Application of dimethyl fumarate in preparation of medicine for preventing and / or treating skeletal muscle atrophy

Abstract

The invention discloses application of dimethyl fumarate in preparation of a medicine for preventing and / or treating skeletal muscle atrophy, and belongs to the field of medicine. It is proved for the first time that DMF can effectively relieve related symptoms of skeletal muscle atrophy caused by cancer cachexia, and a new thought is provided for treatment of skeletal muscle atrophy. The DMF can relieve skeletal muscle atrophy by reducing mitochondrial damage and improving mitochondrial homeostasis imbalance, and the potential mechanism of the DMF is possibly related to the regulation of HIF1alpha, mTOR and other pathways. In addition, DMF can improve cancer cachexia skeletal muscle atrophy and does not inhibit tumor growth, which indicates that DMF directly acts on skeletal muscle instead of tumor. Due to the common end characteristic of mitochondrial homeostasis imbalance and the like in amyotrophy caused by different causes (such as aging and drug toxicity), the adjusting effect of DMF on homeostasis in skeletal muscle can be expanded to be used for preventing and treating other types of amyotrophy, including amyotrophy caused by aging, glucocorticoid, chemotherapy drugs and the like. The invention provides an application basis for preventing and treating skeletal muscle atrophy by DMF.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to the use of dimethyl fumarate in the preparation of medicaments for the prevention and / or treatment of skeletal muscle atrophy. Background Technology

[0002] Skeletal muscular atrophy (SMA) is a common pathological manifestation of many serious diseases, particularly prominent and typical in cancer cachexia. It affects up to 80% of patients with advanced cancer, directly leading to decreased quality of life, reduced treatment tolerance, and increased mortality. However, due to its complex pathogenesis, there are currently no effective treatments, resulting in significant unmet clinical needs. Skeletal muscular atrophy is not unique to cachexia. In aging populations, age-related sarcopenia (ALA) is a major cause of frailty, falls, and disability, severely impacting the health of the elderly. Furthermore, certain commonly used clinical drugs (such as glucocorticoids and chemotherapy drugs) can also induce drug-induced muscular atrophy, a serious side effect that limits their clinical application. Therefore, developing a universal therapy to prevent and treat muscular atrophy has broad market prospects and clinical value.

[0003] Despite different triggers (such as tumor inflammation, aging, or drug toxicity), the various types of skeletal muscle atrophy share a core commonality in their molecular pathways: an imbalance in the homeostasis of skeletal muscle protein synthesis and degradation, particularly the overactivation of the ubiquitin-proteasome and autophagy-lysosome systems, inflammation, and mitochondrial homeostasis imbalance, all of which lead to muscle fiber degradation. However, most existing studies target single triggers and lack universally applicable strategies to intervene in skeletal muscle atrophy by addressing this common pathway. This invention is based on an understanding of this common pathological mechanism.

[0004] Dimethyl fumarate (DMF) is a mature drug approved by the FDA, initially used in clinical trials for multiple sclerosis (MS). Its core mechanism of action in treating MS focuses on immune system regulation. MS is an autoimmune disease of the central nervous system, its pathogenesis stemming from an imbalance in the body's immune tolerance. This imbalance leads to abnormal activation of immune cells such as T cells and B cells, which infiltrate the central nervous system and attack the myelin sheath, triggering inflammatory responses, demyelination, and axonal damage, ultimately resulting in neurological dysfunction. DMF activates the nuclear factor E2-related factor 2 (Nrf2) signaling pathway, upregulates the expression of genes related to antioxidant stress, reduces immune cell-mediated inflammatory damage, inhibits the release of pro-inflammatory cytokines (such as TNF-α and IL-1β), and regulates the balance of immune cell subsets, thereby blocking the destruction of nerve tissue by the immune system and slowing disease progression.

