Application of gefitinib in preparation of medicine for improving and / or treating muscle atrophy

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of gefitinib in preparation of a medicine for improving and / or treating amyotrophy. The invention provides an application of gefitinib in preparation of a medicine for improving and / or treating muscle atrophy. The gefitinib provided by the invention can improve the occurrence of muscular atrophy from the cellular level. Meanwhile, the gefitinib can be used for treating the muscular atrophy caused by fixation of the posterior limbs of the mouse, after the gefitinib is treated, the grasping capacity, the running endurance and the contractility of the extensor of the toe length of the mouse are improved, the weight of the gastrocnemius muscle is increased, the cross sectional area of the gastrocnemius muscle fiber is increased, and the expression of muscular atrophy marker genes Atrogin-1 and MuRF-1 is reduced compared with that of a DMSO treatment group, so that the muscular atrophy can be effectively inhibited. The gefitinib can be applied to preparation of drugs for improving and / or treating muscle atrophy.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the use of gefitinib in the preparation of a drug for improving and / or treating muscle atrophy. Background Art

[0002] Muscle atrophy is caused by the reduction of tissue or organ volume and protein loss due to muscle cell atrophy. Systemic diseases such as heart failure and renal failure, acute physical injuries such as severe burns and major traumas, and diseases such as cancer, as well as factors such as aging, can all cause muscle atrophy. Muscle atrophy can seriously affect the quality of life of patients, increase the incidence of other related diseases in patients, seriously affect people's normal life, and bring a heavy burden to families and society. With the acceleration of aging, muscle atrophy diseases will increasingly become important diseases that endanger the health of the people. Clinically, for the treatment of muscle atrophy, except for exercise rehabilitation training, no effective treatment methods have been found. Therefore, developing new methods to combat muscle atrophy will be beneficial to various clinical conditions. Summary of the Invention

[0003] The purpose of the present invention is to provide the use of gefitinib in the preparation of a drug for improving and / or treating muscle atrophy. Gefitinib can improve various types of muscle atrophy, providing a new drug R & D approach and drug action target for the treatment of muscle atrophy.

[0004] The present invention provides the use of gefitinib in the preparation of a drug for improving and / or treating muscle atrophy.

[0005] As a preferred embodiment, the muscle atrophy includes at least one of disuse muscle atrophy, dexamethasone-induced muscle atrophy, tumor necrosis factor-α-induced muscle atrophy, and angiotensin II-induced muscle atrophy.

[0006] As a preferred embodiment, the disuse muscle atrophy includes muscle atrophy induced by hindlimb immobilization.

[0007] As a preferred embodiment, the efficacy of the drug includes at least one of the following:

[0008] 1) Restoring muscle strength;

[0009] 2) Increasing the weight of the gastrocnemius muscle;

[0010] 3) Increasing the cross-sectional area of gastrocnemius muscle fibers;

[0011] 4) Increasing the diameter of myotubes.

[0012] As a preferred embodiment, the drug is a unit-dose preparation; calculated based on mice, the unit-dose preparation is formulated into a dosage form for gefitinib administration at a dose of 10-50 mg / kg.

[0013] As a preferred embodiment, the gefitinib is the sole active ingredient of the drug.

[0014] As a preferred embodiment, the drug further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include one or more of diluents, buffers, suspensions, emulsions, granules, encapsulants, excipients, fillers, binders, sprays, transdermal absorbents, wetting agents, disintegrants, absorption promoters, surfactants, colorants, flavoring agents, and adsorbent excipients.

[0015] As a preferred embodiment, the dosage form of the drug includes an oral dosage form or an injection.

[0016] The present invention also provides the use of gefitinib in the preparation of a muscle atrophy gene expression inhibitor.

[0017] As a preferred embodiment, the muscle atrophy genes include Atrogin-1 and / or MuRF-1.

[0018] Beneficial effects: The present invention provides the use of gefitinib in the preparation of a drug for improving and / or treating muscle atrophy. The gefitinib of the present invention can restore muscle strength, increase the weight of the gastrocnemius muscle, the cross-sectional area of gastrocnemius muscle fibers, and the diameter of myotubes, and inhibit the expression of muscle atrophy genes. The results of the examples show that in muscle atrophy models induced by dexamethasone (Dex), tumor necrosis factor-α (TNF-α), and angiotensin II (AngII) respectively, the diameter of myotubes after gefitinib treatment is larger than that of the DMSO treatment group, and the expression of Atrogin-1 and MuRF-1 is lower than that of the DMSO treatment group, indicating that gefitinib treatment can improve muscle atrophy at the cellular level. At the same time, based on the mouse model of hindlimb immobilization (SWI), after gefitinib treatment, the grasping ability, running endurance, and extensor digitorum longus muscle contractility of the mice are improved, the weight of the gastrocnemius muscle increases, the cross-sectional area of gastrocnemius muscle fibers increases, and the expression of muscle atrophy marker genes Atrogin-1 and MuRF-1 is lower than that of the DMSO treatment group. Gefitinib can treat muscle atrophy caused by hindlimb immobilization in mice, and gefitinib can be applied to the preparation of a drug for improving and / or treating muscle atrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0020] Figure 1 It is the immunofluorescence detection map of different treatment groups in the Dex-induced myotube model in Example 1; where A is the micrograph, and the scale bar is 50 μm; B is the statistical chart of myotube diameter, *** indicates that the data has extremely significant differences, p < 0.001;

