Use of a traditional Chinese medicine composition in the preparation of a medicament for treating sarcopenia

Abstract

This invention discloses the application of a traditional Chinese medicine composition in the preparation of a drug for treating sarcopenia, wherein the traditional Chinese medicine composition consists of Astragalus membranaceus and Anemarrhena asphodeloides. The study found that the combination of Astragalus membranaceus and Anemarrhena asphodeloides can significantly improve the differentiation function of C2C12 myoblasts induced by high glucose and inhibit lipid deposition, and improve skeletal muscle mass, muscle strength, and motor function in a streptozotocin-induced C57BL / 6 diabetic sarcopenia mouse model. In vitro and in vivo experiments have confirmed that this drug has significant effects in improving muscle atrophy, enhancing motor function, and restoring muscle strength; it can effectively increase the cross-sectional area of ​​muscle fibers, promote myotube fusion, and accelerate damage repair, showing a clear potential to inhibit diabetic muscle atrophy. This provides an effective traditional Chinese medicine compound intervention strategy for the clinical treatment of diabetic sarcopenia and has good prospects for development and application.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of a traditional Chinese medicine composition in the preparation of a drug for treating sarcopenia. Background Technology

[0002] Diabetic sarcopenia is a key pathological process of progressive skeletal muscle loss under chronic hyperglycemia, characterized by a simultaneous decline in muscle mass, strength, and physical function. This complication significantly increases the risk of falls, fractures, and metabolic deterioration. Epidemiological data show that patients with type 2 diabetes have a 2-3 times higher risk of developing sarcopenia compared to non-diabetic individuals. Diabetic sarcopenia significantly increases the risk of weakness, functional impairment, and adverse clinical outcomes, leading to a reduced 5-year survival rate and a median survival time shortened by 2-3 years. Although existing glycemic control strategies can partially manage blood sugar, their effectiveness in improving muscle atrophy is limited. Furthermore, these therapies are associated with dose-limiting toxicities, including gastrointestinal adverse reactions and renal impairment, which affect long-term treatment adherence and patient prognosis.

[0003] Skeletal muscle, as the primary site of glucose metabolism, experiences severe energy crisis under diabetic conditions, with mitochondrial dysfunction being a core characteristic. In a hyperglycemic environment, mitochondrial dynamic imbalance leads to mitochondrial division, producing dysfunctional units that lose their ability to oxidative phosphorylation. This structural damage not only reduces ATP production, resulting in insufficient muscle energy, but also triggers a burst of reactive oxygen species, further inducing cytotoxicity. Crucially, this mitochondrial dysfunction is closely related to lipid metabolism disorders. Impaired fatty acid oxidation reduces lipid clearance efficiency, leading to the abnormal accumulation of lipotoxic metabolites within myofibrils. These lipid substances, in turn, exacerbate mitochondrial damage, creating a vicious cycle that accelerates muscle atrophy. Notably, pharmacological inhibition of these dysregulated metabolic nodes has proven effective in regulating oxidative stress, thus positioning mitochondrial quality and fatty acid oxidation as promising therapeutic targets for preventing the progression of diabetic sarcopenia.

[0004] Traditional Chinese medicine (TCM) herbs are an important source for drug development. In traditional Chinese medicine, Astragalus membranaceus (Huangqi) is a basic herb used to tonify Qi, while Anemarrhena asphodeloides (Zhimu) is a classic Yin-nourishing herb; the two are often combined to invigorate Qi and nourish Yin. However, there are currently no reports of using this herb to treat sarcopenia. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a traditional Chinese medicine composition in the preparation of a drug for treating sarcopenia, thus providing a new and effective approach for the treatment of diabetic sarcopenia.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0007] The application of a traditional Chinese medicine composition in the preparation of a drug for treating sarcopenia, wherein the traditional Chinese medicine composition is composed of Astragalus membranaceus and Anemarrhena asphodeloides.

