Application of 6-shogaol in preparation of medicine for promoting skeletal muscle regeneration and / or repair

Abstract

The invention discloses an application of 6-shogaol in preparation of a medicine for promoting skeletal muscle regeneration and / or repair. The 6-shogaol can activate stationary-stage muscle satellite cells, promote myoblast differentiation and myotube fusion, guide damaged broken end new muscle fiber connection and realize structure repair. Meanwhile, 6-shogaol can inhibit excessive deposition of collagen, reduce scar formation and promote functional regeneration. In the fracture accompanied with muscle injury, the bone healing quality is synergistically enhanced by improving the soft tissue environment and vascularization, new bone formation is increased, and the problem of bone nonunion caused by muscle injury is solved. In a word, the 6-shogaol can solve the pain point that the physical injury of the skeletal muscle is difficult to heal through the dual mechanisms of promoting muscle regeneration and resisting fibrosis, and cooperatively promotes the accompanying bone injury repair. The application is different from conventional osteoporosis treatment or disuse atrophy treatment, and has definite clinical pertinence and application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to the use of 6-shogaol in the preparation of medicaments for promoting skeletal muscle regeneration and / or repair. Background Technology

[0002] Skeletal muscle injury repair is a significant clinical challenge, especially in complex cases involving impaired blood supply, infection, or large-area tissue loss, where the body's own repair capabilities are often insufficient. Currently, while autologous muscle tissue transplantation is considered the gold standard, it faces challenges such as limited donor availability and the risk of secondary injury. Allogeneic transplantation carries the risk of immune rejection and limitations in bioactivity. Biomaterial scaffolds provide structural support, but remain relatively weak in actively regulating cell function, promoting angiogenesis, and improving the local microenvironment. Growth factor drugs, while possessing some repair-promoting effects, often exhibit single-effect, poor stability, and dose-dependent side effects. Therefore, there is an urgent need to develop repair strategies that can comprehensively regulate myogenesis, vascularization, and the immune microenvironment.

[0003] In recent years, the research focus of muscle regeneration medicine has shifted from single-target intervention to the systemic regulation of the continuous process of "inflammation-angiogenesis-muscle regeneration." Under this trend, active ingredients of traditional Chinese medicine with multi-target and holistic regulatory characteristics have attracted attention. 6-Shogaol, an active ingredient derived from gingerol during ginger processing, has high bioavailability. Studies have shown that this component possesses multiple activities, including anti-inflammatory, antioxidant, and angiogenesis-promoting effects. It can influence cell proliferation and differentiation by regulating signaling pathways such as MAPK and NF-κB, demonstrating potential in promoting skeletal muscle regeneration.

[0004] However, current research on the application of 6-shogaol in muscle repair is still relatively lacking, especially regarding its systemic effects and mechanisms in coordinating myogenic differentiation, inhibiting protein degradation, regulating fibrosis, and the inflammatory microenvironment. Therefore, this invention aims to explore the role and molecular mechanisms of 6-shogaol in skeletal muscle repair, in order to provide experimental evidence for the development of novel muscle regeneration treatment strategies. Summary of the Invention

[0005] This invention addresses the problems faced by existing technologies in treating severe skeletal muscle physical injuries (such as trauma and surgical resection), including insufficient muscle fiber regeneration capacity, easy formation of fibrotic scars, and difficulty in healing adjacent fractures due to poor soft tissue repair. It provides the application of 6-shogaol in the preparation of drugs for promoting skeletal muscle regeneration and / or repair.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Provides the use of 6-shogaol in the preparation of medicaments for promoting skeletal muscle regeneration and / or repair.

[0007] Furthermore, the drug is used to treat traumatic skeletal muscle injury accompanied by tissue damage.

[0008] Furthermore, the traumatic skeletal muscle injury is selected from mechanical trauma, crush injury, muscle tear, or surgically induced muscle injury.

[0009] Furthermore, the drug is used as an adjunct treatment for fractures or bone defects accompanied by damage to surrounding skeletal muscles.

