The components of HP were identified using the keywords “shudihuang,” “cangzhu,” “ganjiang,” and “dazhao” across three databases: Traditional Chinese Medicine Systems Pharmacology (TCMSP) (http://tcmspw.com/tcmsp.php, accessed: 2025-03) (17), Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine (http://www.tcmip.cn/TCMIP/index.php, accessed: 2025-03) (18), and A Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (http://bionet.ncpsb.org.cn/batman-tcm/, accessed: 2025-03) (19) databases. Active ingredients were screened using pharmacokinetic core parameters (absorption, distribution, metabolism, excretion (ADME)): oral bioavail-ability (OB) ≥30% and drug similarity (DL) ≥0.18 (8). The corresponding target protein of each active ingredient was then obtained from the TCMSP database. Finally, the UniProt database (https://www.uniprot.org/, accessed: 2025-03) was used to unify the names of the target proteins. Duplicate items were removed, and the data were merged and converted into gene symbols.

Relevant targets for OP were identified by searching the GeneCards (http://www.genecards.org, accessed: 2025-03), OMIM (http://www.omim.org, accessed: 2025-03), and DrugBank (https://go.drugbank.com, accessed: 2025-03) databases with the keyword “Osteoporosis”. Subsequently, the target data were standardized by unifying protein names, deduplicating entries, merging datasets, and converting gene symbols through the UniProt database.

The Venny 2.1 system (https://bioinfogp.cnb.csic.es, accessed: 2025-03) was used to generate a Venn diagram to identify shared targets between HP active components and OP-related targets. Cytoscape 3.7.1 software (http://cytoscape.org) was used to map the relationships between drugs, active components, common targets, and OP, resulting in a drug-active component-target-disease network.

The shared HP-OP targets were uploaded to the STRING database (https://string-db.org, accessed: 2025-03) to construct a protein-protein interaction (PPI) network. The species was limited to Homo sapiens with a medium confidence interaction score threshold (>0.4). Subsequent topological analysis of the network was conducted using Cytoscape 3.7.1, and the CytoNCA plugin was used to calculate the node degree.

Shared HP-OP targets were imported into Metascape (http://metascape.org, accessed: 2025-03) for functional enrichment analysis. Gene ontology (GO) terms (biological process, molecular function, and cellular component) and Kyoto encyclopedia of genes and genomes (KEGG) pathways were analyzed using Homo sapiens settings, and the results were visualized.

Twenty-four 3-month-old female Sprague-Dawley (SD) rats (body weight: 240–300 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (license number: SCXK (Jing) 2021-0006). The rats were sheltered in the SPF-grade laboratory of the Animal Experiment Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine. The environmental conditions included constant temperature and humidity (room temperature: 22–26 °C; relative humidity: 50 ± 5%, a 12-hour light/dark cycle, and ad libitum access to food and water).

According to the 3R principles for laboratory animals (20), 24 female SD rats were randomly divided into four groups (n = 6 per group) using a random number table: NC-Group, model, experimental, and positive control groups. The Experimental Animal Ethics Committee of the Shandong University of Traditional Chinese Medicine (approval number: SDSZYYAWE20231219001) approved this study protocol.

HP was composed of Rehmanniae (熟地黄, 32g), Atractylodes (苍术, 32g), Dried ginger (干姜, 2g), and jujube (大枣, 34g). Herbal pieces were purchased from the pharmacy of the Affiliated Hospital of Shandong University of TCM and authenticated by TCM pharmacists in the hospital. The preparation was conducted in a decoction room, and the four herbs were soaked in 10 volumes of water for 30 min. The mixture was decocted twice (2 h each time), filtered through a gauze, and the filtrates were combined. The resulting solution was at a final concentration of 1 g/mL and stored at 4 °C for later use.

Alendronate Sodium Tablets (Beijing Fuyuan Pharmaceutical Co., Ltd.; National Drug Approval No. H20059029) were used as positive controls.

Micro-Computed Tomography (CT) (Model: NC-200), Multicolor Fluorescence Imaging System (Model: GE Amersham Imager 600), Embedding Machine (Model: JB-P5), Paraffin Microtome (Model: Leica RM2255), Automated Slide Analysis System (Model: HS6), and Bicinchoninic Acid (BCA) Protein Quantification Kit (Catalog No. P0010; Beyotime Biotechnology, Shanghai, China), Anti- hypoxia-inducible factor (HIF)-1α Antibody (cat. no. ab179483; Abcam, Cambridge, UK), anti-AGER/RAGE (cat. no. 83759-5-RR; Proteintech, USA), Anti- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Antibody (cat. no. 10494-1-AP, Proteintech, USA), horseradish Peroxidase (HRP)-Labeled goat anti-rabbit IgG (cat. no. 20536-1-AP, Proteintech, USA).

