紫甘薯汁抗肿瘤活性及诱导人肝癌细胞HepG2凋亡机制研究

目的:研究紫甘薯汁的抗肿瘤活性及其诱导肝癌细胞HepG2凋亡机制。方法:采用Alamar Blue、倒置显微镜、荧光显微镜、扫描电镜、琼脂糖凝胶电泳、RT-PCR和Western blotting法检测不同体积分数的紫甘薯汁对SGC-7901、HO-8910和HepG2细胞的增殖抑制作用及诱导人肝癌细胞HepG2凋亡机制研究。结果:与对照组相比,体积分数5%和10%的紫甘薯汁对SGC-7901、HO-8910和HepG2细胞具有抑制作用,呈体积分数依赖性;作用HepG2细胞48h后,能观察到细胞皱缩和凋亡小体以及凋亡细胞典型的梯状DNA条带。RT-PCR和Western blotting法检测结果显示,紫甘薯汁可以诱导HepG2细胞中Fas、FADD、Caspase-3、Caspase-8和p53 mRNA表达水平上调和蛋白表达量增加,Caspase-3、Caspase-8、Caspase-9的活性裂解片段明显增高且Caspase-3酶活性显著提高。结论:紫甘薯汁体外对肿瘤细胞的增殖具有一定抑制作用且通过细胞表面死亡受体途径诱导HepG2细胞凋亡,同时p53在此凋亡过程中也起着重要的作用。     

灵芝肽诱导人肝癌HepG2细胞凋亡的细胞学观察

目的：研究灵芝肽(Ganoderma lucidum peptides,GLP)在体外对人肝癌HepG2细胞增殖抑制及诱导凋亡的作用。方法：HepG2细胞用含不同浓度GLP培养基培养12、24、36、48、60h,GLP分为5个浓度梯度,分别为0.25、0.5、1、2、4mg/ml,采用噻唑兰(methyl thiazolyl tetrazolium,MTT)法观察细胞生长抑制作用,用可见光显微镜、荧光显微镜观察细胞形态学变化、激光共聚焦(laser scanning confocal microscopy,LSCM)观察细胞内钙离子浓度的变化。结果：MTT法显示灵芝肽能显著抑制人肝癌HepG2细胞的生长,且存在浓度及时间依赖性。镜下可见细胞出现典型凋亡细胞形态学改变：细胞染色质浓缩,细胞体积缩小、核固缩深染、细胞膜出泡、凋亡小体形成;LSCM观察到胞内钙离子浓度明显增加。结论：0.25～4.0mg/ml的GLP体外可显著抑制人肝癌HepG2细胞增殖,存在量效关系和时效关系,能诱导人肝癌HepG2细胞凋亡,并且提示细胞内钙离子浓度的升高可能是细胞凋亡的机制之一。     

蓝莓花色苷对H2O2诱导A549细胞氧化损伤的保护作用

为探究蓝莓花色苷（BA）对H2O2诱导A549细胞氧化损伤的保护作用及其机制,通过MTT法测定A549细胞存活率,采用ELISA法检测细胞内丙二醛（MDA）水平、谷胱甘肽过氧化物酶（GSH-Px）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性,采用DCFH-DA荧光探针检测细胞内活性氧（ROS）水平,采用流式细胞分析仪测定细胞凋亡率,通过RT-PCR技术测定细胞内抗氧化酶和凋亡相关蛋白的mRNA表达量,用免疫印迹法（Western blot）测定凋亡相关蛋白表达量。结果表明：选择H2O2浓度400 μmol/L、处理时间24 h来构建细胞氧化损伤模型。BA质量浓度为30,60,90 mg/mL对A549细胞无毒性作用;BA能显著抑制H2O2造成的A549细胞存活率降低（P<0.05）,同时显著降低细胞凋亡率、MDA和胞内ROS水平（P<0.05）,显著增加GSH-Px、SOD和CAT活性及其相对mRNA表达量（P<0.05）,并显著上调Bcl-2/Bax的比值和下调Cytochrome c、Caspase-9和Caspase-3相对mRNA和蛋白表达量（P<0.05）。BA对H2O2诱导A549细胞氧化损伤具有保护作用,其作用机制可能与抑制细胞凋亡通路相关蛋白的表达,提高胞内抗氧化酶活性,清除胞内过量ROS,降低细胞凋亡率有关。     