[0021] Figure 2 It is the expression map of muscle atrophy marker genes Atrogin-1 and MuRF-1 in different treatment groups of the Dex-induced myotube model in Example 1, *** indicates that the data has extremely significant differences, p < 0.001;

[0022] Figure 3 It is the immunofluorescence detection map of different treatment groups in the TNF-α-induced myotube model in Example 2; where A is the micrograph, and the scale bar is 50 μm; B is the statistical chart of myotube diameter, *** indicates that the data has extremely significant differences, p < 0.001;

[0023] Figure 4 It is the expression map of muscle atrophy marker genes Atrogin-1 and MuRF-1 in different treatment groups of the TNF-α-induced myotube model in Example 2, *** indicates that the data has extremely significant differences, p < 0.001;

[0024] Figure 5 It is the immunofluorescence detection map of different treatment groups in the AngⅡ-induced myotube model in Example 3; where A is the micrograph, and the scale bar is 50 μm; B is the statistical chart of myotube diameter, *** indicates that the data has extremely significant differences, p < 0.001;

[0025] Figure 6 It is the expression map of muscle atrophy marker genes Atrogin-1 and MuRF-1 in different treatment groups of the AngⅡ-induced myotube model in Example 3, ** indicates that the data has extremely significant differences, p < 0.01, *** indicates that the data has extremely significant differences, p < 0.001;

[0026] Figure 7 It is the statistical chart of the grasping ability and running endurance of mice in different treatment groups in Example 4; where A is the grasping ability map of mice; B is the running endurance map of mice; ** in the figure indicates that the data has extremely significant differences, p < 0.01, *** indicates that the data has extremely significant differences, p < 0.001;

[0027] Figure 8 It is the statistical chart of the contractile force of the extensor digitorum longus muscle of mice in different treatment groups in Example 4; where A is the relative contractile force; B is the forced contractile force; ** in the figure indicates that the data has extremely significant differences, p < 0.01, *** indicates that the data has extremely significant differences, p < 0.001;

[0028] Figure 9Statistical chart of the gastrocnemius muscle weight of mice in different treatment groups in Example 4; where A is a picture of the gastrocnemius muscle, B is a chart of the gastrocnemius muscle weight, * indicates significant difference in data, p < 0.05, ** indicates extremely significant difference in data, p < 0.01;

[0029] Figure 10 Statistical chart of the average cross-sectional area of the gastrocnemius muscle of mice in different treatment groups in Example 4; where A is a micrograph of muscle fibers, B is a chart of the cross-sectional area of the gastrocnemius muscle, *** indicates extremely significant difference in data, p < 0.001, and C is a chart of the distribution frequency of different gastrocnemius muscles;

[0030] Figure 11 Expression diagram of muscle atrophy marker genes Atrogin-1 and MuRF-1 in mice in different treatment groups in Example 4, * indicates significant difference in data, p < 0.05, ** indicates extremely significant difference in data, p < 0.01, *** indicates extremely significant difference in data, p < 0.001. Detailed implementation mode

[0031] The present invention provides an application of gefitinib in the preparation of a drug for improving and / or treating muscle atrophy. The present invention uses gefitinib to inhibit the activity of EGFR (epidermal growth factor receptor), thereby alleviating the occurrence of muscle atrophy at the cellular level. The source of the gefitinib in the present invention is not particularly limited. In a specific embodiment of the present invention, the gefitinib is purchased from selleck. The chemical structural formula of the gefitinib in the present invention is shown in Formula I:

[0032]

[0033] As an implementation mode, the muscle atrophy includes at least one of disuse muscle atrophy, dexamethasone-induced muscle atrophy, tumor necrosis factor-α-induced muscle atrophy, and angiotensin II-induced muscle atrophy. In a specific embodiment of the present invention, the muscle atrophy can be disuse muscle atrophy, dexamethasone-induced muscle atrophy, TNFα-induced muscle atrophy, or angiotensin II-induced muscle atrophy.

[0034] As an implementation mode, the disuse muscle atrophy includes muscle atrophy induced by hindlimb fixation.

[0035] As an implementation mode, the efficacy of the drug includes at least one of the following: 1) restoring muscle strength; 2) increasing the weight of the gastrocnemius muscle; 3) increasing the cross-sectional area of the gastrocnemius muscle fibers; 4) increasing the myotube diameter.

[0036] As an implementation mode, the drug is a unit-dose preparation; calculated based on mice, the unit-dose preparation is formulated into a dosage form for gefitinib administration at a dose of 10 - 50 mg / kg.

[0037] As an embodiment, gefitinib is the only active ingredient of the drug.

