Use of urolithin a in improving sarcopenic obesity

Abstract

The application discloses application of urolithin A in improving sarcopenic obesity, and belongs to the technical field of biological medicine. Through high-fat diet induced obesity mouse models and palmitic acid induced C2C12 myotube cell high-fat lipotoxicity models, it is found for the first time that urolithin A can inhibit abnormal weight growth induced by high-fat diet without depending on reducing food intake, improve the decline of forelimb grip strength, quadriceps weight and relative weight, relieve skeletal muscle tissue structure damage and muscle fiber cross-sectional area reduction, and down-regulate Atrogin-1, Murf1, Casp-3 and other muscle atrophy degradation related genes, and up-regulate Myf5, Myog, Myod1 and other myogenesis related factors, thereby providing experimental basis for urolithin A to improve obesity related muscle atrophy.

Description

Technical Field

[0001] This invention relates to the application of urolithin A in improving sarcopenic obesity, and belongs to the field of biomedical technology. Background Technology

[0002] Urolithin A is a small-molecule active metabolite formed by the metabolism of ellagic acid or ellagic tannins from foods such as pomegranates, berries, and nuts by intestinal flora. Previous studies have suggested that urolithin A can promote mitophagy, improve mitochondrial function, reduce inflammation, regulate oxidative stress, and improve energy metabolism, thus possessing potential value in improving aging and metabolic abnormalities.

[0003] Obesity is a chronic metabolic state caused by factors such as a high-fat diet, insufficient exercise, and an imbalance between energy intake and expenditure. It is often manifested as weight gain, excessive accumulation of adipose tissue, and lipid metabolism disorders. Obesity is not only a significant risk factor for diseases such as non-alcoholic fatty liver disease, type 2 diabetes, hypertension, and atherosclerosis, but it can also lead to abnormal lipid deposition in non-adipose tissues such as skeletal muscle, causing skeletal muscle atrophy, insulin resistance, chronic inflammation, and mitochondrial dysfunction. In a long-term state of obesity, skeletal muscle may exhibit changes such as decreased muscle mass, reduced muscle fiber cross-sectional area, weakened muscle strength, decreased exercise capacity, and increased expression of muscle atrophy-related factors, severely affecting the body's motor function and metabolic health.

[0004] However, it's important to note that obesity and obesity-induced skeletal muscle atrophy are not the same. Obesity is primarily characterized by excessive accumulation of adipose tissue, weight gain, dyslipidemia, and insulin resistance. Interventions for obesity typically focus on reducing fat mass, decreasing energy intake, inhibiting lipid absorption, increasing energy expenditure, or improving glucose and lipid metabolism. In contrast, obesity-induced skeletal muscle atrophy (oligomuscular obesity) occurs on top of excessive fat accumulation, further affecting skeletal muscle tissue. This manifests as increased intramuscular lipid deposition, enhanced lipotoxicity, mitochondrial dysfunction, chronic low-grade inflammation, and elevated oxidative stress, ultimately leading to increased muscle protein degradation, suppressed myogenesis, reduced muscle fiber cross-sectional area, and decreased muscle function. Therefore, obesity-induced muscle atrophy is not merely a matter of "excessive weight" or simply "excess fat," but rather the result of the combined effects of abnormal adipose tissue expansion and skeletal muscle metabolic damage. These two factors exacerbate each other; fat infiltrates the muscle, triggering chronic inflammation and further accelerating muscle breakdown.

[0005] Current obesity interventions primarily target weight and fat loss as their main endpoints, such as restricting energy intake, inhibiting lipid absorption, regulating appetite, improving insulin sensitivity, or promoting energy expenditure. These strategies can improve obesity and abnormal glucose and lipid metabolism to some extent, but their core goal is usually weight loss or fat reduction, and they do not necessarily improve skeletal muscle mass simultaneously.

[0006] In fact, some weight loss interventions may reduce lean body mass or skeletal muscle mass while reducing fat mass (Wilding JPH, et al. Impact of semaglutide on body composition in adults with overweight or obesity: exploratory analysis of the STEP 1 study. 2021.). Therefore, for obesity combined with muscle atrophy, relying solely on traditional weight loss or fat reduction strategies may be insufficient: on the one hand, simply reducing weight does not guarantee the recovery of muscle mass and function; on the other hand, if skeletal muscle energy metabolism, mitochondrial function, inflammatory oxidative stress, and protein degradation status are not improved during the intervention, obesity-related muscle atrophy may persist.

[0007] Obesity-induced skeletal muscle atrophy differs from ordinary disuse atrophy. Disuse atrophy primarily occurs under conditions such as prolonged bed rest, immobilization, weightlessness, reduced exercise, or decreased neuromuscular activity, with its core causes being reduced mechanical load and muscle contraction stimulation. Due to the lack of normal stretching and contraction signals in muscles, protein synthesis decreases, and protein degradation pathways such as the ubiquitin-proteasome system and the autophagy-lysosome system are activated, ultimately leading to thinner muscle fibers and decreased muscle strength. Therefore, current interventions for disuse atrophy typically focus on restoring mechanical stimulation and anabolic signals, such as resistance exercise, rehabilitation training, neuromuscular electrical stimulation, and protein or amino acid supplementation.

[0008] In contrast, obesity-related skeletal muscle atrophy exhibits more pronounced metabolic and inflammatory characteristics. Its occurrence is not entirely dependent on muscle usage, but is closely related to lipid overload caused by a high-fat diet, intramuscular lipid deposition, insulin resistance, adipokines imbalance, chronic inflammation, oxidative stress, and mitochondrial dysfunction. In this context, simply increasing mechanical load or supplementing nutrition may offer some assistance in muscle synthesis, but it is difficult to fundamentally resolve lipotoxicity, inflammatory responses, and mitochondrial energy metabolism disorders. Therefore, intervention strategies for obesity-related muscle atrophy need to simultaneously address both fat reduction and muscle preservation. This involves reducing fat accumulation and lipotoxicity while improving skeletal muscle mitochondrial function, inhibiting inflammatory oxidative stress, reducing apoptosis and protein degradation, and promoting muscle repair and regeneration.

