Human Colonic Cell Line Caco-2 Research Articles

Genomic characteristics and virulence of common but overlooked Yersinia intermedia, Y. frederiksenii, and Y. kristensenii in food.

Yersinia intermedia, Y. frederiksenii, and Y. kristensenii are a group of pathogens that are commonly found in food and are often overlooked in terms of their pathogenic potential. This study conducted a systematic and comprehensive genomic analysis of 114 Y. intermedia genomes, 20 Y. frederiksenii genomes, and 65 Y. kristensenii genomes from public database and our previous study. The results showed that these species were most frequently detected in Europe (56.28%, 112/199), followed by in Asia (20.6%, 41/199). Additionally, 33.17% (66/199) genomes were isolated from food. Y. intermedia were grouped into Bayesian analysis of population structure (Baps) groups 3 and 4, demonstrating significant genomic diversity. This species has a high proportion of accessory genes (79.43%), approximately 50% of which have unknown functions, indicating a high degree of genomic plasticity. The three species carried a large number of mobile genetic elements (MGEs), including plasmids such as ColRNAI_1, ColE10_1, Col440II_1, Col440I_1, and Col (Ye4449) _1; insertion sequences (ISs) like MITEYpe1, MITEEc1, and IS1635; genomic islands (GIs); and prophages. In Y. intermedia, the following antibiotics resistance genes (ARGs) were detected: qnrD1 in 3.51% (4/114), aph(3')-Ia in 2.63% (3/114), blaA in 1.75% (2/114), and catA1, vat(F), and tet(C) each in 0.88% (1/114). In Y. kristensenii, vat(F) was present in 98.46% (64/65), blaTEM-116 in 7.69% (5/65), and aph(3')-Ia in 1.54% (1/65). However, only one Y. frederiksenii genome carried vat(F). There were differences in the virulence gene composition of the three species, with Y. kristensenii having the highest number of virulence genes, particularly its complete cytotoxic genes (yaxA and yaxB) and flagellar motor proteins genes (motA and motB). The pathogenic mechanisms of Y. intermedia and Y. frederiksenii were more similar, especially in the carriage of O-antigen related genes. Y. frederiksenii's unique mechanisms also include the yapC gene, which encodes the autotransporter protein YapC from Y. pestis. After co-cultured with human colonic epithelial cell lines Caco-2 and HT-29, Y. intermedia and Y. kristensenii demonstrated different adhesive and invasive capabilities, particularly the Y. intermedia strain y7, which exhibited stronger adhesion and invasion in both cell lines. In strains y118 and y119 of Y. intermedia, an Arg378del mutation in the UreC protein was identified, resulting in the loss of urease activity. Therefore, this study revealed the pathogenic potential of Y. intermedia, Y. frederiksenii, and Y. kristensenii. Future research should focus on identifying their unknown virulence genes and strengthening public food safety measures to mitigate potential risks.

Transport of perfluoroalkyl substances across human induced pluripotent stem cell-derived intestinal epithelial cells in comparison with primary human intestinal epithelial cells and Caco-2 cells

Humans can be exposed to per- and polyfluoroalkyl substances (PFASs) via many exposure routes, including diet, which may lead to several adverse health effects. So far, little is known about PFAS transport across the human intestinal barrier. In the current study, we aimed to assess the transport of 5 PFASs (PFOS, PFOA, PFNA, PFHxS and HFPO-DA) in a human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cell (IEC) model. This model was extensively characterized and compared with the widely applied human colonic adenocarcinoma cell line Caco-2 and a human primary IEC-based model, described to most closely resemble in vivo tissue. The hiPSC-derived IEC layers demonstrated polarized monolayers with tight junctions and a mucus layer. The monolayers consisted of enterocytes, stem cells, goblet cells, enteroendocrine cells, and Paneth cells that are also present in native tissue. Transcriptomics analysis revealed distinct differences in gene expression profiles, where the hiPSC-derived IECs showed the highest expression of intestinal tissue-specific genes relative to the primary IEC-based model and the Caco-2 cells clustered closer to the primary IEC-based model than the hiPSC-derived IECs. The order of PFAS transport was largely similar between the models and the apparent permeability (Papp) values of PFAS in apical to basolateral direction in the hiPSC-derived IEC model were in the following order: PFHxS > PFOA > HFPO-DA > PFNA > PFOS. In conclusion, the hiPSC-derived IEC model highly resembles human intestinal physiology and is therefore a promising novel in vitro model to study transport of chemicals across the intestinal barrier for risk assessment of chemicals.

