Amelioration of CD44-mediated Progression of Experimental Periodontitis in Osteoporotic Mice by Glycitein

Summary

CD44 facilitates disease pathogenesis by orchestrating inflammatory cascades and osteoclastic differentiation via PI3K/Akt pathway activation. Glycitein exerts therapeutic effects on comorbid osteoporosis (OP) + periodontitis (PD) manifestations through targeted attenuation of CD44-mediated signaling.

Abstract

This investigation elucidates the critical molecular determinants associated with the comorbidity of osteoporosis (OP) and periodontitis (PD) through proteomic profiling, while delineating the regulatory function of CD44 in experimental periodontitis in an OP murine model. Phase I involved collecting serum specimens from patients with OP+PD (n = 3) and healthy controls (n = 6) undergoing routine health evaluations at our institution for comparative proteomic analysis. Subsequent translational validation of differentially expressed genes (DEGs) and associated signaling cascades was conducted across clinical specimens and OP+PD murine models. To mechanistically characterize CD44's role in PD progression under osteoporotic conditions, an OP murine model was generated through bilateral ovariectomy, followed by experimental PD induction via ligature placement. Comprehensive assessments included histomorphometric alterations via hematoxylin-eosin staining, microarchitectural bone analysis at the maxillary first molar region using micro-CT, and immunoblotting evaluation of phosphoinositide-3-kinase (PI3K)/Akt pathway components. Parallel network pharmacological screening coupled with molecular docking simulations was executed to identify bioactive constituents of Angelica sinensis with therapeutic potential. Proteomic interrogation identified CST3, A2M, CD44, CDH13, CETP, and VWF as candidate pathogenic mediators in OP+PD pathogenesis. In our hands, gene set enrichment analysis revealed that PI3K/Akt signaling functions as a principal mediator of OP+PD disease progression. Quantitative reverse-transcription PCR-based validation confirmed significant CD44 upregulation in both clinical and experimental OP+PD cohorts. In vivo modulation via CD44 suppression significantly restored periodontal tissue integrity, reduced inflammatory cell infiltration, and strengthened alveolar bone microarchitecture in OP mice, concomitant with PI3K/Akt pathway inhibition. Network pharmacology revealed glycitein as the primary bioactive phytochemical in Angelica sinensis, with CD44 identified as its central molecular target. Glycitein improved alveolar bone structure in OP+PD mice, increasing bone volume fraction (BV/TV), trabecular thickness (Tb.Th), bone mineral density (BMD), and reducing trabecular number (Tb.N), bone surface-to-bone volume ratio (BS/BV), indicating healthier bone quality, mechanistically attributed to CD44 signaling axis attenuation.

Introduction

Periodontitis (PD) is a complex, mixed microbial disease caused by the deposition of pathological biofilms in the gingival sulcus¹. It is characterized by an imbalance in the microecological environment that triggers a pathological inflammatory cascade within the host, ultimately leading to irreversible progressive harm to the cementum, periodontal ligament, and alveolar bone, which are components of the periodontal attachment system. This can result in tooth loosening or even loss². The quantity was standardized for comparability, and the most recent epidemiological data³ indicate that over 1.066 billion individuals globally suffer from severe periodontitis, with an age-standardized prevalence of approximately 12.5%, highlighting considerable variation across international populations.

Osteoporosis (OP), a systemic bone illness, is characterized by a substantial degradation of bone microstructure and a systematic loss of bone mass^4,5. The risk of fractures is greatly increased by this structural weakness, especially in older people. Notably, persons aged 65 and older in Japan had a higher prevalence of periodontal disease than adults between the ages of 30 and 64. Postmenopausal women have a higher incidence of periodontal disease and often experience other health issues^6,7. The study predicts that from 2005 to 2025, the disease burden will increase by over 50%, with the economic cost expected to rise from $17 billion to $25 billion. The study predicts that from 2005 to 2025, the disease burden will increase by over 50%, with the economic cost expected to rise from $17 billion to $25 billion. This trend highlights the significant socioeconomic impact of these periodontal diseases. Additionally, in female patients with periodontal disease and gingivitis, there is a clear positive correlation between the duration of the disease and its chronicity and the increased health risks⁸.

