Assessment of Iron Bioavailability in Ten Kinds of Chinese Wheat Flours Using an in vitro Digestion/Caco-2 cell Model

Abstract Objective To compare iron bioavailability （Fe BV） from ten selected kinds of Chinese wheat flours in order to provide scientific basis for further human trials and enable plant breeding programs to screen biofortified wheat cultivars. Methods An in vitro digestion/Caco-2 cell model was used to assess Fe BV of ten flour samples from six leading Chinese wheat cultivars and the stability of Fe BV in one cultivar was studied across three growing environments. Results Significant differences were observed in both Fe BV and Fe bioavailability per gram of food （Fe BVPG） among cultivars （P〈0.01） grown at the same location with the same flour extraction rate. Zhongyou 9507 and Jingdong 8 had Fe BV 37%-54% and Fe BVP（3 103%-154% higher than the reference control. In the Anyang environment, Zhongyou 9507 had a higher wheat flour-Fe level and Fe BVPG. Differences in Fe BV were detected in cultivars with different flour extraction rates. Conclusion Zhongyou 9507 and Jingdong 8 were identified as the most promising cultivars for further evaluation of efficacy by using human subjects. The growing environments had no effect on Fe BV, but did have a significant effect on Fe BVPG. Fe bioavailabilities in low-extraction （40%） flours were higher than those in high-extraction （78%） flours.

Tartary buckwheat(Fagopyrum tataricum(L.)Gaertn)protein-derived antioxidant peptides:mechanisms of action and structure-activity relationship in Caco-2 cell models

Excessive reactive oxygen species(ROS)can cause oxidative damage and lead to various metabolic disease.Tartary buckwheat(Fagopyrum tataricum(L.)Gaertn)is a new kind of protein-rich functional food,the protein in which has been proved to have good antioxidant capacity.In this study,in order to further explore the antioxidant mechanism of Tartary buckwheat protein,4 peptides(CR-8,LR-8,GK-10 and SR-12)were isolated and identified from it.H2 O2 was used to induce oxidative damage to Caco-2 cells to evaluate antioxidant capacity of these peptides.The results of superoxide dismutase(SOD),total antioxidant capacity(T-AOC)and mitochondrial membrane potential etc.showed that these peptides have superior antioxidant capacity.CR-8 has the best antioxidant capacity.In order to further clarify the antioxidant mechanism of CR-8,metabolomics was used to analyze related metabolites and metabolic pathways.The results showed that after CR-8 intervention,the content of metabolites such as L-acetyl carnitine has increased.This indicated that CR-8 can improve the antioxidant capacity of damaged cells by intervening in multiple metabolic pathways.This also revealed the anti-oxidant mechanism of tartary buckwheat protein.In conclusion,it provided a theoretical basis for further studying the activity of tartary buckwheat portein and utilizing buckwheat resources.

Overloading of differentiated Caco-2 cells during lipid transcytosis induces glycosylation mistakes in the Golgi complex

Overloading the intestine enterocytes with lipids induced alteration of the Golgi complex(GC;Sesorova et al.,2020)and could cause glycosylation errors.Here,using differentiated Caco-2 cells with the established 0[I]blood group phenotype(no expression of the blood antigens A and B[AgA,AgB]under normal conditions)as a model of human enterocytes we examined whether the overloading of these cells with lipids could cause errors in the Golgi-dependent glycosylation.We demonstrated that under these conditions,there were alterations of the GC and the appearance of lipid droplets in the cytoplasm.Rare cells produced AgA and AgB.This suggested that after overloading of enterocytes with lipids,AgA were mistakenly synthesized in individual enterocytes by the Golgi glycosyltransferases.These mistakes could explain why in the absence of AgA and AgB antibodies against them exist in the blood.

