Application of pyrimidinedione compound in preparation of medicine for improving and / or treating amyotrophy

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a pyrimidinedione compound in preparation of a medicine for improving and / or treating amyotrophy. The invention provides application of a pyrimidinedione compound in preparation of a medicine for improving and / or treating amyotrophy. The chemical structural formula of the pyrimidinedione compound is shown as a formula I in the specification. The pyrimidinedione compound (THPD) can recover muscle strength, increase the gastrocnemius muscle weight, the gastrocnemius muscle fiber cross sectional area and the myotube diameter and inhibit expression of muscle atrophy genes, and the compound THPD 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 medical technology, and specifically relates to the use of a pyrimidinedione compound in the preparation of a drug for improving and / or treating muscle atrophy. Background Art

[0002] At present, with the accelerated aging of the population, people's health problems are also in urgent need of resolution. An important health problem facing the aging population is muscle atrophy, which is a common complication and a systemic response to a variety of conditions such as heart failure, cancer, chronic kidney disease, and chronic obstructive pulmonary disease (COPD). It is characterized by the loss of muscle mass and strength, and muscle atrophy reduces the quality of life and survival rate. Muscle atrophy is also a condition observed due to loss of innervation, lack of activity, and aging. Muscle atrophy is caused by abnormal activation of protein degradation and reduced protein synthesis. So far, there is no effective treatment for muscle atrophy except for nutritional supplementation and physical exercise. Therefore, the development of new methods to combat muscle atrophy will benefit a variety of clinical conditions. Summary of the invention

[0003] The object of the present invention is to provide an application of a pyrimidinedione compound in the preparation of a drug for improving and / or treating muscle atrophy, wherein the pyrimidinedione compound can restore muscle strength, increase gastrocnemius muscle weight, gastrocnemius muscle fiber cross-sectional area and myotube diameter, and inhibit the expression of muscle atrophy genes.

[0004] The present invention provides an application of a pyrimidinedione compound in the preparation of a drug for improving and / or treating muscle atrophy. The chemical structural formula of the pyrimidinedione compound is shown in Formula I:

[0005]

[0006] As a preferred embodiment, the muscle atrophy includes disuse muscle atrophy and / or dexamethasone-induced muscle atrophy.

[0007] As a preferred embodiment, the disuse-induced muscle atrophy includes muscle atrophy induced by hind limb immobilization.

[0008] As a preferred embodiment, the efficacy of the drug includes at least one of the following:

[0009] 1) Restore muscle strength;

[0010] 2) Increase the weight of the gastrocnemius muscle;

[0011] 3) Increase the cross-sectional area of ​​gastrocnemius muscle fibers;

[0012] 4) Increase the diameter of myotubes.

[0013] As a preferred embodiment, the drug is a unit dose preparation; the unit dose preparation is formulated into a dosage form for administration of pyrimidinedione compounds at 10 to 50 mg / kg of mouse body weight.

[0014] As a preferred embodiment, the pyrimidinedione compound is the only active ingredient of the drug.

[0015] As a preferred embodiment, the drug also includes a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier includes one or more of a diluent, a buffer, a suspension, an emulsion, a granule, an encapsulation, an excipient, a filler, an adhesive, a spray, a transdermal absorbent, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, a colorant, a flavoring agent and an adsorption carrier.

[0016] As a preferred embodiment, the dosage form of the drug includes oral dosage form and / or injection.

[0017] The present invention also provides an application of a pyrimidinedione compound in the preparation of a muscular atrophy gene expression inhibitor, wherein the chemical structural formula of the pyrimidinedione compound is shown in Formula I.

[0018] As a preferred embodiment, the muscle atrophy gene includes Atrogin-1 and / or MuRF-1.

