Application of GW8510 in preparation of medicine for treating muscular atrophy

Abstract

The invention provides an application of GW8510 in preparation of a medicine for treating amyotrophy. Compared with the prior art, the invention provides a new application of the GW8510 in treating amyotrophy. Experimental research proves that the GW8510 administration can significantly recover the athletic ability of mice with amyotrophy, improve the cross-sectional area of the muscles of the mice and recover the activity of creatine kinase in serum of the mice, so that the phenotype of the amyotrophy of the mice and related biochemical indexes of the amyotrophy are improved; the GW8510 can be used for remarkably recovering cell amyotrophy related gene and protein expression, and improving amyotrophy phenotype through an MSTNPGC1 alpha NRF2 related pathway; the GW8510 can be used for remarkably recovering cell fibrosis and denervation related gene expression and improving fibrosis and denervation degrees in a muscle atrophy model.

Description

Technical Field

[0001] The present invention belongs to the technical field of new drug applications, and in particular relates to the application of GW8510 in the preparation of drugs for treating muscular atrophy. Background Art

[0002] Muscular atrophy (myatrophy; myophagism) refers to the reduction of muscle volume caused by skeletal muscle nutritional disorders, thinning or even disappearance of muscle fibers, etc. The causes of muscular atrophy are mainly divided into neurogenic muscular atrophy, myogenic muscular atrophy and disuse muscular atrophy.

[0003] The symptoms of muscle atrophy are diverse, mainly including: 1. Reduced muscle volume: Patients can usually observe that the affected muscles are significantly thinner than the normal side, and the limb circumference is reduced. For example, the limbs become thinner, the deltoid muscle atrophy appears square shoulders, the hand muscle atrophy is manifested as thinning and flattening of the palm, and the bone morphology is prominent; 2. Muscle weakness: The muscle strength of patients with muscle atrophy will be significantly reduced, resulting in the inability to complete daily activities or requiring greater effort to complete. For example, calf muscle atrophy is mainly manifested as difficulty in squatting and standing; small muscle atrophy first manifests as fine motor disorders, such as interosseous muscle atrophy in the hands will cause difficulty in writing; head and facial muscle atrophy is mainly manifested as weak chewing, swallowing choking, and weak tongue extension; neck and limb proximal muscle atrophy is manifested as weak head lifting, scapula protruding outward, and difficulty lifting the limbs. 3. Motor dysfunction: Due to muscle atrophy and reduced strength, patients may show symptoms such as abnormal gait, unstable standing, and easy falling. Actions such as running, jumping, and climbing stairs will also become more strenuous, and some people may not even be able to complete these actions. 4. Muscle tremor: Some patients experience involuntary tremors when their muscles contract, which may be caused by muscle fiber lesions or abnormal excitement of the nervous system. 5. Abnormal sensation: Patients with muscular atrophy may experience numbness, tingling or pain, especially in the atrophied muscle area. This abnormal sensation may be caused by nerve damage or muscle lesions.

[0004] At present, the treatment methods for muscular atrophy vary according to the cause and condition, mainly including physical therapy, drug therapy, intramuscular injection therapy, nutritional support and surgical treatment. Among them, there are many therapeutic drugs, but there is still a need for further expansion. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides the use of GW8510 in the preparation of drugs for treating muscular atrophy.

[0006] Technical solution: To achieve the above-mentioned invention object, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides use of GW8510, or a composition comprising GW8510, in the preparation of a drug for treating muscular atrophy.

[0008] In a second aspect, the present invention provides the use of GW8510, or a composition comprising GW8510, in the preparation of a drug for improving creatine kinase activity.

[0009] In a third aspect, the present invention provides the use of GW8510, or a composition comprising GW8510, in the preparation of a drug for inhibiting the expression of genes Fbxo32 and Trim63.

[0010] In a fourth aspect, the present invention provides use of GW8510, or a composition comprising GW8510, in the preparation of a drug for inhibiting the expression of MSTN, PGC1α and NRF2 proteins.

[0011] In a fifth aspect, the present invention provides use of GW8510, or a composition comprising GW8510, in the preparation of a drug for inhibiting the expression of genes Acta2 and Tgfb1.

