Hydrogen-rich liquid down-regulates the expressions of inflammatory factors by ultraviolet B-induced human HaCaT keratinocytes through the autophagy pathway / 中华皮肤科杂志

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 groupsblank 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 Beclin 1,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-1 β,IL-6 and HMGB 1,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,II-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 ofautophagy-associated proteins,and hydrogen-rich medium can down-regulate the expressions of inflammatory factors by UVB-induced HaCaT cells through the autophagy pathway.