[0038] As an embodiment, the drug further comprises a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient includes one or more of a diluent, a buffer, a suspension, an emulsion, a granule, a capsule, an excipient, a filler, a binder, a spray, a transdermal absorbent, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, a colorant, a flavoring agent, and an adsorption carrier.

[0039] As an embodiment, the dosage form of the drug includes an oral dosage form or an injection.

[0040] The present invention also provides the use of gefitinib in the preparation of a muscle atrophy gene expression inhibitor. As an embodiment, the muscle atrophy gene includes Atrogin-1 and / or MuRF-1.

[0041] To further illustrate the present invention, the following describes in detail the use of gefitinib provided by the present invention in the preparation of a drug for improving and / or treating muscle atrophy with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] (1) Construction of dexamethasone (Dex) model

[0044] ① Culturing myotube cells:

[0045] A. One day before digesting C2C12 cells, add a 0.1% gelatin solution to the cell culture plate so that its bottom is covered with gelatin, and coat the bottom with gelatin overnight; the next day, examine the gelatin under a microscope and confirm that it is free of contamination for standby. Preparation of gelatin: Weigh 1 g of gelatin, add it to 100 mL of double-distilled water, and prepare a 1% gelatin solution. Sterilize it in an autoclave for 2 h; after the sterilization is completed and cooled to 40 °C, take out the gelatin solution, and filter it through a 0.22-μm filter head while it is hot, that is, obtain a 1% gelatin solution (10× solution). When in use, first dilute the 10× gelatin solution with sterilized water to a 1× solution (i.e., a 0.1% gelatin solution).

[0046] B. Digest C2C12 cells and count them, and dilute the C2C12 cells into a cell suspension of 30,000 cells / mL.

[0047] C. Discard the gelatin solution in the cell culture plate in step A, and quickly add the cell suspension in step B to the culture wells of the cell culture plate. During this process, do not let the bottom gelatin dry out, and use the figure-eight or cross method to shake it horizontally close to the workbench to make the cells evenly distributed in the culture medium, and then put it back into the incubator for culture.

[0048] D. After culturing for 24 - 48 h, observe the cells. Wait until the cells are completely spread out and the confluence reaches 70% - 80%, then change to the differentiation medium to start differentiation. The differentiation medium is based on high - glucose medium (DMEM Corning, #10 - 013 - CV) and also contains: 2% horse serum (Horse Serum Gibco, #16050 - 122) and 1% penicillin - streptomycin (Jiangsu KeyGen Biotech Co., Ltd., KGL2303 - 100); The time for complete differentiation is generally 4 - 5 d, and during this process, change the medium every day. When C2C12 cells differentiate into mature myotubes, they are randomly divided into an experimental group (Dex) and a control group (Con).

[0049] ② Establish a myotube atrophy model: Dissolve Dex powder in absolute ethanol to prepare a 25 mM stock solution and store it at 4°C. Dilute the Dex stock solution to 50 μM with the differentiation medium in the experimental group, and add an equal amount of absolute ethanol to the control group medium. Add the prepared Dex solution and absolute ethanol to the cells according to the grouping. After 24 h, detect the degree of myotube cell atrophy to confirm the successful establishment of the myotube atrophy model.

[0050] (2) Treat the Dex - induced atrophic myotubes with Gefitinib

[0051] ① Gefitinib (ZD1839) purchased from selleck is configured as a Gefitinib stock solution with a final concentration of 5 mM using DMSO.

[0052] ② Randomly divide the experimental group (Dex) in step (1) into 2 groups, denoted as Dex + DMSO group and Dex + Gefitinib group in sequence; Randomly divide the control group (Con) cells in step (1) into 2 groups, denoted as Con + DMSO group and Con + Gefitinib group in sequence. Among them, add the Gefitinib stock solution to the Dex + Gefitinib group and Con + Gefitinib group to make the final concentration of Gefitinib 2 μM, and add an equal amount of DMSO to the Dex + DMSO group and Con + DMSO group, and let it stand for 24 h.

[0053] (3) Immunofluorescence staining

[0054] ① Discard the medium in the cell plate treated for 24 h in step (2), wash it 3 times with 1×PBS, then add 4% paraformaldehyde (PFA, just cover it), place it on a slow shaker, and fix it at room temperature for 20 min.

[0055] ② Discard the PFA, wash it 3 times with 1×PBS, 5 min each time; After washing, add 0.5% Triton X - 100, and permeabilize the membrane at room temperature for 20 min.

[0056] ③ Discard Triton, wash 3 times with 1×PBS for 5 min each time; block with 5% BSA (bovine serum albumin) solution at room temperature for 2 h; dilute the primary antibody MF-20s (purchased from DSHB, #AB2147781) with 5% BSA solution at a ratio of 1:100 and incubate overnight on a slow shaker at 4°C. The 5% BSA solution is prepared with 1×PBS.

[0057] ④ The next day, discard the primary antibody, wash 3 times with 1×PBS for 5 min each time; dilute anti-mouse Alexa Fluor 488-labeled IgG (secondary antibody) with 5% BSA solution at a ratio of 1:200; incubate on a slow shaker at room temperature in the dark for 2 h.

