Search

Introduction: Genistin, as a kind of natural isoflavone glycoside, has good biological activity, and its weak absorption makes it closely related to intestinal flora. However, the roleof theintestinal flora is still unclear andwhether themetabolites produced by the intestinal flora are absorbed systemically is also variable. Methods: Genistin was fermented for 24 h based on fecal bacteria fermentation technology. The components were qualitatively and quantitatively analyzed by HPLC and UHPLC-Q-Exactive Orbitrap Mass spectrometry. The composition of intestinal flora in fermentation samples from fecal bacteria was detected by 16S rRNA sequencing. Five representative probiotics were cultured in vitro and fermented with genistin to determine similarities and differences in genistin metabolites by different bacteria at different times. Finally, the absorption results of metabolites by fermentation were verified by a Caco-2 cell monolayer. Results: The HPLC results of fecal fermentation showed that genistein levels increased from 0.0139 ± 0.0057 mg/mL to 0.0426 ± 0.0251 mg/mL and two new metabolites were produced. A total of 46 metabolites following fecal fermentation were identified, resulting from various biotransformation reaction products, such as decarbonylation, hydroxylation, and methylation. Simultaneously, the 16S rRNA results showed that the intestinal flora changed significantly before and after fermentation and that the intestinal microorganisms in the control (Con) group and the fermentation (Fer) group showed a significant separation trend. Five genera, Lactobacillus, Bifidobacterium, Parabacteroides, Sutterella, and Dorea, were considered the dominant flora for genistin fermentation. The qualitative results of fermentation of genistin by five probiotics at different times showed that there were significant differences in small molecule metabolites by fermentation of different bacteria. Meanwhile, most metabolites could be identified following fecal bacteria fermentation, which verified the importance of the dominant bacteria in the feces for the biotransformation of components. Finally, the absorption results of the metabolites based on the Caco-2 cell monolayer showed that 14 metabolites could be absorbed into the circulation in vivo through the mesentery. Discussion: The small molecule metabolites of genistin by fermentation of fecal bacteria can be well absorbed systemically by the body. These studies provide a reference value for explaining the transformation and absorption of flavonoid glycosides in the intestine. [ABSTRACT FROM AUTHOR]

Cannabidiol (CBD) is a major non-psychotropic phytocannabinoid that exists in the Cannabis sativa plant. CBD has been found to act on various receptors, including both cannabinoid and non-cannabinoid receptors. In addition, CBD has antioxidant effects that are independent of receptors. CBD has demonstrated modulatory effects at different organ systems, such as the central nervous system, immune system, and the gastrointestinal system. Due to its broad effects within the body and its safety profile, CBD has become a topic of therapeutic interest. This literature review summarizes previous research findings with regard to the effect of CBD on the gastrointestinal (GI) system, including its effects at the molecular, cellular, organ, and whole-body levels. Both pre-clinical animal studies and human clinical trials are reviewed. The results of the studies included in this literature review suggest that CBD has significant impact on intestinal permeability, the microbiome, immune cells and cytokines. As a result, CBD has been shown to have therapeutic potential for GI disorders such as inflammatory bowel disease (IBD). Furthermore, through interactions with the gut, CBD may also be helpful in the treatment of disorders outside the GI system, such as non-alcoholic liver disease, postmenopausal disorders, epilepsy, and multiple sclerosis. In the future, more mechanistic studies are warranted to elucidate the detailed mechanisms of action of CBD in the gut. In addition, more well-designed clinical trials are needed to explore the full therapeutic potential of CBD on and through the gut. [ABSTRACT FROM AUTHOR]