[0009] It is evident that current obesity intervention techniques primarily focus on weight loss, reducing fat deposition, or improving glucose and lipid metabolism. Common methods include restricting energy intake, inhibiting lipid absorption, increasing energy expenditure, regulating insulin sensitivity, or improving blood lipid levels. However, obesity-related skeletal muscle atrophy is not simply a weight issue; it also involves skeletal muscle lipotoxicity, energy metabolism disorders, increased muscle protein degradation, and suppressed myogenesis. Therefore, intervention programs targeting only fat reduction or blood sugar control are insufficient to comprehensively address the decline in skeletal muscle mass and function in obese individuals. Consequently, how to improve obesity-induced muscle atrophy is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0010] To address the aforementioned issues, this invention provides the application of urolithin A in improving sarcopenic obesity. Using a high-fat diet-induced obese mouse model and a palmitic acid-induced C2C12 myotube cell hyperlipidemia model, this invention, for the first time, discovers that urolithin A can inhibit abnormal weight gain induced by a high-fat diet without relying on reduced food intake, improve forelimb grip strength, quadriceps muscle weight and relative weight loss, alleviate skeletal muscle tissue structural damage and reduced muscle fiber cross-sectional area, and downregulate muscle atrophy and degradation-related genes such as Atrogin-1, Murf1, and Casp-3, while upregulating myogenesis-related factors such as Myf5, Myog, and Myod1, providing experimental evidence for urolithin A's ability to improve obesity-related muscle atrophy.

[0011] The first objective of this invention is to improve the use of urolithin A in the preparation of products for the prevention, relief and / or improvement of sarcopenic obesity.

[0012] In one implementation, sarcopenic obesity is a special condition in which a large amount of muscle loss and excessive accumulation of body fat coexist, that is, a decrease in skeletal muscle mass and a decline in muscle function, while excessive accumulation of body fat. People in this category may have normal or even excessive weight, but their body composition is very unhealthy. The sarcopenic obesity refers to muscle atrophy caused by obesity.

[0013] In one embodiment, the structural formula of urolithin is shown in Formula I: .

[0014] In one embodiment, the product includes, but is not limited to, one or more of the following: pharmaceuticals, food, health products, feed, and feed additives.

[0015] In one embodiment, the dosage form of the drug is a liquid formulation or a solid formulation. Optionally, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, or oral liquids.

[0016] In one embodiment, the drug further includes pharmaceutically acceptable excipients; The excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

[0017] In one embodiment, the food includes health food, food for special medical purposes, functional food, or pet food; Preferably, the food products include grain products, starch products, vegetable products, fruit products, meat products, poultry products, egg products, and dairy products.

[0018] In one embodiment, the health product also contains acceptable excipients.

[0019] A second objective of the present invention is to improve a medicament for preventing, alleviating and / or improving sarcopenic obesity, said medicament containing urolithin A.

[0020] In one embodiment, the dosage form of the drug is a liquid formulation or a solid formulation. Optionally, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, or oral liquids.

[0021] In one embodiment, the drug further includes pharmaceutically acceptable excipients.

[0022] In one embodiment, the excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

[0023] Beneficial effects (1) This invention proposes a new use of urolithin A in improving sarcopenic obesity, breaking through the limitations of existing obesity interventions that mainly focus on weight and fat metabolism while neglecting skeletal muscle protection.

[0024] (2) This invention demonstrates that urolithiasis A can inhibit abnormal weight gain induced by a high-fat diet without significantly reducing food intake, suggesting that its effect is not simply achieved by reducing food intake.

[0025] (3) This invention demonstrates that urolithin A can improve the decrease in forelimb grip strength, the reduction in quadriceps muscle weight and relative weight, and the decrease in muscle fiber cross-sectional area in obese mice, thus exerting a protective effect on skeletal muscle at both the tissue and functional levels. Specifically, after intervention with urolithin A, compared with the HFD group, the forelimb grip strength of the UA-L group increased by 0.02 N, an increase of approximately 2.1%; the UA-M group increased by 0.18 N, an increase of approximately 18.9%; and the UA-H group increased by 0.28 N, an increase of approximately 29.5%. Among them, the forelimb grip strength of the UA-H group recovered to a level close to that of the NC group, recovering approximately 75.7% of the decrease in grip strength induced by HFD. The quadriceps muscle weight in the NC group was 172.2±18.4 mg, while that in the HFD group decreased to 140.6±14.2 mg, a decrease of 31.6 mg compared to the NC group, a decrease of approximately 18.4%. After urolithin A intervention, compared with the HFD group, the quadriceps muscle weight increased by 3.7 mg in the UA-L group (approximately 2.6%), by 10.1 mg in the UA-M group (approximately 7.2%), and by 26.9 mg in the UA-H group (approximately 19.1%). Regarding the relative weight of the quadriceps muscle, the quadriceps muscle weight / body weight ratio was 6.45 ± 0.71 mg / g in the NC group, decreasing to 4.04 ± 0.46 mg / g in the HFD group, a decrease of 2.41 mg / g compared to the NC group, representing a reduction of approximately 37.4%. After urolithin A intervention, compared with the HFD group, the relative weight of the quadriceps muscle increased by 0.12 mg / g in the UA-L group (approximately 3.0%), by 0.84 mg / g in the UA-M group (approximately 20.8%), and by 2.11 mg / g in the UA-H group (approximately 52.2%). The relative weight of the quadriceps muscle in the UA-H group recovered approximately 87.6% of the HFD-induced decrease.