[0012] In a sixth aspect, the present invention provides use of GW8510, or a composition comprising GW8510, in the preparation of a drug for inhibiting the expression of genes Gadd45a and Ncam1.

[0013] In a seventh aspect, the present invention provides use of GW8510, or a composition comprising GW8510, in the preparation of a medicament for improving the degree of muscle fibrosis and denervation.

[0014] As a specific embodiment, in the composition containing GW8510, GW8510 is the only active ingredient, or GW8510 is one of the active ingredients.

[0015] As a specific embodiment, the composition comprising GW8510 further comprises a pharmaceutically acceptable excipient or carrier.

[0016] Beneficial effects: Compared with the prior art, the present invention provides a new use of GW8510 for the treatment of muscular atrophy. Experimental studies have confirmed that the administration of GW8510 can significantly restore the motor ability of muscular atrophy mice, improve the muscle cross-sectional area of ​​mice, and restore the activity of serum creatine kinase in mice, thereby improving the muscular atrophy phenotype and muscular atrophy-related biochemical indicators of mice; GW8510 can significantly restore the expression of cellular muscular atrophy-related genes and proteins, and improve the muscular atrophy phenotype through the MSTN\PGC1α\NRF2-related pathways; GW8510 can significantly restore the expression of cellular fibrosis and denervation-related genes, and improve the degree of fibrosis and denervation in muscular atrophy models. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 HE staining images of normal mice, glycerol-induced muscular atrophy mice and GW8510-administered mice (A) and statistical quantitative diagram of the cross-sectional area of ​​the tibialis anterior muscle (B).

[0018] Figure 2 This is a statistical quantitative bar graph of serum creatine kinase activity in normal mice, glycerol-induced muscular atrophy mice, and GW8510-administered mice.

[0019] Figure 3 Statistical quantitative bar graphs of grip strength (A) and fatigue tolerance (B) of normal mice, glycerol-induced muscular atrophy mice and GW8510-administered mice.

[0020] Figure 4 Muscle mass (A) and statistical quantitative bar graph (B) of normal mice, denervation-induced muscular atrophy mice and GW8510-administered mice, as well as statistical quantitative bar graph of muscular atrophy-related gene expression (C).

[0021] Figure 5 Statistical quantitative bar graphs of atrophy-related gene expression in gastrocnemius (A) and soleus (B) muscles of normal mice, denervation-induced muscular atrophy mice, and GW8510-administered mice.

[0022] Figure 6 Statistical quantitative bar graphs of muscular atrophy-related gene expression were constructed for normal mouse myoblasts C2C12, dexamethasone-treated cells, and GW8510-treated cells.

[0023] Figure 7 Muscular atrophy-related protein imaging bands (A) and protein expression statistical quantitative bar graphs (B) for normal mouse myoblasts C2C12, dexamethasone-treated muscular atrophy cells, and GW8510-treated cells.

[0024] Figure 8 Statistical quantitative bar graphs of fibrosis-related gene expression were constructed for normal mouse myoblasts C2C12, dexamethasone-treated muscular atrophy cells, and GW8510-treated cells.

[0025] Fig. 9 Statistical quantitative bar graphs of denervation-related gene expression were constructed for normal mouse myoblasts C2C12, dexamethasone-treated muscular atrophy cells, and GW8510-treated cells. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0027] The molecular formula of GW8510 used in the following examples is C 21 H 15 N5O3S2, chemical formula structure:

[0028]

[0029] It is a yellow powder with poor water solubility, which can be dissolved by heating. The average molecular weight is about 449.51 g / mol.

[0030] Example 1

[0031] This experiment revealed that GW8510 can restore the fatigue tolerance of the muscular atrophy mouse model, increase the mouse's grip strength, increase the cross-section of the mouse's muscle fiber tissue and restore the activity of creatine kinase in the mouse's blood.

[0032] Experimental methods:

[0033] 1. Experimental Materials

[0034] 1) Experimental animals: 6-8 week old male ICR mice, weighing about 25 g, were purchased from the Laboratory Animal Department of Peking University Health Science Center. The mice were kept under controlled temperature (21-25°C), humidity (60±5%), and light (12 / 12h light / dark cycle), and had free access to food and water.

