating muscle atrophy include glucocorticoids, vitamins, and immunosuppressants.

[0031] Furthermore, the vitamin medication includes vitamin E and vitamin B1.

[0032] Furthermore, the immunosuppressant drugs include nucleic acid drugs.

[0033] Furthermore, the muscle atrophy includes neurogenic muscle atrophy, myogenic muscle atrophy, disuse muscle atrophy, and senile muscle atrophy.

[0034] The pharmaceutical compositions of the present invention can also be used in combination with other drugs for treating, preventing, reducing, and / or alleviating muscle atrophy. These other compounds can be administered simultaneously with the main active ingredient (e.g., Serpina3n recombinant protein), or even simultaneously in the same composition. Other therapeutic compounds can also be administered alone in a single composition or at a dosage form different from that of the main active ingredient.

[0035] In this invention, the pharmaceutical composition can be formulated for oral, parenteral, or rectal administration. Furthermore, the pharmaceutical composition of this invention can be formulated 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).

[0036] 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 readily determine the optimal dose to be administered, and the optimal dose will vary depending on the specific drug used, the route of administration, the strength of the formulation, and the progression of the disease condition. Furthermore, factors related to the specific patient receiving treatment, including the patient's age, weight, diet, and timing of administration, will necessitate adjustments to the dose to an appropriate therapeutic level.

[0037] According to the specific implementation plan, an effective amount or effective dose refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the disease, disorder, or condition to be treated or related symptoms; (ii) reducing the duration of the disease, disorder, or condition to be treated or related symptoms; (iii) preventing the development of the disease, disorder, or condition to be treated or related symptoms; (iv) causing the remission of the disease, disorder, or condition to be treated or related symptoms; (v) preventing the development or exacerbation of the disease, disorder, or condition to be treated or related symptoms. (vi) to prevent recurrence of the treated disease, disorder, or condition or related symptoms; (vii) to reduce hospitalization of subjects suffering from the treated disease, disorder, or condition or related symptoms; (viii) to reduce the length of hospital stay of subjects suffering from the treated disease, disorder, or condition or related symptoms; (ix) to improve the survival of subjects suffering from the treated disease, disorder, or condition or related symptoms; (xi) to suppress or reduce the treated disease, disorder, or condition or related symptoms in subjects; and / or (xii) to enhance or improve the preventive or therapeutic effects of another therapy.

[0038] The dosage forms of the pharmaceutical compositions described in this invention are dosage forms that are advantageous for administration and are prepared by conventional methods, including but not limited to: non-gastrointestinal dosage forms and gastrointestinal dosage forms. Specific examples include but are not limited to: aqueous injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder inhalers, sustained-release agents, and controlled-release agents.

[0039] In some embodiments, the injectable dosage form includes, but is not limited to, various injectable formulations such as intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and intracavitary injection.

[0040] In some implementations, the cavity drug delivery dosage form includes, but is not limited to: suppositories, aerosols, effervescent tablets, drops, pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.

[0041] In some embodiments, the mucosal drug delivery dosage forms include, but are not limited to: eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive tablets, and films.

[0042] In some embodiments, the skin delivery dosage forms include, but are not limited to: topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.

[0043] A third aspect of the present invention provides a method for evaluating whether a substance to be screened has the effect of preventing and / or treating muscle atrophy.

[0044] Furthermore, the method includes using mouse-derived Serpina3n recombinant protein or human-derived Serpina3 recombinant protein as a positive control to evaluate the efficacy of the drug to be screened.

[0045] Furthermore, 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.

[0046] In some embodiments, the drug to be screened also includes a substance having an amino acid sequence that is 80% identical to that of mouse Serpina3n recombinant protein or human Serpina3 recombinant protein.

[0047] In some embodiments, the substance that promotes the expression or activity of mouse-derived Serpina3n recombinant protein or human-derived Serpina3 recombinant protein refers to any substance that can increase the activity of Serpina3n protein, improve the stability of Serpina3n protein, upregulate the expression of Serpina3n protein, or increase the effective duration of Serpina3n protein action. These substances can all be used in this invention as substances useful for upregulating Serpina3n, thereby being used to prevent or treat muscular atrophy.

