Application of forsythin in preparation of medicine for preventing or treating muscular atrophy

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a novel application of forsythin in preparation of a medicine for preventing and treating amyotrophy. In-vivo and in-vitro experiments prove that the phillyrin can remarkably improve the level of endogenous prostaglandin E2 by inhibiting the activity or expression of 15-hydroxyprostaglandin dehydrogenase in muscular tissues, so that the expression of muscular atrophy key factors FOXO3a and Atrogin-1 is inhibited, and finally the effects of protecting muscle quality and improving muscle functions are achieved. The invention reveals that forsythin has the functions of inhibiting 15-PGDH and relieving muscle atrophy for the first time, and brand new medical application is provided for forsythin. The invention provides a brand-new therapeutic scheme and pharmaceutical composition for treating sarcopenia by using forsythin as an active component and regulating the 15-PGDH / PGE2 pathway. The invention provides an important candidate compound for developing efficient and safe sarcopenia resisting medicines, and has important practical significance.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of forsythoside in the preparation of drugs for the prevention or treatment of muscle atrophy. Background Technology

[0002] Muscle atrophy, particularly sarcopenia, is a degenerative skeletal muscle disease closely related to aging, chronic diseases (such as diabetes and heart failure), and long-term use of medications (such as glucocorticoids). It is characterized by a progressive decline in skeletal muscle mass, strength, and function. Sarcopenia not only severely impacts patients' quality of life and independence but also increases the risk of falls, fractures, and all-cause mortality, becoming an increasingly serious global public health problem. Currently, clinical interventions for muscle atrophy are very limited and have many limitations. Non-pharmacological treatments, such as resistance exercise, have some effect, but are difficult for elderly, frail patients or those with other complications to adhere to and implement. In terms of pharmacological treatment, direct supplementation with anabolic hormones (such as testosterone and growth hormone) can increase muscle mass to some extent, but long-term use carries the risk of serious side effects such as cardiovascular disease and prostate cancer, limiting its widespread clinical application. Therefore, developing novel drugs that can safely and effectively promote muscle protein synthesis and inhibit its degradation is a key issue that urgently needs to be addressed in clinical practice. In recent years, the intrinsic regulatory mechanisms of skeletal muscle have become a research hotspot.

[0003] Prostaglandin E2 (PGE2) has been identified as a key signaling molecule for maintaining skeletal muscle homeostasis. It activates muscle satellite cells, promotes muscle regeneration, and inhibits the protein degradation pathway of the ubiquitin-proteasome system. However, PGE2 is rapidly inactivated in vivo by catalytic degradation by 15-PGDH. Therefore, 15-PGDH, as a key enzyme negatively regulating PGE2 levels, has been considered a promising new target for the treatment of muscle atrophy in recent years. Theoretically, inhibiting 15-PGDH activity could increase endogenous PGE2 levels, thereby mimicking its beneficial effects in promoting muscle regeneration. Currently, although the potential of 15-PGDH as a therapeutic target has been preliminarily recognized, research on finding highly effective, low-toxicity, especially naturally derived, 15-PGDH inhibitors and applying them to the prevention or treatment of muscle atrophy remains lacking.

[0004] To date, there are no reports on the effectiveness of forsythoside in treating muscle atrophy, and whether forsythoside can be used in the preparation of drugs for muscle atrophy remains to be studied. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that, although inhibiting 15-PGDH has been considered a potential strategy for treating muscle atrophy, there is currently a lack of highly effective and safe 15-PGDH inhibitors, especially inhibitors derived from natural products, in order to achieve this treatment strategy.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is: to provide the use of forsythoside in the preparation of drugs for the prevention or treatment of muscle atrophy.

[0007] In the above-mentioned uses, muscle atrophy is characterized by a decrease in skeletal muscle mass or a decline in function.

[0008] In the above-mentioned uses, the muscle atrophy is at least one of sarcopenia, disuse atrophy, or glucocorticoid-induced muscle atrophy.

[0009] Furthermore, the muscle atrophy includes a decrease in muscle mass, a decline in muscle function and exercise endurance, a decrease in muscle fiber area, and a conversion of fast-twitch muscle fibers into slow-twitch muscle fibers.

[0010] In the aforementioned uses, forsythoside works by inhibiting the activity or expression of 15-PGDH.

[0011] Among the aforementioned uses, forsythoside works by increasing the level of endogenous prostaglandin E2.

[0012] The present invention also provides a medicament for the prevention or treatment of muscle atrophy, the medicament comprising a therapeutically effective amount of forsythoside.

