Application of Hirudo medicinalis in the preparation of drugs that promote skeletal muscle cell differentiation and / or prevent muscle atrophy

Abstract

This invention relates to the application of *Hirudo medicinalis* in the preparation of drugs that promote skeletal muscle cell differentiation and / or prevent muscle atrophy, belonging to the field of biomedical technology. This invention is the first to discover and experimentally demonstrate that freeze-dried *Hirudo medicinalis* powder has the activity of promoting myogenic differentiation and preventing muscle atrophy, opening up new applications for traditional Chinese medicine leeches in the prevention and treatment of muscle atrophy, and expanding the medicinal value of *Hirudo medicinalis*.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of Hirudo medicinalis in the preparation of drugs that promote skeletal muscle cell differentiation and / or prevent muscle atrophy. Background Technology

[0002] Chemotherapy-induced muscle atrophy is a common and serious adverse reaction in cancer patients undergoing chemotherapy, and has become a significant clinical problem affecting their quality of life and prognosis. Doxorubicin, an anthracycline antitumor drug, is a widely used chemotherapy agent with significant efficacy against various malignant tumors. However, while exerting its antitumor effects, it can cause significant toxic damage to skeletal muscle tissue. Studies have shown that doxorubicin can lead to skeletal muscle damage through multiple mechanisms, including inducing oxidative stress, mitochondrial dysfunction, and activation of protein degradation pathways. This results in decreased muscle mass, reduced muscle strength, decreased exercise endurance, and physical dysfunction, severely impacting patients' daily living abilities and treatment tolerance.

[0003] The pathogenesis of chemotherapy-related muscle atrophy involves multiple pathological processes, including an imbalance between muscle protein synthesis and degradation, impaired myogenic differentiation capacity, and damage to muscle fiber structure. At the molecular level, chemotherapy drugs can inhibit the expression of myogenic regulatory factors MyoD and Myogenin, downregulate the synthesis of muscle structural proteins such as myosin heavy chain MyHC, and activate protein degradation pathways, accelerating the breakdown of muscle protein, ultimately leading to myotube atrophy and decreased muscle function.

[0004] Currently, there are no specific drugs for the prevention and treatment of muscle atrophy caused by chemotherapy drugs in clinical practice. Existing interventions mainly rely on exercise rehabilitation training and nutritional support therapy, but these suffer from problems such as poor patient compliance, large individual differences, and limited effectiveness. Therefore, finding safe and effective natural drugs that can promote skeletal muscle cell differentiation, maintain muscle protein expression, and improve myotube morphology damage is of great clinical significance for the prevention and treatment of muscle atrophy caused by chemotherapy drugs.

[0005] Hirudo medicinalis is one of the medicinal leech species listed in the Pharmacopoeia of the People's Republic of China. Its traditional effects include breaking up blood stasis, promoting menstruation, and eliminating masses. Modern pharmacological studies have shown that leeches and their extracts possess multiple pharmacological activities, including anticoagulation, antithrombosis, anti-inflammation, improving microcirculation, and antioxidation. Hirudin has been developed into an injectable antithrombotic drug. However, current research on leech-based medicinal materials mainly focuses on cardiovascular and cerebrovascular diseases, and no literature or patents have reported that Hirudo medicinalis or its freeze-dried powder can promote skeletal muscle cell differentiation or improve chemotherapy-induced muscle atrophy. Therefore, developing new applications for freeze-dried Hirudo medicinalis powder in the prevention and treatment of chemotherapy-related muscle atrophy could provide new drug options for the prevention and treatment of muscle atrophy in chemotherapy patients. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of Hirudo medicinalis in the preparation of drugs that promote skeletal muscle cell differentiation and / or prevent muscle atrophy.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Application of Hirudo medicinalis in the preparation of drugs that promote skeletal muscle cell differentiation and / or prevent muscle atrophy.

[0008] Furthermore, the skeletal muscle cells are myoblasts.

[0009] Furthermore, the leech mentioned is lyophilized leech powder.

[0010] Furthermore, the in vitro cell experiments using lyophilized Hirudo medicinalis powder yielded a concentration of 0.05–50 μg / mL, the in vivo intraperitoneal administration dose to mice was 10 mg / kg, and the administration concentration to the Caenorhabditis elegans model was 0.05–0.1 mg / mL.

[0011] Furthermore, the muscle atrophy mentioned is skeletal muscle atrophy induced by chemotherapy drugs.

[0012] Furthermore, the chemotherapy drug mentioned is doxorubicin (Dox).

[0013] The present invention also provides a drug for promoting skeletal muscle cell differentiation and / or preventing muscle atrophy, the active ingredient of which includes lyophilized leech powder.

[0014] Furthermore, the drug dosage form is an injection, powder for injection, tablet, oral liquid, capsule, granule or powder.

[0015] This invention provides the application of *Hirudo medicinalis* in the preparation of drugs that promote skeletal muscle cell differentiation and / or prevent muscle atrophy, wherein the skeletal muscle cells are myoblasts. In vitro, this drug can upregulate the expression of myosin heavy chain (MyHC), myogenin, and myogenic determinant MyoD, increase myotube diameter, and promote myoblast differentiation into mature myotubes. In vivo, it can improve doxorubicin-induced skeletal muscle atrophy in mice and repair muscle fiber damage caused by natural aging nematodes. The chemotherapeutic drug is doxorubicin, which exerts a comprehensive anti-atrophy effect by maintaining the expression of myosin heavy chain (MyHC) and improving myotube and muscle fiber morphological damage.

[0016] This invention provides the application of lyophilized leech powder in the preparation of a drug for preventing and treating myotube atrophy induced by chemotherapy drugs. The chemotherapy drug is doxorubicin. This substance can maintain the expression of myosin heavy chain MyHC and improve myotube morphological damage at the cellular level. On the other hand, it can improve doxorubicin-induced skeletal muscle atrophy in mice, while repairing muscle fiber damage in naturally aging nematodes and improving their motor function. It exerts anti-muscle atrophy effects at multiple levels, including cellular, mammalian, and invertebrate levels.

[0017] The lyophilized leech powder was obtained from whole leeches via freeze-drying and was purchased from Yunnan Yuyao Biopharmaceutical Co., Ltd. The in vitro cell culture concentration of the lyophilized leech powder was 0.05–50 μg / mL, preferably 0.5–5 μg / mL; the intraperitoneal administration dose in mice was 10 mg / kg; and the final concentration for Caenorhabditis elegans administration was 0.05–0.1 mg / mL.

[0018] Compared with the prior art, the beneficial effects of this invention are as follows: This invention is the first to discover and confirm, through multiple experiments using cells and model animals, that freeze-dried leech powder possesses both the bioactivity of promoting myoblast differentiation and preventing chemotherapy-induced muscle atrophy. This opens up a new application direction for traditional medicinal leeches in the prevention and treatment of chemotherapy-related muscle atrophy, and expands the medicinal value and application scope of leeches.