[0058] ⑤ Discard the secondary antibody, wash 3 times with 1×PBS for 5 min each time; dilute Hoechst 33342 with 1×PBS at a ratio of 1:2000; if it is a 12-well plate, add 250 μL to each well; if it is a 24-well plate, add 150 μL to each well, incubate at room temperature in the dark for 20 min; after incubation, wash 3 times with 1×PBS for 5 min each time.

[0059] ⑥ Take pictures using a fluorescence microscope with a 20× objective lens. If it is a 12-well plate, take at least 40 pictures per well; if it is a 24-well plate, take at least 20 pictures per well. Use Image J to count the myotube diameter, and count at least 4 - 5 myotubes per picture. The detection results are shown in Table 1 and Figure 1 。

[0060] Table 1 Myotube diameters of different treatment groups in the Dex-induced myotube model

[0061] Group DMSO Group (μm) Gefitinib Group (μm) Control Group (Con) 25.6647 23.8494 Experimental Group (Dex) 12.8643 22.4445

[0062] According to Table 1 and Figure 1 it can be seen that in the Dex-induced muscle atrophy model, the myotube diameter after Gefitinib treatment is larger than that of the DMSO treatment group, indicating that Gefitinib treatment can improve the occurrence of Dex-induced muscle atrophy.

[0063] (4) Detection by real-time fluorescence quantitative PCR reaction

[0064] A. RNA extraction: Extract the RNA of cells in each treatment group using an RNA extraction kit (FastPure Complex Tissue / Cell Total RNA Isolation Kit, Vazyme, RC113-01).

[0065] B. cDNA Synthesis: ① Use a reverse transcription kit (Thermo-LBID, catalog number K16225) to perform reverse transcription with the RNA in step A as the template to obtain a cDNA reaction solution. The total reverse transcription system is 6 μL: 5 μL of RNA + sterilized water, 0.5 μL of Oligo DT, and 0.5 μL of Random; the reverse transcription reaction conditions are 65 °C for 5 min, 4 °C for ∞ / 2 min. ② Use the cDNA reaction solution in step ① as the template and primers to perform PCR amplification to obtain cDNA. The PCR system is 10 μL: 2 μL of Reaction buffer, 1 μL of 10 mM dNTP, 0.5 μL of RT enzyme, 0.5 μL of RI enzyme, and 4 μL of cDNA reaction solution; the PCR conditions are: 25 °C for 5 min, 42 °C for 1 h, 70 °C for 5 min, 4 °C for ∞.

[0066] C. Real-time Fluorescent Quantitative PCR Reaction for Common Genes: Use a real-time fluorescent quantitative PCR instrument to perform real-time fluorescent quantitative PCR to detect the expression of muscle atrophy marker genes (Atrogin-1 and MuRF-1). The real-time fluorescent quantitative PCR reaction system is shown in Table 2.

[0067] Table 2 Real-time Fluorescent Quantitative PCR Reaction System

[0068] Reagent Usage Amount (μL) SYBRGREEN 4.875 Sense Primer (10 μM) 0.25 Antisense Primer (10 μM) 0.25 cDNA Template 0.125 Nuclease-free water 4.5 Total Volume 10

[0069] The primer sequences for the real-time fluorescent quantitative PCR are as follows:

[0070] Atrogin-1-F (SEQ ID NO.1): 5′-CAGCTTCGTGAGCGACCTC-3′;

[0071] Atrogin-1-R (SEQ ID NO.2): 5′-GGCAGTCGAGAAGTCCAGTC-3′;

[0072] MURF-1-F (SEQ ID NO.3): 5′-GTGTGAGGTGCCTACTTGCTC-3′;

[0073] MURF-1-R (SEQ ID NO.4): 5′-GCTCAGTCTTCTGTCCTTGGA-3′;

[0074] mmu-18s-F (SEQ ID NO.5): 5′-TCAAGAACGAAAGTCGGAGG-3′;

[0075] mmu-18s-R (SEQ ID NO.6): 5′-GGACATCTAAGGGCATCAC-3′.

[0076] The real-time fluorescence quantitative PCR reaction program is as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for 34 cycles; sufficient extension at 72°C for 10 min; 4°C ∞. After the reaction, the Ct value of the fluorescence curve was exported using software, and semi-quantification was performed using the internal reference method. The detection results of the real-time fluorescence quantitative PCR reaction are shown in Table 3 and Figure 2 as follows.

[0077] Table 3 Relative expression levels of atrophy genes in different treatment groups of the Dex-induced myotube model

[0078]

[0079] According to Table 3 and Figure 2 it can be seen that in the muscle atrophy model induced by Dex, the expressions of Atrogin-1 and MuRF-1 after Gefitinib treatment were lower than those in the DMSO treatment group, indicating that Gefitinib treatment can improve the occurrence of Dex-induced muscle atrophy.

[0080] Example 2

[0081] (1) Construction of the tumor necrosis factor-α (TNF-α) model

[0082] ① Culturing myotube cells: Operate according to ① in step (1) of Example 1.

