Purified Glycosaminoglycans from Cooked Haddock May Enhance Fe Uptake Via Endocytosis in a Caco-2 Cell Culture Model

ABSTRACT:  This study aims to understand the enhancing effect of glycosaminoglycans (GAGs), such as chondroitin/dermatan structures, on Fe uptake to Caco-2 cells. High-sulfated GAGs were selectively purified from cooked haddock. An  in vitro  digestion/Caco-2 cell culture model was used to evaluate Fe uptake (cell ferritin formation) from a Fe⁺³-containing solution, and Fe⁺³/ascorbic acid (AA) and Fe⁺³/GAGs mixtures. Mitochondria (MTT test) and endosomal/lysosomal activities (neutral red uptake, NR), intracellular accumulation of reactive oxygen species, and GSH concentration were monitored as biomarkers of the changes of cellular metabolism. Changes in mRNA expression of Fe transporters, divalent metal transporter-1 (DMT1), and duodenal cytochrome-b (DcytB) were also evaluated. The Fe uptake from Fe⁺³/GAGs mixture was up to 1.8-fold higher than from Fe⁺³ alone. Both Fe⁺³ alone and Fe⁺³/AA mixture produced highest increase in MTT conversion. In contrast, cell cultures exposed to the Fe⁺³/GAGs mixture exhibited highest NR uptake values. All Fe-containing solutions tested caused a sharp intramitochondrial accumulation of reactive oxygen species. Cell cultures exposed to the Fe⁺³/GAGs mixture exhibited a more preserved (by 8%) intracellular GSH concentration compared to cultures exposed to Fe⁺³ or Fe⁺³/AA mixture. In addition to cell responses, the mRNA expression of Fe transporters may suggest that Fe could also be internalized into cells by endocytosis in addition to via DMT1 in Fe⁺³/GAGs mixtures. These aspects need to be confirmed in  in vivo  experiments to better establish nutritional interventional strategies.     

Polyphenols bioaccessibility and bioavailability assessment in ipecac infusion using a combined assay of simulated in vitro digestion and Caco-2 cell model

In this report, we investigated for the first time the total polyphenols content (TPC) and antioxidant activity before and after digestion of Carapichea ipecacuanha root infusion, better known as ipecac, prepared at different concentrations. An in vitro digestion system coupled to a Caco-2 cell model was applied to study the bioavailability of antioxidant compounds. The ability of ipecac bioaccessible fractions to inhibit reactive oxygen species (ROS) generation at cellular level was also evaluated. The findings revealed that water volume of 50 mL g⁻¹ of sample provided the maximum yield of extraction of TPC and antioxidant activity. Polyphenols increased in content and activity after digestion and they were highly bioavailable (75% of intestinal absorption). Polyphenols were also present in the residual parts which indicate a possible local activity. Results also suggest that ipecac infusion could represent a promising source of effective bioavailable antioxidants to be exploited in functional foods field.     

妊娠期大鼠补充苏氨酸锌对仔鼠金属硫蛋白基因MT1表达的影响

为了探讨母体补充苏氨酸锌对子代生长性能、金属硫蛋白（metallothionein,MT1）mRNA表达的影响,实验以苏氨酸锌为受试物,将90 只受孕SD大鼠随机分成5 组,并于妊娠期第6～15天进行灌胃补充不同剂量苏氨酸锌。母鼠分娩后将仔鼠进行常规饲养至1 月龄,测量仔鼠的体质量、身长和尾长,将各组仔鼠断颈处死后取肝脏、肾脏、脾脏和胸骨等组织进行RNA的提取并测定MT1基因相对表达量。结果表明：与空白对照组相比,苏氨酸锌各剂量组仔鼠体质量有显著增加（P＜0.05）。与溶媒对照组和空白对照组相比较,苏氨酸锌各剂量组仔鼠的MT1 mRNA表达量高,且差异显著（P＜0.05）,并且1 000 mg/kg苏氨酸锌处理组各组织中MT1的基因表达量要高于其他组。本结果表明：妊娠期补充苏氨酸锌能显著提高仔鼠MT1 mRNA表达水平,并能在一定程度上增加仔鼠体质量。     