[0019] The mechanism of action of this invention is clearly defined. In vitro, the lyophilized powder of Hirudo medicinalis can regulate the key pathway of MyoD-Myogenin-MyHC myoblast differentiation, promoting the differentiation of myoblasts into mature myotubes and increasing the diameter of myotubes, while maintaining normal expression of myosin heavy chain MyHC and repairing myotube morphological damage caused by doxorubicin. In vivo animal experiments show that this product can significantly improve the phenotype of doxorubicin-induced weight loss, limb muscle weakness, and skeletal muscle atrophy in mice. In a naturally aging Caenorhabditis elegans model, it can also effectively improve the decline in nematode motor function and repair damaged muscle fiber structure. This invention has validated its efficacy in multiple models including cells, mammals, and invertebrates, with a clear target and broad application prospects.

[0020] This invention has high safety. Hirudo medicinalis is a medicinal animal species included in the Chinese Pharmacopoeia, with a long history of medicinal use and a clear safety record. The freeze-dried powder preparation process is mature and controllable, and the effective components are fully preserved, making it easy to achieve large-scale production and industrial application.

[0021] This invention provides a new source of natural medicine and a solution for the prevention and treatment of muscle atrophy caused by chemotherapy drugs. Attached Figure Description

[0022] Figure 1 The graph shows the results of detecting the effect of lyophilized leech powder on the viability of C2C12 cells; where A is a bar chart of cytotoxicity detection after treatment with different concentrations of lyophilized leech powder for 48 hours; and B is a bar chart of cell proliferation activity detection after treatment with different concentrations of lyophilized leech powder. Figure 2The figures show the Western blot results of the effect of lyophilized leech powder on the expression of proteins related to C2C12 myoblast differentiation; where A is a diagram of the C2C12 differentiation pattern; B is a Western blot band diagram of MyHC, Myogenin, and MyoD protein expression; C is a bar chart of the relative expression levels of MyoD protein; D is a bar chart of the relative expression levels of Myogenin protein; and E is a bar chart of the relative expression levels of MyHC protein. Figure 3 The image shows the immunofluorescence results of the effect of lyophilized leech powder on the morphology of myotubes in C2C12 myoblasts; where A is the MyHC immunofluorescence staining image and B is the histogram of myotube diameter. Figure 4 The image shows the Western blot results of the effect of lyophilized leech powder on MyHC protein expression in a doxorubicin-induced myotube atrophy model. A represents the Dox-induced myotube atrophy model; B represents the Western blot bands of MyHC protein expression; and C represents the bar chart of relative MyHC protein expression levels. Figure 5 The image shows the immunofluorescence results of the effect of lyophilized leech powder on the morphology of myotube atrophy induced by doxorubicin; where A is the MyHC immunofluorescence staining image; and B is the histogram of myotube diameter. Figure 6 The following figures show the effects of lyophilized leech powder on Dox-induced muscle function and muscle mass in mice; where A is a schematic diagram of the mouse drug modeling test protocol; B is a bar chart of relative body weight of mice; C is a bar chart of grip strength test of mice; and D is a bar chart of wet weight of gastrocnemius muscle of mice. Figure 7 Figure 1 shows the effect of lyophilized leech powder on the cross-sectional area of ​​Dox-induced gastrocnemius muscle fibers in mice. In this figure, A is an immunofluorescence image of gastrocnemius muscle co-stained with Dystrophin and DAPI; B is a statistical bar chart of the average cross-sectional area of ​​muscle fibers in each group; and C is a statistical distribution chart of the frequency of cross-sectional area of ​​gastrocnemius muscle fibers. Figure 8 The results show the effects of freeze-dried Hirudo medicinalis powder on the motor function of naturally aging N2 wild-type nematodes. Among them, A is a schematic diagram of the time scheme for simultaneous culture and drug administration intervention of nematodes; B is a scatter plot of the number of body flexion times of nematodes in each group in 30 seconds; C is a scatter plot of the number of pharyngeal pump beats of nematodes in each group in 30 seconds. Figure 9 Figure 1 shows the effect of freeze-dried leech powder on the morphology of myofiber muscle fibers in the naturally aging RW1596 transgenic nematode strain. In the figure, A is a GFP fluorescence imaging image of a single muscle fiber; B is an overall fluorescence observation image of the nematode body wall muscle; and C is a quantitative statistical bar chart of myofiber fluorescence intensity. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the embodiments.

[0024] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be obtained by purchase.

[0025] This invention provides the application of *Hirudo medicinalis* in the preparation of drugs that promote skeletal muscle cell differentiation and / or prevent muscle atrophy, preferably using lyophilized *Hirudo medicinalis* powder. This invention comprehensively utilizes in vitro cell models, in vivo mouse models, and *C. elegans* models to verify efficacy, demonstrating that lyophilized *Hirudo medicinalis* powder can promote myoblast differentiation, regulate the balance between muscle synthesis and differentiation, and effectively alleviate chemotherapy-induced muscle atrophy at multiple levels, including cellular, mammalian, and invertebrate stages.

[0026] Preferably, the muscle atrophy is chemotherapy-induced muscle atrophy. In a specific embodiment of the present invention, doxorubicin is used to induce the construction of a C2C12 mature myotube atrophy model and a mouse skeletal muscle atrophy model, while a naturally aging Caenorhabditis elegans model is used for synergistic verification.

[0027] The efficacy of the product includes at least one of the following: upregulating the expression of myosin heavy chain MyHC, myogenic differentiation factor MyoD, and Myogenin; promoting the differentiation of myoblasts into mature myotubes, increasing the diameter of myotubes, maintaining the integrity of myotube morphology, and antagonizing myotube damage caused by chemotherapy drugs; improving the phenotype of weight loss, limb muscle weakness, and skeletal muscle atrophy induced by doxorubicin in mice; improving the decline in motor function in naturally aging Caenorhabditis elegans and repairing damaged muscle fiber structure.

[0028] The lyophilized leech powder was obtained from whole leeches via freeze-drying and was purchased from Yunnan Yuyao Biopharmaceutical Co., Ltd. The in vitro cell culture concentration of the lyophilized leech powder was 0.05–50 μg / mL, preferably 0.5–5 μg / mL; the in vivo intraperitoneal administration dose to mice was 10 mg / kg; and the administration concentration in a Caenorhabditis elegans model was 0.05–0.1 mg / mL.

[0029] 1. Experimental consumables and their sources The details are shown in Table 1.

[0030] Table 1

[0031] 2. Experimental reagent formula: (1) Lyophilized leech powder solution: Lyophilized leech powder is dissolved in dimethyl sulfoxide (DMSO), filtered with filter paper, and then filtered with a 0.22μm disposable syringe filter, and dispensed for use.

[0032] (2) 10×PBS buffer: Weigh 80g NaCl, 2g KCl, 36.3g Na2HPO4·12H2O and 2.4g KH2PO4, then use ddH2O to make up to 1L, autoclave and store at room temperature.

[0033] (3) 1×PBST buffer: Measure 1L of 1×PBS buffer, add 1mL of Tween-20, shake well and store at room temperature for later use.