[0010] Furthermore, the route of administration of the drug is selected from oral, transdermal, intramuscular, subcutaneous, or intravenous injection.

[0011] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention demonstrates for the first time that 6-shogaol can significantly activate quiescent myosatellite cells in vitro, promoting myoblast differentiation and myotube fusion. Unlike "anti-atrophy" drugs that simply increase muscle fiber thickness, 6-shogaol can induce the generation of new muscle fibers from damaged ends, achieving the connection and repair of physical structures.

[0012] In muscle injury repair, scar formation is a major obstacle to functional recovery. This invention is the first to discover that 6-shogaol can effectively inhibit excessive collagen deposition at the site of injury and reduce the formation of non-functional scar tissue, thereby guiding muscles to undergo "functional regeneration" rather than "scar healing".

[0013] For fractures accompanied by muscle damage, 6-shogaol promotes local vascularization by improving the "soft tissue envelope" around the fracture. Although its core role is in muscle repair, a healthy muscle environment significantly improves the healing quality of bone defects, increases new bone formation and bone microstructural parameters, thereby solving the clinical challenge of nonunion caused by muscle damage.

[0014] In summary, 6-shogaol addresses the challenge of slow healing of physical injuries to skeletal muscle through a dual mechanism of promoting muscle regeneration and combating fibrosis, while also synergistically promoting the repair of accompanying bone damage. This application differs from conventional treatments for osteoporosis or disuse atrophy, demonstrating clear clinical relevance and practical value. Attached Figure Description

[0015] Figure 1 This figure shows the results of detecting the effect of different concentrations of 6-shogaol on the differentiation of mouse C2C12 myotube cells stimulated by dexamethasone (100 μM) in an embodiment of the present invention, in order to evaluate its promoting effect on myogenic differentiation during skeletal muscle regeneration. in: Figure 1 Figure A shows the immunofluorescence results of the effect of different concentrations of 6-shogaol on the differentiation of mouse C2C12 myotube cells stimulated with dexamethasone (100 μM). Figure 1 B is the corresponding bar chart.

[0016] Figure 2 This is a diagram showing the detection results of 6-shogaol on the expression of related proteins in mouse C2C12 myotube cells stimulated with dexamethasone (100 μM) in an embodiment of the present invention; wherein: Figure 2 A shows the Western Blot results of different concentrations of 6-shogaol on the expression of myogenic differentiation-related proteins (MyOD, MYH) and protein metabolism-related factors (MuRF1, Atrogin-1), reflecting its role in regulating protein synthesis and degradation during skeletal muscle repair. Figure 2 B shows a bar chart illustrating the relative expression levels of the above proteins in each treatment group.

[0017] Figure 3 The image shown is a Masson staining result from an embodiment of the present invention, demonstrating the ameliorative effect of 6-shogaol on gastrocnemius muscle fibrosis in LLC tumor-bearing mice, suggesting its anti-fibrotic potential in skeletal muscle tissue repair.

[0018] Figure 4 The figure shown is an ELISA detection result from an embodiment of the present invention, demonstrating the regulatory effect of 6-shogaol on the level of the inflammatory factor IL-6 in the serum of LLC tumor-bearing mice, reflecting its effect on improving the inflammatory microenvironment during skeletal muscle regeneration.

[0019] Figure 5 The figure shown is an RNA-seq sequencing result diagram from an embodiment of the present invention, which illustrates the volcano plot analysis of differentially expressed genes in the gastrocnemius muscle of LLC tumor-bearing mice by 6-shogaol, revealing its regulatory role in gene expression profiles during skeletal muscle repair.

[0020] Figure 6 The image shown is an RNA-seq sequencing result diagram from an embodiment of the present invention, displaying a heatmap analysis of differentially expressed genes in the gastrocnemius muscle of LLC tumor-bearing mice by 6-shogaol, further illustrating its role in skeletal muscle regeneration-related signaling pathways.

