Palmatine enhances osteogenesis in hADMSCs via antioxidant enzyme upregulation and early RUNX2 activation

Palmatine, a protoberberine alkaloid with recognized anti-inflammatory and anti-resorptive activities, has recently attracted interest as a potential modulator of bone homeostasis; however, its direct osteo-inductive effects on human adipose-derived mesenchymal stem cells (hADMSCs) remain undefined. Given the accessibility, proliferative efficiency, and clinical relevance of hADMSCs, identifying natural small molecules capable of enhancing their osteogenic differentiation is of considerable therapeutic significance. This study investigated the osteogenic potential of Palmatine using a multi-level in vitro approach. Cytotoxicity assays established a non-toxic working concentration. Osteogenic differentiation was assessed by quantifying alkaline phosphatase (ALP) activity, calcium deposition, and matrix mineralization using Alizarin Red and von Kossa staining. Mechanistic insight was gained by evaluating antioxidant enzyme activity (superoxide dismutase, catalase) and profiling key osteogenic genes via real-time RT-PCR. Low-dose Palmatine (1 μM) enhanced hADMSC viability, whereas higher concentrations displayed dose-dependent cytotoxicity. Functional assays demonstrated that Palmatine significantly increased ALP activity and calcium content at both early (Day 7) and late (Day 14) stages. Transcriptionally, Palmatine promoted early upregulation of Runt-related transcription factor 2, osteocalcin, and osteonectin, followed by robust induction of ALP and collagen type I at later stages, reflecting canonical osteogenic progression. Furthermore, Palmatine augmented antioxidant capacity through early elevation of superoxide dismutase and later activation of catalase, suggesting a redox-associated mechanism that stabilizes osteogenic signaling and supports matrix maturation. Overall, these findings provide the first comprehensive evidence that Palmatine functions as a potent, multi-level osteo-inductive molecule in hADMSCs, highlighting its promise as a natural candidate for redox-modulated bone regenerative strategies.