Network pharmacologyActive compound screening

The active compounds of Cuscuta chinensis and Dioscorea opposita were retrieved by integrating multiple databases, including the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP; http://lsp.nwu.edu.cn/tcmsp.php) (Ru et al. 2014), SymMap Database (http://www.symmap.org) (Wu et al. 2019), and Herb Bioactive Compounds Database (Herb; http://www.herb.org) (Fang et al. 2021). On the basis of ADME (absorption, distribution, metabolism, excretion) properties, screening thresholds were set as oral bioavailability (OB) ≥ 30% and drug likelihood (DL) ≥ 0.18 to obtain candidate compounds.

Target annotation and integration

Candidate compounds were subjected to target annotation and standardization via the UniProt database (https://www.uniprot.org), with species limited to Homo sapiens. Redundant entries were removed to establish the drug target set. Aging-related targets were acquired by searching the GeneCards (https://www.genecards.org), DrugBank (https://go.drugbank.com), Online Mendelian Inheritance in Man (OMIM, https://www.omim.org), Therapeutic Target Database (TTD, https://db.idrblab.org/ttd), and Pharmacogenomics Knowledgebase (PharmGKB, https://www.pharmgkb.org) databases (Wang et al. 2025). The search keywords included “aging”, “antiaging”, and “senescence”. After standardization and deduplication, the aging-related target set was constructed. The Venny 2.1 online tool (https://bioinfogp.cnb.csic.es/tools/venny) was used to analyze the intersection targets between the drug and disease.

Protein‒protein interaction (PPI) network construction

The intersection targets were imported into the STRING database (https://string-db.org), with the species set to Homo sapiens and a confidence score threshold > 0.4. Disconnected nodes were hidden to obtain the PPI data. The PPI network was visualized via Cytoscape software (v3.10.2). Six topological parameters, degree centrality, betweenness centrality, closeness centrality, eigenvector centrality, local average connectivity, and network centrality, were calculated via the built-in Network Analyzer plugin. Core targets, defined as those with values above the median for all six parameters, were selected to construct the core PPI network (Li et al. 2011).

Functional enrichment analysis

Gene Ontology (GO) enrichment analysis for the core targets was performed on the basis of the biological process (BP), molecular function (MF), and cellular component (CC) categories via the clusterProfiler R package (v4.0) (Yu et al. 2012). The significance threshold was set at a Benjamini‒Hochberg adjusted p value < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted via the DAVID 2021 online platform (https://david.ncifcrf.gov), with significant pathways selected at p < 0.05 (Huang et al., 2009).

Drugs and reagents

Cuscuta chinensis Lam. (Batch No. 231101CP292) and Dioscorea opposita Thunb. (Batch No. 240101CP017) were provided and pharmacognostically authenticated by the Pharmacy Department of the Traditional Chinese Medicine Hospital of Binhai New Area (Tianjin, China). D-gal was purchased from Sigma‒Aldrich (St. Louis, MO, USA; Cat. No. G0750). Vitamin E (VE, α-tocopherol acetate) was obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China; Cat. No. V8010). Prior to use, D-gal was dissolved in sterile 0.9% physiological saline for injection. VE was dissolved in distilled water containing 1% (v/v) Tween 80 for oral gavage.

Experimental animals and ethics

Specific pathogen-free (SPF) male C57BL/6 mice (8 weeks old, body weight 24–26 g) were purchased from Sipeifu Biotechnology Co., Ltd. (Beijing, China). The mice were acclimatized for one week under standard barrier-sustained conditions (temperature: 22 ± 1 °C, relative humidity: 50 ± 10%, 12 h/12 h light/dark cycle) with ad libitum access to autoclaved standard rodent chow and sterile water. All experimental procedures were strictly performed in accordance with internationally recognized principles of humane animal research (the 3Rs: Replacement, Reduction, Refinement) and adhered to guidelines including the Guide for the Care and Use of Laboratory Animals (National Research Council, NRC). The study protocol was formally reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of our institution (Ethics Approval No.: 2023 − 0317), thereby ensuring the welfare and rights of the animals were fully protected throughout the entire experimental period.