[0019] Beneficial effects: The present invention provides an application of a pyrimidinedione compound in the preparation of a drug for improving and / or treating muscle atrophy, and the chemical structural formula of the pyrimidinedione compound is shown in Formula I. The pyrimidinedione compound (THPD) of the present invention can restore muscle strength, increase gastrocnemius muscle weight, gastrocnemius muscle fiber cross-sectional area and myotube diameter, and inhibit the expression of muscle atrophy genes. The results of the embodiment show that in the Dex-induced muscle atrophy model, the myotube diameter after THPD treatment is larger than that of the DMSO-treated group, and the expression of Atrogin-1 and MuRF-1 is lower than that of the DMSO-treated group, indicating that THPD treatment can improve the occurrence of Dex-induced muscle atrophy. At the same time, after treatment with the compound THPD in the mouse model based on hindlimb fixation (SWI), the mouse's grasping ability, running endurance and contraction force of the long extensor digitorum were improved, the gastrocnemius weight increased, the gastrocnemius muscle fiber cross-sectional area increased, and the expression of muscle atrophy marker genes Atrogin-1 and MuRF-1 was lower than that of the DMSO-treated group. The compound THPD can treat muscle atrophy caused by hind limb fixation in mice, and the compound THPD can be used in preparing drugs for improving and / or treating muscle atrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 The immunofluorescence staining of cells after THPD treatment in Example 1 is a representative image; A is a microscopic image, with a scale of 50 μm; B is a statistical graph of myotube diameters, ** indicates that the data are significantly different, p < 0.01, *** indicates that the data are significantly different, p < 0.001;

[0022] Figure 2 This is the expression diagram of muscle atrophy marker genes in cells after THPD treatment in Example 1, ** indicates that the data are significantly different, p < 0.01;

[0023] Figure 3 Schematic diagram of the THPD injection treatment of SWI model mice in Example 2;

[0024] Figure 4 The figures are statistical graphs of the grasping ability and running endurance of SWI mice after injection of THPD in Example 2; A is a graph of the grasping ability of mice; B is a graph of the running endurance of mice; ** in the figure indicates that the data are significantly different, p < 0.01;

[0025] Figure 5 This is a statistical diagram of the contraction force of the extensor digitorum longus muscle of SWI mice after injection of THPD in Example 2; A is the relative contraction force; B is the tetanic contraction force; *** in the figure indicates that the data are significantly different, p < 0.001;

[0026] Figure 6 The weight of the gastrocnemius muscle of the SWI mice after injection of THPD in Example 2 is statistically shown; A is a picture of the gastrocnemius muscle; B is a graph of the gastrocnemius muscle weight; ** indicates that the data are significantly different, p < 0.01;

[0027] Figure 7 The figure is a statistical diagram of the average cross-sectional area of ​​the gastrocnemius muscle of the SWI mice after injection of THPD in Example 2; A is a micrograph of muscle fibers; B is a cross-sectional area diagram of the gastrocnemius muscle, ** indicates that the data are significantly different, p < 0.01; C is a distribution frequency diagram of gastrocnemius muscles with different cross-sectional areas;

[0028] Figure 8 This is the expression diagram of the muscle atrophy marker gene in SWI mice after injection of THPD in Example 2. ** indicates that the data have significant differences, p < 0.01. DETAILED DESCRIPTION

[0029] The present invention provides an application of a pyrimidinedione compound in the preparation of a drug for improving and / or treating muscle atrophy. The chemical structural formula of the pyrimidinedione compound is shown in Formula I:

[0030]

[0031] The pyrimidinedione compound (6-{[benzyl(cyclopropyl)amino]methyl}-1,2,3,4-tetrahydropyrimidine-2,4-dione, C 15 H 17 N3O2, THPD) is a small molecule chemical inhibitor. The present invention does not specifically limit the source of the pyrimidine dione compound. In a specific embodiment of the present invention, the pyrimidine dione compound is purchased from Enamine Ltd, Cat: Z287226108.