[0035] 2) Experimental drug: GW8510, dose 2 mg / kg

[0036] 3) Experimental reagents: Glycerol (Sevier Biotechnology, GC205008)

[0037] 2. Experimental content

[0038] 1) Establishment of glycerol-induced muscle atrophy model: ICR mice were adaptively fed for one week and intraperitoneally injected with GW8510 for 6

[0039] Weeks later, a glycerol-induced muscle atrophy mouse model was established. Glycerol was mixed with sterile saline in a 1:1 ratio, and then 100 μL was injected intramuscularly into the tibialis anterior muscle of mice. Five days later, behavioral indicators of mice and histochemical and other experimental measurements were performed.

[0040] 2) Fatigue tolerance test of glycerol mice: Before the formal experiment, ICR mice were placed in a fatigue rotarod instrument and rotated at a constant speed of 15 rpm for a pre-experiment so that the mice could get familiar with the environment.

[0041] The time it takes to fall while exercising on the fatigue rotarod instrument. Each mouse was tested three times, and the average time to fall was calculated. 3) Mouse grip strength measurement: Before the formal experiment, ICR mice were placed on the grip strength instrument, and the tail of the mouse was grasped and the hand was pulled upward.

[0042] Pull back to allow the mice to get familiar with the experimental environment. During the formal experiment, record the indicators displayed by the mouse gripping force meter, test each mouse 5-7 times, and calculate the average gripping force.

[0043] 4) Histochemical assay: Isolate the mouse tibialis anterior muscle, fix the tissue, embed, slice and perform HE staining. 5) Serum creatine kinase activity assay: Collect mouse blood, place at room temperature for 30 minutes, centrifuge at 3500 rpm,

[0044] After 10 minutes, the upper layer of serum was collected and the serum enzyme activity was determined using a creatine kinase (CK) kit.

[0045] 6) Establishment of denervated muscle atrophy model: The denervated muscle atrophy model was established. The mice were kept for 8 weeks.

[0046] Four weeks after intraperitoneal injection of GW8510, the mice were anesthetized with isoflurane, the sciatic nerve of the right hind limb was transected, the wound was sutured with absorbable sutures, and the temperature was kept at 37 degrees Celsius until the mice woke up. The weight and physiological condition of the mice were recorded after surgery, and their muscles, movements and abilities were evaluated. The mice were euthanized 7 days after surgery. After the death of the model mice, the gastrocnemius, tibialis anterior, soleus, quadriceps femoris and extensor digitorum longus of the hind limbs were dissected to detect the muscle indexes of the mice.

[0047] 7) Tissue RNA extraction and qRT-PCR detection of muscular atrophy gene expression: About 20 mg of gastrocnemius and tibialis anterior muscle tissue was extracted, and the supernatant was transferred for subsequent RNA extraction after homogenization. RNA extraction was performed according to the instructions of the tissue cell RNA microextraction kit (Meiji Bio). 350 μL RTL LysisBuffer, and then an equal amount of μL RNABinding Buffer was added. Transfer the mixture to the purification column and centrifuge at 13000 rpm for 1 min. Add 350 μL Buffer RW1 to the column and centrifuge at 13000 rpm for 1 min.

[0048] Add 500μL Buffer RW2 (diluted with ethanol) to the column and centrifuge at 13000rpm for 1min. Repeat once. Transfer the column to a 1.5ml centrifuge tube. Add 30μL RNase Free Water to the center of the column membrane. Let stand for 1min. Centrifuge at 13000rpm for 1min. The resulting solution is muscle tissue RNA.

[0049] a) cDNA synthesis: Prepare the reverse transcription reaction system according to the following components

[0050]

[0051] The reaction conditions are as follows:

[0052]

[0053] The cDNA obtained from the reaction was used for subsequent qPCR experiments.

[0054] b) qPCR reaction: cDNA was diluted in deionized water at a ratio of 1:5 and mixed by vortex. The qPCR reaction system (20 μL) was prepared according to the following components:

[0055]

[0056] The qPCR reaction program was set up as follows:

[0057]

[0058]

[0059] Gapdh was used as the internal reference gene, and the relative quantification method of gene expression was 2-ΔΔ CT Way.