[0083] ② Construction of the myotube atrophy model: A. Dissolve TNF-α powder in sterilized ddH 2 O to prepare a stock solution of 100 μg / mL, and aliquot it into 2 mL EP tubes and store at -20°C. B. Dilute the TNF-α stock solution to 100 ng / mL with differentiation medium in the experimental group, and add an equal amount of sterilized ddH 2 O to the control group. Add the prepared medium to the cells according to the grouping, and detect the degree of myotube cell atrophy after 48 h to confirm the successful construction of the myotube atrophy model.

[0084] (2) Treatment of TNF-α-induced atrophic myotubes with Gefitinib

[0085] ① Gefitinib (ZD1839) was purchased from selleck and a Gefitinib stock solution with a final concentration of 5 mM was prepared using DMSO.

[0086] ②Randomly divide the experimental group (TNF-α) in step (1) into 2 groups, denoted as TNF-α+DMSO group and TNF-α+Gefitinib group in sequence; randomly divide the control group (Con) cells in step (1) into 2 groups, denoted as Con+DMSO group and Con+Gefitinib group in sequence. Among them, Gefitinib stock solution is added to the TNF-α+Gefitinib group and Con+Gefitinib group to make the final concentration of Gefitinib 2 μM, and an equal amount of DMSO is added to the TNF-α+DMSO group and Con+DMSO group, and then left standing for 24 h.

[0087] (3) Immunofluorescence staining

[0088] Operate according to step (3) of Example 1, and the detection results are shown in Table 4 and Figure 3 as follows.

[0089] Table 4 Myotube diameters of different treatment groups in the TNF-α-induced myotube model

[0090] Group DMSO Group (μm) Gefitinib Group (μm) Control Group (Con) 26.5241 26.4139 Experimental Group (TNF-α) 11.7325 27.2918

[0091] According to Table 4 and Figure 3 it can be seen that in the muscle atrophy model induced by TNF-α, the myotube diameter after Gefitinib treatment is larger than that of the DMSO treatment group, indicating that Gefitinib treatment can improve the occurrence of muscle atrophy induced by Dex.

[0092] (4) Detection by real-time fluorescence quantitative PCR reaction

[0093] Operate according to step (3) of Example 1, and the detection results of the real-time fluorescence quantitative PCR reaction are shown in Table 5 and Figure 4 .

[0094] Table 5 Relative expression levels of atrophy genes in different treatment groups of the TNF-α-induced myotube model

[0095]

[0096] According to Table 5 and Figure 4 it can be seen that in the muscle atrophy model induced by TNF-α, the expressions of Atrogin-1 and MuRF-1 after Gefitinib treatment are lower than those of the DMSO treatment group, indicating that Gefitinib treatment can improve the occurrence of muscle atrophy induced by TNF-α.

[0097] Example 3

[0098] (1) Construction of angiotensin II (Ang II) model

[0099] ① Culturing myotube cells: Operate according to ① in step (1) of Example 1.

[0100] ② Constructing the myotube atrophy model:

[0101] A. Prepare a 5 mM stock solution of Ang II with Ang II buffer solution, dispense it into 2 mL EP tubes, wrap them with tin foil, and store at -20 °C. The Ang II buffer solution is 0.9% normal saline prepared with 0.006% acetic acid solution.

[0102] B. Dilute the Ang II stock solution to 500 nM with differentiation medium in the experimental group, add an equal amount of Ang II buffer solution to the control group medium, add the prepared medium to the cells according to the grouping, wrap the cell plate with tin foil, and note that the whole operation process should be carried out under light-proof conditions. After 48 h, detect the degree of myotube cell atrophy to confirm the successful construction of the myotube atrophy model.

[0103] (2) Treating Ang II-induced atrophic myotubes with Gefitinib

[0104] ① Purchase Gefitinib (ZD1839) from selleck and prepare a Gefitinib stock solution with a final concentration of 5 mM using DMSO.

[0105] ② Randomly divide the experimental group (Ang II) in step (1) into 2 groups, denoted as Ang II + DMSO group and Ang II + Gefitinib group in sequence; randomly divide the control group (Con) cells in step (1) into 2 groups, denoted as Con + DMSO group and Con + Gefitinib group in sequence. Among them, add the Gefitinib stock solution to the Ang II + Gefitinib group and Con + Gefitinib group to make the final concentration of Gefitinib 2 μM, and add an equal amount of DMSO to the Ang II + DMSO group and Con + DMSO group, and let it stand for 24 h.

[0106] (3) Immunofluorescence staining

[0107] Operate according to step (3) of Example 1, and the detection results are shown in Table 6 and Figure 5 as follows.

[0108] Table 6 Myotube diameters of different treatment groups in the Ang II-induced myotube model

[0109] Group DMSO Group (μm) Gefitinib Group (μm) Control Group (Con) 30.7088 29.5682 Experimental Group (AngⅡ) 11.2246 28.3419

[0110] According to Table 6 and Figure 5It can be seen that in the Ang II-induced muscle atrophy model, the myotube diameter after Gefitinib treatment was larger than that in the DMSO treatment group, indicating that Gefitinib treatment could improve the occurrence of Ang II-induced muscle atrophy.

