RNA Sequencing Reveals that Thrombospondin-4/Signal Transducer and Activator of Transcription 3 Signaling Reduces Odontoblast Apoptosis.

Abstract

Introduction: Bacteria from dental caries and their toxic products can induce apoptosis of odontoblasts via sustained and intense stimulation. In this study, we profiled the transcriptomes of healthy and carious human odontoblastic layer and investigated the role of thrombospondin-4 (TSP-4) in reducing odontoblast apoptosis under inflammatory conditions.

Methods: In the human clinical model, odontoblastic layers isolated from healthy and carious teeth were subjected to RNA sequencing . Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to investigate gene expression profiles. Differentially expressed genes (DEGs) were validated with reverse transcription quantitative real-time PCR (RT-qPCR). Further, western blot and immunofluorescence were used to detect TSP-4 expression. The mouse caries model was used to confirm TSP-4 expression in vivo. In the human odontoblastic-like cells (hOBLCs) model, cells were treated with lipopolysaccharide, small interfering RNA targeting TSP-4, recombinant human TSP-4, Stattic, and small interfering RNA targeting signal transducer and activator of transcription 3 (STAT3). TSP-4 and caspase-3 expression and STAT3 phosphorylation were assessed using western blot. Apoptosis was evaluated by flow cytometry analysis.

Results: In the human clinical model, RNA sequencing results identified 779 DEGs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that DEGs were enriched in the extracellular matrix organization. TSP-4 expression was increased in carious human dental pulp and in mouse carious dental pulp. In the hOBLCs model, lipopolysaccharide treatment increased TSP-4 expression. TSP-4 knockdown suppressed STAT3 phosphorylation and promoted apoptosis, while recombinant human TSP-4 treatment increased STAT3 phosphorylation and decreased apoptosis, which was abolished by Stattic or STAT3 knockdown.

Conclusions: Gene expression profiles of the odontoblastic layer in healthy and carious human teeth were identified. TSP-4 was found to regulate apoptosis in hOBLCs via STAT3 signaling. This study may deepen our understanding of dental pulp inflammation and offer a potential therapeutic target for preventing odontoblast apoptosis and subsequent damage during caries progression.