Use of shihubine in the preparation of a medicament for treating sarcopenia and / or chemical muscle injury

Abstract

This invention discloses the application of dendrobine in the preparation of drugs for treating sarcopenia and / or chemically induced muscle injury, belonging to the field of biomedical technology. Using an 18-month-old aging mouse model and a BaCl2-induced acute skeletal muscle injury model, combined with a primary muscle stem cell isolation and culture system, this invention demonstrates for the first time that dendrobine can target muscle stem cells, activating mitochondrial autophagy by upregulating the level of phosphorylated ubiquitin (Ser65) at Ser65 and the protein expression ratio of LC3B-II / LC3B-I, and restoring the pathologically downregulated glycolysis rate in aging muscle stem cells to normal levels. This breaks the vicious cycle of metabolic-oxidative stress caused by aging / injury, maintaining the self-renewal and differentiation balance of the muscle stem cell pool. This invention provides a novel treatment strategy and drug candidate for sarcopenia and chemically induced muscle injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of dendrobine in the preparation of drugs for treating sarcopenia and / or chemical muscle damage. Background Technology

[0002] The iliac muscle accounts for approximately 30% to 40% of body weight, and its regenerative capacity primarily relies on muscle stem cells (MuSCs), also known as muscle satellite cells, located between the basement membrane and sarcolemma of muscle fibers. Under normal physiological conditions, MuSCs are usually in a resting state, but after muscle injury, they can be rapidly activated into the cell cycle, proliferating, differentiating, and fusing to form new muscle fibers, while simultaneously self-renewing to maintain the homeostasis of the stem cell pool. However, under aging or pathological conditions, the activation capacity of MuSCs significantly declines, leading to impaired muscle regeneration and subsequently triggering a series of muscle-related diseases. Among these, sarcopenia and chemically induced muscle injury are two representative muscle dysfunctions, both of which lack effective targeted therapies in clinical practice.

[0003] Sarcopenia, also known as muscle loss, is an age-related syndrome characterized by a decline in skeletal muscle mass, strength, or physical function. Its core features include reduced muscle mass, decreased muscle strength, and / or impaired physical function. The occurrence of sarcopenia is associated with factors such as age, nutritional status, physical activity level, polypharmacy, chronic inflammation, hormone levels, acute and chronic diseases (such as diabetes, chronic obstructive pulmonary disease, cirrhosis, and cancer), and geriatric syndromes (such as depression, frailty, and cognitive impairment). Currently, there are no specific drugs for targeted treatment in clinical practice.

[0004] Chemical muscle injury is another important type of muscle disease. Chemical toxins (such as barium chloride and certain biological toxins) can directly lead to muscle fiber necrosis, inflammatory infiltration, and muscle dysfunction. Clinically, chemical muscle injury can occur in various scenarios, including adverse drug reactions, exposure to environmental toxins, and iatrogenic injuries. Currently, treatment mainly relies on anti-inflammatory drugs for symptomatic relief, and there is a lack of effective treatments that can actively promote muscle regeneration and repair.

[0005] Dendrobine is one of the typical alkaloids found in Dendrobium officinale, and its chemical structure belongs to the tetracyclic sesquiterpene type alkaloids. Existing studies have shown that dendrobine has various pharmacological activities, including anti-inflammatory, antioxidant, and neuroprotective effects.

[0006] For the treatment of sarcopenia, there are existing patent applications related to natural products. For example, Chinese invention patent application CN117899063A discloses the use of oat alkaloids in the preparation of products for the prevention and / or treatment of muscle atrophy-related diseases (including sarcopenia), whose mechanism of action involves inhibiting the expression of muscle atrophy-related proteins such as FoxO1. However, oat alkaloids and dendrobines differ fundamentally in chemical structure, source, and pharmacological mode of action, and their therapeutic efficacy needs further improvement.

[0007] In summary, the existing technologies have the following shortcomings: (1) there is a lack of clinical treatment drugs for sarcopenia, and there is an urgent need for safe and effective new treatment strategies; (2) there is a lack of effective means to promote active regeneration and repair of chemically induced muscle damage; and (3) research on dendrobine in the field of muscle stem cell regeneration regulation and muscle damage treatment is still incomplete. Therefore, developing new uses for dendrobine in the treatment of sarcopenia and chemically induced muscle damage has important clinical significance and application prospects. Summary of the Invention

[0008] Based on the shortcomings of the prior art, this invention provides the application of dendrobine in the preparation of drugs for treating sarcopenia and / or chemical muscle damage, and confirms through pharmacodynamic experiments that dendrobine can significantly enhance the regenerative capacity of muscle stem cells, improve age-related muscle function decline and muscle repair after chemical damage, and provide a new drug strategy for the treatment of sarcopenia and chemical muscle damage.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0012] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0013] In a first aspect, the present invention provides the use of dendrobine in the preparation of a medicament for treating sarcopenia and / or chemically induced muscle damage.