[0008] This invention reveals that the Astragalus and Anemarrhena asphodeloides herb pair can significantly improve the differentiation function of C2C12 myoblasts induced by high glucose and inhibit lipid deposition, as well as improve skeletal muscle mass, strength, and motor function in a streptozotocin-induced C57BL / 6 diabetic sarcopenia mouse model. Mechanistic studies show that this herb pair can reverse the vicious cycle of "energy crisis-lipotoxicity" in diabetic skeletal muscle by effectively restoring mitochondrial dynamics balance and promoting fatty acid β-oxidation. In vitro and in vivo experiments have confirmed that this herb pair can significantly improve protein and gene expression levels in diabetic sarcopenia, demonstrating significant effects in improving muscle atrophy, enhancing motor function, and restoring muscle strength. Therefore, the Astragalus-Anemarrhena asphodeloides herb pair provided by this invention can effectively increase the cross-sectional area of ​​muscle fibers, promote myotube fusion, and accelerate damage repair, possessing a clear potential to inhibit diabetic muscle atrophy. It provides an effective traditional Chinese medicine compound intervention strategy for the clinical treatment of diabetic sarcopenia, expands the modern scientific connotation of the "tonifying qi and nourishing yin" treatment principle, and has good prospects for development and application.

[0009] Preferably, the mass ratio of Astragalus membranaceus to Anemarrhena asphodeloides in this traditional Chinese medicine composition is 2:1.

[0010] Preferably, the sarcopenia is diabetic sarcopenia. More preferably, the diabetic sarcopenia is diabetic-associated sarcopenia of the Qi and Yin deficiency type.

[0011] Preferably, the diabetic sarcopenia is type 2 diabetic sarcopenia.

[0012] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0013] This invention reveals that the Astragalus and Anemarrhena asphodeloides herb pair can significantly improve the differentiation function of C2C12 myoblasts induced by high glucose and inhibit lipid deposition, as well as improve skeletal muscle mass, strength, and motor function in a streptozotocin-induced C57BL / 6 diabetic sarcopenia mouse model. Mechanistic studies show that this herb pair can reverse the vicious cycle of "energy crisis-lipotoxicity" in diabetic skeletal muscle by effectively restoring mitochondrial dynamics balance and promoting fatty acid β-oxidation. In vitro and in vivo experiments have confirmed that this herb pair can significantly improve protein and gene expression levels in diabetic sarcopenia. Therefore, the Astragalus-Anemarrhena asphodeloides herb pair provided by this invention has a clear potential to inhibit diabetic muscle atrophy, offering an effective traditional Chinese medicine compound intervention strategy for the clinical treatment of diabetic sarcopenia, expanding the modern scientific connotation of the "tonifying qi and nourishing yin" treatment principle, and has good prospects for development and application. Attached Figure Description

[0014] Figure 1The effects of different drug treatments on the results of skeletal muscle functional behavior (weighted swimming) tests in mice;

[0015] Where Swimming time (of control) represents swimming time (compared to the blank control group), STZ represents streptozotocin, AM represents astragalus, AA represents anemarrhena, and MET represents metformin, the same below;

[0016] Figure 2 The effects of different drug treatments on the results of skeletal muscle functional behavior (suspension test) in mice;

[0017] Wherein, Climbing pole (of control) represents the results of the pole climbing test (compared to the blank control group);

[0018] Figure 3 The effects of different drug treatments on the results of skeletal muscle functional behavior (grip strength test) in mice;

[0019] Here, Grip (of control) represents the grip test result (compared to the blank control group);

[0020] Figure 4 HE staining and myosin heavy chain immunohistochemical staining results of mouse skeletal muscle after different drug treatments;

[0021] Among them, MHC myosin heavy chain represents a scale bar of 200 μm;

[0022] Figure 5 A bar chart showing the quantitative analysis results of lipid deposition in mouse skeletal muscle after different drug treatments;

[0023] Wherein, Relative expression of CEBP (of control) represents the protein expression level of CEBP (relative to the control group), and the same applies below;