[0005] The pathogenesis of muscle atrophy differs fundamentally from that of MS: muscle atrophy originates from the interaction between tumor / senescent cells and the host microenvironment. Tumor / senescent cells release large amounts of muscle atrophy-related factors, simultaneously triggering systemic metabolic reprogramming in the host. This leads to skeletal muscle mitochondrial dysfunction (such as decreased mitochondrial membrane potential, increased oxidative stress, and disordered energy metabolism), overactivation of the autophagy pathway (abnormal degradation of intracellular functional proteins and organelles), and hyperactivity of the ubiquitin-proteasome pathway (accelerating myofibril protein degradation). Ultimately, this disrupts the dynamic balance between myofibril protein synthesis and degradation, resulting in muscle atrophy. The core driving factors, key pathological processes, and affected tissue systems are completely different between the two. Currently, there is an urgent need for a drug or method to treat muscle atrophy. Summary of the Invention

[0006] The purpose of this invention is to provide the application of dimethyl fumarate (DMF) in the preparation of drugs for the prevention and / or treatment of skeletal muscle atrophy, in order to solve the problems existing in the prior art. This invention demonstrates for the first time that DMF can effectively alleviate symptoms related to skeletal muscle atrophy caused by cachexia, providing a new approach to the treatment of skeletal muscle atrophy.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides the use of dimethyl fumarate in the preparation of medicaments for the prevention and / or treatment of skeletal muscle atrophy.

[0009] Optionally, the skeletal muscle atrophy includes tumor cachexia-related skeletal muscle atrophy, age-related skeletal muscle atrophy, or drug-induced skeletal muscle atrophy.

[0010] Optionally, the dimethyl fumarate can improve muscle strength.

[0011] Optionally, the dimethyl fumarate can increase muscle mass and muscle mass index.

[0012] Optionally, the muscle index includes the gastrocnemius index, tibialis anterior index, quadriceps index, soleus index, total muscle index, and epididymal fat index.

[0013] Optionally, the dimethyl fumarate can improve the reduction in muscle fiber area.

[0014] Optionally, the dimethyl fumarate can reduce adipose tissue loss.

[0015] Optionally, the dimethyl fumarate can improve mitochondrial homeostasis imbalance.

[0016] Optionally, the dimethyl maleate can regulate the HIF1α and mTOR pathways.

[0017] The present invention also provides a medicament for improving skeletal muscle atrophy, the medicament comprising dimethyl fumarate and pharmaceutically acceptable excipients;

[0018] The excipients include at least one of the following: diluent, filler, excipient, binder, wetting agent, disintegrant, absorption promoter, surfactant, adsorbent carrier, lubricant, and fragrance.

[0019] The present invention discloses the following technical effects:

[0020] This invention demonstrates for the first time that DMF can effectively alleviate symptoms associated with skeletal muscle atrophy caused by cancer cachexia, providing a new approach to the treatment of skeletal muscle atrophy.

[0021] Dimethyl fumarate (DMF) may alleviate skeletal muscle atrophy by reducing mitochondrial damage and improving mitochondrial homeostasis imbalance. Its potential mechanism may be related to DMF regulation of pathways such as HIF1α and mTOR. The results obtained in this invention will provide valuable research clues and theoretical basis for the clinical application of dimethyl fumarate (DMF) in inhibiting skeletal muscle atrophy. As an FDA-approved drug, DMF, through drug repurposing, could become a promising candidate for the treatment of skeletal muscle atrophy. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 To improve muscle function impairment in LLC tumor-bearing mice by administering DMF; A: Animal experimental protocol; B: Comparison of grip strength changes in mice of different groups on day 0; C: Comparison of grip strength changes in mice of different groups on day 7; D: Comparison of grip strength changes in mice of different groups on day 14; E: Scoring criteria for mouse suspension test; 0-5 represents scores from 0 to 5; F: Forelimb suspension test scores in mice of different groups; G: Forelimb suspension time in mice of different groups; H: Limb suspension time in mice of different groups.

[0024] Figure 2 DMF administration improves muscle atrophy in LLC tumor-bearing mice; A: Changes in body weight of mice in each group; B: Cumulative food intake of mice in each group; C: Schematic diagram of tumor size of mice in each group; D: Changes in tumor volume of mice in each group; E: Body weight of mice after tumor removal in each group; F: Comparison of gastrocnemius muscle index of mice in each group; G: Comparison of quadriceps muscle index of mice in each group; H: Comparison of tibialis anterior muscle index of mice in each group; I: Comparison of soleus muscle index of mice in each group; J: Comparison of total muscle index of mice in each group; K: Comparison of epididymal fat of mice in each group.