白花蛇舌草对HepG2人肝癌细胞Bcl-2和Caspase-3表达的影响

目的：研究白花蛇舌草对HepG2人肝癌细胞Bcl-2和Caspase-3表达的影响。方法：MTT检测白花蛇舌草提取液（EHD）对HeDG2细胞增殖的抑制作用和免疫组化SP法检测Bcl-2和Caspase-3蛋白表达。结果：EHD在一定浓度和时间内能抑制HepG2细胞的生长,并具有剂量和时间依赖性;EHD（160mg／m;_）作用HepG2细胞48h后,Bcl-2的表达水平下降,而CaspaseJ的表达水平升高。结论：EHD（160mg／mL）对HepG2细胞具有抑制增殖和诱导凋亡作用,婪机制可能与相关基因BcL2、 Casnase-3的袁主太调控有美．     

豌豆肽缓解胰岛素抵抗形成效果探究

目的:探究豌豆肽对缓解人肝癌细胞胰岛素抵抗(insulin resistance,IR)形成的作用。方法:体外采用高浓度胰岛素诱导人肝癌细胞HepG2建立IR细胞模型,利用葡萄糖氢化酶过氧化氢酶法(GOP-POD)测定不同浓度豌豆肽对胰岛素诱导前后HepG2/IR细胞的葡萄糖含量及消耗量变化;MTT比色法检测豌豆肽对发生胰岛素抵抗细胞的毒性影响;采用流式细胞术(Flow cytometry,FCM)手段检测细胞中胰岛素受体(InsR)及凋亡促进蛋白Caspase-3的表达。结果:胰岛素诱导48 h建立的HepG2/IR抵抗模型最为稳定,其葡萄糖消耗量与正常HepG2细胞相比降低10%。利用不同浓度豌豆肽对胰岛素诱导的HepG2细胞进行作用时,其葡萄糖消耗量提高1.31~1.68倍,InsR的表达水平提高1.19~1.34倍,凋亡蛋白Caspase-3阳性率表达增加。结论:豌豆肽对肝细胞内胰岛素抵抗的形成具有一定缓解作用。     

黄酮对AAPH诱导的HepG2细胞氧化应激的作用

研究部分黄酮对AAPH诱导的HepG2细胞氧化应激的作用。建立AAPH诱导HepG2细胞氧化应激的模型,比较对照组和实验组细胞内的氧化自由基ROS的含量。结果表明,用不同浓度的黄酮处理HepG2细胞1h后,经过相同浓度的氧化剂作用后,细胞内自由基ROS的含量相对空白对照组均发生变化。这些选择性的黄酮对于AAPH诱导的HepG2细胞氧化应激具有促进或者抑制作用,其作用机制可能清除HepG2细胞内ROS以及与细胞内氧化酶和抗氧化酶系统的作用有关。     

酒糟粗提物对肝癌细胞HepG2增殖的抑制作用

为了解酒糟粗提物对人体肝癌细胞株HepG2细胞增殖和凋亡的影响,首先采用液相色谱-质谱联用仪（LC-MS）分析酒糟粗提物成分,再利用不同浓度的酒糟提取物分别处理HepG2细胞,采用RTCA检测其对HepG2细胞增殖的影响,采用流式细胞术检测其对HepG2细胞周期的影响,采用实时荧光定量PCR检测其对CyclinA、CDK2、Bax、Bcl-2基因表达的影响,采用Western Blot检测其对9种凋亡相关蛋白表达量的影响。研究发现浓香型白酒酒糟粗提物主要含有43种物质,酒糟粗提物可明显抑制HepG2细胞的增殖且呈浓度依赖;酒糟粗提物可以显著下调S期相关周期蛋白CyclinA及其激酶CDK2的mRNA及蛋白的表达,同时使抗凋亡蛋白Bcl-2的mRNA及蛋白的表达减少,促凋亡蛋白Bax的mRNA及蛋白的表达增加（P<0.05）;CYTC、Cleaved-Caspase-9及Cleaved-Caspase-3表达量逐渐升高（P<0.05）,Caspase-9、Caspase-3表达量逐渐降低（P<0.05）。结果表明酒糟粗提物含多种活性物质,可抑制HepG2细胞的增殖,并通过激活介导凋亡的线粒体通路诱导肝癌HepG2细胞发生凋亡。     