[0024] Figure 6 A bar chart showing the statistical results of cross-sectional area of ​​mouse muscle fibers after different drug treatments;

[0025] Among them, the average cross sentional area (μm) 2） This represents the average cross-sectional area of ​​a muscle fiber;

[0026] Figure 7 A bar chart showing the immunofluorescence intensity results of C2C12 myoblast differentiation status after different drug-containing serum treatments;

[0027] Wherein, Relative Fluorescence Intensity of MHC represents the MHC fluorescence intensity (relative to the control group); High glucose represents high glucose induction; the same applies below;

[0028] Figure 8 A bar chart showing the statistical results of C2C12 myoblast differentiation index after different drug-containing serum treatments;

[0029] The differentiation index (100%) represents the cell differentiation index, expressed as a percentage.

[0030] Figure 9 A bar chart showing the results of CT-8 staining analysis of lipid valve area in C2C12 cells after treatment with different drug-containing serums;

[0031] Figure 10 A bar chart showing the results of Oil Red O staining for lipid deposition in C2C12 myoblasts after treatment with serum containing different drugs;

[0032] Relative lipid drop content refers to the amount of lipid droplets in the cell (relative to the control group).

[0033] Figure 11 A bar chart showing the migration ability of C2C12 myoblasts after treatment with serum containing different drugs;

[0034] Wound healing (relative fold) refers to the ability of cells to migrate and proliferate in the scratched area (relative fold).

[0035] Figure 12 Bar chart showing Nile Red staining results of lipid deposition in C2C12 myoblasts after treatment with serum containing different drugs;

[0036] Here, Nile Red (of control) represents the Nile Red staining result (relative to the control group). Detailed Implementation

[0037] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0038] In the embodiments of this invention, all experimental procedures were repeated at least three times, and the data were statistically analyzed and plotted using GraphPad Prism 9.0 software. Difference analysis employed either the t-test (Student's T test) or one-way ANOVA, with all values ​​expressed as "mean ± SD".

[0039] Example 1: Effects of Astragalus and Anemarrhena on Improving STZ-Induced Diabetic Sarcopenia in Mice

[0040] 1. Animals and Experimental Protocol

[0041] Male C57 / BL6J mice aged 8-10 weeks (purchased from Hangzhou Medical College, Hangzhou, China), weighing 25-30g, were selected. Before the formal experiment, the mice were pre-acclimatized for 7 days under standard conditions: temperature 24±2℃, 12-hour light / dark cycle, and free access to standard food and water.

[0042] Sixty SPF-grade C57BL / 6 mice were randomly divided into the following six groups (n=10 per group):

[0043] Blank control group: fed with normal maintenance feed + gavage with an equal volume of physiological saline;

[0044] Model group: High-fat diet feeding + streptozotocin (STZ, intraperitoneal injection) induction;

[0045] AM single-drug group: After successful modeling, Astragalus extract was administered. The Astragalus extract was prepared as follows: Astragalus slices were added to 8 times (v / w) of boiling water, heated under reflux for 2 hours, and the extract was filtered and freeze-dried (-50℃, 48 h) and stored at 4℃ for later use. When administering the extract, a suspension was prepared with distilled water and administered by gavage once daily at a volume of 10 mL / kg for 8 consecutive weeks.

[0046] AA single-drug group: After successful model establishment, Anemarrhena asphodeloides extract was administered. The preparation method of Anemarrhena asphodeloides extract was as follows: Anemarrhena asphodeloides slices were taken, and 8 times the volume (v / w) of boiling water was added. The mixture was heated under reflux for 2 hours. The extract was filtered, freeze-dried (-50℃, 48 h), and stored at 4℃ for later use. When administering the extract, a suspension was prepared with distilled water and administered by gavage at a volume of 10 mL / kg once daily for 8 consecutive weeks.

