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 relation...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 FI value 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 A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell 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.展开更多
A high sensitive method based on liquid chromatography tandem mass spectrometry（LC-MS/MS） was developed and validated for the study of permeability of danshensu（DS） and paeoniflorin（PF） in Caco-2 intestinal abso...A high sensitive method based on liquid chromatography tandem mass spectrometry（LC-MS/MS） was developed and validated for the study of permeability of danshensu（DS） and paeoniflorin（PF） in Caco-2 intestinal absorption model. The DS and PF were extracted from cell culture by vacuum-lyophilizing and then separated on a Zorbax Stable Bond C18 column with 0.1% acetic acid aqueous solution and methanol as mobile phase. Detection was carried out by negative electrospray ionization（ESI ） with selected reaction monitoring（SRM） mode. The apparent permeability coefficients（Papp） of DS and PF in Caco-2 cell medium were calculated and the effects of verapamil on the coefficients Papp of the two test compounds were also illustrated. The permeability of PF was much better than that of DS when the two compounds were administrated individually. Co-administration of DS and PF led to the decrease of the transport from apical side to basolateral side for both the compounds. However, the transport in the contrary direction were accelerated. It was also observed that verapamil could accelerate the transport of the test compounds from apical side to basolateral side. However, the absorption-enhanced effect of verapamil was attenuated when DS and PF were co-administrated. These observations suggest that both passive diffusion and active efflux involved in P-gp would effect the passage of DS and PF across Caco-2 cell monolayer. At the same time, the co-administration of DS and PF to an alteration of transport behavior, which suggests that the interaction must be taken into account when ‘n-in-one＇ samples were used in Caco-2 intestinal model.展开更多
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： Bot...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 too. 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.展开更多
AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The tran...AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations (】 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION :Transepithelial transport of putrescine across Caco2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.展开更多
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 b...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.展开更多
AIM： To understand the digestive stability and mechanism of release and intestinal uptake of pea ferritin iron in caco-2 cell line model.METHODS： Pea seed ferritin was purified using salt fractionation followed by g...AIM： To understand the digestive stability and mechanism of release and intestinal uptake of pea ferritin iron in caco-2 cell line model.METHODS： Pea seed ferritin was purified using salt fractionation followed by gel filtration chromatography.The bioavailability of ferritin iron was assessed using coupled in vitro digestion/Caco-2 cell model in the presence or absence of ascorbic acid and phytic acid.Caco-2 cell ferritin formation was used as a surrogate marker of iron uptake. Structural changes of pea ferritin under simulated gastric pH were characterized using electrophoresis, gel filtration and circular dichroism spectroscopy.RESULTS： The caco-2 cell ferritin formation was significantly increased （P 〈 0.001） with FeSO4 （19.3±9.8 ng/mg protein） and pea ferritin （13.9 ± 6.19 ng/mg protein） compared to the blank digest （3.7 ± 1.8 ng/mg protein）. Ascorbic acid enhanced while phytic acid decreased the pea ferritin iron bioavailability. However,either in the presence or absence of ascorbic acid, the ferritin content of caco-2 cells was significantly less with pea ferritin than with FeSO4. At gastric pH, no band corresponding to ferritin was observed in the presence of pepsin either on native PAGE or SDS-PAGE. Gel filtration chromatography and circular dichroism spectroscopy revealed a pH dependent loss of quaternary and secondary structure.CONCLUSION： Under gastric conditions, the iron core of pea ferritin is released into the digestive medium due to acid induced structural alterations and dissociation of protein. The released iron interacts with dietary factors leading to modulation of pea ferritin iron bioavailability,resembling the typical characteristics of non-heme iron.展开更多
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...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.展开更多
Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATC...Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATCCj cell line.Methods:Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization.Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR.Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out.In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line.Reverse transcription — PCR was carried out to detect level of expression of p53 in Caco-2 cell line.Results:HF.I displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99%and 97%respectively.The multiple sequence alignment of the two fungal isolates showed that,the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of Slst to 399th base pairs,88th to 525th base pairs respectively.While those in the ITS genes were identified with the nucleotide regions of 88th to 463rd and Slst to 274th.The two isolates showed IC<sup><</sup>sub>50</sub> value with（6.24±5.21） and（9.84±0.36） μ g/mL) concentrations respectively at 28h.Reverse transcription- PCR indicated that these cells showed high level of expression for p53 mRNA.Conclusions:The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans;new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt.Treatment with the two isolates caused P53 expression in Caco-2 cell line.These two isolates can be used as an anticancer agents.展开更多
