Supplementary file 1_Ectoine attenuates H2O2-Induced cellular senescence in human keratinocytes and endothelial cells by modulating the p53/p21 and p16 pathways.docx

BackgroundEctoine ((S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a major compatible solute found in halophilic microorganisms from salt lakes. The anti-cellular senescence effect and skin safety of Ectoine on H2O2-induced oxidative stress senescence in HaCaT cells and EA. hy926 endothelial cells were evaluated through a series of in vitro assays.
MethodsAn oxidative stress senescence model was established using H2O2 in HaCaT and EA. hy926 cells pretreated with various concentrations of Ectoine. Cell viability was assessed using the CCK-8 assay, proliferative capacity was evaluated with the EdU assay, and senescence status was determined by SA-β-gal staining. Intracellular ROS levels were measured using a DCFH probe, and cell death was analysed by flow cytometry. The expression of senescence-related markers was evaluated at the transcriptional and protein levels: The mRNA levels of TP53, CDKN1A (encoding p21), CDKN2A (encoding p16), MMP2, and MMP9 were measured by qRT‒PCR, while their corresponding protein products (p53, p21, and p16) were analysed by Western blotting. Lamin B1 expression was examined by immunofluorescence.
ResultsExposure to H2O2 successfully induced cellular senescence, as evidenced by increased SA-β-gal activity, elevated ROS levels, and upregulated expression of senescence-associated markers (TP53, CDKN1A, CDKN2A, MMP2, and MMP9), along with decreased Lamin B1 expression. Ectoine pretreatment significantly attenuated these senescence phenotypes in a concentration-dependent manner, with 0.50 μmol/L identified as the most effective concentration. At this dosage, Ectoine enhanced cell viability, reduced ROS accumulation, and suppressed cell death without causing cytotoxicity. Mechanistically, Ectoine downregulated the expression of the p53/p21 and p16 pathway components, thereby inhibiting cell cycle arrest.
ConclusionEctoine exerts potent anti-senescence effects in H2O2-induced models of skin-related cell senescence, primarily by modulating the p53/p21 and p16 signalling pathways and reducing oxidative damage. These findings may support further exploration of its potential application in anti-cellular senescence research.