摄食DHA-磷脂酰丝氨酸对发育期小鼠体组织DHA水平的影响

目的：比较研究断乳后干预二十二碳六烯酸-磷脂酰丝氨酸（docosahexaenoic acid-phosphatidylserine, DHA-PS）和DHA-甘油三酯（DHA-triglyceride,DHA-TG）对发育期ICR小鼠体组织DHA含量以及脂肪酸组成的影 响。方法：母鼠孕期及哺乳期喂饲n-3多不饱和脂肪酸（n-3 polyunsaturated fatty acids,n-3 PUFAs）缺乏饲料,子代 雄小鼠断乳后随机分为3 组,分别喂饲n-3 PUFAs缺乏饲料（n-3缺乏组）、DHA-TG饲料（DHA-TG组）、DHA-PS 饲料（DHA-PS组）,每组8 只,喂养2 周后检测小鼠脑皮质、精巢、肝脏、红细胞中脂肪酸组成和DHA含量。结 果：补充DHA-PS和DHA-TG可以使发育期小鼠各组织中DHA含量显著增加（P＜0.05）,二十二碳五烯酸和花生四 烯酸含量显著降低（P＜0.05）。DHA-TG组精巢中DHA含量明显高于DHA-PS组;DHA-PS组肝脏TG、磷脂和红细 胞中DHA水平显著高于DHA-TG组（P＜0.05）,而两组脑皮质DHA水平无显著性差异（P＞0.05）。结论：膳食补 充DHA-PS和DHA-TG均可明显提高发育期小鼠各组织中DHA水平,但二者的组织蓄积特性不同,DHA-PS可以更 高效地提高小鼠肝脏和红细胞中DHA水平。     

哺乳期干预甘油三酯型和磷脂型DHA对仔鼠体组织DHA水平的影响

研究哺乳期母鼠干预甘油三酯型DHA(DHA-TG)和磷脂型DHA(DHA-PL)对仔鼠体组织脂肪酸组成及二十二碳六烯酸(DHA)水平的影响。母鼠妊娠期给予n-3多不饱和脂肪酸(n-3PUFAs)缺乏饲料,产仔后分别给予n-3 PUFAs缺乏饲料(缺乏组)、含DHA-TG饲料(DHA-TG组)和含DHA-PL饲料(DHA-PL组),饲喂至仔鼠3周龄断乳。测定仔鼠脑皮质、肝脏、血清和红细胞总脂中脂肪酸组成和DHA含量。与缺乏组相比,哺乳期干预DHA-TG和DHA-PL后仔鼠各组织中DHA和n-3 PUFAs含量均显著升高,二十二碳五烯酸(DPA)、花生四烯酸(AA)、∑n-6 PUFAs与∑n-3 PUFAs比值均显著降低。其中DHA-PL组仔鼠脑皮质和红细胞总脂中DHA水平显著高于DHA-TG组,而肝脏和血清中DHA水平两组之间无显著性差异。总之,哺乳期母鼠补充DHA-TG和DHA-PL均可显著提高仔鼠各组织中DHA水平,其中DHA-PL对脑皮质和红细胞中DHA的补充效果更佳。     

n-3多不饱和脂肪酸对结肠癌细胞HCT116抗增殖作用

目的:研究n-3多不饱和脂肪酸在结肠癌细胞内的代谢与转化,深入探究脂肪酸代谢调节因子LTB₄、PGE₂、LXA₄对结肠癌细胞HCT116的毒性作用。方法:选用结肠癌细胞HCT116为研究对象,采用不同浓度n-3多不饱和脂肪酸(ALA、EPA、DHA)和阳性药物5-氟尿嘧啶(以下简称5-FU)处理细胞,检测细胞存活率、细胞凋亡、细胞脂滴聚积、细胞脂肪酸组成变化,研究代谢因子LTB₄、PGE₂、LXA₄表达水平及其对细胞增殖影响。结果:ALA、EPA、DHA和5-FU对HCT116细胞的生长具有显著的抑制作用和诱导凋亡作用(p<0.05),ALA(200 μmol/L)、EPA(160 μmol/L)、DHA(120 μmol/L)诱导凋亡率分别为7.9%、21.8%、29.2%。ALA、EPA、DHA促进HCT116细胞内脂滴积聚状态,提高不饱和脂肪酸比例,降低LTB₄积累,提高LXA₄/PGE₂比率,使癌细胞趋向于抗炎状态。结论:n-3多不饱和脂肪酸(ALA、EPA、DHA)可通过促进抗炎因子LXA₄分泌,抑制促炎因子LTB₄生成,进而对HCT116细胞产生杀伤作用。     

