Abstract: Objective  To investigate the effects of thioredoxin-interacting protein (TXNIP) on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) under lipopolysaccharide (LPS)-induced inflammatory homeostatic imbalance, and to explore potential homeostasis regulatory mechanisms. Methods  Human PDLSCs were isolated, cultured, and identified. The optimal concentration of the TXNIP inhibitor SRI37330 was determined using cell counting kit-8 (CCK-8) method and real time quantitative reverse transcription polymerase chain reaction (RT-qPCR). PDLSCs were cultured under Blank group (normal culture), SRI37330 group (addition of SRI37330 to normal culture medium), LPS group (LPS added under conventional culture conditions), LPS + SRI37330 group (LPS-treated PDLSCs with SRI37330), LPS + osteogenic medium (OM) group (LPS-treated PDLSCs with OM), and LPS + OM + SRI37330 group (LPS-treated PDLSCs with both osteogenic medium and SRI37330). Alkaline phosphatase (ALP) staining and activity assays were used to assess early mineralization levels in each group, and alizarin red S (ARS) staining were employed to evaluate mineralized matrix formation. RT-qPCR and Western blotting (WB)  were employed to analyses measure expression levels of osteogenic-related genes and proteins. The potential signaling pathways that TXNIP regulates the inflammatory-osteogenic homeostasis transition in PDLSCs were analyzed with  RNA transcriptome sequencing. Results  PDLSCs with favorable morphology and growth status were successfully cultured. Flow cytometry validated that these cells were positive for mesenchymal stem cell markers, and negative for hematopoietic markers. Based on CCK8 assays and RT-qPCR results, SRI37330 with a concentration of 0.6  μmol/L was selected to treat PDLSCs in subsequent experiments. ALP staining, ALP activity detection, ARS staining, RT-qPCR, and WB results showed that LPS-induced inflammatory homeostatic imbalance could inhibit the osteogenic differentiation of PDLSCs, while the addition of TXNIP inhibitor SRI37330 could significantly reverse the inhibition of osteogenic differentiation ability of PDLSCs. RNA transcriptome sequencing analysis showed that SRI37330 may promote the reconstruction of osteogenic homeostasis in PDLSCs under inflammatory stimulation by regulating the mTOR and AMPK signaling pathway. Conclusion  Under inflammatory homeostatic imbalance, TXNIP may inhibit the osteogenic differentiation ability of PDLSCs through the mTOR and AMPK pathway. Targeted regulation of TXNIP contributes to the restoration of bone homeostasis in inflammatory microenvironments and provides a new therapeutic strategy for periodontal tissue regeneration from the perspective of homeostasis medicine.

Key words: periodontal ligament stem cells, inflammatory microenvironment, osteogenic differentiation, thioredoxin-interacting protein, lipopolysaccharide, SRI37330, homeostasis medicine