多种抗氧化剂对NIH3T3细胞紫外损伤的保护作用研究
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摘要
随着环境污染的日益严重,到达地球表面的紫外线日益增多,紫外线辐射会导致皮肤细胞的活性氧自由基增多,促进皮肤光老化、癌变、白内障、免疫抑制等一系列疾病的发生,因此寻找高效的抗氧化剂是预防和治疗紫外损伤的重要手段。本研究以NIH3T3细胞为实验材料,建立了紫外辐射对NIH3T3细胞的损伤模型,采用MTT方法和激光扫描共聚焦显微镜技术研究不同浓度的姜黄素、槲皮素、石杉碱甲对NIH3T3细胞的紫外损伤保护作用。
结果表明,姜黄素预处理不但可以降低紫外损伤造成的细胞死亡率(p＜0.05)、减少细胞内活性氧的堆积(p＜0.05),而且对细胞线粒体膜电位具有保护作用(p＜0.01);此外,姜黄素对紫外损伤细胞的保护作用与浓度呈正相关。实验结果得出姜黄素可以清除细胞内的活性氧自由基,维持细胞的线粒体膜电位,提高紫外辐照细胞的存活率。
通过不同浓度的槲皮素的预处理,紫外损伤后的细胞内活性氧自由基含量明显降低(p＜0.05),阻止了细胞膜电位下降(p＜0.05),提高了细胞存活率(p＜0.05);且都呈现出较强的槲皮素浓度依赖性。结果表明紫外损伤可诱导细胞内产生大量的活性氧自由基,使细胞线粒体膜电位下降,降低细胞的存活率;槲皮素预处理,可以清除细胞内的活性氧自由基,维持细胞的线粒体膜电位,抑制细胞凋亡。
经过不同浓度的石杉碱甲的预处理后的NIH3T3细胞,经紫外损伤后,其细胞死亡率降低(p＜0.05)、细胞内活性氧的含量减少(p＜0.01),而且起到了对细胞线粒体膜电位的保护作用(p＜0.01);同时,石杉碱甲预处理后的NIH3T3细胞的细胞核形态相对保持完整,细胞凋亡过程中的关键蛋白caspase-3的表达量下降(p＜0.01),bcl-2的表达量增加(p＜0.01),实验结果表明,石杉碱甲可以有效的清除NIH3T3细胞内的活性氧自由基,保护细胞线粒体的膜电位,通过调控蛋白表达,抑制由紫外辐射诱导的NIH3T3细胞凋亡,提高细胞存活率。
Studies have shown that chronic repeated exposures to sunlight are epidemiologically shown to be the main cause of skin cancers, skin aging, erythema, photoaging, immune suppression. At the cellular level, UV radiation causes significant oxidative stress due to generation of reactive oxygen species such as singlet oxygen, hydroxyl radical, superoxide anion, and hydrogen peroxide, then, our study set up a damage model with NIH3T3 cells by exposure it to UV radiation; investigate the antioxidative effects of curcumin, quercetin and huperzine A against the damage produced by exposing to UV in the cells.
For curcumin and quercetin, the vitality of cells was determined by MTT assay; the membrane potential (MMP) across the inter membrane of the mitochondrial and the ROS level in the cells were evaluated by the laser scanning confocal microscopy. The study find out that exposure cells to UV caused a marked increase in ROS, and a decrease in mitochondrial membrane potential (MMP). Pre-incubation of NIH3T3 cells with curcumin and quercetin decrease the intracellular accumulation of ROS, and these two antioxidants caused a marked increase of MMP in 3T3 cells, they can protect cells against UV irradiation, increase the cell viabilty.
For huperzine A, we use MTT assay to examine the vitality of cells and LSCM to determine the MMP, intracellular ROS level, cell karyotype, caspase-3 protein expression and bcl-2 expression. We find out that huperzine A can decrease the UV induced intracellular accumulation of ROS, increase the MMP of cells, keep the cell karyotype integrity; inhibit the UV induced apoptosis by regulate protein caspase-3 and bcl-2 expression, increase the viability of cells.
结果表明,姜黄素预处理不但可以降低紫外损伤造成的细胞死亡率(p＜0.05)、减少细胞内活性氧的堆积(p＜0.05),而且对细胞线粒体膜电位具有保护作用(p＜0.01);此外,姜黄素对紫外损伤细胞的保护作用与浓度呈正相关。实验结果得出姜黄素可以清除细胞内的活性氧自由基,维持细胞的线粒体膜电位,提高紫外辐照细胞的存活率。
通过不同浓度的槲皮素的预处理,紫外损伤后的细胞内活性氧自由基含量明显降低(p＜0.05),阻止了细胞膜电位下降(p＜0.05),提高了细胞存活率(p＜0.05);且都呈现出较强的槲皮素浓度依赖性。结果表明紫外损伤可诱导细胞内产生大量的活性氧自由基,使细胞线粒体膜电位下降,降低细胞的存活率;槲皮素预处理,可以清除细胞内的活性氧自由基,维持细胞的线粒体膜电位,抑制细胞凋亡。
经过不同浓度的石杉碱甲的预处理后的NIH3T3细胞,经紫外损伤后,其细胞死亡率降低(p＜0.05)、细胞内活性氧的含量减少(p＜0.01),而且起到了对细胞线粒体膜电位的保护作用(p＜0.01);同时,石杉碱甲预处理后的NIH3T3细胞的细胞核形态相对保持完整,细胞凋亡过程中的关键蛋白caspase-3的表达量下降(p＜0.01),bcl-2的表达量增加(p＜0.01),实验结果表明,石杉碱甲可以有效的清除NIH3T3细胞内的活性氧自由基,保护细胞线粒体的膜电位,通过调控蛋白表达,抑制由紫外辐射诱导的NIH3T3细胞凋亡,提高细胞存活率。
Studies have shown that chronic repeated exposures to sunlight are epidemiologically shown to be the main cause of skin cancers, skin aging, erythema, photoaging, immune suppression. At the cellular level, UV radiation causes significant oxidative stress due to generation of reactive oxygen species such as singlet oxygen, hydroxyl radical, superoxide anion, and hydrogen peroxide, then, our study set up a damage model with NIH3T3 cells by exposure it to UV radiation; investigate the antioxidative effects of curcumin, quercetin and huperzine A against the damage produced by exposing to UV in the cells.
