何首乌二苯乙烯苷抑制长波紫外线诱导的HaCaT细胞氧化应激损伤
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摘要
目的探讨二苯乙烯苷对长波紫外线(UVA)照射HaCaT细胞氧化应激损伤的保护作用。方法从何首乌中提取二苯乙烯苷。UVA照射(18J／cm2)人角质形成细胞HaCaT构建体外细胞模型。MTT法测定细胞活力；酶生化法测定胞质SOD, GSH-px活性及MDA水平：人氧化应激与抗氧化PCR芯片检测细胞内氧化应激及抗氧化相关基因mRNA的变化,并通过实时定量PCR验证芯片结果。结果在18J/cm2UVA照射下,给定浓度范围内的二苯乙烯苷能剂量依赖性提高胞质SOD、GSH-px活力,降低胞质MDA水平：基因芯片检测发现二苯乙烯苷可明显改变部分氧化应激及抗氧化基因的表达。通过实时定量PCR验证,基因芯片结果真实、可靠。结论二苯乙烯苷对UVA辐射HaCaT细胞的氧化应激损伤具有一定的保护作用。其机制与清除自由基、提高抗氧化酶活性、改变相关基因表达有关。
Objective To investigate the stilbene glucoside's protective ability to UVA-induced oxidative damage to HaCaT keratinocytes. Methods Stilbene glycoside was isolated from Polygonum multiflorum. The UVA radiant intensity was 18J/cm2, and human keratinocytes HaCaT were used as an experimental in vitro model. To test the cell viability by MTT; Enzyme biochemical method determined the cytoplasmic SOD, GSH-px activity and MDA levels. The Human Oxidative Stress and Antioxidant Defense RT2 ProfilerTM PCR Array was used to detect the related intracellular gene's mRNA changes, and its results were validated by Real-time PCR. Results Under 18J/cm2 UVA radiation, within the scope of a given concentration of stilbene glucoside can dose-dependently increase SOD, GSH-px activity and decrease MDA level in the cytoplasm; gene chip detected that stilbene glucoside can significantly change some oxidative and antioxidative genes expression, and PCR Array's results were validated conformably by Real-time PCR. Conclusion Stilbene glycoside have some protective effect on UVA-induced oxidative injury to HaCaT keratinocytes. Its mechanism may be related to the elimination of free radicals, the increasing of SOD and GSH levels, changs in some related genes expression.
Objective To investigate the stilbene glucoside's protective ability to UVA-induced oxidative damage to HaCaT keratinocytes. Methods Stilbene glycoside was isolated from Polygonum multiflorum. The UVA radiant intensity was 18J/cm2, and human keratinocytes HaCaT were used as an experimental in vitro model. To test the cell viability by MTT; Enzyme biochemical method determined the cytoplasmic SOD, GSH-px activity and MDA levels. The Human Oxidative Stress and Antioxidant Defense RT2 ProfilerTM PCR Array was used to detect the related intracellular gene's mRNA changes, and its results were validated by Real-time PCR. Results Under 18J/cm2 UVA radiation, within the scope of a given concentration of stilbene glucoside can dose-dependently increase SOD, GSH-px activity and decrease MDA level in the cytoplasm; gene chip detected that stilbene glucoside can significantly change some oxidative and antioxidative genes expression, and PCR Array's results were validated conformably by Real-time PCR. Conclusion Stilbene glycoside have some protective effect on UVA-induced oxidative injury to HaCaT keratinocytes. Its mechanism may be related to the elimination of free radicals, the increasing of SOD and GSH levels, changs in some related genes expression.
引文
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[7]苗明三,方晓艳.制何首乌多糖对衰老模型小鼠抗氧化作用的研究[J].中药药理与临床.2002,18(5)：23-24
[8]李艳,余君.何首乌水提液对老化模型大鼠脑组织内抗氧化酶表达的影响[J].湖北民族学院学报.医学版.2007,24(2)：15-17
[9]宋士军,李芳芳,岳华等.何首乌的抗衰老作用研究[J].河北医科大学学报2003,24(2)：90-91
[10]Valencia A, Kochevar IE. Noxl-based NADPH oxidase is the major source of UVA-induced reactive oxygen species in human keratinocytes[J]. J Invest Dermatol, 2008,128(1):214-22.
[11]Emri G, Horkay I, Remenyik E. The role of free radicals in the UV-induced skin damage [J]. Photoaging. Orv Hetil,2006,147(16):731-5.
[12]Van Laethem A, Nys K, Van Kelst S, et al. Apoptosis signal regulating kinase-1 connects reactive oxygen species to p38 MAPK-induced mitochondrial apoptosis in UVB-irradiated human keratinocytes[J]. Free Radic Biol Med,2006,41(9):1361-71
[13]Kovacs D, Raffa S, Flori E, et al. Keratinocyte growth factor down-regulates intracellular ROS production induced by UVB [J]. J Dermatol Sci,2009, 54(2):106-13.
[14]Yao J, Liu Y, Wang X, et al. UVB radiation induces human lens epithelial cell migration via NADPH oxidase-mediated generation of reactive oxygen species and up-regulation of matrix metalloproteinases [J]. Int J Mol Med.2009 Aug; 24(2):153-9.
[15]Van Laethem A, Nys K, Van Kelst S, et al.The UV (Ribotoxic) stress response of human keratinocytes involves the unexpected uncoupling of the Ras-extracellular signal-regulated kinase signaling cascade from the activated epidermal growth factor receptor[J]. Mol Cell Biol,2002,22(15):5380-94
[16]Iordanov MS, Choi RJ, Ryabinina OP, et al.Apoptosis signal regulating kinase-1 connects reactive oxygen species to p38 MAPK-induced mitochondrial apoptosis in UVB-irradiated human keratinocytes [J]. Free Radic Biol Med,2006,41(9):1361-71.
[17]Wang Q, Turlington A, Heo S, et al. Extracellular matrix activity and caveolae events contribute to cell surface receptor activation that leads to MAP kinase activation in response to UV irradiation in cultured human keratinocytes [J]. Int J Mol Med,2005,15(4):633-40.
18] Cooper SJ, Bowden GT. Ultraviolet B regulation of transcription factor families: roles of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) in UVB-induced skin carcinogenesis [J]. Curr Cancer Drug Targets,2007,7(4):325-34.
[19]居永芳,朱威.皮肤光老化的研究进展[J].中华现代皮肤科学杂志.2005,2(2)：676-678
[20]MARTA WRONA, WITOLD KORYTOWSKI, MAGORZATA ROZANOWSKA, et al. COOPERATION OF ANTIOXIDANTS IN PROTECTION AGAINST PHOTOSENSITIZED OXIDATION; Free Radical Biology & Medicine, 2003,35(10):1319-1329
[21]Heng-long Hu, R.J. Forsey, Tony J. Blades, et al.Antioxidants may contribute in the fight against ageing:an in vitro model. Mechanisms of Ageing and Development, 2000,12:1217-230
[22]Hoppe U, Bergemann J, Diembeck W, et al. Coenzyme Q10:a cutaneous antioxidant and energizr [J]. Biofactom.1999,(241):371.
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