[0026] Regarding the cross-sectional area of ​​muscle fibers, after urolithin A intervention, compared with the HFD group, the cross-sectional area of ​​muscle fibers in the UA-L group increased by 167.7 μm², an improvement of approximately 9.6%; the UA-M group increased by 654.0 μm², an improvement of approximately 37.5%; and the UA-H group increased by 864.2 μm², an improvement of approximately 49.5%. Among them, the cross-sectional area of ​​muscle fibers in the UA-H group recovered approximately 88.5% of the decrease induced by HFD.

[0027] (4) This invention demonstrates that urolithin A can downregulate Atrogin-1, Murf1, and Casp-3, and upregulate Myf5, Myog, and Myod1, thereby regulating the balance between muscle protein degradation and myogenesis at the molecular level.

[0028] (5) This invention further demonstrates, through a palmitic acid-induced C2C12 myotube cell model, that urolithin A can reduce lipid droplet deposition, improve myotube atrophy-like morphological changes, increase ATP levels, and reduce abnormal lactate accumulation. Specifically, regarding ATP content, compared with the NC group, the PA group showed a decrease of 0.8884 nmol / mg protein, a reduction of approximately 42.8%; compared with the Ve group, the PA group showed a decrease of 1.0402 nmol / mg protein, a reduction of approximately 46.7%. After intervention with urolithin A, the ATP content in the UA group increased to 2.0965±0.4866 nmol / mg protein. Compared with the PA group, the ATP content in the UA group increased by 0.9103 nmol / mg protein, an increase of approximately 76.7%; and the ATP level in the UA group was close to that in the NC and Ve groups, suggesting that urolithin A can significantly improve PA-induced insufficient ATP production in myotube cells.

[0029] Regarding lactate concentration, compared with the NC group, the lactate concentration in the PA group increased by 1.0181 mM, an increase of approximately 18.6%. After intervention with urolithin A, the lactate concentration in the UA group decreased to 6.0561 ± 0.0696 mM. Compared with the PA group, the lactate concentration in the UA group decreased by 0.4355 mM, a decrease of approximately 6.7%. Urolithin A restored approximately 42.8% of the lactate increase induced by PA, indicating that urolithin A can alleviate PA-induced abnormal lactate accumulation to some extent.

[0030] (6) The present invention forms a technical evidence chain that combines an animal obesity model and a cell lipotoxicity model, which can be used for the development of drugs, health foods, special dietary foods, sports nutrition foods or dietary supplements. Attached Figure Description

[0031] Figure 1 The effect of urolithin A on body weight changes in high-fat diet-induced obese mice.

[0032] Figure 2 The effect of urolithin A on energy intake in high-fat diet-induced obese mice.

[0033] Figure 3 The effect of urolithin A on forelimb grip strength in high-fat diet-induced obese mice.

[0034] Figure 4 The effect of urolithin A on the weight and relative weight of the quadriceps femoris muscle in high-fat diet-induced obese mice is shown in Figure 1. (A) shows the effect of urolithin A on the weight of the quadriceps femoris muscle in high-fat diet-induced obese mice; (B) shows the effect of urolithin A on the relative weight of the quadriceps femoris muscle in high-fat diet-induced obese mice.

[0035] Figure 5The effect of urolithin A on the H&E staining morphology of the quadriceps femoris muscle in mice with high-fat diet-induced obesity.

[0036] Figure 6 The effect of urolithin A on the cross-sectional area of ​​quadriceps muscle fibers in mice with high-fat diet-induced obesity.

[0037] Figure 7 The effect of urolithin A on the expression of genes related to quadriceps muscle atrophy and degradation induced by a high-fat diet in obese mice is shown in the figure. (A) is the relative expression level of Atrogin-1; (B) is the relative expression level of Murft; and (C) is the relative expression level of Casp.

[0038] Figure 8 The effect of urolithin A on the expression of genes related to quadriceps myogenesis in obese mice induced by a high-fat diet is shown in the figure. (A) represents the relative expression level of Myf5; (B) represents the relative expression level of Myog; and (C) represents the relative expression level of Myod1.

[0039] Figure 9 The effects of urolithin A on lipid deposition and cell morphology in palmitic acid-induced C2C12 myotube cells.

[0040] Figure 10 The effect of urolithin A on palmitic acid-induced ATP and lactate levels in C2C12 myotube cells is shown in Figure (A). Figure (B) shows the effect of urolithin A on palmitic acid-induced ATP in C2C12 myotube cells.

[0041] Figure 11 The effect of urolithin A on the expression of genes related to palmitic acid-induced muscle atrophy and degradation in C2C12 myotube cells is shown in the figure. (A) is the relative expression level of Atrogin-1; (B) is the relative expression level of Murft; and (C) is the relative expression level of Casp.