[0025] Figure 3 DMF administration improves muscle atrophy in LLC tumor-bearing mice; A: Tibialis anterior muscle fiber area and area distribution in each group of mice; B: MYOG mRNA expression level; C: MURF1 mRNA expression level; D: Atrogin1 mRNA expression level;

[0026] Figure 4 DMF administration improves muscle atrophy in LLC tumor-bearing mice: A: Representative WB bands representing the expression levels of proteins related to muscle synthesis (MYHC) and muscle atrophy (Atrogin1 and MURF1); B: Statistical plot corresponding to A;

[0027] Figure 5 DMF administration improves mitochondrial damage in the muscles of LLC tumor-bearing mice; A: Transmission electron microscopy images of mice in each group; green arrows indicate extensive mild expansion of the sarcoplasmic reticulum (SPR), red arrows indicate numerous autophagosomes containing incompletely degraded organelles, blue arrows indicate decreased matrix electron density of mitochondria (M) compared to the WT group, and yellow arrows indicate uneven I-band Z-line and A-band M-line; B: Expression bands of autophagy-related proteins (mTOR, P62, LC3) and their corresponding statistical graphs; C: Expression bands of proteins related to mitochondrial division (Drp1 and FIS1) and synthesis (MFN1 and OPA1) and their corresponding statistical graphs; D: Expression bands of proteins related to mitochondrial biosynthesis (PGC1α and Nrf1) and their corresponding statistical graphs.

[0028] Figure 6 For proteomics prediction of potential drug targets for DMF; A: Venny plot of differentially expressed proteins among normal group, model group and drug-treated group; B: Volcano plot of differentially expressed proteins between drug-treated group and model group; C: Cluster plot of differentially expressed proteins between drug-treated group and model group;

[0029] Figure 7 KEGG enrichment analysis of differentially expressed proteins between the drug administration group and the model group;

[0030] Figure 8 GO enrichment analysis for differentially expressed proteins between the drug-treated group and the model group;

[0031] Figure 9 The image shows the expression levels of proteins (HIF1α, HK2, PDK1, Glut1, LDHA) related to the core proteomics pathway HIF1α signaling pathway (A) and their corresponding statistical graphs (B). Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Example 1

[0038] The C57BL / 6J wild-type mice used in this invention were purchased from Sigma-Aldrich (Beijing) Biotechnology Co., Ltd., and all mice were raised in the same environment; DMF was provided by Sigma-Aldrich, catalog number 242926-25G.

[0039] In this embodiment, all animal studies were conducted under the guidance of the Experimental Animal Center of the Ethics Review Committee of Shihezi University.

[0040] 1. Model construction for muscle atrophy caused by cancer cachexia

[0041] After stable passage of mouse LLC Lewis lung cancer cells for 3 generations, the cells were digested with an appropriate amount of trypsin and collected. After centrifugation at 5000 rpm for 5 min, the cells were washed again with DMEM medium and centrifuged 2-3 times. DMEM was then prepared to contain 1.25 × 10⁻⁶ cells / mL. 7 Cell suspension at 100 cells / mL. 0.2 mL of LLC cell suspension was subcutaneously injected into the right forelimb axilla of mice (i.e., 2.5 × 10⁶ cells per mouse). 6 The control group received 200 μL of physiological saline at the same location (cells). Experimental groups were formed 7 days after inoculation, when a tumor the size of a grain of rice could be palpated at the inoculation site. Food intake and body weight were monitored every two days, tumor size was measured every three days, and muscle grip strength, limb grip strength, and forelimb grip strength were measured to assess the effect of DMF administration on cancer cachexia symptoms. The experimental procedure is as follows: Figure 1 A.

[0042] 2. Experimental Grouping

[0043] Normal control group (CON): 10 mice were injected subcutaneously with physiological saline (200 μL) under the right forelimb axilla as a control. After 7 days, they were administered physiological saline of 1% DMSO by gavage for 13 days, once a day.

[0044] Normal drug control group (CON+H-DMF): A total of 10 mice were injected subcutaneously with physiological saline (200 μL) in the axilla of the right forelimb as a control. After 7 days, they were administered 0.1 mg / 10g of DMF at a concentration of 1 mg / mL by gavage for 13 days, once a day.

[0045] LLC cachexia group (LLC): 10 mice were injected subcutaneously into the right forelimb axilla with 2.5 × 10⁻⁶ mmol / L. 6 200 μL of LLC cells were inoculated into the tumor. Tumor length and width were measured every 3 days after inoculation, and tumor volume was calculated. After 7 days of tumor inoculation, the tumor cells were administered a 1% DMSO saline solution by gavage once daily for 13 days.