Preparation of TSZW-containing media

Cuscuta chinensis was processed by soaking it in glutinous rice wine for 12 h and then air-drying it for 12 h; this immersion‒drying cycle was repeated three times. After low-temperature stir-frying until slightly yellow, the mixture was pulverized and mixed with Dioscorea opposita powder at a 1:2 (w/w) ratio. Based on the human daily dose of Cuscuta chinensis (12 g) documented in Baopuzi, the mouse equivalent dose was calculated using the body surface area (BSA)-based dose equivalence method (conversion factor: 9.1) (Reagan-Shaw et al. 2008). The resulting daily mouse doses were 12.9 mg for Cuscuta chinensis and 25.8 mg for Dioscorea opposita. Considering the average daily food intake (5 g/d, determined from preliminary experiments), the medium-dose diet contained 247 mg Cuscuta chinensis and 494 mg Dioscorea opposita per 100 g of feed. Low- and high-dose diets were formulated at 10-fold gradients: low-dose (24.7 mg Cuscuta chinensis + 49.4 mg Dioscorea opposita/100 g) and high-dose (2.47 g Cuscuta chinensis + 4.94 g Dioscorea opposita/100 g).

The basal diet used was an SPF-grade mouse maintenance feed (Model: SPF-R02-001) produced by Sipeifu (Beijing) Biotechnology Co., Ltd. (Beijing, China; https://spfbiotech.com/), with a production license number of SCXK (Jing) 2019-0010. Its basic composition included corn, soybean meal, fish meal, lysine, multivitamins, mineral trace elements, and amino acids. All formulations were subjected to standardized mixing and pelleting processes by Sipeifu Biotechnology Co., Ltd. (Beijing, China) to ensure homogeneous distribution. The final products were hot-air-dried at 60 °C, vacuum-packed, and stored at 4 °C in a light-protected environment.

D-gal-induced aging model and group interventions

Following acclimatization, the animals were randomly divided into six groups (n = 20/group): the normal control (CTL), D-gal model (D-gal), low/medium/high-dose TSZW (TSZW-L/M/H), and VE (positive control) groups. The total experimental duration was 17 weeks. For dietary intervention, the CTL, D-gal, and VE groups were provided with the standard basal diet throughout the study, whereas the TSZW groups received corresponding low/medium/high-dose formulated diets. For aging induction, all groups except the CTL group were administered a daily subcutaneous injection of D-gal at 150 mg/kg from weeks 1 to 8 (Cao et al. 2025). For positive control intervention, the VE group underwent daily oral gavage with a VE suspension (prepared in distilled water containing 1% Tween-80) at 50 mg/kg from weeks 1 to 17 (Kong et al. 2018). During the same period, the CTL, D-gal, and TSZW groups underwent daily oral gavage with an equal volume of the vehicle (distilled water containing 1% Tween-80).

Behavioral assessments were conducted during the final week (week 17) of the intervention to evaluate changes in neurobehavioral and motor functions. At the end of the intervention period, all mice were anesthetized. Blood samples were collected from the abdominal aorta, after which euthanasia was performed via cervical dislocation under anesthesia in compliance with ethical guidelines. Liver and brain tissues were immediately harvested. A portion of the harvested tissue samples was rapidly frozen and stored at −80 °C, while the remaining portion was fixed in 4% paraformaldehyde. The wet weights of the gastrocnemius muscle, spleen, and thymus were also measured to calculate the respective organ indices (organ index = organ wet weight/body weight × 100%).

Behavioral assessmentsOpen field test (OFT)

The mice were individually placed at the center of a 30 cm × 30 cm open-field arena. Their spontaneous activity was recorded for 5 min using an overhead camera system linked to analysis software (Beijing Zhi Shu Duo Bao Biotechnology Co., Ltd.). The system automatically tracked and analyzed each mouse’s movement trajectory and behavioral performance in the open field. The analyzed parameters included total distance traveled, average speed, activity index, and number of resting bouts (Han et al. 2025).

