Pakhawadee Palungwachira1, Salunya Tancharoen2, Pornpen Dararat2, Thamthiwat Nararatwanchai1
1Department of Dermatology School of Anti-Aging and Regenerative Medicine, Mae Fah Luang University, Chiangrai, Thailand.
2Department of Pharmacology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand.
Background: Oxidative stress, cellular toxicity, and inflammation lead to skin damage, which results in premature skin aging. Recently, anthocyanins (ANT) have received much attention as dietary anti-oxidants involved in the prevention of oxidative damage. Materials and Methods: This study investigated the effects of ANT extracted from black rice (Oryza sativa L.) on the survival of rat dermal fibroblasts (RDFs) after oxidative stress-induced cellular damage by hydrogen peroxide (H2O2) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We further investigated the apoptosis-inducing effects of ANT using 4′,6-diamidino-2-phenylindole and Annexin V staining. The effect of ANT extract on autophagy was confirmed by reverse transcription polymerase chain reaction of the autophagy-related microtubule-associated protein 1B light chain 3 (LC3-II) and ffluorescence microscopy of the LC3-II protein. Results: The high-performance liquid chromatography results indicated the presence of cyanidin-3-O-glucoside in both extracts. The study demonstrated that the addition of crude or purified ANT extract before H2O2 treatment increased RDF cell viability. Pretreatment with ANT decreased the number of cells exhibiting dense chromatin fragments and DNA condensation, which are characteristics of apoptotic cell death. ANT decreased the number of late apoptotic/necrotic (Annexin + and propidium iodide (PI) +) cells and early apoptotic (Annexin V + and PI-) cells. Furthermore, ANT inhibited the H2O2-mediated induction of LC3-II gene expression in RDFs. Conclusion: The contribution of autophagy induction to the protective effects of ANT was verified by the observed decrease in the mRNA and protein expression of LC3-II. These results suggest the therapeutic potential of polyphenolic compounds extracted from O. sativa L. in oxidative damage-induced skin aging.
Keywords: Anthocyanin, autophagy, cell death, microtubule-associated protein 1B light chain 3 (LC3-II), Oryza sativa L., oxidative stress.