[0021] Figure 7 The figure shown is an RNA-seq sequencing result diagram in an embodiment of the present invention, which shows the enrichment analysis results of differentially expressed genes in the gastrocnemius muscle of LLC tumor-bearing mice by 6-shogaol, suggesting its functional pathways involved in skeletal muscle repair.

[0022] Figure 8 The figure shown is an RNA-seq sequencing result diagram in an embodiment of the present invention, which shows the KEGG pathway analysis results of 6-shogaol on the gastrocnemius muscle of LLC tumor-bearing mice, revealing its comprehensive role in skeletal muscle regeneration, including inflammation regulation, angiogenesis, and myogenic differentiation-related pathways. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0024] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0025] Example 1: In vitro cell experiments This embodiment uses cell experiments to verify the regulatory effect of 6-shogaol on myogenic differentiation and protein metabolism at the skeletal muscle cell level.

[0026] 1.1 Experimental Methods: Mouse fibroblast C2C12 cells were induced to differentiate into myotube cells with 2% horse serum. After differentiation, the C2C12 myotube cells were co-cultured with different concentrations (5 μM, 10 μM, 20 μM) of 6-shogaol and 100 μM dexamethasone for 24 hours.

[0027] (1) To assess myoblast differentiation, after treatment, the cells were fixed, permeabilized and blocked, and then incubated with myosin heavy chain (MyHC) primary antibody at 4°C overnight; after washing, they were incubated with fluorescent secondary antibody for 2 hours; then the cell nuclei were stained with DAPI and mounted, observed and photographed under a fluorescence microscope, and the fluorescent areas were statistically analyzed.

[0028] (2) To detect the expression of relevant proteins, after treatment, total protein was extracted using RIPA lysis buffer, and the concentration was determined by the BCA method. Samples were then balanced and subjected to Western blotting. The proteins detected included myoblast differentiation-related factors (MyOD, MYH) and protein metabolism-related factors (MuRF1, Atrogin-1). 1.2 Experimental Results: Depend on Figure 1 It is evident that the area of ​​C2C12 myotubes decreased significantly after dexamethasone treatment; however, the area of ​​MyHC-labeled myotubes gradually increased under different concentrations of 6-shogaol treatment, suggesting that 6-shogaol can improve dexamethasone-induced differentiation impairment and exhibits a concentration-dependent effect.

[0029] Western Blot results are as follows: Figure 2 As shown in Figure A, different concentrations of 6-shogaol significantly regulate the expression of the aforementioned proteins. Statistical analysis is as follows: Figure 2 As shown in Figure B, dexamethasone treatment decreased the expression levels of MyOD and MYH, while increasing the expression levels of MuRF1 and Atrogin-1. With 6-shogaol intervention, the expression levels of MyOD and MYH were restored, while the expression levels of MuRF1 and Atrogin-1 decreased in a concentration-dependent manner. These results indicate that 6-shogaol can promote protein synthesis and inhibit excessive protein degradation, thereby improving the skeletal muscle repair process.

[0030] Example 2 Animal Experiment This embodiment uses a tumor-bearing mouse model to verify the comprehensive regulatory effects of 6-shogaol on skeletal muscle repair, fibrosis, inflammation, and transcriptome at the whole animal level.

[0031] 2.1 Experimental Methods 2.1.1 Experimental Materials and Animal Preparation This study used SPF-grade healthy adult male C57BL / 6 mice, aged 6-8 weeks and weighing 18-22g. All mice were housed in a standard SPF-grade laboratory animal facility (Animal Facility of Shanghai Sixth People's Hospital, Animal Ethics No.: DWSY 2024-0143), with the ambient temperature maintained at 22-25°C and the relative humidity at 50%-60%, and a 12-hour light / dark cycle implemented.