[0046] LLC cachexia DMF low-dose group (2.5 mg / kg, LLC+L-DMF): 10 mice were injected subcutaneously into the right forelimb axilla with 2.5 × 10 mg / kg DMF. 6 200 μL of LLC cells were inoculated into the tumor. The length and width of the tumor were measured every 3 days after tumor inoculation, and the tumor volume was calculated. After 7 days of tumor inoculation, DMF at a concentration of 0.25 mg / mL (0.1 mL / 10 g) was administered by gavage once daily for 13 days.

[0047] LLC cachexia DMF medium-dose group (5 mg / kg, LLC+M-DMF): 10 mice were injected subcutaneously into the right forelimb axilla with 2.5 × 10 mg / kg DMF. 6200 μL of LLC cells were inoculated into the tumor. The length and width of the tumor were measured every 3 days after tumor inoculation, and the tumor volume was calculated. After 7 days of tumor inoculation, DMF at a concentration of 0.5 mg / mL (0.1 mL / 10 g) was administered by gavage once daily for 13 days.

[0048] High-dose LLC cachexia DMF (10 mg / kg, LLC+H-DMF): 10 mice were injected subcutaneously into the right forelimb axilla with 2.5 × 10 mg / kg DMF. 6 200 μL of LLC cells were inoculated into the tumor. The length and width of the tumor were measured every 3 days after tumor inoculation, and the tumor volume was calculated. After 7 days of tumor inoculation, DMF at a concentration of 1 mg / mL (0.1 mL / 10 g) was administered by gavage once daily for 13 days.

[0049] 3. Behavioral testing of mouse muscle grip strength

[0050] 3.1 Suspension Experiment

[0051] A length of wire was stretched and fixed approximately 60 cm off the ground. A mouse was suspended from the wire, its two front paws gripping it. The mouse was observed to see if it had sufficient strength to grip the wire with its hind paws. A score of 0 was given if the mouse immediately fell; 1 point was given if the mouse merely hung on the wire without any attempt to climb; 2 points were given if the mouse hung on the wire and showed an attempt to climb, but its hind paws did not grip the wire; 3 points were given if one hind paw gripped the wire; 4 points were given if both hind paws gripped the wire; and 5 points were given if both hind paws gripped the wire and the mouse crawled along it. The test was repeated three times, and the average value was recorded. The time each mouse held the wire was recorded; any time exceeding 5 minutes was recorded as 300 seconds.

[0052] 3.2 Mouse grip strength test

[0053] After weighing and recording the mice's weight, a gripping force test was performed on each mouse: After calibrating the gripping force tester, the measurement button was pressed. The mouse was placed on the gripping board, and its tail was grasped and gently pulled back. Once the mouse had a firm grip, a uniform force was applied and pulled back. The instrument automatically recorded the mouse's gripping force value. The average of three measurements was taken as the mouse's gripping force. For standardized comparison, the gripping force test results were evaluated using gripping force / body weight as the assessment standard. Gripping forces were measured on days 0, 7, and 14.

[0054] 3.3 Mouse limb gripping strength test

[0055] Grasp the mouse cage cover with all four limbs, rotate the cage cover 180°, and record the time it takes for all four limbs to grip the cage cover. If it takes more than 5 minutes, record it as 300 seconds.

[0056] 4. Western blot (WB) experiment

[0057] This will be conducted after the experiment is completed.

[0058] 4.1 Sample Preparation

[0059] Approximately 50 mg of mouse gastrocnemius muscle tissue was weighed into a new EP tube pre-placed on ice. A clean steel ball was added, followed by 500 μL of protein lysis buffer (high-potency RIPA lysis buffer: protease inhibitor: phosphatase inhibitor volume ratio = 100:2:1). The tube was then placed in a tissue homogenizer and homogenized for 1 min (3 times) at 4°C. After standing at 4°C for 30 min, the homogenate was centrifuged at 12000 g for 15 min at 4°C. The supernatant was used to determine the protein concentration using the BCA method. The gastrocnemius muscle tissue sample was diluted 40-fold, and the cell sample was diluted 20-fold for concentration determination. 4× loading buffer and RIPA lysis buffer were added to adjust the loading concentration to 2 ng / μL. The tube was sealed with sealing film, heated in boiling water for 10 min, and then aliquoted. A portion of the samples were analyzed, and the remaining samples were stored at -80°C to avoid repeated freeze-thaw cycles.