Novel object recognition test (NOR)

The mice were acclimated to a 40 cm cubic arena containing two identical ceramic cups (familiar objects) for 5 min. After 24 h, one cup was replaced with a novel plastic cube. Sessions were recorded from a vertical angle via a mounted camera (Beijing Zhi Shu Duo Bao Biotechnology Co., Ltd., Beijing, China). Exploration time (defined as the nose actively approaching within 2 cm of the object with the head orientation directed toward it) was manually recorded with 0.1 s precision. The following parameters were calculated: time spent exploring the familiar object (Tfamiliar), time spent exploring the novel object (Tnovel), and recognition index (RI) = [Tnovel/(Tnovel + Tfamiliar)] × 100%.

Forelimb grip strength test (FGST)

The mice were positioned to grasp a triangular pull bar connected to a digital force gauge (Beijing Zhi Shu Duo Bao Biotechnology Co., Ltd., Beijing, China) with their forepaws. The tail was gently pulled horizontally until grip release occurred. The peak tension force at release was recorded. Three consecutive trials were performed per mouse, with the mean value used for statistical analysis (Han et al. 2025).

Detection of serum and tissue biochemical markersDetection of serum routine biochemical parameters

Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), creatinine (Cr), urea, total cholesterol (TC), and triglyceride (TG) were quantified via enzymatic colorimetric kits. Serum was obtained by centrifuging whole blood samples at 4 °C and 3000 rpm for 10 min. Assays were performed strictly according to the manufacturers’ instructions: ALT (Cat. No. C009-2-1) and AST (Cat. No. C010-2-1) from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); TG (Cat. No. E-BC-K261-M), TC (Cat. No. E-BC-K109-M), Cr (Cat. No. E-BC-K188-M), and Urea (Cat. No. E-BC-K183-M) from Elabscience Biotechnology Co., Ltd., (Wuhan, China). The absorbance was measured via a Bio-Rad microplate reader (Hercules, CA, USA), and the concentrations were determined on the basis of standard curves.

Detection of serum inflammatory cytokines

The serum levels of TNF-α, interleukin-1 beta (IL-1β), and IL-6 were quantified via commercial enzyme-linked immunosorbent assay (ELISA) kits (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China; TNF-α: Cat. No. SEKM-0034; IL-1β: Cat. No. SEKM-0002; IL-6: Cat. No. SEKM-0007) following the manufacturer’s protocols. The absorbance was measured via the aforementioned Bio-Rad microplate reader, and the concentrations were interpolated from standard curves.

Detection of tissue OS markers

Liver and brain tissues were homogenized in ice-cold phosphate-buffered saline (PBS) and centrifuged at 4 °C and 10,000 rpm for 5 min to obtain supernatants. The protein concentration of the supernatant was determined via the bicinchoninic acid (BCA) assay. Reactive oxygen species (ROS) levels in the homogenates were measured via a commercial assay kit (Wuhan Giled Biotechnology Co., Ltd., Wuhan, China; Cat. No. J25190). The activities of total superoxide dismutase (SOD, Cat. No. E-BC-K019-M) and glutathione peroxidase (GSH-Px, Cat. No. E-BC-K096-S), as well as malondialdehyde (MDA, Cat. No. E-BC-K025-M) levels, were quantified via colorimetric kits (Elabscience Biotechnology Co., Ltd., Wuhan, China). All tissue marker assays were performed according to the manufacturers’ instructions. The absorbance was measured via a Bio-Rad microplate reader, and the results were calculated on the basis of standard curves.

