The Mechanistic Study of Melatonin Regulation of
p16Ink4a
and Oct4 in Anti‐Cellular Senescence
Yang Xu, Cong Zhang, Li Wang, Xiaoyu Chen ABSTRACT
Melatonin is a multifunctional indole hormone with established roles in anti‐aging and tissue regeneration. Although it has been shown to modulate key senescence‐associated markers such as p16Ink4a (p16) and OCT4, the underlying molecular mechanisms remain incompletely understood. MC3T3‐E1 cells were treated with melatonin to evaluate its effects on p16 and OCT4 expression using Western blot and RT‐qPCR. Isothermal titration calorimetry (ITC) and molecular docking were employed to investigate protein–protein interactions and structural binding patterns. VDR knockdown experiments were performed to assess functional causality. In addition, osteogenic differentiation was evaluated in bone marrow‐derived mesenchymal stem cells (BMSCs) using alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining. Melatonin significantly downregulated p16 and upregulated OCT4 expression without affecting cell viability. ITC analysis revealed that melatonin does not directly bind to p16 or OCT4, but both proteins interact with the vitamin D receptor (VDR) with moderate affinity. Molecular docking further supported stable binding conformations between VDR and p16/OCT4. Functionally, VDR knockdown disrupted the balance of p16 and OCT4 expression and impaired osteogenic differentiation, as evidenced by decreased ALP activity and reduced mineralized nodule formation. These findings identify VDR as a critical mediator of melatonin signalling and reveal a novel VDR‐centered regulatory axis linking senescence and stemness pathways. This mechanism provides new insights into the anti‐senescence and pro‐regenerative effects of melatonin and suggests potential therapeutic strategies for aging‐related diseases such as osteoporosis.