[0060] 4.2 Electrophoretic separation

[0061] Gel Preparation: Prepare a separating gel of appropriate concentration based on the molecular weight of the target protein. Generally, the larger the molecular weight of the target protein, the lower the concentration of the separating gel used, and vice versa. After adding the separating gel to the electrophoresis rack, slowly pour purified water along the glass plate to seal the gel. Once the separating gel has solidified, discard the purified water, add an appropriate amount of stacking gel, insert the corresponding comb, and after the upper layer of gel has solidified, pour in 1× electrophoresis buffer for later use.

[0062] Slowly pull the comb vertically upwards and add samples from left to right according to the group. The protein content of each sample added is 20-40 μg. Add different volumes of protein markers to the wells next to the first and last sample wells for labeling. Do not use markers in the first and last wells.

[0063] Electrophoresis: Add an appropriate amount of 1× electrophoresis buffer, assemble the electrophoresis apparatus, set the voltage to 65 V, and after about 30 minutes of electrophoresis, when the sample runs out of the stacking gel and is flattened into a straight line, adjust the voltage to 120 V and electrophore until the bromophenol blue reaches the bottom edge of the glass plate. Stop electrophoresis, turn off the power, and perform membrane transfer.

[0064] 4.3 Immunoblotting

[0065] Transfer: After electrophoresis, cut a PVDF membrane to the appropriate size and soak it in methanol for 1 min to activate it. Assemble the transfer apparatus in the following order: black gel, red membrane, red clip, sponge pad, transfer filter paper, PVDF membrane, gel, filter paper, sponge pad, black clip. Use a roller to remove excess air bubbles, ensuring the transfer assembly is airtight. Place the transfer apparatus in the electrotransfer tank, add 1× electrotransfer buffer, set the current to 300 mA, and place the electrotransfer tank in ice water for transfer. The transfer time is determined based on the molecular weight of the target protein.

[0066] Blocking: After the transfer is complete, remove the PVDF membrane and place it in a pre-prepared blocking solution containing 5% skim milk powder (1×TBST). Shake gently on a shaker at room temperature for 2 hours.

[0067] Primary antibody incubation: After blocking, place the PVDF membrane in 1×TBST and gently shake and rinse 4 times on a shaker for 5 minutes each time. After washing, add the corresponding primary antibody and incubate overnight at 4°C.

[0068] Secondary antibody incubation: After primary antibody incubation, remove the PVDF membrane and wash it four times in 1×TBST solution, 5 min each time. After washing, according to the species of the primary antibody, place the PVDF membrane into the pre-prepared secondary antibody of the corresponding species and incubate gently on a shaker at room temperature for 45 min-60 min.

[0069] 4.4 Development and Exposure

[0070] After the secondary antibody incubation, the PVDF membrane was washed 5 times with 1×TBST, 5 min each time. According to the ECL luminescence solution instructions, solution A and solution B were mixed at a 1:1 volume ratio, and an appropriate amount of the mixed luminescence solution was added to the PVDF membrane. Automatic exposure was performed using a fully automated chemiluminescence imaging system.

[0071] 4.5 Data Analysis

[0072] Image J software was used to quantify the protein expression of the bands.

[0073] 5. H&E measurement of the surface area of ​​the tibialis anterior muscle fibers in mice.

[0074] 1) Tissue sections: The fixed mouse skeletal muscle was embedded in paraffin to make paraffin sections, and baked at 60℃ for 30 min.

[0075] 2) Dewaxing and dehydration: The tissue was dewaxed three times with xylene for 15 minutes each time; the sections were then treated with 100% ethanol, 95% ethanol, 85% ethanol and 75% ethanol for 5 minutes each to dehydrate the tissue sections.

[0076] 3) Rinse the sections repeatedly with tap water, then rinse twice with deionized water; stain the tissue with hematoxylin-eosin (HE);

[0077] 4) Staining the nucleus: Use hematoxylin to stain the nucleus of the tissue sections for 2 minutes, then rinse with tap water;

[0078] 5) Differentiation: Differentiate in hydrochloric acid-ethanol solution for a few seconds, then rinse with warm water to turn blue again for a few seconds;

[0079] 6) Rinse the slices with tap water, then rinse twice with deionized water;

[0080] 7) Eosin staining: Immerse the tissue section in eosin staining solution for 20-30 seconds to stain the cytoplasm;

[0081] 8) Rinse the slices with tap water, then rinse twice with deionized water;

[0082] 9) Dehydration: Dehydration using a gradient of 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol;

[0083] 10) Transparent: Clear the solution three times with xylene;

[0084] 11) Mounting: Mount with neutral resin, taking care to avoid air bubbles;

[0085] 12) Image acquisition: After drying, observe and photograph under a microscope. The cytoplasm appears red and the nucleus appears blue.