[0014] The chemical name of the dendrobine is Dendrobine, and its molecular formula is C. 16 H 25 NO2 has the following chemical structure: .

[0015] Dendrobine can enhance mitochondrial autophagy flux in muscle stem cells and upregulate glycolytic metabolism rate, restoring metabolic homeostasis of muscle stem cells in aging or damaged states, thereby enhancing muscle regeneration capacity.

[0016] Preferably, the dendrobine can upregulate the expression level of phosphorylated ubiquitin (Ser65) at the Ser65 site in muscle stem cells, promoting the selective clearance of damaged mitochondria.

[0017] Preferably, the dendrobine enhances the autophagic flux of muscle stem cells by increasing the protein expression ratio of LC3B-II / LC3B-I and decreasing the level of the autophagy substrate p62.

[0018] Dendrobine can increase PAX7. + The number of muscle stem cells was increased by Ki67. + Proportion of proliferating cells.

[0019] Dendrobine can increase the cross-sectional area of ​​muscle fibers, reduce post-injury fibrosis, and / or upregulate myoblast gene expression.

[0020] The chemically induced muscle damage is barium chloride-induced muscle damage.

[0021] The dosage form of the drug is an oral preparation or an injectable preparation.

[0022] Preferably, the oral preparation includes granules, capsules, tablets, or oral liquid.

[0023] The drug comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is dendrobine.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention reveals for the first time the use of dendrobine in the preparation of drugs for the treatment of sarcopenia and / or chemical muscle damage, filling the research gap of dendrobine in the field of muscle stem cell regeneration regulation and muscle damage treatment.

[0025] (2) This invention reveals for the first time that dendrobine exerts its therapeutic effect by regulating the mitochondrial metabolic reprogramming of muscle stem cells, specifically by activating mitochondrial autophagy (upregulating the LC3B-II / I ratio, Phospho-Ubiquitin Ser65 level, and reducing p62 level), restoring the glycolytic metabolic rate of aging muscle stem cells, and reducing oxidative stress levels. Attached Figure Description

[0026] Figure 1 The following figure shows the effect of dendrobine on the motor ability of aging mice in Example 1: A is the grip strength of the mice; B is the number of free wheel laps of the mice within 24 hours; C is the muscle mass of the mice.

[0027] Figure 2 This is the effect of dendrobine on the expression level of myogenic genes in aging mice, as shown in Example 1.

[0028] Figure 3 The figure shows the effect of dendrobine on the number of muscle stem cells in aging mice in Example 1 (staining results and statistical graphs of PAX7, a marker of muscle stem cells in two groups of mice).

[0029] Figure 4 The following is a graph showing the effect of dendrobine on the motor ability of mice with muscle damage in Example 2: A represents the grip strength level of the mice; B represents the number of free wheel laps of the mice within 24 hours.

[0030] Figure 5 The figure shows the effect of dendrobine and oat alkaloids on muscle function (difference in the ratio of tibialis anterior muscle to body weight) in mice with barium chloride injury in Example 2.

[0031] Figure 6 The following is a graph showing the effect of dendrobine and oat alkaloids on the accumulation of fibroblasts in muscle injury in Example 2: A is Sirius red staining; B is the statistical analysis graph of A.

[0032] Figure 7 Figure 2 shows the effect of dendrobine on damaged mouse muscle stem cells in Example 2: A represents the flow cytometry analysis of CD34. + Figure showing the difference in the number of muscle stem cells; B represents the immunofluorescence analysis of PAX7. + A graph showing the difference in the number of muscle stem cells.

[0033] Figure 8 Figure 2 shows the effect of dendrobine on muscle proliferation in injured mice in Example 2: A is the Ki67 immunohistochemical staining image; B is the statistical graph of Figure A.

[0034] Figure 9 Figure A shows the results of transcriptomic analysis of muscles in the dendrobine intervention group and control group of aging mice in Example 3: Figure A shows the gene pathways upregulated by GO analysis in transcriptomics; Figure B shows the pathways enriched by GSEA.

[0035] Figure 10 This is a graph showing the difference in expression of related genes in the muscle transcriptomics of the dendrobine intervention group and the control group in Example 3.

[0036] Figure 11 The graph shows the differences in expression of autophagy-related proteins in the muscles of mice treated with dendrobine in Example 3: A shows the changes in protein levels of LC3BII / LC3BI and p62; B shows the changes in protein levels of Phospho-Ubiquitin (Ser65).

[0037] Figure 12 The graph shows the differences in metabolic and ROS levels of muscle stem cells between the dendrobine intervention group and the control group in Example 4: A represents the change in glycolysis rate of muscle stem cells; B represents the ROS level of muscle stem cells.