A perfusion host-microbe bioreactor (HMB) system that captures dynamic interactions of secreted metabolites between epithelial cells cocultured with a human gut anaerobe.

The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes.

Treatment with S-adenosylmethionine ameliorates irinotecan-induced intestinal barrier dysfunction and intestinal microbial disorder in mice

This study aimed to investigate the protective effects of S-adenosylmethionine (SAM) on irinotecan-induced intestinal barrier dysfunction and microbial ecological dysregulation in both mice and human colon cell line Caco-2, which is widely used for studying intestinal epithelial barrier function. Specifically, this study utilized Caco-2 monolayers incubated with 7-ethyl-10-hydroxycamptothecin (SN-38) as well as an irinotecan-induced diarrhea model in mice.Our study found that SAM pretreatment significantly reduced body weight loss and diarrhea induced by irinotecan in mice. Furthermore, SAM inhibited the increase of intestinal permeability in irinotecan-treated mice and ameliorated the decrease of Zonula occludens-1(ZO-1), Occludin, and Claudin-1 expression. Additionally, irinotecan treatment increased the relative abundance of Proteobacteria compared to the control group, an effect that was reversed by SAM administration. In Caco-2 monolayers, SAM reduced the expression of reactive oxygen species (ROS) and ameliorated the decrease in transepithelial electrical resistance (TER) and increase in fluorescein isothiocyanate–dextran 4000 Da (FD-4) flux caused by SN-38. Moreover, SAM attenuated changes in the localization and distribution of ZO-1and Occludin in Caco-2 monolayers induced by SN-38 and protected barrier function by inhibiting activation of the p38 MAPK/p65 NF-κB/MLCK/MLC signaling pathway. These findings provide preliminary evidence for the potential use of SAM in treating diarrhea caused by irinotecan.

Bile Acid Derivatives Effectively Prevented High-Fat Diet-Induced Colonic Barrier Dysfunction.

Bile acids (BAs) have recently emerged as important regulators of many physiological and pathological processes. However, the change of colonic BAs induced by high-fat diet (HFD) and their effects on colonic barrier function remain to be further elucidated. C57BL/6 mice are divided into two groups and feed 12weeks with diets differing for fat content. Higher levels of serum diamine oxidase (DAO) activity, endotoxin (ET), and d-lactate (d-LA) are observed in HFD-fed mice, indicating an increase in intestinal permeability. Real-time quantitative PCR and western blot analyses demonstrate that HFD downregulates tight junction proteins (TJs, including zonula-occludens 1 [ZO-1], Occludin, and Claudin1) and Muc2 expression in the colon. The colonic BA profiles are analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). HFD induces an increase in primary BAs but a decrease in secondary BAs. In human colonic cell line Caco-2, secondary BAs (deoxycholic acid [DCA], lithocholic acid [LCA], their 3-oxo- and iso- derivates) upregulate the expression of TJs and counteract DSS-induced increase in intestinal permeability at physiological concentrations. IsoDCA and isoLCA are the most effective ones. Moreover, supplementation of isoDCA or isoLCA also effectively prevents HFD-induced colonic barrier dysfunction in mice. These results demonstrate that secondary BAs (especially isomerized derivatives) may be important protectors for the colonic barrier function.

