富硒油茶籽油对H_2O_2诱导HaCaT细胞氧化损伤的保护作用研究
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摘要
探究油茶籽油对过氧化氢诱导人永生化表皮细胞(HaCaT)氧化损伤的保护作用。以油茶果经物理压榨所得油茶籽油为材料,以体外培养的HaCaT细胞为试验对象,试验分为空白对照组、过氧化氢(H_2O_2)模型损伤组、阳性对照组(VC)、处理组(油茶籽油5～500μg/m L预处理),以细胞内细胞存活率(凋亡率)、SOD、GSH-PX、ROS及其Nrf2基因表达水平为综合评价指标,评价油茶籽油的抗氧化性。结果表明:当加入油茶籽油对细胞进行预保护后,质量浓度50～200μg/m L的油茶籽油对损伤细胞的存活率在70%～82%之间,较H_2O_2模型损伤组的高。50μg/m L油茶籽油处理组细胞凋亡率为4. 49%,较H_2O_2模型损伤组降低了44. 77%;细胞内SOD水平(22. 09 U/mg)、GSH-PX水平(64. 50 U/mg)较H_2O_2模型损伤组升高了46. 68%、63. 66%,且存在明显差异(P <0. 05);而ROS水平显著下降,与H_2O_2模型损伤组(88. 27)相比细胞内平均荧光强度下降了37. 66%;此时Nrf2基因相对表达量为1. 13,较模型组提高了101. 78%;各指标均较优于阳性对照50μg/m L VC处理组的效果。说明油茶籽油对H_2O_2造成的氧化损伤具有一定的预保护作用,且好于相同质量浓度的VC处理效果,可进一步将其开发成相应的天然有效抗氧化剂产品。
The protective effect of oil-tea camellia seed oil on the human immortalized epidermal cells( HaCaT) injuried by hydrogen peroxide was studied. The oil-tea camellia seed oil from Camellia oleifera obtained by physical pressing was used as material. HaCaT cells cultured in vitro were used as the experimental objects. The experiment was divided into control group,H_2O_2 model damage group,positive control group( VC),and treatment group( oil-tea camellia seed oil,5-500 μg/mL),using cell survival rate( apoptosis rate) in cell,SOD level,GSH-PX level,ROS level and Nrf2 gene expression as evaluation indexes to evaluate the antioxidant activity. The results showed that when oil-tea camellia seed oil was added to pre-protect the cells, the survival rate of damaged cells was70%-82% for 50-200 μg/mL of oil-tea camellia seed oil,higher than H_2O_2 model group.The apoptosis rate of 50 μg/mL oil-tea camellia seed oil treatment group was 4. 49%,reducing by44. 77% compared with H_2O_2 model damage group. Compared with H_2O_2 model damage group,the levels of intracellular SOD( 22. 09 U/mg) and GSH-PX( 64. 50 U/mg) increased by 46. 68% and63. 66% respectively,with significant differences( P < 0. 05). While the level of ROS decreased significantly,the mean fluorescence intensity in cells decreased by 37. 66% compared with H_2O_2 model damage group( 88. 27). At the same time,the relative expression of Nrf2 gene was 1. 13,increasing by101. 78% compared with H_2O_2 model damage group. All indexes were better than those of positive control 50 μg/mL VCtreatment group. It indicated that oil-tea camellia seed oil had a certain pre-protective effect on oxidative damage caused by H_2O_2,better than the same mass concentration of VC. It could be further developed into a natural and effective antioxidant product.
The protective effect of oil-tea camellia seed oil on the human immortalized epidermal cells( HaCaT) injuried by hydrogen peroxide was studied. The oil-tea camellia seed oil from Camellia oleifera obtained by physical pressing was used as material. HaCaT cells cultured in vitro were used as the experimental objects. The experiment was divided into control group,H_2O_2 model damage group,positive control group( VC),and treatment group( oil-tea camellia seed oil,5-500 μg/mL),using cell survival rate( apoptosis rate) in cell,SOD level,GSH-PX level,ROS level and Nrf2 gene expression as evaluation indexes to evaluate the antioxidant activity. The results showed that when oil-tea camellia seed oil was added to pre-protect the cells, the survival rate of damaged cells was70%-82% for 50-200 μg/mL of oil-tea camellia seed oil,higher than H_2O_2 model group.The apoptosis rate of 50 μg/mL oil-tea camellia seed oil treatment group was 4. 49%,reducing by44. 77% compared with H_2O_2 model damage group. Compared with H_2O_2 model damage group,the levels of intracellular SOD( 22. 09 U/mg) and GSH-PX( 64. 50 U/mg) increased by 46. 68% and63. 66% respectively,with significant differences( P < 0. 05). While the level of ROS decreased significantly,the mean fluorescence intensity in cells decreased by 37. 66% compared with H_2O_2 model damage group( 88. 27). At the same time,the relative expression of Nrf2 gene was 1. 13,increasing by101. 78% compared with H_2O_2 model damage group. All indexes were better than those of positive control 50 μg/mL VCtreatment group. It indicated that oil-tea camellia seed oil had a certain pre-protective effect on oxidative damage caused by H_2O_2,better than the same mass concentration of VC. It could be further developed into a natural and effective antioxidant product.
