矢车菊素-3-O-葡萄糖苷对H_2O_2诱导细胞氧化损伤的保护作用
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摘要
目的:研究矢车菊素-3-O-葡萄糖苷(C3G)对人胚肾HEK-293细胞氧化损伤的保护作用。方法:通过检测细胞活力、活性氧(ROS)水平和丙二醛(MDA)水平考察C3G对氧化损伤细胞的影响,并采用Western blot法和q RTPCR检测Nrf2和Keap1的蛋白表达量和m RNA表达量变化。结果表明:C3G预处理氧化应激细胞后,细胞活力显著高于H_2O_2损伤组(p <0.05),并呈浓度依赖性提高; H_2O_2损伤组的DCF荧光强于对照组,ROS相对含量增加到近2倍,1.25、5、20μmol/L C3G预处理后细胞荧光强度逐渐减弱,ROS相对量呈浓度依赖性降低; C3G预处理后细胞MDA水平呈浓度依赖性降低; 1.25、5、20μmol/L C3G处理后,Nrf2的m RNA表达和蛋白表达量均呈浓度依赖性上调,Keap1蛋白表达量显著低于H_2O_2损伤组(p <0.05),5和20μmol/L C3G对Keap1的m RNA下调作用明显。结论:矢车菊素-3-O-葡萄糖苷对HEK-293细胞有保护作用,可降低细胞ROS和MDA水平,其作用机制可能是通过激活Nrf2/Keap1信号通路以抵抗H_2O_2导致的细胞氧化损伤。
Objective: To investigate the inhibitive effect of Cyanidin-3-O-glucoside (C3G) on oxidative stress in human embryonic kidney cells (HEK-293). Methods: Cell viability,MDA level and the level of intracellular reactive oxygen species (ROS) were investigated.Quantitative real-time PCR and Western blotting were used to detect the expression of Nrf2 and Kelch-like ECH associated protein 1 (Keap1).Results: After pretreatment with C3G,the cell viability was significantly higher than that of the injury group and increased in a concentration-dependent manner (p < 0.05). The fluorescence intensity of oxidative DCF in injured group was increased compared with control group,and the ROS content of injured group was twice greater then control group. The fluorescence intensity of cells which pretreated with C3G was gradually weakened. The ROS content of C3G group decreased in a concentration-dependent manner.After pretreatment of C3G,the MDA levels decreased in a concentration-dependent manner. Furthermore,the protein and m RNA expressions of Nrf2 were significantly enhanced by 1.25,5 and 20 μmol/L of C3G (p < 0.05).The protein expression of Keap1 was significantly lower than that of injury group,and the m RNA expression of Keap1 was significantly down-regulated by 5 and 20 μmol/L of C3G. Conclusion: These results demonstrated that C3G protect HEK-293 cells against H_2O_2-induced oxidative stress through reducing intracellular ROS and MDA,as well as activating Nrf2/Keap1 signaling pathway.
Objective: To investigate the inhibitive effect of Cyanidin-3-O-glucoside (C3G) on oxidative stress in human embryonic kidney cells (HEK-293). Methods: Cell viability,MDA level and the level of intracellular reactive oxygen species (ROS) were investigated.Quantitative real-time PCR and Western blotting were used to detect the expression of Nrf2 and Kelch-like ECH associated protein 1 (Keap1).Results: After pretreatment with C3G,the cell viability was significantly higher than that of the injury group and increased in a concentration-dependent manner (p < 0.05). The fluorescence intensity of oxidative DCF in injured group was increased compared with control group,and the ROS content of injured group was twice greater then control group. The fluorescence intensity of cells which pretreated with C3G was gradually weakened. The ROS content of C3G group decreased in a concentration-dependent manner.After pretreatment of C3G,the MDA levels decreased in a concentration-dependent manner. Furthermore,the protein and m RNA expressions of Nrf2 were significantly enhanced by 1.25,5 and 20 μmol/L of C3G (p < 0.05).The protein expression of Keap1 was significantly lower than that of injury group,and the m RNA expression of Keap1 was significantly down-regulated by 5 and 20 μmol/L of C3G. Conclusion: These results demonstrated that C3G protect HEK-293 cells against H_2O_2-induced oxidative stress through reducing intracellular ROS and MDA,as well as activating Nrf2/Keap1 signaling pathway.
引文
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[22]Yan F,Chen Y,Azat R,et al.Mulberry anthocyanin extract ameliorates oxidative damage in Hep G2 cells and prolongs the lifespan of caenorhabditis elegans through MAPK and Nrf2Pathways[J].Oxidative Medicine and Cellular Longevity,2017,2017:7956158.
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[24]宋柬,廖小军,胡小松,等.矢车菊素_3_葡萄糖苷干预丙烯酰胺细胞毒性的体外模型筛选[J].中国食品学报,2017,17(9):11-19.
[2]Reuter S,Gupta S C,Chaturvedi M M,et al.Oxidative stress,inflammation,and cancer:How are they linked?[J].Free Radical Biology and Medicine,2010,49(11):1603-1616.
