The blue color represents collagen fiber

The blue color represents collagen fiber. the potential of caffeine in the protection of skin disease. oxidative stress model. Here we report that low dose of caffeine (1-10 M) inhibits AAPH- or UV-induced skin cell senescence through activating the A2AR/SIRT3/AMPK-mediated autophagy. These Filgotinib results illustrate the molecular mechanisms underlying the protective effect of caffeine against oxidative stress-induced skin damage. Results AAPH induces cellular senescence To explore strategies that can ameliorate oxidative stress-induced skin aging, we first established senescence models in human A375 melanoma cells and mouse NIH3T3 fibroblasts by AAPH. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) survival assay showed that AAPH inhibited the proliferation of both A375 (Figure ?Figure11A) and NIH3T3 (Figure ?Figure11F) cells in a time- and concentration-dependent manner. To understand how AAPH inhibited cell proliferation, we analyzed cell cycle and cell death by propidium iodide (PI) and PI/Annexin V staining, respectively. AAPH at doses below 4 mM rarely affected the cell cycle (Figure S1) nor induced cell death (Figure S2). However, when the dose was increased to 8 mM and above, AAPH changed the cell cycle profile (mostly G2/M phase arrest, Figure S1) and induced cell death (apoptotic and non-apoptotic, Figure S2). These results suggest a bipartite growth inhibitory effect of AAPH: at high dose, it induces cell cycle arrest and cell death; at low dose, AAPH is generally nontoxic and therefore inhibits cell proliferation through as-yet unidentified mechanisms (P 0.05), confirming the role of oxidative stress in AAPH-induced cell growth inhibition. Open in a separate window Figure 1 Senescence cell models induced by AAPH. (A) The cell growth inhibitory effect of AAPH on A375 cells determined by the MTT assay. (B) Effects of NAC (1 mM) on AAPH-induced A375 cell growth inhibition. (C) SA -Gal staining in A375 cells. Representative images of cells treated with 1 mM AAPH and NAC are shown. Scale bar = 40 m. The ratio of SA -Gal positive cells was presented in the panel. (D) A375 cells were treated with 1 mM of AAPH for 48 h, stained with the anti-K9M-H3-Alexa Fluor 488 antibodies and co-stained with DAPI. Black and white images were used for DAPI and K9M-H3 to better visualize the Filgotinib punctate structures of SAHF. Yellow represented co-localization of DAPI and Alexa Flour 488. Scale bar = 5 m. Quantitation of SAHF-positive cells is shown on the 0.01vs.Control group, ? 0.05 and ?? 0.01 AAPH group. Subsequently, we asked if AAPH could induce senescence in skin cells. To this end, we analyzed three widely accepted senescence markers. First, we measured senescence-associated -galactosidase (SA -Gal) activity. The results show that AAPH dramatically increased the SA -Gal activity in A375 (Figure ?Figure11C, 0.01). Third, we examined activation of p53 and p21, as the p53-p21 pathway not only regulates cell cycle arrest and cell death, but also plays a critical role in senescence induction 35. AAPH significantly increased the protein level of p21 and elevated p53 phosphorylation in A375 cells (Figure ?Figure11E), indicating activation of this pathway. Co-treatment with NAC reversed the AAPH-induced activation of the MGMT p53-p21 pathway (Figure ?Figure11E, 0.01). These results suggest that AAPH induces cellular senescence in transformed skin cells in a manner dependent on oxidative stress. Caffeine inhibits AAPH-induced oxidative stress and senescence Caffeine had been shown to Filgotinib inhibit oxidative stress-induced vascular endothelial cell senescence 13. We found that caffeine at 2.5-10 M significantly attenuated the growth inhibitory effect of AAPH in NIH3T3 cells (Figure S3A). This prompted us to ask whether caffeine could suppress AAPH-induced cellular senescence. We found that caffeine indeed inhibited AAPH-induced increase in the SA -Gal activity in A375 (Figure ?Figure22A) and NIH3T3 cells (Figure S3C). Further, caffeine Filgotinib suppressed AAPH-induced.