Use of timosaponin A-III in preparation of a drug for treating sarcopenia

Abstract

This invention discloses the application of Anemarrhena saponin A-III in the preparation of drugs for treating sarcopenia. This invention is the first to discover that Anemarrhena saponin A-III can significantly promote the growth of C2C12 myocytes into myotubes, improve autophagy function, and reduce lipid deposition; it can strengthen muscle function, alleviate the morphology of the gastrocnemius muscle in sarcopenia model mice, and increase muscle fiber area, thereby effectively combating sarcopenia. This provides an effective approach for the treatment of sarcopenia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Anemarrhena saponin A-III in the preparation of drugs for the treatment of sarcopenia. Background Technology

[0002] Sarcopenia is a systemic skeletal muscle failure disorder characterized by a continuous loss of skeletal muscle mass, strength, and function, increasing the risk of adverse outcomes such as weakness, falls, disability, and even death, resulting in a heavy disease burden. Currently, there are no officially approved drugs for the treatment of sarcopenia worldwide. Interventions for sarcopenia mainly include nutritional support (N-acetylcysteine ​​+ glycine) and resistance training. However, the use of drugs such as N-acetylcysteine ​​+ glycine lacks specificity, has an overly broad mechanism of action, and low bioavailability.

[0003] Recent studies have revealed that lipid metabolism disorders play a significant role in the pathogenesis of sarcopenia: in aging or pathological states, significant ectopic lipid deposition in skeletal muscle cells can damage the normal physiological structure and function of muscles. Lipophagy, a form of selective autophagy, is a crucial process in which cells specifically recognize and transport lipid droplets to lysosomes for degradation, and is essential for maintaining intracellular lipid homeostasis and energy balance.

[0004] Anemarrhena asphodeloides has the effects of clearing heat and purging fire, nourishing yin and moistening dryness. Existing pharmacological studies have shown that the main active ingredient in Anemarrhena asphodeloides, anemarrhenasaponin A-III, has anti-inflammatory, antioxidant, immunomodulatory, and autophagy-regulating pharmacological activities. However, there are currently no reports, either domestically or internationally, on the specific applications of anemarrhenasaponin A-III in improving skeletal muscle atrophy and treating sarcopenia. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Anemarrhena saponin A-III in the preparation of sarcopenia treatment drugs, thereby providing an effective approach for the treatment of sarcopenia.

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

[0007] Application of Anemarrhena saponin A-III in the preparation of drugs for the treatment of sarcopenia.

[0008] This invention is the first to discover that Anemarrhena saponin A-III can significantly promote the growth of C2C12 myocytes into myotubes, improve autophagy function, and reduce lipid deposition; it can strengthen muscle function, alleviate the morphology of the gastrocnemius muscle in sarcopenia model mice, and increase the area of ​​muscle fibers, thereby effectively combating sarcopenia.

[0009] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0010] This invention is the first to discover that Anemarrhena saponin A-III can significantly promote the growth of C2C12 myocytes into myotubes, improve autophagy function, and reduce lipid deposition; it can also strengthen muscle function, alleviate the morphology of the gastrocnemius muscle in sarcopenia model mice, and increase muscle fiber area, thereby effectively combating sarcopenia. This provides an effective approach for the treatment of sarcopenia. Attached Figure Description

[0011] Figure 1 The results of laser confocal microscopy observation of C2C12 myoblasts after immunofluorescence staining under different drug treatments;

[0012] Wherein, MHC represents myosin heavy chain, Dex represents dexamethasone, TA-III represents anemarrhena saponin A-III, NAC represents N-acetylcysteine, GLY represents glycine, and the same applies below;

[0013] Figure 2 Figure showing the effect of anemarrhena saponin A-III on improving dexamethasone-induced myotube atrophy at C2C12.

[0014] Wherein, Con represents the blank control, and the same applies below;

[0015] Figure 3 The results of inverted microscopy observation of C2C12 myoblasts after Oil Red O staining under different drug treatments;

[0016] Oil Red O represents Oil Red O staining;

[0017] Figure 4 The results of the analysis on the regulatory effect of Anemarrhena saponin A-III on dexamethasone-induced C2C12 autophagy;

[0018] Wherein, Oil Red O Area (%) represents the percentage of the Oil Red O stained area;

[0019] Figure 5 The results of laser confocal microscopy observation of autophagosome expression in C2C12 myoblasts under different drug treatments;

[0020] Figure 6 The results of the analysis on the regulatory effect of Anemarrhena saponin A-III on dexamethasone-induced C2C12 lipid deposition;

[0021] Among them, GFP-LC3 Fluorescent represents the fluorescence intensity of the GFP-LC3 protein;

[0022] Figure 7 Bar chart showing the effect of Anemarrhena saponins A-III on weight-bearing swimming in dexamethasone-induced sarcopenia model mice;

[0023] Among them, weight-loaded swimming (min) represents the duration (in minutes) of swimming with added weight.