Western blot

Western blotting was performed as previously described (He et al. 2024). Briefly, proteins were extracted from tissues via RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China; Cat. No. P0013B). After quantification via the BCA method, equal amounts of protein were separated via SDS‒PAGE and transferred to PVDF membranes (MilliporeSigma, MA, USA; Cat. No. ISEQ00010). The membranes were blocked with 5% skim milk for 1 h, incubated with primary antibodies at 4 °C overnight, and then incubated with HRP-conjugated secondary antibodies (Proteintech Group, Wuhan, China; Cat. No. SA00001-2; RRID: AB_2722564) at room temperature for 1 h. Imaging was performed via an enhanced chemiluminescence (ECL) detection system (Zeta Life, California, USA). Antibody information is listed in Table S1.

Hematoxylin‒eosin (HE) staining

Paraffin Sect. (4 μm thick) were dewaxed and hydrated, stained with hematoxylin solution (Beyotime Biotechnology, Shanghai, China; Cat. No. C0107) for 5 min, rinsed with running water to remove floating dye, differentiated with 1% hydrochloric acid‒ethanol for 1 s, and then blued with saturated lithium carbonate solution for 30 s. After washing, the sections were immersed in eosin solution (Beyotime Biotechnology, Shanghai, China; Cat. No. C0109) for 4 min, sequentially dehydrated through an ethanol gradient, cleared in xylene three times (with each step lasting 5 min), and mounted with neutral gum. The staining results were observed, and images were captured via an optical microscope (Olympus BX53, Tokyo, Japan) to evaluate tissue morphology and pathological changes.

Immunohistochemistry (IHC)

IHC staining was performed as previously described (Han et al. 2025). Briefly, paraffin sections were sequentially dewaxed, hydrated, subjected to antigen retrieval, and permeabilized with 0.3% Triton X-100 for 10 min. Nonspecific sites were blocked with serum, after which the sections were incubated with primary antibodies at 4 °C overnight. The sections were then incubated with secondary antibodies (ZSBio Co., Ltd., Beijing, China; Cat. No. PV-6000) at 37 °C for 1 h. DAB chromogenic development was performed, followed by washing with PBS. Nuclei were counterstained with hematoxylin, rinsed, and blued in running water. The sections were dehydrated through an ethanol gradient, cleared in xylene, and mounted. The staining results were observed and recorded via an optical microscope (Olympus BX53, Tokyo, Japan) to analyze histopathological features. Antibody details are provided in Supplementary Table S1.

Immunofluorescence

Brain tissue sections were fixed, permeabilized, and blocked. The samples were then sequentially incubated with primary antibodies and CoraLite® Plus 594-conjugated secondary antibodies (Proteintech Group, Wuhan, China; Cat. No. RGAM004; RRID: AB_3073502). After DAPI staining (Beyotime Biotechnology, Shanghai, China; Cat. No. C1006), the sections were mounted with antifade reagent and imaged via a Zeiss confocal microscope (Carl Zeiss AG, Germany) for analysis (Han et al. 2025).

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT‒qPCR)

The experimental procedure was conducted as previously described (He et al. 2024). Total RNA was isolated via a Promega RNA extraction kit (Beijing, China; Cat. No. LS1040). The RNA concentration and purity were determined via a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), with A260/A280 ratios between 1.8 and 2.0 considered acceptable. RT‒qPCR was performed as follows: reverse transcription was conducted via a Takara PrimeScript RT reagent Kit (Shiga, Japan; RR037A) to synthesize cDNA. The cDNA was mixed with primers (Table S2) and Premix (Solarbio Life Sciences, Beijing, China; SR1121) and then amplified in a LightCycler 480 real-time PCR system (Roche, Basel, Switzerland) under the following conditions: 95 °C for 10 min, followed by 35 cycles of 95 °C for 15 s and 60 °C for 1 min. GAPDH was used as an internal reference gene. Relative gene expression was calculated via the 2^(-ΔΔCt) method.

Statistical analysis

Statistical analyses were performed via GraphPad Prism 9. The quantitative data are expressed as the means ± SDs. Group differences were assessed by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Statistical significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001. The sample sizes are provided in the figure legends.