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 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.

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 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.

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.

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

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.

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.

Quantitative Comparison of Bile Acid Distribution and Intestinal Transport from Native Cow-bezoar and Artificial and in vitro Cultured Substitutes using Caco-2 Cell Monolayer Model

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

The combination of sitagliptin and bee honey extract potentiates the anti-proliferative properties of 5-fluorouracil on Caco-2 cell line without detectable inflammatory events

Background:Colon cancer originates in the colon,specifically the large intestine.It carries a poor prognosis and high mortality rate due to late diagnosis and migration.Objective:Here our objective was to evaluate the anticancer effects of sitagliptin(Sita)in colon cancer using Caco-2 cells.Additionally,we examined the role of bee honey extract in modulating cancer cell division and necrotic events commonly observed during drug treatments.Methods:We monitored cell viability rate to evaluate the effect of bee honey extract compared to 5-fluorouracil(5-Fu),Sita,and their combinations.To gain further foresights into the implicated molecular interaction,we assessed the expression outline of Raf-1 and MEK,as proliferation effectors.Additionally,we examined the expression outline of p53 and Caspase 3,which are associated with programmed cell death(PCD),through western blot analysis.Results:We identified the Raf-1 expression pattern as a likely target for the drug combination and bee honey extract(HE),which effectively controlled colon cancer cell proliferation.Our study demonstrates that honey extract,either alone or in combination with drugs,can induce PCD by restoring the p53 and CASP-3 proteins.This was accompanied by a synergistic effect on the production of apoptotic cytokines,particularly interlukine-6(IL-6)and IL-8,in cancer cells.Moreover,the treatment modulated the levels of pro-inflammatory cytokines,including IL-1𝛼and IL-1𝛽and anti-inflammatory cytokines,including IL-4 and IL-10.Conclusion:Our findings shed light on honey extract and its combinations with 5-Fu and Sita in stimulating PCD and modulating cytokine production in Caco-2 cells.

红毛藻多糖抑制大肠杆菌黏附Caco-2细胞的机制

基于体外人结肠腺癌细胞(Caco-2)单层模型,研究红毛藻多糖(Bangia fusco-purpurea polysaccharide,BFP)对大肠杆菌(Escherichia coli)黏附Caco-2细胞的影响以及潜在作用机制。采用CFDA-SE荧光标记技术分析BFP对E.coli黏附Caco-2细胞单层的影响;利用实时聚合酶链式反应分析BFP对Caco-2细胞表面整合素β1和E.coli黏附素fimH及E.coli黏附Caco-2细胞导致细胞产生的炎症因子(白细胞介素(interleukin,IL)-1β、IL-8、肿瘤坏死因子(tumor necrosis factor,TNF)-α)和细胞紧密连接蛋白(闭锁小带蛋白-1(zonula occludens-1,ZO-1)与Occludin)的基因表达情况,最终基于Western Blot分析验证ZO-1、Occludin蛋白表达。结果表明,BFP在质量浓度为400~800μg/mL时能够显著抑制E.coli黏附Caco-2细胞单层;BFP主要通过下调Caco-2细胞表面整合素β1和E.coli黏附素基因fimH的表达抑制细菌的黏附。此外,BFP可显著抑制E.coli及其培养上清液诱导Caco-2细胞产生的炎症细胞因子基因表达的上调、紧密连接蛋白(ZO-1与Occludin)及其基因表达的下调。综上所述,BFP能够抑制E.coli对Caco-2细胞单层的黏附,实验结果可为其开发新型的抗菌产品以及BFP的高值利用和精深加工提供参考。

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。