For curcumin and quercetin, the vitality of cells was determined by MTT assay; the membrane potential (MMP) across the inter membrane of the mitochondrial and the ROS level in the cells were evaluated by the laser scanning confocal microscopy. The study find out that exposure cells to UV caused a marked increase in ROS, and a decrease in mitochondrial membrane potential (MMP). Pre-incubation of NIH3T3 cells with curcumin and quercetin decrease the intracellular accumulation of ROS, and these two antioxidants caused a marked increase of MMP in 3T3 cells, they can protect cells against UV irradiation, increase the cell viabilty.
For huperzine A, we use MTT assay to examine the vitality of cells and LSCM to determine the MMP, intracellular ROS level, cell karyotype, caspase-3 protein expression and bcl-2 expression. We find out that huperzine A can decrease the UV induced intracellular accumulation of ROS, increase the MMP of cells, keep the cell karyotype integrity; inhibit the UV induced apoptosis by regulate protein caspase-3 and bcl-2 expression, increase the viability of cells.
引文
[1]Seji kondo.The roles of cytokines in photoaging[J].Journal of Dermatological Science.2000,23 sup(1):S30-S36.
[2]Yasuhiro Matsumura,Honnavara N,Ananthaswamy.Short-term and long-term cellular and molecular events following UV irradiation of skin:implications for molecular medicine.[J],expert reviews in molecular medicine.2002,12:1-22.
[3]David Gourassian,Fafen Gad,Mina Yaar,et.al.Mechanisms and implications of the age-associated decrease in DNA repair capacity[J].The FASEB Journal.2000,14:1325-1334.
[4]杜华.p53在调节细胞周期阻滞和细胞凋亡中的作用及其机制[J].国外医学·放射医学核医学分册,2002,26(2):79-83.
[5]Byeong Hyeok Choi,Chang Gun Kim,Yoongho Lim.Curcumin down-regulates the multidrug-resistance mdr1b gene by inhibiting the PI3K/Akt/NFjB pathway,Cancer Letters2008,259:111-118.
[6]Abuarqoub,H,Green,C.J,Foresti,R.Curcumin reduces cold storage-induced damage in human cardiac myoblasts.Experimental and Molecular Medicine,2007.39(2):139-148.
[7]M.T.Huang,T.Lysz,T.Ferraro.Inhibitory effects of curcumin on in vitro lipoxygenase and cyclooxygenase activities in mouse epidermis.Cancer Res.1991,5:813-819.
[8]Annelyse Duvoix,Romain Blasius,Sylvie Delhalle.Chemopreventive and therapeutic effects of curcumin.Cancer Letters.2005,223:181-190.
[9]S.Shishodia,P.Potdar,C.G.Gairola.Curcumin(diferutoylmethane)down-regulates cigarette smoke-induced NF-kappaB activation through inhibition of IkappaBalpha kinase in human lung epithelial cells:correlation with suppression of COX-2,MMP-9 and cyclin D1.Carcinogenesis 2003,24:1269-1279.
[10]T.T.Phan,P.See,S.T.Lee.Protective effects of curcumin against oxidative damage on skin cells in vitro:its implication for wound healing,[J].Trauma.2001,51:927-931.
[11]K.Saja,Mani Shankar Babu,D.Karunagaran.Anti-inflammatory effect of curcumin involves down-regulation of MMP-9 in blood mononuclear cells.[J]International Immunopharmacology.2007,7:1659-1667.
[12]Toda,S.,Miyase,T.,Arichi,H.Antioxidative components isolated from rhizome of Curcuma longa.[J].Chemical and Pharmaceutical Bulletin 33(4):1725-1728.
[13] Ya-Fan Liao, Hui-Chih Hung, Tzyh-Chyuan Hour. Curcumin induces apoptosis through an ornithine decarboxylase-dependent pathway in human promyelocytic leukemia HL-60 cells. [J]. Life Sciences 2008,82:367-375.
[14] Cohly, H.H., Taylor, A., Angel, M.F. Effect of turmeric, turmerin and curcumin on H2O2-induced renal epithelial (LLCPK1) cell injury. [J]. Free Radical Biology and Medicine 24 (1):49-54.
[15] G.P. Lim, T. Chu, F. Yang. The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse, [J]. Neurosci. 2001,21:8370-8377.
[16]Calabrese, V., Butterfield, D.A., Stella, A.M. Nutritional antioxidants and the heme oxygenase pathway of stress tolerance: novel targets for neuroprotection in Alzheimer's disease. [J]Italian Journal of Biochemistry 2003.52(4): 177-181.
[17] Yang, F., Lim, G.P., Begum, A.N. Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. Journal of Biological Chemistry 2005.280(7):5892-5901.
[18]Singh, S.V., Hu, X., Srivastava, S.K. Mechanism of inhibition of benzo[a]pyrene-induced forestomach cancer in mice by dietary curcumin. [J].Carcinogenesis 1998.19 (8):1357-1360.
[19]Aggarwal, B.B., Kumar, A., Bharti, A.C. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Research 2003.23 (lA):363-398.
[20] Jeremy James Johnson, Hasan Mukhtar. Curcumin for chemoprevention of colon cancer. [J]. Cancer Letters 2007,255:170-181.