PUFAs in Fish: Extraction,Fractionation, Importance in Health

ABSTRACT: Polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are currently in demand in the pure form and actively being studied to understand their potential roles in human health. Arachidonic acid, 20:4 (n‐6), and DHA, 22:6 (n‐3), are important in normal neurodevelopment and visual function. Infants fed formula often have low blood lipid 20:4 (n‐6) and 22:6 (n‐3). Consumption of fish oils may increase the 20:5 (n‐3) (EPA) and 22:6 (n‐3) (DHA) in human blood. Some marine fish oils contain higher amounts of arachidonic acid, EPA, and DHA. PUFA contents in different marine fishes and methods for their extraction and fractionation, in terms of fatty acid constituents in the form of methyl esters, are covered in this review. Emphasis is given to the fractionations of EPA and DHA by means of supercritical fluid extractions (SFE). The advantages of SFE compared to conventional methods are discussed in this review. PUFAs are usually extracted at about 10 to 30 MPa and at 40 to 80 °C. SFE is a promising and currently the best technique to extract PUFAs, especially EPA and DHA, from marine and freshwater fish.     

Fatty acid composition in some selected marine fish species living in Turkish waters

The muscle fatty acid (FA) composition of 12 selected marine fish species living in Turkish waters was determined. FA profiles of muscle were compared in terms of total and individual saturated (SFAs) and unsaturated fatty acids. Total SFA, monounsaturated fatty acid (MUFA), n‐3 polyunsaturated fatty acid (n‐3 PUFA) and n‐6 polyunsaturated fatty acid (n‐6 PUFA) contents as well as n‐3/n‐6 FA and eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratios differed (P < 0.01) in all fish species. Palmitic acid (16:0) among total SFAs, oleic acid (18:1n‐9) among MUFAs, DHA (22:6n‐3) among n‐3 PUFAs and 18:2n‐6 and 20:4n‐6 among n‐6 PUFAs were the most abundant FAs. The ratio of n‐3/n‐6 PUFA contents, indicating the availability of n‐3 PUFAs that are beneficial for human health, ranged from 2.67 to 12.61. The EPA/DHA ratio was between 0.17 and 0.83 in the marine fish species studied. Copyright © 2005 Society of Chemical Industry     

Physiological Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA)—A Review

Marine lipids have long been documented to be the major source of polyunsaturated fatty acids (PUFAs), especially n-3 fatty acids such as eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3). Both EPA and DHA have been documented to have significant influence on biochemical and physiological changes in the body. Although these long chain PUFA exert positive influences on human nutrition and health, there are also some controversies pertaining to the functioning of these n-3 PUFAs including the extent of their requirement by the body. As marine lipids have been thoroughly reviewed often, the present review mainly focuses on works related to physiological effects of EPA and DHA.     

Physiological Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA)—A Review

Marine lipids have long been documented to be the major source of polyunsaturated fatty acids (PUFAs), especially n-3 fatty acids such as eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3). Both EPA and DHA have been documented to have significant influence on biochemical and physiological changes in the body. Although these long chain PUFA exert positive influences on human nutrition and health, there are also some controversies pertaining to the functioning of these n-3 PUFAs including the extent of their requirement by the body. As marine lipids have been thoroughly reviewed often, the present review mainly focuses on works related to physiological effects of EPA and DHA.     

