Metformin combats high glucose-induced  damage  to the osteogenic differentiation of human periodontal ligament  stem  cells via inhibition of MAPK pathway mediated through NPR3

Abstract Background High glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) has long been a challenge to periodontal regeneration for diabetic individuals. Metformin is an anti-hyperglycemic drug that exhibiting abundant biological activities associated with cell metabolism and downstream tissue regeneration. However, how metformin combats damage to PDLSC osteogenic potential under high glucose and the underlying mechanisms remain unknown. Methods Osteogenic differentiation of PDLSCs was assessed by Alkaline phosphatase (ALP) staining, ALP activity, Alizarin red staining and quantitative assay, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. RNA-seq analysis was performed to screen target genes of metformin, and the effects of target genes were confirmed using lentivirus transfection. Western blot analysis was also used to detect the protein level of underlying signaling pathways. Results Osteogenic differentiation of PDLSCs under high glucose was decreased and metformin addition enhanced the capacity of differentiation. Furthermore, natriuretic peptide receptor 3 (NPR3) was upregulated in PDLSCs under high glucose and downregulated after metformin addition. Additionally, we demonstrated that upregulation of NPR3 compromised the metformin-enhanced PDLSC osteogenic differentiation through activating the MAPK pathway, and that inhibition of p38 MAPK or Erk1/2 pathway enhanced the osteogenic differentiation of PDLSCs with NPR3 upregulation. Conclusions The present study suggests that metformin may enhance the osteogenic differentiation of PDLSCs under high glucose via downregulation of NPR3 and inhibition of its downstream MAPK pathway. This is the first report identifying the involvement of NPR3-mediated MAPK pathway in the metformin-enhanced osteogenic differentiation, indicating that NPR3 antagonists, such as metformin, may be feasible therapeutics for periodontal tissue regeneration in diabetic individuals.