[21] Joanna Skommer, Donald Wlodkowic, Jukka Pelkonen. Cellular foundation of curcumin-induced apoptosis in follicular lymphoma cell lines.[J].Experimental Hematology 2006,34:463-474.
[22]陈文娟.姜黄素对恶性肿瘤细胞的调控.临床血液学杂志.1999,12(5). 238-241.
[23]Ramirez-Tortosa MC, Mesa MD, Aguilera MC, et al. Oral administration of a turmeric extract inhibits LDL oxidation and has hypocholesterolemic effects in rabbits with experimental atterosclerosis.Atherosclerosis.1999,147(2):371-373.
[24]Dickinson DA,Iles KE,Zhang H. Curcumin alters EpRE and AP-1 binding complexes and elevates glutamatecysterine ligase expressin.[J].FASEB J 2003,17:473-475.
[25]Reddy AC, Lokesh BR. Studies on apices of principles as antioxidants in theinhibition of lipid peroidation of rat liver microsomes.[J]. Mol Cell Biochem 1992,111:117-124.
[26]Agnes W. Boots, Guido R.M.M. Haenen, Aalt Bast. Health effects of quercetin: From antioxidant to nutraceutical. European Journal of Pharmacology,2008.
[27]Charles S. Bestwick, Lesley Milne. Quercetin modifies reactive oxygen levels but exerts only partial protection against oxidative stress within HL-60 cells. Biochimica et Biophysica Acta 2001,1528:49-59.
[28]Lonneke C. Wilms, Jos C.S. Kleinjans. Discriminative protection against hydroxyl and superoxide anion radicals by quercetin in human leucocytes in vitro. Toxicology in Vitro2008,22:301-307.
[29]Ulrike Kaindl, Isgard Eyberg, The dietary antioxidants resveratrol and quercetin protect cells from exogenous pro-oxidative damage. Food and Chemical Toxicology 2008,46:1320-1326.
[30]Hanasaki,Y., Ogawa,S., Fukui,S. The correlation between active oxygens scavenging and antioxidative effects of flavonoids. Free Radic Biol Med. 1994.16:845-850.
[31]Cushnie, T. P., Lamb, A. J. Antimicrobial activity of flavonoids. Int J Antimicrob Agents. 2005.26:343-356.
[32]van Acker, S. A., Tromp, M. N., Haenen, G. R. M. M., Flavonoids as scavengers of nitric oxide radical. Biochem Biophys Res Commun. 1995. 214, 755-759.
[33]Haenen, G. R. M. M., Bast, A. Nitric oxide radical scavenging of flavonoids. Methods Enzymol. 1999.301:490-503.
[34]Haenen, G. R. M. M., Paquay, J. B., Korthouwer, R. E., Bast, A. Peroxynitrite scavenging by flavonoids. Biochem Biophys Res Commun.l997.236:591-593.
[35]Heijnen, C. G., Haenen, G. R. M. M., van Acker, F. A., van der Vijgh, W. J., Bast, A. Flavonoids as peroxynitrite scavengers: the role of the hydroxyl groups. Toxicol In Vitro. 2001.15:3-6.
[36]Szeto Y T, Collins A R, Benziel F F. Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure.[J]. Mutat Res,2002,20(1/2):31-38
[37]Bla siak J, Trzeciak A, Gasiorowska A, et al. Vitamin C and quercetin modulate DNA- damaging effect of N- methyl- N' - nitro- Nnitrosoguanidine(MNNG)[J]. Plant Foods Hum.Nutr.2002,57(1):53-61.
[38]Jalili, T., Carlstrom, J., Kim, S., Freeman, D. Quercetin-supplemented diets lower blood pressure and attenuate cardiac hypertrophy in rats with aortic constriction. J Cardiovasc Pharmacol. 2006.47:531-541.
[39] Jin, N. Z., Zhu, Y. P., Zhou, J. W. Preventive Effects of Quercetin against Benzo[a]pyrene-Induced DNA Damages and Pulmonary Precancerous Pathologic Changes in Mice. Basic Clin Pharmacol Toxicol. 2006. 98:593-598.
[40]Jurado, J., Alejandre-Duran, E., Alonso-Moraga, A., Pueyo, C. Study on the mutagenic activity of 13 bioflavonoids with the Salmonella Ara test.Mutagenesis. 1991.6:289-295.
[41]Silva, I. D., Gaspar, J., da Costa, G. G., Rodrigues, A. S. Chemical features of flavonols affecting their genotoxicity.Potential implications in their use as therapeutical agents.Chem Biol Interact.2000.124:29-51.
[42]Rueff,J.,Gaspar,J.,Laires,A.Structural requirements for mutagenicity of flavonoids upon nitrosation.A structure-activity study.Mutagenesis.1995.10:325-328.
[43]翟莺莺,周蕾.槲皮素抗肿瘤作用的研究.现代临床医学生物工程学杂志2005.11(1):18-20.
[44]朱蕾,何丽娅.槲皮素抗癌作用的分子机制.武汉科技大学学报(自然科学版).2004.6:194-196.
[45]肖东,顾振纶,朱寿彭.槲皮素的抗肿瘤作用与相关基因的调控.中国野生植物资源.1996.4:5-8.
[46]牛丕业,宫智勇,谭皓.槲皮素抑制HSP70表达对紫外线C致DNA损伤的影响.工业卫生与职业病.2007,33(2):82-84.
[47]张永刚,张杰,范红.槲皮素对肺腺癌细胞系A549细胞中凋亡相关因子Bcl-2表达的影响.四川医学.2007,28(10):1071-1073.
[48]Read,M.A.Flavonoids:naturally occurring anti-inflammatory agents.Am J Pathol.1995.147:235-237.