Transport mechanism of hydroxy-propyl-beta-cyclodextrin modified solid lipid nanoparticles across human epithelial cells for the oral absorption of antileishmanial drugs

BackgroundIn this study, the transport mechanism of fluorescently labeled hydroxypropyl beta-cyclodextrin (HPβ-CD) modified SLNs loaded with Amphotericin B (AmB) and Paromomycin (PM) have been investigated by using in vitro human epithelial cell model of a human colonic adenocarcinoma cell line (Caco-2). MethodsFabrication of HPβ-CD modified fluorescently labeled AmB and PM-loaded SLNs (HPꞵ-CD-FITC-DDSLNs) was performed by using the emulsion solvent evaporation method. Caco-2 cells were used to investigate different endocytosis and exocytosis pathways to be followed by the nanoparticles. Intracellular co-localization of nanoformulation with different organelles was investigated. ResultsThe toxicity studies have shown the biocompatible nature of the modified lipid nanoparticles. The average particle size and PDI of HPꞵ-CD-FITC-DDSLNs were found to be 187 ± 2.3 nm and 0.31 respectively. The most prevalent endocytosis mechanisms were shown to be macropinocytosis and caveolae (lipid raft) dependent pathways. The Golgi complex and endoplasmic reticulum are the confirmed destinations of HPꞵ-CD-FITC-DDSLNs in the Caco-2 cell monolayer, even though lysosomes have been shown to escape and play a minimal role during nanoparticle transport. ConclusionHPꞵ-CD-DDSLNs were found to be biocompatible and safe for delivering hydrophobic as well as hydrophilic drugs through an oral route to target the RES system for the treatment of visceral leishmania. General significanceUnderstanding the process underlying the transport of modified solid lipid nanoparticles for oral drug delivery could be useful for many medicines with low solubility, permeability, and stability.

Polyphenol Content of Green Pea (Pisum sativum L.) Hull under In Vitro Digestion and Effects of Digestive Products on Anti-Inflammatory Activity and Intestinal Barrier in the Caco-2/Raw264.7 Coculture Model.

Green pea hulls are a byproduct of the processing of green pea and are rich in phenolic substances. In the present study, in vitro digestion, human colonic adenocarcinoma cell line (Caco-2) monolayer, and the Caco-2/macrophage cell lines of the murine origin (Raw264.7) coculture model were established to investigate the release of polyphenols, absorption, and transport of digestive products and their effects on inflammation and intestinal barrier. During the digestive process, polyphenols were constantly released from the pea hulls, reaching the maximum amount in the small intestine (total phenolic content (TPC): 5.41 ± 0.04 mg gallic acid (GAE)/g dry weight (DW)), and the digestive products (800 μg/mL) could reduce the secretion of NO (50.9%), IL-6 (50.6%), and TNF-α (24.6%) and inhibit the mRNA expression of cyclooxygenase-2 (COX-2) (37.2%) and inducible nitric oxide synthase (iNOS) (91.1%) compared with the lipopolysaccharide (LPS) group. A total of 12 phenolic components were quantified by ultraperformance liquid chromatography-linear ion trap orbitrap tandem mass spectrometry (UHPLC-LTQ-OrbiTrap-MS) technology. Kaempferol trihexoside in digestive products could be absorbed and transported (1.25 ± 0.13 ng quercetin/mL). The digestive products could promote the expression of claudin-1 (210.8%), occludin (64.9%), and zonulin occludin-1 (ZO-1) (52.0%) compared with the LPS group and exert anti-inflammatory effects after being absorbed. The results indicated that pea hull polyphenols could be continuously released and absorbed to play a positive role in protecting the intestinal barrier and anti-inflammatory activity.

Effects of norepinephrine on colonic tight junction protein expression during heat stress.