[0042] Figure 12 The effect of urolithin A on the expression of myogenic genes induced by palmitic acid in C2C12 myotube cells is shown in the figure. (A) represents the relative expression level of Myf5; (B) represents the relative expression level of Myog; and (C) represents the relative expression level of Myod1. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0044] The sources of the raw materials, reagents, and instruments involved in the following examples are as follows: Urolithin A, palmitic acid, and BSA were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the CAS number for urolithin is 1143-70-0; its structural formula is as follows:

[0045] The eosin and hematoxylin staining solutions were purchased from Beijing Solarbio Science & Technology Co., Ltd.; 4% paraformaldehyde was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; and the ATP and L-lactic acid reagent kits were purchased from Xiamen Huijia Biotechnology Co., Ltd. The HBS-1101 ELISA reader was purchased from Nanjing Detie Biotechnology Co., Ltd.; the real-time quantitative PCR instrument was purchased from Bio-Rad Laboratories, USA; the Leica 1150H paraffin embedding machine, CM1950 cryostat, and HistoCore MULTICUT paraffin microtome were all Leica equipment. RNA extraction kits, reverse transcription kits, and amplification kits were purchased from Nanjing Novizan Biotechnology Co., Ltd. Six-week-old SPF-grade male C57BL / 6J mice were purchased from Cavens Laboratory Animal Co., Ltd. The animals were housed at the Animal Experiment Center of Jiangnan University under the following conditions: temperature 25±2℃, relative humidity 60±10%, 12-hour diurnal light cycle, and free access to purified water. The basic diet used in the experiments was provided by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., and the high-fat diet was provided by Changzhou Shusan Biotechnology Co., Ltd. Animal experimental procedures strictly followed the guidelines for the protection of laboratory animals. C2C12 cells, 10% fetal bovine serum, 1% penicillin-streptomycin, high-glucose DMEM medium, and 2% horse serum DMEM were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0046] In this embodiment, "% (w / v)" refers to the percentage of mass to solution volume (g / mL).

[0047] Example 1: Effects of urolithin A on high-fat diet-induced mice 1. Establishment of a mouse model After all mice had adapted to the environment for one week, they were randomly divided into 5 groups of 6 mice each: blank group (NC group), high-fat model group (HFD group), low-dose urolithin A group (UA-L group), medium-dose urolithin A group (UA-M group), and high-dose urolithin A group (UA-H group).

[0048] Specifically as follows: Preparation of urolithin A solution: The usual oral administration volume for mice is 0.2 mL. Prepare 5 mL solutions for each group. Weigh 5 mg, 12.5 mg, and 25 mg of urolithin A respectively, add an appropriate amount of physiological saline, mix thoroughly, and bring the volume to 5 mL to obtain the urolithin A solutions required for administration of 10, 25, and 50 mg / kg.

[0049] Control group (NC group): From day 1 to day 84, the animals were fed a normal diet. From day 29 to day 84, they were given 0.2 mL of physiological saline solution by gavage. High-fat model group (HFD group): From day 1 to day 84, the diet was high-fat. From day 29 to day 84, 0.2 mL of physiological saline solution was administered by gavage. Low-dose urolithin A group (UA-L group): From day 1 to day 84, the diet was high-fat. From day 29 to day 84, 0.2 mL of urolithin A solution was administered by gavage (gavage dose: 10 mg / kg·d). Urolithin A medium-dose group (UA-M group): From day 1 to day 84, the diet was high-fat. From day 29 to day 84, 0.2 mL of urolithin A solution was administered by gavage (gavage dose: 25 mg / kg·d). High-dose urolithin A group (UA-H group): From day 1 to day 84, the diet was high-fat. From day 29 to day 84, 0.2 mL of urolithin A solution was administered by gavage (gavage dose: 50 mg / kg·d). Each cage is fed 50g of feed, which is changed every three days. The total energy of the regular feed is 3500 kcal / kg, with fat providing 10% of the energy; the total energy of the high-fat feed is 5000 kcal / kg, with fat providing 60% of the energy.

[0050] 2. Effects of urolithin A on body weight and food intake in mice induced by a high-fat diet During the establishment of the mouse model in step 1, the weight of each group of mice was recorded at 8:00 AM every Monday. The weight of the feed added was accurately weighed each time the mice were fed, and the weight of the remaining feed was weighed at the next feeding. The difference between the two weighings was used to calculate the weekly food intake. The effects of urolithin A on the weight changes and food intake of high-fat diet-induced obese mice were determined.

[0051] The measurement results are as follows Figure 1 as well as Figure 2 As shown in Tables 1 and 2: Table 1. Effects of urolithin A on body weight induced by a high-fat diet in mice.

[0052] Table 2. Measurement of average daily food intake of mice during the experiment.

[0053] The results show that: After 12 weeks of intervention, significant differences in body weight were observed among the different groups of mice. Under the high-fat diet intervention, weight gain was observed in all experimental groups, with the HFD group showing a significantly higher weight gain than the NC group fed a conventional diet. The remaining mice fed a high-fat diet showed lower weight gain than the HFD group, indicating that urolithin A gavage under a high-fat diet can effectively control weight gain in a dose-dependent manner. The UA-H group showed the most significant inhibitory effect on body weight gain, suggesting that UA can effectively alleviate the obesity phenotype induced by a high-fat diet.

[0054] Daily food intake data showed no statistically significant difference among the experimental groups. This indicates that there was no significant difference in food intake among the groups, ruling out the influence of differences in food intake on body weight changes. Daily energy intake data for each group of mice during the experimental period showed no significant differences in weekly daily energy intake among the groups.

[0055] Based on the above data on food intake, it can be suggested that the effect of urolithiasis A gavage on reducing animal weight is not achieved by reducing food intake, but by improving the body's energy metabolism and promoting the rational use or consumption of energy, thereby achieving the effect of weight reduction.

[0056] 3. Effects of urolithin A on grip strength induced by a high-fat diet in mice Mice were treated using the method described in step 1. After treatment (i.e., 12 weeks after intervention), the forelimb grip strength of mice in each group was measured using a small animal grip dynamometer. Before the experiment, the grip dynamometer was zeroed and calibrated, and the testing environment was kept quiet and stable. During the test, the experimenter gently held the proximal end of the mouse's tail, allowing its forelimbs to naturally grasp the metal grid of the grip dynamometer, and then pulled it backward at a uniform speed in the horizontal direction. The maximum tension value displayed by the instrument when the mouse's forelimb released the grid was recorded as the forelimb grip strength, expressed in grams per force (g).