[0038] The comparison between the groups in the figure is as follows. P<0.05, P<0.01, P<0.001, P<0.0001. Detailed Implementation

[0039] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0040] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0041] Example 1: Dendrobine improves muscle function and increases muscle mass and stem cell count in aging mice. 1. Experimental Materials 1.1 Test Drug Dendrobine was purchased from Shanghai TargetMol Company, and the purity of all samples was 99.70%.

[0042] 1.2 Reagents and Consumables Trizol, reverse transcription kit, and qPCR premix kit were all purchased from Nanjing Novizan Biotechnology Co., Ltd.; PAX7 antibody was purchased from Chengdu Zhengneng Biotechnology Co., Ltd.; multicolor staining kit was purchased from Hunan Aifang Biotechnology Co., Ltd. Mouse grip strength tester (Puyan); small animal treadmill (Anhui Zhenghua).

[0043] 2. Laboratory animals and grouping 18-month-old male C57BL / 6J aging mice, weighing 25-30g, were housed in an SPF barrier at an ambient temperature of 22±2℃ and a relative humidity of 40-60%, with a 12h light / 12h dark cycle.

[0044] After weighing, the mice were randomly divided into two groups: a control group (normal drinking water) and a dendrobine group (dendrobine administered via drinking water at a dose of 0.1 mg / kg / day). The experiment lasted for 10 weeks, with body weight and food intake measured weekly.

[0045] 3. Detection indicators 3.1 Motor ability testing After drug administration, the grip strength of the mice's limbs was measured using a small animal grip strength tester, with each mouse measured three times and the average value taken. The mice's voluntary movement ability was assessed using a free running wheel test, and the number of wheel laps over 24 hours was recorded.

[0046] 3.2 Muscle mass testing The muscle content of mice was detected using a small animal body fat percentage analyzer (MRI). After the experiment, the mice were euthanized by cervical dislocation, and the tibialis anterior muscle (TA) was collected and weighed wetly.

[0047] 3.3 Real-time quantitative qPCR detection Total RNA was extracted from cells in each group using the TRIzol-phenol-chloroform method. RNA concentration and purity were detected using NanoDrop. RNA was reverse transcribed into cDNA using a reverse transcription kit. Expression levels of muscle regeneration-related genes were detected using a qPCR premix kit.

[0048] 3.4 PAX7 Immunofluorescence Staining (1) Sample preparation: Paraffin sectioning: The sections were sequentially immersed in xylene I for 15 min → xylene II for 15 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 95% ethanol for 5 min → 85% ethanol for 5 min → 75% ethanol for 5 min, and then washed with distilled water.

[0049] (2) Antigen retrieval: Tissue slides were placed in a retrieval box filled with pH 6.0 citric acid retrieval buffer and microwaved on medium heat for 8 min, then turned off for 8 min, and then microwaved on medium-low heat for 7 min for antigen retrieval. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time.

[0050] (3) Blocking endogenous peroxidase: Add peroxidase blocking solution to the slide and incubate at room temperature in the dark for 15 min. Place the slide in PBS (pH 7.4) buffer and wash it three times on a decolorizing shaker for 5 min each time.

[0051] (4) Non-specific target blocking: After the section is slightly dried, the tissue is evenly covered with normal goat serum and blocked at room temperature for 30 minutes.

[0052] (5) Incubation of specific primary antibody: Gently shake off the blocking solution, drop the primary antibody PAX7 diluted with antibody diluent onto the slide, and incubate the slide flat in a light-proof humidified box at 4°C overnight.

[0053] (6) Incubation with Polymer-HRP secondary antibody: Place the slide in PBS (pH 7.4) buffer and wash it three times on a decolorizing shaker for 5 min each time. Shake the slide slightly dry, add Polymer-HRP secondary antibody of the same species as the primary antibody to cover the tissue, incubate at room temperature in the dark for 30 min, and wash three times with PBS for 5 min each time.

[0054] (7) TSA fluorescent dye staining: 50 μL of TSA-570 fluorescent dye (ready-to-use type) was directly added to the section and evenly covered the tissue. The mixture was reacted at room temperature for 10 min, and then washed three times with PBS for 5 min each time.

[0055] (8) DAPI counterstaining of cell nuclei and mounting: After the slide is dried, drop a drop of anti-fluorescence quenching mounting medium (containing DAPI) onto the sample, gently cover with a coverslip, and wait 5 minutes before observation.

[0056] (9) Microscopic examination and photography: The slides were observed and images were acquired under a fluorescence microscope / confocal microscope / multichannel fluorescence scanner / multispectral imaging system.

