Abstract Skeletal muscle aging frequently leads to a reduction in muscle mass and strength, significantly compromising the quality of life in elderly individuals. Skeletal muscle dysfunction during aging is widely recognized to be closely linked to chronic inflammation, oxidative stress and mitochondrial dysfunction. In this study, we confirmed the successful synthesis of M12 (muscle homing peptide)-modified EGCG (Epigallocatechin gallate) liposomes and validated their specific targeting to skeletal muscle through immunofluorescence analysis and in vivo imaging in small animal models. Both in vivo and in vitro experiments demonstrated that M12EGLP effectively suppressed the expression of inflammatory markers such as TNF-α and IL-6, thereby alleviating oxidative stress and restoring mitochondrial function in skeletal muscle. These effects ultimately contributed to the improvement of skeletal muscle dysfunction in aging mice. We have developed M12-modified EGCG liposomes (M12EGLP), a targeted drug delivery system capable of specifically accumulating in skeletal muscle, thereby enhancing the bioavailability and therapeutic potential of EGCG. M12EGLP enhances the exercise capacity of aging mice by reducing skeletal muscle inflammation, which subsequently alleviates oxidative stress and improves mitochondrial function. Therefore, as a novel and targeted drug delivery system, M12EGLP may provide a promising therapeutic strategy for the clinical management of age-related skeletal muscle dysfunction. Keywords: Epigallocatechin gallate, Liposome, Aging, Skeletal muscle, Inflammation Graphical abstract [37]Image 1 [38]Open in a new tab Highlights * • We prepared M12EGLP, which possess skeletal muscle targeting capability and exhibit excellent biosafety. * • M12EGLP enhances skeletal muscle function by mitigating inflammation and oxidative stress levels in aging skeletal muscle. * • M12EGLP may offer innovative approaches for the clinical management of aging-induced skeletal muscle dysfunction. 1. Introduction With advancing age, there is a progressive decline in skeletal muscle mass and function, which increases the risk of falls and fractures among the elderly and significantly compromises their quality of life. Inflammation and oxidative stress are widely recognized as key contributors to age-related skeletal muscle dysfunction [[39]1,[40]2]. Accumulating evidence indicates that elevated secretion of pro-inflammatory cytokines such as IL-6 and TNF-α in aging skeletal muscle critically inhibits myogenesis and accelerates muscle dysfunction [[41]3,[42]4]. The abnormal accumulation of these cytokines within skeletal muscle leads to increased levels of reactive oxygen species (ROS), further exacerbating muscle deterioration [[43]5,[44]6]. Therefore, targeting the inflammatory response represents a promising therapeutic strategy for ameliorating age-related skeletal muscle dysfunction. Unfortunately, no effective clinical interventions have been approved to date. However, natural compounds such as ursolic acid, resveratrol and epigallocatechin gallate hold significant therapeutic potential due to their anti-inflammatory and antioxidant properties [[45][7], [46][8], [47][9]]. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, has attracted considerable scientific interest due to its potent antioxidant and anti-inflammatory properties [[48]10,[49]11]. It is widely investigated for the treatment of neurodegenerative diseases, age-related disorders and cancers [[50]12]. However, the limited solubility and high susceptibility to oxidation of EGCG significantly reduce its bioavailability, thereby hindering its clinical application [[51]13]. Accumulating evidence suggests that polymer-based, lipid-based, inorganic, and carbon-based nanomaterials can markedly enhance drug stability and bioavailability [[52]14]. Among these, liposomes demonstrate superior cellular uptake and surface modifiability, making them a promising and biocompatible platform for in vivo drug delivery [[53]15]. Nevertheless, the lack of specific targeting ability in conventional liposomes restricts their utility in precision medicine. Therefore, surface modification of liposomes to achieve targeted delivery represents a promising strategy for improving therapeutic efficacy. Notably, muscle homing peptide (M12) has shown significant potential for skeletal muscle-specific targeting, owing to its capacity to bind bioactive molecules or receptors on the surface of muscle cells [[54]16]. In our study, we developed an M12-modified, EGCG-loaded