[0032] As an embodiment, the muscle atrophy includes disuse muscle atrophy and / or dexamethasone-induced muscle atrophy. In a specific embodiment of the present invention, the muscle atrophy can be dexamethasone-induced muscle atrophy, and can also be disuse muscle atrophy. As an embodiment, the disuse muscle atrophy includes hind limb immobilization-induced muscle atrophy.

[0033] As an embodiment, the efficacy of the drug includes at least one of the following: 1) restoring muscle strength; 2) increasing gastrocnemius muscle weight; 3) increasing gastrocnemius muscle fiber cross-sectional area; 4) increasing myotube diameter.

[0034] As an embodiment, the drug is a unit dose preparation; the unit dose preparation is formulated into a dosage form for administering 10 to 50 mg / kg of mouse body weight of the pyrimidinedione compound.

[0035] As an embodiment, the pyrimidinedione compound is the only active ingredient of the drug. As an embodiment, the drug further comprises a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier comprises one or more of a diluent, a buffer, a suspension, an emulsion, a granule, an encapsulation, an excipient, a filler, an adhesive, a spray, a transdermal absorbent, a wetting agent, a disintegrant, an absorption accelerator, a surfactant, a colorant, a flavoring agent, and an adsorption carrier.

[0036] As an embodiment, the dosage form of the drug includes an oral dosage form and / or an injection.

[0037] The present invention also provides an application of a pyrimidinedione compound in preparing a muscular atrophy gene expression inhibitor, wherein the chemical structural formula of the pyrimidinedione compound is shown in Formula I. As an embodiment, the muscular atrophy gene includes Atrogin-1 and / or MuRF-1.

[0038] In order to further illustrate the present invention, the application of a pyrimidinedione compound provided by the present invention in the preparation of a drug for improving and / or treating muscle atrophy is described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Establishment of a cellular muscle atrophy model

[0041] (1) Dexamethasone (Dex) model construction

[0042] ① One day before the differentiation of C2C12 cells, add 0.1% gelatin solution to the cell culture plate so that the bottom is covered with gelatin. The bottom is coated with gelatin overnight. The next day, inspect the gelatin under a microscope to confirm that it is free of contamination and set aside.

[0043] Preparation of gelatin: weigh 1g gelatin and add it to 100mL double distilled water to prepare 1% gelatin. After sterilization in an autoclave for 2h; after sterilization to 40℃, take out the gelatin solution and filter it with a 0.22μm filter head while it is hot to obtain a 1% gelatin solution (10× solution). When using, first dilute the 10× gelatin solution with sterile water to a 1× solution (i.e., 0.1% gelatin solution).

[0044] ② Digest the C2C12 cells, count them, and dilute them into a cell suspension of 30,000 cells / mL.

[0045] ③Discard the gelatin solution in the cell culture plate in step ①, and quickly add the cell suspension in step ② to the culture wells. Do not let the gelatin at the bottom dry out during this process, use the figure 8 or cross method, shake it horizontally against the workbench to evenly distribute the cells in the culture medium, and return it to the incubator for culture.

[0046] ④ After culturing for 24 to 48 hours, observe the cells, and when the cells are fully spread and the degree of confluence reaches 70% to 80%, replace them with differentiation medium to start differentiation; the time for differentiation to be completed is generally 4 to 5 days, and the medium can be changed every day during this process. The differentiation medium is based on a high-glucose medium (DMEM Corning, #10-013-CV) as the basic medium, and also contains: 2% horse serum (Horse Serum Gibco, #16050-122) and 1% penicillin-streptomycin (Jiangsu KeyGen Biotechnology Co., Ltd., KGL2303-100).

[0047] ⑤ Prepare Dex powder into a 25mM stock solution with anhydrous ethanol and store at 4°C. When C2C12 cells differentiated into mature myotubes, they were randomly divided into an experimental group (Dex) and a control group (Control). The experimental group used differentiation medium to dilute the Dex stock solution to 50μM, and the control group culture medium was added with an equal amount of anhydrous ethanol. The prepared Dex solution and anhydrous ethanol were added to the cells according to the grouping. After 24 hours, the degree of myotube cell atrophy was detected to construct a myotube atrophy model.