[3]Prestera T,Talalay P,Alam J,et al.Parallel induction of heme oxygenase-1 and chemoprotective phase 2 enzymes by electrophiles and antioxidants:Regulation by upstream antioxidant-responsive elements(ARE)[J].Molecular medicine(Cambridge,Mass),1995,1(7):827-837.
[4]Kensler T W,Wakabayash N,Biswal S.Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway[M].Annual Review of Pharmacology and Toxicology,Palo Alto:Annual Reviews,2007:89-116.
[5]Cimino F,Ambra R,Canali R,et al.Effect of cyanidin-3-O-glucoside on UVB-induced response in human keratinocytes[J].Journal of Agricultural and Food Chemistry,2006,54(11):4041-4047.
[6]龚辉,傅丽,李澜奇,等.超声对花色苷稳定性影响及其降解动力学[J].食品科学,2018,39(9):67-75.
[7]Khoo H E,Azlan A,Tang S T,et al.Anthocyanidins and anthocyanins:Colored pigments as food,pharmaceutical ingredients,and the potential health benefits[J].Food&Nutrition Research,2017,61(1):1361779.
[8]Azzini E,Giacometti J,Russo G L.Antiobesity effects of anthocyanins in preclinical and clinical studies[J].Oxidative Medicine and Cellular Longevity,2017,2017:2740364.
[9]Kong J.Analysis and biological activities of anthocyanins[J].Phytochemistry,2003,64(5):923-933.
[10]Liu D,Zhang T,Chen Z,et al.The beneficial effect of ginsenosides extracted by pulsed electric field against hydrogen peroxide-induced oxidative stress in HEK-293 cells[J].Journal of Ginseng Research,2017,41(2):169-179.
[11]Liu J B,Chen Z F,He J,et al.Anti-oxidative and antiapoptosis effects of egg white peptide,Trp-Asn-Trp-Ala-Asp,against H2O2-induced oxidative stress in human embryonic kidney 293 cells[J].Food Funct,2014,5(12):3179-3188.
[12]Xu J M,Hao Z M,Gou X B,et al.Imaging of reactive oxygen species burst from mitochondria using laser scanning confocal microscopy[J].Microsc Res Tech,2013,76(6):612-617.
[13]Speciale A,Anwar S,Canali R,et al.Cyanidin-3-O-glucoside counters the response to TNF-alpha of endothelial cells by activating Nrf2 pathway[J].Molecular Nutrition&Food Research,2013,57(11):1979-1987.
[14]邵佩兰徐,郭晓丹,郑安然,等.红枣色素对H2O2诱导血管内皮细胞损伤的保护作用[J].食品科学,2017,38(9):173-178.
[15]Naval M V,Gomez-Serranillos M P,Carretero M E,et al.Neuroprotective effect of a ginseng(Panax ginseng)root extract on astrocytes primary culture[J].Journal of Ethnopharmacology,2007,112(2):262-270.
[16]Rossetto M,Vanzani P,Mattivi F,et al.Synergistic antioxidant effect of catechin and malvidin 3-glucoside on free radical-initiated peroxidation of linoleic acid in micelles[J].Arch Biochem Biophys,2002,408(2):239-245.
[17]Lee J S,Kim Y R,Song I G,et al.Cyanidin-3-glucoside isolated from mulberry fruit protects pancreatic beta-cells against oxidative stress-induced apoptosis[J].Int J Mol Med,2015,35(2):405-412.
[18]Chen W,Li Y,Bao T,et al.Mulberry fruit extract affords protection against ethyl carbamate-induced cytotoxicity and oxidative stress[J].Oxidative Medicine and Cellular Longevity,2017,2017:1594963.
[19]Fratantonio D,Speciale A,Ferrari D,et al.Palmitateinduced endothelial dysfunction is attenuated by cyanidin-3-O-glucoside through modulation of Nrf2/Bach1 and NF-kappa Bpathways[J].Toxicology letters,2015,239(3):152-160.
[20]Bhullar K S,Rupasinghe H P.Antioxidant and cytoprotective properties of partridgeberry polyphenols[J].Food Chemistry,2015,168:595-605.
[21]Speciale A,Canali R,Chirafisi J,et al.Cyanidin-3-O-glucoside protection against TNF-alpha-induced endothelial dysfunction:Involvement of nuclear factor-kappa B signaling[J].Journal of Agricultural and Food Chemistry,2010,58(22):12048-12054.
[22]Yan F,Chen Y,Azat R,et al.Mulberry anthocyanin extract ameliorates oxidative damage in Hep G2 cells and prolongs the lifespan of caenorhabditis elegans through MAPK and Nrf2Pathways[J].Oxidative Medicine and Cellular Longevity,2017,2017:7956158.
[23]Mattera R,Benvenuto M,Giganti M G,et al.Effects of polyphenols on oxidative stress-mediated injury in cardiomyocytes[J].Nutrients,2017,9(5).
[24]宋柬,廖小军,胡小松,等.矢车菊素_3_葡萄糖苷干预丙烯酰胺细胞毒性的体外模型筛选[J].中国食品学报,2017,17(9):11-19.