[0034] (4) Membrane regeneration solution: 15g glycine + 1g SDS; dissolve in deionized water to 1L, adjust pH to 2.2 and then add 10mL Tween-20.

[0035] (5) 10× Electrophoresis buffer: Weigh 10g SDS, 144g glycine and 30.2g Tris-base, then adjust the volume to 1L with ddH2O and store at room temperature.

[0036] (6) 10× transfer buffer: Weigh 144g glycine and 30.3g Tris-base, then dissolve them in 800mL ddH2O, and then bring the volume up to 1L with ddH2O. Store at room temperature. When needed, dilute to 1× transfer buffer according to the ratio of 10× transfer buffer: ddH2O: anhydrous ethanol = 2:7:1.

[0037] (7) 30% Acrylamide / 0.8% Methylenebisacrylamide (30%AB): Weigh 145g acrylamide and 5g methylenebisacrylamide, then use ddH2O to make up to 500mL. After complete dissolution, filter with filter paper and avoid storing at room temperature.

[0038] (8) 4×Tris-HCl / SDS (pH=6.8): Weigh 30.25g Tris-base, then dissolve it in 300mL ddH2O, adjust the pH to 6.8 with concentrated hydrochloric acid, then make up to 500mL, add 0.4g SDS, and store at room temperature.

[0039] (9) 4×Tris-HCl / SDS (pH=8.8): Weigh 91g Tris-base, then dissolve it in 300mL ddH2O, adjust the pH to 8.8 with concentrated hydrochloric acid, then make up to 500mL, add 1g SDS, and store at room temperature.

[0040] (10) 10% ammonium persulfate (10% AP): Weigh 1g of ammonium persulfate, then dissolve it in 10mL of ddH2O, dispense it into 1.5mL centrifuge tubes, and store it at -20℃.

[0041] (11) 6× protein denaturation loading buffer: Weigh 0.93g dithiothreitol DTT, 1.2mg bromophenol blue, measure 3mL glycerol, 7mL 4×Tris-HCl / SDS, and finally weigh 1g SDS until fully dissolved. Aliquot into 1.5mL centrifuge tubes and store at -20℃ for later use.

[0042] (12) 5% (m / v) skim milk powder blocking solution: Weigh 2.5g of skim milk powder and dissolve it in 50mL of 1×PBST buffer. Note that it should be prepared and used immediately.

[0043] (13) 0.5% (m / v) BSA blocking solution: Weigh 0.25g BSA and dissolve it in 50mL 1×PBST buffer. Note that it should be prepared and used immediately.

[0044] (14) 5% (m / v) BSA blocking solution: Weigh 2.5g of BSA and dissolve it in 50mL of 1×PBST buffer. Note that it should be prepared and used immediately.

[0045] (15) Complete culture medium: Take 5 mL of fetal bovine serum into a centrifuge tube and bring the volume up to 50 mL with DMEM high glucose medium.

[0046] (16) Differentiation medium: Take 1 mL of horse serum into a centrifuge tube and bring the volume up to 50 mL with DMEM high glucose medium.

[0047] (17) MTS detection reagent: high sugar DMEM medium and MTS solution are prepared at a volume ratio of 5:1.

[0048] (18) Nematode synchronization lysis buffer: 750 μL 5mol / L NaOH + 2 mL ddH2O + 1 mL NaClO.

[0049] (19) NGM solid medium (NGM plate): Weigh 2g tryptone, 16g agar powder, 2.4g NaCl, and 0.8mL 5mg / mL cholesterol. Then, use ddH2O to make up to 800mL. After high temperature and high pressure sterilization, add 800μL 1mol / L CaCl2, 800μL 1mol / L MgSO4, and 20mL 1mol / L PK buffer to a clean bench. Shake well and pour the plate. After solidification, wrap it with plastic wrap and store it at 4℃ for later use.

[0050] (20) PK buffer: Weigh 59.5g KH2PO4 and 14.09g Na2HPO4·12H2O, then make up to 500mL with ddH2O, autoclave, and store at room temperature.

[0051] (21) M9 buffer: Weigh 3g KH2PO4, 15.12g Na2HPO4·12H2O, and 5g NaCl, then bring the volume to 1L with ddH2O, autoclave, and then add 1mL of 1mol / L MgSO4 in a clean bench and let it stand at room temperature for later use.

[0052] (22) 5mg / mL cholesterol: Weigh 5mg cholesterol and dissolve it in 1mL of anhydrous ethanol. Store at 4℃ for later use.

[0053] (23) LB liquid culture medium: Weigh 10g NaCl, 10g Tryptone and 5g yeast extract, then make up to 1L with ddH2O, sterilize at high temperature and high pressure and store at 4℃ for later use.

[0054] 3. Main experimental equipment The details are shown in Table 2.

[0055] Table 2

[0056] 4. Example Example 1: Establishment of C2C12 cell culture system and screening of safe concentration of lyophilized leech powder in vitro This embodiment details the culture and passage procedures for C2C12 cells and uses the MTS method to screen the safe intervention concentration of lyophilized leech powder, laying the foundation for subsequent experiments.

[0057] 1. Establishment of cell culture system 1.1 Cell resuscitation A. Remove the C2C12 cell cryopreservation tubes from the liquid nitrogen tank and immediately place them in a 37°C incubator to thaw completely within 1 minute, obtaining a cell suspension.

[0058] B. Sterilize the cryovials by thoroughly wiping the outer wall with 75% (v / v) ethanol. In a clean bench, transfer the entire cell suspension to a 15 mL centrifuge tube containing 5 mL of pre-warmed 37°C complete culture medium.

[0059] C. Centrifuge at 1000 rpm for 5 minutes and carefully discard the supernatant. Add 1 mL of complete culture medium to the cell pellet and gently resuspend by pipetting. Transfer the cell suspension to a 90 mm cell culture dish and add 9 mL of complete culture medium.

[0060] D. Gently shake the culture dish in a cross shape to distribute the cells evenly, and incubate in a 37°C, 5% CO2 incubator. After 24 hours, discard the old culture medium and replace it with 10 mL of fresh complete culture medium, and continue culturing.

[0061] 1.2 Cell passage A. Observe daily under an inverted microscope. When the cell density reaches 80%~90%, passage the cells.

[0062] B. Discard the old culture medium, add 3 mL of 1×PBS buffer preheated to 37°C, gently shake to wash the cell surface, and then discard.

[0063] C. Add 3 mL of trypsin-EDTA solution (0.25%:0.02%), gently shake to cover the entire bottom of the bottle, and place in a 37°C incubator for digestion for 3 minutes.

[0064] D. Under a microscope, when the intercellular spaces increase and the cell shape becomes rounded, immediately add 1 mL of complete culture medium to stop the digestion.

[0065] E. Gently and repeatedly pipette the bottom of the dish until all cells detach and a uniform cell suspension is formed.

[0066] F. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm and 25 °C for 5 minutes, discard the supernatant, add 3 mL of complete culture medium to resuspend the cells, and obtain the resuspended cell solution.