[0048] (2) THPD treatment of Dex-induced atrophic myotubes

[0049] ① THDP powder (purchased from Enamine Ltd, Cat: Z287226108, relative molecular mass 271.31) was prepared into a THDP stock solution with a final concentration of 5 mM using 737 μL of DMSO.

[0050] ② The control group in step (1) was randomly divided into a THDP group and a DMSO group, namely, a Control+THPD group and a Control+DMSO group; the experimental group was randomly divided into a THDP group and a DMSO group, namely, a Dex+THPD group and a Dex+DMSO group. THDP stock solution was added to the Control+THPD group and the Dex+THPD group, respectively, so that the final concentration of THDP was 50 μM; an equal amount of DMSO was added to the Control+DMSO group and the Dex+DMSO group, respectively; after adding THPD or DMSO, the cells were allowed to stand for 24 hours.

[0051] (3) Immunofluorescence staining

[0052] ① Discard the culture medium in the cell plate treated for 24 hours in step (2), wash 2-3 times with 1×PBS, add 4% paraformaldehyde (PFA), cover it, place it on a slow shaker, and fix it at room temperature for 20 minutes.

[0053] ② Discard PFA and wash with 1×PBS for 3 times, 5 min each time; after washing, add 0.5% polyethylene glycol octylphenyl ether (Triton) and break the membrane at room temperature for 20 min.

[0054] ③ Discard Triton, wash with 1×PBS 3 times, 5 min each time; block with 5% BSA at room temperature for 2 h, dilute MF-20s (primary antibody, DSHB, MF-20-s, #AB2147781) with 5% BSA at a ratio of 1:100, and incubate at 4°C overnight. The 5% BSA was prepared with 1×PBS.

[0055] ④ On the next day, discard the primary antibody and wash three times with 1×PBS, 5 min each time; dilute the anti-mouse AlexaFluor488-labeled IgG (secondary antibody) with 5% BSA at a ratio of 1:200; incubate at room temperature on a slow shaker in the dark for 2 h.

[0056] ⑤Discard the secondary antibody, wash with 1×PBS for 3 times, 5 min each time; dilute Hoechst33342 with 1×PBS at a ratio of 1:2000; incubate at room temperature in the dark for 20 min; after incubation, wash with 1×PBS for 3 times, 5 min each time.

[0057] ⑥ Use a fluorescence microscope to take pictures, and use a 20x 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 diameter of myotubes, and count at least 4 to 5 myotubes per picture. The test results are shown in Tables 1 and Figure 1 , where A is a microscopic picture with a scale of 50 μm; B is a statistical graph of myotube diameters, ** indicates that the data are significantly different, p < 0.01, *** indicates that the data are significantly different, p < 0.001.

[0058] Table 1 Myotube diameters in different treatments

[0059] Group DMSO group (μm) THPD group (μm) Control 32.763 34.200 Experimental group (Dex) 21.534 26.742

[0060] According to Table 1 and Figure 1 It can be seen that in the Dex-induced muscle atrophy model, the diameter of the myotubes after THPD treatment was larger than that of the DMSO-treated group, indicating that THPD treatment can improve the occurrence of Dex-induced muscle atrophy.

[0061] (4) Real-time fluorescence quantitative PCR reaction detection

[0062] A. RNA extraction: RNA from cells in each treatment group was extracted using an RNA extraction kit (FastPure Complex Tissue / Cell Total RNA Isolation Kit, Vazyme, RC113-01).