[0057] 4. Statistical Methods All data were analyzed using Prism 9 (GraphPad) and ImageJ software. Normality was tested using the Shapiro-Wilk test. For two normally distributed datasets, an unpaired two-tailed t-test was used; for non-normally distributed datasets, the Mann-Whitney test was used. For datasets with more than two groups, one-way ANOVA was performed, with p < 0.05 considered statistically significant.

[0058] 5. Experimental Results 5.1 Dendrobine enhances the motor function of aging mice After 10 weeks of administration, compared with the control group, the grip strength of senescent mice in the dendrobine group was significantly improved (P<0.05). Figure 1 (A) The results of the free running wheel test showed that the number of running laps in the Dendrobium alkaloid group was significantly greater than that in the control group within 24 hours (P<0.05). Figure 1 (B in the middle).

[0059] 5.2 Dendrobine increases muscle mass in aging mice Nuclear magnetic resonance imaging results are as follows Figure 1 As shown in C, the muscle weight of aging mice in the dendrobine group was significantly increased compared to the control group (P<0.01). The qPCR results are shown below. Figure 2 As shown, the expression levels of myogenic genes Myf5, Myf6, and Myogenin (Myog) in the muscle tissue of aging mice in the dendrobine group were significantly upregulated (P<0.05).

[0060] 5.3 Dendrobine increases the number of muscle stem cells in aging mice PAX7 immunofluorescence staining results are as follows: Figure 3 As shown, PAX7 in the muscles of aging mice in the dendrobine group + The number of muscle stem cells was significantly higher than that in the control group (P<0.01).

[0061] Example 2: Dendrobine alleviates muscle damage caused by barium chloride (BaCl2). 1. Experimental Materials 1.1 Test Drug Dendrobine was purchased from Shanghai TargetMol Company, with a purity of 99.70%; oat alkaloids were also purchased from Shanghai TargetMol Company, with a purity of 99.76%.

[0062] 1.2 Reagents and Consumables Trizol, reverse transcription kit, and qPCR premix kit were all purchased from Nanjing Novizan Biotechnology Co., Ltd.; PAX7 antibody was purchased from Chengdu Zhengneng Biotechnology Co., Ltd.; Ki67 antibody was purchased from Cell Signaling Technology, Inc.; Sirius Red staining kit was purchased from Savill Biotechnology Co., Ltd.; multicolor staining kit and immunohistochemistry kit were both purchased from Hunan Aifang Biotechnology Co., Ltd.; CD34-AF700, CD45-PreCP-Cy5.5, CD31-PreCP-Cy5.5, and Sca-1-PE-Cy7 flow cytometry antibodies were all purchased from BIOLEGEND (Beijing) Biotechnology Co., Ltd.

[0063] 2. Experimental Methods 2.1 Laboratory Animals Eight-week-old male C57BL / 6J mice, weighing 20-25g, were fed under the same conditions as in Example 1.

[0064] 2.2 Construction of BaCl2 muscle injury model Mice were anesthetized with isoflurane, fixed in a supine position, and the hair on the anterolateral aspect of one lower leg was removed and disinfected with iodine. Using a 1 mL insulin syringe (29G), 50 μL of BaCl2 solution (1.2%) was slowly injected into the center of the tibialis anterior muscle belly at three selected sites (insertion depth 3-5 mm, avoiding bone). After a short pause, the needle was quickly withdrawn, and the puncture site was gently pressed to prevent leakage. Temporary lameness occurred on the injected limb postoperatively, which subsided within several hours.

[0065] 2.3 Grouping and Administration After modeling, mice were randomly divided into 6 groups: control group (normal drinking water), barium chloride group (1.2% intramuscular injection), dendrobine group (dendrobine 0.5 mg / kg, administered via drinking water), barium chloride (1.2% intramuscular injection) + dendrobine group (dendrobine 0.5 mg / kg, administered via drinking water), oat alkaloid group (0.5 mg / kg, administered via drinking water), and barium chloride (1.2% intramuscular injection) + oat alkaloid group (0.5 mg / kg, administered via drinking water). Administered medication for 7 consecutive days post-surgery, with the medication changed daily. Mice were allowed free access to water and food during this period.

[0066] 3. Detection indicators 3.1 Motor ability testing On the 7th day after surgery, the grip strength and free running wheel movement ability of mice were tested using the same method as in Example 1.

[0067] 3.2 Muscle mass testing The muscle content of mice was detected using a small animal body fat percentage analyzer (MRI). After the experiment, the mice were euthanized by cervical dislocation, and the tibialis anterior muscle (TA) was collected and weighed wetly.

[0068] 3.3 Collection of mouse muscle tissue At the end of the experiment, the mice were fasted for 12 hours, and their weight was measured. The mice were then euthanized by cervical dislocation, and the tibial lateral muscle was collected. A portion was cryopreserved, another portion was fixed in paraformaldehyde for 24 hours, embedded in paraffin, sectioned, and used for multicolor staining. The remaining portion was digested to prepare a single-cell suspension for flow cytometry analysis.