[0024] Figure 8 Bar chart showing the effect of Anemarrhena saponins A-III on grip strength in dexamethasone-induced sarcopenia model mice;

[0025] Wherein, Grip strength (g) represents grip strength (grams);

[0026] Figure 9 Bar chart showing the effect of Anemarrhena saponins A-III on suspension in dexamethasone-induced sarcopenia model mice;

[0027] Wherein, Wire hang(s) represents the suspension duration (in seconds);

[0028] Figure 10 The results of myoskeletal ultrasound examination in mice under different drug treatments;

[0029] B-scan refers to B-mode basic ultrasound imaging;

[0030] Figure 11 The effect of Anemarrhena saponin A-III on the gastrocnemius muscle area and weight in dexamethasone-induced sarcopenia model mice;

[0031] Wherein, Maximum Area / Weight represents the maximum area / weight;

[0032] Figure 12 The results of H&E staining of myotube sections from mice under different drug treatments;

[0033] Figure 13 For H&E statistical analysis charts;

[0034] Where % of myotubes represents the percentage of muscle fibers; Myotubes Area represents the area of ​​muscle fibers.

[0035] Figure 14 This is a statistical analysis graph of muscle fibers;

[0036] CSA represents the cross-sectional area of ​​the muscle fiber. 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] Example 1

[0039] 1. Dexamethasone-induced in vitro model of sarcopenia

[0040] C2C12 myoblasts were placed in DMEM medium containing antibiotics and 10% fetal bovine serum and cultured routinely in an incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested, grouped as follows, and treated with the appropriate drugs for 24 hours before subsequent experiments.

[0041] ① Blank group: The same volume of diluent as the drug-treated group;

[0042] ② Model group: 1μM Dextramethasone;

[0043] ③ 0.01μM TA-III (Anemarrhena saponin) group: 1μM Dex + 0.01μM TA-III;

[0044] ④0.1μM TA-III group: 1μM Dex+0.1μM TA-III;

[0045] ⑤1μM TA-III group: 1μM Dex+1μM TA-III,

[0046] ⑥ Positive drug group: 1μM NAC (N-acetylcysteine) + 1μM GLY (glycine).

[0047] 2. Immunofluorescence analysis

[0048] Cells from each group were seeded into culture plates containing coverslips. The slides were fixed with 4% paraformaldehyde for 15 min and washed three times with PBS. Cells were permeated with 0.1% Triton X-100 at room temperature for 10 min and washed three times with PBS. 5% BSA was added to the slides, and the plates were blocked at room temperature for 1 h. The blocking solution was discarded, and diluted primary antibody (MHC) was added to each slide, which was incubated overnight at 4°C. The slides were washed three times with TBST. Diluted goat anti-mouse IgG (Alexa Fluor® 488) secondary antibody was added, and the slides were incubated at room temperature for 1 h and washed three times with TBST. For nucleus staining, DAPI was added, and the slides were incubated at room temperature in the dark for 10 min and washed three times with PBS. After mounting and detection, slides were mounted with an anti-fluorescence quencher, and images were observed and analyzed under a laser confocal microscope. Results are as follows: Figure 1 and Figure 2 As shown.

[0049] from Figure 1 and Figure 2As can be seen, compared with the blank control group, the expression level of MHC (myosin heavy chain) in the model group cells was significantly reduced, while after TA-III intervention, the MHC expression level of the cells was significantly increased. Among them, the MHC levels in the 0.1 μM TA-III group and the 1 μM TA-III group were better than those in the positive drug group. This indicates that TA-III has a significant ameliorative effect on dexamethasone-induced C2C12-induced myotube atrophy and promotes myotube growth.

[0050] 3. Oil Red O staining analysis

[0051] Cells from each group were seeded into culture plates with coverslips and gently rinsed 1-2 times with PBS. 4% paraformaldehyde was added, and the cells were fixed at room temperature for 30 minutes. The fixative was discarded, and the cells were gently washed twice with distilled water or PBS. 60% isopropanol was added to infiltrate the cells, and the cells were incubated at room temperature for 30 seconds to 1 minute. Freshly prepared Oil Red O working solution was added, just enough to cover the bottom of the wells, and the cells were incubated at room temperature in the dark for 30 minutes. Immediately, 60% isopropanol was added to initiate differentiation, and the cells were gently agitated for a few seconds. The cells were then gently washed 2-3 times with distilled water or PBS to remove residual isopropanol, and images were taken using an inverted microscope. The fluorescence images were analyzed, and the results are as follows: Figure 3 and Figure 4 As shown.