[49]Manjeet,K.R.,Ghosh,B.Quercetin inhibits LPS-induced nitric oxide and tumor necrosis factor-alpha productionin murine macrophages.Int J Immunopharmacol.1999.21:435-443.
[50]Geraets,L.,Moonen,H.J.J.,Brauers,K.Dietary flavones and flavonoles are inhibitors of poly(ADPribose)polymerase-1 in pulmonary epithelial cells.J Nutr.2007,137:2190-2195.
[51]Genevieve Bureau,Fanny Longpre,M.-G.Martinoli.Resveratrol and quercetin,two natural polyphenols,reduce apoptotic neuronal cell death induced by neuroinflammation.Journal of Neuroscience Research.2008,8(2):403-410.
[52]Wai Mun Loke,Julie M.Proudfoot,Scott Stewart,Allan J.McKinley.Metabolic transformation has a profound effect on anti-inflammatory activity of flavonoids such as quercetin:Lack of association between antioxidant and lipoxygenase inhibitory activity,b i o c h e m i c a l pharma c o logy.2008,75:10 45-1053.
[53]肖东,顾振纶,槲皮素对血小板激活时胞浆游离钙的影响.苏州医学院学报,1995,5(1):40-42.
[54]McKinney,M.,Miller,J.H.,Yamada,F.,Tuckmantel,W.and Kozikowski,A.P.,Potencies and stereoselectivities of enantiomers of huperzine A for inhibition of rat cortical acetylcholinesterase,Eur.J.Pharmacol.,203(1991)303-305.
[55]Hai Yan Zhang,Yan Qi Liang,Xi Can Tang,Xu Chang He,Dong Lu Bai.Stereoselectivities of enantiomers of huperzine A in protection againstβ-amyloid 25-35-induced injury in PC12 and NG108-15 cells and cholinesterase inhibition in mice.Neuroscience Letters 317(2002)143-146.
[56]Selkoe DJ.Toward a comprehensive theory for Alzheimer's disease.Hypothesis:Alzheimer's disease is caused by the cerebral accumulation and cytotoxicity of amyloid beta-protein[J].Ann N Y Acad Sci,2000,924:17-25.
[57]Reena S.Shah,Hyoung-Gon Lee,Zhu Xiongwei,George Perry,Mark A.Smith,Rudy J.Castellani.Current approaches in the treatment of Alzheimer's disease.Biomedicine & Pharmacotherapy xx(2008)1-9.
[58]张均田.老年痴呆的发病机理及治疗策略[J].药学学报,2000,35(8):6351.
[59]Hai Yan Zhang and Xi Can Tang.Neuroprotective effects of huperzine A:new therapeutic targets for neurodegenerative disease.TRENDS in Pharmacological Sciences 2006.Vol.27 No.12.
[60]Hong-Yu Zhang.One-compound-multiple-targets strategy to combat Alzheimer's disease.FEBS Lett.,2005,579:5260-5264.
[61]Youdim,M.B.et al.Multi-functional drugs for various CNS targets in the treatment of neurodegenerative disorders.Trends Pharmacol.Sci.2005.26,27-35.
[62]王月娥,岳冬贤,唐希灿.石杉碱甲的抗胆碱酯酶作用.中国药理学报,1986,7(2):110.
[63]Wang R,Xiao X Q,Tang X C.Huperzine A attenuates hydrogen peroxide-induced apoptosis by regulating expression of apoptosis-related genes in rat PC12 cells.[J].Neuro Report,2001,12(12):2629-2634.
[64]周永其等,石杉碱甲对氧自由基的影响.中国野生植物资源.2004,23(2):44-45.
[65]王润生,冯征,张均田.细胞凋亡的分子生化机制及其在Alzheimer's病发病中的作用.中国药学杂志,2001;36(5):289.
[66]Xiao X Q,Zhang H.Huperzine A attenuates amyloid β-peptide fragment 25-35-induced apoptosis in rat cortical neurons via inhibiting reactive oxygen species formation and caspase-3 activation[J].J Neurosci Res,2002,67:30-36.
[67]Zhou J,Tang XC.Huperzine A attenuates apoptosis and mitochondria-dependent caspase-3 in rat cortical neurons.FEBS Lett,2002;526(1-3):21.
[68]Ved H,Koenig M L,Dave J R,et al.Huperzine A,a potential therapeutic agent for dementia,reduces neuronal cell death caused by glutamate[J].Neuro Report,1997,8(4):963.
[69]Zhang J M,HU G Y.Huperzine A,a nootropic alkaloid,inhibits N-methyl-D -aspeartate-induced current in rat dissociated hippocampal neuouns[J].Neuroscience, 2001,105(3):663-669.
[70]Zangara,A.The psychopharmacology of huperzine A:an alkaloid with congnitive enhancing and neuroprotective properties of interest in the treatment of Alzheimer's disease.Pharmacology,Biochemistry and Behavior 2003.75:675-686.
[71]方蓉,李芳秋,武建国.MTT比色法的条件探讨.临床检验杂志,2003,21(1):34-35.
[72]郑永唐,贲昆龙.测定细胞存活和增殖的MTT方法的建立[J].免疫学杂志.1992.8(4):266-269.
[73]Berridge MV,Tan AS.Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT):subcellular localization,substrate dependence,and involvement of mitochondrial electron transport in MTT reduction.Arch Biochem Biophys.1993,303(2):474-82.
[74]Zhang J,Liu Z,Jing P,Li GJ.A Method of measurement mitochondrial membrane potential[J].J Capi Univ Med Sci,2006.27(1):124-125.
[张杰,刘祯,景鹏,李国君.细胞线粒体膜电位的测量方法.首都医科大学学报,2006.27(1):124-125.]
[75]Armstrong BK,Kricker A.The epidemiology of UV induced skin cancer[J].J Photochem Photobiol B,2001,63(1-3)8-18.