36种蔬菜抑制肝癌HepG2和结肠腺癌Caco-2细胞增殖活性评价

为明确36种中国主要消费蔬菜多酚提取物的总酚含量和抑制人肝癌细胞Hep G2和人结肠腺癌细胞Caco-2增殖活性,分别采用Folin-Ciocalteu法确定了蔬菜提取物的总酚含量,采用亚甲基蓝法确定了其抗Hep G2和Caco-2细胞增殖的活性,分析了总酚含量与抗Hep G2和Caco-2细胞增殖活性之间的相关性。结果表明,36种蔬菜每百克中没食子酸当量:芥蓝(973.09±31.29)μmol/hg,莲藕(920.55±29.00)μmol/hg,它们的总酚含量最高;丝瓜(44.94±4.16)μmol/hg,其总酚含量最低。在可测出抗增殖EC50值的蔬菜中,韭菜(15.96±0.88)mg/m L和蒜苔(17.54±0.03)mg/m L,它们的抗Hep G2细胞增殖的活性最强;上海青(390.52±17.63)mg/m L和空心菜(394.25±11.89)mg/m L,它们的活性最弱。韭菜抗Caco-2细胞增殖的活性最强,为(21.08±1.67)mg/m L;青尖椒的活性最弱,为(390.17±4.78)mg/m L。这些蔬菜的抗Hep G2和Caco-2细胞增殖活性与其总酚含量相关性均不显著(R2=0.027 1,p0.05;R2=0.151 3,p0.05),表明蔬菜的抗肿瘤活性不能单从其多酚含量的高低来推测。     

基于Caco-2细胞模型的3种大豆肽降胆固醇能力研究

为了探究具有降胆固醇生物活性的大豆多肽在机体内的转运吸收机制,本研究以3种降胆固醇大豆肽VAWWMY(VY-6)、LPYPR(LR-5)、WGAPSL(WL-6)为研究对象,以22-NBD胆固醇胶束孵育的Caco-2细胞模型为高胆固醇模型,探究这3种多肽抑制胆固醇溶解,影响细胞吸收胆固醇与抑制胆固醇正向转运的能力。研究结果表明,在抑制胆固醇溶解率方面:WL-6多肽浓度为0.5 mmol/L时,在3种多肽中的抑制率最高,达14.2%,之后随浓度升高抑制率降低;在抑制细胞对胆固醇吸收方面:VY-6多肽浓度为2 mmol/L时,抑制率最大为43.1%,LR-5多肽浓度为2.5 mmol/L时,抑制率最大为45.7%,WL-6多肽浓度为2 mmol/L时,抑制率最大为45.93%,WL-6多肽在3种大豆肽中的抑制能力最强,且高于辛伐他汀组;在抑制胆固醇转运能力方面:WL-6多肽浓度为2 mmol/L时,可抑制30.7%的胆固醇转运,抑制率最高。综上可知WL-6多肽在3种大豆多肽中的降胆固醇能力最强。     

胶束化对Caco-2上皮细胞叶黄素吸收和转运的影响

本试验为研究胶束化促进叶黄素肠上皮细胞转运的特性,采用体外消化模型探究胶束化处理对叶黄素生物利用度的影响以及Caco-2细胞模型测定胶束化对叶黄素肠细胞摄取、表观渗透系数和细胞内吞的影响。结果显示:随浓度升高胶束化叶黄素生物可给率先增大后减小,浓度为6×10^-5 mol/L时胶束化叶黄素的生物可给率最高,是叶黄素单体的1.42倍;叶黄素胶束化显著促进了其细胞吸收量,细胞内积累量是单体的2.6倍。表观渗透系数(P_app)测定表明胶束化叶黄素累积转运分数大于1.5%,且被动扩散为其跨膜输送主要途径;胶束化后,P_app(B→A)与P_app(A→B)比值明显降低。进一步通过细胞内吞抑制实验发现制霉菌素(Nystain)、3-羟基-2-萘甲酸[(3,4-二羟基苯基)亚甲基]酰肼(Dynasore)均能抑制胶束化叶黄素的转运(P<0.05),而5-(N-乙基-N-异丙基)阿米洛利(EIPA)无显著抑制作用(P>0.05)。以上研究结果表明,胶束化处理显著促进了叶黄素的生物利用度,其在肠细胞中的跨膜吸收途径以被动扩散为主,兼具网格蛋白介导和小窝/脂筏蛋白介导的细胞内吞途径。     