After one week of acclimatization, rats in the model, experimental, and positive control groups were anesthetized using an intraperitoneal injection of Zoletil (15 mg/kg) + Xylazine Hydrochloride (5 mg/kg). After successful anesthesia, the dorsal area was shaved and disinfected. A longitudinal incision (approximately 1.5 cm) was made below the ribs on both sides of the spine. The subcutaneous tissues and muscles were bluntly dissected to expose the ovaries. Both fallopian tubes were ligated, and bilateral ovariectomy was conducted to establish an ovariectomized OP model. Hemostasis, irrigation, and disinfection were completed, followed by layered suturing of the muscles and skin. Postoperatively, the rats received intramuscular penicillin (8×104U/rat) for 3 consecutive days to prevent infection.

Drug interventions commenced 7 days after modeling. Based on body surface area conversion (21), the experimental group received 10.43 g/kg/day of HP decoction through oral gavage. The positive control group was administered 7.35 mg/kg alendronate sodium (prepared by suspending finely ground tablets in purified water to 2 mg/mL, 3.675 mL/kg) once a week. The NC-Group and model groups received equivalent volumes of saline daily through oral gavage. All treatments were administered for 12 weeks.

After gavage, the rats were euthanized by cervical dislocation. The hair of their bodies was shaved, and the entire body was disinfected by immersing the rats in 75% alcohol. The left and right femurs were then harvested. For the right femur, it was fixed with 4% paraformaldehyde fixative at 4 °C for over 48 h, followed by Micro-CT imaging analysis. Subsequently, the femur was decalcified with 10% EDTA decalcification solution at 4 °C for 4 weeks, and paraffin tissue sections were prepared. The left femur was immediately immersed in liquid nitrogen for quick freezing and stored at -80 °C for future use.

The right femurs from each group was subjected to micro-CT scanning. Then, three-dimensional reconstruction was conducted using NRecon software (version 1.20.3.0; Bruker MicroCT, Belgium) on selected regions of interest. Quantitative analysis of bone microstructure parameters was conducted with CT Analyzer (version 1.20.3.0) under standardized thresholds. BMD, BV/TV, and trabecular number (Tb.N) were calculated.

Paraffin-embedded right femur samples were sectioned into 3.0–4.0 μm-thick slices using a microtome. The sections were stained with hematoxylin and eosin (H&E) per the manufacturer’s protocol. Histopathological evaluation of the trabecular bone morphology and quantity was conducted using a light microscope.

The left femurs of rats in each group were taken, sequentially crushed into a mortar, rapidly ground into a powder with multiple additions of liquid nitrogen, mixed with lysis buffer, and oscillated with a homogenizer at 4 °C. Oscillation was conducted once every 5 min for six times, and then centrifuged for 10 min. The supernatant was collected to extract proteins, and the protein concentration was determined using the BCA method. Loading buffer was added, and the mixture was boiled for 10 min for denaturation. Twenty micrograms of protein were loaded, subjected to Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis, and electrotransferred to a polyvinylidene fluoride membrane. The membrane was blocked with 50 g/L skim milk solution at room temperature for 3 h, incubated with primary antibody at 4 °C overnight, washed, and incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. After washing, the protein bands were visualized using electrochemiluminescence. GAPDH was used as an internal reference, and Image J analysis software was used to analyze the gray values of the bands in each group.

ADME-based screening produced 30 qualifying HP constituents: rehmanniae (two components), atractylodes (nine components), dried ginger (six components), and jujubae (19 components) (Table 1). Target proteins were annotated using UniProt, and 228 unique targets remained after deduplication.

A Search for “Osteoporosis” identified 5,936 targets in GeneCards, 45 in OMIM, and 115 in the Drug Bank. These datasets were consolidated, standardized using UniProt, and deduplicated, resulting in 6,032 non-redundant targets for further analysis.

Venny 2.1 identified 178 shared HP-OP targets (Figure 1A), which were designated as key therapeutic targets. A drug-ingredient-target-disease network was constructed in Cytoscape 3.7.1 (Figure 1B) using these targets, HP drugs, bioactive ingredients, and OP nodes. The network (213 nodes, 601 edges) used color coding: green (HP drugs), purple (bioactives), blue (targets), and red (OP), with edges indicating functional interactions. The diagram shows that the effective ingredients of HP intervene in OP by acting on key targets.