Previous studies indicated that OP might promote the development of PD and vice versa due to common risk factors for both diseases, such as aging, smoking, and inadequate calcium and vitamin D intake. In the postmenopausal female population, severe OP could be accompanied by reduced mandibular mineral content, which might be associated with deficient levels of periodontal attachment⁹. It has been demonstrated that OP-associated reduced bone density might accelerate PD-induced alveolar bone resorption, thereby favoring the invasion of periodontal pathogens¹⁰. Among a range of age- and physiological change-related health challenges, PD and OP are particularly prominent with the acceleration of global population aging and the significant increase in the number of menopausal women. Therefore, clarifying the relevant indicators and their associations is of crucial importance for disease prevention, treatment, and prognostic assessment. Motivated by these aspects, this study used proteomics to screen key proteins, with an emphasis on CD44-mediated PI3K/Akt signaling, in the development of OP+PD, exploring the regulatory roles and possible mechanisms of key proteins using an experimental PD model in OP mice. Further screening of targeted therapeutic agents present in Angelica sinensis-a traditional Chinese medicinal herb widely used in clinical practice for its anti-inflammatory and bone-protective properties, and previously reported to alleviate bone loss and modulate inflammatory responses in osteoporosis and periodontal disease. Guided by network pharmacology and molecular docking, glycitein was identified as the primary bioactive phytochemical and a rational candidate for OP+PD intervention.

Protocol

All animal experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals. The study protocol was reviewed and approved by the Animal Ethics Committee. All efforts were made to minimize animal suffering.

This study aims to evaluate the role of CD44-mediated regulation of the PI3K/Akt pathway in osteoclastic differentiation and inflammatory progression during comorbid osteoporosis and periodontitis (OP+PD) and determine whether glycitein, a bioactive compound from Angelica sinensis, can therapeutically modulate these effects. The rationale stems from emerging evidence linking CD44 overexpression to pathological bone resorption and immune dysregulation in OP+PD^11,12,13. By integrating proteomics, in vivo modeling, and network pharmacology, the protocol systematically investigates molecular mechanisms and therapeutic interventions to mitigate disease progression¹⁴.

The sample size for in vivo experiments was based on previous studies and power analysis, resulting in n = 6 per group, which ensures adequate statistical power to detect significant differences (P < 0.05) across treatment conditions. Glycitein was administered at 50 mg/kg and 100 mg/kg dosages, suspended in 5% DMSO in olive oil, and delivered via daily oral gavage. Mice were monitored over 8 weeks post-induction of PD. Ligature placement to induce periodontal inflammation utilized 5-0 silk thread, and bilateral ovariectomy was performed under 0.3% pentobarbital sodium anesthesia (0.5 mL/100 g)¹⁵. Additionally, molecular assays, including RT-qPCR, western blotting, and ELISA, were conducted using validated protocols¹⁶, and histological evaluations, such as hematoxylin-eosin (H&E) and methylene blue staining, followed established timelines and staining durations.

All experimental procedures involving chemical reagents, staining agents, and biological specimens were conducted following standard biosafety protocols to minimize health risks. Hazardous materials such as formalin, EDTA, methylene blue, and acid fuchsin were handled with personal protective equipment (PPE), including gloves, masks, and safety goggles. All waste materials, including used reagents and contaminated disposables, were collected in designated biohazard containers and disposed of through certified hazardous waste disposal services by institutional and environmental safety guidelines. Figure 1 shows the overall experimental workflow.

Clinical data collection
In our study, we systematically collected and comprehensively analyzed baseline clinical data and detailed medical histories of three OP+PD patients and six control subjects, including demographic and lifestyle factors, as well as anatomical and metastatic profiles. In addition, rigorous confirmation of the test result for HPV infection (positive or negative) was done. The progression stage or recurrence pattern of metastatic lesions was recorded in detail. During the first 7 days of immunotherapy, patients' blood samples were systematically collected and standardized, including biochemical indicators such as blood routine tests. Routine blood tests were performed in strict accordance with standard procedures to accurately evaluate key parameters such as platelets, lymphocytes, and neutrophils. Serum albumin was measured in parallel, and liver function was assessed by quantitative analysis of globulin concentration index.

Patients
Bone mineral density assessment and participant stratification
We conducted a systematic comparison of bone density measurements in the lumbar region and femoral neck. All examinations were performed by experienced radiologists in our department, using precision equipment to accurately quantify bone density values. To ensure standardized sample collection, participants were required to fast for at least 8 h before the sampling. By integrating bone density data with comprehensive periodontal assessment data, we strictly categorized the subjects into either the osteoporosis with periodontitis (OP+PD) group or the healthy control group (HC), ensuring that the sex ratio was perfectly matched between the two groups. The sex-matching refers to the equal distribution of females between the OP+PD and healthy control groups. However, only female participants were included in this study to focus on postmenopausal osteoporosis; there were no male subjects in either group. The study adhered to the ethical guidelines of the Declaration of Helsinki, with all participants signing formal informed consent forms, fully protecting their rights.