This work aimed to evaluate the Caco‐2 cells as a model to study the epithelial transport of intestinal lipid extracts subjected to in vitro digestion, to establish a standard protocol for the determination of bioaccessibility and bioavailability of fatty acids in meat matrix, especially in those enriched with ω‐3 (eicosapentaenoic (EPA) and docosahexaenoic acids (DHA)). Samples were first subjected to in vitro digestion, and then, the intestinal extract was incubated with Caco‐2 cells. A first trial was conducted to select the most influencing variables on the fatty acid transport during Caco‐2 cell incubation: fat quantity on the intestinal extract, incubation time, and shaking. Then, a second experiment was carried out to determine the influence of these variables, being the fat quantity and the incubation time the most influencing factors on the transport and bioavailability of fatty acids. The effect of shaking was not so marked but seemed to improve the bioavailability of saturated fatty acids. This study also allows establishing the most suitable conditions: intestinal extracts with 30 mg of fat, longer incubation times (8 h), and shaking, achieving active and passive fatty acid transport without compromising the integrity of the Caco‐2 cell monolayer. The accurate results obtained for major and minor fatty acids, especially EPA and DHA are remarkable, due to the interest in these bioactive compounds. Thus, this study provides a combined protocol based on static in vitro digestion followed by Caco‐2 cell incubation to assess the bioaccessibility and bioavailability of fatty acids in meat samples. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the anticancer potential of Cymbopogon citratus extract. Methods: GC-MS analysis was used to identify phytocomponents in the methanolic extract of Cymbopogon citratus. A fractionation method was employed to isolate and assess the bioactivity of different fractions and their cytotoxic activities against cancer cell lines HCT116, LoVo, Caco-2, and HT-29 were investigated. A dual staining method with acridine orange and ethidium bromide was used to assess the effect of the extract on cell apoptosis. Additionally, the expression levels of Bax and TP53 were quantified using realtime PCR in Caco-2 cells treated with the ethyl acetate fraction of Cymbopogon citratus extract. A protein array was employed to profile key pro- and anti-apoptotic proteins in Caco-2 cells. Moreover, molecular docking studies were conducted to investigate the interactions between key compounds of Cymbopogon citratus extract and specific apoptosis-related protein domains (PDB IDs: 7wql and 4bkx). Results: A significant growth inhibition was observed in Caco-2 cells treated with Cymbopogon citratus extract. Among the seven fractions of the plant extract, the ethyl acetate fraction showed the highest cytotoxicity against Caco-2 cells with an IC50 value of (6.16 ± 0.01) μg/mL. The immunofluorescence assay showed that the ethyl acetate fraction could induce apoptosis of Caco-2 cells. Moreover, the fraction upregulated the gene expressions of Bax and TP53 in a dose-dependent manner. The docking analysis demonstrated the interaction of five compounds isolated from the ethyl acetate fraction with key proteins in Caco-2 cells, indicating their anticancer properties. Conclusions: Cymbopogon citratus extract shows anticancer activity against Caco-2 cells by inducing apoptosis. It may be a promising candidate for the treatment of colon cancer, which needs further investigation.

Abstract Chronic non-communicable diseases (CNCDs) pose a significant public health challenge. Addressing this issue, there has been a notable breakthrough in the prevention and mitigation of NCDs through the use of antioxidants and anti-inflammatory agents. In this study, we aim to explore the effectiveness of Eupatorium adenophora Spreng leaves (EASL) as an antioxidant and anti-inflammatory agent, and its potential applications. To construct a cellular model of oxidative damage and inflammation, Caco-2 cells were treated with tert-butyl hydroperoxide (t-BHP). The biocompatibility of EASL-AE with Caco-2 cells was assessed using the MTT assay, while compatibility was further verified by measuring LDH release and the protective effect against oxidative damage was also assessed using the MTT assay. Additionally, we measured intracellular oxidative stress indicators such as ROS and 8-OHdG, as well as inflammatory pathway signalling protein NFκB and inflammatory factors TNF-α and IL-1β using ELISA, to evaluate the antioxidant and anti-inflammatory capacity of EASL-AE. The scavenging capacity of EASL-AE against free radicals was determined through the DPPH Assay and ABTS Assay. Furthermore, we measured the total phenolic, total flavonoid, and total polysaccharide contents using common chemical methods. The chemical composition of EASL-AE was analyzed using the LC–MS/MS technique. Our findings demonstrate that EASL-AE is biocompatible with Caco-2 cells and non-toxic at experimental levels. Moreover, EASL-AE exhibits a significant protective effect on Caco-2 cells subjected to oxidative damage. The antioxidant effect of EASL-AE involves the scavenging of intracellular ROS, while its anti-inflammatory effect is achieved by down-regulation of the NFκB pathway. Which in turn reduces the release of inflammatory factors TNF-α and IL-1β. Through LC–MS/MS analysis, we identified 222 compounds in EASL-AE, among which gentianic acid, procaine and L-tyrosine were the compounds with high antioxidant capacity and may be the effective constituent for EASL-AE with antioxidant activity. These results suggest that EASL-AE is a natural and high-quality antioxidant and anti-inflammatory biomaterial that warrants further investigation. It holds great potential for applications in healthcare and other related fields.