[0052] from Figure 3 and Figure 4 As can be seen, compared with the blank control group, the proportion of Oil Red O region in the model group cells was significantly increased, while it decreased to varying degrees after TA-III administration. The proportion of Oil Red O region in the 1 μM TA-III group was better than that in the positive control group. This indicates that TA-III regulates the autophagy induced by dexamethasone in C2C12 cells, helping to improve the autophagy function of cells.

[0053] 4. Fluorescent probe detection method

[0054] To assess autophagosome expression, C2C12 myoblasts were stimulated with the drug and then co-incubated with GFP-LC3B for labeling. The results were observed under a laser confocal microscope, and the images were analyzed. The results are as follows: Figure 5 and Figure 6 As shown.

[0055] from Figure 5 and Figure 6As can be seen, compared with the blank control group, the fluorescence intensity of GFP-LC3B protein in the model group cells was significantly reduced, indicating increased intracellular lipid deposition; while after TA-III administration, the fluorescence intensity of GFP-LC3B protein in the cells was significantly increased, comparable to that in the positive drug group. This indicates that TA-III regulates the abnormal lipid deposition induced by dexamethasone in C2C12 cells, thereby reducing lipid deposition.

[0056] Example 2

[0057] 1. Dexamethasone-induced establishment of a mouse model of sarcopenia

[0058] The sarcopenia model was induced by continuous intraperitoneal injection of dexamethasone (25 mg / kg / d) in C57BL / 6J mice as an in vivo research model. The experimental group was simultaneously treated with TA-III (1, 10 mg / kg / d) or NAC+ GLY by gavage for a total of 14 days.

[0059] 2. Behavioral tests

[0060] (1) Grip strength test: The forelimb strength of mice was measured using a grip strength meter.

[0061] (2) Weighted swimming test: A weight equivalent to 5% of the mouse's body weight was attached to a fixed position on the mouse's tail. Each weighted mouse was placed in water at a temperature maintained at 25°C and swam until fatigue, and the swimming time was recorded.

[0062] (3) Suspension experiment:

[0063] (a) Grasping and placing

[0064] The experimenter put on gloves, gently picked up the mouse, and placed it in the center of a horizontal wire.

[0065] 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.

[0066] (b) Observation and timing

[0067] 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.

[0068] The results are as follows Figure 7 , Figure 8 and Figure 9 As shown.

[0069] from Figure 7 , Figure 8 and Figure 9As can be seen, TA-III can significantly improve muscle strength and enhance muscle function in dexamethasone-induced sarcopenia model mice, as evidenced by increased grip strength, prolonged weight-bearing swimming time, and shortened hanging climbing time.

[0070] 3. Musculoskeletal ultrasound examination

[0071] After anesthesia, the mice's hind limbs were extended naturally. Basic B-mode ultrasound imaging was performed using a 20 MHz linear array transducer. The transducer was placed vertically on the surface of each mouse's hind limb to minimize muscle pressure and obtain relatively accurate elasticity data. The length and thickness of the gastrocnemius muscle were measured under B-mode basic ultrasound imaging. Results are as follows: Figure 10 and Figure 11 As shown.

[0072] from Figure 10 and Figure 11 As can be seen, compared with the blank control group, the gastrocnemius muscle area and weight of the model group mice were significantly reduced, while TA-III intervention could significantly alleviate the gastrocnemius muscle morphology and increase the muscle fiber area in sarcopenia model mice, thus having an anti-sarcopenia effect.

[0073] 4. H&E (hematoxylin-eosin) staining

[0074] First, the fixed gastrocnemius muscle paraffin sections were sequentially immersed in xylene I and II for 10-15 minutes each to dewax them. Then, they were gradually rehydrated by treating with a gradient of alcohols (100%, 95%, 80%, 70% ethanol) for 5 minutes each, until a water-washed state was achieved. Next, the sections were stained with hematoxylin for 3-8 minutes to stain the cell nuclei. After rinsing with tap water, they were quickly immersed in 1% hydrochloric acid alcohol for 2-5 seconds to remove background staining, followed by a 10-15 minute rinse with tap water to re-blue. Then, the sections were stained with eosin for 30 seconds to 2 minutes to stain the cytoplasm, and quickly washed away any remaining staining with tap water. Finally, the stained sections were dehydrated by a gradient of alcohols (70% to 100%), cleared with xylene, covered with neutral resin, and sealed with a coverslip for observation under a microscope. Results are as follows: Figure 12 , Figure 13 and Figure 14 As shown.

[0075] From H&E results ( Figure 12 , Figure 13 and Figure 14 As shown in the figure, Dex leads to a decrease in the size of mouse muscle fibers and a reduction in the proportion of muscle fiber area, while TA-III can thicken muscle fibers and increase the area of ​​muscle fibers.

Claims

1. Application of Anemarrhena saponin A-III in the preparation of drugs for the treatment of sarcopenia.

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