Diagnostic parameters
Osteoporosis was defined per the Chinese Guidelines for Diagnosis and Management of Primary Osteoporosis (2022)¹⁷, with diagnostic thresholds set at T-score ≤ -2.5 standard deviations (SD) for lumbar (L1-L4) or hip BMD. Periodontitis staging followed the 2018 World Workshop Classification of Periodontal Diseases¹⁸, with inclusion restricted to Grade A, Stage II-III cases. Inclusion criteria: Postmenopausal females aged 50-65 years, dual diagnosis of OP and PD meeting the above criteria, absence of autoimmune disorders, no history of malignant neoplasms, normal cardiopulmonary, hepatic, and renal function. Exclusion criteria: Declined participation by patient or family, recent (≤6 months) pharmacotherapy for OP or PD.

Proteomic profiling of serum samples
Serum samples from three OP+PD patients and six control subjects were collected for proteomic analysis. Relative protein quantification was achieved through standard procedures, and differentially expressed proteins were defined as those with P < 0.05 and fold change > 1.5¹⁹.

Gene set enrichment analysis (GSEA)
GSEA was used to identify key signaling pathways dysregulated between the control and OP+PD cohorts. Raw transcriptomic data were first transformed using log2 conversion and normalized via quantile normalization to remove batch effects. Gene sets with multiple biological functions were screened from the Molecular Signatures Database (MSigDB), and statistical significance was assessed using the non-parametric Wilcoxon rank-sum test. Enrichment results were reported using normalized enrichment scores and false discovery rates (FDR < 0.25).

Weighted gene co-expression network analysis (WGCNA)
WGCNA was used to identify gene modules significantly associated with OP+PD phenotypes. A soft-threshold power of 12 was selected to achieve a scale-free network topology (R² > 0.85), and Pearson correlation analysis was used to assess relationships between module eigengenes and clinical traits. Functional enrichment analyses were conducted to uncover relevant biological pathways in strongly correlated modules.

Establishment of ovariectomy-induced osteoporotic murine models
This study involved 116 SPF-grade female mice. Before the experiments, the animals were housed under controlled temperature and humidity conditions for a 7 day adaptation phase, during which they were given standardized rodent chow and distilled water. All animal handling procedures strictly followed the ethical protocols approved by the Ethics Committee of Xi'an Jiaotong University Health Science Center, under approval ID 0609. The study was registered under project number [2024-054].

Bilateral oophorectomy (OVX) surgical protocol:
Mice were anesthetized via intraperitoneal injection of 0.3% pentobarbital sodium (0.5 mL/100 g body weight). Dorsal fur was depilated surgically, and animals were positioned in dorsal recumbency on a heated, sterile surgical platform. After aseptic preparation with 75% ethanol, bilateral paravertebral incisions (1 cm lateral to the spine) were made at the level of the renal fat pads. The ovarian bursa was exteriorized, and the oviduct was ligated proximal to the ovary using 5-0 silk sutures, followed by excision of bilateral ovaries, associated adipose tissue, and partial oviductal structures. Musculofascial and cutaneous layers were closed sequentially with 4-0 absorbable sutures. Postoperative antisepsis was performed with 75% ethanol.

Sham surgery controls:
Sham-operated mice underwent identical procedural steps, including ovarian exposure and periovarian adipose tissue resection, without ovarian extirpation.

Postoperative management:
Postanesthetic recovery was facilitated using circulating water heating pads until full ambulation. Analgesia was maintained via subcutaneous carprofen administration (5 mg/kg daily) for 72 h postoperatively, supplemented with prophylactic penicillin G (50,000 IU/kg) for 48 h to prevent surgical site infections. Body mass was monitored weekly during the 6-week osteoporotic modeling phase, with exclusion criteria applied to animals exhibiting >20% weight loss or clinical distress.

Construction of experimental OP+PD animal model
The experimental PD model was developed based on the previously established OP animal model. To avoid thread breakage and ensure smooth insertion between the molars, 5-0 silk suture was used for ligation. During the procedure, the mouse's mouth corner was gently retracted, and the thread was grasped with forceps and carefully placed at the cervical region between the first and second molars. The wire and the tip of the forceps were kept level to minimize trauma to the buccal mucosa, thereby reducing the risk of bleeding or mortality. The other hand gently pulled the opposite end of the wire, guiding it gradually into the interproximal space between the molars. Once the thread was properly positioned, a section near the buccal side was clamped with forceps and carefully passed between the second and third molars using the same technique. Gentle downward traction ensured smooth passage of the silk. Finally, the thread was tied securely around the second molar at the mid-cervical region. The mice were subsequently allowed to recover.