引文
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[13]CHANG Y L,DAE-OK K.Comprehensive study on vitamin C equivalent antioxidant capacity(VCEAC)of various polyphenolics in scavenging a free radical and its structural relationship[J].Crit Rev Food Sci Nutr,2004,44(4):253-273.
[14]SUN Y,CHENG Z,HUANG S,et al.Selenium polysaccharide SPMP-2a from pleurotus geesteranus alleviates H2O2-induced oxidative damage in Ha Ca T cells[J].Biom Res Int,2017,2017:1-9.
[15]李振洁,彭丽倩,江娜,等.枸杞多糖对紫外线辐射Ha Ca T细胞急性损伤中Nrf2/Bach1通路的影响[J].中国皮肤性病学杂志,2017(11):1169-1174.
[16]霍仕霞,彭晓明,高莉,等.高良姜素对过氧化氢诱导的A375细胞氧化损伤后Nrf2、γ-GCS基因表达的影响[J].中国中医药信息杂志,2015,22(11):69-72.
[2]宋英强,杨粉莉,杨博,等.我国油茶种植环境适宜性评价初步研究[J].山东农业大学学报(自然科学版),2015(2):180-188.
[3]ZHU Y,ZHONG H Y,SUN H Z,et al.Development of quantitative analysis of fatty acid for monitoring changes of fatty acid profile of camellia seed oil[J].Adv Mater Res,2012,554-556:1202-1210.
[4]吕建云,孙丰霞,耿越.山茶油中4种功能性成分的测定[J].食品安全质量检测学报,2014(6):1641-1646.
[5]冯秋瑜,宋宁,黄慧学,等.山茶油的药用研究进展[J].中国实验方剂学杂志,2016(10):215-220.
[6]FLORIDAJAMES G D,SIMPSON R,DAVISON G,et al.Exercise,free radical metabolism,and aging:cellular and molecular processes[J].Oxid Med Cel Long,2016,2016:1-2.
[7]VINCENT H K,TAYLOR A G.Biomarkers and potential mechanisms of obesity-induced oxidant stress in humans[J].Int J Obes,2006,30(3):400-418.
[8]ASEERVATHAM G S,SIVASUDHA T,JEYADEVI R,et al.Environmental factors and unhealthy lifestyle influence oxidative stress in humans-an overview[J].Environ Sci Pollut Res,2013,20(7):4356-4369.
[9]方丽君,林颖彬,黄恩,等.Calpain抑制剂ALLN对H2O2诱导的661W细胞损害的影响[J].中国药理学通报,2018(2):181-185.
[10]SMITS E,BURVEENICH C,HEYNEMAN R.Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood[J].Vet Immunol Immunopathol,1997,56(3/4):259-269.
[11]徐鹰,黄贤华,唐丹.大豆甙元对氧自由基所致PCl2细胞毒作用的影响[J].中药药理与临床,2007,23(5):78-80.
[12]宋菲,王挥,唐敏敏,等.天然椰子油提取物对H2O2诱导的HSF细胞氧化损伤的保护作用[J].日用化学工业,2017,47(9):517-521.
[13]CHANG Y L,DAE-OK K.Comprehensive study on vitamin C equivalent antioxidant capacity(VCEAC)of various polyphenolics in scavenging a free radical and its structural relationship[J].Crit Rev Food Sci Nutr,2004,44(4):253-273.
[14]SUN Y,CHENG Z,HUANG S,et al.Selenium polysaccharide SPMP-2a from pleurotus geesteranus alleviates H2O2-induced oxidative damage in Ha Ca T cells[J].Biom Res Int,2017,2017:1-9.
[15]李振洁,彭丽倩,江娜,等.枸杞多糖对紫外线辐射Ha Ca T细胞急性损伤中Nrf2/Bach1通路的影响[J].中国皮肤性病学杂志,2017(11):1169-1174.
[16]霍仕霞,彭晓明,高莉,等.高良姜素对过氧化氢诱导的A375细胞氧化损伤后Nrf2、γ-GCS基因表达的影响[J].中国中医药信息杂志,2015,22(11):69-72.