[0067] G. Add 9 mL of complete culture medium and 1 mL of cell resuspending solution to a 90 mm cell culture dish. Mix well by cross-shaking, then return to a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for further culture.

[0068] 2. Cell viability assay (MTS method) 2.1 Experimental Procedure A. After trypsin digestion of C2C12 cells in logarithmic growth phase, digestion was terminated with complete culture medium and the cells were resuspended. The cell density was adjusted to 1.0 × 10⁻⁶ cells using a hemocytometer. 4 cells / mL.

[0069] B. Administer the cell suspension at 100 μL per well (i.e., 1.0 × 10⁻⁶). 3 Cells / wells were precisely seeded into the central 60 wells of a 96-well cell culture plate, and 100 μL of 1×PBS (or cell-free medium) was added to the outer 36 wells to reduce edge effects.

[0070] C. Place the inoculated culture plate in a constant temperature incubator at 37℃, 5% CO2 and saturated humidity for 8 hours to allow the cells to adhere fully.

[0071] D. Carefully aspirate the original culture medium from each well. Add complete culture medium containing a series of concentrations of lyophilized leech powder (0.05, 0.5, 5, 50 μg / mL) to the experimental groups, with 6 technical replicate wells for each concentration. Add an equal volume of complete culture medium (containing 0 μg / mL lyophilized leech powder) to the blank control group.

[0072] E. Return the culture plates to a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 24, 48, 72, 96, and 120 hours, respectively. At each detection time point, add 100 μL of MTS cell proliferation assay reagent to each well.

[0073] F. Wrap the culture plate in aluminum foil to protect it from light and continue incubation at 37°C for 1.5 hours.

[0074] G. Use a multi-functional microplate reader to measure the absorbance of each well at a wavelength of 490 nm.

[0075] 2.2 Experimental Results The results are as follows Figure 1 As shown in A, the lyophilized leech powder showed no significant toxicity to C2C12 cells within the concentration range of 0.05–50 μg / mL: cell viability in all treatment groups was not lower than that in the blank control group, and cell viability in the 0.05–5 μg / mL concentration groups was significantly higher than that in the blank control group, while there was no statistically significant difference between the 50 μg / mL group and the blank control group. Figure 1 As shown in Figure B, the proliferation curve results indicate that the lyophilized leech powder had no inhibitory effect on cell proliferation within this concentration range, with the 0.5 μg / mL and 5 μg / mL concentrations showing the best proliferative effects. These two concentrations were selected as the intervention concentrations for subsequent experiments.

[0076] Example 2: Evaluation of the efficacy of lyophilized leech powder in promoting myogenic differentiation and myotube formation in C2C12 cells. This embodiment details the specific operations and results of verifying the effect of lyophilized leech powder on promoting myogenic differentiation of C2C12 cells using Western blotting and immunofluorescence techniques.

[0077] 1. Experimental Grouping Control group: cultured in normal differentiation medium, without drug intervention.

[0078] Experimental group: lyophilized leech powder solution was added to the differentiation medium to a final concentration of 0.5 μg / mL and 5 μg / mL, respectively.

[0079] 2. Induction of differentiation of mature myotubes A. Take C2C12 cells in the logarithmic growth phase with a cell density of 80%~90%, digest and resuspend them according to the cell passage method in section 1.2 of Example 1, and prepare a uniform single-cell suspension; take 200μL of the cell suspension and seed it into a 6-well cell culture plate, add 2mL of complete culture medium to each well, mix well, and then place it in a constant temperature incubator at 37℃, 5% CO2, and saturated humidity.

[0080] B. Place the culture plate in a 37°C, 5% CO2 incubator and observe the cell growth status under an inverted microscope every 24 hours.

[0081] C. When the cell density reaches 70%~80%, discard the original complete culture medium and gently wash the cells twice with 1×PBS pre-warmed to 37°C to completely remove serum residue.

[0082] D. Grouped administration: The experimental group was given differentiation medium, and lyophilized leech powder solution was added to a final concentration of 0.5 and 5 μg / mL, respectively; the control group used an equal volume of differentiation medium without lyophilized leech powder. The volume of medium per well was 2 mL.

[0083] E. Return the cells to the incubator and continue culturing, designating this day as Day 0 of differentiation. From this point onward, discard the old culture medium daily and replace it with fresh differentiation medium containing or without the appropriate concentration of lyophilized leech powder, continuing the induction process for 6 days. This will induce C2C12 cells to form mature myotubes.

[0084] F. After 6 days of differentiation, the culture medium was discarded and the sample was washed with 1×PBS.

[0085] 3. Western blot detection of myoblast-related protein expression 3.1 Sample Preparation After 6 days of differentiation, the culture medium was discarded and the cells were washed with 1×PBS. Then, 200 μL of 3× protein loading buffer was added directly to each well, and cells were scraped off with a cell scraper. The resulting cell lysate suspension was collected into a centrifuge tube and heated in a 100°C metal bath for 10 minutes to denature the proteins. The resulting denatured protein samples were stored at -80°C for Western blotting analysis.

[0086] 3.2 Glue preparation Select the appropriate concentration of SDS-PAGE separating gel based on the molecular weight of the target protein: use an 8% separating gel for MyHC detection and a 10% separating gel for MyoD and Myogenin detection. Prepare the corresponding concentration of separating gel solution according to the Immunoway SDS-PAGE Gel formula. Add 7 mL of separating gel solution to the gel casting plate, cover the liquid surface with 200 μL of isopropanol to isolate it from air, and let it stand at room temperature for 30 minutes until the separating gel is completely solidified. Discard the upper layer of isopropanol, prepare a 5% stacking gel solution according to the formula, add it to the gel casting plate and insert a 10-well sample comb, and let it stand at room temperature for 30 minutes until the stacking gel is completely solidified.

[0087] 3.3 SDS-PAGE electrophoresis and membrane transfer A. Remove the denatured protein sample prepared above from the -80℃ ultra-low temperature freezer, thaw it naturally at room temperature, and then briefly centrifuge it. Place the sample in a metal bath and heat it again at 100℃ for 5 minutes, then immediately transfer it to ice to cool, and after a brief centrifugation for 10 seconds, vortex it to mix it for later use.

[0088] B. Use a pipette to draw 10 μL of the sample after vortexing A and slowly add it to the loading well of the SDS-PAGE gel. Add 10 μL of macromolecular protein marker MF290 plus to the adjacent well as a reference.

[0089] C. In 1× electrophoresis buffer, start electrophoresis at a constant voltage of 80V. After the bromophenol blue indicator enters the separating gel, increase the voltage to 120V and continue electrophoresis until the bromophenol blue leading edge reaches the bottom edge of the gel, at which point the electrophoresis is terminated.

[0090] D. After electrophoresis, carefully remove the gel and equilibrate it with a PVDF membrane that has been pre-activated with anhydrous methanol for 30 seconds in 1× transfer buffer pre-cooled at 4°C for 15 minutes.

[0091] E. Use the wet transfer method, assembling the transfer clamp in the order of "cathode-filter paper-gel-PVDF membrane-filter paper-anode" to ensure no air bubbles between layers. Place the transfer device in an ice bath environment and transfer at a constant voltage of 100V for 60~120 minutes.