[0060] 8) Statistical analysis: The muscle cross-sectional area was measured from the HE staining images of muscle tissue using Image J. Statistical analysis was performed using GraphPad Prism 9.0. One-way analysis of variance was used for significance test for results of more than two groups, with P < 0.05 considered statistically significant.

[0061] 3. Experimental results

[0062] Glycerol-induced muscle cross-sectional area in mice decreased significantly, and GW8510 was able to restore the muscle cross-sectional area of ​​mice ( Figure 1 In addition, the statistical quantitative results showed that glycerol induced a decrease in the cross-sectional area of ​​the tibialis anterior muscle in mice in the range of 1000-4000 μm 2 GW8510 can restore the cross-sectional area of ​​tibialis anterior muscle to 1000-4000 μm 2 quantity( Figure 1 In addition, glycerol induction significantly reduced the activity of serum creatine kinase in mice, while GW8510 treatment significantly improved the activity of creatine kinase in mice ( Figure 2 ). The results of behavioral experiments showed that glycerol induced a decrease in the grasping strength and fatigue tolerance of mice, while GW8510 could restore the grasping strength and fatigue tolerance of mice ( Figure 3 The results of the denervation-induced mouse model showed that the gastrocnemius (GC) and soleus (SOL) muscles had a significant increase in mass after administration ( Figure 4 ). In addition, RNA from the two tissues was extracted for muscle atrophy gene detection. The results showed that GW8510 administration significantly inhibited the expression of Fbxo32 and Trim63 genes in the two tissues. Myog is an upstream transcription factor of muscle atrophy genes Fbxo32 and Trim63, and GW8510 treatment can significantly reduce its gene expression ( Figure 5). The above results show that GW8510 administration can significantly restore the motor ability of mice with muscle atrophy, improve the muscle cross-sectional area of ​​mice, and restore the activity of serum creatine kinase in mice, thereby improving the muscle atrophy phenotype and muscle atrophy-related biochemical indicators of mice.

[0063] Example 2

[0064] This experiment showed that GW8510 reduced the expression of muscular atrophy-related molecular markers in mouse myoblasts C2C12 cells.

[0065] Experimental methods

[0066] 1. Experimental Materials

[0067] 1) Experimental cells: mouse myoblasts (Pnosai, CL-0044)

[0068] 2) Experimental reagents: special horse serum (Solebo, S9050), high-glucose DMEM cell culture medium (Punosai), 0.25% trypsin (Zhongke Maichen), dexamethasone (Taoshu Biotechnology, T1076), tissue cell RNA micro-extraction kit (Meiji Biotechnology, R4012), StarScript III one-tube de-genomic reverse transcription premix (Kangrun Biotechnology, A230), Hieff Universal Blue qPCRSYBR Green Master Mix (Yisheng Bio, 11184ES), Myog primary antibody (Santa Cruz, sc-52903), Fbx32 primary antibody (Abcam, ab168372), Trim63 primary antibody (ABclonal, A3101)

[0069] 2. Experimental content

[0070] 1) Cell culture: Mouse myoblast C2C12 cells were cultured in high-glucose DMEM medium (10% FBS + 1% penicillin-streptomycin mixture) and placed at 37°C and 5% carbon dioxide for culture and passage.

[0071] Cells within 10 generations can be used for subsequent experiments

[0072] 2) Cell differentiation: Mouse myoblasts C2C12 were cultured in 6-well plates and then differentiated into myotubes to simulate the in vitro muscle atrophy model. When the cell density reached 100%, myotube differentiation medium (high-glucose DMEM medium containing 2% special horse serum and 1% penicillin-streptomycin mixture) was added, and the medium was changed every 48 hours, and differentiation was induced twice.

[0073] 3) Construction of in vitro muscular atrophy cell model and drug treatment: After the differentiation of mouse myoblasts was completed, muscular atrophy was induced and drug treatment was performed. There were three groups in this experiment: control group (cells were treated with DMSO), model group (cells were treated with 10μM dexamethasone), and drug treatment group (cells were treated with 10μM dexamethasone and 2μM GW8510). Cells were harvested after 24 hours of treatment and subsequent experiments were performed.