茶多酚协同荷叶碱的抗氧化活性及其对结肠癌细胞的抑制作用

目的：探讨茶多酚协同荷叶碱的体外抗氧化活性及茶多酚单独对结肠癌细胞Caco-2的毒性作用。方法：采用二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）法、2,2’-联氮双（3-乙基苯并噻唑啉-6-磺酸）二铵盐（2,2’-azinobis（3-ethylbenzothi azoline-6-sulfonic acid）ammonium salt,ABTS）法检测茶多酚、荷叶碱及二者协同对DPPH自由基、ABTS＋•的清除作用;采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法检测茶多酚、荷叶碱及二者混合物对Cu2＋/H2O2体系诱导的牛血清白蛋白（bovine serum albumin,BSA）氧化损伤的保护作用;采用噻唑蓝（methyl thiazolyl tetrazolium,MTT）法,AO/EB、DAPI荧光染色法检测茶多酚对Caco-2细胞的毒性。结果：茶多酚、荷叶碱具有清除DPPH自由基、ABTS＋•及保护BSA蛋白氧化损伤的作用且呈剂量依赖性,两者之间存在显著的协同效应;1 mg/mL茶多酚作用24 h能显著降低Caco-2细胞活力,诱导Caco-2细胞发生凋亡。结论：茶多酚能有效地清除DPPH自由基、ABTS＋•,抑制蛋白氧化,且与其他活性成分具有协同效应;茶多酚能抑制Caco-2细胞的活力。     

体外消化/Caco-2 细胞模型评价面粉锌生物利用率

采用体外消化/Caco-2 细胞模型对10 种小麦粉进行锌生物利用率比较。以锌吸收率(Caco-2 细胞中的锌含量占加入到细胞的面粉消化液总锌量比例)反映锌生物利用率(Zn bioavailability,Zn BV)。发现不同面粉间锌生物利用率差异具有统计学意义,出粉率78% 的安阳中优9507 是面粉锌吸收率和可吸收锌量都高的品种,它将进入下一步人体实验进行营养功效的验证。     

基于Caco-2细胞的外源核酸吸收模型的建立及其吸收机制

基于人结肠癌细胞系Caco-2细胞建立外源核酸的吸收模型,并在此模型的基础上探究质粒DNA的肠道吸收机制。在Transwell孔膜上培养Caco-2细胞21 d后,通过透射电子显微镜观察细胞形态、监测培养期间跨膜电阻（transepithelial electrical resistance,TEER）值、测定荧光黄透过率评价细胞模型是否成功建成;通过细胞毒性实验探究质粒对细胞生长的影响;将质粒DNA加入细胞模型中进行双向转运实验,探究质粒的肠道吸收规律;在低温和添加特异性抑制剂条件下进行质粒转运实验,进一步推测质粒的肠道吸收机制。结果表明,细胞分化形态良好、TEER值及荧光黄表观透过系数（apparent permeability coefficient,Papp）均符合要求,细胞模型可用于转运实验;质粒对细胞生长无显著抑制作用,可用于转运实验;随着时间的延长,质粒在模型两方向上的转运均呈逐渐饱和趋势,Papp（肠腔侧（apical,AP）→基底侧（basolateral,BL））远大于Papp（BL→AP）,提示质粒的转运受到某种载体蛋白的介导,膜介导的跨细胞运输方式可能参与其中;在低温和添加特异性抑制剂条件下,质粒的转运均受到显著抑制,进一步表明该过程是一个跨细胞方式的主动运输过程。     

Availability of iron from milk-based formulas and fruit juices containing milk and cereals estimated by in vitro methods (solubility,dialysability) and uptake and transport by Caco-2 cells