[0086] 6. Observation of mitochondrial damage in the tibialis anterior muscle of mice using transmission electron microscopy.

[0087] 1) Tissue Sampling and Fixation: Determine the sampling site for fresh tissue, minimizing mechanical damage such as traction, contusion, and compression. The tissue volume should generally not exceed 1mm × 1mm × 1mm. Quickly immerse the tissue in electron microscopy fixation solution at 4℃ for 2-4 hours. After centrifuging the cells to the bottom of the tube, cell clumps the size of mung beans should be visible. Discard the culture medium and add electron microscopy fixation solution, fixing at 4℃ for 2-4 hours. Rinse three times with 0.1 M phosphate-buffered saline (PBS, pH 7.4), 15 min each time.

[0088] 2) Post-fixation: Fix with 1% osmium tetroxide in 0.1M phosphate buffered PBS (pH 7.4) at room temperature (20℃) for 2 h. Wash three times with 0.1M phosphate buffered PBS (pH 7.4), 15 min each time.

[0089] 3) Dehydration: The tissues were sequentially immersed in 50%-70%-80%-90%-95%-100%-100% alcohol for dehydration, each time for 15 minutes.

[0090] 4) Infiltration: A 1:1 mixture of acetone and 812 embedding agent was infiltrated overnight, and pure 812 embedding agent was infiltrated overnight.

[0091] 5) Encapsulation: Polymerize at 60℃ for 48 h.

[0092] 6) Slicing: Use a slicer to slice 60-80 nm ultrathin sections.

[0093] 7) Staining: Uranium-lead double staining (2% uranium acetate saturated aqueous solution, lead citrate, each staining for 15 min), and the sections are dried at room temperature overnight.

[0094] 8) Observe under a transmission electron microscope and collect and analyze images.

[0095] 7. qRT-PCR detection of mRNA expression levels

[0096] Total RNA was extracted from mouse gastrocnemius muscle tissue using the Trizol method. The RNA was then reverse transcribed into cDNA using a reverse transcription kit and stored at -80°C for later use. Amplification was performed according to the kit instructions (PerfectStart). ® The reaction mixture (20 μL) was prepared using UniversalGreen qPCR SuperMix-AQ631-01 (from TransGen Biotech): 10.0 μL TBGreen Premix Ex Taq II, 0.8 μL each of forward and reverse primers (nucleotide sequences shown in Table 1), 2 μL cDNA, and 7.2 μL ddH2O. A two-step PCR reaction was performed under the following conditions: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, for a total of 40 cycles. GAPDH was used as the internal reference gene. -ΔΔCt The method calculates the relative expression level of the target gene.

[0097] Table 1 Primer Sequences

[0098]

[0099] 8. Results

[0100] 8.1 DMF administration improves muscle damage in LLC tumor-bearing mice

[0101] The muscle grip strength of mice in each group was tested on days 0, 7, and 14 after tumor growth. The results showed that the model group had a significant decrease in muscle grip strength on days 7 and 14 after tumor growth (P<0.5), and the drug improved this phenomenon. Figure 1 China B- Figure 1On day 0, there were no significant differences in muscle grip strength among the groups (P>0.5). Specific numerical changes are shown in Table 2. Continuous administration revealed that the drug significantly (P<0.5) improved the reduction in grip strength in the forelimbs and quadriplegs of tumor-bearing mice. Figure 1 China E- Figure 1 The specific numerical changes (H) are shown in Table 3. All the above results demonstrate that DMF improves muscle function impairment in LLC tumor-bearing mice in a dose-dependent manner.

[0102] Table 2. Changes in grip strength (N / g) of mice in each group on days 0, 7, and 14.

[0103]

[0104] Note: Compared with the LLC group, P<0.05 is marked with *, P<0.01 with **, and P<0.001 with ***. The same applies to the following table.