[0013] Among the aforementioned drugs, muscle atrophy refers to a type of disease characterized by a decrease in skeletal muscle mass or a decline in muscle function.

[0014] Among the aforementioned drugs, muscle atrophy is characterized by a decrease in skeletal muscle mass or a decline in function.

[0015] Among the aforementioned drugs, forsythoside works by inhibiting the activity or expression of 15-PGDH.

[0016] Among the aforementioned drugs, forsythoside works by increasing the level of endogenous prostaglandin E2.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention demonstrates through in vivo animal experiments that forsythoside significantly improves dexamethasone-induced muscle atrophy and functional decline in mice, increasing muscle weight and muscle fiber cross-sectional area. At the molecular mechanism level, this invention reveals for the first time that forsythoside effectively inhibits the protein expression of 15-PGDH in muscle tissue, significantly increases the level of endogenous PGE2, and subsequently downregulates the expression of key muscle atrophy markers FOXO3a and Atrogin-1. These findings indicate that forsythoside has outstanding effects in treating muscle atrophy by targeting and inhibiting 15-PGDH and regulating the PGE2 pathway, providing a clear candidate compound and theoretical basis for the development of novel anti-sarcopenia drugs. Attached Figure Description

[0019] Figure 1 The following figures show the representative muscle wet weights of mice in each group after 14 days of intervention in Example 1: (A) Comparative images of representative muscle samples from the four groups of mice; (BE) Bar charts comparing the representative muscle wet weights of the four groups of mice. Compared with the NC control group, Dex treatment significantly reduced the wet weight / body weight ratios of the gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus muscles in mice, by 25%, 29%, 22%, and 30%, respectively (***p < 0.001, n=3). Treatment with forsythoside (50mg / mL / d) significantly restored the wet weight / body weight ratios of the gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus muscles in mice, with recovery levels approaching 90% of the normal group (**p < 0.001, n=3).

[0020] Figure 2 The following is a comparison of the dwell time of mice in each group during the behavioral experiments in Example 2: (A) The results of the single-bar suspension test showed that, compared with the NC control group, the hanging time of mice treated with Dex was significantly reduced (***p < 0.001, n=3). Forsythoside treatment significantly prolonged the hanging time of mice (**p < 0.01, n=3), restoring it to the normal level; (B) The results of the rotarod fatigue test showed that, compared with the NC control group, the dwell time of mice treated with Dex was drastically reduced (***p < 0.001, n=3), while the dwell time of mice treated with forsythoside was significantly increased (p < 0.01, n=3).

[0021] Figure 3The images shown are HE staining images of gastrocnemius muscle fibers in each group of mice in Example 3: (A) HE staining images of gastrocnemius muscle fibers in each group of mice (scale bar: 50 μm); (B) Bar chart showing the comparison of cross-sectional area (CSA) of gastrocnemius muscle fibers in different treatment groups; compared with the NC control group, the gastrocnemius muscle fibers of mice treated with DEX were significantly thinner and the gaps between them were larger, and the CSA was significantly reduced (****p < 0.0001, n=3); the treatment with forsythoside caused the CSA of muscle fibers to recover (**p < 0.01, n=3), close to the level of the normal group.

[0022] Figure 4 The following are immunofluorescence images from Example 4: (A) Immunofluorescence staining images of gastrocnemius muscle in each group of mice; (BD) Bar charts showing the quantitative analysis of the ratio of fast to slow muscle fibers in each group of mice. There was no significant difference in the total number of muscle fibers among the groups. The normal group was dominated by fast muscle fibers. Compared with the NC control group, the proportion of fast muscle fibers did not change significantly after Dex treatment. Treatment with forsythoside significantly decreased the proportion of fast muscle fibers and correspondingly significantly increased the proportion of slow muscle fibers (***p < 0.001, n=3), suggesting that forsythoside can significantly promote the conversion of fast muscle fibers to oxidized slow muscle fibers.

[0023] Figure 5 The following are immunohistochemical images from Example 5: (A) Expression images of HPGD (15-PGDH), PTGER4, and PTGER2 proteins in mice of each group (scale bar: 50 μm); compared with the NC control group, the positive area rates of 15-PGDH (HPGD) and EP2 (PTGER2) increased in the Dex-treated group (***P < 0.001, n=3); conversely, the positive area rate of EP4 (PTGER4) decreased (***P < 0.001, n=3). After intervention with forsythoside, the positive area rates of 15-PGDH and EP2 were significantly reduced to normal levels (**P < 0.01, n=3).