[0057] Each mouse was measured five times consecutively, with appropriate intervals between each measurement to avoid fatigue. If a mouse failed to grasp the grid effectively, only one forelimb contacted the grid, or exhibited turning, jumping, or hindlimb-assisted grasping, that data was discarded and the measurement was repeated. Only the valid measurements were used for statistical analysis.

[0058] The measurement results are as follows Figure 3 And as shown in Table 3: Table 3. Effects of urolithin A on grip strength induced by a high-fat diet in mice.

[0059] The results show: After 12 weeks of intervention, there were significant differences in forelimb grip strength among the groups of mice. Compared with the NC group, the forelimb grip strength of mice in the HFD group was significantly reduced, suggesting that a long-term high-fat diet can lead to a decrease in skeletal muscle strength in mice, indicating that obesity induced by a high-fat diet can cause skeletal muscle dysfunction and muscle atrophy-related phenotypes.

[0060] After intervention with urolithin A, the forelimb grip strength of mice in all treatment groups increased to varying degrees compared with the HFD group, indicating that urolithin A can improve the decline in skeletal muscle strength induced by a high-fat diet. Among them, the forelimb grip strength of mice in the UA-H group showed the most significant recovery, suggesting that high-dose urolithin A has a better effect on improving skeletal muscle function decline and muscle atrophy in obese individuals.

[0061] 4. Effect of urolithin A on muscle weight induced by a high-fat diet in mice Mice were treated using the method described in step 1. After treatment (i.e., 12 weeks after intervention), the mice were weighed and their weight recorded. Following animal ethics requirements, the mice were euthanized. Immediately after euthanasia, the mice were placed supine on a dissection table. The skin of the hind limbs was cut open and the subcutaneous tissue was dissected to fully expose the anterior femoral muscle group. Subsequently, the surrounding fascia and connecting tissues were carefully separated along the anatomical location of the quadriceps femoris muscle. Using ophthalmic scissors and forceps, the quadriceps femoris muscle was completely removed from its origin and insertion points.

[0062] During tissue collection, handle the tissue gently to avoid excessive stretching, squeezing, or incomplete cutting, which could affect tissue quality and weighing results. Remove visible non-muscle components such as fat, blood vessels, fascia, and connective tissue from the extracted quadriceps muscle tissue. Then, gently rinse the tissue surface with pre-cooled physiological saline to remove any residual blood, and blot dry with filter paper to obtain quadriceps muscle tissue isolated from different groups of mice. Immediately afterward, weigh the quadriceps muscle using an electronic balance, record the data, measure the weight change of the quadriceps muscle in each group of mice, and calculate the quadriceps muscle weight to body weight ratio for each group.

[0063] The measurement results are as follows Figure 4 And as shown in Table 4: Table 4. Effects of urolithin A on muscle weight induced by a high-fat diet in mice.

[0064] The results show: Compared with the NC group, the quadriceps muscle weight of mice in the HFD group was significantly reduced, suggesting that obesity induced by a high-fat diet can lead to a decrease in hind limb skeletal muscle mass in mice and cause obesity-related muscle atrophy phenotype. After intervention with urolithin A, the quadriceps muscle weight of mice in each dose of urolithin A group increased to varying degrees compared with the HFD group, indicating that urolithin A can improve the decrease in skeletal muscle mass induced by a high-fat diet. Among them, the quadriceps muscle weight recovery in the UA-H group was the most significant, with a better improvement effect than the low-dose and medium-dose groups, suggesting that high-dose urolithin A has a more significant protective effect against the decrease in quadriceps muscle mass under obesity.

[0065] 5. Effects of urolithin A on muscle morphology in mice induced by a high-fat diet Quadriceps femoris muscle tissue isolated from different groups of mice in step 4 was gently rinsed with PBS to remove surface blood and impurities, and then cut into tissue blocks approximately 4 mm × 4 mm in size. The tissue blocks were then fixed in pre-cooled 4% paraformaldehyde fixative for 24 h. After fixation, the tissues were dehydrated, cleared, and embedded in paraffin according to standard histological procedures, and paraffin sections approximately 5 μm thick were prepared. After dewaxing in xylene, the sections were rehydrated sequentially with ethanol of different concentrations, followed by rinsing with distilled water. After rehydration, the nuclei were stained with hematoxylin, rinsed with distilled water, and then briefly differentiated in differentiation medium to remove background staining. The sections were then thoroughly washed again with distilled water. After nuclear staining, eosin staining was added to counterstain the cytoplasm and myofibril structures. After counterstaining, the sections were rapidly dehydrated sequentially with ethanol in a gradient, and then cleared in xylene. Finally, a neutral resin was used for mounting. After the mounting medium solidified, images of the quadriceps femoris tissue morphology were observed and collected under an optical microscope to evaluate the arrangement, size, and skeletal muscle atrophy of each group of mice.

[0066] The measurement results are as follows Figure 5 , Figure 6 As shown, the results indicated that the quadriceps muscle fibers in the NC group mice had regular edges, clear structure, and were tightly packed with relatively uniform morphology, suggesting that the quadriceps muscle tissue structure of normal mice was intact. Compared with the NC group, the quadriceps muscle fibers in the HFD group mice showed significant abnormalities in morphology, characterized by irregular edges, sparse and disordered arrangement, enlarged interfiber spaces, and reduced fiber size and number compared to the NC group, suggesting that obesity induced by a high-fat diet can lead to damage to the quadriceps muscle tissue structure and skeletal muscle atrophy. After intervention with urolithin A, the morphology of the quadriceps muscle tissue in all treatment groups was improved compared to the HFD group. Among them, the UA-H group showed the most significant improvement, with the muscle fibers being more tightly packed than those in the HFD group, the morphology of the muscle fibers becoming more regular, and the size and number of muscle fibers approaching those in the NC group, suggesting that high-dose urolithin A can effectively improve the quadriceps muscle tissue structure damage induced by a high-fat diet.