Iron solubility, dialysability and transport and uptake (retention + transport) by Caco-2 cells as indicators of iron availability have been estimated in the in vitro gastrointestinal digests of infant foods (adapted, follow-up and toddler milk-based formulas and fruit juices containing milk and cereals (FMC)). Low correlation coefficients (in all cases R-squared ? 37.1%) were obtained between iron solubility or dialysability versus transport or uptake efficiency – a fact emphasizing the importance of incorporating Caco-2 cell cultures to in vitro systems in order to adapt the conditions to those found in in vivo assays. The highest uptake efficiency corresponded to FMC (25.6–26.1%) and toddler formulas (32.1–41.9%), the samples with the highest ascorbic acid contents and ascorbic acid/iron molar ratios. In addition, the toddler formulas contained caseinphosphopeptides with the cluster sequence SpSpSpEE, representing the binding site for minerals. In adapted formulas, greater iron uptake efficiency was obtained for the formulation containing ferrous lactate (22.7%) versus ferrous sulfate (4.7%).     

杜仲叶乙醇提取物的降糖作用机理

为了阐明杜仲叶的降血糖功效及其机制,采用酶-抑制剂模型,筛选高抑制率的杜仲叶乙醇提取物并判断其对α-葡萄糖苷酶的抑制类型;借助Caco-2人结肠腺癌细胞模型,研究杜仲叶20%乙醇解吸物对α-葡萄糖苷酶活性及葡萄糖转运蛋白的影响;气相色谱-质谱联用分析杜仲叶20%乙醇解吸物主要成分。杜仲叶20%乙醇解吸物可竞争性地抑制α-葡萄糖苷酶,其抑制常数Ki值为32.90 mg/mL。它对Caco-2细胞中的α-葡萄糖苷酶有较强抑制作用,其IC50为0.57 mg/mL,且对Caco-2细胞摄取葡萄糖也有一定抑制作用,1 mg/mL时抑制率可达26.25%。气相色谱-质谱分析表明杜仲叶20%乙醇解吸物主要有DL-异柠檬酸内酯、百里酚、儿茶素、2,6-二羟基-苯甲酸和3,4-二羟基-苯丙烯酸等。这些结果表明,杜仲叶乙醇提取物可以通过抑制α-葡萄糖苷酶活性,降低葡萄糖吸收来实现降血糖的效果。     

Supplemental inulin does not enhance iron bioavailability to Caco-2 cells from milk- or soy-based,probiotic-containing,yogurts but incubation at 37 °C does

The in vitro effects of supplemental inulin (4%) on iron (Fe) availability in two different probiotic-containing yogurts were examined. Milk or soy-based yogurts, with and without inulin, were incubated (37 °C) for 48 h or without any incubation before comparison by an in vitro gastrointestinal digestion/Caco-2 cell culture model was used to assess iron bioavailability. The dialysable Fe fraction, cell ferritin formation, and cell associated Fe were monitored. Supplemental inulin decreased dialysable Fe only in non-incubated milk-based yogurt. In both yogurts incubation by itself increased dialysable Fe, and inulin increased the latter only in soy-based yogurt. Cellular ferritin concentration were higher after exposure to non-incubated milk-based than soy-based yogurt, although, after incubation the latter induced the highest ferritin formation. These data suggest that inulin does not have a direct effect on Fe bioavailability in the small intestine, and that probiotic bacteria play an enhancing role on Fe bioavailability.     

Bioavailability of iron and zinc from multiple micronutrient fortified beverage premixes in Caco-2 cell model

Abstract: Iron and zinc deficiencies are the most prevalent nutrient deficiencies worldwide. They often coexist as the dietary factors, especially phytate, which impairs iron absorption also affects zinc absorption. Therefore, suitable strategies are required to control multiple micronutrient deficiencies in populations that subsist on high‐phytate foods such as the whole wheat flour based Indian bread (chapatti). The objective of the study, therefore, was to test the bioavailability of iron and zinc in 2 multiple micronutrient beverage premixes in the absence and presence of chapatti. The premix‐1 contained iron, zinc, and vitamin A while premix‐2 contained all micronutrients in premix‐1, plus folic acid and ascorbic acid. Ferritin induction and ⁶⁵Zn uptake were assessed using coupled in vitro digestion/Caco‐2 cell line model as the surrogate markers of iron and zinc bioavailability, respectively. The results show that iron bioavailability from premixes‐1 and 2 was similar in the absence of chapatti. However, premix‐2 showed significantly higher iron bioavailability compared to premix‐1 in the presence of chapatti. In contrast, the zinc uptake was similar from both premixes‐1 and 2 in the absence or presence of chapatti. These results suggest that both the premixes provide bioavailable minerals, but premix‐2 appears to be promising in the presence of foods that have high phytate.     