[76]Kim TY,Kripke ML,Ullrich SE.Immunosuppression by factors released from UV-irradiated epidermal cells selective effects on the generation of contact and delayed hypersensitivity after exposure to UVA or UVB radiation[J].J Invest Dermatol,1990,94(1):26-32.
[77]Shimmura S,Suematsu M,Shimoyama M,Tsubota K,Oguchi Y,Ishimura Y.Subthreshold UV radiation-induced peroxide formation in cultured corneal epithelial cells:the protective effects of lactoferrin[J].Exp Eye Res,1996.63(5):519-526.
[78]Kondo S,Mamada A,Yamaguchi J,Fukuro S.Protective effect of dl-alpha-tocopherol on the cytotoxicity of ultraviolet B against human skin fibroblasts in vitro[J].Photodermatol Photoimmunol Photomed,1990.7(4):173-177.
[79]Maia Campos PM,Gianeti MD,Kanashiro A,Lucisano-Valim YM,Gaspar LR.In vitro antioxidant and in vivo photoprotective effects of an association of bioflavonoids with liposoluble vitamins[J].Photochem Photobiol,2006.82(3):683-688.
[80]Petit PX,Lecoeur H,Zorn E.Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis[J].J Cell Biol,1995.130(1):157-67.
[81]修梅红,彭建新,洪学珠.杆状病毒诱导凋亡昆仲细胞中的线粒体反应和Ca~(2+)的变化[J].科学通报,2005,50(12):1213-1219。
[82]Virginie Cl'ement,Isabelle Dunand-Sauthier,Maciej Wiznerowicz.UV-induced apoptosis in XPG-deficient fibroblasts involves activation of CD95 and caspases but not p53.dna repair.2007,6:602-614.
[83]Zerihun Assefa,An Van Laethem,Marjan Garmyn.Ultraviolet radiation-induced apoptosis in keratinocytes:On the role of cytosolic factors.Biochimica et Biophysica Acta.2005,1755:90-106.
[84]Susanna Zierler,Barbara Klein,Tanja Furtner.Ultraviolet irradiation-induced apoptosis does not trigger nuclear fragmentation but translocation of chromatin from nucleus into cytoplasm in the microglial cell-line,BV-2.B R A I N R E S E A R C H.2006,1121(1):12-21.
[85]Li Li Tang,Rui Wang,Xi Can Tang.Huperzine A protects SHSY5Y neuroblastoma cells against oxidative stress damage via nerve growth factor production.European Journal of Pharmacology.2005,519:9-15.
[86]Xiao Qiu Xiao,Jian Wei Yang,Xi Can Tang.Huperzine A protects rat pheochromocytoma cells against hydrogen peroxide-induced injury.Neuroscience Letters.1999,275:73-76.
[87]Xiao Qiu Xiao,Rui Wang,Yi Fan Han.Protective effects of huperzine A on β-amyloid25-35 induced oxidative injury in rat pheochromocytoma cells.Neuroscience Letters.2000,286:155-158.
[88]Cost ntini P,J acotot E,Decuadin D,et al.Mitochondrion as anovel target of anticancer chemotherapy.J Natl Caner Inst,2000,92(13):1042-1053.
[89]Jin Zhou,Yan Fu,Xi Can Tang.Huperzine A and donepezil protect rat pheochromocytoma cells against oxygen-glucose deprivation.Neuroscience Letters.2001,306:53-56.
[90]Zhi Fei Wang,Xi Can Tang.Huperzine A protects C6 rat glioma cells against oxygen-glucose deprivation-induced injury.FEBS Letters.2007,581:596-602.
[91]Hai Yan Zhang,Xi Can Tang.Huperzine A attenuates the neurotoxic effect of staurosporine in primary rat cortical neurons.Neuroscience Letters.2003,340:91-94.
[2]Yasuhiro Matsumura,Honnavara N,Ananthaswamy.Short-term and long-term cellular and molecular events following UV irradiation of skin:implications for molecular medicine.[J],expert reviews in molecular medicine.2002,12:1-22.
[3]David Gourassian,Fafen Gad,Mina Yaar,et.al.Mechanisms and implications of the age-associated decrease in DNA repair capacity[J].The FASEB Journal.2000,14:1325-1334.
[4]杜华.p53在调节细胞周期阻滞和细胞凋亡中的作用及其机制[J].国外医学·放射医学核医学分册,2002,26(2):79-83.
[5]Byeong Hyeok Choi,Chang Gun Kim,Yoongho Lim.Curcumin down-regulates the multidrug-resistance mdr1b gene by inhibiting the PI3K/Akt/NFjB pathway,Cancer Letters2008,259:111-118.
[6]Abuarqoub,H,Green,C.J,Foresti,R.Curcumin reduces cold storage-induced damage in human cardiac myoblasts.Experimental and Molecular Medicine,2007.39(2):139-148.
[7]M.T.Huang,T.Lysz,T.Ferraro.Inhibitory effects of curcumin on in vitro lipoxygenase and cyclooxygenase activities in mouse epidermis.Cancer Res.1991,5:813-819.
[8]Annelyse Duvoix,Romain Blasius,Sylvie Delhalle.Chemopreventive and therapeutic effects of curcumin.Cancer Letters.2005,223:181-190.
[9]S.Shishodia,P.Potdar,C.G.Gairola.Curcumin(diferutoylmethane)down-regulates cigarette smoke-induced NF-kappaB activation through inhibition of IkappaBalpha kinase in human lung epithelial cells:correlation with suppression of COX-2,MMP-9 and cyclin D1.Carcinogenesis 2003,24:1269-1279.
[10]T.T.Phan,P.See,S.T.Lee.Protective effects of curcumin against oxidative damage on skin cells in vitro:its implication for wound healing,[J].Trauma.2001,51:927-931.
