Single-cell sequencing reveals that specnuezhenide protects against osteoporosis via activation of METTL3 in LEPR+ BMSCs.

ABSTRACT

BACKGROUND: Osteoporosis (OP) has garnered significant attention due to its substantial morbidity and mortality rates, imposing considerable health burdens on societies worldwide. However, the molecular mechanisms underlying osteoporosis pathogenesis remain largely elusive, and the available therapeutic interventions are limited. Therefore, there is an urgent need for innovative strategies in the treatment of osteoporosis. PURPOSE: The primary objective of this study was to elucidate the molecular mechanisms underlying osteoporosis pathogenesis using single-cell RNA sequencing (scRNA-seq), thereby proposing novel therapeutic agents. METHODS: The mice osteoporosis model was established through bilateral ovariectomy. Micro-computed tomography (µCT) and hematoxylin and eosin (H&E) staining were employed to assess the pathogenesis of osteoporosis. scRNA-seq was utilized to identify and analyze distinct molecular mechanisms and sub-clusters. Gradient dilution analysis was used to obtain specific sub-clusters, which were further validated by immunofluorescence staining and flow cytometry analysis. Molecular docking and cellular thermal shift assay (CETSA) were applied for screening potential agents in the TCMSPs database. Alkaline phosphatase (ALP) activity and alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Osteogenic organoids analysis was employed to assess the proliferation and sphere-forming ability of BMSCs. Quantitative real-time PCR (qRT-PCR) and western blot analysis were conducted to investigate signaling pathways. Wound healing assay and tube formation analysis were employed to evaluate the angiogenesis of endothelial cells. RESULTS: The scRNA-seq analysis revealed the crucial role of LEPR+ BMSCs in the pathogenesis of osteoporosis, which was confirmed by immunofluorescence staining of the epiphysis. Subsequently, the LEPR+ BMSCs were obtained by gradient dilution analysis and identified by immunofluorescence staining and flow cytometry. Accordingly, specnuezhenide (Spe) was screened and identified as a potential compound targeting METTL3 from the TCMSPs database. Spe promoted bone formation as evidenced by µ-CT, and H&E analysis. Additionally, Spe enhanced the osteogenic capacity of LEPR+ BMSCs through ALP and ARS assay. Notably, METTL3 pharmacological inhibitors S-Adenosylhomocysteine (SAH) attenuated the aforementioned osteo-protective effects of Spe. Particularly, Spe enhanced the LEPR+ BMSCs-dependent angiogenesis through the secretion of SLIT3, which was abolished by SAH in LEPR+ BMSCs. CONCLUSION: Collectively, these findings suggest that Spe could enhance the osteogenic potential of LEPR+ BMSCs and promote LEPR+ BMSCs-dependent angiogenesis by activating METTL3 in LEPR+ BMSCs, indicating its potential as an ideal therapeutic agent for clinical treatment of osteoporosis.