Stress induced by changes in the internal or external environment in humans and animals leads to intestinal epithelial damage, in a manner that is associated with impaired intestinal barrier function. However, the role of the stress hormone norepinephrine (NE) in impairments in barrier function remains poorly understood. In the present study, a rat heat-exposed model was used to observe changes in the tight junction proteins Occludin and zonula occludens-1 (ZO-1), in addition to those in protease-activated receptor 2 (PAR-2) and transient receptor potential ankyrin 1 channel (TRPA1) in colon. The levels of plasma NE were detected using an ELISA kit. Different concentrations of NE were used to culture the human colon cell line Caco-2 for 6 and 24 h to investigate the cell viability using Cell Counting Kit-8 assay, whilst the expression levels of Occludin, ZO-1, PAR-2 and TRPA1 were examined using western blotting and immunofluorescence in Caco-2 cells and immunohistrochemistry in rat colon tissues. Although there was no clear histological damage to the rat colonic mucosa, there were decreased expression levels of tight junction proteins Occludin and ZO-1 after heat exposure. In addition, PAR-2 expression was increased by heat exposure. It was found that TRPA1 expression was concentrated to the luminal surface of the colon in the heat exposed group compared with that in the control group. After the administration of increasing concentrations of NE for 6 h, treatment did not affect cell viability. Furthermore, after application of NE for 24 h, cell viability gradually increased as the NE concentration was elevated from 10 to 100 µM. However, no significant increase in viability was observed when the cells were treated with 120 and 160 µM NE. Occludin expression was decreased when 10 µM NE was applied for 6 or 24 h. By contrast, 60 µM NE significantly downregulated Occludin expression in the 6 h group, but caused an insignificant decrease in the 24 h group. It was found that ZO-1 expression was upregulated after treatment with 10 µM NE for 6 h, whilst downregulation was observed after treatment with 10 µM NE for 24 h. PAR-2 protein expression was increased after application of NE for both 6 and 24 h, but not after treatment with 60 µM NE. In addition, TRPA1 expression was not affected by the treatment of NE, but increased positive staining was observed on the luminal side of the mucosa, which appeared to be concentrated in the cells of the luminal side in the rat colon after heat exposure. Collectively, the present results suggested that expression of tight junction proteins Occludin and ZO-1, in addition to that of PAR-2, can be regulated by NE, which may contribute to impairments in barrier function observed during heat stress.

Piezo1 regulates intestinal epithelial function by affecting the tight junction protein claudin-1 via the ROCK pathway

AimsDefective tight junctions (TJs) can induce intestinal epithelial dysfunction, which participates in various diseases such as irritable bowel syndrome. However, the mechanisms of TJ defects remain unclear. Our study revealed the role of Piezo1 in regulating intestinal epithelial function and TJs. Materials and methodsThe human colonic adenocarcinoma cell line Caco-2 were cultured on Transwell plate to form an epithelial barrier in vitro, and Piezo1 expression was manipulated using a lentivirus vector. Epithelial function was evaluated by measuring transepithelial electronic resistance (TEER) and 4-kDa FITC-dextran (FD4) transmission. TJ proteins (claudin-1, occludin, ZO-1) were evaluated by RT-PCR, western blot, and immunostaining analysis. Potential signal pathways, including the ROCK and Erk pathways, were detected. Moreover, to explore the regulatory effect of Piezo1 activity on epithelial function, inhibitors (ruthenium red, GsMTx4) and an agonist (Yoda1) were introduced both ex vivo and in vitro. Key findingsAlteration of Piezo1 expression altered epithelial function and the expression of the tight junction protein claudin-1. Piezo1 expression regulated phosphorylated ROCK1/2 expression, whereas interference on ROCK1/2 prevented the regulation of claudin-1 by Piezo1. In both Caco-2 monolayer and mouse colon epithelium, Piezo1 activity directly modulated epithelial function and permeability. SignificancePiezo1 negatively regulates epithelial barrier function by affecting the expression of claudin-1. Such regulation may be achieved partially via the ROCK1/2 pathway. Moreover, activating Piezo1 can induce epithelial dysfunction.

Cytochrome P450 expression, induction and activity in human induced pluripotent stem cell-derived intestinal organoids and comparison with primary human intestinal epithelial cells and Caco-2 cells

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by β-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.