[0048] The fourth aspect of the present invention provides a method for relieving and / or inhibiting myotube cell atrophy in vitro for non-therapeutic purposes.

[0049] Furthermore, the method includes the following steps: treating myotube cells with an effective amount of mouse-derived Serpina3n recombinant protein or human-derived Serpina3 recombinant protein or a pharmaceutical composition containing an effective amount of mouse-derived Serpina3n recombinant protein or human-derived Serpina3 recombinant protein.

[0050] The fifth aspect of the present invention provides a system for alleviating and / or inhibiting myotube cell atrophy or for alleviating and / or inhibiting skeletal muscle.

[0051] Furthermore, the system includes a treatment unit for treating myotube cells or skeletal muscle with a therapeutically effective amount of mouse-derived Serpina3n recombinant protein or human-derived Serpina3 recombinant protein.

[0052] In this invention, the term "therapeutic effective amount" refers to an amount sufficient to achieve the desired outcome 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 subject's disease state, age, sex, and weight. As will be understood by those skilled in the art, dosage or treatment regimens can be adjusted to provide an optimal therapeutic response.

[0053] In this invention, the subject of drug treatment is defined as any member of the animal kingdom, typically a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domesticated and farm animals, as well as zoo, sporting, or pet animals such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. As a preferred embodiment, the subject of treatment is a human.

[0054] In some specific embodiments, the system provided by the present invention includes a computer-readable storage medium for storing a computer-readable program or instructions, which, when executed by a processor, enable the therapeutic unit in the system of the present invention to perform its functions.

[0055] In some implementations, the system may be a user's electronic device or a computer system remotely located relative to the electronic device.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0057] The terms “comprising,” “including,” or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. The indefinite articles “a” and “an” preceding an element or component of the invention are not restrictive in terms of the number of elements or components (i.e., the number of times they appear). Therefore, “an” or “an” should be interpreted as including one or at least one, and singular elements or components also include plural forms, unless the quantity clearly refers only to the singular. “A plurality” means at least two, such as two, three, etc., unless otherwise expressly specified.

[0058] The beneficial effects of this invention are: This invention is the first to discover and confirm the novel use of mouse-derived Serpina3n recombinant protein or human-derived Serpina3 recombinant protein as a therapeutic agent for muscular atrophy. It provides a new direction for the research and development of therapeutic agents for muscular atrophy and is expected to become a safe and effective drug for the treatment of muscular atrophy, with broad application prospects in the field of muscular atrophy treatment. Attached Figure Description

[0059] Figure 1 The figure shows the effect of recombinant Serpina3n protein on senescent myotube cells; Figure 1 A shows the Myh staining pattern of myotube cells; Figure 1 B and C are quantitative diagrams of the length and diameter of the myotube, respectively; Figure 1 Figure D shows the expression of Atrogen1, a marker gene for muscle atrophy, as detected by Western Blot. Figure 2 The figure shows the effect of recombinant Serpina3n protein on dexamethasone-induced myotube cell atrophy; in which... Figure 2A shows Myh staining and quantitative measurements of length and diameter of myotube cells; Figure 2 Figure B shows the expression of Atrogen1 and Murf1, marker genes for muscle atrophy, as detected by Western Blot. Figure 3 The figure shows the effect of recombinant Serpina3n protein on age-related skeletal muscle atrophy; among which, Figure 3 Figure A shows the weight results 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 gripping strength and endurance results of mice in the PBS injection group and the Serpina3n recombinant protein injection group, respectively. Figure 4 The figure shows the effect of recombinant Serpina3n protein on disuse-induced skeletal muscle atrophy in mice; among them... Figure 4 A is a flowchart of the manufacturing process for each group of mouse models; Figure 4 B shows the weight results of four types of muscle fibers in different groups of mice; Figure 4 C shows the results of grip strength and endurance for each group of mice; Figure 4 Figure D shows the expression of Atrogen1 and Murf1, marker genes for muscle atrophy, in mice of each group as detected by Western Blot.