[0092] 3.4 Blocking and Antibody Incubation F. After the transfer is complete, remove the PVDF membrane and place it in 1×PBST blocking solution containing 5% (w / v) skim milk. Block it on a horizontal shaker at 60-80 rpm at room temperature for 2 hours.

[0093] G. After blocking, rinse the membrane three times with 1×PBST solution for 5 minutes each time. Cut the membrane into corresponding band regions according to the molecular weight of the target protein and incubate them in the corresponding primary antibody working solutions. Dilute the primary antibodies with 1×PBST solution at the following volume ratios: anti-MyHC antibody (1:100), anti-MyoD antibody (1:500), anti-Myogenin antibody (1:500), and anti-GAPDH antibody (1:20000). Incubate the membrane and corresponding primary antibody working solutions at 4°C on a shaker at 50 rpm overnight (12-14 hours).

[0094] H. The primary antibody was recovered the next day, and the membrane was washed three times with 1×PBST solution for 10 minutes each time. The membrane was then placed in HRP goat anti-mouse secondary antibody IgG and incubated on a shaker at room temperature for 1 hour at a volume ratio of 1:5000. (Secondary antibodies for MyHC, MyoD, Myogenin, and GAPDH were all mouse antibodies).

[0095] I. After the secondary antibody incubation, wash the membrane three times with 1×PBST solution for 10 minutes each time. Mix ECL chemiluminescence reagent A and B in a 1:1 (v / v) ratio, and then add the mixture evenly to the protein side of the PVDF membrane. Acquire the signal in the chemiluminescence imaging system.

[0096] J. The gray values ​​of the target protein band and the internal control GAPDH band were quantitatively analyzed using ImageJ image analysis software. The ratio of the gray values ​​of the target band to the internal control band was used as the relative expression level of the protein.

[0097] 3.5 Test Results The results are as follows Figure 2 As shown, compared with the control group, the protein expression levels of MyHC, MyoD, and Myogenin were significantly upregulated after intervention with lyophilized leech powder. This result confirms that lyophilized leech powder can effectively promote the myogenic differentiation process of C2C12 cells.

[0098] 4. MyHC immunofluorescence staining for myotube morphology 4.1 Staining Procedure After 6 days of differentiation of A.C2C12 myoblasts, the culture medium was discarded and the cells were washed with 1×PBS.

[0099] B. Cell fixation and permeabilization: Discard the culture medium and wash the cells three times with 1×PBS; add 4% (w / v) paraformaldehyde (PFA) and fix at room temperature for 15 minutes; discard the paraformaldehyde and wash three times with 1×PBS (5 minutes each time); add 0.02% (v / v) Triton X-100 in PBS solution and permeabilize at room temperature for 15 minutes, then wash with 1×PBS for 10 minutes.

[0100] C. Blocking and primary antibody incubation: Discard 1×PBS, add 0.5% (m / v) BSA in 1×PBS blocking solution and block at room temperature for 2 hours; discard the blocking solution, add MYH2 primary antibody working solution diluted with 1×PBS at a volume ratio of 1:300, and incubate overnight in a humidified chamber at 4°C.

[0101] D. Secondary antibody incubation and nuclear staining: Recover the primary antibody and wash three times with 1×PBST (5 minutes each time); add FITC-labeled secondary antibody working solution diluted with PBST at a volume ratio of 1:300, and incubate at room temperature in the dark for 2 hours; discard the secondary antibody and wash three times with 1×PBST (5 minutes each time); add DAPI staining solution diluted with 1×PBST at a volume ratio of 1:5000, and incubate at room temperature in the dark for 10 minutes.

[0102] E. Image acquisition: After washing three times with 1×PBS, add 500μL of 1×PBS to each well to cover the cells. Use a high-content imaging system to acquire MYH2 (green fluorescence) and DAPI (blue fluorescence) images under a 20x objective lens.

[0103] 4.2 Observation Results The results are as follows Figure 3 As shown, the myotube diameter in the lyophilized leech powder treatment group was significantly increased compared to the control group. Quantitative analysis indicated that the average myotube size increased in a concentration-dependent manner after administration of the lyophilized leech powder. This morphological evidence is consistent with the upregulation of MyHC protein expression detected by Western blot, jointly demonstrating that the lyophilized leech powder can effectively promote myogenic differentiation and myotube formation in C2C12 cells.

[0104] Example 3: Protective effect of lyophilized leech powder against doxorubicin (Dox)-induced atrophy and morphological damage of mature C2C12 myotubes. This embodiment details the protective effect of lyophilized leech powder against doxorubicin-induced mature myotube atrophy, which was evaluated using Western blotting and immunofluorescence techniques.

[0105] 1. Experimental Grouping Control group: Normal differentiation culture medium, no drug treatment.

[0106] Dox model group: 200nM Dox processing.

[0107] Dox + Hirudo medicinalis group: 200 nM Dox + 0.5, 5 μg / mL lyophilized Hirudo medicinalis powder solution.

[0108] 2. Induction of differentiation of mature myotubes A. Take C2C12 cells in the logarithmic growth phase with a cell density of 80%~90%, digest and resuspend them according to the cell passage method in section 1.2 of Example 1, and prepare a uniform single-cell suspension; inoculate 200μL of the cell suspension into a six-well cell culture plate, and add 2mL of complete culture medium to each well.

[0109] B. Place the culture plate in a 37°C, 5% CO2 incubator and observe the cell growth status under an inverted microscope every 24 hours.

[0110] C. When the cell density reaches 70%~80%, discard the original complete culture medium and gently wash the cells twice with 1×PBS pre-warmed at 37°C to completely remove serum residue.

[0111] D. Replace all three groups with high-glucose DMEM differentiation medium containing 2% (v / v) horse serum, with a medium volume of 2 mL per well.

[0112] E. Return the cells to the incubator and continue culturing, marking this day as Day 0 of differentiation. From this point onward, discard the old culture medium daily and replace it with fresh high-glucose DMEM differentiation medium containing 2% (v / v) horse serum. Continue to induce differentiation for 6 days, allowing C2C12 cells to form mature myotubes.

[0113] F. After 6 days of differentiation, the culture medium was discarded and the sample was washed with 1×PBS.

[0114] 3. Construction and drug administration of the myotube atrophy model G. After C2C12 myoblasts differentiated into mature myotubes after 6 days, the culture medium in the control group was replaced with fresh differentiation medium. The Dox model group was replaced with high-glucose DMEM differentiation medium containing 2% (v / v) horse serum at a concentration of 200 nM Dox. The Dox + Hirudo medicinalis group was replaced with high-glucose DMEM differentiation medium containing 2% (v / v) horse serum at a concentration of 200 nM Dox, and Hirudo medicinalis lyophilized powder solution was added to a final concentration of 0.5 μg / mL and 5 μg / mL. The culture medium volume per well was 2 mL, and induction continued for 2 days.