Roles of N-terminal Annexin A2 phosphorylation sites and miR-206 in colonic adenocarcinoma

AimsAnnexin A2 (ANXA2) is closely associated with tumor malignancy and its N-terminus includes a vital domain for its function. The aims are to explore the correlation between the sites (Tyr23, Ser1, Ser11 and Ser25) in the domain and its roles. Main methodsWe re-expressed ANXA2 with mutated sites in ANXA2-deleted human colonic adenocarcinoma cell line caco2 (ANXA2-/-caco2). A series of analyses were used to determine the correlation of each site with ANXA2 activation, cell malignancy enhancement and motility-associated microstructural development. Bioinformatics and luciferase reporter assays were employed to validate ANXA2-targeted miRNAs. Key findingsThe in vitro results showed that all single and multiple mutations of the ANXA2 N-terminal sites inhibited ANXA2 phosphorylation at different levels and subsequently inhibited the proliferation, motility, and polymerization of F-actin and β-tubulin in caco2 cells. Motility-associated microstructures were significantly remodeled when these sites were mutated. The forced expression of miR-206 significantly suppressed the proliferation, motility and epithelial-mesenchymal transition (EMT) of caco2 cells. The in vivo results showed that all the ANXA2 N-terminal site mutations and forced expression of miR-206 significantly inhibited tumor growth. Overall, this study demonstrated that the sites of the ANXA2 N-terminus, especially Tyr23, play crucial roles in maintaining the high malignancy of colonic adenocarcinoma. Furthermore, miR-206 targets ANXA2 and plays a role as a cancer suppressor in colonic adenocarcinoma. SignificanceOur study provided evidence that further elucidates the molecular mechanism of ANXA2 and its roles in colonic adenocarcinoma and suggested potential targets of ANXA2 for colonic adenocarcinoma therapy by using miR-206 as a novel strategy.

Heat Shock Protein 27 Regulates the Inflammatory Response of Intestinal Epithelial Cells by the Nuclear Factor-κB Pathway.

The specific and accurate pathogenesis of diarrhea-type irritable bowel syndrome is still unclear. We explored the mechanism of heat shock protein 27 (HSP27) in diarrhea-type irritable bowel syndrome to identify the key targets for the disease. The human colonic epithelial cell lines Caco-2 and NCM460 were pretreated with KRIBB3 (a phosphorylation inhibitor of HSP27) and then stimulated with lipopolysaccharide for different times. The apoptosis ratios of Caco-2 and NCM460 cells were examined with Annexin V/PI assays. Cell growth was determined using the cell counting kit-8 assay, and the expression levels of IL-1β and IL-6 in the cell supernatant were analyzed by ELISA. In addition, the expression levels of HSP27 and the nuclear factor-κB (NF-κB) signaling pathway were examined by Western blot assay. Stimulation with lipopolysaccharide promoted the expression of HSP27 in colonic epithelial cells. HSP27 was phosphorylated at serine 78 and 82 after exposure to LPS. Apoptosis, growth inhibition, and inflammatory factor expression of lipopolysaccharide-induced colonic epithelial cells were greatly exacerbated by KRIBB3 treatment. In addition, KRIBB3 inhibited the phosphorylation of IκB-α and the activation of NF-κB. Gene silencing by small interfering RNA indicated that phosphorylation of HSP27 may regulate the NF-κB pathway. HSP27 plays an important role in the inflammatory response of intestinal human colonic epithelial cells. HSP27 may protect intestinal epithelial cells against damage by regulating the NF-κB pathway.

Effects of Hydrogenized Water on Intracellular Biomarkers for Antioxidants, Glucose Uptake, Insulin Signaling and SIRT 1 and Telomerase Activity

