High-throughput phenotypic screening identifies the phytochemical melissic acid that mitigates SHED cellular senescence via the ADCY5/cAMP/CREB axis

Introduction Cellular senescence drives aging and age-related diseases, posing a critical barrier to stem cell-based regeneration. While selective elimination of senescent cells (senolysis) remains a theoretically promising strategy, safe natural senotherapeutic agents are limited. Human exfoliated deciduous tooth stem cells (SHED) possess high regenerative potential yet are vulnerable to senescence, compromising their therapeutic utility. Methods We performed a phenotypic screen of 800 natural compounds in replicatively senescent SHED. The hit compound, the phytochemical melissic acid, was then validated in two independent senescence models—replicative exhaustion and doxorubicin-induced stress. Melissic acid (5–20 μM) was assessed for cytotoxicity and anti-senescence efficacy via SA-β-Gal staining and senescence marker expression. Transcriptomic profiling and mechanistic assays were performed to dissect the underlying molecular pathways. Results Melissic acid exhibited minimal cytotoxicity but significantly reduced SA-β-Gal positivity and senescence markers in both models. Transcriptomic profiling revealed marked upregulation of ADCY5 and coordinated changes in cAMP signaling, autophagy, and lipid metabolism genes. Mechanistically, melissic acid increased CREB Ser133 phosphorylation, enhanced Beclin-1 expression, and reduced p62/SQSTM1, indicating activation of cAMP/CREB-dependent autophagy. It also decreased lipid peroxidation, restored mitochondrial membrane potential, and relieved G0/G1 arrest via upregulation of Cyclin D1 and Rb Ser780 phosphorylation. Discussion Collectively, melissic acid alleviates SHED senescence through the ADCY5/cAMP/CREB-autophagy axis, promoting autophagic clearance, mitochondrial recovery, and cell-cycle re-entry—thereby ameliorating senescence-associated phenotypes in senescent stem cells. This study identifies melissic acid as a natural senomorphic candidate for stem cell function restoration and provides a mechanistic link between cAMP-dependent autophagy and amelioration of stem cell aging, offering a potential strategy to overcome stem cell exhaustion and regenerative limits imposed by cellular senescence.