Quantitative reverse transcription-polymerase chain reaction (RT-qPCR)
The Table of Materials lists specific forward and reverse primer sequences for RT-qPCR targeting genes related to inflammation, coagulation, and cell adhesion, including CD44, CDH13, VWF, and GAPDH. The primer sequences used for RT-qPCR analysis are summarized in Table 1.

Western blot analysis
The expression characteristics of key signaling pathway proteins in periodontal tissues were systematically studied using western blotting. The periodontal tissues were subjected to freeze-thaw homogenization using RIPA buffer, containing protease and phosphatase inhibitors, to efficiently extract total protein. After high-speed centrifugation (12,000 × g, 4 °C, 10 min) to separate the lysate, the supernatant was used to accurately measure protein concentration with a BCA kit, providing reliable data for subsequent experiments.

For each experimental group, 30 µg of total protein were loaded and separated through a 10% SDS-PAGE system. The resolved proteins were then transferred onto PVDF membranes using a semi-dry blotting method. To minimize background interference, membranes were blocked with 5% non-fat dry milk for h at ambient temperature. Subsequently, the membranes were kept at 4 °C in a stable environment for further processing.

The membranes were then incubated with specific primary antibodies (see Table of Materials) for 2 h at °C. On the following day, TBST buffer was used for thorough washing, and membranes were then treated with HRP-conjugated secondary antibodies at a 1:10,000 dilution for 1.5 hours at room temperature. All immunodetection steps were performed in strict accordance with standardized protocols to ensure the consistency and validity of the signaling pathway analysis²⁰.

By using enhanced chemiluminescence (ECL) substrates to capture signals and high-resolution imaging equipment to precisely locate protein bands, the optical density values of the bands were quantitatively analyzed using ImageJ software. Ultimately, by systematically comparing the relative expression levels of target proteins/β-Actin in each group with the control group, the activation degree and dynamic changes of the PI3K/Akt pathway under different interventions were accurately assessed.

Enzyme-linked immunosorbent assay (ELISA)
Using ELISA kits (see Table of Materials), this study achieved precise quantification of various inflammatory cytokines, including TNF-α, IL-6, IL-1β, and IL-4, in serum samples. It also evaluated concentrations of TRAP5b, ALP, and TGF-β as markers of osteoclast activity. A 96-well plate precoated with capture antibodies was incubated at 25 °C with serum samples diluted 1:10 in PBS for 2 h, allowing antigens to bind specifically to the immobilized capture antibodies. After incubation, unbound substances were removed via three washes using PBS-T (phosphate-buffered saline with Tween-20). Subsequently, biotin-labeled detection antibodies were added and incubated for 1 hour at the same temperature. Next, streptavidin-HRP was introduced to bind the biotinylated antibodies, and the mixture was incubated for an additional 20 min at room temperature under light-protected conditions. The enzymatic reaction was then terminated using the stop solution, and absorbance was measured at 450 nm using a microplate reader to determine cytokine concentrations.

Hematoxylin & Eosin (H&E) staining
Standard methods were used to perform H&E staining on paraffin-embedded sections of periodontal tissue to analyze microstructural changes. The sample preparation process involved initial fixation in a 4% paraformaldehyde solution for 24 h, followed by graded ethanol dehydration (70%, 80%, 95%, and 100%) and clearing in xylene, and finally embedding in paraffin to produce 4 µm-thick sections²¹. After dewaxing in xylene, the samples were rehydrated through descending ethanol concentrations (100% to 70%). The staining process began with a 5 min immersion in hematoxylin (Meisner's), followed by rinsing under running water, differentiation in 1% acid alcohol in ethanol, and another rinse. Sections were then stained with 0.5% eosin for 10 s to complete the protocol.

After staining, slides were dehydrated again in a graded ethanol series, cleared in xylene, and sealed with mounting medium. Observations were made under a microscope equipped with 10x and 40x objectives for high-resolution image acquisition. Histological analysis assessed the structural integrity of periodontal connective tissue, inflammatory cell infiltration, and alveolar bone resorption morphology. The level of inflammation was scored using an improved Loe-Silness index, with two pathologists blinded to sample identity independently evaluating the samples via a semi-quantitative scale to ensure objectivity and reproducibility²².