[0069] 3.4 Flow Cytometry Analysis of Mouse Muscle Stem Cells The obtained tibialis muscle tissue was washed three times with PBS and transferred to a sterile culture dish. The muscle was repeatedly minced until it became a paste-like consistency with no visible tissue fragments. It was then transferred to a 15 mL centrifuge tube, and 4 mL of enzyme digestion solution was added per adult mouse tibia muscle. The mixture was incubated at 37°C with shaking at 120 rpm for 60 minutes. Gentle vortexing was performed every 15 minutes to promote separation. An equal volume of DMEM containing 10% FBS was added to terminate the enzyme reaction. The entire suspension was passed sequentially through a 70 μm cell filter on ice and placed in a 50 mL centrifuge tube. The filter was rinsed with washing buffer, and the filtrate was collected. The tube was centrifuged at 350 × g for 10 minutes at 4°C. The pellet was resuspended in 1 mL of erythrocyte lysis buffer, incubated at room temperature for 5 minutes, and 10 times the volume of washing buffer was added to terminate the reaction. The tube was then centrifuged at 300 × g for 5 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in 100 μL of staining buffer, counted, and 1 × 10⁻⁶ cells were collected. 7 Cells were added to 40 μL of the muscle stem cell staining antibody mix and stained at 4°C for 30 min. 1 mL of washing buffer was added, and the cells were centrifuged at 300 × g for 5 min at 4°C, discarding the supernatant. The pellet was resuspended in 500 μL of staining buffer, filtered through a 300-mesh filter, and analyzed by flow cytometry. The muscle stem cell staining antibody mix consisted of CD34-AF700, CD45-PreCP-Cy5.5, CD31-PreCP-Cy5.5, and Sca-1-PE-Cy7.

[0070] 3.5 Sirius Red staining Paraffin sectioning: Immerse sections sequentially in environmentally friendly dewaxing solution I for 10 min → environmentally friendly dewaxing solution II for 10 min → environmentally friendly dewaxing solution III for 10 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → anhydrous ethanol III for 5 min → wash with distilled water. Soak sections in Sirius scarlet stain A in a 65°C oven for 30 min; wash sections with water until the yellow color fades, spin dry, and immerse in Sirius scarlet stain B for 2 min; wash with water, spin dry, and immerse in Sirius scarlet stain C for 30 min; quickly wash with water for 1-2 seconds, then quickly rinse and dehydrate in three separate tanks of anhydrous ethanol for 3 seconds each; clear sections in clean xylene for 5 min, and mount with neutral resin. Scan with a digital slide scanner.

[0071] 3.6 PAX7 Immunofluorescence Staining (Multicolor Staining Kit) Same as step 2.5 in Example 1.

[0072] 3.7 Ki67 Immunohistochemical Staining (1) Sample preparation: Paraffin sectioning: The sections were placed in xylene I for 15 min → xylene II for 15 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 95% ethanol for 5 min → 85% ethanol for 5 min → 75% ethanol for 5 min → washed with distilled water.

[0073] (2) Antigen retrieval: Tissue sections were placed in a retrieval box filled with pH 6.0 citrate retrieval buffer and microwaved on medium heat for 8 min, then turned off for 8 min, and then microwaved on medium-low heat for 7 min for antigen retrieval. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time. The slides were permeabilized with 0.3% Triton X-100 for 10 min, and then washed twice with PBS (pH 7.4) for 5 min each time.

[0074] (3) Blocking endogenous peroxidase: Add peroxidase blocking solution to the slide and incubate at room temperature in the dark for 15 min. Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time.

[0075] (4) Non-specific target blocking: After the section is slightly dried, the tissue is evenly covered with normal goat serum and blocked at room temperature for 30 minutes.

[0076] (5) Incubation of specific primary antibody: Gently shake off the blocking solution, drop the primary antibody PAX7 diluted with antibody diluent onto the slide, and incubate the slide in a light-proof humidified box at 4°C overnight.

[0077] (6) Incubation with Polymer-HRP secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time. Shake the slide slightly dry, add Polymer-HRP secondary antibody of the same species as the primary antibody to cover the tissue, incubate at room temperature in the dark for 30 min, and wash three times with PBS for 5 min each time.

[0078] (7) DAB color development: Freshly prepared DAB working solution is added to cover the tissue and observed under a microscope in real time. When a brownish-yellow positive signal appears and the background is relatively light, the tissue is immediately immersed in distilled water to stop the reaction.

[0079] (8) Counterstain with hematoxylin solution for 30s, wash with water, differentiate with hydrochloric acid alcohol for 2s, and return to blue with warm water for 5min.