[0024] Figure 6The following table shows the expression levels of FOXO3A, Atrogin-1, p-mTOR, mTOR, and PGC-1α detected by Western blotting in Example 6: (A) Representative Western Blot bands of FOXO3A, Atrogin-1, p-mTOR, mTOR, PGC-1α, and the internal control GAPDH. (BG) is a bar chart showing the quantitative statistical analysis of the relative expression levels of the aforementioned proteins and the p-mTOR / mTOR ratio. Regarding protein degradation, compared with the normal NC group, Dex treatment significantly reduced the p-FOXO3a / FOXO3a ratio and upregulated Atrogin-1, suggesting that overactivation of the FOXO3a–Atrogin-1 axis is a key factor in Dex-induced enhanced muscle protein degradation. Forsythoside treatment significantly increased the p-FOXO3a / FOXO3a ratio and downregulated Atrogin-1 expression (***p < 0.001, n=3). Detailed Implementation

[0025] This invention constructs a dexamethasone-induced mouse model of muscle atrophy and functional decline. Mice are then treated with forsythoside (50 mg / kg of forsythoside solution injected intraperitoneally daily) for 14 days. After intervention, the mice are sacrificed and the overall phenotype, tissue morphology, fiber type, and molecular signaling pathways of the muscles in each treatment group are observed.

[0026] This invention, through the aforementioned experiments, reveals that forsythoside can reverse dexamethasone-induced muscle mass loss in mice. Forsythoside can also restore dexamethasone-induced muscle function and exercise endurance in mice. Furthermore, forsythoside can alleviate dexamethasone-induced reduction in muscle fiber area in mice. It can also induce the conversion of fast-twitch muscle fibers to slow-twitch muscle fiber types in mice, a result consistent with the aforementioned improvement in exercise endurance, providing evidence at the muscle fiber type level for its functional improvement. Mechanistic experiments revealed that the core mechanism of action of forsythoside is through inhibiting 15-PGDH, restoring the PGE2-EP4 signaling axis, thereby activating PGC-1α to improve mitochondrial function, and inhibiting FOXO3a-Atrogin1-mediated protein degradation.

[0027] This invention reveals for the first time that forsythoside can be used as a drug to prevent or treat muscle atrophy, providing a new drug option and treatment approach for the prevention or treatment of muscle atrophy, and also providing a new use for forsythoside, which has great application prospects.

[0028] The specific implementation of the present invention will be explained and described below through examples, but this does not mean that the scope of protection of the present invention is limited to the scope described in the examples.

[0029] The C57BL / 6 mice used in the examples were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and all other reagents or materials were commercially available products.

[0030] Example 1: Comparison of muscle mass loss in mice under different treatment groups

[0031] After one week of acclimatization, the mice were randomly divided into four groups (n=8 / group) according to their body weight, with an intervention period of 14 days.

[0032] Normal control group: Daily intraperitoneal injection of sterile saline (10 mL / kg) and oral gavage of 0.1% ethanol solution (50 μL / animal).

[0033] Dexamethasone model group (DEX): Daily intraperitoneal injection of dexamethasone solution (5 mg / kg) and oral gavage of 0.1% ethanol solution (50 μL / animal);

[0034] Phlyrin intervention group: Daily intraperitoneal injection of dexamethasone solution (5 mg / kg) and intraperitoneal injection of forsythoside solution (50 mg / kg);

[0035] Rapamycin positive control group (LP): Daily intraperitoneal injection of dexamethasone solution (5 mg / kg) and intraperitoneal injection of rapamycin solution (1 mg / kg).

[0036] During the intervention period, the mice’s food intake, water intake and mental state were recorded daily, and they were weighed every 3 days to adjust the dosage volume to ensure accurate dosing.

[0037] 1. Collection of representative muscle tissue samples from mice

[0038] (1) After the last administration, the mice were fasted for 12 hours and allowed free access to water to eliminate the interference of food residue on body weight and metabolic indicators.

[0039] (2) After weighing the mouse, the cervical spine dislocation method was used to euthanize the mouse: Place the mouse on a flat operating table, fix the back of its head with the thumb and index finger of the left hand; pinch the base of the tail with the right hand and pull it backward quickly, while pushing forward with the left hand until the cervical spine is felt to dislocate; after confirming that breathing and heartbeat have stopped, dissect it immediately.

[0040] (3) Positioning and skin incision: Fix the mouse in a supine position on the dissecting board; disinfect the skin of the hind legs with 75% ethanol; cut the skin along the midline of the lower limbs, gently peel off the subcutaneous fascia, and fully expose the hind limb muscles.

