摘要
目的:研究甘草素(liquiritigenin,LQ)对长波紫外线(UVA)诱导的正常人表皮角质形成细胞(NHEK)氧化损伤的保护作用,并探讨其光老化的保护作用机制。方法:建立12 J·cm^(-2)剂量的UVA诱导的NHEK细胞的光老化模型。WST⁃8(CCK⁃8)比色法测定细胞增殖活力;β⁃半乳糖苷酶染色法检测细胞衰老水平;流式细胞仪检测活性氧(ROS)含量;荧光显微镜观察线粒体膜电位的变化;试剂盒检测细胞中超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GSH)、丙二醛(MDA)的活性。蛋白免疫印迹法(Western blot)检测相关蛋白表达水平;免疫荧光法(immunofluorescence)探究核因子E2相关因子2(Nrf2)和Keap⁃1在细胞核内表达及相互作用。结果:与UVA组相比,20,50,100μmol·L^(-1)甘草素呈浓度依赖性地抑制UVA诱导的细胞衰老、ROS和MDA的生成、线粒体膜电位的下降以及基质金属蛋白酶(MMP⁃1,MMP⁃3,MMP⁃9)的合成;显著提高细胞增殖活力、抗氧化酶活性(SOD,GSH)和Ⅰ型胶原蛋白(COL1A1)的生成(P<0.05)。在蛋白表达水平上,与UVA组相比,甘草素能够促进Nrf2和SOD⁃2的表达水平升高,抑制Keap⁃1的表达水平降低(P<0.05)。共定位分析表明,12 J·cm^(-2)剂量的UVA辐照导致NHEK细胞Keap⁃1与Nrf2结合减弱,核内Nrf2表达降低,抑制其转录活性;100μmol·L^(-1)甘草素能够维持Keap⁃1与Nrf2结合,促进Nrf2在核内表达,激活其转录活性。结论:甘草素能够通过Keap⁃1/Nrf2信号通路,促进Nrf2核转录,增强抗氧化能力保护UVA诱导的NHEK细胞氧化损伤,为明确其作为皮肤光老化保护剂提供了理论依据。
Objective:To study the protective effect of liquiritigenin(LQ)on oxidative damage of normal human epidermal keratinocytes(NHEK)induced by long⁃wave ultraviolet(UVA),and to explore its protective mechanism of photoaging.Methods:A photoaging model of NHEK cells induced by UVA was established at a dose of 12 J·cm^(-2).Cell proliferation activity was determined using WST⁃8(CCK⁃8)colorimetric method,and the optimal effective concentration of the drug was screened.β⁃Galactosidase staining was used to detect the level of cell senescence.The activity oxygen species(ROS)were detected by flow cytometry.Fluorescence microscopy was used to observe changes in mitochondrial membrane potential.The kits detecting the activities of superoxide dismutase(SOD),glutathione reductase(GSH)and malondialdehyde(MDA)were applied in cells.Western blotting was used to detect related proteins,and immunofluorescence to explore the expression and interaction of Nrf2 and Keap⁃1 in the nucleus.Results:Compared with the UVA group,liquiritigenin at concentrations of 20,50,and 100μmol·L^(-1)inhibited UVA⁃induced cell senescence,with the generation of ROS and MDA,the decrease of mitochondrial membrane potential,and the synthesis of matrix metalloproteinases(MMP⁃1,MMP⁃3,MMP⁃9)in a concentration⁃dependent manner.Cell proliferation activity,antioxidant enzyme activity(SOD,GSH)and the production of type I collagen significantly increased(P<0.05).In terms of protein expression,compared with UVA group,liquiritigenin promoted the expression levels of Nrf2 and SOD⁃2,and inhibited that of Keap⁃1(P<0.05).Co⁃localization analysis showed that UVA irradiation at a dose of 12 J·cm^(-2)weakened the binding of Keap⁃1 to Nrf2 in NHEK cells,decreased the protein expression and transcriptional activity of Nrf2 in the nucleus.The binding of Keap⁃1 to Nrf2 was maintained by LQ at 100μmol·L^(-1).It also promoted Nrf2 expression in nucleus and activated its transcriptional activity.Conclusion:Liquiritigenin can promote the nuclear trans
作者
卢琦
邹林峰
高远真
叶婷
李梦娇
张钰坤
梁冰
原阳
孙文社
邢东明
LU Qi;ZOU Lin-feng;GAO Yuan-zhen;YE Ting;LI Meng-jiao;ZHANG Yu-kun;LIANG Bing;YUAN Yang;SUN Wen-she;XING Dong-ming(School of Basic Medicine,Qingdao University,Qingdao266071,China;Cancer Institute of The Affiliated Hospital of Qingdao University and Qingdao Cancer Institute,Qingdao266071,China;School of Life Science,Tsinghua University,Beijing100091,China)
出处
《中国新药杂志》
CAS
CSCD
北大核心
2022年第20期2056-2066,共11页
Chinese Journal of New Drugs