[0060] Figure 5 Figure showing the effect of human Serpina3 recombinant protein on dexamethasone-induced myotube cell atrophy; Figure 5 Figure A shows the expression of Atrogen1 and Murf1, marker genes for muscle atrophy in atrophic myotubes, as detected by RT-PCR. Figure 5 Figure B shows the expression of Atrogen1 and Murf1, marker genes for muscle atrophy in atrophic myotubes, as detected by Western blotting. Detailed Implementation

[0061] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0062] Therefore, the present invention is intended to cover such modifications and variations that fall within the scope of the appended claims and their equivalent scope. Other objects, features, and aspects of the present invention are disclosed in the following detailed description or are obvious therefrom. 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.

[0063] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. In the quantitative tests of the following examples, three repeated experiments are set, and the results are averaged.

[0064] Example 1 Serpina3n recombinant protein can alleviate the atrophy of senescent myotubes 1. Experimental method 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 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 ultrasonic treatment was performed. 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, and then the protein was eluted by loading it onto a nickel column using a wash buffer (50 mM Tris-HCl, 150 mM NaCl, 150 mM imidazole). The collected protein was further purified and separated on a Superdex™ 75 Increase chromatography column (Cytiva, 29148721). The purified Serpina3n recombinant protein was dialyzed to remove imidazole, and the purified Serpina3n recombinant protein was stored at -80 °C.

[0065] Culture and induction of differentiation of mouse myoblast line C2C12: (1) C2C12 cell proliferation stage: During the proliferation stage, C2C12 cells are cultured in complete medium DMEM containing 10% fetal bovine serum and 2% penicillin / streptomycin mixture. The cell density should not be too high during the culture period. Cell passage is required when the cell density reaches 50%-60%.

[0066] (2) Inducing C2C12 cells to undergo myoblast differentiation: When the density of C2C12 cells reaches 90%-100%, they are induced to differentiate using complete culture medium DMEM (called differentiation medium) containing 2% horse serum and 2% penicillin / streptomycin mixture. Fresh differentiation medium is replaced every two days until they differentiate into mature myotubes (generally, they differentiate into mature myotubes in 7 days).

[0067] Mature myotube cells differentiated for 7 days were stained with Myh. Senescent myotube cells differentiated for 11 days were stained with Myh after adding 10 ng and 50 ng of recombinant Serpina3n (SPN) protein to their culture medium. The control group culture medium was supplemented with Vehicle empty control liquid PBS. The length and diameter of the stained myotube cells were then quantified. The expression of the marker gene Atrogen1 for muscle atrophy in senescent myotubes was detected by Western blotting.

[0068] Western Blotting: Cell samples were lysed for 1 hour in lysis buffer (50 mM tris, pH 7.5, 150 mM NaCl, 0.5% NP-40) with added protease inhibitors, followed by SDS-PAGE and wet transfer. After transfer, the membrane was blocked with 5% skim milk powder for 1 hour, then incubated with primary antibody overnight at 4°C. After incubation, the membrane was washed 3 times with washing buffer for 10 minutes each time, followed by incubation with the corresponding secondary antibody, and then washed 3 times with washing buffer for 10 minutes each time. Finally, the colorimetric reaction was performed using horseradish peroxidase chemiluminescence.

[0069] 2. Experimental Results Myh staining was performed on mature myotube cells differentiated for 7 days and senescent myotube cells differentiated for 11 days. The results are as follows: Figure 1 As shown in Figure A, the length and diameter of the stained myotubes were quantified, and the results are shown in Figure A. Figure 1As shown in B and C, the myotubes of Day 11 showed shortened length and thinner diameter, indicating atrophy in aging myotubes. Treatment with two different concentrations of recombinant Serpina3n protein (SPN) to the myotubes of Day 11 both prolonged myotube length and increased diameter, indicating that the myotube atrophy was alleviated. Simultaneously, Western blotting analysis revealed a decreasing trend in the expression of Atrogen1, a marker gene for muscle atrophy in aging myotubes. Figure 1 (D) This result supports the idea that SPN can alleviate the atrophy of senescent myotube cells.