[0067] Results of muscle fiber cross-sectional area measurement: Figure 6 And as shown in Table 5: Table 5. Effects of urolithin A on the cross-sectional area of ​​muscle fibers in mice induced by a high-fat diet.

[0068] The results show: The cross-sectional area of ​​quadriceps muscle fibers in the HFD group was significantly lower than that in the NC group, indicating that a high-fat diet can induce quadriceps muscle fiber atrophy. After intervention with urolithin A, the cross-sectional area of ​​muscle fibers in all dose groups increased to varying degrees compared with the HFD group, with the UA-H group showing the most significant improvement.

[0069] 6. Effects of urolithin A on gene expression in skeletal muscle of mice induced by a high-fat diet Quadriceps femoris muscle tissues isolated from different groups of mice in step 4 were used to extract total RNA. The effect of urolithin A on gene expression in high-fat diet-induced skeletal muscle of mice was determined using the following method: Quadriceps femoris muscle tissues isolated from different groups of mice in step 4 were used for total RNA extraction, reverse transcription, and RT-qPCR detection using the Novizan kit; the specific detection methods were as follows: Take an appropriate amount of frozen quadriceps femoris muscle tissue, add RNA lysis buffer and homogenize thoroughly. Follow the instructions of the Novizan Total RNA Extraction Kit to obtain total RNA through lysis, phase separation or adsorption column purification, washing and elution steps. Subsequently, the RNA concentration and purity are detected using an ultra-micro spectrophotometer, requiring an A260 / A280 ratio between 1.8 and 2.1. RNA meeting the requirements is used for subsequent experiments.

[0070] Take an equal amount of RNA and use the Novizan reverse transcription kit to synthesize cDNA. Add RNA template, genomic DNA removal reagent, reverse transcriptase mixture and RNase-free ddH2O to the reaction system. Perform genomic DNA removal and reverse transcription reaction according to the instructions. Store the obtained cDNA at -20℃ for later use.

[0071] RT-qPCR was performed using the Novizan SYBR Green qPCR kit. Each reaction system included 2×qPCR Master Mix, forward and reverse primers, cDNA template, and ddH2O. The reaction program was 95℃ pre-denaturation for 30 s, followed by 40 cycles: 95℃ for 10 s, 60℃ for 30 s. Melting curve analysis was performed after the reaction to confirm amplification specificity. Each sample was prepared in triplicate, with β-actin used as an internal control gene.

[0072] After the reaction, the Ct values ​​for each sample were recorded, and the relative gene expression level was calculated as 2^-ΔΔCt. The primer sequences used are shown in Table 6. All qPCR assays were performed using a real-time quantitative PCR instrument.

[0073] Table 6 Primer sequences for RT-PCR of skeletal muscle tissue

[0074] The measurement results are as follows Figure 7 , Figure 8 And as shown in Table 7: Table 7. Relative mRNA expression levels of different genes

[0075] The results show: Compared with the NC group, the expression levels of muscle atrophy and degradation-related genes Atrogin-1, Murf1, and Casp-3 in the quadriceps femoris muscle of mice in the HFD group were significantly increased, suggesting that obesity induced by a high-fat diet can activate molecular pathways related to skeletal muscle protein degradation and atrophy. Simultaneously, the expression levels of myogenesis-related factors Myf5, Myog, and Myod1 in the quadriceps femoris muscle of mice in the HFD group were decreased, indicating that a high-fat diet can inhibit skeletal muscle formation and repair processes. After intervention with urolithin A, the expression levels of Atrogin-1, Murf1, and Casp-3 in the quadriceps femoris muscle of mice in each dose of urolithin A group were lower than those in the HFD group, while the expression levels of Myf5, Myog, and Myod1 were higher, indicating that urolithin A can inhibit the upregulation of muscle atrophy and degradation-related genes induced by a high-fat diet and promote the recovery of myogenesis-related gene expression. With increasing urolithin A intervention dose, the expression of muscle atrophy and degradation-related genes further decreased, and the expression of myogenesis-related factors further increased, with the UA-H group showing the most significant improvement. The above results indicate that urolithin A can regulate the abnormal expression of muscle atrophy and myogenesis-related genes in the quadriceps femoris muscle of obese mice induced by a high-fat diet. By downregulating Atrogin-1, Murf1, and Casp-3 and upregulating Myf5, Myog, and Myod1, it can improve the state of enhanced skeletal muscle protein breakdown and suppressed myogenesis in obese patients, thereby playing a role in alleviating obesity-related skeletal muscle atrophy.

[0076] Example 2: Effect of urolithin A on palmitic acid-induced C2C12 myotube cells 1. Establishment of cell models (1) Raw material preparation A hyperlipidemic injury model of C2C12 myotube cells was established using sodium palmitate (PA). Before the experiment, a 50 mM sodium palmitate stock solution was prepared with water and thoroughly combined with BSA (albumin) solution. Specifically, the 50 mM sodium palmitate stock solution was thoroughly mixed with 22.2% (w / v) BSA solution at a volume ratio of 1:9 to obtain a final concentration of 5 mM PA-BSA complex stock solution. Before use, the 5 mM PA-BSA complex stock solution was diluted 10-fold with high-glucose DMEM medium to achieve a final PA concentration of 0.5 mM and a final BSA concentration of 2% (w / v).