[11]K.Saja,Mani Shankar Babu,D.Karunagaran.Anti-inflammatory effect of curcumin involves down-regulation of MMP-9 in blood mononuclear cells.[J]International Immunopharmacology.2007,7:1659-1667.
[12]Toda,S.,Miyase,T.,Arichi,H.Antioxidative components isolated from rhizome of Curcuma longa.[J].Chemical and Pharmaceutical Bulletin 33(4):1725-1728.
[13] Ya-Fan Liao, Hui-Chih Hung, Tzyh-Chyuan Hour. Curcumin induces apoptosis through an ornithine decarboxylase-dependent pathway in human promyelocytic leukemia HL-60 cells. [J]. Life Sciences 2008,82:367-375.
[14] Cohly, H.H., Taylor, A., Angel, M.F. Effect of turmeric, turmerin and curcumin on H2O2-induced renal epithelial (LLCPK1) cell injury. [J]. Free Radical Biology and Medicine 24 (1):49-54.
[15] G.P. Lim, T. Chu, F. Yang. The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse, [J]. Neurosci. 2001,21:8370-8377.
[16]Calabrese, V., Butterfield, D.A., Stella, A.M. Nutritional antioxidants and the heme oxygenase pathway of stress tolerance: novel targets for neuroprotection in Alzheimer's disease. [J]Italian Journal of Biochemistry 2003.52(4): 177-181.
[17] Yang, F., Lim, G.P., Begum, A.N. Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. Journal of Biological Chemistry 2005.280(7):5892-5901.
[18]Singh, S.V., Hu, X., Srivastava, S.K. Mechanism of inhibition of benzo[a]pyrene-induced forestomach cancer in mice by dietary curcumin. [J].Carcinogenesis 1998.19 (8):1357-1360.
[19]Aggarwal, B.B., Kumar, A., Bharti, A.C. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Research 2003.23 (lA):363-398.
[20] Jeremy James Johnson, Hasan Mukhtar. Curcumin for chemoprevention of colon cancer. [J]. Cancer Letters 2007,255:170-181.
[21] Joanna Skommer, Donald Wlodkowic, Jukka Pelkonen. Cellular foundation of curcumin-induced apoptosis in follicular lymphoma cell lines.[J].Experimental Hematology 2006,34:463-474.
[22]陈文娟.姜黄素对恶性肿瘤细胞的调控.临床血液学杂志.1999,12(5). 238-241.
[23]Ramirez-Tortosa MC, Mesa MD, Aguilera MC, et al. Oral administration of a turmeric extract inhibits LDL oxidation and has hypocholesterolemic effects in rabbits with experimental atterosclerosis.Atherosclerosis.1999,147(2):371-373.
[24]Dickinson DA,Iles KE,Zhang H. Curcumin alters EpRE and AP-1 binding complexes and elevates glutamatecysterine ligase expressin.[J].FASEB J 2003,17:473-475.
[25]Reddy AC, Lokesh BR. Studies on apices of principles as antioxidants in theinhibition of lipid peroidation of rat liver microsomes.[J]. Mol Cell Biochem 1992,111:117-124.
[26]Agnes W. Boots, Guido R.M.M. Haenen, Aalt Bast. Health effects of quercetin: From antioxidant to nutraceutical. European Journal of Pharmacology,2008.
[27]Charles S. Bestwick, Lesley Milne. Quercetin modifies reactive oxygen levels but exerts only partial protection against oxidative stress within HL-60 cells. Biochimica et Biophysica Acta 2001,1528:49-59.
[28]Lonneke C. Wilms, Jos C.S. Kleinjans. Discriminative protection against hydroxyl and superoxide anion radicals by quercetin in human leucocytes in vitro. Toxicology in Vitro2008,22:301-307.
[29]Ulrike Kaindl, Isgard Eyberg, The dietary antioxidants resveratrol and quercetin protect cells from exogenous pro-oxidative damage. Food and Chemical Toxicology 2008,46:1320-1326.
[30]Hanasaki,Y., Ogawa,S., Fukui,S. The correlation between active oxygens scavenging and antioxidative effects of flavonoids. Free Radic Biol Med. 1994.16:845-850.
[31]Cushnie, T. P., Lamb, A. J. Antimicrobial activity of flavonoids. Int J Antimicrob Agents. 2005.26:343-356.
[32]van Acker, S. A., Tromp, M. N., Haenen, G. R. M. M., Flavonoids as scavengers of nitric oxide radical. Biochem Biophys Res Commun. 1995. 214, 755-759.
[33]Haenen, G. R. M. M., Bast, A. Nitric oxide radical scavenging of flavonoids. Methods Enzymol. 1999.301:490-503.
[34]Haenen, G. R. M. M., Paquay, J. B., Korthouwer, R. E., Bast, A. Peroxynitrite scavenging by flavonoids. Biochem Biophys Res Commun.l997.236:591-593.
[35]Heijnen, C. G., Haenen, G. R. M. M., van Acker, F. A., van der Vijgh, W. J., Bast, A. Flavonoids as peroxynitrite scavengers: the role of the hydroxyl groups. Toxicol In Vitro. 2001.15:3-6.
[36]Szeto Y T, Collins A R, Benziel F F. Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure.[J]. Mutat Res,2002,20(1/2):31-38
[37]Bla siak J, Trzeciak A, Gasiorowska A, et al. Vitamin C and quercetin modulate DNA- damaging effect of N- methyl- N' - nitro- Nnitrosoguanidine(MNNG)[J]. Plant Foods Hum.Nutr.2002,57(1):53-61.
[38]Jalili, T., Carlstrom, J., Kim, S., Freeman, D. Quercetin-supplemented diets lower blood pressure and attenuate cardiac hypertrophy in rats with aortic constriction. J Cardiovasc Pharmacol. 2006.47:531-541.
