中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
CHINESE JOURNAL OF IMMUNOLOGY
2009年
11期
991-995
,共5页
张春晶%顾立刚%牛旭艳%王甫%彭桂英
張春晶%顧立剛%牛旭豔%王甫%彭桂英
장춘정%고립강%우욱염%왕보%팽계영
人参皂苷Rg1%氧化应激%NF-κB%双荧光素酶报告系统
人參皂苷Rg1%氧化應激%NF-κB%雙熒光素酶報告繫統
인삼조감Rg1%양화응격%NF-κB%쌍형광소매보고계통
Ginsenoside Rg1%Oxidative stress%Nuclear factor-kappa B (NF-κB)%Dual-luciferase reporter system
目的:观察人参皂苷Rg1对H_2O_2诱导HEK293T细胞损伤过程中NF-κB转录活性的影响,探讨人参皂苷Rg1的抗氧化机制.方法:H_2O_2模拟氧化应激条件,MTT法和台盼蓝染色法检测人参皂苷Rg1对细胞生长的影响;以DCFH-DA为探针流式细胞仪检测细胞内ROS水平;利用双荧光素酶顺式报告系统,检测人参皂苷Rg1对荧光素酶报告基因NF-κB-Luc相对荧光素酶值的影响,以检测人参皂苷Rg1是否具有下调H_2O_2诱导的NF-κB转录激活的作用.结果:H_2O_2诱导损伤的293T细胞存活率随H_2O_2浓度的增高而下降,相同H_2O_2浓度下,人参皂苷Rg1预保护组细胞存活率较损伤组显著提高(P＜0.05);H_2O_2处理后细胞内自由基明显增加,其荧光强度增加了40%～50%,而给予有效浓度的人参皂苷Rg1后,荧光强度降低35%～40%;与正常对照组比较,H_2O_2模拟氧化应激条件下,HEK293T细胞中NF-κB荧光素酶报告活性明显升高(P＜0.05),而在人参皂苷Rg1预保护组则明显受到抑制(P＜0.05).结论:人参皂苷Rg1对H_2O_2所致的细胞氧化应激损伤具有明显的保护作用,其可能机制是有效地清除了细胞内过多的自由基,下调了转录因子NF-κB的转录活性,继而抑制了NF-κB通路的激活.
目的:觀察人參皂苷Rg1對H_2O_2誘導HEK293T細胞損傷過程中NF-κB轉錄活性的影響,探討人參皂苷Rg1的抗氧化機製.方法:H_2O_2模擬氧化應激條件,MTT法和檯盼藍染色法檢測人參皂苷Rg1對細胞生長的影響;以DCFH-DA為探針流式細胞儀檢測細胞內ROS水平;利用雙熒光素酶順式報告繫統,檢測人參皂苷Rg1對熒光素酶報告基因NF-κB-Luc相對熒光素酶值的影響,以檢測人參皂苷Rg1是否具有下調H_2O_2誘導的NF-κB轉錄激活的作用.結果:H_2O_2誘導損傷的293T細胞存活率隨H_2O_2濃度的增高而下降,相同H_2O_2濃度下,人參皂苷Rg1預保護組細胞存活率較損傷組顯著提高(P＜0.05);H_2O_2處理後細胞內自由基明顯增加,其熒光彊度增加瞭40%～50%,而給予有效濃度的人參皂苷Rg1後,熒光彊度降低35%～40%;與正常對照組比較,H_2O_2模擬氧化應激條件下,HEK293T細胞中NF-κB熒光素酶報告活性明顯升高(P＜0.05),而在人參皂苷Rg1預保護組則明顯受到抑製(P＜0.05).結論:人參皂苷Rg1對H_2O_2所緻的細胞氧化應激損傷具有明顯的保護作用,其可能機製是有效地清除瞭細胞內過多的自由基,下調瞭轉錄因子NF-κB的轉錄活性,繼而抑製瞭NF-κB通路的激活.
목적:관찰인삼조감Rg1대H_2O_2유도HEK293T세포손상과정중NF-κB전록활성적영향,탐토인삼조감Rg1적항양화궤제.방법:H_2O_2모의양화응격조건,MTT법화태반람염색법검측인삼조감Rg1대세포생장적영향;이DCFH-DA위탐침류식세포의검측세포내ROS수평;이용쌍형광소매순식보고계통,검측인삼조감Rg1대형광소매보고기인NF-κB-Luc상대형광소매치적영향,이검측인삼조감Rg1시부구유하조H_2O_2유도적NF-κB전록격활적작용.결과:H_2O_2유도손상적293T세포존활솔수H_2O_2농도적증고이하강,상동H_2O_2농도하,인삼조감Rg1예보호조세포존활솔교손상조현저제고(P＜0.05);H_2O_2처리후세포내자유기명현증가,기형광강도증가료40%～50%,이급여유효농도적인삼조감Rg1후,형광강도강저35%～40%;여정상대조조비교,H_2O_2모의양화응격조건하,HEK293T세포중NF-κB형광소매보고활성명현승고(P＜0.05),이재인삼조감Rg1예보호조칙명현수도억제(P＜0.05).결론:인삼조감Rg1대H_2O_2소치적세포양화응격손상구유명현적보호작용,기가능궤제시유효지청제료세포내과다적자유기,하조료전록인자NF-κB적전록활성,계이억제료NF-κB통로적격활.
Objective:To observe the influence of ginsenoside Rg1 on transcriptional activation of NF-κB induced by hydrogen peroxide (H_2O_2) in 293T cell,and probe into the antioxidant mechanism of ginsenoside Rg1.Methods:In the experiment,cells was exposed to H_2O_2 after pretreatment with Rg1,cell proliferation and cytotoxicity studies were detected by MTT and Trypan blue.The quantities of generation of intracellular reactive oxygen species (iROS) was analyzed by flow cytometric analysis measured with fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA).NF-κB-responsive element-luciferase reporter gene was transfected and dual-luciferase cis-reporting systems were used to assay the transcriptional activity of NF-κB under the stimulated circumstance of oxidative stress induced by H_2O_2.Results:The results of MTT showed that ginsenoside Rg1 apparently protected the proliferation of 293T cell,which were repressed by H_2O_2 (P＜0.05).The results by trypan blue showed that H_2O_2 stimulated substantial cytotoxicity.This effect was markedly attenuated by treatment with ginsenoside Rg1.Oxidant production,measured as the fluorescence of dichlorofluorescein,was significant increased by 40%-50% through H_2O_2 stimulation.The decrease in iROS generation was significant blocked by 35%-40% through Rg1 and antioxidant.The relative luciferase reporter assay of NF-κB was apparently improved by H_2O_2-induced(P＜0.05),but Ginsenoside Rg1 significantly repressed the relative value of luciferase (P＜0.05).Conclusion:Ginsenoside Rg1 has the obvious protective function from the damage of oxidative stress damage,whose possible mechanism is to eliminate excessive free radicals of the cells effectively,to reduce transcriptional activation of nuclear factor kappa B(NF-κB),and subsequently to suppress the NF-κB circuit activation.