[0063] B. cDNA synthesis:

[0064] ① Using a reverse transcription kit (Thermo Scientific TM , Product No. K1622) reverse transcribes the RNA in step A as a template to obtain a cDNA reaction solution. The total reverse transcription system is 6 μL: RNA + sterile water 5 μL, Oligo (dT) 18Primer 0.5 μL, Random primer 0.5 μL; the reverse transcription reaction conditions are 65°C 5 min, 4°C∞ / 2 min.

[0065] ② Using the cDNA reaction solution in step ① as a template, perform PCR amplification to obtain cDNA. The PCR system is 10 μL: Reaction buffer 2 μL, 10 mM dNTP 1 μL, RT enzyme 0.5 μL, RI enzyme 0.5 μL, cDNA reaction solution 4 μL; the PCR conditions are: 25°C 5 min, 42°C 1 h, 70°C 5 min, 4°C∞.

[0066] C. Real-time fluorescence quantitative PCR reaction of common genes: Fluorescence quantitative PCR was performed using 2×rTaq enzyme containing SYBR Green I nucleic acid molecule dye to detect the expression of muscle atrophy marker genes (Atrogin-1 and MuRF-1). The real-time fluorescence quantitative PCR reaction system is shown in Table 2.

[0067] Table 2 Real-time fluorescence quantitative PCR reaction system

[0068] Reagents Usage amount (μL) SYBRGREEN 4.875 Sense primer (10 μM) 0.25 Antisense primer (10 μM) 0.25 cDNA template 0.125 Nuclease-freewater 4.5 Total volume 10

[0069] The primer sequences of the real-time fluorescence 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′-GCTCAGTCTTTCTGTCCTTGGA-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 procedure is: 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, 34 cycles; full extension at 72°C for 10 min; 4°C∞. After the reaction, the fluorescence curve Ct value was derived using software, and semi-quantification was performed using the internal reference method. The real-time fluorescence quantitative PCR reaction detection results are shown in Table 3 and Figure 2 As shown, Figure 2 ** indicates that the data are significantly different, p < 0.01.

[0077] Table 3 Expression of muscle atrophy specific ubiquitinase in cells

[0078]

[0079] According to Table 3 and Figure 2 It can be seen that in the Dex-induced muscle atrophy model, the expression of Atrogin-1 and MuRF-1 after THPD treatment was lower than that in the DMSO-treated group, indicating that THPD treatment can improve the occurrence of Dex-induced muscle atrophy.

[0080] Example 2

[0081] (1) Establishment of the SWI (Spiral Wire Immobilization) mouse muscle atrophy model

[0082] ① Male mice aged 8 to 10 weeks were selected and randomly divided into a muscular atrophy model SWI group (SWI) and a muscular atrophy model control group (sham);

[0083] ② Use a cotton swab to dip a small amount of depilatory cream and gently apply it on the two hind limbs of the mice in the muscular atrophy model SWI group to remove the surface hair;

[0084] ③ After hair removal, 4% chloral hydrate (10 μL / g) was injected intraperitoneally for anesthesia;

[0085] ④ After the mouse is anesthetized, wrap the wire around the two hind limbs of the mouse to make them unable to move. At the same time, observe whether the toes of the mouse change color to ensure that there is no ischemia in the two limbs;

[0086] ⑤ After the mice regained consciousness naturally, they were placed in cages separately to construct the SWI mouse muscle atrophy model.

[0087] (2) Application of THPD in the treatment of muscular atrophy

[0088] ① The muscular atrophy model SWI group in step (1) was randomly divided into a DMSO injection group and a THPD injection group, namely, the DMSO injection group + muscular atrophy model control group, and the THPD injection group + muscular atrophy model control group; the muscular atrophy model control group was randomly divided into a DMSO injection group and a THPD injection group, namely, the DMSO injection group + muscular atrophy model SWI group, and the THPD injection group + muscular atrophy model SWI group.