[0115] H. After induction, discard the differentiation medium and wash with 1×PBS.

[0116] I. If Western blotting analysis is to be performed subsequently, add 200 μL of 3× protein loading buffer directly to each well after step H, scrape the cells with a cell scraper, transfer the resulting suspension to a centrifuge tube, heat in a 100°C metal bath for 10 minutes to denature the protein, and store the resulting sample at -80°C.

[0117] J. If immunofluorescence staining is to be performed subsequently, the immunofluorescence staining step described in Example 2 should be performed directly after step H.

[0118] 4. Western blot detection of MyHC protein expression 4.1 Detection Method The Western blotting procedure described in Example 2 was followed. Anti-MyHC antibody and anti-GAPDH antibody were used as primary antibodies. The primary antibodies were diluted with 1×PBST solution at a volume ratio of: anti-MyHC antibody (1:100), anti-GAPDH antibody (1:20000). HRP goat anti-mouse secondary antibody IgG was used, diluted with 1×PBST at a volume ratio of 1:5000.

[0119] 4.2 Test Results like Figure 4 As shown, compared with the control group, the gray value of the MyHC protein band in the Dox model group decreased significantly, indicating that myotubular protein degradation and atrophy occurred; the expression of MyHC protein in the Dox + Hirudo medicinalis group was significantly higher than that in the Dox model group, indicating that the lyophilized Hirudo medicinalis powder can effectively maintain the expression of muscle-specific proteins.

[0120] 5. Immunofluorescence staining to detect myotube morphology 5.1 Staining Method The immunofluorescence staining procedure described in Example 2 was followed. The primary antibody was anti-MYH2 antibody, diluted 1×PBST solution at a volume ratio of 1:300. The secondary antibody was FITC-conjugated goat anti-mouse IgG, diluted 1×PBST solution at a volume ratio of 1:300, used to label the muscle tubes. DAPI was diluted 1×PBST solution at a volume ratio of 1:5000 for nuclear staining. Images were acquired using a high-content imaging system.

[0121] 5.2 Observation Results The results are as follows Figure 5 As shown: Control group: The muscle tubes were thick, continuous, neatly arranged, and intact in shape.

[0122] Dox model group: Myotubes became significantly thinner, with breakage and fragmentation, and the diameter of myotubes decreased significantly.

[0123] The Dox + Hirudo medicinalis group showed significant improvement in myotube atrophy, reduced rupture, and significantly better myotube diameter and integrity compared to the model group.

[0124] Example 4: Protective effect of lyophilized leech powder against doxorubicin (Dox)-induced muscle atrophy and morphological damage in mice. This embodiment details the specific procedures and results of verifying the improvement of muscle atrophy in mice by measuring muscle weight, detecting grip strength, and using immunofluorescence technology.

[0125] 1. Mouse experimental model The animal model used in this experiment was a 3-month-old wild-type (WT) mouse with a C57BL / 6J background. All mice were housed in individually ventilated cages, provided with ample food and water, and their bedding was changed weekly to maintain a suitable living environment. The temperature in the animal room was strictly controlled at 22±2℃, and the relative humidity was maintained at 50±10%, with a 12-hour light-dark cycle implemented daily (light period 08:00-20:00). The experimental protocol has been approved by the Experimental Animal Ethics Committee of Nanchang University.

[0126] 2. Pretreatment of mice and preparation of experimental drugs 2.1 Pretreatment of mice Twelve healthy male C57BL / 6J mice were selected, and after the auricles were disinfected with iodine, they were tagged and numbered. Their initial body weight (average weight of mice was 30g) was recorded. The mice were then randomly divided into three groups according to their weight, and the average body weight of each group was controlled to be not significantly different.

[0127] Mice were divided into groups and recorded daily on day 0 of the experiment. The drugs were administered intraperitoneally on days 3, 6, 9, 12, and 15, with a single injection volume of 100 μL for each group, for a total of 5 administrations. The administration regimen is as follows: Control group: Intraperitoneal injection of an equal volume of 0.9% (w / v) normal saline; Dox model group: 1.5 mg / mL Dox working solution was injected intraperitoneally, and the dosage was 5 mg / kg; Dox + Hirudo medicinalis group: Dox and lyophilized leech powder were administered intraperitoneally in combination, with Dox dosage of 5 mg / kg and lyophilized leech powder dosage of 10 mg / kg.

[0128] 2.2 Preparation of test drugs Preparation of lyophilized leech powder drug solution: Weigh 300 mg of lyophilized leech powder, dissolve it in 10% (v / v) DMSO and bring the volume to 10 mL to obtain a 30 mg / mL stock solution; use 0.9% (w / v) physiological saline as the diluent and dilute it 10 times according to the stock solution: physiological saline = 1:9 volume ratio to obtain a 3 mg / mL lyophilized leech powder drug solution; the average weight of mice is 30 g, the single injection of mice is 100 μL, the single dose is 0.3 mg, and the dosage is 10 mg / kg.

[0129] Preparation of Dox working solution: Weigh 150 mg Dox and dissolve it in 1 mL DMSO to prepare a 150 mg / mL stock solution; dilute with physiological saline at a volume ratio of 1:99 (stock solution to physiological saline), dilute 100 times to obtain a final concentration of 1.5 mg / mL Dox working solution. Inject 100 μL per ampoule, with a dose of 0.15 mg, corresponding to a dose of 5 mg / kg.

[0130] Dox + Hirudo medicinalis combination solution: Prepare using the two stock solutions mentioned above, with a total volume of 100 μL per administration vial. Prepare 500 μL of combination solution each time: Take 5 μL of Dox stock solution, 50 μL of Hirudo medicinalis stock solution, and 445 μL of 0.9% (w / v) physiological saline, mix well to obtain 500 μL of combination solution for later use.

[0131] 3. Mouse limb grip strength test Throughout the experimental period, mouse body weight was measured and recorded at fixed times daily. Grasping force testing was divided into two phases: adaptation training and formal testing. For three consecutive days (days 13, 14, and 15), mice were placed on the gripping device at fixed times each day to minimize the impact of stress on the results. Formal gripping force testing was conducted on day 16, avoiding drug administration. During testing, the mouse's tail was gently held, guiding its forepaws to firmly grasp the metal sensor grid of the gripping device. Once the grip was stable, the mouse was pulled horizontally and at a constant speed until its paws completely detached from the grid. The device automatically recorded the peak gripping force. Five consecutive tests were performed on each mouse, with appropriate intervals (e.g., 30 minutes) between tests to allow for sufficient rest. After removing the maximum and minimum values ​​from each set, the arithmetic mean of the remaining values ​​was taken as the mouse's final gripping force.

[0132] 4. Sampling of mouse skeletal muscle and preparation of frozen sections 4.1 Sampling of mouse skeletal muscle samples After all indicators were tested, samples were taken at low temperatures, and the entire process was carried out on an ice surface covered with plastic wrap (4°C). Mice were anesthetized, weighed, and euthanized. The gastrocnemius, soleus, and tibialis anterior muscles were separated and weighed separately. OCT embedding medium was pre-filled into the embedding cassette and pre-cooled at 4°C for 2 hours to remove bubbles. The left skeletal muscle of the mouse was immersed in the pre-cooled embedding medium and refrigerated at 4°C to remove bubbles.