liposome (M12EGLP). This formulation demonstrates enhanced skeletal muscle targeting, thereby increasing the bioavailability of EGCG in skeletal muscle and subsequently improving its therapeutic efficacy. Our findings show that M12EGLP modulates the microenvironment of aging skeletal muscle by suppressing inflammation, alleviating oxidative stress, and enhancing mitochondrial function. Moreover, it promotes the expression of myokines, leading to the amelioration of age-related skeletal muscle dysfunction both in vivo and in vitro ([55]Scheme 1). This research provides a novel and precise therapeutic strategy for targeting skeletal muscle to combat age-associated muscle decline. Scheme 1. [56]Scheme 1 [57]Open in a new tab The preparation and mechanism of M12EGLP. 2. Materials and methods 2.1. Materials and reagents Epigallocatechin gallate (EGCG, purity >99.5 %) was procured from MACKLIN. C2C12 cells were sourced from the Cell Bank of the Chinese Academy of Sciences. The Oil Red O staining kit, β-galactosidase staining kit, mitochondrial membrane potential (JC-1) detection kit, and reactive oxygen species (ROS) detection kit were obtained from Beyotime Biotechnology. Hematoxylin-eosin (H&E) staining kits, malondialdehyde (MDA) content determination kits, superoxide dismutase (SOD) activity measurement kits, total glutathione (GSH) content determination kits Na^+-K^+-ATPase and ATP kits, Mitochondrial respiratory chain complexes I and IV kits were acquired from Solarbio (Beijing, China). Antibodies against HO-1, Nrf2, IL-6, IL-10, TNF-α, β-tubulin, p53, p38 and MYHⅠ/Ⅱ were purchased from Affinity Biosciences (Jiangsu, China). Myostatin (GDF8) and GAPDH antibodies were obtained from Proteintech, while MyoD and MyoG antibodies were sourced from Santa Cruz Biotechnology. 2.2. Network pharmacological analysis Based on data from the TCMSP database ([58]https://tcmspw.com/tcmsp.php), CNKI ([59]http://www.cnki.net), and PubMed ([60]https://pubmed.ncbi.nlm.nih.gov), we conducted a preliminary screening of the potential activity of EGCG. We utilized PubChem ([61]https://pubchem.ncbi.nlm.nih.gov) to retrieve its chemical properties and employed the SwissTargetPrediction database ([62]http://www.swisstargetprediction.ch) to predict potential targets for the active ingredients, selecting those with a target score greater than 0. For aging-related skeletal muscle dysfunction, we retrieved gene information from GeneCards ([63]https://www.genecards.org) and DisGeNET ([64]https://www.disgenet.org), removing any duplicate data. The drug targets and active gene data were imported into Venny2.1.0 ([65]https://bioinfogp.cnb.csic.es/tools/venny). The intersection of drug-related genes and disease-associated genes was identified. This cross-gene data was subsequently imported into STRING ([66]https://cn.string-db.org), with the species restricted to “human,” to construct protein-protein interaction (PPI) networks, and TSV format files were exported. These TSV files were then imported into Cytoscape 3.9.0 software for PPI network topology analysis, focusing on nodes with centrality measures greater than two medians, one median, and those close to or exceeding the median centrality. For functional enrichment analysis, the cross-gene dataset was uploaded to DAVID ([67]https://david.ncifcrf.gov), using “human” as the background organism and setting a significance threshold of p < 0.05 for GO and KEGG pathway analyses. Finally, the results were visualized and analyzed using the microbial system platform ([68]http://www.bioinformatics.com.cn), which provided insights into biological processes (GO-BP), cellular components (GO-CC), molecular functions (GO-MF), and associated signaling pathways (KEGG). The 2D structure document of EGCG downloaded from PubChem was opened with ChemOffice18.0, and MinimizeEnergy was selected for energy optimization and saved. The corresponding human target proteins of EGCG and the key target genes of aging skeletal muscle were downloaded from the RCSB protein database ([69]https://www.rcsb.org/). PyMOL1.0 and AutoDockTools1.5.6 software were used for molecular docking and visualization of key targets. 