Abstract:

Zhang Bingxin, Xing Weibin, Fu Guojun, Chen Hongguang Department of Dermatology, First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China （Zhang BX）; Department of Dermatology, Tianjin Fifth Centre Hospital, Tianjin 300450, China （Xing WB）; Department of Dermatology, Cangzhou People′s Hospital, Cangzhou 061000, Hebei, China （Fu GJ）; Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin 300052, China （Chen HG） Corresponding author: Xing Weibin, Email: xingweibin111@163.com 【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.