Hydrogen has been shown in several clinical trials to be completely safe without adverse events and there are no warnings in the literature of its toxicity or adverse effects during long-term exposure. Molecular hydrogen has proven useful and convenient as a novel antioxidant and modifier of gene expression in many conditions where oxidative stress and changes in gene expression result in cellular damage. Our intracellular biomarker studies have shown that a hydrogenized water drink formula containing 2.6 ppm dissolved hydrogen was able to penetrate cellular membranes and function as an antioxidant in human liver cells (HePG2) utilizing the Cellular Antioxidant Assay (CAA). This assay uses the protection of a florescent probe as a marker for cellular damage by reactive oxygen species (ROS), such as peroxyl radical, and compares this to the known antioxidant standard, Quercetin. Using this system oxidative damage was reduced in a dose-dependent manner. One ml of hydrogenized water was found to possess antioxidant capacity equivalent to 0.05 µmole of quercetin. When examined in a human colon cell line (Caco-2 cells), hydrogenized water demonstrated a dose- and time-dependent permeability inhibition of an intracellular fluorescent glucose derivative (2-NBDG), indicating decreased glucose uptake. In another study, the impact of hydrogenized water on Akt phosphorylation (Ser473), a biomarker for insulin signaling, was monitored in human skeletal muscle cells. The hydrogenized water treatment markedly elevated the level of phosphorylation of Akt (Ser473) in a dose-dependent manner. The anti-aging effects of hydrogenized water were examined utilizing SIRT1 expression as a biomarker of aging in human umbilical cells (HUVECs). Hydrogenized water increased dose-dependent SIRT1 gene expression. Hydrogenized water also increased telomerase activity (an anti-aging biomarker in HUVEC cells) up to 148% when cells were treated with media containing 25% hydrogenized water formula. Increased telomerase activity caused by hydrogenized water may be able to protect telomeres from degradation, suggesting the possible use of hydrogenized water in therapeutic interventions of age-related diseases. These studies show that commercial hydrogenized water improved the levels or activities of a few intracellular biomarkers specific for antioxidant activity, glucose uptake, insulin signaling and SIRT 1 and telomerase activities. Industrial Relevance: The molecular hydrogen used in this study indicates that certain commercial sources of hydrogenized water can provide similar antioxidant and gene expression modifications seen in other sources of molecular hydrogen. The biomarkers evaluated here lend well to hydrogenized water’s biological activity relating to health conditions and aging.

Evaluation of Caco-2 cells response to Listeria monocytogenes virulence factors by RT-PCR

Listeria monocytogenes expresses various virulence factors enabling the invasion and multiplying in host cells, and together induces cytokines transcription. In order to explore the relationship between virulence factors of L. monocytogenes wild-type EGD-e and cellular response in human colonic epithelial cell line(Caco-2), we constructed mutant strains with in-frame deletions of critical virulence genes of inlA, inlB, hly, actA and virulence regulatory factor prfA from EGD-e, respectively. Compared with EGD-e, mutant strains showed significantly decreased invasion and apoptosis in Caco-2 cells. However, mutant strains were capable to evoke cytokines transcription of interleukin-8 (IL-8), mononuclear chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β), interleukin-6 (IL-6) and CXCL-2 production in Caco-2 cells. Interestingly, EGD-e Δhly-infected Caco-2 cells showed a significant decrease of IL-6, IL-8 and MCP-1 transcription compared with EGD-e at 1 h post-infection. Simultaneously, EGD-e ΔinlB-infected cells showed a decrease in IL-6 transcription, while EGD-e ΔactA-infected cells reflected a decrease in MCP-1 transcription. Virulence genes play a role in inflammatory transcription, but the interaction between pathogenic bacteria and the host cells predominates in inflammatory transcription. Overall, the data showed cellular response of Caco-2 cells infected with EGD-e mutant strains.

Lactobacillus rhamnosus GG supernatant enhance neonatal resistance to systemic Escherichia coli K1 infection by accelerating development of intestinal defense

The objective of this study was to determine whether Lactobacillus rhamnosus GG culture supernatant (LCS) has a preventive effect against gut-derived systemic neonatal Escherichia coli (E. coli) K1 infection. The preventive effects were evaluated in human colonic carcinoma cell line Caco-2 and neonatal rat models. Our in vitro results showed that LCS could block adhesion, invasion and translocation of E. coli K1 to Caco-2 monolayer via up-regulating mucin production and maintaining intestinal integrity. In vivo experiments revealed that pre-treatment with LCS significantly decrease susceptibility of neonatal rats to oral E. coli K1 infection as reflected by reduced bacterial intestinal colonization, translocation, dissemination and systemic infections. Further, we found that LCS treated neonatal rats have higher intestinal expressions of Ki67, MUC2, ZO-1, IgA, mucin and lower barrier permeability than those in untreated rats. These results indicated that LCS could enhance neonatal resistance to systemic E. coli K1 infection via promoting maturation of neonatal intestinal defense. In conclusions, our findings suggested that LCS has a prophylactic effect against systemic E. coli K1 infection in neonates. Future studies aimed at identifying the specific active ingredients in LCS will be helpful in developing effective pharmacological strategies for preventing neonatal E. coli K1 infection.