Antioxidant Activity and Protective Effects of Alcalase-Hydrolyzed Soybean Hydrolysate in Human Intestinal Epithelial Caco-2 Cells

Soybeans are known as a promising source of bioactive peptides.However,knowledge on the antioxidant behaviors of soybean protein hydrolysate(SPH)in the human intestinal epithelium is limited.In this study,SPH was prepared with Alcalase and subsequently ultrafiltered into four peptide fractions as SPH-I(<3 kDa),SPH-II(3~5 k Da),SPH-III(5~10 k Da)and SPH-IV(>10 kDa).The antioxidant properties of SPH and membrane fractions were investigated using different chemical assays and their protective effects against oxidative stress were evaluated using H2 O2-stressed human intestinal Caco-2 cells.Results showed that SPH-I exhibited the strongest 2,2-diphenyl-1-picrylhydrazyl(DPPH)radical scavenging activity(IC50=2.56 mg/m L)and reducing capacity while SPH-III had the best metal ion-chelating activity(IC50=0.29 mg/m L).Both SPH and the peptide fractions dose-dependently suppressed intracellular reactive oxygen species(ROS)accumulation induced by H2O2 in Caco-2 cells,but the strongest inhibitory effect was observed for SPH-I.Amino acid(AA)results revealed that SPH-I was rich in hydrophobic and antioxidant AAs,which could contribute to its stronger antioxidant properties.Additionally,SPH-I protected Caco-2 cells from H2O2-induced oxidative stress via inhibiting lipid peroxidation and stimulating antioxidant enzyme activities.These results suggest that SPH-I and constitutive peptides can be beneficial ingredients with antioxidant properties and protective effects against ROS-mediated intestinal injury.

Effect of dephytinization on bioavailability of iron,calcium and zinc from infant cereals assessed in the Caco-2 cell model

AIM:To test the effect of the dephytinization of three different commercial infant cereals on iron, calcium, and zinc bioavailability by estimating the uptake, retention, and transport by Caco-2 cells.METHODS:Both dephytinized (by adding an exogenous phytase) and non-dephytinized infant cereals were digested using an in vitro digestion protocol adapted to the gastrointestinal conditions of infants younger than 6 mo. Mineral cell retention, transport, and uptake from infant cereals were measured using the soluble fraction of the simulated digestion and the Caco-2 cells. RESULTS: Dephytinization of infant cereals significantly increased (P<0.05) the cell uptake efficiency (from 0.66%-6.05% to 3.93%-13%), retention (from 6.04%-16.68% to 14.75%-20.14%) and transport efficiency (from 0.14%-2.21% to 1.47%-6.02%), of iron, and the uptake efficiency (from 5.0%-35.4% to 7.3%-41.6%) and retention (from 4.05%-20.53% to 14.45%-61.3%) of zinc, whereas calcium only cell uptake showed a significant increase (P<0.05) after removing phytate from most of the samples analyzed. A positive relationship (P<0.05) between mineral solubility and the cell uptake and transport efficiencies was observed.CONCLUSION: Removing phytate from infant cereals had a beneficial effect on iron and zinc bioavailability when infant cereals were reconstituted with water. Since in developing countries cereal-based complementary foods for infants are usually consumed mixed with water, exogenous phytase additions could improve the nutritional value of this weaning food.

Effects of extracellular iron concentration on calcium absorption and relationship between Ca^(2+) and cell apoptosis in Caco-2 cells

AIM: To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. METHODS: Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry.RESULTS: Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase Ⅺ and dispase Ⅰ. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 μmol/L) increased the FI of Caco-2 cells from 27.50±13.18 (control,n = 150) to 35.71±13.99 (n = 150, P＜0.01), 72.19±35.40 (n = 150, P＜0.01) and 211.34±29.03 (n = 150, P＜0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 μmol/L). The FIvalue of Caco-2 cells treated by FAC was 185.85±33.77 (n = 150, P＜0.01), 122.73±58.47 (n = 150, P＜0.01), and 53.29±19.82 (n = 150, P＜0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187 (0.1, 1.0 and 10 μmol/L) increased the FI of Caco-2 cells from 40.45±13.95 (control, n = 150) to 45.19±21.95 (n = 150, P＜0.01), 89.87±43.29 (n = 150,P＜0.01) and 104.64±51.07 (n = 150, P＜0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with indicating that the increase in the positive apoptotic cell~ increased from 0.32% to 0.69%, 0.90% and 1.10%, number was positively correlated with [Ca2+]i.CONCLUSION: Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.