[0111] (4) Detection by real-time fluorescence quantitative PCR reaction

[0112] Performed according to the operation steps of Example 1 (3), and the results of real-time fluorescence quantitative PCR reaction are shown in Table 7 and Figure 6 .

[0113] Table 7 Relative expression levels of atrophy genes in different treatment groups of the Ang II-induced myotube model

[0114]

[0115] According to Table 7 and Figure 6 it can be seen that in the Ang II-induced muscle atrophy model, the expressions of Atrogin-1 and MuRF-1 after Gefitinib treatment were lower than those in the DMSO treatment group, indicating that Gefitinib treatment could improve the occurrence of Ang II-induced muscle atrophy.

[0116] Example 4

[0117] Establishment of the SWI (Spiral Wire Immobilization) mouse muscle atrophy model

[0118] ① Select male mice aged 8 - 10 weeks and randomly divide them into the muscle atrophy model SWI group (SWI) and the muscle atrophy model control group (sham);

[0119] ② Dip a cotton swab in a small amount of depilatory cream and gently apply it to the two hind limbs of the mice in the muscle atrophy model SWI group to remove the surface hair;

[0120] ③ After removing the hair, intraperitoneally inject 4% chloral hydrate (10 μL / g mouse body weight) for anesthesia;

[0121] ④ After the mice are anesthetized, wind iron wires around the two hind limbs to make the hind limbs of the mice immobile. At the same time, observe that the toes of the mice do not change color to ensure that the two limbs are not ischemic; ⑤ After the mice naturally regain consciousness, cage them separately to construct the SWI mouse muscle atrophy model.

[0122] (2) Application of Gefitinib in the treatment of muscle atrophy

[0123] ① Randomly divide the SWI group of the muscle atrophy model in step (1) into a DMSO injection group and a Gefitinib injection group, namely: DMSO injection + SWI group of the muscle atrophy model, Gefitinib injection group + SWI group of the muscle atrophy model; randomly divide the control group of the muscle atrophy model into a DMSO injection group and a Gefitinib injection group, namely: DMSO injection group + control group of the muscle atrophy model, Gefitinib injection group + control group of the muscle atrophy model. ② Dissolve 250 mg of Gefitinib (ZD1839, purchased from selleck) in 4 mL of DMSO solution to prepare a stock solution (concentration 62.5 mg / mL). Take 320 μL of the stock solution, add 2560 μL of PEG300, mix well until clear, then add 320 μL of Tween 80, mix well until clear, and then add 3200 μL of ddH 2 O, mix well until clear to obtain a Gefitinib solution, and use it immediately after preparation. ③ Intragastrically administer DMSO or Gefitinib to mice at a dose of 25 mg / kg / day for a total of 21 days. ④ After the intragastric administration is completed, test the grasping ability, running endurance, and extensor digitorum longus muscle contractility of the mice; after the test is completed, sacrifice the mice and isolate the gastrocnemius muscle and tibialis anterior muscle, and analyze the change in the weight of the gastrocnemius muscle; cut the middle part of the removed gastrocnemius muscle transversely and embed it in OCT, and store it at -80 °C; take the muscle sample embedded in OCT for frozen sectioning. When performing frozen sectioning, use a cryostat to cut 10 μm thin slices and stick them on glass slides. After the frozen sectioning is completed, put the frozen sections into -80 °C for storage; perform WGA staining on the frozen sections to statistically analyze the change in the size of muscle fibers; finally, extract the RNA of the gastrocnemius muscle tissue, and use fluorescence quantitative PCR to detect the expression changes of the muscle atrophy marker genes Atrogin-1 and MuRF-1. Judge whether injecting Gefitinib can improve the occurrence of muscle atrophy induced by SWI at the animal level according to the test results.

[0124] A. Detection of the grasping ability of mice:

[0125] First, place the mice on a tensile force measuring instrument for 3 minutes for adaptive adjustment, then let the mice grasp the metal rod on the measuring instrument, and then hold the mice's tails and pull the metal rod horizontally backward. At this time, the tensile force measuring instrument will display the tensile force value of the mice, and three effective values need to be recorded for each mouse.

[0126] When detecting the grasping force of the right hind limb, wrap the right hind limb of the mice with tape so that it cannot grasp the metal rod on the tensile force measuring instrument, and then perform the above measurement method. The detected value is the tensile force of the remaining three limbs. Repeat three times to calculate the average value, and then subtract the average value of the grasping force of the three limbs from the average value of the grasping force of the four limbs to obtain the grasping force value of the right hind limb. During the measurement, the grouping needs to be disrupted for blind testing, and the test results are shown in Table 8 and Figure 7 A in.

[0127] Table 8 Mouse Grip Ability

[0128] Group DMSO Group (g) Gefitinib Group (g) Muscle Atrophy Model Control Group (sham) 49.9443 49.0274 Muscle Atrophy Model SWI Group (SWI) 25.6667 34.2083

[0129] As can be seen from Table 8 and Figure 7 A, in the SWI group of the amyotrophic model, the grip ability of mice in the Gefitinib group was improved compared with that of mice in the DMSO injection group.