[0105] Table 3. Changes in grip strength of forelimbs and limbs in mice of each group

[0106]

[0107] 8.2 DMF administration improves muscle atrophy in LLC tumor-bearing mice

[0108] The results showed that after 13 days of continuous administration, DMF had no effect on mouse body weight or cumulative food intake. Figure 2 China A- Figure 2 (B), and also had no significant effect on tumor growth changes ( Figure 2 C- Figure 2 (D), but the decrease in tumor-bearing mice after tumor removal was significantly improved, and this was dose-dependent ( Figure 2 (E). Continuous administration to mice for 13 days also improved, in a dose-dependent manner, the reduction in muscle mass and loss of adipose tissue in tumor-bearing mice. Figure 2 China F- Figure 2 (K). This improvement is also reflected in the change in body weight after tumor removal, indicating that its weight-protective effect is mainly contributed by the maintenance of muscle and adipose tissue (specific numerical changes are shown in Table 4). Meanwhile, H&E staining results showed that the drug improved the reduction in muscle fiber area in tumor-bearing mice (K). Figure 3 Similarly, corresponding results were obtained at the mRNA and protein levels of genes related to muscle atrophy and muscle synthesis. Figure 3 China B- Figure 3 China D and Figure 4The results were evident: DMF significantly improved muscle atrophy in tumor-bearing mice without affecting tumor size, indicating that its action targets skeletal muscle directly rather than the tumor, and can specifically regulate skeletal muscle homeostasis. Given the common characteristics of skeletal muscle atrophy caused by different etiologies (such as cancer cachexia, aging, and drug toxicity) in the end-stage pathology, the regulatory effect of this compound on skeletal muscle homeostasis can be reasonably extended to the prevention or treatment of other types of skeletal muscle atrophy, including age-related skeletal muscle atrophy and skeletal muscle atrophy caused by glucocorticoids, chemotherapy drugs, etc.

[0109] Table 4. Changes in muscle index and epididymal fat index in tumor-bearing mice.

[0110]

[0111] 8.3 DMF improves mitochondrial damage and maintains mitochondrial homeostasis

[0112] Electron microscopy results showed that, under transmission electron microscopy, in the normal group: local myofibril bundles were broken, as indicated by the black arrows; the mitochondrial (M) double membrane structure was clear, with lamellar cristae visible, and the cristae were arranged in roughly parallel lamellar patterns; the matrix electron density was high; and the sarcoplasmic reticulum (SPR) showed no significant expansion. In the model group: the I-band Z-line and A-band M-line were irregular; the mitochondrial (M) matrix electron density was lower than in the normal group; a large number of autophagosomes containing incompletely degraded organelles were also visible; and the sarcoplasmic reticulum (SPR) showed slight expansion. In the low-dose group: local myofibril bundles were broken; the I-band Z-line and A-band M-line were irregular; the mitochondrial (M) matrix electron density was lower; a small number of autophagosomes containing incompletely degraded organelles were also visible; and a small amount of sarcoplasmic reticulum (SPR) showed slight expansion. In the medium-dose group: muscle fibers were neatly and tightly arranged, and myofilaments were neatly and tightly arranged to form sarcomeres, with no breaks observed; the mitochondrial (M) matrix electron density was lower; and the sarcoplasmic reticulum (SPR) showed no significant expansion. High dose: Local myofibril bundles are broken, the mitochondrial (M) double membrane structure is clear, lamellar cristae are visible, and the cristae are arranged in roughly parallel lamellar patterns; the matrix electron density is high; the sarcoplasmic reticulum (SPR) shows no significant expansion. In summary, LLC leads to severe damage to the ultrastructure of skeletal muscle (myofibrils, Z-lines, H-bands, etc.); DMF intervention can improve this damage in a dose-dependent manner. Medium and high doses of DMF can significantly restore the order and integrity of the skeletal muscle ultrastructure, and high-dose DMF has no significant toxicity to normal mouse skeletal muscle. Figure 5 (A) Localized autophagy was observed in the skeletal muscle of mice in the LLC group, and the autophagy was improved after treatment with DMF.

[0113] Based on the above results, this embodiment further examined the effect of DMF on the expression of autophagy-related proteins in LLC tumor-bearing mice. The results are as follows: Figure 5As shown in Figure B, compared with the normal group, the p-mTOR / mTOR level in the gastrocnemius muscle of LLC mice was significantly decreased, while the LC3-II / LC3-I ratio and P62 protein level were significantly increased, and these changes were statistically significant. DMF treatment significantly upregulated the p-mTOR / mTOR level in the gastrocnemius muscle of LLC tumor-bearing mice and inhibited the upregulation of the LC3II / LC3I ratio and P62 protein level.