[0133] 4.2 Preparation of frozen sections of mouse skeletal muscle After placing the left skeletal muscle in a foil-wrapped embedding mold, add OCT embedding agent to completely immerse the tissue, refrigerate at 4°C and thoroughly remove air bubbles, then adjust the muscle tissue to be arranged longitudinally; use liquid nitrogen for quick freezing to form the muscle, avoiding liquid nitrogen from entering the mold or contaminating the embedding agent during the quick freezing process to prevent the tissue from freezing and cracking, and after freezing, transfer it to a -80°C freezer for long-term storage.

[0134] Pre-treatment of sections: The embedded blocks were placed in a -20℃ cryostat for 1 hour to equilibrate. The temperature of the microtome was set to -18℃ to -22℃, and sections were cut to a thickness of 7μm. The sections were then attached to adhesive glass slides. After all sections were cut, the glass slide samples were stored in a -80℃ freezer in the dark.

[0135] 4.3 Results of mouse body weight, limb grip strength, and skeletal muscle wet weight tests After continuous drug administration and intervention, changes in body weight, limb grip strength, and skeletal muscle wet weight were measured in mice in each group. The results are as follows: Figure 6 As shown.

[0136] Compared with the control group, mice in the Dox model group showed a significant decrease in body weight, a marked reduction in limb grip strength, and a significant decrease in gastrocnemius muscle wet weight, indicating that doxorubicin can effectively induce the pathological phenotypes of skeletal muscle atrophy, muscle weakness, and emaciation in mice. Compared with the Dox model group, the Dox + Hirudo medicinalis group showed a significant reduction in body weight, a marked recovery in limb muscle strength, and an effective increase in gastrocnemius muscle wet weight, suggesting that lyophilized Hirudo medicinalis powder can effectively improve the symptoms of doxorubicin-induced skeletal muscle atrophy, muscle weakness, and body weight loss in mice.

[0137] 5. Immunofluorescence staining of frozen sections of skeletal muscle 5.1 Immunofluorescence staining procedure A. Rewarming: Remove the slices from the -80℃ freezer and air dry at room temperature for 30 minutes.

[0138] B. Fixation: Fix the sections in pre-cooled (-20℃) acetone for 10 minutes, then rinse 3 times with 1×PBS.

[0139] C. Hydrophobic zone delineation: When the slide surface is slightly dry, but the sample is not completely dry, if necessary, use lens paper to wipe the non-sample blank areas dry. Use an immunohistochemical pen to draw a circle around the tissue specimen to form a hydrophobic barrier and prevent the subsequent reaction solution from being lost.

[0140] D. Blocking and primary antibody incubation: In a humidified chamber, add 5% (v / v) BSA blocking solution at room temperature and block for 1 hour. Add Dystrophin primary antibody working solution diluted with 2.5% (v / v) BSA at a volume ratio of 1:500 and incubate overnight in a humidified chamber at 4°C.

[0141] E. Rinsing and secondary antibody incubation: On the second day, remove the slides and rinse them three times with 1×PBS for 5 minutes each time. Then add Cy3-labeled goat anti-rabbit IgG secondary antibody working solution diluted 1×PBST at a volume ratio of 1:200 and incubate at room temperature in the dark for 1 hour.

[0142] F. DAPI staining: Rinse three times with 1×PBS for 5 minutes each time, then incubate with DAPI staining solution diluted 1×PBST at a volume ratio of 1:5000 for 10 minutes at room temperature in the dark.

[0143] G. Mounting: Wash the slides three times with 1×PBS for 5 minutes each time, and let them air dry naturally; take 10 μL of fluorescent mounting medium that has been preheated in a 60℃ water bath and add it next to the tissue, then slowly cover it with a coverslip and remove any air bubbles to complete the mounting.

[0144] H. Image Acquisition and Analysis: After acquiring sample images using the Olympus VS200 digital slide scanning system or fluorescence microscope, quantitative analysis was performed based on the Halo AI digital pathology analysis system.

[0145] 5.2 Results of Detection of Muscle Fiber Morphology and Cross-sectional Area in Mouse Skeletal Muscle Dystrophin immunofluorescence staining of the gastrocnemius muscle was completed, and the morphology and cross-sectional area of ​​the muscle fibers were quantitatively analyzed. The results are as follows: Figure 7 As shown.

[0146] Compared with the control group, the gastrocnemius muscle fibers of mice in the Dox model group were thinner, with a significantly reduced average cross-sectional area and a significantly increased proportion of small-diameter muscle fibers, indicating obvious muscle fiber atrophy. Compared with the Dox model group, the cross-sectional area of ​​muscle fibers in mice in the Dox + Hirudo medicinalis group was significantly restored, the proportion of microfibrils decreased, and the integrity of muscle fiber morphology was significantly improved, indicating that the lyophilized Hirudo medicinalis powder can effectively alleviate Dox-induced muscle fiber atrophy in mice.

[0147] Example 5: Protective effect of freeze-dried leech powder on improving myofibrous atrophy in naturally aging Caenorhabditis elegans. 1. Culture and preparation of OP50 Escherichia coli bacterial suspension Select a single colony of purified Escherichia coli OP50 and inoculate it into a 15 mL sterile centrifuge tube containing 10 mL of LB liquid medium. Incubate at 37°C with shaking at 220 r / min for 8–12 h. Once the LB liquid medium is visibly turbid and the bacteria have reached the logarithmic growth phase, the seed culture is obtained.

[0148] Subsequently, at a volume ratio of 1:30, 1 mL of the above seed culture was transferred to a 50 mL sterile centrifuge tube containing 30 mL of fresh LB liquid medium. Under the same conditions of 37°C and 220 r / min, the culture was shaken vigorously for 5–8 h. The resulting Escherichia coli OP50 culture was stored at 4°C. During storage, 30 mL of the culture was placed in one 50 mL sterile centrifuge tube, and 3–5 tubes were prepared each time. These were then used for NGM plate spreading to feed Caenorhabditis elegans.

[0149] 2. Nematode culture A. Under aseptic conditions, spread 100 μL of Escherichia coli OP50 bacterial suspension evenly in the center of a 60 mm NGM plate to form an inoculation area with a diameter of about 2 cm.

[0150] B. After smearing the bacteria, the NGM plates are air-dried in a clean bench for 15 minutes, and then transferred to a 37°C constant temperature incubator for inverted incubation for 12-16 hours.

[0151] C. Place the NGM plates that have completed bacterial culture in a 20℃ constant temperature incubator for 2 hours to equilibrate, and obtain pretreated NGM plates for later use.

[0152] D. Use platinum wire to pick up healthy nematodes in their growth period and transfer them to the edge of the pretreated NGM plate OP50 mycelial growth.