[0080] (9) Dehydrate the slides sequentially with 75%, 85%, 95%, and 100% ethanol solutions for 2 minutes each, and clear them twice with xylene for 5 minutes each time. Mount the slides with neutral resin, air dry them in a ventilated place, and then examine them under a microscope.

[0081] 4. Statistical Methods All data were analyzed using Prism 9 (GraphPad) and ImageJ software. Normality was tested using the Shapiro-Wilk test. For two normally distributed datasets, an unpaired two-tailed t-test was used; for non-normally distributed datasets, the Mann-Whitney test was used. For datasets with more than two groups, one-way ANOVA was performed, with p < 0.05 considered statistically significant.

[0082] 5. Experimental Results 5.1 Dendrobine enhances motor function in mice with muscle injury Mice with barium chloride-induced muscle injury were treated with either dendrobine (dendrobine group) or plain drinking water (control group). After one week, grip strength and motor function were assessed in both groups. Results showed that the grip strength of the limbs in the dendrobine group was significantly improved. Figure 4 In section A; the free-running wheel test was used to compare the free movement ability of the two groups of mice. The results showed that the mice in the dendrobine group ran more laps per unit activity time. (See section A) Figure 4 B in the middle.

[0083] 5.2 Dendrobine increases muscle mass in mice with muscle injury The results of the TA weight to body weight ratio are as follows: Figure 5 As shown, the weight of mice with muscle damage in the dendrobine group was significantly increased compared with the control group (P<0.05), while there was no significant difference between the oat alkaloid group and the control group.

[0084] 5.2 Dendrobine reduces muscle fibrosis in mice with muscle injury Sirius red staining was performed on muscle tissue sections from mice in the dendrobine group and the control group, respectively, to detect the effect of dendrobine on the degree of fibrosis after muscle injury. Results are as follows: Figure 6 As shown, dendrobine significantly reduces collagen deposition caused by muscle damage and prevents fibroblasts from replacing damaged muscle cells that cannot regenerate in time.

[0085] 5.3 Dendrobine increases the regenerative capacity of muscle stem cells in mice with muscle injury. Muscle injury repair primarily relies on the regenerative capacity of muscle stem cells. To further determine the effect of dendrobine on muscle stem cells, flow cytometry analysis and PAX7 staining (a marker for muscle stem cells) were performed on barium chloride-damaged muscle tissue. Flow cytometry results showed that the dendrobine group had CD45... - CD31 - Sca1 - and CD34 + The number of cells was significantly higher than that in the control group, as shown in [the original text]. Figure 7In section A, PAX7 staining of the same tissue section yielded the same results, see... Figure 7 B in the sample. Meanwhile, the Ki67 immunohistochemical results are as follows: Figure 8 As shown, the dendrobine group had more muscle cells in the proliferative phase.

[0086] Example 3: Dendrobine activates mitochondrial autophagy in aging muscle tissue 1. Experimental Materials 1.1 Test Drug Same as Example 1.

[0087] 1.2 Reagents and Consumables LC3B antibody, P62 antibody, and Vinculin antibody were purchased from Cell Signaling Technology, Inc.; Phospho-Ubiquitin (Ser65) antibody and PVDF membrane were purchased from Merck Millipore; RIPA lysis buffer, protease inhibitor, and BCA protein concentration assay kit were purchased from Beyotime Biotechnology Co., Ltd.; ECL chemiluminescent substrate was purchased from Thermo Fisher Scientific.

[0088] 2. Experimental Methods 2.1 Animal grouping and administration Same as step 2.1 in Example 1.

[0089] 3. Indicator Testing 3.1 Protein Extraction Take 50 mg of tibialis anterior muscle tissue from each of the two groups of mice in Example 1, add 400 μL of RIPA lysis buffer containing protease inhibitor, homogenize, centrifuge at 12000 rpm for 5 min at 4 °C, and collect the supernatant. Determine the protein concentration by BCA method and adjust the loading volume to be consistent.

[0090] 3.2 Western Blot Protein samples were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane; blocked with 5% skim milk at room temperature for 1 h; LC3B antibody (1:1000), p62 antibody (1:1000), Phospho-Ubiquitin (Ser65) antibody (1:500), and Vinculin antibody (1:5000) were added respectively, and incubated overnight at 4°C; the membrane was washed three times with TBST; incubated with secondary antibody at room temperature for 1 h; ECL chemiluminescence imaging was performed, and images were acquired using a gel imaging system. Using Vinculin as an internal control, the band grayscale values ​​were analyzed using ImageJ software.