[0047] AM-AA drug pair group: After successful modeling, Astragalus-Anemarrhena mixed extract (based on crude drug weight, it is a mixture of Astragalus extract and Anemarrhena extract in a mass ratio of 2:1) was administered by gavage once a day at a volume of 10 mL / kg for 8 consecutive weeks.

[0048] Positive drug group: After successful modeling, metformin was administered, prepared as a 1 mM solution with physiological saline, and administered by gavage once daily at a volume of 10 mL / kg for 8 consecutive weeks.

[0049] After successful model establishment, each treatment group received intervention at the corresponding dosage. The AM single-drug group, AA single-drug group, and AM-AA drug pair group received the same amount of raw drug (2 g / kg / d), while the blank group and model group received an equal volume of physiological saline. This treatment lasted for 8 weeks. Skeletal muscle function and histological examinations were performed at the end of the experimental period.

[0050] 2. Behavioral tests

[0051] (1) Weighted swimming test: A weight equivalent to 5% of the mouse's body weight was attached to a fixed position on the tail of the mouse. Each weighted mouse was placed in water at a temperature maintained at 25°C and swam until fatigue, and the swimming time was recorded.

[0052] (2) Suspension experiment:

[0053] (a) Grasping and placing

[0054] The experimenter put on gloves, gently picked up the mouse, and placed it in the center of a horizontal wire.

[0055] Guide the mouse to grasp the metal wire with both front paws. Once the mouse has a firm grip, slowly release its paws and start timing simultaneously.

[0056] (b) Observation and timing

[0057] End point of timing: Record the time from when the mouse starts to grasp the wire to when it reaches the end of the wire. Stop timing when the mouse climbs to the end of the wire.

[0058] (3) Grip strength test: The forelimb strength of mice was measured using a grip strength meter.

[0059] The results are as follows Figure 1 , Figure 2 and Figure 3 As shown.

[0060] from Figure 1 , Figure 2 and Figure 3 As can be seen, AM monotherapy, AA monotherapy, and AM-AA drug pair can all significantly improve muscle strength and enhance muscle function in streptozotocin-induced sarcopenia model mice, as manifested as prolonged weight-bearing swimming time, shortened hanging climbing time, and improved grip strength.

[0061] 2. Hematoxylin and eosin (HE) staining

[0062] Mouse gastrocnemius muscle tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5 μm sections. The sections were stained with hematoxylin and eosin (HE) using a kit (G1076, Xavier Biosciences). The tissue sections were observed under an APERIOVERSA 8 microscope (Leica, Germany). Results are as follows: Figure 4 As shown.

[0063] Figure 4HE staining results showed that the muscle tissue in the model group exhibited obvious disordered arrangement, uneven size, and widened gaps in muscle fibers. AM, AA, and AM-AA treatment groups all improved muscle fiber morphology and structure, with the AM-AA treatment group showing the most significant effect; the muscle fibers were neatly arranged with clear striations, and interstitial edema and inflammatory infiltration were significantly reduced.

[0064] 3. Immunohistochemistry

[0065] Mouse gastrocnemius muscle sections were immunostained with the following primary antibodies: anti-MHC and anti-CEBP, and imaged under a confocal laser scanning microscope (LSM800, Zeiss, Oberkochen, Germany). Results are as follows: Figure 4 and Figure 5 As shown.

[0066] from Figure 4 The results show that the MHC staining intensity in the muscle fibers of the diabetic model group was significantly reduced, indicating a large loss of contractile proteins. In contrast, the AM-AA drug pair group showed the most significant enhancement of MHC staining, suggesting that this drug pair can effectively maintain the structural integrity and contractile function of muscle fibers.

[0067] from Figure 5 The results showed that CEBP expression in the nuclei of myocytes was significantly upregulated in the diabetic model group, indicating abnormally active adipogenic differentiation. The AM-AA drug pair showed a strong inhibitory effect, suggesting that this drug pair can effectively reverse the process of ectopic lipid deposition in diabetic skeletal muscle.