Gastric digestion of pea ferritin and modulation of its iron bioavailability by ascorbic and phytic acids in caco-2 cells

瞄准：在 caco-2 房间线模型理解版本和豌豆含铁听铁的肠的举起的消化稳定性和机制。方法：豌豆种子含铁锡用分别由胶化过滤层析跟随了的盐被净化。含铁听铁的简历可获得性当面用联合在试管内 digestion/Caco-2 房间模型被估计或抗坏血酸和 phytic 的缺席。Caco-2 房间含铁听形成被用作铁举起的一个代理人标记。在模仿的胃的 pH 下面的豌豆含铁锡的结构的变化用电气泳动，胶化过滤和圆形的二色性光谱学被描绘。结果：caco-2 房间含铁听形成显著地被增加(P 【 0.001 ) 与 FeSO4 (19.3 +/- 9.8 ng/mg 蛋白质) 并且豌豆含铁锡(13.9 +/- 6.19 ng/mg 蛋白质) 与空白的文摘相比(3.7 +/- 1.8 ng/mg 蛋白质) 。当 phytic 减少了时，提高的抗坏血酸豌豆含铁听铁简历可获得性。然而，也当面或抗坏血酸的缺席， caco-2 房间的含铁听内容是显著地有豌豆含铁锡的更少比与 FeSO4。在胃的 pH，相应于含铁锡的乐队都没在本国的页或 SDS 页上面对胃朊酶任何一个被观察。胶化过滤层析和圆形的二色性光谱学揭示了第四级、第二等的结构的 pH 依赖者损失。结论：在胃的条件下面，豌豆含铁锡的铁核心由于酸被释放进消化媒介蛋白质的导致的结构的改变和分离。释放的铁与导致豌豆含铁听铁简历可获得性的调整的饮食的因素交往，类似于 non-heme 铁的典型特征。

Hormonal regulation of dipeptide transporter(PepT1)in Caco-2 cells with normal and anoxia/reoxygenation management

AIM: To determine the regulation effects of recombinant human growth hormone (rhGH) on dipeptide transporter (PepT1) in Caco-2 cells with normal culture and anoxia/reoxygenation injury.METHODS: A human intestinal cell monolayer (Caco-2) was used as the in vitro model of human small intestine and cephalexin as the model substrate for dipeptide transporter (PepT1). Caco-2 cells grown on Transwell membrane filters were preincubated in the presence of rhGH in the culture medium for 4 d, serum was withdrawn from monolayers for 24 h before each experiment. The transport experiments of cephalexin across apical membromes were then conducted;Caco-2 cells grown on multiple well dishes (24 pore) with normal culture or anoxia/reoxygenation injury were preincubated with rhGH as above and uptake of cephalexin was then measured.RESULTS: The transport and uptake of cephelaxin across apical membranes of Caco-2 cells after preincubation with rhGH were significantly increased compared with controls (P=0.045, 0.0223). Also, addition of rhGH at physiological concentration (34 nM) to incubation medium greatly stimulates cephalexin uptake by anoxia/reoxygenation injuried Caco-2 cells (P=0.0116), while the biological functions of PepT1 in injured Caco-2 cells without rhGH were markedly downregulated. Northem blot analysis showed that the level of PepT1 mRNA of rhGH-treated injured Caco-2cells was greatly increased compared to controls.CONCLUSION: The present results of rhGH stimulating the uptake and transport of cephalexin indicated that rhGH greatly upregulates the physiological effects of dipeptide transporters of Caco-2 cells. The alteration in the gene expression may be a mechanism of regulation of PepT1. In addition, Caco-2 cells take up cephalexin by the Proton-dependent dipeptide transporters that closely resembles the transporters present in the intestine. Caco-2 cells represent an ideal cellular model for future studies of the dipeptide transporter.

Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells： an insight into the efflux-metabolism alliance

Aim The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide（CLA） enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients （Papp） of （ - ） and （ ＋ ）CLA were higher in the ab- sorptive direction than those in the secretory direction with efflux ratios（ER） of 0. 709 ± 0.411 and 0. 867± 0. 250 （ Х10^-6 -1 cm · s ）, respectively. Their bidirectional transports were significantly reduced by （75.6 ± 87.5）% af- ter treatment with verapamil （ a P-glycoprotein inhibitor） that increased the rate of metabolism by CYP3 A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of （ - ） and （ ＋ ） CLA with ERs being 2. 934 ± 1. 432 and 1. 877 ± 0. 148 （ Х 10^-6 cm/s） respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enanti- omers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following （ - ） or （ ＋ ）-isomer treatment alone. Furthermore, the uptake of （ - ）CLA was more than that of （ ＋ ）CLA in the transfected cells. Incubation with ketoeonazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexametha- sone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharma- cology of （？） CLA as a candidate drug for treatment of Alzheimer＇ s disease.

Availability and toxicity of Fe(II) and Fe(III) in Caco-2 cells

The objective of the present study was to compare the toxicity and availability of Fe(II) and Fe(III) to Caco-2 cells. Cellular damage was studied by measuring cell proliferation and lactate dehydrogenase (LDH) release. The activities of two major antioxidative enzymes [superoxide dismutase (SOD) and glutathione peroxidase (GPx)] and differentiation marker (alkaline phosphatase) were determined after the cells were exposed to different levels of iron salts. The cellular iron concentration was investigated to evaluate iron bioavailability. The results show that iron uptake of the cells treated with Fe(II) is significantly higher than that of the cells treated with Fe(III) (P<0.05). Fe(II) at a concentration >1.5 mmol/L was found to be more effective in reducing cellular viability than Fe(III). LDH release investigation suggests that Fe(II) can reduce stability of the cell membrane. The activities of SOD and GPx of the cells treated with Fe(II) were higher than those of the cells treated with Fe(III), although both of them increased with raising iron supply levels. The results indicate that both Fe(II) and Fe(III) could reduce the cellular antioxidase gene expression at high levels.

Nutritional properties of Europen eel(Anguilla anguilla)bone peptide-calcium and its apoptosis effect on Caco-2 cells

The present study aimed at making a rational usage for European eel bone by-products by preparing Europen eel bone peptide chelated calcium(EBPC-Ca).Nutritional properties and bioactivity of EBPC-Ca were evaluated.Results showed that nutritional properties of calcium ions will cause intra-and inter-molecular folding and aggregation of peptide to uniformly form EBPC-Ca chelate.The chelated compound of EBPC and calcium ion triggered a strong apoptosis in heterogeneous human epithelial colorectal adenocarcinoma(Caco-2)in concentration-and time-dependent manners.Western blot analysis revealed that the EBPC-Ca induced apoptosis may be the result of a blocked autophagy flux through mitochondrial-dependent pathway.Additionally,the increase in FGF-23 protein expression inhibited the absorption of calcium ions and alleviated cell apoptosis.It was also found that the cell apoptosis occurs with significant increases in the levels of reactive oxygen species(ROS)and Ca^(2+)in the cells,indicating the anti-tumor potential of EBPC-Ca may involve multiple channels.

Intestinal Absorption of Ergostane and Lanostane Triterpenoids from Antrodia cinnamomea Using Caco-2 Cell Monolayer Model

Antrodia cinnamomea is a precious medicinal mushroom.It exhibits promising therapeutic effects on cancer,intoxication,hypertension,hepatitis,and inflammation.Its major bioactive constituents are ergostane and lanostane triterpenoids.In this study,we used intestinal Caco-2 cell monolayer model to reveal the intestinal absorption property of 14 representative triterpenoids from A.cinnamomea.The bidirectional transport through the monolayer at different time points was monitored by a fully validated LC/MS/MS method.In the case of pure compounds,ergostanes 5(25R-antcin H),6(25Santcin H)and 10(25R-antcin B)could readily pass through the Caco-2 cell layer,whereas lanostanes 13(dehydroeburicoic acid)and 14(eburicoic acid)could hardly pass through.When the cells were treated with A.cinnamomea extract,antcins A,B,C,H and K(1–6 and 9–11)were absorbed via passive transcellular diffusion,and showed high PAB and PBA values(>2.5×10^(-5) cm/s).Meanwhile,the lanostanes dehydrosulphurenic acid(8),15a-acetyldehydrosulphurenic acid(12),13 and 14 exhibited poor permeability.Transport features of these compounds were consistent with their pharmacokinetic behaviors in rats.This study could also be helpful in predicting the intestinal absorption of A.cinnamomea in human.