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 inv...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.展开更多
Objective This study was conducted to examine the absorption and translocation of conjugated bile acids(BAs)in Calculus bovis and its substitutes to detect differences in these materials.Methods A Caco-2 monolayer cel...Objective This study was conducted to examine the absorption and translocation of conjugated bile acids(BAs)in Calculus bovis and its substitutes to detect differences in these materials.Methods A Caco-2 monolayer cell model was used to compare the apparent permeability coefficient(Papp)value and efflux ratio(ER)of BAs in natural cow-bezoar(NCB),artificial cow-bezoar(ACB),and in vitro cultured cow-bezoar(Ivt-CCB).Papp and ER values were determined by liquid chromatography-mass spectrometry.Samples were separated on an analytical column.Results The distribution of BAs in NCB was significantly different from that in ACB and Ivt-CCB.The percentages of conjugated BAs were significantly higher in NCB than in the two substitutes.The distribution differences of conjugated and unconjugated BAs can be used to distinguish costly NCB from relatively inexpensive substitutes.Conclusion The transport characteristics of BAs in Ivt-CCB were more consistent with NCB than with ACB,even when the proportions of BAs in Ivt-CCB were closer to those of ACB.展开更多
Based on the high-energy-resolution fluorescence spectrometer on the BL14W1 beamline at Shanghai Synchrotron Radiation Facility,an in-situ high-energyresolution X-ray absorption spectroscopy technique,with an in-situ ...Based on the high-energy-resolution fluorescence spectrometer on the BL14W1 beamline at Shanghai Synchrotron Radiation Facility,an in-situ high-energyresolution X-ray absorption spectroscopy technique,with an in-situ heating cell,was developed.The high-energyresolution fluorescence detection for X-ray absorption near-edge spectroscopy(HERFD-XANES) was tested in a UO_2 oxidation experiment to measure the UL_3-edge,with higher signal-to-noise ratio and higher-energy-resolution than conventional XANES.The technique has potential application for in-situ study of uranium-based materials.展开更多
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]bl...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.展开更多
AIM： To investigate the possible involvement of 25-hydroxyvitamin D3-1cx-hydroxylase [1α-25（OH）2D3] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells. METHODS： Caco-2 cells were incub...AIM： To investigate the possible involvement of 25-hydroxyvitamin D3-1cx-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 umol/L 25（OH）2D3 or with 1 umol/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 provide 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 forthe treatment of colon cancer.展开更多
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 tw...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.展开更多
基金Supported by the Natural Science Foundation of Hebei Province,No. 303158 Education Department Foundation of Hebei Province,No. 2002136
文摘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 FI value 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 A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell 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.
基金the National Basic Research Priorities Program of China(No. 2005CB523402)the Natural Science Foundation of Zhejiang Province,China(No.Y204418)the Program for New Century Excellent Talents in University of China (No.NCET-06-0515)
文摘A high sensitive method based on liquid chromatography tandem mass spectrometry（LC-MS/MS） was developed and validated for the study of permeability of danshensu（DS） and paeoniflorin（PF） in Caco-2 intestinal absorption model. The DS and PF were extracted from cell culture by vacuum-lyophilizing and then separated on a Zorbax Stable Bond C18 column with 0.1% acetic acid aqueous solution and methanol as mobile phase. Detection was carried out by negative electrospray ionization（ESI ） with selected reaction monitoring（SRM） mode. The apparent permeability coefficients（Papp） of DS and PF in Caco-2 cell medium were calculated and the effects of verapamil on the coefficients Papp of the two test compounds were also illustrated. The permeability of PF was much better than that of DS when the two compounds were administrated individually. Co-administration of DS and PF led to the decrease of the transport from apical side to basolateral side for both the compounds. However, the transport in the contrary direction were accelerated. It was also observed that verapamil could accelerate the transport of the test compounds from apical side to basolateral side. However, the absorption-enhanced effect of verapamil was attenuated when DS and PF were co-administrated. These observations suggest that both passive diffusion and active efflux involved in P-gp would effect the passage of DS and PF across Caco-2 cell monolayer. At the same time, the co-administration of DS and PF to an alteration of transport behavior, which suggests that the interaction must be taken into account when ‘n-in-one＇ samples were used in Caco-2 intestinal model.
基金Supported by Fundación Séneca,0578/PI/07,Consejería de Educación, Ciencia a Investigación de la Comunidad Autónoma de la Región de Murcia,CONSOLIDER FUN-C-FOOD.Nuevos ingredientes funcionales para mejorar la salud
文摘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 too. 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.
文摘AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations (】 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION :Transepithelial transport of putrescine across Caco2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.
基金funded by the HarvestPlus China(#8231)Xihua University programs(R0910507)the Key Laboratory of Food Biotechnology,Xihua University
文摘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.