Radioactive Permeability Studies of Doxycycline Hyclate from Microemulsion and Solution

Doxycycline Hyclate (DOX) is an antibacterial drug which is member of the second tetracycline group and the absorption of DOX is reduced about 20% in the presence of skimmed milk. Therefore new drug delivery system can be advisable for DOX to reduce the food and drug interaction and improve the bioavailability. The aim of the present study is to prepare a microemulsion system of DOX which could result in reducing in food interaction and an improvement of oral bioavailability by increasing the drug's permeability. For this purposes, DOX was radiolabeled with 99m Tc. Radiochemical purity was determined with radioactive thin layer chromatography (RTLC) studies. Permeability of DOX from 99m Tc-DOX solution ( 99m Tc-DOX-S) and 99m Tc-DOX loaded microemulsion ( 99m Tc-DOX-M) was investigated with in vitro cell culture studies by using human colonic adenocarcinoma cell line (Caco-2). The radioactivity of 99m Tc-DOX-M for the apical to basolateral direction (P app (A→B)) and basolateral to apical direction (P app (B→A)) were found higher than 99m Tc-DOX-S. Based on the in vitro cell culture studies, this dosage form is a promising formulation as an alternative for oral drug delivery of DOX.

α-Linolenic acid (ALA) is an anti-inflammatory agent in inflammatory bowel disease

Studies suggest that consumption of omega-3 (n-3) polyunsaturated fatty acids (PUFA) plays a protective role in inflammatory bowel disease; however, the use of plant-derived oils rich in α-linolenic acid (ALA) has not been widely investigated. The aims of this study were to test the effects of two different sources of (n-3) PUFA, fish and plant-derived oils, in two animal models of experimental colitis and to determine whether the (n-3) PUFA-enriched diets could ameliorate the inflammatory status. Rats were fed diets rich in corn, fish or sage oil with or without vitamin A supplementation for 3weeks then colitis was induced by adding dextran sodium sulfate to the drinking water or by injecting 2,4,6-trinitrobenzene sulfonic acid. We show that colitic rats fed the sage oil diets had a lower inflammatory response, improved histological repair and had less necrotic damage in the mucosa when compared to the corn and fish oil groups. Colonic damage and myeloperoxidase activity were significantly lower. Colonic mRNA levels of pro-inflammatory genes including interleukin IL-6, cyclooxygenase 2 and tumor necrosis factor α were markedly down-regulated in rats fed fish and sage oils compared to control. These results were supported by experiments in the human colonic epithelial cell line Caco-2, where ALA supplementation was shown to be effective in inhibiting inflammation induced by IL-1β by down-regulating mRNA levels of pro-inflammatory genes including IL-8, COX2 and inducible nitric oxide synthase. Taken together, these results suggest that plant-derived oil rich in ALA could ameliorate the inflammatory damage in colitis.

DNA damage and oxidative stress induced at low doses by the fungicide hexachlorobenzene in human intestinal Caco-2 cells