[0089] ② In the DMSO injection group + muscular atrophy model control group and the DMSO injection + muscular atrophy model SWI group, DMSO was gavaged into the mice every day at a dose of 25 mg / kg / day, for a total of 28 days; in the THPD injection group + muscular atrophy model control group and the THPD injection group + muscular atrophy model SWI group, THPD was gavaged into the mice every day at a dose of 25 mg / kg / day, for a total of 28 days.

[0090] ③After gavage, test the mice's grasping ability, running endurance, and contraction force of the extensor digitorum longus muscle; after the test, separate the gastrocnemius and tibialis anterior muscles, and analyze the changes in gastrocnemius muscle weight; embed the middle part of the removed gastrocnemius muscle in OCT and freeze it at -80°C; take the OCT-embedded muscle sample for frozen sectioning, use a freezing slicer to cut 10 mm slices during ice cutting, stick them on a glass slide, and freeze the frozen sections at -80°C after ice cutting; perform WGA staining on the frozen sections to statistically analyze the changes in muscle fiber size; finally, extract RNA from the gastrocnemius muscle tissue, and use fluorescent quantitative PCR to detect the expression changes of muscle atrophy marker genes Atrogin-1 and MuRF-1. See the experimental process for details. Figure 3 Based on the test results, it is determined whether the injection of THPD can improve the occurrence of SWI-induced muscle atrophy at the animal level.

[0091] A. Mouse grasping ability test:

[0092] First, place the mouse on the tension measuring instrument for 3 minutes for adaptive adjustment. Then let the mouse grab the metal rod on the measuring instrument. Grasp the mouse's tail and pull the metal rod horizontally backward. At this time, the tension measuring instrument will display the mouse's tension value. Three valid values ​​must be recorded for each mouse.

[0093] When testing the gripping force of the right hind limb, the right hind limb of the mouse was wrapped with tape so that it could not grasp the metal rod on the tension measuring instrument, and then the above method was used for measurement. The detected value was the tension of the remaining three limbs. After repeating three times to find the average value, the average gripping force of the three limbs was subtracted from the average gripping force of the four limbs to obtain the gripping force value of the right hind limb. When measuring, the groups need to be disrupted for blind testing. The test results are shown in Table 4 and Figure 4 Middle A.

[0094] Table 4. Grasping ability of mice

[0095] Group DMSO group (g) THPD group (g) Muscular atrophy model control group (sham) 54.0 55.3 Muscular atrophy model SWI group (SWI) 37.5 47.7

[0096] According to Table 4 and Figure 4 As can be seen from Figure A, in the SWI group of the muscular atrophy model, the grasping ability of the THPD group mice was improved compared with that of the DMSO-injected group mice.

[0097] B. Mouse running endurance test:

[0098] In the experiment, mice were placed on a small animal treadmill to assess their exercise capacity. 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 they began formal exercise 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 speed and duration of the mice were recorded so that the farthest running distance of the mice could be calculated based on these records. The results are shown in Tables 5 and Figure 4 As shown in B.

[0099] Table 5 Running distance of mice

[0100] Group DMSO group (m) THPD group (m) Muscular atrophy model control group (sham) 595.9 604.0 Muscular atrophy model SWI group (SWI) 409.5 506.6

[0101] According to Table 5 and Figure 4 As can be seen from B, in the SWI group of the muscular atrophy model, the running endurance of the THPD group mice was improved compared with that of the DMSO-injected group mice.

[0102] C. Contraction force test of mouse extensor digitorum longus muscle:

[0103] To measure the tension contraction force of the muscle, the EDL (extensor digitorum longus) muscle was fixed between the force sensor and the adjustable hook by a non-absorbable silk thread. The muscle was incubated in 37°C oxygenated saline for 1 to 2 minutes. The oxygenated saline was Krebs buffer, which consisted of: NaCl 137×10 -3 M, KCl 5×10 -3 M, CaCl22×10 -3 M、MgSO41×10 -3 M, NaH2PO4 1×10 -3 M, NaHCO3 24×10 -3 M, glucose 11×10 -3 M, pH = 7.35.