Upregulation of 25-hydroxyvitamin D_3-1α-hydroxylase by butyrate in Caco-2 cells

AIM: To investigate the possible involvement of 25-hydroxyvitamin D3-1α-hydroxylase [1α-25(OH)2D3] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells.METHODS: Caco-2 cells were incubated either with 3 mmol/L butyrate and 1 μmol/L 25(OH)2D3 or with 1μmol/L 1α-25(OH)2D3 for various time intervals ranging from 0 to 72 h. Additionally, cells were co-incubated with butyrate and either 25(OH)2D3 or 1α-25(OH)2D3.1α-25(OH)2D3 mRNA was determined semi-quantitatively using the fluorescent dye PicoGreen. Immunoblotting was used for the detection of 1α-25(OH)2D3 protein.Finally, enzymatic activity was measured by ELISA.RESULTS: Both butyrate and 1α-25(OH)2D3 stimulated differentiation of Caco-2 cells after a 48 h incubation period, while 25(OH)2D3 had no impact on cell differentiation. Synergistic effects on differentiation were observed when cells were co-incubated with butyrate and vitamin D metabolite. Butyrate transiently upregulated 1α-25(OH)2D3 mRNA followed by a timely delayed protein upregulation. Coincidently, enzymatic activity was enhanced significantly. The induction of the enzyme allowed for comparable differentiating effects of both vitamin D metabolites.CONCLUSION: Our experimental data pr ovide a further mechanism for the involvement of the vitamin D signaling pathway in colonic epithelial cell differentiation by butyrate. The enhancement of 1α-25(OH)2D3 followed by antiproliferative effects of the vitamin D prohormone in the Caco-2 cell line suggest that 25(OH)2D3 in combination with butyrate may offer a new therapeutic approach for the treatment of colon cancer.

Influence of magnetic iron oxide nanoparticles on red blood cells and Caco-2 cells

The interactions of two types of cells (red blood cells, Caco-2 cells) with magnetic iron oxide nanoparticles (non-grafted, citrate-grafted, dendrimer-grafted) of 11 nm in size have been investigated. We focused on two important physiological parameters of the cells, the intracellular pH and the intracellular Ca2+ content. The results show that the nanoparticles do not have a significant influence on the pH and Ca2+ content of Caco-2 cells. The Ca2+ content of red blood cells is also not affected but the intracellular pH is slightly reduced.

Evaluation of Chito-Oligosaccharide(COS)in Vitro and in Vivo:Permeability Characterization in Caco-2 Cells Monolayer and Pharmacokinetics Properties in Rats

Chito-oligosaccharide(COS)had shown a variety of biological activities and potential biomedical implications.The present study investigated the pharmacokinetics,bioavailability,and in vitro absorption of COS with degrees of polymerization(DPs)2-7 and explored the influence of DPs on them.From Caco-2 cell permeation studies,COS were low permeability compounds with no directional effects,suggesting a low in vivo absorption mediated by facilitation diffusion and paracellular absorption.After an intragastrical administration to rats,COS2 showed the highest systemic exposure in six oligosaccharides.The bioavailability of COS2-7 was 7.33%,6.11%,4.67%,4.13%,4.02%,0.99%,respectively.Differences in bioavailability for each COS correlated to structural variations,with high DPs contributing to a decrease in bioavailability.In conclusion,COS could be absorbed by the intestinal tract both in vitro and in vivo.The very low oral bioavailability of COS could be due to low permeability.DPs can affect absorption and bioavailability of COS2-7.This study provided evidence for the absorption characteristics of COS2-7 to help us better understanding the pharmacological actions.