[0041] (4) Gastrocnemius muscle (GAS) separation: Lift the Achilles tendon with forceps and cut the connective tissue along the edge of the gastrocnemius muscle; separate upwards to the femoral condyle attachment point and completely dissect the muscle; immediately place it in pre-cooled PBS to rinse and remove blood stains.

[0042] (5) Soleus muscle (SOL) separation: Gently turn the gastrocnemius muscle to the outside to expose the soleus muscle underneath; cut the tendons at both ends longitudinally along the muscle belly, carefully dissect to avoid damaging blood vessels; remove completely and place in PBS.

[0043] (6) Tibialis anterior (TA) separation: cut the anterolateral fascia of the lower leg; separate the muscle along the anterior border of the tibia and cut its insertion point at the base of the metatarsal bone; dissect upward to the proximal attachment point of the tibia.

[0044] (7) Separation of extensor digitorum longus (EDL): Gently separate the tibialis anterior muscle to reveal the lateral EDL; separate along the muscle belly, taking care to protect the distal bifurcation tendon; remove completely and place in PBS.

[0045] (8) Weighing and recording of tissues: Place the muscle on filter paper and gently blot dry the surface liquid; place the muscle piece by piece and record the wet weight (unit: mg); weigh each muscle piece 3 times and take the average value as the final wet weight value.

[0046] Experimental results are as follows Figure 1 As shown, the results indicated that compared with the NC control group, Dex treatment significantly reduced the wet weight / body weight ratio of the gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus muscles in mice, by 25%, 29%, 22%, and 30%, respectively (***p < 0.001, n=3). Treatment with forsythoside (50 mg / mL / d) significantly restored the wet weight / body weight ratio of the gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus muscles in mice, with the recovery level approaching 90% of the normal group (**p < 0.001, n=3).

[0047] This demonstrates that forsythoside can reverse dexamethasone-induced muscle mass loss in mice.

[0048] Example 2: Changes in muscle function and exercise endurance in sarcopenic mice

[0049] 2. Behavioral experiments

[0050] (1) To test the gripping force of the mouse's forelimbs, we conducted a single-bar suspension experiment. The single bar specifications were: 2 mm in diameter, 30 cm in length, and 30 cm above the ground.

[0051] (2) To reduce stress response, adaptive training was conducted before the experiment (for 2 consecutive days): the mouse was lifted by its tail and its forelimbs were gently placed on a bar about 30 cm above a cage with bedding. The mouse was released after it had a stable grip. The training was repeated for 1 minute each time, twice a day.

[0052] (3) Formal experiment: Record the time from hanging to falling of the mouse. To ensure experimental consistency and reduce mouse stress, the maximum hanging time was set at 60s. If the mouse did not fall within 60s, the experiment was terminated and its score was recorded as 60s. Each mouse was tested three times a day, with an interval of at least 30 minutes between each test, and the longest hanging time was used for statistical analysis.

[0053] (4) To assess the changes in muscle endurance in the limbs of mice, we conducted a rotarod fatigue test. The rotarod fatigue tester used in this experiment was from Shanghai Xinruan Co., Ltd.

[0054] (5) Adaptation training before the experiment (3 consecutive days): The mice were placed on a rotarod (3 cm in diameter) and trained at a constant speed of 4 rpm for 2 min / time, with an interval of 10 min, for a total of 5 times / day, to eliminate abnormal movement caused by fear.

[0055] (6) For the formal experiment, the rotation speed was linearly increased from 4 rpm to 40 rpm (acceleration rate 0.5 rpm / s), and the time it took for the mouse to fall from the rotator was recorded (maximum observation time 300s, if it did not fall, it was counted as 300s). The test was repeated 3 times (with an interval of 30 min), and the average value was used for statistical analysis.

[0056] Experimental results are as follows Figure 2 As shown, the results indicated that compared with the NC control group, the suspension time of mice treated with Dex was significantly reduced (***p < 0.001, n=3). Forsythoside treatment significantly prolonged the suspension time of mice (**p < 0.01, n=3), restoring it to normal levels; (B) The rotundus fatigue test results showed that compared with the NC control group, the rotundus dwell time of mice treated with Dex decreased sharply (***p < 0.001, n=3), while the rotundus dwell time of mice treated with forsythoside significantly increased (p < 0.01, n=3).

[0057] This demonstrates that forsythoside can restore dexamethasone-induced muscle function and exercise endurance in mice.