[0091] 3.3 Transcriptomics Sequencing Transcriptional sequencing was performed on the muscle tissue of each group of mice (sequencing company: Beijing Novogene Technology Co., Ltd.). RNA was isolated from adipose tissue using TRI reagent (Sigma-Aldrich) according to the manufacturer's instructions. Library construction and sequencing were performed by Novogene in China. The steps were as follows: RNA samples for transcriptome analysis were first pretreated with DNase, and then processed according to Illumina's instructions, including the use of oligo(dT) magnetic beads to isolate polyA-tailed mRNA (polyA+ mRNA) from total RNA. Fragmentation buffer containing divalent cations was added to break the mRNA into short fragments of 200 to 700 nucleotides in length. Using these short fragments as templates, first-strand cDNA was synthesized using random hexamer primers. Subsequently, second-strand cDNA was synthesized using a buffer containing dNTPs, RNase H, and DNA polymerase I. After purification, the products were used to prepare the ends and add A-tails using the QIAquick PCR purification kit (Qiagen) and elution buffer. Purified cDNA fragments were ligated to sequencing adapters, and suitable fragments were selected for PCR amplification by gel electrophoresis. Quantitative analysis and quality control of the sample libraries were performed using an Agilent 2100 bioanalyzer and an Applied Biosystems StepOnePlus real-time quantitative PCR system. Paired-end cDNA libraries were constructed and sequenced at Novogene in China using the NovaSeq X Plus platform. Sequencing reads were aligned to the mouse reference genome (mm10) using HISAT software. Differentially expressed genes (DEGs) between genotypes were calculated using standard bioinformatics analysis procedures, and hierarchical clustering maps of differentially expressed genes among samples were generated based on the DEGs.

[0092] 4. Statistical Methods All data were analyzed using Prism 9 (GraphPad) and ImageJ software, and the Shapiro-Wilk test was used to test for normality. For two normally distributed datasets, an unpaired two-tailed t-test was used; for non-normally distributed datasets, the Mann-Whitney test was used; for datasets with more than two groups, one-way ANOVA was used, and p < 0.05 was considered statistically significant.

[0093] 5. Experimental Results 5.1 Dendrobine enhances autophagic flux in muscle tissue After a 10-week intervention, transcriptomic analysis of the muscles in both groups of mice showed that dendrobine could promote muscle proliferation and differentiation, and simultaneously increase the level of mitochondrial autophagy in aging muscles. Figure 9and Figure 10 Western blot results showed that, compared with the control group, the LC3B-II / LC3B-I ratio in muscle tissue of the dendrobine group was significantly increased (P<0.05), while the level of autophagy substrate p62 protein was significantly decreased (P<0.01). Figure 11 (A) The increase in the LC3B-II / I ratio and the decrease in p62 together indicate that dendrobine activates the full autophagic flux, rather than just the accumulation of autophagosomes.

[0094] 5.2 Dendrobine specifically activates mitophagy Phospho-Ubiquitin (Ser65) is a marker of PINK1 activation and phosphorylation of ubiquitin, a key signal for initiating mitophagy. Western blot results showed that the level of Phospho-Ubiquitin (Ser65) in muscle tissue of the dendrobine group was significantly upregulated compared with the control group (P<0.05). Figure 11 The B in the figure indicates that dendrobine can specifically promote the labeling and clearance of damaged mitochondria in aging skeletal muscle.

[0095] In summary, dendrobine activates ubiquitin phosphorylation at the Ser65 site, initiates the PINK1 / Parkin-mediated mitophagy pathway, enhances mitochondrial quality control in aging muscle tissue, clears dysfunctional mitochondria, and provides metabolic support for maintaining muscle stem cell function.

[0096] Example 4: Dendrobine reverses metabolic disorders in aging muscle stem cells and reduces oxidative stress 1. Experimental Materials 1.1 Test Drug Dendrobine, purity 99.70%.

[0097] 1.2 Reagents and Consumables DMEM / F10 medium, horse serum, fetal bovine serum, and penicillin antibodies were all purchased from Gibco Life Sciences; poly-D-lysine and extracellular matrix (ECM) were purchased from Sigma-Aldrich; the Seahorse XF glycolysis rate assay kit was purchased from Agilent Technologies; the reactive oxygen species detection kit (DCFH-DA) was purchased from Beyotime Biotechnology Co., Ltd.; and the CD34-AF700, CD45-PreCP-Cy5.5, CD31-PreCP-Cy5.5, and Sca-1-PE-Cy7 flow cytometry antibodies were all purchased from BIOLEGEND (Beijing) Biotechnology Co., Ltd.