Permeation of roxarsone and its metabolites increases caco-2 cell proliferation

The benzenearsonate, Roxarsone, has been used since 1944 as an antimicrobial, growth-promoting poultry feed additive. USGS and EPA report that Roxarsone (4-hydroxy-3-nitrobenzenearsonate) and metabolites, including AHBA (3-amino-4-hydroxybenzenearsonate), contaminate waterways at greater than 1100 tons annually. To assess human impact of these organic arsenic water contaminants, it was important to study their potential absorption. The human adenocarcinoma cell line, Caco-2, is a model for intestinal absorption. We found proliferative effects on Caco-2 cells at micromolar levels of these compounds, as monitored by [3H]-thymidine incorporation into DNA. Flow cytometry cell cycle analysis confirmed accumulation in S phase from 21% (control) to 36% (24 hour exposure to 10 μM AHBA). Confluent Caco-2 cells grown on collagen-coated Transwell plates were dosed on the apical side. After exposure, media from apical and basolateral sides were collected separately. Following removal of FBS by 30K centrifugal filtration, the benzenearsonates in the collected media were analyzed by HPLC. Analyses were at wavelengths in the ultraviolet/visible range where the absorbance values were linear with respect to concentration. Concentrations were calculated by comparison with analytically-prepared commercial standards. Results from cells dosed at 10 μM for 24 hours with AHBA, Roxarsone, or Acetarsone indicated 6%-29% permeation occurring from apical to basolateral side, modeling absorption across intestinal epithelium to the circulatory system. Benzenearsonate feed additives are frequently applied in combination with antibiotics, raising additional health concerns. We conclude that micromolar levels of these benzenearsonates are adequate to stimulate Caco-2 cell proliferation.

染料木素-川芎嗪共晶在Caco-2细胞模型中的转运研究

旨在考察染料木素(genistein,GEN)、川芎嗪(tetramethylpyrazine,TMP)、染料木素-川芎嗪共晶(GEN-TMP)及染料木素与川芎嗪的物理混合物(GEN+TMP)在Caco-2细胞模型中的转运特征;建立Caco-2细胞模型,并以细胞跨膜电阻和标志物渗漏检查等指标进行验证,采用高效液相色谱法,考察并计算安全浓度下药物的累积转运量、表观渗透系数和外排率,并探讨P糖蛋白(P-gp)抑制剂维拉帕米、乳腺癌耐药蛋白(breast cancer resistant protein,BCRP)抑制剂KO143和多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)抑制剂MK571对转运的影响。结果显示,Caco-2细胞模型完整性与功能性良好,GEN浓度为40μg/mL时,GEN、TMP、GEN-TMP以及GEN+TMP的细胞存活率分别为90.06%、84.21%、97.60%和89.37%;GEN、TMP、GEN-TMP和GEN+TMP的表观渗透系数(P_(app))大于1.0×10^(-6)cm/s,属于吸收良好药物;GEN-TMP中GEN的累积转运量和P_(app)值分别为(2.78±0.11)μg和(8.61±0.33)×10^(-6)cm/s,比GEN的(1.92±0.15)μg和(5.96±0.47)×10^(-6)cm/s提高了44.79%和44.46%;GEN+TMP中GEN的累积转运量和P_(app)值与GEN无显著性差异。GEN只受BCRP的外排作用,GEN-TMP中的GEN同时受到P-gp和BCRP的外排作用,TMP、GEN-TMP和GEN+TMP中的TMP均受到MRP2的外排作用。结果表明,相同浓度下,GEN-TMP的细胞存活率高于GEN+TMP。GEN-TMP的吸收强于GEN和GEN+TMP中的GEN,共晶受外排蛋白的作用区别于GEN和GEN+TMP中的GEN,研究工作为共晶的转运研究提供了借鉴和参考。