Methylene blue staining
This study employed the methylene blue staining method on standardized, processed maxillary alveolar bone sections to quantify the degree of bone resorption. The experimental procedure began with tissue fixation: periodontal tissues were fixed in 10% neutral formalin solution for 24 h, followed by a 21 day decalcification treatment in 10% EDTA (pH 7.4) solution. After decalcification, the samples were paraffin-embedded and prepared into 4 µm-thick tissue sections, which were then mounted onto silanized slides. The section pretreatment process included xylene dewaxing, gradient ethanol hydration, and incubation in a 60 °C water bath. Slides were then stained with 0.1% methylene blue solution for 15 min.

After staining, the sections were washed for 1 min using distilled water that had been preheated to 60 °C, ensuring effective removal of excess dye without damaging tissue structure. They were then counterstained with 0.1% acid fuchsin for 5 min to enhance contrast between tissue structures. This staining protocol provided consistent and reliable visualization for quantitative analysis of alveolar bone resorption²³. Finally, slides were dehydrated using graded ethanol, cleared in xylene, and mounted. The stained bone tissue was analyzed under a light-field microscope, and high-resolution images were acquired to precisely assess alveolar bone edge resorption for subsequent morphological evaluation.

Micro-computed tomography (Micro-CT) analysis
Micro-CT scanning was performed to assess 3D microstructural changes in the alveolar bone of OP+PD model mice, specifically targeting the maxillary first molar region. After 8 weeks of postoperative monitoring, the mice were humanely euthanized via CO₂ inhalation, a method suitable for preserving maxillofacial structures. Maxillary specimens were then harvested and fixed in 4% paraformaldehyde at 4 °C for 48 h²⁴.

Micro-CT analysis was performed with the reconstructed DICOM images of the maxillary region imported into the software by selecting the dataset corresponding to the scanned specimens. A standard region of interest (ROI) was defined manually as the mesial buccal one-third portion of the root of the maxillary first molar by outlining the alveolar bone structure on sagittal and coronal views, and approximately 50 consecutive slices were typically included to fully cover the region.

To separate trabecular bone from surrounding soft tissue and background, a density threshold of 180-220 mgHA/cm³ was applied. Segmentation was then performed to generate a binary structure representing only the trabecular bone. Morphometric analysis was carried out using the Bone Morphometry Module within the software. Parameters were automatically calculated, including bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone surface-to-bone volume ratio (BS/BV), and bone mineral density (BMD). Following the analysis, numerical results were exported as spreadsheets.

These measurements were used to evaluate the extent of microstructural damage in alveolar bone associated with the OP+PD pathological condition and to assess the therapeutic efficacy of glycitein in restoring bone quality. This approach allowed for a high-resolution, reproducible, and quantitative evaluation of structural integrity in the periodontal bone environment, offering key morphological insights into treatment outcomes.

Network pharmacology profiling
To systematically investigate the bioactive components of traditional Chinese medicine (TCM) that may modulate key biological pathways relevant to comorbid osteoporosis and periodontitis (OP+PD), this study employed a network pharmacology approach to analyze Angelica sinensis. This plant was selected based on its historical use in TCM for managing bone-related disorders and inflammation, including symptoms associated with both osteoporosis and chronic gum disease. In the compound screening phase, active ingredients of Angelica sinensis were retrieved using TCMSP and TCMID databases [database details moved to ToM], and filtered using oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥0.18 criteria. Compound-target interactions were predicted using STITCH, with target site validation via DisGeNET and OMIM. Pathway enrichment analysis was conducted using the DAVID 2021 platform to identify KEGG signaling pathways potentially modulated by the selected compounds. To visualize molecular interactions, a "compound-target-pathway" network was constructed based on topological indices (degree centrality, betweenness centrality). The analysis identified glycitein, a natural flavonoid, as a compound with favorable pharmacokinetic properties and strong connectivity to CD44, suggesting potential therapeutic relevance in OP+PD. However, glycitein has not been clinically approved for the treatment of osteoporosis or periodontitis; these findings are based on in silico predictions and provide a theoretical framework for subsequent in vivo validation experiments ²⁵.