基金Supported by a grant No. BT/PR6728/AGR/02/334/2005 from the Department of Biotechnology, Government of India to KMN and RP, SB is supported by a Research Fellowship from the Indian Council of Medical Research
文摘AIM： To understand the digestive stability and mechanism of release and intestinal uptake of pea ferritin iron in caco-2 cell line model.METHODS： Pea seed ferritin was purified using salt fractionation followed by gel filtration chromatography.The bioavailability of ferritin iron was assessed using coupled in vitro digestion/Caco-2 cell model in the presence or absence of ascorbic acid and phytic acid.Caco-2 cell ferritin formation was used as a surrogate marker of iron uptake. Structural changes of pea ferritin under simulated gastric pH were characterized using electrophoresis, gel filtration and circular dichroism spectroscopy.RESULTS： The caco-2 cell ferritin formation was significantly increased （P 〈 0.001） with FeSO4 （19.3±9.8 ng/mg protein） and pea ferritin （13.9 ± 6.19 ng/mg protein） compared to the blank digest （3.7 ± 1.8 ng/mg protein）. Ascorbic acid enhanced while phytic acid decreased the pea ferritin iron bioavailability. However,either in the presence or absence of ascorbic acid, the ferritin content of caco-2 cells was significantly less with pea ferritin than with FeSO4. At gastric pH, no band corresponding to ferritin was observed in the presence of pepsin either on native PAGE or SDS-PAGE. Gel filtration chromatography and circular dichroism spectroscopy revealed a pH dependent loss of quaternary and secondary structure.CONCLUSION： Under gastric conditions, the iron core of pea ferritin is released into the digestive medium due to acid induced structural alterations and dissociation of protein. The released iron interacts with dietary factors leading to modulation of pea ferritin iron bioavailability,resembling the typical characteristics of non-heme iron.
基金Shanghai Natural Science Foundation(20ZR1455800)the National Science Foundation of China(31871805)+1 种基金China Agriculture Research System(CARS-08-D2)Shanghai Municipal Education Commission(Plateau Discipline Construction Program)。
文摘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.
文摘Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATCCj cell line.Methods:Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization.Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR.Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out.In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line.Reverse transcription — PCR was carried out to detect level of expression of p53 in Caco-2 cell line.Results:HF.I displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99%and 97%respectively.The multiple sequence alignment of the two fungal isolates showed that,the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of Slst to 399th base pairs,88th to 525th base pairs respectively.While those in the ITS genes were identified with the nucleotide regions of 88th to 463rd and Slst to 274th.The two isolates showed IC<sup><</sup>sub>50</sub> value with（6.24±5.21） and（9.84±0.36） μ g/mL) concentrations respectively at 28h.Reverse transcription- PCR indicated that these cells showed high level of expression for p53 mRNA.Conclusions:The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans;new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt.Treatment with the two isolates caused P53 expression in Caco-2 cell line.These two isolates can be used as an anticancer agents.
文摘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.
基金support from the Youth Development Research Foundation of NIFDC(2014A1)National Science and Technology Major Project(2017ZX09101002-002-008)the National Natural Science Foundation of China(No.81403171 and No.81603517)
文摘Objective This study was conducted to examine the absorption and translocation of conjugated bile acids(BAs)in Calculus bovis and its substitutes to detect differences in these materials.Methods A Caco-2 monolayer cell model was used to compare the apparent permeability coefficient(Papp)value and efflux ratio(ER)of BAs in natural cow-bezoar(NCB),artificial cow-bezoar(ACB),and in vitro cultured cow-bezoar(Ivt-CCB).Papp and ER values were determined by liquid chromatography-mass spectrometry.Samples were separated on an analytical column.Results The distribution of BAs in NCB was significantly different from that in ACB and Ivt-CCB.The percentages of conjugated BAs were significantly higher in NCB than in the two substitutes.The distribution differences of conjugated and unconjugated BAs can be used to distinguish costly NCB from relatively inexpensive substitutes.Conclusion The transport characteristics of BAs in Ivt-CCB were more consistent with NCB than with ACB,even when the proportions of BAs in Ivt-CCB were closer to those of ACB.
基金supported by the National Nature Science Foundation of China(Nos.11175244 and U1532259)
文摘Based on the high-energy-resolution fluorescence spectrometer on the BL14W1 beamline at Shanghai Synchrotron Radiation Facility,an in-situ high-energyresolution X-ray absorption spectroscopy technique,with an in-situ heating cell,was developed.The high-energyresolution fluorescence detection for X-ray absorption near-edge spectroscopy(HERFD-XANES) was tested in a UO_2 oxidation experiment to measure the UL_3-edge,with higher signal-to-noise ratio and higher-energy-resolution than conventional XANES.The technique has potential application for in-situ study of uranium-based materials.
文摘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.
基金Supported by the Else Kroner-Fresenius Foundation, Bad Homburg, Germany
文摘AIM： To investigate the possible involvement of 25-hydroxyvitamin D3-1cx-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 umol/L 25（OH）2D3 or with 1 umol/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 provide 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 forthe treatment of colon cancer.
文摘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.