[0074] 4) RNA extraction and qRT-PCR detection of muscular atrophy gene expression:

[0075] a) RNA extraction: RNA extraction was performed according to the instructions of the tissue cell RNA micro-extraction kit (Meiji Bio). Wash the 12-well cell culture plate once with PBS, add 100μL RTL Lysis Buffer, and then add 100μL RNA Binding Buffer. Transfer the mixture to the purification column and centrifuge at 13000rpm for 1min. Add 350μL Buffer RW1 to the column and centrifuge at 13000rpm for 1min. Add 500μL Buffer RW2 (diluted with ethanol) to the column and centrifuge at 13000rpm for 1min. Repeat once. Transfer the column to a 1.5ml centrifuge tube. Add 30μL RNase Free Water to the center of the column membrane. Let stand for 1min. Centrifuge at 13000rpm for 1min. The resulting solution is cellular RNA.

[0076] b) cDNA synthesis: Prepare the reverse transcription reaction system according to the following components:

[0077]

[0078]

[0079] The reaction conditions are as follows:

[0080]

[0081] The cDNA obtained from the reaction was used for subsequent qPCR experiments.

[0082] c) qPCR reaction: dilute cDNA in deionized water at a ratio of 1:5 and vortex mix.

[0083] Components for qPCR reaction system (20 μL):

[0084]

[0085] The qPCR reaction program was set up as follows:

[0086]

[0087] Gapdh was used as the internal reference gene, and the relative quantification method of gene expression was 2 -ΔΔCT Way.

[0088] 5) Western blot detection of dystrophin and related pathway protein expression:

[0089] a) Cell lysis: Wash the cells in a 12-well cell culture plate twice with PBS, add 100 μL RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors) to each well, and grind the cells thoroughly on ice to ensure complete cell lysis.

[0090] b) Protein quantification: The protein quantification process was performed according to the instructions for use of the BCA protein concentration assay kit (Biyuntian Biotechnology). Take 25 mg / ml protein standard and dilute it to a final concentration of 2 mg / ml protein standard. At the same time, prepare the BCA working solution at a ratio of 50:1. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of protein standard to the standard wells of the 96-well plate, and add PBS buffer to 20 μL. The protein sample to be tested was diluted 10 times with PBS, and 20 μL was added to each well; 200 μL of BCA working solution was added to all wells, gently aspirated and mixed, and placed in a 37°C incubator for 20 hours. Read the absorbance of each well at a wavelength of 562 nm on the microplate reader, draw a standard curve, and calculate the protein concentration of the sample to be tested. Subsequently, 25 μL of 5× Loading Buffer was added to the protein lysate, the metal bath was 100°C, boiled for 10 minutes, and stored in a -20°C refrigerator.

[0091] c) Protein electrophoresis and membrane transfer: Prepare 10% separation gel and 5% concentration gel, load 10 μg per well, and keep constant voltage

[0092] Electrophoresis was performed at 80V for about 2 hours. Then the membrane was transferred to NC membrane at a constant current of 200mA for 80 minutes. d) Antibody incubation: The target protein antibody was diluted 1:1000, and the internal reference protein antibody was diluted 1:5000 and incubated at 4°C.

[0093] Overnight. Then wash the membrane 6 times with TBST, 5 min each time. Dilute the corresponding species HRP-labeled secondary antibody 1:5000 and incubate at room temperature for 1 hour. Wash the membrane with TBST and then develop.

[0094] e) Data analysis: The grayscale values ​​of Western Blot results were counted using Image J software.