[0068] 4. Measurement of cross-sectional area of ​​gastrocnemius muscle

[0069] Mouse gastrocnemius muscle tissue sections were stained with hematoxylin and eosin, and then observed and images were acquired under a microscope. At 200x magnification, five samples were randomly selected, with 20 muscle fibers from each sample. The cross-sectional area of ​​each muscle fiber was calculated using ImageJ software, and the average value was calculated to determine whether there were differences in the cross-sectional area of ​​muscle fibers under different treatments. Results are as follows: Figure 6 As shown.

[0070] from Figure 6 As can be seen, compared with the blank control group, the average cross-sectional area of ​​the gastrocnemius muscle of mice in the model group was significantly reduced. Compared with the model group, the cross-sectional area of ​​the gastrocnemius muscle of mice in the AM single drug group, AA single drug group and AM-AA drug pair group was significantly increased, and the AM-AA drug pair group showed better intervention effect.

[0071] Example 2: The effect of serum containing Astragalus membranaceus and Anemarrhena asphodeloides on improving high glucose-induced cell damage.

[0072] 1. Preparation of serum containing Astragalus and Anemarrhena asphodeloides

[0073] Ten male C57 / BL6J mice weighing 25-30g were randomly divided into a blank control group, an AM group, an AA group, and an AM-AA group, with five mice in each group. The AM, AA, and AM-AA groups (AM to AA in a mass ratio of 2:1) were administered the drug by gavage daily, while the blank control group was administered the same dose of physiological saline orally daily for seven consecutive days. Blood samples were collected two hours after the last administration. The blood was allowed to stand at room temperature for two hours, and the serum supernatant was separated by centrifugation. The serum was then inactivated at 56°C for 30 minutes, sterilized by passing it through a 0.22μm filter membrane, and stored at -80°C.

[0074] 2. Cell culture and grouping

[0075] C2C12 cells were purchased from the Type Culture Collection Committee of the Chinese Academy of Sciences and cultured in DMEM medium containing 2% FBS (11995065, Thermo Fisher Scientific) at 37°C and 5% CO2. To study the protective effects of AM and AA against high glucose-induced damage, the cells were grouped and treated for 24 hours as follows: (1) normal control group; (2) high glucose model group (25 mmol / L); (3) high glucose + 0.1% AM-containing serum group; (4) high glucose + 0.1% AA-containing serum group; (5) high glucose + 0.1% AM + AA combined serum group; (6) high glucose + MET group as a positive control; (7) blank serum control group, treated with 0.1% blank control serum to exclude the influence of serum components themselves. The drug-containing serum was obtained by gavage administration of the corresponding Chinese herbal extracts to mice. After 24 hours of treatment, all cells were collected for downstream analysis.

[0076] 3. Immunofluorescence staining

[0077] C2C12 cells were seeded into cell culture slides. After complete cell adhesion, the cells were washed with PBS, fixed in 4% paraformaldehyde at room temperature for 30 min, washed again, and permeabilized with 0.1% Triton X-100 at room temperature for 15 min. Then, 3% bovine serum albumin (BSA) was added for blocking for 1 h, followed by overnight incubation at 4°C with the following primary antibodies: MHC (BF8012, Jiangsu Qinke Biotechnology Research Center Co., Ltd.) and CT-8 (PB2553, Wuhan Sanying Biotechnology Co., Ltd.). Secondary antibody labeled with Alexa Fluor was added for 1 h. Cell nuclei were counterstained with DAPI (C1005, Shanghai Beyotime Biotechnology Co., Ltd.), mounted with neutral resin, and images were acquired using a Zeiss LSM510 META confocal microscope and analyzed using ZEN imaging software. Results are as follows: Figure 7 , Figure 8 and Figure 9 As shown.

[0078] Depend on Figure 7 and Figure 8 It was observed that after induction under high glucose conditions, the immunofluorescence intensity of MHC and the myotube fusion index in C2C12 cells were significantly reduced, confirming the inhibitory effect of diabetic conditions on myogenic differentiation. After intervention with AM, AA, or AM-AA drug pairs, MHC protein expression and myotube fusion in high glucose-activated myoblasts were significantly restored; among them, the intervention effect of the AM-AA drug pair was significantly higher than that of AM or AA alone, reaching a level comparable to or even higher than the positive control.