2.3. Preparation of M12EGLP To prepare EGLP-NH2, 200 mg of soybean lectins, 40 mg of cholesterol, 40 mg of EGCG, and 5 mg of DSPE-PEG-NH2 (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine) were dissolved in 4 mL of absolute ethanol under stirring conditions. Subsequently, 20 mL of water at 37 °C was slowly injected using a syringe at a constant rate while maintaining continuous stirring for 4 h. The ethanol was then removed by rotary evaporation. Blank liposomes (LP) without EGCG and DSPE-PEG-NH2 were prepared using the same method and EGLP without DSPE-PEG-NH2 was also obtained. NHS and EDC (5 mg/mL) were added to EGLP-NH2 to activate the embedded amino groups. Finally, M12EGLP was synthesized by dissolving M12 in water and incubating it with activated EGLP-NH2 for 6 h at room temperature. 2.4. Characterization UV–visible spectroscopic data were acquired using a PerkinElmer Lambda 605S UV–Visible spectrophotometer at ambient temperature. The crystallographic structures of the samples were analyzed via X-ray diffraction (XRD) using a Shimadzu instrument (Kyoto, Japan) and differential scanning calorimetry (DSC). Additionally, dynamic light scattering (DLS) measurements were conducted with a Malvern Nano ZS90 instrument (Worcestershire, UK) to determine the particle size distribution. Morphological features of the samples were examined by transmission electron microscopy (TEM) utilizing a Tecnai G2F30 microscope (FEI, USA). 2.5. In vitro drug release studies Drug release experiments were conducted using a shaker apparatus. The dissolution medium consisted of 300 mL of phosphate-buffered saline (PBS) at 37 °C. Aliquots (2 mL) were withdrawn at predetermined time intervals (5, 15, 30, 45, 60, 90 min and 2, 4, 6, 8, 12, 18, 24 h), and immediately filtered through a 0.22 μm microporous membrane. An equivalent volume of fresh PBS was added to maintain a constant dissolution medium volume. The concentration of the released drug was quantified using a UV–visible spectrophotometer at a wavelength of 272 nm. 2.6. Hemolysis assay The hemolysis assay was conducted to evaluate the blood compatibility of materials. Whole blood was collected from healthy male Sprague-Dawley (SD) rats via orbital sinus puncture and stored in heparinized anticoagulant tubes. The blood samples were washed twice with phosphate-buffered saline (PBS) at 3000 rpm for 15 min. Following removal of the supernatant, erythrocytes were resuspended in PBS to achieve a final concentration of 2 %. A volume of 200 μL of the 2 % erythrocyte suspension was incubated with 200 μL of material solutions at various concentrations (EGCG: 5, 10, 20, 50, 100, 200, 300 μg/mL) for 4 h at 37 °C. After incubation, the samples were centrifuged at 3000 rpm for 15 min, and the absorbance of the supernatant was measured at 570 nm using a microplate reader. Phosphate-buffered saline (PBS) and distilled water (ddH[2]O) served as negative and positive controls, respectively. The hemolysis rate (%) was calculated as follows: (OD[sample] - OD[PBS])/(OD[ddH2O] - OD[PBS]). 2.7. Cell culture C2C12 cells, obtained from the Cell Bank of the Chinese Academy of Sciences, were maintained in growth medium (DMEM supplemented with 10 % fetal bovine serum and 1 % penicillin-streptomycin) at 37 °C in a humidified atmosphere containing 5 % CO2. Differentiation was induced using differentiation medium (DMEM supplemented with 2 % horse serum and 1 % penicillin-streptomycin). To induce cellular senescence, the cells were exposed to D-galactose (20 g/L) for 24 h, followed by treatment with EGCG, EGLP, or M12EGLP for an additional 24 h. The experimental groups included Control, D-gal, EGCG (D-gal + EGCG), EGLP (D-gal + EGLP) and M12EGLP (D-gal + M12EGLP). 2.8. CCK-8 assay C2C12 cells were seeded in a 96-well plate at a density of 10,000 cells per well. Equal volumes of varying concentrations of EGCG, EGLP, and M12EGLP were added to each well (including blank and control groups). The plate was then incubated in a humidified atmosphere containing 5 % CO[2] at 37 °C for 12 h. Subsequently, 10 μL of CCK-8 reagent was added to each well and the plate was further incubated at 37 °C for 30 min. Finally, absorbance was measured at 450 nm using a microplate reader. 2.9. Cellular uptake experiments FITC was incubated with M12EGLP or EGLP-NH2 overnight to achieve fluorescent labeling. The cellular uptake of M12EGLP by C2C12 cells was assessed by quantifying the fluorescence intensity using confocal laser scanning microscopy (CLSM). C2C12 cells were incubated with either EGLP or M12EGLP solutions for 3 h. Following fixation and staining, CLSM images were acquired, and the fluorescence intensity of each sample was analyzed using ImageJ software. 