[0077] Urolithin A was dissolved in DMSO (dimethyl sulfoxide) to prepare a 50 mM urolithin A stock solution, approximately 11.4 g / L, which was stored protected from light. During the experiment, the urolithin A stock solution was diluted to a final concentration of 50 μM with high-glucose DMEM medium. Since DMSO was used as the solvent for the urolithin A stock solution, the final concentration of DMSO in the UA group culture system was 0.1% (v / v). To eliminate the influence of DMSO on the experimental results, a Vehicle group was set up as a solvent control group. An equal volume of DMSO was added to the Vehicle group to achieve the same final concentration of 0.1% (v / v).

[0078] Mouse myoblast cell line C2C12 was used at approximately 1.0 × 10⁻⁶ cells per cell line. 4 pcs / cm 2 The cells were seeded at a density of approximately 1.0 × 10⁶ cells per well in 6-well cell culture plates. 5 Cells were added to 2 mL of high-glucose DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin and streptomycin, equivalent to approximately 5.0 × 10⁶ cells. 4 Cells were cultured at 37°C in a 5% (v / v) CO2 incubator. When cell confluence reached approximately 80%–90%, the culture was stopped, and the cells were immediately replaced with high-glucose DMEM induction medium according to the experimental group conditions for differentiation and drug administration.

[0079] (2) Model building C2C12 cells were divided into four groups: control group (NC), solvent control group (Ve), sodium palmitate group (PA), and urolithin A group (UA). The treatment methods for each group are as follows: Control group (NC): The culture medium of mouse myoblast line C2C12 cells after the end of proliferation culture in step (1) was replaced with high glucose DMEM induction medium containing 2% (v / v) horse serum and 2% (w / v) BSA, without the addition of PA and DMSO; Solvent control group (Ve): The culture medium of mouse myoblast C2C12 cells after the end of proliferation culture in step (1) was replaced with high-glucose DMEM induction medium containing 2% (v / v) horse serum, 2% (w / v) BSA and 0.1% (v / v) DMSO, without the addition of PA and urolithin A; Sodium palmitate group (PA): The culture medium of mouse myoblast C2C12 cells after the end of proliferation culture in step (1) was replaced with high-glucose DMEM induction medium containing 2% (v / v) horse serum, 0.5 mM PA and 2% (w / v) BSA to establish a palmitic acid-induced lipotoxic injury model. Urolithin A group (UA): The culture medium of mouse myoblast C2C12 cells after the end of proliferation culture in step (1) was replaced with high-glucose DMEM induction medium containing 2% (v / v) horse serum, 0.5 mM PA, 2% (w / v) BSA, 50 μM urolithin A and 0.1% (v / v) DMSO, to observe the effect of urolithin A on the improvement of PA-induced lipotoxic damage and abnormal myotube differentiation in C2C12 cells.

[0080] During differentiation and drug treatment, cells from each group were cultured in a 37℃, 5% (v / v) CO2 incubator after the culture medium was changed. Fresh induction medium was replaced for each group every 2 days, and the differentiation treatment was continued for 6 days until morphologically mature multinucleated myotubes were formed. After treatment, cells (C2C12 myotube cells) or cell culture supernatant from different groups were collected for subsequent detection of lipid droplet deposition, myotube morphology, ATP content, lactate concentration, and related molecular indicators.

[0081] 2. Effects of urolithin A on palmitic acid-induced lipid deposition and cell morphology in C2C12 myotube cells. The deposition of neutral lipids and lipid droplets in C2C12 myotube cells after different treatments obtained in step (2) of step 1 was detected by Oil Red O staining. The specific method is as follows: Take the C2C12 myotube cells from different treatment groups obtained in step (2) of step 1, discard the culture medium, and gently wash the cells 2-3 times with PBS to remove residual culture medium and free lipids. Then add an appropriate amount of 4% paraformaldehyde fixative to each well and fix at room temperature for 20-30 min. After fixation, discard the fixative and wash the cells 2-3 times with PBS. Then add freshly prepared and filtered Oil Red O working solution and stain at room temperature in the dark for 15-30 min. After staining, discard the staining solution and gently wash the cells with distilled water or PBS to remove unbound dye. If necessary, 60% isopropanol can be used for rapid differentiation to reduce background staining. After washing, observe and photograph the red lipid droplet deposition in the cells of each group under a microscope.

[0082] Oil Red O staining results of C2C12 myotube cells in different groups are as follows: Figure 9 As shown, the results indicated that no lipid droplets were present in the C2C12 cells of the NC and Ve groups, and the cell morphology remained intact. After treatment with 0.5 mM PA, numerous red lipid droplets appeared in the cells, and the staining intensity increased, indicating that PA could induce significant lipid deposition in C2C12 cells, successfully establishing a high-lipotoxicity cell model.

[0083] Meanwhile, the PA group showed certain abnormalities in cell morphology, with a tendency for myotubes to narrow and atrophy-like morphological changes, suggesting that PA-induced lipid overload may further damage the C2C12 myotube structure. After UA intervention, PA-induced lipid droplet deposition was reduced, and the number and staining intensity of intracellular red lipid droplets were lower than in the PA group. Simultaneously, the myotube morphology in the UA group was improved compared to the PA group, with a reduced tendency for narrowing and some relief of atrophy-like morphological changes.

[0084] 3. Effects of urolithin A on energy metabolism in C2C12 myotube cells induced by high palmitic acid levels The ATP levels in C2C12 cells of each group were detected using an ATP assay kit, as follows: Take the C2C12 myotube cells obtained from step (2) of step 1, discard the culture medium, gently wash the cells with pre-cooled PBS, and add ATP lysis buffer to fully lyse the cells. After lysis, collect the cell lysate, centrifuge according to the kit instructions, and use the supernatant for ATP detection. During detection, add the ATP standard and sample to the detection plate in sequence, followed by the ATP detection working solution. After reacting in the dark, measure the signal value using an ELISA reader or chemiluminescence analyzer. Calculate the ATP content in the sample based on the ATP standard curve.