[39] Jin, N. Z., Zhu, Y. P., Zhou, J. W. Preventive Effects of Quercetin against Benzo[a]pyrene-Induced DNA Damages and Pulmonary Precancerous Pathologic Changes in Mice. Basic Clin Pharmacol Toxicol. 2006. 98:593-598.
[40]Jurado, J., Alejandre-Duran, E., Alonso-Moraga, A., Pueyo, C. Study on the mutagenic activity of 13 bioflavonoids with the Salmonella Ara test.Mutagenesis. 1991.6:289-295.
[41]Silva, I. D., Gaspar, J., da Costa, G. G., Rodrigues, A. S. Chemical features of flavonols affecting their genotoxicity.Potential implications in their use as therapeutical agents.Chem Biol Interact.2000.124:29-51.
[42]Rueff,J.,Gaspar,J.,Laires,A.Structural requirements for mutagenicity of flavonoids upon nitrosation.A structure-activity study.Mutagenesis.1995.10:325-328.
[43]翟莺莺,周蕾.槲皮素抗肿瘤作用的研究.现代临床医学生物工程学杂志2005.11(1):18-20.
[44]朱蕾,何丽娅.槲皮素抗癌作用的分子机制.武汉科技大学学报(自然科学版).2004.6:194-196.
[45]肖东,顾振纶,朱寿彭.槲皮素的抗肿瘤作用与相关基因的调控.中国野生植物资源.1996.4:5-8.
[46]牛丕业,宫智勇,谭皓.槲皮素抑制HSP70表达对紫外线C致DNA损伤的影响.工业卫生与职业病.2007,33(2):82-84.
[47]张永刚,张杰,范红.槲皮素对肺腺癌细胞系A549细胞中凋亡相关因子Bcl-2表达的影响.四川医学.2007,28(10):1071-1073.
[48]Read,M.A.Flavonoids:naturally occurring anti-inflammatory agents.Am J Pathol.1995.147:235-237.
[49]Manjeet,K.R.,Ghosh,B.Quercetin inhibits LPS-induced nitric oxide and tumor necrosis factor-alpha productionin murine macrophages.Int J Immunopharmacol.1999.21:435-443.
[50]Geraets,L.,Moonen,H.J.J.,Brauers,K.Dietary flavones and flavonoles are inhibitors of poly(ADPribose)polymerase-1 in pulmonary epithelial cells.J Nutr.2007,137:2190-2195.
[51]Genevieve Bureau,Fanny Longpre,M.-G.Martinoli.Resveratrol and quercetin,two natural polyphenols,reduce apoptotic neuronal cell death induced by neuroinflammation.Journal of Neuroscience Research.2008,8(2):403-410.
[52]Wai Mun Loke,Julie M.Proudfoot,Scott Stewart,Allan J.McKinley.Metabolic transformation has a profound effect on anti-inflammatory activity of flavonoids such as quercetin:Lack of association between antioxidant and lipoxygenase inhibitory activity,b i o c h e m i c a l pharma c o logy.2008,75:10 45-1053.
[53]肖东,顾振纶,槲皮素对血小板激活时胞浆游离钙的影响.苏州医学院学报,1995,5(1):40-42.
[54]McKinney,M.,Miller,J.H.,Yamada,F.,Tuckmantel,W.and Kozikowski,A.P.,Potencies and stereoselectivities of enantiomers of huperzine A for inhibition of rat cortical acetylcholinesterase,Eur.J.Pharmacol.,203(1991)303-305.
[55]Hai Yan Zhang,Yan Qi Liang,Xi Can Tang,Xu Chang He,Dong Lu Bai.Stereoselectivities of enantiomers of huperzine A in protection againstβ-amyloid 25-35-induced injury in PC12 and NG108-15 cells and cholinesterase inhibition in mice.Neuroscience Letters 317(2002)143-146.
[56]Selkoe DJ.Toward a comprehensive theory for Alzheimer's disease.Hypothesis:Alzheimer's disease is caused by the cerebral accumulation and cytotoxicity of amyloid beta-protein[J].Ann N Y Acad Sci,2000,924:17-25.
[57]Reena S.Shah,Hyoung-Gon Lee,Zhu Xiongwei,George Perry,Mark A.Smith,Rudy J.Castellani.Current approaches in the treatment of Alzheimer's disease.Biomedicine & Pharmacotherapy xx(2008)1-9.
[58]张均田.老年痴呆的发病机理及治疗策略[J].药学学报,2000,35(8):6351.
[59]Hai Yan Zhang and Xi Can Tang.Neuroprotective effects of huperzine A:new therapeutic targets for neurodegenerative disease.TRENDS in Pharmacological Sciences 2006.Vol.27 No.12.
[60]Hong-Yu Zhang.One-compound-multiple-targets strategy to combat Alzheimer's disease.FEBS Lett.,2005,579:5260-5264.
[61]Youdim,M.B.et al.Multi-functional drugs for various CNS targets in the treatment of neurodegenerative disorders.Trends Pharmacol.Sci.2005.26,27-35.
[62]王月娥,岳冬贤,唐希灿.石杉碱甲的抗胆碱酯酶作用.中国药理学报,1986,7(2):110.
[63]Wang R,Xiao X Q,Tang X C.Huperzine A attenuates hydrogen peroxide-induced apoptosis by regulating expression of apoptosis-related genes in rat PC12 cells.[J].Neuro Report,2001,12(12):2629-2634.
[64]周永其等,石杉碱甲对氧自由基的影响.中国野生植物资源.2004,23(2):44-45.
[65]王润生,冯征,张均田.细胞凋亡的分子生化机制及其在Alzheimer's病发病中的作用.中国药学杂志,2001;36(5):289.