[0104] Before the experiment, the optimal muscle length was determined and recorded. The dynamic muscle control software (Aurora Scientific, ASI600A) was used to measure and control the tetanic contraction according to the standard experimental protocol. Tetanic contraction induction parameters: initial delay 0.5s; frequency: 120hz; duration 0.3s. The test results are shown in Table 6 and Figure 5 As shown, Figure 5 A is relative contraction force, B is tetanic contraction force, *** indicates that the data are significantly different, p < 0.001.

[0105] Table 6 Relative contraction force of the extensor digitorum longus muscle of mice

[0106] Group DMSO group (mN / mm) THPD group (mN / mm) Muscular atrophy model control group (sham) 24.6 23.7 Muscular atrophy model SWI group (SWI) 13.2 18.3

[0107] According to Table 6 and Figure 5 It can be seen that in the muscular atrophy model SWI group, the contraction force of the extensor digitorum longus muscle of the THPD group mice was improved compared with that of the DMSO-injected group mice.

[0108] D. Gastrocnemius muscle weight test:

[0109] When sampling mice, the gastrocnemius muscles of the mouse hind limbs were completely removed, photographed, and weighed and recorded using an analytical balance. The results are shown in Table 7 and Figure 6 As shown, Figure 6 A is the picture of gastrocnemius muscle, B is the weight picture of gastrocnemius muscle, ** indicates that the data are significantly different, p < 0.01.

[0110] Table 7 Gastrocnemius muscle weight of mice

[0111] Group DMSO group (g) THPD group (g) Muscular atrophy model control group (sham) 0.140 0.154 Muscular atrophy model SWI group (SWI) 0.062 0.090

[0112] According to Table 7 and Figure 6 It can be seen that in the muscular atrophy model SWI group, the gastrocnemius muscle weight of the THPD group mice was improved compared with that of the DMSO-injected group mice.

[0113] E. WGA (wheat germ agglutinin) detection in mouse muscle tissue:

[0114] Take the frozen sections out of the -80℃ refrigerator and place them in a wet box to rewarm at room temperature for 20 minutes; wash them 3 times with 1×PBS, 5 minutes each time, wipe the water drops around the tissue on the sections with a paper towel to avoid wiping off the tissue, and then circle the tissue with an immunohistochemical pen; add 4% PFA to the tissue with a Baxter tube to cover the tissue, fix it at room temperature for 30 minutes, wash it 3 times with 1×PBS, 5 minutes each time; wipe the water drops with a paper towel after washing. After drying, dilute the WGA stain with 1×PBS at a ratio of 1:200, dilute the Hoechst dye at a ratio of 1:2000, mix and dilute the WGA and Hoechst dyes, cover the tissue with the stain with a Baxter tube under light-proof conditions, and co-stain at room temperature for 60 minutes. Finally, wash it 3 times with 1×PBS, 5 minutes each time, wipe it dry with a paper towel, and seal it with 50% glycerol. Cover the slide with a coverslip and store it in an opaque slide box at 4℃. Random photos were taken using a fluorescence microscope, with a 20x objective lens. At least 20 photos were taken for each tissue section, and the muscle fiber cross-sectional area was counted using Image J. At least 500 muscle fibers were counted for each mouse sample. The results are shown in Tables 8 and Figure 7 , where A is the muscle fiber micrograph, B is the gastrocnemius cross-sectional area graph, ** indicates that the data are significantly different, p < 0.01, and C is the distribution frequency graph of different gastrocnemius muscles.

[0115] Table 8 Cross-sectional area of ​​gastrocnemius muscle of mice

[0116] Group DMSO group (μm) THPD group (μm) Muscular atrophy model control group (sham) 3271.837 3234.730 Muscular atrophy model SWI group (SWI) 2057.489 2607.096

[0117] According to Table 8 and Figure 7 It can be seen that in the SWI group of muscular atrophy model, the cross-sectional area of ​​the gastrocnemius muscle fibers in the DMSO-injected mice was significantly smaller, and injection of THPD could significantly improve muscle atrophy.

