Abstract
<title>Abstract</title> <p> Fracture healing is a complex biological process in which osteoblast differentiation plays a central role, yet the molecular mechanisms governing this process remain incompletely understood. The PI3K/Akt/mTOR signaling pathway and autophagy are critical regulators of osteoblast function, but the relationship between their transcriptional and post-translational regulation during human osteoblast differentiation has not been characterized. <italic>Eupolyphaga sinensis</italic> Walker (ES) is a medicinal arthropod traditionally used in East Asian medicine for promoting fracture healing; however, its molecular targets in osteoblasts remain largely undefined. This study aims to elucidate the regulatory dynamics of the PI3K/Akt/mTOR–autophagy axis during osteoblast differentiation and to identify the molecular targets of ES constituents. Single-cell RNA sequencing data of human primary osteoblasts (GSE147390) were analyzed using Seurat for clustering and cell-type annotation, AUCell for pathway activity scoring, scTenifoldKnk for in silico gene knockout, and Slingshot for pseudotime trajectory inference. A protein–protein interaction (PPI) network was constructed using STRING and analyzed with igraph. Compound–target profiling of ES constituents was performed using DrugBank, followed by molecular docking with AutoDock Vina. Cross-species ortholog mapping between human and rabbit was conducted via Ensembl Compara. Western blot analysis was performed in rabbit osteoblasts to validate the effects of ES on PI3K/Akt/mTOR pathway phosphorylation and autophagy markers. Unsupervised clustering of 8,526 human primary osteoblasts resolved six transcriptionally distinct cell types along a differentiation continuum. AUCell scoring revealed that PI3K/Akt/mTOR pathway and autophagy core gene signatures were transcriptionally invariant across osteoblast subtypes (Spearman ρ = −0.0058; P = 0.60). Virtual knockout of MTOR perturbed ribosomal protein gene expression without inducing transcriptional changes in autophagy-related genes. Pseudotime trajectory analysis confirmed stable autophagy gene expression throughout differentiation. PPI network topological analysis identified ribosomal proteins as all ten top-ranked hub genes, with RPS6—the direct phosphorylation substrate of mTORC1 effector kinase S6K1—ranking third by degree centrality (degree = 162). Integrative network pharmacology and molecular docking revealed that multiple ES constituents, including adenosine and protocatechuic acid, target AKT1 and mTOR with favorable binding affinities. Western blot validation demonstrated that ES treatment markedly suppressed P-PI3K, P-AKT, and P-mTOR levels in rabbit osteoblasts without altering total protein expression, and modulated autophagy markers including the LC3-II/I ratio and Beclin-1. The PI3K/Akt/mTOR–autophagy axis is predominantly regulated at the post-translational level during human osteoblast differentiation, with ribosomal proteins serving as the dominant signaling hubs. ES constituents polypharmacologically target this axis, providing a mechanistic rationale for its traditional use in fracture repair. </p>