[0070] Example 2: Recombinant Serpina3n protein can alleviate dexamethasone-induced myotube cell atrophy. 1. Experimental Methods Long-term use of the anti-inflammatory drug dexamethasone (DEX) can lead to muscle atrophy as a side effect. Pretreated mature myotube cells were treated with 80 μM dexamethasone for 24 hours, resulting in a drug-induced atrophy phenotype characterized by reduced myotube length and diameter. In this case, two concentrations of SPN recombinant protein (10 ng and 50 ng) were used for treatment. The control group received Vehicle empty control PBS. Myh staining and Western blotting were then performed.

[0071] 2. Experimental Results The results of myotube length and diameter after SPN treatment are as follows: Figure 2 As shown in Figure A, treatment with both concentrations of SPN alleviated both the length and diameter of the myotubes. Simultaneously, Western blotting analysis revealed a decreasing trend in the expression of Atrogen1 and Murf1, marker genes for muscle atrophy in senescent myotubes. Figure 2 (B) This result supports the idea that SPN can alleviate dexamethasone-induced myotube cell atrophy.

[0072] Example 3: Recombinant Serpina3n protein can alleviate age-related skeletal muscle atrophy. 1. Experimental Methods Mice husbandry and drug administration: 20-month-old aged C57BL / 6 mice were purchased from Beijing Spefair Pharmaceutical Co., Ltd., and 8-week-old young C57BL / 6 mice were purchased from Beijing Vital River Pharmaceutical Co., Ltd. All animal experimental procedures were approved by the Animal Ethics Committee of Peking Union Medical College. All mice used in the experiment were housed and bred in SPF-grade animal facilities, with the indoor temperature strictly controlled at 21-25℃, humidity at 50%-60%, and 12 hours of light followed by 12 hours of darkness. The feed, water, and bedding used by the mice underwent strict high-temperature sterilization. Mice feed and drinking water were replenished or changed daily, and bedding was changed twice a week. The growth and development of the mice were closely monitored throughout the rearing process.

[0073] The drug administration regimen for 20-month-old aged C57BL / 6 mice was as follows: 50 ng of recombinant Serpina3 protein was injected orally into the right hind leg muscle of the mice three times a week for three consecutive weeks. After three weeks, the mice were sacrificed, and four types of muscle fibers from the injected hind limb skeletal muscles were isolated: tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (Gas), and soleus (Sol). The weight of each type of muscle fiber was compared between the PBS control group and the SPN group, and grip strength and endurance tests were performed on the two groups of mice.

[0074] 2. Experimental Results The weight results of various muscle fibers in mice injected with PBS and SPN are as follows: Figure 3 As shown in Figure A, the weights of the extensor digitorum longus (EDL) and soleus (Sol) muscles in the injection group mice were significantly increased, indicating increased muscle mass and improved atrophy. The grip strength of the two groups of mice was tested. Figure 3 B) and endurance ( Figure 3 The test in C) showed that the grip strength and endurance of the SPN-injected mice were enhanced after injection, while there was no change in the PBS control group, indicating that the muscle function of the SPN-injected mice was enhanced.

[0075] Example 4: Recombinant Serpina3n protein can alleviate disuse atrophy of skeletal muscle in mice. 1. Experimental Methods Induction of muscular atrophy in 8-week-old young C57BL / 6 mice and drug administration regimen: Three groups of mice were designed for the experiment. One group served as a control group, receiving no treatment. The other two groups had their right hind legs bound with bandages to induce disuse skeletal muscle atrophy. After 10 days of binding, the mice were injected three times a week with either 50 ng SPN or Vehicle empty control liquid PBS for 3 consecutive weeks. After three weeks, the mice in all three groups were sacrificed, and four types of muscle fibers from the injected hind limb skeletal muscles were isolated: tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (Gas), and soleus (Sol). The weight of each type of muscle fiber was compared. Before sacrifice, endurance and grip strength tests were performed on the three groups of mice, and the expression of the marker genes for muscle atrophy, Atrogen1 and Murf1, in the skeletal muscles of the three groups of mice was detected using Western blotting.