2.10. Flow cytometry analysis C2C12 cells were induced with 2 % horse serum. Subsequently, the cells were incubated with FITC-labeled EGLP or M12EGLP solutions for a period of 3 h. Following incubation, the cells were washed three times with PBS to ensure complete removal of unbound FITC. The cells were then digested using 0.25 % trypsin-EDTA, centrifuged and resuspended in PBS. Finally, the cell suspension was transferred to a flow cytometry tube and analyzed by flow cytometry. We used FSC-A/SSC-A to exclude fragmented cells, then used single gating (FSC-H and FSC-A), and finally used FITC and gating to select cells labeled with FITC as the uptake cells. 2.11. Animal experiments Eight-week-old male C57BL/6 mice were procured from Liaoning Changsheng Biological Co., Ltd. The animals were housed under controlled conditions with ad libitum access to water and standard chow, maintained at a constant temperature and humidity, and exposed to a 12-h light/dark cycle. After one week of acclimatization, nine mice were randomly assigned to the control group and continued on a normal diet. Body weights were recorded weekly. The remaining mice received intraperitoneal injections of D-galactose (D-gal) at a dose of 400 mg/kg for eight weeks to induce aging. Mice in the treatment groups were administered EGCG (10 mg/kg) via tail vein injection every other day for six weeks. The experimental groups included: Control, D-gal, EGLP (D-gal + EGLP), and M12EGLP (D-gal + M12EGLP). All procedures adhered to the guidelines set forth by the Animal Ethics Committee of Jinzhou Medical University (Issue No. 230911). 2.12. SA-β-gal staining Fresh gastrocnemius muscle tissue from the Control and D-gal groups were taken for frozen sectioning and stained with the SA-β-gal kit according to the kit's instructions. The stained sections were observed under a fluorescence microscope. 2.13. In vivo targeting and uptake assays To evaluate the targeting efficacy of M12EGLP, a dose of 10 mg/kg was administered via tail vein injection in mice. At predetermined time points (3, 6, 9, 12, 18, and 24 h post-injection), gastrocnemius muscle, heart, liver, spleen, lung and kidney tissues were harvested. Fluorescence signals from these tissues were quantified at excitation/emission wavelengths of 488 nm/525 nm using an in vivo imaging system (IVIS Spectrum, PerkinElmer). For further analysis, gastrocnemius muscle sections were processed as follows: they were rewarmed to room temperature for 1 h, washed three times with PBS, permeabilized with 0.3 % Triton X-100 for 15 min and washed again three times with PBS. Sections were then blocked with diluted sheep serum (1:10) for 2 h, incubated with the primary antibody overnight at 4 °C, followed by three washes with PBS. Subsequently, sections were incubated with the secondary antibody for 2 h at room temperature and washed three times with PBS. Nuclei were stained with DAPI, and coverslips were mounted. Fluorescence intensity was analyzed using ImageJ software. 2.14. Measurement of ROS Cell: C2C12 cells were first induced with D-gal for 24 h. Subsequently, the cells were incubated with PBS, EGCG, EGLP, and M12EGLP for 12 h. The cell culture medium was then replaced, and 10 μM of the DCFH-DA (2′,7′-Dichlorofluorescein diacetate) probe was added. Cells were incubated at 37 °C for 30 min. Following this, the cells were washed three times with PBS to ensure complete removal of the DCFH-DA probe before observation under an inverted microscope. Gastrocnemius muscle: The frozen sections of gastrocnemius muscle were thawed for 1 h, washed with PBS three times, loaded with DHE probe, and incubated at 37 °C for 30 min. The sections were washed with PBS three times to thoroughly remove the DHE probe. The fluorescence intensity of each group was observed under an inverted microscope and analyzed using ImageJ software. 2.15. Measurement of mitochondrial membrane potential Cell: Following a 24-h induction of C2C12 cells with D-gal, the cells were subsequently treated with PBS, EGCG, EGLP, and M12EGLP for an additional 12 h. The culture medium was then replaced, and the mitochondrial membrane potential was assessed using the JC-1 probe by incubating the cells at 37 °C for 30 min. After incubation, the cells were washed three times with PBS to ensure complete removal of the JC-1 probe, followed by observation under an inverted microscope. Gastrocnemius muscle: Take the gastrocnemius muscle tissues from each group, add the gastrocnemius muscle tissue extract, homogenize, extract mitochondria using the mitochondrial extraction kit, then add the JC-1 probe, incubate at 37 °C for 30 min, wash three times with PBS to thoroughly remove the JC-1 probe, and determine JC-1 by chemiluminescence. 