羊血红蛋白水解物中新型抗氧化肽的纯化、鉴定和对H_(2)O_(2)损伤Caco-2细胞保护作用

以新鲜羊血为原料制备羊血红蛋白抗氧化肽,采用葡萄糖凝胶G-25色谱、DEAE葡萄糖凝胶A-50阴离子交换色谱、反相高效液相色谱和液相色谱-串联质谱对羊血红蛋白水解物进行分离纯化和鉴定,通过研究自由基清除能力、模拟胃肠消化后的稳定性及对H2O2诱导的Caco-2细胞保护作用评价羊血红蛋白肽的抗氧化活性。结果表明,从羊血红蛋白粗肽中纯化并鉴定出Ala-Tyr-Glu-Val-Asp(AYEVD)、Phe-His-Thr-Met-Glu(FHTME)、Ser-PheMet-Tyr-Glu-Lys(SFMYEK)3种新型抗氧化活性肽,分子质量分别为595.60、663.74、803.92 Da;其中AYEVD的1,1-二苯基-2-三硝基苯肼自由基清除能力最强;AYEVD在模拟胃肠道消化后显示出更强的抗氧化活性。此外,AYEVD还能抑制H2O2诱导Caco-2细胞的氧化损伤,显著减少活性氧积累、抑制早期细胞凋亡和丙二醛的形成,并提高细胞内抗氧化酶活性。该研究可为羊血红蛋白肽作为新型抗氧化剂应用于功能食品提供理论依据。

清肠温中方含药血清调控NLRP6途径对Caco-2/HT29-MTX细胞共培养模型MUC2表达的影响

目的:研究清肠温中方含药血清调控NLRP6途径影响Caco-2/HT29-MTX细胞共培养模型MUC2分泌的作用机制。方法:使用SD大鼠制备清肠温中方含药血清;Caco-2/HT29-MTX细胞共培养构建单层肠上皮细胞模型;IL-1β刺激共培养细胞模拟体外肠道炎症模型;si-NLRP6质粒转染干扰NLRP6,研究清肠温中方作用机制。qPCR检测NLRP6、IL-18的mRNA表达水平;Western blot检测NLRP6、IL-18蛋白表达水平;免疫荧光染色观察MUC2表达。结果:正常培养的Caco-2/HT29-MTX细胞中,NLRP6敲除后,MUC2表达明显减少。清肠温中方含药血清增加IL-1β诱导的细胞炎症模型中NLRP6和IL-18 mRNA及蛋白表达水平(P<0.05,P<0.01),且一定程度上抵消si-NLRP6的抑制作用。与模型组相比,清肠温中方含药血清干预后,促进MUC2蛋白表达恢复,si-NLRP6后一定程度上削弱了清肠温中方的作用。结论:清肠温中方含药血清通过调控NLRP6信号途径,进一步调节MUC2分泌,修复UC黏液屏障损伤。

天冬氨酸和谷氨酸及其甘氨酸二肽调控Caco-2细胞钙离子吸收的研究

为了探究天冬氨酸和谷氨酸及其甘氨酸二肽对钙离子吸收的影响机理,本文利用等温滴定量热技术、电化学、Caco-2细胞模型结合量子化学计算(密度泛函理论)对天冬氨酸和谷氨酸及其甘氨酸二肽与钙离子的相互作用进行了探究。结果表明,天冬氨酸和谷氨酸通过焓和熵驱动的放热反应与钙离子形成复合物,而其形成的二肽则是通过熵驱动的吸热反应与钙离子结合。此外,二肽比单独的氨基酸表现出更强的钙离子结合能力。量子化学计算表明,氨基酸/二肽中的羧基为钙离子的主要结合位点,在碱性条件下,会导致钙离子结合位点转移,氨基也可以参与钙离子结合。Caco-2细胞吸收实验表明,天冬氨酸和谷氨酸及其二肽均可促进钙的吸收,其中甘氨酸-谷氨酸二肽(Gly-Glu)的促进效果最好,促钙吸收率为1.33±0.115。此外,促钙吸收能力与钙离子结合能力之间没有明确的相关性,与其电子亲和力的关系更为密切。研究结果解释了天冬氨酸、谷氨酸及其甘氨酸二肽与钙之间的相互作用,为进一步开发钙补充剂提供科学依据。