Molecular docking validation
Protein structures of CD44 (PDB ID: 1UUH) and PI3K (PDB ID: 4L23) were retrieved from the RCSB Protein Data Bank (https://www.rcsb.org). Ligand structures (glycitein, ligustilide) were optimized through energy minimization using a molecular force field. Docking simulations were conducted with a genetic algorithm under the following parameters: 100 runs, population size 150, and 25×10⁶ energy evaluations. Binding pockets were defined based on crystallographic ligand coordinates (CD44: 20×20×20 ų; PI3K: 25×25×25 ų). Resultant docking poses were ranked by binding energy (ΔG, kcal/mol), and reproducibility was validated using a root-mean-square deviation (RMSD) threshold of <2.0 Å.

Therapeutic evaluation of gycitein in OP+PD murine models
Glycitein (purity ≥ 98%) was obtained from a certified biochemical supplier in analytical grade for in vivo experiments is a natural isoflavone known to be present in Angelica sinensis, with reported anti-inflammatory and osteoprotective properties²⁶. Osteoporotic periodontitis was induced in 11-week-old female C57BL/6 mice through bilateral ovariectomy (OVX) and ligature-induced periodontal inflammation. Mice were randomized into four cohorts (n = 6 per group):

Sham: Sham surgery + vehicle (5% dimethyl sulfoxide [DMSO] in olive oil, daily oral gavage)
OP+PD: Disease model + vehicle
OP+PD + Low-dose glycitein (50 mg/kg): Disease model + glycitein suspension in vehicle
OP+PD + High-dose glycitein (100 mg/kg): Disease model + glycitein suspension in vehicle

Statistical analysis
Inferential statistical tests were performed, and visual data representations were created for graph generation and data visualization. Numerical data were expressed as mean ± SD. Group comparisons were assessed through two-tailed unpaired t-tests, considering values with &l 0.05 as statistically significant. These methods ensured data robustness and enhanced the reliability of subsequent analytical outcomes.

Results

Proteomic profiling of OP+PD cohorts
Quantitative proteomic analysis of serum from OP+PD patients (n=3) and healthy controls (n=6) identified dysregulated proteins implicated in disease pathogenesis. Comparative analysis revealed nine downregulated proteins (APOL1, CST3, IGLV3-27, IGHV3-33, PF4, CLEC3B, SRGN, C6, SELL) and six upregulated proteins (IGLV9-49, A2M, VWF, CETP, CD44, CDH13) in OP+PD sera versus controls (Figure 2A,B). Weighted Gene Co-Expres...

Discussion

This study proposes a translational research framework that combines proteomic profiling, gene expression validation, in vivo animal modeling, and network pharmacology to explore the molecular mechanisms underlying the comorbidity of osteoporosis and periodontitis (OP+PD). The central hypothesis is that CD44 modulates the PI3K/Akt signaling axis, thereby exacerbating inflammatory bone loss in OP+PD conditions, and that glycitein, a flavonoid from Angelica sinensis, may attenuate this process by inhibiti...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ELISA kits	Shanghai Enzyme Link Biotechnology		Quantification of inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-4, ALP, TRAP5b, TGF-β, VWF, CD44)
RNA extraction kit	Shanghai Yesheng Biotechnology		Serum RNA extraction
cDNA synthesis kit	Shanghai Yesheng Biotechnology		High-activity reverse transcription
qPCR kit	Shanghai Yesheng Biotechnology		Quantitative PCR amplification
Antibodies (primary/secondary)	Cell Signaling Technology / Abcam / Hangzhou Xianli Biotechnology / Beijing Zhongshan Jinqiao Biotechnology		Specific primary antibodies; β-actin internal control; goat anti-rabbit IgG secondary antibody
Histology reagents	Beyotime Biotechnology / Shanghai Shangshang		Hematoxylin–eosin and methylene blue staining
Chemiluminescent substrate	Yakken Biotechnology		Western blot detection
Micro-CT system	Scanco Medical AG (Viva CT40)		High-resolution microstructural analysis
LC-MS/MS system	Thermo Scientific Q Exactive HF-X		High-resolution proteomic analysis
Software: MaxQuant (v2.1.0), Andromeda	Broad Institute		Proteomics data preprocessing
Software: GSEA (v4.3.2)	Broad Institute		Pathway enrichment analysis
Software: R (v4.0.3), WGCNA (v1.73), pheatmap (v1.0.12)	R Foundation		Statistical computing, network analysis, clustering, heatmaps
Software: AutoDock 4.2, AutoDockTools 1.5.7	The Scripps Research Institute		Molecular docking and grid definition
Software: Open Babel 3.1.1	Open Babel project		Ligand structure optimization
Software: PyMOL 2.5.2	Schrödinger, LLC		Molecular docking visualization