2.16. Detection of oxidative stress indicators Take the C2C12 cells or gastrocnemius muscle tissues from each group for the detection of SOD activity, MDA level and GSH level, and measure them by ultraviolet spectrophotometry. Collect the gastrocnemius muscle extract for the kits (SOD, MDA, GSH). Use the microplate reader for the detection. 2.17. Detection of ATP and Na ^+ -K^+-ATPase Take the C2C12 cells or gastrocnemius muscle tissues from each group and use the Na^+-K^+-ATPase and ATP content kit for detection. Follow the kit's instructions to measure the ATP content and Na^+-K^+-ATPase activity. 2.18. Detection of mitochondrial respiratory chain complex activity Take the gastrocnemius muscle tissues from each group and use the Mitochondrial respiratory chain complexes I and IV kit for detection. Follow the kit's instructions to measure the Mitochondrial respiratory chain complexes I and IV activity. 2.19. Behavioral experiments 2.19.1. Hounder experiment In this experiment, the hounder apparatus was utilized to evaluate the motor coordination and grip strength of mice. Mice were placed on the hounder with their two forelimbs, and subsequently released to observe any behavioral changes. Scoring criteria were as follows: falling within 0–10 s (0 points), remaining on the hounder for more than 10 s (1 point), two hind limbs clinging to the hounder (2 points), three limbs clinging to the hounder (3 points), all four limbs clinging to the hounder (4 points), and tail curling with crawling from one end of the hounder to the other (5 points). 2.19.2. Grid suspension experiment Mice were positioned on a grid, which was subsequently inverted. The latency period until the mice fell from the grid was meticulously recorded. The maximum time limit is set at 10 min. 2.19.3. Slope experiment The mouse was positioned on one side of a smooth plastic plate, and the plate was gradually tilted to form an angle between the plate and the horizontal surface. The angle at which the mouse was unable to maintain its position on the plate for 5 s and began to slip downward was recorded. 2.19.4. Swimming endurance experiment The mice were placed in a 30 cm high plastic container filled with water to a level where they could not reach the bottom. The time at which the mice sank to the bottom for 10 s was recorded as the endpoint and the duration of fatigue-induced swimming was documented. 2.20. Gastrocnemius muscle weight ratio Mice were euthanized via an overdose of 3 % sodium pentobarbital anesthesia, following which the heart, liver, spleen, lung, kidney, and gastrocnemius muscles were excised. The dimensions and mass of the gastrocnemius muscles were meticulously recorded. The treatment protocol was standardized relative to the individual body weight of each mouse. 2.21. Hematoxylin-eosin (H&E) staining Mice were anesthetized with 3 % sodium pentobarbital and subsequently perfused with 4 % paraformaldehyde (PFA) via cardiac puncture. Harvested tissues, including the heart, liver, spleen, lung, kidney and gastrocnemius muscle, were fixed in 4 % PFA, processed for paraffin embedding, sectioned and subjected to routine deparaffinization. The sections were stained following the manufacturer's protocol and examined under a light microscope. 2.22. Oil Red O staining The harvested gastrocnemius muscle tissue was fixed in 4 % PFA and subsequently processed for frozen sectioning. The frozen sections were stained following the protocol provided in the Oil Red O staining kit. Microscopic examination was conducted to observe the stained sections. 2.23. Immunofluorescence Cells and tissues were washed three times with PBS for 5 min each. Triton X-100 (1 % for cells and 3 % for tissues) was applied in the dark for 15 min, followed by three additional washes with PBS for 5 min each. Blocking was performed for 2 h using Zhongshan Jinqiao blocking solution for cells and 10 % goat serum for tissues. The following primary antibodies were added at the indicated dilutions: IL-6 (1:200), IL-10 (1:200), TNF-α (1:200), Nrf2 (1:200), HO-1 (1:200), MYH Ⅰ (1:200), MYH Ⅱ (1:200), p53 (1:200), p38 (1:200), and β-tubulin (1:1000). Samples were incubated overnight at 4 °C and then washed three times with PBS for 5 min each. After recovering the primary antibody, a secondary antibody (1:1000) was added and incubated at room temperature for 2 h. Following three more PBS washes for 5 min each, DAPI was added for nuclear staining, and samples were observed under a fluorescence microscope. 