Context: Hexachlorobenzene (HCB), a persistent chlorinated organic chemical, could be detected in human tissues in several countries of the world. Human exposure to Persistent Organic Pollutants (POPs) occurring primarily through diet, HCB and its metabolites are therefore supposed to interact directly with intestinal mucosa.Objective: The aim of this study was to investigate the possible effects of low doses of HCB on DNA integrity, cellular viability, differentiation and oxidative status in vitro in human colonic carcinoma cell line Caco-2.Materials and methods: Cells were exposed to increasing doses of HCB for 14 days to assess the cytotoxic, genotoxic and oxidative properties of this compound. The involvement of oxidative stress in the observed effects was evaluated by co exposure of Caco-2 cells with HCB and α-tocopherol.Results: Exposure of Caco-2 cells to HCB resulted in a dose-dependent cytotoxicity, DNA damages and alterations of the cell layer integrity and the barrier function. Moreover, exposure of Caco-2 cells to HCB led to an enhancement of H2O2 production and to an increased activity of antioxidant enzymes. In addition, Co exposure of Caco-2 cells to HCB and α-tocopherol reversed the effects observed in cells exposed to HCB alone.Conclusion: These results suggested that HCB effects on Caco-2 cells could be linked, at least in part, to its pro-oxidative potential.

Multidrug resistance-associated protein 2 is involved in the efflux of Aconitum alkaloids determined by MRP2-MDCKII cells.

Aconitum alkaloids mainly contain highly toxic aconitine (AC), mesaconitine (MA), and hypaconitine (HA) and less toxic benzoylaconine (BAC), benzoylmesaconine (BMA), benzoylhypaconine (BHA), aconine, mesaconine, and hypaconine. The efflux transporters including P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) can efflux toxicants to prevent poisoning. Our previous publication has proved that P-gp and BCRP contributed to the efflux of AC, MA and HA, which is demonstrated in the human colonic adenocarcinoma cell lines (Caco-2 cells), Mardin-Darby canine kidney cell lines transfected with MDR1 or BCRP (MDR1-MDCKII and BCRP-MDCKII cells). However, the role of MRP2 remains uncertain. The MRP2-MDCKII cells were used to determine the efflux ratios (Er) and intracellular amounts of Aconitum alkaloids. In addition, the importance of MRP2 was further investigated with or without the MRP2 inhibitor, LTC4. The Er values of AC, MA, HA, BAC, BMA and BHA in MRP2-MDCKII cells (6.4 ± 0.3, 5.9 ± 0.5, 2.2 ± 0.2, 1.6 ± 0.3, 1.7 ± 0.2 and 1.9 ± 0.2 respectively) were significantly higher than those in MDCKII cells, which were close to 1. In the presence of LTC4, the Er values of AC, MA, HA, BAC, BMA and BHA were reduced to approximately 1 and their intracellular amounts were also significantly increased in MRP2-MDCKII cells. MRP2 was involved in the efflux of AC, MA, HA, BAC, BMA and BHA, which would be useful for the safe application of these components or their herbs.

Molecular mechanisms associated with PTHrP-induced proliferation of colon cancer cells.

Parathyroid Hormone-related Protein (PTHrP) is normally produced in many tissues and is recognized for its endocrine, paracrine, autocrine and intracrine modes of action. PTHrP is also implicated in different types of cancer and its expression correlates with the severity of colon carcinoma. Using the human colon cell line Caco-2 we recently obtained evidence that PTHrP, through a paracrine pathway, exerts a protective effect under apoptotic conditions. However, if exogenous PTHrP is able or not to induce the proliferation of these intestinal tumor cells is not known. We found that PTHrP treatment increases the number of live Caco-2 cells. The hormone induces the phosphorylation and nuclear translocation of ERK 1/2, α p38 MAPK, and Akt, without affecting JNK phosphorylation. In addition, PTHrP-dependent ERK phosphorylation is reverted when PI3K activity was inhibited. Following MAPKs nuclear translocation, the transcription factors ATF-1 and CREB were activated in a biphasic manner. In addition PTHrP induces the translocation into the nucleus of β-catenin, protein that plays key role in maintaining the growth and proliferation of colorectal cancer, and increases the amount of both positive cell cycle regulators c-Myc and Cyclin D. Studies with ERK1/2, α p38 MAPK, and PI3K specific inhibitors showed that PTHrP regulates Caco-2 cell proliferation via these signaling pathways. In conclusion, the results obtained in this work expand our knowledge on the role of exogenous PTHrP in intestinal tumor cells and identify the signaling pathways that are involved in the mitogenic effect of the hormone on Caco-2 cells.