[0130] B Detection of Mouse Running Endurance:

[0131] In the experiment, the mice were placed on a small animal treadmill to evaluate their motor ability. First, the mice were allowed to walk on the treadmill at a speed of 5 m / min for 5 min to adapt to the treadmill; then the formal exercise started at a speed of 15 m / min. Then, the speed was increased by 1 m / min every 4 min until the mice were exhausted; at this time, the running distance of the mice was recorded, and the results are shown in Table 9 and Figure 7 B as shown.

[0132] Table 9 Mouse Running Distance

[0133] Group DMSO Group (m) Gefitinib Group (m) Muscle Atrophy Model Control Group (sham) 595.337 577.65 Muscle Atrophy Model SWI Group (SWI) 377.04 453.147

[0134] As can be seen from Table 9 and Figure 7 B, in the SWI group of the amyotrophic model, the running endurance of mice in the Gefitinib group was improved compared with that of mice in the DMSO injection group.

[0135] C. Detection of the Contractile Force of Mouse Extensor Digitorum Longus:

[0136] To measure the tension contractile force of the muscle, the EDL (extensor digitorum longus) muscle was fixed between a force sensor and an adjustable hook by a non-absorbable silk thread. The muscle was incubated in oxygenated saline at 37 °C for 1-2 min. The oxygenated saline is Krebs buffer, and the specific components are: NaCl 137×10 -3 M, KCl 5×10 -3 M, CaCl 2 2×10 -3 M, MgSO 4 1×10 -3 M, NaH 2 PO 4 1×10 -3 M, NaHCO 3 24×10 -3 M and glucose 11×10 -3 M, pH = 7.35.

[0137] Before the experiment, determine the optimal muscle length and record the muscle length. Use dynamic muscle control software (Aurora Scientific, ASI600A) to measure and control tetanic contractions according to the standard experimental protocol. Tetanic contraction induction parameters: initial delay 0.5 s; frequency: 120 Hz; duration 0.3 s. The detection results are shown in Table 10 and Figure 8 as follows.

[0138] Table 10 Relative contractile force of extensor digitorum longus muscle in mice

[0139] Group DMSO Group (mN / mm) Gefitinib Group (mN / mm) Muscle Atrophy Model Control Group (sham) 24.1248 23.2307 Muscle Atrophy Model SWI Group (SWI) 12.7854 17.9375

[0140] According to Table 10 and Figure 8 it can be seen that in the SWI group of the muscle atrophy model, compared with the mice in the DMSO injection group, the contractile force of the extensor digitorum longus muscle in the Gefitinib group of mice was improved.

[0141] D. Gastrocnemius muscle weight detection:

[0142] When sampling mice, remove the gastrocnemius muscle of the hindlimb of the mouse intact, take a photo and weigh it with an analytical balance and record it. The results are shown in Table 11 and Figure 9 as follows, where Figure 9 A is a picture of the gastrocnemius muscle, B is a graph of the gastrocnemius muscle weight, ** indicates that the data has significant differences, p < 0.01.

[0143] Table 11 Gastrocnemius muscle weight of mice

[0144] Group DMSO Group (g) Gefitinib Group (g) Muscle Atrophy Model Control Group (sham) 0.1595 0.1432 Muscle Atrophy Model SWI Group (SWI) 0.1208 0.1363

[0145] According to Table 11 and Figure 9 it can be seen that in the SWI group of the muscle atrophy model, compared with the mice in the DMSO injection group, the gastrocnemius muscle weight of the Gefitinib group of mice was improved.

[0146] E. Detection of WGA (wheat germ agglutinin) in mouse muscle tissue:

[0147] Take the frozen sections out of the -80 °C refrigerator and place them in a wet box for rewarming at room temperature for 20 min; wash them 3 times with 1×PBS for 5 min each time. Use a tissue paper to dry the water droplets around the tissue on the sections, taking care not to wipe off the tissue. Then, use an immunohistochemistry pen to circle the tissue. Drop 4% PFA onto the tissue with a Pasteur pipette to cover the tissue. After fixing at room temperature for 30 min, wash 3 times with 1×PBS for 5 min each time. After the washing is completed, dry the water droplets with a tissue paper. After drying, dilute the WGA staining solution with 1×PBS at a ratio of 1:200 and dilute the Hoechst dye at a ratio of 1:2000. Mix and dilute the WGA and Hoechst dyes to obtain a mixed staining solution. Under light-proof conditions, cover the tissue with the mixed staining solution using a Pasteur pipette and co-stain for 60 min at room temperature. Finally, wash 3 times with 1×PBS for 5 min each time, dry with a tissue paper, and mount the sections with 50% glycerol. After covering with a coverslip, place the slides in an opaque slide box and store at 4 °C. Randomly take pictures using a fluorescence microscope, select a 20× objective lens, take at least 20 pictures for each tissue section, and use Image J to statistically analyze the cross-sectional area of muscle fibers. Statistically analyze at least 500 muscle fibers for each mouse sample. The detection results are shown in Table 12 and Figure 10 as follows.