[0032] 2.1.2 Drug Preparation Accurately weigh an appropriate amount of 6-shogaol and dissolve it thoroughly in 2% dimethyl sulfoxide (DMSO). After the drug is completely dissolved, slowly add physiological saline (0.9% NaCl) to dilute and mix well to prepare a series of concentrations of drug solution for intraperitoneal injection. The control group uses physiological saline containing the same proportion (2%) of DMSO as a solvent control.

[0033] 2.1.3 Establishment of LLC tumor-bearing mouse model Lewis lung cancer (LLC) cells in the logarithmic growth phase and in good condition were harvested, digested with trypsin, resuspended in sterile PBS buffer, and the cell density was adjusted to 1*10^6 cells / year. 6Cells / mL. The right axilla of male C57BL / 6 mice was disinfected, and 0.1 mL of cell suspension was subcutaneously inoculated into each mouse. Tumor formation was observed daily after inoculation. Subcutaneous tumor modeling was considered successful when the tumor nodules grew to an average volume of approximately 100 mm³. A cachexia model was considered successful when the mice's net weight decreased by more than 10% compared to the normal group at the experimental endpoint, and progressive weight loss not due to reduced food intake was observed. After successful modeling, mice were randomly assigned to receive 10 mg / kg of 6-shogaol or a control treatment for 4 consecutive weeks. After the experiment, serum and gastrocnemius muscle tissue were collected from the mice for the following analysis: (1) The serum inflammatory factor IL-6 level in LLC tumor-bearing mice was detected by ELISA; (2) Masson staining was performed on the gastrocnemius muscle tissue to observe the degree of muscle fibrosis; (3) Total RNA was extracted from gastrocnemius muscle tissue for RNA-seq transcriptome sequencing, and differentially expressed genes were analyzed by volcano plot analysis, cluster heatmap analysis, GO functional enrichment analysis and KEGG pathway enrichment analysis.

[0034] 2.2 Experimental Results like Figure 4 As shown, the serum IL-6 level in the 6-shogaol treatment group decreased significantly, suggesting that it can effectively alleviate the inflammatory microenvironment and provide favorable conditions for skeletal muscle repair.

[0035] like Figure 3 As shown, the degree of fibrosis in the gastrocnemius muscle of the 6-shogaol treatment group was significantly reduced, suggesting that it has an anti-fibrotic effect in skeletal muscle repair and further promotes muscle fiber regeneration.

[0036] like Figure 5 As shown in the volcano plot results, compared with the control group, the 6-shogaol treatment group had a large number of differentially expressed genes in the gastrocnemius muscle, involving pathways related to muscle differentiation, inflammatory response and energy metabolism.

[0037] like Figure 6 As shown in the heatmap of differential gene clustering, the treatment group and the control group were clearly separated in terms of transcriptional patterns, suggesting that 6-shogaol has a systemic regulatory effect on skeletal muscle gene expression.

[0038] like Figure 7 As shown, the GO enrichment results indicate that differentially expressed genes are mainly concentrated in biological processes related to muscle development, protein synthesis, angiogenesis, and immune regulation.

[0039] like Figure 8 As shown, KEGG pathway analysis results indicate that differentially expressed genes are significantly enriched in signaling pathways such as MAPK, PI3K / Akt, and NF-κB, which are closely related to inflammation relief, angiogenesis, and myogenic differentiation.

[0040] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

The use of 1,6-shogaol in the preparation of drugs for promoting skeletal muscle regeneration and / or repair.

2. The application according to claim 1, characterized in that, The drug is used to treat traumatic skeletal muscle injuries accompanied by tissue damage.

3. The application according to claim 2, characterized in that, The traumatic skeletal muscle injury is selected from mechanical trauma, crush injury, muscle tear, or surgically induced muscle injury.

4. The application according to claim 1, characterized in that, The drug is used as an adjunct treatment for fractures or bone defects accompanied by damage to surrounding skeletal muscles.

5. The application according to claim 1, characterized in that, The route of administration for the drug is selected from oral, transdermal, intramuscular, subcutaneous, or intravenous injection.

Images