2.24. Western Blot analysis was conducted as follows RIPA lysis buffer was utilized to extract proteins from cells and tissues in each group. Protein concentrations were quantified using the BCA Protein Assay Kit. Proteins were separated via SDS-PAGE and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5 % skim milk powder. Primary antibodies against Nrf2 (1:1000), HO-1 (1:1000), TNF-α (1:1000), IL-6 (1:1000), IL-10 (1:1000), MyoD (1:1000), MyoG (1:1000), Myostatin (GDF8) (1:1000), β-tubulin (1:50000), and GAPDH (1:50000) were incubated overnight at 4 °C. After washing, the corresponding secondary antibodies, including anti-mouse IgG (1:10000) and anti-rabbit IgG (1:500), were added and incubated for 2 h at room temperature on a shaker. Protein expression levels were visualized using Super ECL detection reagent. 2.25. Statistical analysis All data from the experiments were subjected to statistical analysis using GraphPad Prism 8.0 software and are presented as mean ± SD. Differences among multiple groups were evaluated using one-way analysis of variance (ANOVA). Comparisons between groups were made by LSD-t or Tambane's analysis. if the variance was not uniform, Kruskal-Wallis test was used. p < 0.05 indicated that the difference was statistically significant. 3. Results 3.1. Exploring the mechanism of EGCG in alleviating skeletal muscle aging based on network pharmacology and molecular docking The therapeutic effect of EGCG on aging skeletal muscle likely involves multiple pathways and molecular targets. Through systematic database searches, we identified 178 target genes for EGCG, 2070 target genes associated with skeletal muscle dysfunction and 1609 genes related to aging. A total of 72 overlapping genes were identified between EGCG and aging skeletal muscle ([70]Fig. 1A). These 72 overlapping genes were then imported into the STRING database to construct a protein-protein interaction (PPI) network ([71]Fig. 1B). The PPI network data from STRING were subsequently imported into Cytoscape software to generate both a protein interaction network and an EGCG-target-disease network diagram ([72]Fig. 1C). Finally, the overlapping genes were analyzed using the DAVID database to perform Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis ([73]Fig. 1D and E). In the GO enrichment analysis, 559 biological processes, 61 cellular components, and 96 molecular functions were significantly enriched. The EGCG targets involved in the treatment of aging skeletal muscle were primarily associated with cell proliferation, response to reactive oxygen species, DNA-binding transcription factor activity, phosphatase binding, and transcription regulatory complexes. KEGG pathway enrichment analysis revealed a total of 159 signaling pathways, predominantly including endocrine resistance, MAPK signaling, PI3K-AKT signaling, and cancer-related pathways. These findings suggest that EGCG may exert its therapeutic effects on aging skeletal muscle through multiple molecular mechanisms and signaling pathways. Additionally, molecular docking studies were conducted to investigate the binding affinity of EGCG to potential molecular targets in aging skeletal muscle. The results indicated that the binding energies between EGCG and TNF-α as well as IL-6 were both less than −5 kcal/mol, demonstrating strong binding affinities. These results suggested that the therapeutic efficacy of EGCG on aging skeletal muscle may be mediated, at least in part, through interactions with TNF-α and IL-6 ([74]Fig. 1F and G). Fig. 1. [75]Fig. 1 [76]Open in a new tab Network pharmacological analysis and molecular docking of EGCG and aging skeletal muscle dysfunction. (A) Venn diagram illustrating the intersection genes between EGCG and aging skeletal muscle dysfunction. (B) Key therapeutic targets identified from the protein-protein interaction (PPI) network. In this network, edges represent associations between target genes, while nodes represent individual target genes. Nodes with a higher number of connected edges have a greater degree centrality and are likely to be central in the network. (C) Visualization of key protein interaction networks. The node size and color intensity in the graph correlate positively with the degree centrality, where larger and darker nodes indicate higher connectivity. (D) Gene Ontology (GO) enrichment analysis of potential targets for EGCG in treating aging skeletal muscle dysfunction. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of potential targets for EGCG in treating aging skeletal muscle dysfunction. (F) Binding mode of EGCG with TNF-α. (G) Binding mode of EGCG with IL-6. (For interpretation of the references to color in this