[0085] The L-lactic acid content in the cell culture supernatant of each group was detected using an L-lactic acid assay kit, as follows: Take the C2C12 myotube cells obtained in step (2) of step 1, collect the culture supernatant from each group, and dilute them according to the kit requirements. Then add the standard and sample to the detection well, add the corresponding reaction solution, mix thoroughly, and react at the specified temperature. After the reaction, use an ELISA reader to measure the absorbance value at the specified wavelength, and calculate the L-lactic acid concentration in the sample according to the standard curve.

[0086] ATP test results as follows Figure 10 (A) and as shown in Table 8: Table 8

[0087] The results show that: Compared with the NC and Ve groups, the ATP content of C2C12 cells in the PA group was significantly reduced, indicating that 0.5 mM PA treatment can inhibit cellular energy metabolism, suggesting that PA can impair mitochondrial function and energy metabolism in C2C12 myotube cells. After UA intervention, the ATP content in the UA group was higher than that in the PA group, indicating that UA can improve PA-induced ATP deficiency and improve energy metabolism.

[0088] Lactic acid test results as follows Figure 10 (B) and Table 8 show that the lactate level in the PA group was higher than that in the NC and Ve groups, indicating that PA treatment enhanced glycolytic metabolism in C2C12 cells, and that the cells relied more on glycolysis for compensatory energy supply due to impaired mitochondrial oxidative energy supply. After UA intervention, the lactate level in the UA group was lower than that in the PA group, indicating that UA could alleviate PA-induced abnormal lactate accumulation and restore the cellular energy metabolism state. These results suggest that the protective effect of UA against PA-induced muscle atrophy-like damage in C2C12 cells may be related to improving energy metabolism and mitochondrial function.

[0089] 4. Effects of urolithin A on palmitic acid-induced expression of C2C12 myotube genes The mRNA expression levels of muscle atrophy and degradation-related genes and myogenesis-related factors in C2C12 myotubes of each group were detected by real-time quantitative PCR, as follows: After cell treatment, the culture medium was discarded, and the cells were gently washed twice with pre-cooled PBS to remove residual culture medium and serum. Then, following the instructions of the Novizan Total RNA Extraction Kit, an appropriate amount of RNA lysis buffer was added to each well to fully lyse the cells. After mixing by pipetting, the lysis buffer was collected and extracted using an adsorption column followed by washing and elution steps to obtain total RNA. Reverse transcription and RT-qPCR were performed using the method described in step 6 of Example 1, with the genes and related primers as shown in Table 6.

[0090] The measurement results are as follows Figure 11 , Figure 12 And as shown in Table 9: Table 9. Relative mRNA expression levels of different genes

[0091] The results showed little overall difference between the NC and Ve groups, indicating that 2% BSA and DMSO themselves had little effect on muscle atrophy and myogenesis-related gene expression in C2C12 cells. Compared with the NC and Ve groups, the PA group showed significantly increased expression of muscle atrophy degradation-related genes in C2C12 cells, with upregulated mRNA expression levels of Atrogin-1, Murf1, and Casp-3, suggesting that 0.5 mM PA treatment can activate molecular pathways related to muscle protein degradation in C2C12 cells and induce muscle atrophy-like molecular changes. After UA intervention, the expression levels of Atrogin-1, Murf1, and Casp-3 in the UA group were significantly lower than those in the PA group, indicating that UA can inhibit PA-induced upregulation of muscle atrophy degradation-related genes, thereby alleviating PA-induced muscle atrophy-like damage in C2C12 cells.

[0092] Further analysis of myogenesis-related factor expression revealed that the mRNA expression levels of Myf5, Myog, and Myod1 were lower in the PA treatment group than in the NC and Ve groups, indicating that PA inhibits myogenesis and muscle differentiation in C2C12 cells. After UA intervention, the expression of myogenic factors in the UA group was significantly increased, suggesting that UA can restore or enhance the expression of myogenesis-related genes suppressed under PA conditions.

[0093] In summary, PA treatment leads to changes in muscle atrophy-related molecules in C2C12 cells, manifested as upregulation of muscle degradation-related genes such as Atrogin-1, Murf1, and Casp-3, and downregulation of myogenic factors such as Myf5, Myog, and Myod1. UA intervention can reverse these abnormal changes, by inhibiting the expression of muscle atrophy-degrading genes and promoting the recovery of myogenic gene expression. These results indicate that UA has a mitigating effect on PA-induced muscle atrophy-like injury in high-fat C2C12 cells.

[0094] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Application of urolithin A in the preparation of products for the prevention, relief and / or improvement of sarcopenic obesity.

2. The application according to claim 1, characterized in that, The products include, but are not limited to, one or more of the following: pharmaceuticals, food, health products, feed, and feed additives.

3. The application according to claim 2, characterized in that, The drug is in the form of a liquid or solid formulation. Optionally, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, or oral liquids.

4. The application according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients; The excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

5. The application according to claim 2, characterized in that, The food products mentioned include health foods, foods for special medical purposes, functional foods, or pet foods. Preferably, the food products include grain products, starch products, vegetable products, fruit products, meat products, poultry products, egg products, and dairy products.

6. The application according to claim 2, characterized in that, The health products also contain acceptable excipients.

7. A drug for preventing, alleviating, and / or improving sarcopenic obesity, characterized in that, The drug contains urolithiasis A.

8. The medicament according to claim 7, characterized in that, The drug is in the form of a liquid or solid formulation. Optionally, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, or oral liquids.

9. The drug according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

10. The medicament according to claim 9, characterized in that, The excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

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