[0098] 2. Experimental Methods 2.1 Isolation and sorting of primary muscle stem cells (MuSCs) Hind limb muscles (tibialis anterior, gastrocnemius, etc.) from 18-month-old senescent C57BL / 6J mice were collected, washed three times with PBS, and minced into a paste. Enzyme digestion buffer (collagenase II + dispersin) was added, and the mixture was incubated at 37°C with shaking for 60 min. Digestion was terminated by adding DMEM containing 10% FBS. The cells were then passed through 70μm and 40μm cell filters sequentially. The cells were centrifuged at 4°C, 400×g for 10 min, and treated with erythrocyte lysis buffer for 5 min. After resuspending the cells, flow cytometry antibodies (CD34-AF700, CD45-PreCP-Cy5.5, CD31-PreCP-Cy5.5, and Sca-1-PE-Cy7) were added, and staining was performed at 4°C for 30 min. After washing and resuspending, CD45 cells were separated using a BD FACS Aria flow cytometer. - CD31 - Sca1 - and CD34 + Cells, specifically muscle stem cells (MuSCs).

[0099] 2.2 In vitro culture and drug administration of MuSCs The isolated MuSCs were seeded into pre-coated culture plates and cultured using a coating method consisting of first applying poly-D-lysine (0.1 mg / mL) followed by treatment with extracellular matrix (ECM, 25 mg / mL). The growth medium (GM) used was Ham's F10 medium containing 10% horse serum and 1% penicillin-dextrose antibody. Cells were divided into two groups: a control group (DMSO) and a dendrobine group (10 μM dendrobine). Cells were cultured for 48 hours before analysis.

[0100] 2.3 Detection of glycolysis rate using Seahorse XF Add hydration solution to the Seahorse XFe96 assay plate 24 hours in advance and hydrate in a CO2-free incubator at 37°C. Prepare Seahorse XF assay solution (DMEM + 1mM pyruvate + 2mM glutamine + 10mM glucose, pH 7.4) on the day of assay. Cultured MuSCs are placed at 1.5 × 10⁶ cells per well. 4 Cells were seeded in Seahorse cell plates, with four wells at the apex serving as background controls. Glycolysis rates were detected using a Seahorse XFe96 analyzer, with drugs (glucose, oligomycin, 2-DG) added sequentially. Extracellular acidification rate (ECAR) was recorded in real-time, and glycolysis rate values ​​were calculated according to the kit instructions.

[0101] 2.4 Detection of intracellular ROS levels Collect MuSCs after 48 h of drug treatment, dilute the DCFH-DA probe with PBS at a ratio of 1:1000, suspend the cells in the probe solution, and incubate at 37 °C for 20 min (inverting and mixing every 3-5 min); wash 3 times with PBS, and detect fluorescence intensity (488 nm excitation / 525 nm emission) by flow cytometry.

[0102] 3. Statistical Methods Same as Example 1.

[0103] 4. Experimental Results 4.1 Dendrobine specifically upregulates the glycolysis rate of senescent MuSCs Glycolysis is a crucial energy metabolism mechanism for maintaining stemness in stem cells. Seahorse assays showed that after treatment with 10 μM dendrobine for 48 h, the glycolysis rate of MuSCs in the dendrobine group significantly increased, rising by approximately 50% compared to the control group (P < 0.01). Figure 12 The A in the figure indicates that dendrobine can reverse the decline in glycolytic capacity of MuSCs caused by aging.

[0104] 4.2 Dendrobine reduces oxidative stress levels in aging MuSCs Flow cytometry results showed that the intracellular ROS fluorescence intensity of MuSCs in the dendrobine group was significantly reduced by approximately 50% compared to the control group (P<0.01). Figure 12 The B in the figure indicates that dendrobine effectively alleviates the damage of age-related oxidative stress to MuSCs.

[0105] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The use of dendrobine in the preparation of drugs for the treatment of sarcopenia and / or chemically induced muscle damage.

2. The application according to claim 1, characterized in that, The dendrobine can enhance mitochondrial autophagy flux in muscle stem cells and upregulate glycolytic metabolic rate.

3. The application according to claim 2, characterized in that, The dendrobine can upregulate the expression level of phosphorylated ubiquitin at the Ser65 site in muscle stem cells.

4. The application according to claim 2, characterized in that, The dendrobine can increase the protein expression ratio of LC3B-II / LC3B-I and reduce the level of the autophagy substrate p62.

5. The application according to claim 1, characterized in that, Dendrobine can increase PAX7 + The number of muscle stem cells was increased by Ki67. + Proportion of proliferating cells.

6. The application according to claim 1, characterized in that, Dendrobine can increase the cross-sectional area of ​​muscle fibers, reduce post-injury fibrosis, and / or upregulate myoblast gene expression.

7. The application according to claim 1, characterized in that, The chemically induced muscle damage is barium chloride-induced muscle damage.

8. The application according to claim 1, characterized in that, The drug is available in oral or injectable form.

9. The application according to claim 8, characterized in that, The oral preparations include granules, capsules, tablets, or oral liquids.

10. The application according to any one of claims 1-9, characterized in that, The drug comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is dendrobine.

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