[0153] E. Place the culture plate inoculated with nematodes in a 20°C constant temperature incubator and culture it in the dark. Replace the plate with fresh pretreated NGM every 2-3 days until the nematodes enter the oviposition period.

[0154] 3. Nematode synchronization treatment: A. Use M9 buffer to rinse oviposition adults from the surface of NGM plates into 15 mL sterile centrifuge tubes.

[0155] B. Centrifuge the centrifuge tube at 20°C and 1000 rpm for 1 minute, carefully discard the supernatant, and retain the nematode precipitate.

[0156] C. Repeat the washing steps (i.e., steps A to B) three times to ensure that the bacteria residue attached to the nematode's body surface is completely removed.

[0157] D. Add 1 mL of freshly prepared nematode synchronization lysis buffer to the precipitate and immediately place it on a vortex shaker and shake vigorously for 3 minutes.

[0158] E. After confirming under a stereomicroscope that most of the adult worm body walls have lysed and the eggs have been fully released, add 10 mL of LM9 buffer to terminate the lysis reaction.

[0159] F. Centrifuge the centrifuge tube at 3500 rpm for 1.5 minutes, discard the supernatant, and retain the egg precipitate at the bottom.

[0160] G. Resuspend the precipitate in 5 mL of M9 buffer, centrifuge and discard the supernatant, and repeat the washing process three times.

[0161] H. Finally, resuspend the eggs in 2 mL of M9 buffer and place the centrifuge tube in a 20°C incubator for 16–20 hours of static incubation.

[0162] I. After hatching, the hatching status of L1 stage larvae was confirmed under a stereomicroscope, and synchronized larvae were collected for subsequent experiments.

[0163] 4. Drug treatment A. The nematodes were cultured in a standardized manner at a constant temperature of 20℃ throughout their entire life cycle.

[0164] The first day of synchronization culture was calculated from the end of hatching in step H of the above 3. nematode synchronization treatment. Then, Caenorhabditis elegans (young adult stage) that had been synchronized to day 5 was taken and transferred to a fresh NGM plate containing 25 μg / mL 5-fluoro-2'-deoxyuridine using platinum wire (FUDR was added to inhibit nematode oviposition and exclude interference from newborn larvae in the experimental results). The first motor function test was performed under a stereomicroscope, and the frequency of body bending and the number of pharyngeal pump movements were recorded as data for the young control group.

[0165] B. After the nematodes entered the natural senescence stage on day 10, they were randomly divided into a blank senescence control group and a lyophilized leech powder administration group. The lyophilized leech powder solution was prepared by serially diluting Escherichia coli OP50 bacterial solution to obtain three final concentrations of 0 mg / mL (blank senescence control group), 0.05 mg / mL (lyophilized leech powder administration group), and 0.1 mg / mL (lyophilized leech powder administration group). 30 nematodes were placed on each NGM plate, and three biological parallels were set up for each concentration. The drug intervention was carried out continuously at 20°C in the dark for 48 hours. C. After 48 hours of continuous drug intervention, the motility function of nematodes in each group was measured on the 12th day of culture.

[0166] 5. Motor function testing 5.1 Motor Function Testing A. Transfer the nematodes to fresh NGM plates and wash away any residual bacteria on their bodies using M9 buffer; B. Body sway measurement: M9 buffer solution was added to the measuring slide, and nematodes (N2 wild-type nematodes were used in this experiment) were introduced. The number of complete body flexions of the nematode within 30 seconds was counted under a stereomicroscope. The criterion for judging body flexion was that the nematode completed one complete sinusoidal reciprocating sway along its long axis.

[0167] C. Measurement of pharyngeal pump frequency: The same nematode was transferred to the edge of a pretreated NGM plate OP50 bacterial motility layer. The pharynx was focused under a stereomicroscope, and the number of pharyngeal pump contractions within 30 seconds was observed and recorded. Pharyngeal pump contraction was defined as one complete relaxation-contraction cycle of the pharynx.

[0168] 5.2 Test Results like Figure 8 As shown, compared with the young control group, the number of body wiggles in the aging blank control group was significantly reduced in 30 seconds. After intervention with lyophilized leech powder, the body wiggle ability of the nematodes was significantly restored, and the improvement effect gradually increased with the increase of drug concentration. In addition, the frequency of pharyngeal pump contraction in the aging blank control group was significantly lower than that in the young group; intervention with lyophilized leech powder can significantly reverse the decline of pharyngeal pump function caused by aging and improve the pharyngeal pump movement level of aged nematodes.

[0169] 6. Observation of muscle fiber structure 6.1 Morphology and quantitative fluorescence detection of nematode myofibrils A. Treat the RW1596 transgenic line (myo-3p::GFP) according to methods 1-4 above. Collect nematodes on day 5 (young adult stage) and day 12 (senescent stage), respectively, wash with M9 buffer, and anesthetize with 10 mM imidazole solution; B. The anesthetized nematodes were transferred to a glass slide containing a 2% (m / v) agarose pad, and the GFP fluorescence signal images of their body wall muscles were observed and acquired using a Zeiss upright fluorescence microscope (LSM800). C. Use ImageJ software to perform quantitative fluorescence intensity analysis on the acquired images, measure the average fluorescence intensity of the myofibril region, assess the myosin expression level, and observe the continuity and morphological structure of the myofibril arrangement.

[0170] 6.2 Test Results The results are as follows Figure 9 Fluorescence imaging showed that, compared with the young control group, the aging blank control group had disordered muscle fiber arrangement, fiber breakage and damage, and significantly reduced muscle fiber fluorescence intensity. The intervention of lyophilized leech powder could improve the age-induced muscle fiber structural damage, enhance the overall fluorescence expression level of muscle fibers, repair the integrity of muscle fibers, and showed a certain concentration-dependent trend.

[0171] The combined results of three experimental groups—C2C12 cells, mice, and naturally aging nematodes—confirmed that lyophilized leech powder can antagonize doxorubicin-induced myotube atrophy and maintain muscle-specific protein expression in vitro; in vivo, it can improve the reduction in body weight, muscle weakness, and skeletal muscle fiber atrophy in doxorubicin-induced mice, while also improving the reduced motor function and repairing damaged muscle fiber structure in naturally aging nematodes. Multiple models have confirmed that lyophilized leech powder has a clear protective effect against muscle atrophy induced by the chemotherapy drug doxorubicin.

[0172] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Application of Hirudo medicinalis in the preparation of drugs for the prevention and treatment of muscle atrophy.

2. The application according to claim 1, characterized in that, The leech mentioned is freeze-dried leech powder.

3. The application according to claim 2, characterized in that, The in vitro cell experiments using lyophilized Hirudo medicinalis powder were conducted at concentrations of 0.05–50 μg / mL, the in vivo intraperitoneal administration dose to mice was 10 mg / kg, and the administration concentration to the Caenorhabditis elegans model was 0.05–0.1 mg / mL.

4. The application according to claim 1, characterized in that, The muscle atrophy mentioned is skeletal muscle atrophy induced by chemotherapy drugs.

5. The application according to claim 4, characterized in that, The chemotherapy drug mentioned is doxorubicin.

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