中华皮肤科杂志 ›› 2016, Vol. 49 ›› Issue (2): 123-127.

• 论著 • 上一篇    下一篇

富氢液通过自噬途径下调中波紫外线诱导的HaCaT细胞炎症因子的表达

张秉新1,邢卫斌2,付国俊2,陈红光3   

  1. 1. 天津中医药大学第一附属医院皮肤科(联系人:朱学骏)
    2. 河北省沧州市人民医院皮肤科
    3. 天津医科大学
  • 收稿日期:2015-04-20 修回日期:2015-06-18 出版日期:2016-02-15 发布日期:2016-02-04
  • 通讯作者: 邢卫斌 E-mail:xingweibin111@163.com
  • 基金资助:

    Nrf2/ARE 信号通路在氢气治疗重度脓毒症中的作用及机制研究;从线粒体动力学角度研究氢气治疗重度脓毒症的具体机制

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

  • Received:2015-04-20 Revised:2015-06-18 Online:2016-02-15 Published:2016-02-04
  • Contact: wei-bin xing E-mail:xingweibin111@163.com

摘要:

目的 探讨氢气是否能通过自噬途径调节中波紫外线(UVB)诱导的HaCaT细胞炎症因子的表达。 方法 将培养的HaCaT细胞分为空白对照组(不做任何处理),氢气组(仅用富氢培养液培养),1、10、50 mJ/cm2 UVB组,1、10、50 mJ/cm2 UVB + 氢气组,50 mJ/cm2 UVB + 3甲基腺嘌呤(3MA)组,50 mJ/cm2 UVB + 雷帕霉素组,50 mJ/cm2 UVB + 3MA + 氢气组,50 mJ/cm2 UVB + 雷帕霉素 + 氢气组。细胞经过不同处理培养12 h后,噻唑蓝(MTT)法检测细胞增殖,LDH试剂盒检测乳酸脱氢酶(LDH),细胞提取蛋白检测自噬蛋白LC3和Beclin1的表达,上清液用ELISA检测炎症因子肿瘤坏死因子(TNF)α、白细胞介素(IL)-1β、HMGB1和IL-6的水平。 结果 与空白对照组相比,UVB诱导的HaCaT细胞LDH释放增多,细胞活力下降,自噬蛋白LC3和Beclin1表达增加,炎症因子TNF-α、IL-1β、HMGB1和IL-6释放增加(均P < 0.05)。与UVB组相比,氢气可减少LDH释放,提高细胞活力,自噬蛋白LC3和Beclin1表达进一步增加,炎症因子TNF-α、IL-1β、HMGB1和IL-6表达减少(均P < 0.05)。与50 mJ/cm2 UVB组相比,50 mJ/cm2 UVB + 3MA组炎症因子TNF-α、IL-1β、HMGB1和IL-6表达进一步增加(P < 0.05),而50 mJ/cm2 UVB + 雷帕霉素组炎症因子TNF-α、IL-1β、HMGB1和IL-6表达减少(P < 0.05)。与50 mJ/cm2 UVB + 氢气组相比,50 mJ/cm2 UVB + 氢气 + 3MA组炎症因子TNF-α、IL-1β、HMGB1和IL-6表达增加(P < 0.05)。 结论 UVB照射可以诱发自噬蛋白表达增加 ;富氢液可以下调UVB诱导的HaCaT细胞炎症因子的表达,而这一过程是通过激活自噬途径实现的。

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.