[0079] Depend on Figure 9 It was found that the AM-AA drug pair regulates intracellular lipid metabolism under high glucose conditions. Compared with the high glucose model group, the intracellular BODIPY493 / 503 fluorescence intensity was significantly reduced after intervention with AM, AA, or AM-AA. The intervention effect of the AM-AA drug pair was significantly higher than that of AM or AA alone, reaching a level comparable to the positive control; this confirms the ability of the AM-AA drug pair to reduce abnormal lipid droplet accumulation in myoblasts.

[0080] 4. Oil Red O staining

[0081] Oil Red O staining kit (C0157S, Shanghai Beyotime Biotechnology Co., Ltd.) was used to detect lipid deposition in C2C12 cells. C2C12 cells were fixed with 4% paraformaldehyde solution for 10 min. Staining buffer was added to the sample for 20 s, and then the sample was removed by aspiration. The sample was stained with Oil Red O working solution for 20 min. Staining buffer was added, and the sample was allowed to stand for 30 s. The sample was then washed with PBS and observed under a microscope. The results are shown below. Figure 10 As shown.

[0082] Figure 10 Oil Red O staining results showed that a large number of orange-red lipid droplets were aggregated in C2C12 myoblasts in the high-glucose model group, indicating severe lipid deposition. After intervention with AM, AA, or AM-AA drug pairs, the number and area of ​​intracellular lipid droplets were significantly reduced, with the intervention effect of AM-AA drug pair being significantly higher than that of AM or AA alone.

[0083] 5. Cell migration

[0084] C2C12 cells were seeded in 6-well plates, allowed to grow to confluence, and then scratched. Images were acquired using a microscope, and the width of each scratch was measured using ImageJ software. Results are as follows: Figure 11 As shown.

[0085] Figure 11The results of the cell scratch assay showed that high glucose conditions significantly inhibited the migration ability of C2C12 cells. Compared with the model group, treatment with AM-AA significantly accelerated the closure speed of the scratches, and its migration-promoting effect was better than that of AM or AA single-drug intervention.

[0086] 6. Quantitative analysis of neutral lipids

[0087] Cells were fixed and stained using a Nile Red reagent kit (C2051S, Shanghai Beyotime Biotechnology Co., Ltd.) to detect the accumulation of neutral lipids in the cytoplasm. Fluorescence intensity was measured using a Zeiss LSM510 META microscope with 543 nm excitation and 598 nm emission filter, and the fluorescence intensity was quantitatively analyzed. Results are as follows: Figure 12 As shown.

[0088] Depend on Figure 12 Nile red staining revealed that a high-glucose environment induced significant lipid accumulation in C2C12 cells, indicating that diabetes leads to lipotoxic accumulation in skeletal muscle and the progression of muscle atrophy. Intervention with AM-AA significantly reduced high-glucose-induced intracellular lipid deposition, with an intervention effect comparable to the positive control and superior to AM or AA monotherapy.

Claims

1. Use of a traditional Chinese medicine composition in the preparation of a medicament for treating sarcopenia, characterized in that, The aforementioned traditional Chinese medicine composition consists of Astragalus membranaceus and Anemarrhena asphodeloides.

2. The application as described in claim 1, characterized in that, The mass ratio of Astragalus membranaceus to Anemarrhena asphodeloides is 2:

1.

3. The application as described in claim 1, characterized in that, The sarcopenia mentioned is diabetic sarcopenia.

4. The application as described in claim 3, characterized in that, The diabetic sarcopenia described herein is a type of diabetes-associated sarcopenia with both Qi and Yin deficiency.

5. The application as described in claim 3, characterized in that, The diabetic sarcopenia mentioned refers to type 2 diabetic sarcopenia.

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