| Abstract |
The polyphenol derivative 3,4-dihydroxybenzalacetone (DBL) is the primary antioxidative component of the medicinal folk mushroom Chaga (Inonotus obliquus (persoon) Pilat). In this study, we investigated whether the antioxidative effect of DBL could propagate to recipient cells via secreted components, including extracellular vesicles (EVs), after pre-exposing SH-SY5Y human neuroblastoma cells to DBL. First, we prepared EV-enriched fractions via sucrose density gradient ultracentrifugation using conditioned medium from SH-SY5Y cells exposed to 100 µM hydrogen peroxide (H2O2) for 24 h, with and without 1 h of 5 µM DBL pre-treatment. CD63 immuno-dot blot analysis demonstrated that fractions with density of 1.06-1.09 g/cm3 had CD63-like immuno-reactivities. Furthermore, the 2,2-diphenyl-1-picrylhydrazyl assay revealed that the radical scavenging activity of fraction 11 (density of 1.06 g/cm3), prepared after 24-h H2O2 treatment, was significantly increased compared to that in the control group (no H2O2 treatment). Notably, 1 h of 5 µM DBL pre-treatment or 5 min of heat treatment (100 °C) diminished this effect, although concentrating the fraction by 100 kDa ultrafiltration enhanced it. Overall, the effect was not specific to the recipient cell types. In addition, the uptake of fluorescent Paul Karl Horan-labeled EVs in concentrated fraction 11 was detected in all treatment groups, particularly in the H2O2-treated group. The results suggest that cell-to-cell communication via bioactive substances, such as EVs, in conditioned SH-SY5Y cell medium, propagates the H2O2-induced radical scavenging effect, whereas pre-conditioning with DBL inhibits it.
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