Acidolysis and glyceride synthesis reactions using fatty acids with two Pseudomonas lipases having different substrate specificities

Enzymatic acidolysis and glyceride synthesis using polyunsaturated fatty acids (PUFAs) with lipases from Pseudomonas fluorescens HU380 (HU-lipase), P. fluorescens AK102 (AK-lipase), and Candida rugosa (CR-lipase) were studied. The acidolysis of triolein with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in n-hexane was evaluated with lipases immobilized on Celite 545. HU-lipase showed the highest incorporation rate at a low temperature (10 degrees C) with either EPA or DHA as the acyl donor, and the rate decreased with increasing reaction temperature. At 45 degrees C, the rates for EPA and DHA were 7.1 and 0.5 relative to those at 10 degrees C, respectively. The EPA incorporation rate was even higher at a low temperature (10 degrees C), and the DHA incorporation rate increased with decreasing temperature. Although AK-lipase showed the reverse tendency for incorporation rate, the DHA incorporation rate increased with increasing reaction temperature with both PUFAs. HU-lipase reacted well with PUFAs such as DHA, EPA, arachidonic acid (AA), mead acid (MA), and dihomo-gamma-linolenic acid (DGLA) on acidolysis and glyceride synthesis. The reactivities of AK-lipase toward these PUFAs except for DGLA, i.e., MA, AA, EPA, and DHA, were low for both reactions. The unique substrate specificities of the lipases from the Pseudomonas strains will enable us to use these lipases for the modification of fats and oils containing PUFAs such as fish oil.     

Oral administration of docosahexaenoic acid attenuates colitis induced by dextran sulfate sodium in mice

Scope: Dietary supplementation of n‐3 PUFAs, containing docosahexaenoic acid (DHA), modulates the symptoms of colitis. Hence, we investigated the effects of oral administration of pure DHA and the therapeutic agent sulfasalazine (SAL) on chemically induced colitis in mice, and analyzed the expression levels of DHA‐responsive genes in colonic tissue using cDNA arrays. Methods and results: Colitis in BALB/c mice was induced by feeding 5% dextran sulfate sodium (DSS) in drinking water for 7 days. DHA (30 mg/kg/day, DHA) or SAL (100 mg/kg/day, SAL) was administered orally throughout the treatment along with DSS. The DHA‐treated group showed significant reduction of the weight loss and colon shortening compared to the DSS‐treated colitis group. In contrast, SAL treatment was effective in reducing colon shortening, stool consistency and bleeding scores. DHA and SAL treatments also significantly reduced the changes in inflammation of the colon, and reversed the increase in myeloperoxidase activity induced by DSS. Among DSS‐responsive genes, those for inflammatory cytokines (IL‐1β, CD14 antigen and tumor necrosis factor receptor superfamily, member 1b), membrane remodeling genes (matrix metalloproteinase‐3, ‐10 and ‐13) and acute phase proteins (S100 calcium‐binding protein A8), which were increased by DSS, were downregulated by DHA or SAL treatment. Conclusions: DHA was effective in alleviating DSS‐induced colitis in mice, partly by modulating the expression levels of genes involved in colitis.     

Enzymatic purification of polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) have various physiological functions. Of these, ethyl eicosapentaenoate is industrially purified and used as a medicine. Other PUFAs, such as docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), and arachidonic acid (AA), are also expected to be used as pharmaceutical agents; however, their industrial purification processes have not been established. Because PUFAs are highly unstable against heat and oxidation, we attempted to purify them by taking advantage of their enzymatic reactions. When free fatty acids (FFAs) originating from PUFA-containing oil were selectively esterified with lauryl alcohol (LauOH) using a lipase acting on a desired PUFA very weakly, the PUFA was efficiently enriched in the FFA fraction. In addition, when selective alcoholysis of ethyl esters originating from PUFA-containing oil with LauOH was carried out, the PUFA ethyl ester (EtPUFA) was enriched to a desired purity in the unreacted ethyl ester fraction. These reaction mixtures contain LauOH, PUFA (EtPUFA), and lauryl esters, and their molecular weights are different from one another. Hence, PUFA or EtPUFA can be easily separated by conventional distillation. Selective esterification increased the purity of DHA, GLA, and n-6 PUFAs rich in AA to 91, 98, and 96 wt%, respectively. Selective alcoholysis was also effective for increasing the purity of ethyl docosahexaenoate to 90 wt%.