Hydrogen-rich liquid down-regulates the expressions of inflammatory factors by ultraviolet B-induced human HaCaT keratinocytes through the autophagy pathway

Abstract

Objective To investigate whether hydrogen can regulate the expressions of inflammatory factors by ultraviolet B (UVB) -induced human HaCaT keratinocytes through the autophagy pathway. Methods Cultured HaCaT keratinocytes were divided into several groups: blank control group receiving no treatment, hydrogen group cultured in hydrogen-rich medium, three UVB groups irradiated with UVB at 1, 10, 50 mJ/cm2 respectively, three UVB + hydrogen groups irradiated with UVB at 1, 10, 50 mJ/cm2 respectively followed by culture in hydrogen-rich medium, UVB + 3MA group pretreated with the autophagy inhibitor 3MA for 1 hour followed by UVB radiation at 50 mJ/cm2, UVB + rapamycin group pretreated with the autophagy activator rapamycin for 1 hour followed by UVB radiation at 50 mJ/cm2, UVB + 3MA+ hydrogen group pretreated with 3MA for 1 hour followed by UVB radiation at 50 mJ/cm2 and culture in hydrogen-rich medium, UVB + rapamycin + hydrogen group pretreated with rapamycin for 1 hour followed by UVB radiation at 50 mJ/cm2 and culture in hydrogen-rich medium. After additional culture with or without hydrogen for 12 hours, methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity, Western-blot analysis to measure the expressions of autophagy-associated protein 1 light chain 3 (LC3) and Beclin1, and enzyme-linked immunosorbent assay (ELISA) to measure the supernatant levels of inflammatory factors including tumor necrosis factor (TNF) -α, interleukin (IL) -1β, IL-6 and high mobility group protein B1 (HMGB1) , and a test kit was used to determine the level of lactate dehydrogenase (LDH) . Results Compared with the blank control group, the 10- and 50-mJ/cm2 UVB groups showed significantly increased release of LDH, expressions of LC3 and Beclin1 and supernatant levels of TNF-α, IL-1β, IL-6 and HMGB1, but decreased cellular proliferative activity (all P < 0.05) . Hydrogen significantly attenuated the release of LDH, down-regulated the supernatant levels of TNF-α, IL-1β, IL-6 and HMGB1, but up-regulated cellular proliferative activity as well as LC3 and Beclin1 expressions in the 10- and 50-mJ/cm2 UVB + hydrogen groups compared with the 10- and 50-mJ/cm2 UVB groups respectively (all P < 0.05) . In addition, the levels of TNF-α, IL-1β, IL-6 and HMGB1 were significantly higher in the 50-mJ/cm2 UVB + 3MA group than in the 50-mJ/cm2 UVB group, and higher in the 50-mJ/cm2 UVB + 3MA + hydrogen group than in the 50-mJ/cm2 UVB + hydrogen group, but lower in the 50-mJ/cm2 UVB + rapamycin group than in the 50-mJ/cm2 UVB group (all P < 0.05) . Conclusion UVB radiation can increase the expressions of autophagy-associated proteins, and hydrogen-rich medium can down-regulate the expressions of inflammatory factors by UVB-induced HaCaT cells through the autophagy pathway. Key words: Ultraviolet rays; Autophagy; Chemokines; Hydrogen; HaCaT cells