[0058] Example 3: Changes in the cross-sectional area of ​​gastrocnemius muscle fibers in each group of mice

[0059] 3. HE staining

[0060] (1) Baking: Place the paraffin slices on a baking machine at 60-65℃ and bake for at least 30-60 minutes to melt the paraffin and firmly adhere it to the glass slide.

[0061] (2) Xylene I: Place the slices in fresh xylene and soak for 10-15 minutes.

[0062] (3) Xylene II: Transfer to another xylene tank and soak again for 10-15 minutes. Continue until the tissue area is completely transparent to the naked eye.

[0063] (4) Anhydrous ethanol I: Transfer to 100% ethanol, soak for 3-5 minutes, and wash away xylene.

[0064] (5) Anhydrous ethanol II: Transfer to another 100% ethanol tank and soak again for 3-5 minutes.

[0065] (6) Gradient hydration: 95% ethanol: soak for 3-5 min. 80% ethanol: soak for 3-5 min. 70% ethanol: soak for 3-5 min.

[0066] (7) Rinse with running water: Rinse the slices with running tap water for 1-2 minutes to completely remove ethanol.

[0067] (8) Soaking in distilled water: Soak in distilled water for 1-2 minutes to prepare for staining.

[0068] (9) Hematoxylin staining: Immerse the sections completely in Harris hematoxylin staining solution for 3-8 minutes (the specific time needs to be optimized based on the freshness of the staining solution and the room temperature of the preliminary experiment).

[0069] (10) Rinse with running water: Gently rinse away the surface color with running water for about 10-20 seconds.

[0070] (11) Hydrochloric acid ethanol differentiation: Immerse the slices in 1% hydrochloric acid ethanol (prepared with 70% alcohol), quickly lift and insert them several times (about 1-5 seconds), and remove them immediately. Under a microscope, observe until the cell nuclei are purplish-red and the background is almost colorless.

[0071] (12) Rinse with running water: Rinse the slides with running water for 10-15 minutes to completely remove the acid. The slides should turn blue at this time.

[0072] (13) Blue enhancement solution: The slices can be immersed in dilute ammonia (0.5%) or warm PBS for a while to enhance the blue effect, and then rinsed with running water again.

[0073] (14) Eosin staining: Immerse the slide in eosin staining solution for 30 seconds to 3 minutes (the specific time needs to be determined by preliminary experiments).

[0074] (15) Quick wash: Quickly dip and wash in distilled water for 1-2 seconds to remove surface color.

[0075] (16) Gradient dehydration: 70% ethanol: rapid insertion and withdrawal several times. 80% ethanol: rapid insertion and withdrawal several times. 95% ethanol I: soak for 1-2 min. 95% ethanol II: soak for 1-2 min. Anhydrous ethanol I: soak for 3-5 min. Anhydrous ethanol II: soak for 3-5 min.

[0076] (17) Transparency: Xylene I: Soak for 3-5 minutes until the tissue becomes transparent. Xylene II: Soak for 3-5 minutes.

[0077] (18) Covering:

[0078] Remove the slide from the xylene and wipe away any excess liquid from the back of the slide and around the sample with a paper towel, ensuring the tissue surface remains moist. Place a drop of neutral resin in the center of the tissue. Slowly place the coverslip on one side, avoiding air bubbles. Allow the mounting medium to spread naturally and evenly.

[0079] (19) Drying and observation: Place the sealed slides in a ventilated place to air dry horizontally. After the mounting medium has solidified, it can be observed under an optical microscope.

[0080] Experimental results are as follows Figure 3 As shown, the results indicated that compared with the NC control group, the gastrocnemius muscle fibers of mice treated with DEX were significantly thinner and the gaps between them were larger, and the CSA was significantly reduced (****p < 0.0001, n=3); while the treatment with forsythoside caused the CSA of the muscle fibers to recover (**p < 0.01, n=3), approaching the level of the normal group.

[0081] This shows that forsythoside can alleviate the reduction in muscle fiber area induced by dexamethasone in mice.

[0082] Example 4: Distribution ratio of fast and slow muscle fibers in each group of mice

[0083] 4. Immunofluorescence staining

[0084] (1) Dewaxing paraffin sections to water: The sections were placed in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then washed with distilled water.

[0085] (2) Antigen retrieval: During this process, excessive evaporation of the buffer solution should be prevented, and the slide should not be dried. After natural cooling, place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time.

[0086] (3) Blocking endogenous peroxidase: Place the slide in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Then place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time.

[0087] (4) Serum blocking: Add 3% BSA evenly to the histochemistry zone and block at room temperature for 30 min. (Use rabbit serum for primary antibody derived from goat, and BSA for primary antibody derived from other sources.)

