A Novel Standardized Propolis Extract Modulates Oxidative Stress-Induced Senescence in Human Dermal Fibroblasts by Suppressing SASP and Modulating Cell Cycle-Associated Gene Expression: A Preliminary In Vitro Investigation

Background Cellular senescence, often driven by oxidative stress, is a fundamental driver of skin aging ("inflammaging"). While natural extracts are promising senomodulators, traditional propolis extracts suffer from inconsistent composition and problematic solvents. We have developed a novel, non-alcoholic solvent system based on Polyethylene Glycol (PEG) 400 and lecithin, which, through a chemoselective process, yields a Standardized Propolis Extract (SPE) significantly enriched in key phenolic bioactives (p-coumaric acid, trans-ferulic acid, caffeic acid, and CAPE). Objective To investigate the in vitro biological efficacy of this novel SPE in modulating core senescence pathways, thereby providing preliminary molecular insights into its clinically observed anti-aging effects. Methods Human Dermal Fibroblasts (HDFs) were pre-treated with non-cytotoxic doses of SPE (0.01%, 0.05%) or Rapamycin (3µM) as a control. Senescence was induced using a validated stress-induced premature senescence (SIPS) protocol (200µM H ₂ O ₂ for 2 hours). After six days, gene expression for key senescence (CDKN2A/p16, CDKN1A/p21), SASP (IL-6, IL-8), and cell cycle (CDK4, CDK2, CCNE1) markers was quantified via qPCR. A secondary study on Mesenchymal Stem Cells (MSCs) under starvation stress assessed Senescence-Associated β-galactosidase (SA-β-gal) activity. Results The 0.05% SPE concentration was determined to be the optimal non-cytotoxic dose. In H ₂ O ₂ -stressed HDFs, this 0.05% SPE treatment demonstrated a potent anti-inflammatory effect, significantly suppressing the key SASP marker IL-6 (Fold Change: -7.78, p = 0.003). This effect was comparable in magnitude to the Rapamycin control (FC: -8.1, p = 0.003). Critically, 0.05% SPE uniquely and strongly upregulated the cell cycle-associated gene CDK4 mRNA (FC: +6.71, p = 0.002), an effect not observed with Rapamycin. SPE also moderately upregulated the repair-associated p21 marker (FC: +2.33). In the exploratory MSC model, 0.01% SPE qualitatively reduced SA-β-gal activity compared to the stressed control. Conclusion The novel SPE, standardized by its unique extraction process, demonstrates a potent and distinct senomodulatory profile. It effectively uncouples the senescence-associated inflammatory response (suppressing IL-6) from permanent cell cycle arrest by promoting a pro-regenerative transcriptional profile (upregulating CDK4 mRNA), consistent with senomorphic activity. This dual action offers a potential mechanistic explanation for the extract's clinically proven anti-wrinkle efficacy, positioning it as an advanced, evidence-based ingredient for dermo-cosmetic formulations targeting inflammaging.