[0118] F. Real-time fluorescence quantitative PCR reaction detection of common genes:

[0119] (1) Extraction of tissue RNA: ① Clean scissors, tweezers, and steel balls with 75% ethanol; add steel balls and 1 mL Trizol to a 2 mL EP tube; quickly cut rice-sized gastrocnemius tissue and place it in a 2 mL EP tube (pre-cooled on ice); homogenize the gastrocnemius tissue using a tissue homogenizer at 50 Hz for 5 min. After homogenization, remove the small steel balls with a magnet and lyse the sample on ice for 10 min. ② After lysis, add 1 / 5 volume of trioxane to the EP tube, vortex for 10 s, and let stand at room temperature for 5 min; after stratification, use a low-temperature high-speed centrifuge at 4°C, 12,000 x g, and centrifuge for 15 min; transfer the supernatant water phase in the EP tube after centrifugation to a new enzyme-free 1.5 mL EP tube, add an equal volume of isopropanol, mix gently up and down, and let stand at room temperature for 10 min; after standing, centrifuge at 4°C, 12,000 x g, and 20 min. ③ After centrifugation, white RNA is seen at the bottom or side. Carefully use a pipette to slowly suck out the supernatant; add 1 mL of 75% ethanol, turn it upside down to float the precipitate, and wash off the isopropanol; centrifuge at 4°C, 12000xg, and 10 minutes, and use a pipette to suck out the ethanol; blow dry the remaining ethanol in the RNA in the clean bench, add 20-30 μL of DEPC water to dissolve the RNA, and place it at 4°C to dissolve for 30 minutes. Store at -20°C.

[0120] (2) cDNA synthesis: Perform the procedure according to step (4) B in Example 1.

[0121] (3) Common gene real-time fluorescence quantitative PCR reaction: Fluorescence quantitative PCR was performed using a fluorescence quantitative PCR instrument 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 were the same as those in step (4) of Example 1. The detection results are shown in Tables 9 and Figure 8 .

[0122] Table 9 Expression of muscular atrophy-specific ubiquitinase in mice

[0123]

[0124] According to Table 9 and Figure 8 It can be seen that in the SWI-induced muscle atrophy model, the expression of Atrogin-1 and MuRF-1 after THPD treatment was lower than that in the DMSO-treated group, indicating that THPD treatment can improve the occurrence of SWI-induced muscle atrophy.

[0125] In summary, THPD treatment can improve the occurrence of Dex-induced muscle atrophy and can treat muscle atrophy caused by hind limb immobilization in mice. Therefore, the compound THPD can be used to prepare drugs for improving and / or treating muscle atrophy.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a pyrimidinedione compound in the preparation of a drug for improving and / or treating muscle atrophy, characterized in that: The chemical structural formula of the pyrimidinedione compound is shown in Formula I:

2. The use according to claim 1, characterized in that: The muscle atrophy includes disuse muscle atrophy and / or dexamethasone-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 medicine is a unit dose preparation; the unit dose preparation is formulated into a dosage form for administering pyrimidinedione compounds at 10 to 50 mg / kg of mouse body weight.

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

7. The use according to claim 1, characterized in that: The drug also includes a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier includes one or more of a diluent, a buffer, a suspension, an emulsion, a granule, an encapsulation, an excipient, a filler, an adhesive, a spray, a transdermal absorbent, a wetting agent, a disintegrant, an absorption promoter, a surfactant, a colorant, a flavoring agent and an adsorption carrier.

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

9. Use of a pyrimidinedione compound in the preparation of a muscular atrophy gene expression inhibitor, characterized in that: The chemical structural formula of the pyrimidinedione compound is shown in Formula I.

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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