[0088] (5) Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, and incubate the slide in a humidified chamber at 4°C overnight.

[0089] (6) Add secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add the secondary antibody (HRP-labeled) of the corresponding species to the primary antibody to cover the tissue and incubate at room temperature for 50 minutes.

[0090] (7) DAB staining: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. The positive result is brownish-yellow. Rinse the slide with tap water to stop the staining.

[0091] (8) Counterstaining cell nuclei: Counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin blue solution, and rinse with running water.

[0092] (9) Dehydration and mounting: Place the slides in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and make them transparent. Remove the slides from the xylene and let them dry slightly. Then mount them with mounting glue.

[0093] (10) Image acquisition: Observe and acquire images under a fluorescence microscope.

[0094] Experimental results are as follows Figure 4 As shown in the results, there was no significant difference in the total number of muscle fibers among the groups. The normal group was dominated by fast-twitch muscle fibers. Compared with the NC control group, the proportion of fast-twitch muscle fibers did not change significantly after Dex treatment. After treatment with forsythoside, the proportion of fast-twitch muscle fibers decreased significantly, while the proportion of slow-twitch muscle fibers increased significantly (***p < 0.001, n=3).

[0095] This shows that forsythoside induces the conversion of fast-twitch muscle fibers to slow-twitch muscle fibers in mice. This result is consistent with the aforementioned improvement in exercise endurance and provides evidence at the level of muscle fiber type for its functional improvement.

[0096] Example 5: Expression of 15-PGDH, PTGER4, and PTGER2 in mice of different treatment groups

[0097] 5. Immunohistochemical detection

[0098] (1) Dewaxing paraffin sections to water: The sections were placed in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then washed with distilled water.

[0099] (2) Antigen retrieval: See the table above for retrieval instructions. During this process, excessive evaporation of the buffer solution should be prevented, and the slides should not be dried out. After natural cooling, place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 minutes each time. (The retrieval solution and conditions should be determined based on the tissue.)

[0100] (3) Blocking endogenous peroxidase: Place the slide in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Then place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time.

[0101] (4) Serum blocking: Add 3% BSA evenly to the histochemistry zone and block at room temperature for 30 min. (Use rabbit serum for primary antibody derived from goat, and BSA for primary antibody derived from other sources.)

[0102] (5) Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, and incubate the slide in a humidified chamber at 4°C overnight.

[0103] (6) Add secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add the secondary antibody (HRP-labeled) of the corresponding species to the primary antibody to cover the tissue and incubate at room temperature for 50 minutes.

[0104] (7) DAB staining: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. The positive result is brownish-yellow. Rinse the slide with tap water to stop the staining.

[0105] (8) Counterstaining cell nuclei: Counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin blue solution, and rinse with running water.

[0106] (9) Dehydration and mounting: Place the slides in 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and make them transparent. Remove the slides from the xylene and let them dry slightly. Then mount them with mounting glue.

[0107] (10) Microscopic examination: The results are interpreted under a white light microscope.

[0108] Experimental results are as follows Figure 5 As shown, the results indicated that compared with the NC control group, the positive area rates of 15-PGDH (HPGD) and EP2 (PTGER2) were increased in the Dex-treated group (***P < 0.001, n=3); conversely, the positive area rate of EP4 (PTGER4) decreased (***P < 0.001, n=3). After forsythoside intervention, the positive area rates of both 15-PGDH and EP2 were significantly reduced to normal levels (**P < 0.01, n=3).

[0109] This demonstrates that forsythoside reverses the intramuscular prostaglandin imbalance caused by dexamethasone by inhibiting 15-PGDH, degrading PGE2, and simultaneously upregulating EP4 receptors to reconstruct local PGE2-EP4 signaling coupling.

[0110] Example 6: Expression of FOXO3A, Atrogin-1, p-mTOR, mTOR, and PGC-1α in mice of different treatment groups

[0111] 6. Western Blot Detection

[0112] (1) Wash the tissue block 2-3 times with pre-cooled PBS to remove blood stains, cut it into small pieces and place it in a homogenization tube, add 2 4mm homogenization beads, add 10 times the tissue volume of lysis buffer (add various protease inhibitors a few minutes before use), set the homogenization program to homogenize; (if you need to increase the protein concentration, you can reduce the volume of lysis buffer appropriately)

[0113] (2) Take out the homogenized tube after homogenization and place it on ice for 30 minutes in lysis buffer. Shake it every 5 minutes to ensure complete tissue lysis.

[0114] (3) Centrifuge at 12000 rpm and 4℃ for 10 min, and collect the supernatant, which is the total protein solution.