Investigation of the cytotoxicity of food-grade nanoemulsions in Caco-2 cell monolayers and HepG2 cells

Nanoemulsions represent one of the emerging formulations for nutraceutical delivery. However, the possible toxicity associated with the small droplet size (diameter <200 nm) is still unknown. In this study, three nanoemulsions emulsified by modified starch, Tween 20 and whey protein isolate, respectively, were prepared and their cytotoxicity was examined by comparing with the corresponding micron-sized emulsions. Caco-2 cell monolayers were used to mimic the small intestine epithelium. Integrity of the cell membrane and tight junctions was tested by measuring the lactate dehydrogenase leakage and transepithelial electrical resistance, respectively. All three nanoemulsions did not reveal significant difference from their micron-sized counterparts, suggesting no apparent toxicity of the nanoemulsions on the small intestine. Meanwhile, the possible hepatic toxicity was investigated using MTT assay on HepG2 cells. It was found that nanoemulsions made with modified starch and whey protein isolate, but not Tween 20, affected the cell viability/proliferation more than did the micron-sized emulsions. Further in vivo investigation is required to examine the possible hepatic toxicity of nanoemulsions.     

儿童配方奶粉对Caco-2细胞钙转运及对成骨细胞增殖、矿化的影响

为评价某市售儿童成长配方奶粉的补钙和促进骨骼生长能力,以常见市售纯牛奶A作为对照,运用人结肠腺癌细胞（Caco-2）单层模型,研究其对钙离子转运的作用;运用成骨细胞（MC3T3-E1）矿化模型,研究其对成骨细胞增殖与矿化的影响。Caco-2细胞模型结果表明,各剂量组儿童配方奶粉组的钙转运量均高于市售纯牛奶A,但相较空白组无显著性差异（p<0.05）。成骨细胞增殖实验结果表明,5~50 μg/mL的儿童配方奶粉对成骨细胞均有促进增殖作用,增殖速度均在30%以上;并测定细胞碱性磷酸酶活性,当儿童配方奶粉质量浓度为50 μg/mL时,酶活达7.79 U/g蛋白,相较空白组极显著提高（p<0.01）,较市售纯牛奶A的效果更稳定。成骨细胞矿化结果表明,5~50 μg/mL儿童配方奶粉能显著促进成骨细胞产生矿化结晶,50 μg/mL时茜素红吸光值均为0.180,对比空白组提高50%以上,较市售纯牛奶A矿化程度更稳定。综上实验结果,儿童配方奶粉虽对钙在小肠上皮的转运吸收无明显促进作用,但很可能通过促进骨生长而对儿童生长发育起作用。     

红毛藻多糖对H2O2诱导的Caco-2细胞氧化损伤的保护作用

目的:研究红毛藻多糖(Bangia fusco-purpurea polysaccharide,BFP)对H2O2诱导的人结肠腺癌(Caco-2)细胞氧化应激损伤的保护作用.方法:构建过氧化氢(H2O2)诱导的Caco-2细胞氧化损伤模型,通过形态学观察和测定细胞裂解液中的抗氧化物水平及相关酶活力,评价BFP对Caco...     

Tea components influencing bioavailability of fluoride and potential transport mechanism in the Caco-2 cell line model

Previous work showed that the bioavailability of fluoride in dark tea was lower than NaF solution. However, limited information is available indicating the effects of tea components on the fluoride bioavailability. In this study, the effects of the components in tea on the bioavailability of fluoride were evaluated in the Caco-2 cell line model. Additionally, the mechanism of effect of aluminium on fluoride transport was investigated. The result showed that 10–100 μm of epigallocatechin gallate (EGCG) could not influence fluoride transport. Al³⁺ significantly decreased fluoride transport in both apical-basolateral and basolateral-apical directions. Moreover, aluminium could form different forms of aluminium fluoride complexes, which were transported through Caco-2 cells by different pathways. F⁻ transport was mainly dependent on the paracellular pathway and active transport involving Cl⁻ channels. The paracellular pathway played a predominant role in transport of AlF₃. The paracellular pathway and active transport both participated in AlF₂⁺ transport.     

