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....

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 overexpres....

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