[0076] 2. Experimental Results Modeling diagram as follows Figure 4 As shown in Figure A. The weight results for various muscle fibers are as follows. Figure 4 As shown in Figure B, compared to the control group, all four muscle fibers in the two SPN groups or Vehicle groups were reduced, indicating successful establishment of the muscle atrophy model. Simultaneously, compared to the PBS injection group, the SPN injection group showed a significant increase in the fiber mass of the extensor digitorum longus (EDL), gastrocnemius (Gas), and soleus (Sol) muscles, indicating that muscle atrophy was alleviated. Grasping strength and endurance tests were performed on the mice, and the results are as follows... Figure 4 As shown in Figure C, the grip strength and endurance of mice in the SPN injection group were stronger than those in the Vehicle empty-load injection group, indicating that SPN injection enhanced skeletal muscle function. Western blotting analysis of the expression of Atrogen1 and Murf1, marker genes for muscle atrophy in the skeletal muscle of the three groups of mice, also showed that SPN injection improved disuse-induced skeletal muscle atrophy. Figure 4 D).

[0077] Example 5: Recombinant human SerpinA3 protein can alleviate dexamethasone-induced atrophy of human muscle fibers. 1. Experimental Methods 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 number NP_001076.2. The deleted 25 amino acid sequences at the N-terminus are MERMLPLLALGLLAAGFCPAVLCHP) was constructed onto the pET-28a(+) expression vector containing a His tag. SerpinA3 containing the His tag was expressed in Transetta (DE3) (Transgene, CD801-02), cultured at 37 °C in LB medium, and IPTG with a final concentration of 1 mM was added when the OD 600 was approximately 0.6 to induce protein expression, and culturing was continued at 37 °C for 12 - 16 hours; after protein expression, the bacterial cells were lysed using a lysis solution (50 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole), and ultrasonic treatment was performed. The lysis solution 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, and 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™ 75 Increase chromatography column (Cytiva, 29148721). The imidazole was removed from the purified SerpinA3 recombinant protein by dialysis, and the purified SerpinA3 recombinant protein was stored at -80 °C. Human muscle stem cells were purchased from 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 until mature, the pre-differentiated and mature myotube cells were treated with 80 μM dexamethasone for 24 hours to cause a drug-induced atrophy phenotype with reduced myotube length and thinner diameter.

[0078] 2. Experimental results 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) for treatment for 12 hours achieved an effect of alleviating atrophy ( Figure 5 ). At the same time, RT-PCR ( Figure 5 A) and Western Blotting ( Figure 5Method B) was used to detect the expression of Atrogen1 and Murf1, marker genes for muscle atrophy in atrophic myotubes. The results showed a decreasing trend in these two markers of muscle atrophy, which supports the idea that human SPN can alleviate dexamethasone-induced atrophy of human myotube cells.

[0079] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An application of a recombinant protein, characterized in that, The recombinant protein is either mouse-derived Serpina3n recombinant protein or human-derived Serpina3 recombinant protein. The application is selected from either 1) or 2): 1) Application in the preparation of products for treating muscular atrophy; 2) Application in the preparation of reagents for relieving and / or inhibiting skeletal muscle atrophy caused by aging in vitro; The muscle atrophy is classified as disuse atrophy, age-related muscle atrophy, and dexamethasone-induced muscle atrophy. The products include pharmaceuticals; The mouse-derived Serpina3n recombinant protein is based on the wild-type Serpina3n protein with 24 amino acid sequences deleted from the N-terminus. The wild-type Serpina3n protein has the accession number NP_033278.2 on NCBI, and the deleted N-terminal 24 amino acid sequences are AFIAALGLLMAGICPAVLCFPDGT. The recombinant human Serpina3 protein is based on the wild-type Serpina3 protein with 25 amino acid sequences deleted from the N-terminus. The wild-type Serpina3 protein has the NCBI accession number NP_001076.2, and the deleted N-terminal 25 amino acid sequences are MERMLPLLALGLLAAGFCPAVLCHP.

2. The application according to claim 1, characterized in that, The dosage forms of the drug include granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions.

3. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

5. The application according to claim 1, characterized in that, The drug also includes other substances for treating muscle atrophy.

6. The application according to claim 5, characterized in that, Other substances used to treat muscle atrophy include glucocorticoids, vitamins, and immunosuppressants.

7. The application according to claim 6, characterized in that, The vitamin medications mentioned include vitamin E and vitamin B1.

8. The application according to claim 6, characterized in that, The immunosuppressant drugs include nucleic acid drugs.

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