The overlapping targets of HP and OP were imported into the STRING platform to create a PPI network (Figure 2A). This network included 177 nodes and 4281 edges. Cytoscape 3.7.1 topological analysis prioritized biologically significant hub genes using the degree of centrality (DC) calculated using CytoNCA. A filtered subnetwork (Figure 2B) visualizes the target importance through graduated node sizes and colors. A bar chart was created following the top 20 target proteins ranked by DC (Figure 2C). As shown in the bar chart, RAC-alpha serine/threonine-protein kinase (Akt1) ranks among the top targets. Therefore, Akt1 is a key target of HP for treating OP.

The intersecting targets of HP and OP were uploaded to the Metascape platform for GO functional enrichment analysis; the top 20 items were selected for analysis, and a bar chart was drawn (Figure 3A). Using the screening criteria of Min Overlap: 3, P-Value Cutoff: 0.01, and Min Enrichment: 1.5, KEGG pathway enrichment analysis was conducted, the top 20 representative pathways were selected as key pathways, and they were visualized by drawing a bubble chart (Figure 3B). GO functional analysis revealed that HP regulates OP mainly by responding to hormones in biological processes, acting on membrane rafts in cellular components, and binding to transcription factors in molecular functions. KEGG pathway enrichment analysis of the intersecting targets revealed that the primary pathways involved included pathways in cancer, lipid, and atherosclerosis, the Advanced Glycation End products/Receptor for Advanced Glycation End products (AGE-RAGE) signaling pathway in diabetic complications, chemical carcinogenesis-receptor activation, and the HIF-1 signaling pathway.

Three-dimensional reconstruction of the distal femur using micro-CT revealed that compared to the NC-Group, the model group showed significant reductions in trabecular bone quantity, disrupted continuity, and enlarged trabecular spacing. Conversely, the experimental and positive control groups showed increased trabecular bone density, improved trabecular continuity, and reduced trabecular spacing than the model group (Figures 5A–D).

Quantitative analysis of the bone microstructure showed that the model group had significantly lower trabecular BMD, Tb.N, and BV/TV than the NC group did (P = 0.002, 0.000, 0.002 < 0.05, r=0.95, 0.99, 0.97, respectively). Compared to the model group, the BMD, Tb.N, and BV/TV of rats in the experimental group were significantly higher (P = 0.000, 0.004, 0.021< 0.05, r=0.99, 0.93, 0.83, respectively). In the positive control group, the BMD and Tb.N of rats showed an upward trend (P = 0.001, 0.022 < 0.05, r=0.95, 0.83, respectively), while the upward trend of BV/TV was not statistically significant (P = 0.170 > 0.05, r= 0.56) (Figures 5E–G).

Compared with the control group, the model group showed significantly reduced trabecular bone quantity, thinned trabecular morphology, widened and fragmented intertrabecular spaces, and disrupted trabecular networks. Conversely, the experimental and positive control groups showed increased trabecular bone quantity, thickened trabecular morphology, enhanced continuity, and partial restoration of the trabecular network compared with the model group. Notably, the experimental group showed markedly thickened trabecular morphology, while the positive control group showed improved trabecular quantity and more pronounced network restoration (Figures 5H–K).

The expression of the AGE-RAGE signaling pathway and HIF-1 signaling pathway proteins in bone tissue were examined to further validate the results of the network pharmacology KEGG analysis and investigate the potential mechanism by which HP improves OP. The results showed (Figures 5L–N) that, compared to the NC-Group, the expression of HIF-1 signaling pathway proteins in the model group was significantly decreased (P = 0.028 < 0.05, r=0.81), while the expression of AGE-RAGE signaling pathway proteins significantly increased (P = 0.000 < 0.05, r=0.98). Compared with the model group, the expression of HIF-1 signaling pathway proteins in the experimental group increased (P = 0.005 < 0.05, r=0.92), and the expression of AGE-RAGE signaling pathway proteins was significantly decreased (P = 0.028 < 0.05, r=0.81); in the positive control group, the expression of HIF-1 signaling pathway proteins increased (P = 0.036 < 0.05, r=0.98), and the expression of AGE-RAGE signaling pathway proteins was significantly decreased (P = 0.033 < 0.05, r=0.79). The experimental group showed a more significant effect on upregulating the proteins of the HIF-1 signaling pathway and a more obvious effect on downregulating the expression of AGE-RAGE signaling pathway proteins. These results indicate that HP can activate the HIF-1 signaling pathway and inhibit the AGE-RAGE signaling pathway, which may be a potential mechanism for treating OP with HP.