AS2863619 boosts osteogenesis in periodontal ligament stem cells and mitigates inflammatory impairment

Aim: Periodontal ligament stem cells (PDLSCs) represent a promising source for the regeneration of periodontal tissues. However, these cells may experience functional alterations during in vitro expansion and culture, as well as under inflammatory conditions. In this study, we investigated AS2863619 (AS), a synthetic small molecule with potential applications in immune Disease Research, to evaluate its impact on the osteogenic differentiation potential of PDLSCs and to elucidate its underlying mechanisms.

Methods: The effect of AS on the proliferation and cytotoxicity of PDLSCs was evaluated using commercially available kits. The osteogenic potential of PDLSCs was assessed through Alkaline Phosphatase (ALP) activity staining or Alizarin Red S (ARS) staining to detect mineralized nodules. Inflammatory injury in PDLSCs was induced by treating them with recombinant human tumor necrosis factor-alpha (rhTNF-α). Transcriptome Sequencing was employed to investigate the potential target of AS for rescuing osteogenic differentiation from inflammatory injury. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were conducted to determine the expression levels of osteogenic gene markers and associated signaling pathways. Various small molecule inhibitors, agonists, and recombinant proteins were utilized to modulate relevant signaling pathways as specified in the text.

Results: The proliferation of PDLSCs was inhibited by AS at 250 nM after 1 day of culture, and at 125 nM after 3 and 5 days of culture. However, no significant cytotoxicity was observed at concentrations below 1 μM. Additionally, AS significantly enhanced the ALP activity of PDLSCs and accelerated the formation time of mineralized nodules, while suppressing their adipogenic differentiation ability. Furthermore, AS promoted the levels of bone morphogenetic protein (BMP)2 and phosphorylated (p)-SMAD1/5 without significant effects on p-ERK, p-P38, and p-JNK levels. Moreover, AS effectively augmented rhTNF-α-inhibited ALP activity in PDLSCs and attenuated rhTNF-α-induced components involved in retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. Notably, Poly(I:C), an activator of melanoma differentiation-associated 5 (MDA5) and RIG-I receptors, rather than interferon-stimulated gene 15 (ISG15) protein, hindered the reparative effect of AS on inflammatory injury during osteogenic differentiation.

Conclusions: AS is a potent small molecule that effectively enhances the osteogenic potential of PDLSCs, potentially by augmenting BMP2/SMAD signaling. Additionally, AS effectively mitigates inflammation-induced impairment of osteogenic potential in PDLSCs by partially modulating the MDA5/RIG-I receptors-mediated RLR signaling pathway.

BMP2/SMAD signaling pathway; Inflammation; Osteogenic differentiation; Periodontal ligament stem cells; RIG-I-like receptor signaling pathway.