[66]Xiao X Q,Zhang H.Huperzine A attenuates amyloid β-peptide fragment 25-35-induced apoptosis in rat cortical neurons via inhibiting reactive oxygen species formation and caspase-3 activation[J].J Neurosci Res,2002,67:30-36.
[67]Zhou J,Tang XC.Huperzine A attenuates apoptosis and mitochondria-dependent caspase-3 in rat cortical neurons.FEBS Lett,2002;526(1-3):21.
[68]Ved H,Koenig M L,Dave J R,et al.Huperzine A,a potential therapeutic agent for dementia,reduces neuronal cell death caused by glutamate[J].Neuro Report,1997,8(4):963.
[69]Zhang J M,HU G Y.Huperzine A,a nootropic alkaloid,inhibits N-methyl-D -aspeartate-induced current in rat dissociated hippocampal neuouns[J].Neuroscience, 2001,105(3):663-669.
[70]Zangara,A.The psychopharmacology of huperzine A:an alkaloid with congnitive enhancing and neuroprotective properties of interest in the treatment of Alzheimer's disease.Pharmacology,Biochemistry and Behavior 2003.75:675-686.
[71]方蓉,李芳秋,武建国.MTT比色法的条件探讨.临床检验杂志,2003,21(1):34-35.
[72]郑永唐,贲昆龙.测定细胞存活和增殖的MTT方法的建立[J].免疫学杂志.1992.8(4):266-269.
[73]Berridge MV,Tan AS.Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT):subcellular localization,substrate dependence,and involvement of mitochondrial electron transport in MTT reduction.Arch Biochem Biophys.1993,303(2):474-82.
[74]Zhang J,Liu Z,Jing P,Li GJ.A Method of measurement mitochondrial membrane potential[J].J Capi Univ Med Sci,2006.27(1):124-125.
[张杰,刘祯,景鹏,李国君.细胞线粒体膜电位的测量方法.首都医科大学学报,2006.27(1):124-125.]
[75]Armstrong BK,Kricker A.The epidemiology of UV induced skin cancer[J].J Photochem Photobiol B,2001,63(1-3)8-18.
[76]Kim TY,Kripke ML,Ullrich SE.Immunosuppression by factors released from UV-irradiated epidermal cells selective effects on the generation of contact and delayed hypersensitivity after exposure to UVA or UVB radiation[J].J Invest Dermatol,1990,94(1):26-32.
[77]Shimmura S,Suematsu M,Shimoyama M,Tsubota K,Oguchi Y,Ishimura Y.Subthreshold UV radiation-induced peroxide formation in cultured corneal epithelial cells:the protective effects of lactoferrin[J].Exp Eye Res,1996.63(5):519-526.
[78]Kondo S,Mamada A,Yamaguchi J,Fukuro S.Protective effect of dl-alpha-tocopherol on the cytotoxicity of ultraviolet B against human skin fibroblasts in vitro[J].Photodermatol Photoimmunol Photomed,1990.7(4):173-177.
[79]Maia Campos PM,Gianeti MD,Kanashiro A,Lucisano-Valim YM,Gaspar LR.In vitro antioxidant and in vivo photoprotective effects of an association of bioflavonoids with liposoluble vitamins[J].Photochem Photobiol,2006.82(3):683-688.
[80]Petit PX,Lecoeur H,Zorn E.Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis[J].J Cell Biol,1995.130(1):157-67.
[81]修梅红,彭建新,洪学珠.杆状病毒诱导凋亡昆仲细胞中的线粒体反应和Ca~(2+)的变化[J].科学通报,2005,50(12):1213-1219。
[82]Virginie Cl'ement,Isabelle Dunand-Sauthier,Maciej Wiznerowicz.UV-induced apoptosis in XPG-deficient fibroblasts involves activation of CD95 and caspases but not p53.dna repair.2007,6:602-614.
[83]Zerihun Assefa,An Van Laethem,Marjan Garmyn.Ultraviolet radiation-induced apoptosis in keratinocytes:On the role of cytosolic factors.Biochimica et Biophysica Acta.2005,1755:90-106.
[84]Susanna Zierler,Barbara Klein,Tanja Furtner.Ultraviolet irradiation-induced apoptosis does not trigger nuclear fragmentation but translocation of chromatin from nucleus into cytoplasm in the microglial cell-line,BV-2.B R A I N R E S E A R C H.2006,1121(1):12-21.
[85]Li Li Tang,Rui Wang,Xi Can Tang.Huperzine A protects SHSY5Y neuroblastoma cells against oxidative stress damage via nerve growth factor production.European Journal of Pharmacology.2005,519:9-15.
[86]Xiao Qiu Xiao,Jian Wei Yang,Xi Can Tang.Huperzine A protects rat pheochromocytoma cells against hydrogen peroxide-induced injury.Neuroscience Letters.1999,275:73-76.
[87]Xiao Qiu Xiao,Rui Wang,Yi Fan Han.Protective effects of huperzine A on β-amyloid25-35 induced oxidative injury in rat pheochromocytoma cells.Neuroscience Letters.2000,286:155-158.
[88]Cost ntini P,J acotot E,Decuadin D,et al.Mitochondrion as anovel target of anticancer chemotherapy.J Natl Caner Inst,2000,92(13):1042-1053.
[89]Jin Zhou,Yan Fu,Xi Can Tang.Huperzine A and donepezil protect rat pheochromocytoma cells against oxygen-glucose deprivation.Neuroscience Letters.2001,306:53-56.
[90]Zhi Fei Wang,Xi Can Tang.Huperzine A protects C6 rat glioma cells against oxygen-glucose deprivation-induced injury.FEBS Letters.2007,581:596-602.
[91]Hai Yan Zhang,Xi Can Tang.Huperzine A attenuates the neurotoxic effect of staurosporine in primary rat cortical neurons.Neuroscience Letters.2003,340:91-94.