[0148] Table 12 Cross-sectional area of mouse gastrocnemius muscle

[0149] Group DMSO Group (μm) Gefitinib Group (μm) Muscle Atrophy Model Control Group (sham) 2981.04 2999.32 Muscle Atrophy Model SWI Group (SWI) 2257.38 2546.9

[0150] According to Table 12 and Figure 10 it can be seen that in the SWI group of the muscle atrophy model, the cross-sectional area of the gastrocnemius muscle fibers of the mice in the DMSO injection group was significantly smaller, and injecting Gefitinib could significantly improve muscle atrophy.

[0151] F. Detection by real-time fluorescence quantitative PCR reaction of common genes:

[0152] (1) Extraction of tissue RNA: ① Clean scissors, forceps, and steel beads with 75% ethanol; add steel beads and 1 mL of Trizol to a 2 mL EP tube; quickly cut a gastrocnemius muscle tissue the size of a rice grain and place it in a 2 mL EP tube (pre-cooled on ice); homogenize the gastrocnemius muscle tissue using a tissue homogenizer at 50 HZ for 5 min. After homogenization, use a magnet to remove the small steel beads, and let the sample lysate on ice for 10 min. ② After lysis, add 1 / 5 volume of chloroform to the EP tube, vortex for 10 s, and let it stand at room temperature for 5 min; after stratification appears, use a low-temperature high-speed centrifuge at 4°C, 12000 x g, and centrifuge for 15 min; transfer the upper aqueous phase in the EP tube after centrifugation to a new enzyme-free 1.5 mL EP tube, add an equal volume of isopropanol, gently mix up and down, and let it stand at room temperature for 10 min; after standing, centrifuge at 4°C, 12000 x g, and 20 min. ③ After centrifugation, white RNA can be seen at the bottom or side. Carefully use a pipette to slowly aspirate all the supernatant; add 1 mL of 75% ethanol, turn it upside down to make the precipitate float and wash away the isopropanol; centrifuge at 4°C, 12000 x g, and 10 min, and use a pipette to aspirate the ethanol; dry the residual ethanol in the RNA in a laminar flow hood, add 20 μL of DEPC water to dissolve the RNA, and dissolve it at 4°C for 30 min. Store at -20°C.

[0153] (2) cDNA synthesis: Perform according to the operation of B in step (4) of Example 1.

[0154] (3) Real-time fluorescence quantitative PCR reaction for common genes: Use a fluorescence quantitative PCR instrument to perform fluorescence quantitative PCR to detect the expression of muscle atrophy marker genes (Atrogin-1 and MuRF-1). The real-time fluorescence quantitative PCR reaction system, program settings, and primer information are the same as those in step (4) of Example 1. The detection results are shown in Table 13 and Figure 11 .

[0155] Table 13 Expression levels of mouse muscle atrophy marker genes Atrogin-1 and MuRF-1

[0156]

[0157] According to Table 13 and Figure 11 It can be seen that in the muscle atrophy model induced by SWI, after Gefitinib treatment, the expression of Atrogin-1 and MuRF-1 was lower than that in the DMSO treatment group, indicating that Gefitinib treatment can improve the occurrence of muscle atrophy induced by SWI.

[0158] In summary, Gefitinib treatment can improve the occurrence of muscle atrophy induced by Dex, TNF-α, and AngⅡ, and can treat muscle atrophy caused by hindlimb fixation in mice. Therefore, the compound Gefitinib can be applied to the preparation of drugs for treating muscle atrophy.

[0159] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of gefitinib in the preparation of a drug for improving and / or treating muscle atrophy.

2. The use according to claim 1, characterized in that: The muscle atrophy includes at least one of disuse muscle atrophy, dexamethasone-induced muscle atrophy, tumor necrosis factor-α-induced muscle atrophy and angiotensin II-induced muscle atrophy.

3. The use according to claim 2, characterized in that: The disuse-induced muscle atrophy includes muscle atrophy induced by hind limb immobilization.

4. The use according to claim 1, characterized in that: The efficacy of the drug includes at least one of the following: 1) Restore muscle strength; 2) Increase the weight of the gastrocnemius muscle; 3) Increase the cross-sectional area of ​​gastrocnemius muscle fibers; 4) Increase the diameter of myotubes.

5. The use according to claim 1, characterized in that: The drug is a unit dose preparation; based on mice, the unit dose preparation is formulated into a dosage form of 10 to 50 mg / kg for administration of gefitinib.

6. The use according to claim 1, characterized in that: The gefitinib is the only active ingredient of the drug.

7. The use according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include one or more of diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents and adsorption excipients.

8. The use according to claim 1, characterized in that: The dosage form of the drug includes oral dosage form or injection.

9. Application of gefitinib in the preparation of muscular atrophy gene expression inhibitors.

10. The use according to claim 9, characterized in that: The muscle atrophy genes include Atrogin-1 and / or MuRF-1.

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