[0115] (4) Extraction of plasma and nuclear proteins (nuclear protein and cytoplasmic protein extraction kit)

[0116] (5) Solution preparation: Thaw the three reagents in the kit at room temperature and place them on ice immediately after thawing, then mix well. Take an appropriate amount of cytoplasmic protein extraction reagent A for later use, and add PMSF a few minutes before use to make the final concentration of PMSF 1mM. Take an appropriate amount of nuclear protein extraction reagent for later use, and add PMSF a few minutes before use to make the final concentration of PMSF 1mM;

[0117] (6) Add 200µL of cytoplasmic protein extraction reagent A containing PMSF to every 20µL of cell pellet;

[0118] (7) Swirl at the highest speed for 5 seconds to completely suspend and disperse the cell precipitate; (If the cell precipitate is not completely suspended and dispersed, the swirling time can be appropriately extended.)

[0119] (8) Ice bath for 10-15 minutes;

[0120] (9) Add 10 µL of cytoplasmic protein extraction reagent B. Vortex vigorously at maximum speed for 5 seconds, then incubate on ice for 1 minute;

[0121] (10) The highest speed violent vortex was 5s, and centrifuged at 12,000-16,000g for 5min at 4℃;

[0122] (11) Immediately aspirate the supernatant into a pre-cooled EP tube; this is the extracted cytoplasmic protein. It can be used immediately or frozen.

[0123] (12) For the precipitate, completely aspirate the remaining supernatant and add 50µL of nuclear protein extraction reagent with added PMSF;

[0124] (13) Vortex at the highest speed for 15-30 seconds to completely suspend and disperse the cell pellet. Then return it to the ice bath and vortex at the highest speed for 15-30 seconds every 1-2 minutes for a total of 30 minutes;

[0125] (14) Centrifuge at 134℃ for 10 min at 12,000-16,000g;

[0126] (15) Immediately aspirate the supernatant into a pre-cooled EP tube; this is the extracted nuclear protein. It can be used immediately or frozen at -80°C.

[0127] (16) Protein denaturation: Add 5* reduced protein loading buffer to the protein solution at a ratio of 4:1, denature in boiling water bath for 15 min, and store in a -20℃ refrigerator for later use.

[0128] (17) SDS-PAGE electrophoresis

[0129] (18) Clean the glass plate;

[0130] (19) Glue preparation and sample loading;

[0131] (20) After the glass plate has dried naturally, put a concave glass plate and a flat glass plate into a pair, put them into the glue applicator, insert the inclined plate to fix the glass plate, check whether the bottom is aligned, and avoid glue leakage.

[0132] (21) Prepare the electrophoresis gel according to the instructions. After the gel has solidified, remove the gel casting machine and carefully pull out the comb. You are ready to start electrophoresis.

[0133] (22) After placing the gel casting apparatus into the electrophoresis tank, add sufficient electrophoresis buffer and then load the sample for electrophoresis. Stop electrophoresis when the bromophenol blue is about 1 cm from the bottom and proceed with the membrane transfer.

[0134] (23) Transfer membrane;

[0135] (24) Prepare 6 sheets of 7×9cm filter paper and a PVDF (0.45um) membrane of appropriate size. The PVDF membrane should be activated with ethanol before use.

[0136] (25) Place the transfer clamp, two sponges, a glass rod, filter paper and activated PVDF membrane in a basin containing the transfer solution.

[0137] (26) Open the clip. The left side is white and the right side is black. Add a sponge and three layers of filter paper to each side.

[0138] (27) Carefully peel off the separating gel and place it on filter paper. Attach the PVDF membrane to the gel, ensuring there are no air bubbles between the membrane and the gel. Cover the membrane with three sheets of filter paper and remove any air bubbles. Finally, cover with another layer of sponge.

[0139] (28) Transfer conditions: 350mA constant current, 30min.

[0140] (29) Immune response;

[0141] (30) Place the transferred film into an incubation tank containing TBST, rinse it quickly once, then add 5% milk, place it on a decolorizing shaker, and seal it at room temperature for 30 minutes.

[0142] (31) According to the antibody instructions, dilute the primary antibody, pour out the blocking solution in the incubation tank, add the prepared primary antibody, and incubate at 4°C on a shaker overnight (shake slowly).