[0095] 3. Experimental results

[0096] To construct a muscle atrophy cell model, mouse myoblasts C2C12 were differentiated into myotubes by horse serum and then treated with 10 μM dexamethasone. The experimental results showed that dexamethasone treatment significantly upregulated the expression of muscle atrophy genes Fbxo32 and Trim63, while GW8510 administration significantly downregulated the expression of the two genes ( Figure 6 Myog is an upstream transcription factor of the muscular atrophy genes Fbxo32 and Trim63. GW8510 treatment can significantly reduce its gene expression ( Figure 6 ), while dexamethasone treatment had no effect on Myog gene expression. In addition, protein results showed that Myog, Fbxo32 and Trim63 protein expressions were significantly increased under dexamethasone treatment, while GW8510 treatment could significantly restore the expression of the three proteins ( Figure 7 In addition, GW8510 significantly improved the expression of MSTN, PGC1α, and NRF2 proteins, all of which are known pathways related to the muscular atrophy phenotype ( Figure 7 ). The above results show that GW8510 can significantly restore the expression of muscular atrophy-related genes and proteins in cells, and improve the muscular atrophy phenotype through the MSTN\PGC1α\NRF2-related pathway.

[0097] Experimental Example 3

[0098] This experiment showed that GW8510 significantly reduced the degree of fibrosis and denervation in the muscle atrophy model.

[0099] Experimental methods:

[0100] 1. Experimental Materials

[0101] 1) Experimental cells: mouse myoblasts (Pnosai, CL-0044)

[0102] 2) Experimental reagents: special horse serum (Solebo, S9050), high glucose DMEM cell culture medium (Punosai), 0.25% trypsin (Zhongke Maichen), dexamethasone (Taoshu Biotechnology, T1076), tissue cell RNA micro-extraction kit (Meiji Biotechnology, R4012), StarScript III one-tube de-genomic reverse transcription premix (Kangrun Biotechnology, A230), Universal Blue qPCRSYBR Green Master Mix (Yisheng Biotechnology, 11184ES)

[0103] 2. Experimental content

[0104] 1) Cell culture, differentiation, construction of in vitro muscle atrophy model and drug administration were the same as those in Experimental Example 2 (1)-(3)

[0105] content

[0106] 2) RNA extraction and qRT-PCR detection of muscular atrophy gene expression were the same as in Experimental Example 2 (4)

[0107] 3. Experimental results

[0108] The experimental results in this section are used to demonstrate that GW8510 can improve the degree of fibrosis and denervation in muscle atrophy cell models. Dexamethasone treatment upregulated the expression of muscle fibrosis genes Acta2 and Tgfb1, although there was no significant difference, but GW8510 administration significantly downregulated the expression of the two genes ( Figure 8 In addition, dexamethasone treatment significantly upregulated the expression of muscle denervation genes Gadd45a and Ncam1, while GW8510 administration significantly downregulated the expression of the two genes ( Fig. 9 ). The above results show that GW8510 can significantly restore the expression of cell fibrosis and denervation-related genes, proving that GW8510 improves the degree of fibrosis and denervation in the muscle atrophy model.

[0109] The implementation modes of the present invention have been described in detail above in conjunction with specific embodiments, but the present invention is not limited to the above-mentioned implementation modes, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. Use of GW8510 or a composition comprising GW8510 in the preparation of a drug for treating muscular atrophy.

2. Use of GW8510 or a composition comprising GW8510 in the preparation of a drug for improving creatine kinase activity.

3. Use of GW8510 or a composition comprising GW8510 in the preparation of a drug for inhibiting the expression of genes Fbxo32 and Trim63.

4. Use of GW8510 or a composition comprising GW8510 in the preparation of a drug for inhibiting the expression of MSTN, PGC1α and NRF2 proteins.

5. Use of GW8510 or a composition comprising GW8510 in the preparation of a drug for inhibiting the expression of genes Acta2 and Tgfb1.

6. Use of GW8510 or a composition comprising GW8510 in the preparation of a drug for inhibiting the expression of genes Gadd45a and Ncam1.

7. Use of GW8510 or a composition comprising GW8510 in the preparation of a drug for improving muscle fibrosis and denervation.

8. The use according to any one of claims 1 to 7, characterized in that: In the composition containing GW8510, GW8510 is used as the only active ingredient, or GW8510 is used as one of the active ingredients.

9. The use according to any one of claims 1 to 7, characterized in that: The composition comprising GW8510 also includes a pharmaceutically acceptable excipient or carrier.

Images