[0114] Studies have shown that mitochondrial homeostasis plays a crucial role in skeletal muscle atrophy. This example uses Western blotting (WB) to verify the effect of DMF on mitochondrial homeostasis in the skeletal muscle of LLC tumor-bearing mice. The results are as follows: Figure 5 As shown in Figure C, compared with the normal group, the protein levels of mitochondrial fusion-related factors OPA1 and MFN1 in the gastrocnemius muscle of LLC mice were significantly downregulated, while the protein expression of mitochondrial division-related factors FIS1 and DRP1 showed no significant change. DMF intervention significantly improved the decrease in OPA1 and MFN1 protein levels in the gastrocnemius muscle of LLC tumor-bearing mice, but had no significant effect on the protein expression of FIS1 and DRP1. Meanwhile, mitochondrial biosynthesis plays a crucial role in skeletal muscle atrophy. The effect of DMF on mitochondrial biosynthesis in the skeletal muscle of LLC tumor-bearing mice was detected by Western blotting. The results are as follows: Figure 5 As shown in Figure D, compared with the LLC group, DMF significantly promoted the expression of PGC1α and Nrf1 proteins in the gastrocnemius muscle of LLC tumor-bearing mice.

[0115] In summary, DMF can improve cachexia-induced muscle atrophy by reducing mitochondrial damage induced by cancer cachexia and maintaining mitochondrial homeostasis.

[0116] 8.4 DMF improves muscle atrophy by regulating multiple pathways

[0117] Proteomics results showed that, compared with the model group, the drug-treated group upregulated 39 proteins and downregulated 120 proteins. Figure 6 China A- Figure 6 The presence of C indicates that the drug acts on multiple proteins. KEGG and GO enrichment analyses (…) Figure 7 and Figure 8 The results showed that DMF administration activated the HIF1α, MAPK, oxidative stress, and mTOR signaling pathways, providing insights into the potential mechanism by which DMF exerts its anti-muscle atrophy effect.

[0118] 8.5 DMF improves muscle atrophy by regulating the HIFα signaling pathway

[0119] Based on proteomics results, this invention selected the top-ranked HIF1α signaling pathway for experimental verification, and found that DMF administration significantly improved the downregulation of the HIF1α signaling pathway caused by LLC. Figure 9 ).

[0120] Based on the above experimental results, this invention demonstrates for the first time that DMF can effectively alleviate symptoms related to skeletal muscle atrophy caused by cancer cachexia. DMF may reduce mitochondrial damage and improve mitochondrial homeostasis imbalance, thereby alleviating skeletal muscle atrophy caused by cachexia. Its potential mechanism may be related to the activation of multiple pathways such as HIF1α, mTOR, and MAPK by DMF.

[0121] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of dimethyl fumarate in the preparation of drugs for the prevention and / or treatment of skeletal muscle atrophy.

2. The application as described in claim 1, characterized in that, The skeletal muscle atrophy includes cachexia-related skeletal muscle atrophy, age-related skeletal muscle atrophy, or drug-induced skeletal muscle atrophy.

3. The application as described in claim 1, characterized in that, The dimethyl fumarate can improve muscle strength.

4. The application as described in claim 1, characterized in that, The dimethyl fumarate can increase muscle mass and muscle mass index.

5. The application as described in claim 3, characterized in that, The muscle indices include the gastrocnemius index, tibialis anterior index, quadriceps index, soleus index, total muscle index, and epididymal fat index.

6. The application as described in claim 1, characterized in that, The dimethyl fumarate can improve the reduction of muscle fiber area.

7. The application as described in claim 1, characterized in that, The dimethyl fumarate can reduce the loss of adipose tissue.

8. The application as described in claim 1, characterized in that, The dimethyl fumarate can improve mitochondrial homeostasis imbalance.

9. The application as described in claim 1, characterized in that, The dimethyl fumarate can regulate the HIF1α and mTOR pathways.

10. A drug for improving skeletal muscle atrophy, characterized in that, The drug includes dimethyl fumarate and pharmaceutically acceptable excipients; The excipients include at least one of the following: diluent, filler, excipient, binder, wetting agent, disintegrant, absorption promoter, surfactant, adsorbent carrier, lubricant, and fragrance.

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