[0143] (32) Recover the primary antibody, quickly wash the membrane three times with TBST, then add TBST, place on a decolorizing shaker for rapid elution, 5 min each time, and wash three times;

[0144] (33) Dilute the secondary antibody with TBST at a ratio of 1:5000, then add it to the incubation tank, place it on a shaker and shake it slowly, and incubate at room temperature for 30 minutes;

[0145] (34) Quickly rinse the membrane three times with TBST, then add TBST again, place it on a decolorizing shaker and wash quickly for 5 minutes each time, for a total of three washes.

[0146] (35) Chemiluminescence: Mix ECL A and B solutions in a 1:1 ratio and set aside. Take out the eluted PVDF membrane and place it on absorbent paper to slightly dry the liquid on the membrane. Place the membrane on the shelf of the chemiluminescence instrument, add the mixed ECL luminescent solution, and let the liquid completely submerge the membrane. After reacting for 1 minute, use absorbent paper to dry the excess liquid on the membrane, place it in the chemiluminescence instrument, and start chemiluminescence according to the preset program. After exposure is complete, save the original image as a TIFF file.

[0147] (36) Gray value quantization and result analysis.

[0148] Experimental results are as follows Figure 6 As shown, the results indicated that, in terms of protein degradation, compared with the normal NC group, Dex treatment significantly reduced the p-FOXO3a / FOXO3a ratio and upregulated Atrogin-1, suggesting that overactivation of the FOXO3a–Atrogin-1 arm is a key step in Dex-induced enhanced muscle protein degradation. Forsythoside treatment significantly increased the p-FOXO3a / FOXO3a ratio and downregulated Atrogin-1 expression (***p < 0.001, n=3).

[0149] This demonstrates that forsythoside enhances mitochondrial biogenesis and oxidative metabolism by remodeling the mTOR–PGC-1α pathway, thereby comprehensively improving the Dex-induced sarcopenia phenotype.

[0150] The above series of examples systematically demonstrated, from multiple levels including overall phenotype, tissue morphology, fiber type, and molecular signaling pathways, that forsythoside can effectively prevent and treat glucocorticoid-induced sarcopenia. Its core mechanism involves inhibiting 15-PGDH, restoring the PGE2-EP4 signaling axis, thereby activating PGC-1α to improve mitochondrial function, and inhibiting FOXO3a-Atrogin1-mediated protein degradation.

[0151] This invention reveals for the first time that forsythoside can be used as a drug to prevent or treat muscle atrophy, providing a new drug option and treatment approach for the prevention or treatment of muscle atrophy, and also providing a new use for forsythoside, which has great application prospects.

Claims

1. Use of forsythosides in the preparation of drugs for the prevention or treatment of muscle atrophy.

2. The use of forsythoside according to claim 1 in the preparation of a medicament for the prevention or treatment of muscle atrophy, characterized in that: The muscle atrophy described above is characterized by a decrease in skeletal muscle mass or a decline in function.

3. The use of forsythoside according to claim 2 in the preparation of a medicament for the prevention or treatment of muscle atrophy, characterized in that: The muscle atrophy mentioned includes at least one of sarcopenia, disuse atrophy, or glucocorticoid-induced secondary muscle atrophy.

4. The use of forsythoside according to claim 2 in the preparation of a medicament for the prevention or treatment of muscle atrophy, characterized in that: Muscle atrophy includes a decrease in muscle mass, a decline in muscle function and exercise endurance, a decrease in muscle fiber area, and a conversion of fast-twitch muscle fibers into slow-twitch muscle fibers.

5. The use of forsythoside according to claim 1 in the preparation of a medicament for the prevention or treatment of muscle atrophy, characterized in that: The forsythoside exerts its effect by inhibiting the activity or expression of 15-hydroxyprostaglandin dehydrogenase.

6. The use of forsythoside according to claim 1 in the preparation of a medicament for the prevention or treatment of muscle atrophy, characterized in that: The forsythoside works by increasing the level of endogenous prostaglandin E2.

7. A drug for preventing or treating muscle atrophy, characterized in that: The drug contains a therapeutically effective amount of forsythoside.

8. The medicament for preventing or treating muscular atrophy according to claim 7, characterized in that: The muscle atrophy described above is characterized by a decrease in skeletal muscle mass or a decline in function.

9. The medicament for preventing or treating muscular atrophy according to claim 7, characterized in that: The muscle atrophy mentioned includes at least one of sarcopenia, disuse atrophy, or glucocorticoid-induced secondary muscle atrophy.

10. The medicament for preventing or treating muscular atrophy according to claim 7, characterized in that: Muscle atrophy includes a decrease in muscle mass, a decline in muscle function and exercise endurance, a decrease in muscle fiber area, and a conversion of fast-twitch muscle fibers into slow-twitch muscle fibers.

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