Transepithelial transport of 6-O-caffeoylsophorose across Caco-2 cell monolayers

The aim of this study was to clarify the transport behaviour and mechanism of caffeic acid analogue bearing a sugar-moiety, 6-O-caffeoylsophorose (CS), in Caco-2 cells. The absorption of CS was investigated by its transport across Caco-2 cell monolayers using a high-performance liquid chromatography-time-of-flight-mass spectrometry (LC–TOF-MS). The permeation of CS was concentration-dependent and reached the plateau at >6 mM. The apparent permeability (P_app) of CS in the apical-to-basolateral direction was 5.4 × 10⁻⁷ cm/s, while in the reversed direction the P_app value was significantly reduced (1.9 × 10⁻⁷ cm/s). CS transport was competitively inhibited by phloretin, an inhibitor of monocarboxylic acid transporter (MCT). Benzoic acid, an MCT substrate, also reduced CS transport. A less significant change of CS transport was observed across Caco-2 cell monolayers pretreated with quercetin, a suppressor of tight-junction. These findings strongly indicate that CS, a caffeic acid analogue bearing sophorose moiety, can be transported across Caco-2 cell monolayers via the MCT pathway.     

Assessing Potential Effects of Inulin and Probiotic Bacteria on Fe Availability from Common Beans (Phaseolus vulgaris L.) to Caco-2 Cells

ABSTRACT:  Inulin, a prebiotic, may enhance intestinal Fe absorption. Our objective was to assess the effects of supplemental inulin and 2 probiotic bacteria ( B. infantis and L. acidophillus ) on Fe availability to Caco-2 cells from common white and red beans ( Phaseolus vulgaris L.). Cooked beans were mixed or not with supplemental inulin (4%, w/w), and then subjected to simulated gastrointestinal digestion (pepsin, pH 2; pancreatin, pH 7.2). Subsequently, the digests were incubated overnight with and without B. infantis or L. acidophilus . Ferritin formation in Caco-2 cells was used to evaluate Fe uptake. Total soluble phenols (Folin–Ciocalteau) and phytate (HPLC-electrochemical detection) were quantified, and the flavonoids profile (HPLC-PDA/UV detection) was monitored in the digests. Supplemental inulin did not affect Fe uptake from white nor red beans. Incubation with B. infantis increased total soluble phenols (TSP) in the digests and decreased Fe uptake. Incubation with L. acidophilus decreased TSP in the digest and increased Fe uptake. Variations in Fe uptake were not associated with soluble phytate concentrations in the digests. The largest change in flavonoids profile were found in the digests incubated with L. acidophilus , which decreased the soluble concentration of astragalin (kaempferol-3-O-glucoside). These results suggest that certain probiotics could increase Fe uptake from common beans.     

笛鲷鱼鳞源功能多肽对Caco-2细胞氧化应激损伤的保护作用

本实验利用650μmol/L H₂O₂构建Caco-2细胞氧化应激损伤模型,探究笛鲷鱼鳞源功能多肽(crimson snapper scale peptides,CSSPs)对Caco-2细胞氧化应激损伤的保护作用。通过测定细胞上清液乳酸脱氢酶(lactate dehydrogenase,LDH)活力和白细胞介素-8(interleukin-8,IL-8)质量浓度、胞内抗氧化酶活力和丙二醛(malondialdehyde,MDA)含量、胞内活性氧(reactive oxygen species,ROS)相对含量、相关凋亡基因(Caspase-3、Caspase-9、Bax和Bcl-2)的相对表达量,从细胞氧化还原状态和细胞凋亡两方面阐明CSSPs对损伤Caco-2细胞的抗氧化作用。结果显示,CSSPs能够通过抗氧化防御系统来减轻H₂O₂对Caco-2细胞的损伤,主要包括增加胞内抗氧化酶活性以抑制LDH释放、胞内ROS积累、MDA及IL-8的生成。此外,CSSPs抑制氧化应激损伤诱导的细胞凋亡与线粒体...