Cannabidiol (CBD) is a major non-psychotropic phytocannabinoid that exists in the Cannabis sativa plant. CBD has been found to act on various receptors, including both cannabinoid and non-cannabinoid receptors. In addition, CBD has antioxidant effects that are independent of receptors. CBD has demonstrated modulatory effects at different organ systems, such as the central nervous system, immune system, and the gastrointestinal system. Due to its broad effects within the body and its safety profile, CBD has become a topic of therapeutic interest. This literature review summarizes previous research findings with regard to the effect of CBD on the gastrointestinal (GI) system, including its effects at the molecular, cellular, organ, and whole-body levels. Both pre-clinical animal studies and human clinical trials are reviewed. The results of the studies included in this literature review suggest that CBD has significant impact on intestinal permeability, the microbiome, immune cells and cytokines. As a result, CBD has been shown to have therapeutic potential for GI disorders such as inflammatory bowel disease (IBD). Furthermore, through interactions with the gut, CBD may also be helpful in the treatment of disorders outside the GI system, such as non-alcoholic liver disease, postmenopausal disorders, epilepsy, and multiple sclerosis. In the future, more mechanistic studies are warranted to elucidate the detailed mechanisms of action of CBD in the gut. In addition, more well-designed clinical trials are needed to explore the full therapeutic potential of CBD on and through the gut.

The purpose of this study was to evaluate the physicochemical properties of whey protein hydrolysate and determine changes in absorption rate due to enzymatic hydrolysis. The molecular weight distribution analysis of whey protein concentrate (WPC) and low-molecule whey protein hydrolysate (LMWPH) using the Superdex G-75 column revealed that LMWPH is composed of peptides smaller than those in WPC. Fourier-transform infrared spectroscopy indicated differences in peak positions between WPC and LMWPH, suggesting hydrolysis-mediated changes in secondary structures. Moreover, LMWPH exhibited higher thermal stability and faster intestinal permeation than WPC. Additionally, oral LMWPH administration increased serum protein content at 20 min, whereas WPC gradually increased serum protein content after 40 min. Although the total amount of WPC and LMWPH absorption was similar, LMWPH absorption rate was higher. Collectively, LMWPH, a hydrolysate of WPC, has distinct physicochemical properties and enhanced absorptive characteristics. Taken together, LMWPH is composed of low-molecular-weight peptides with low antigenicity and has improved absorption compared to WPC. Therefore, LMWPH can be used as a protein source with high bioavailability in the development of functional materials. [ABSTRACT FROM AUTHOR]

Background: The use of conventional animal testing to determine how medications affect a biological system comes with many challenges. In recent years, a variety of techniques to achieve these goals were presented, one of these is cell line creation technology that has dramatically increased in usefulness and efficiency of drug discovery researches. Cell culture is the term used to describe the removal of cells from an animal or plant and their subsequent cultivation in a lab setting. The Caco-2 model is commonly used in the early stages of drug discovery to make decisions about the permeability and/or absorption of tested chemicals. It is obvious that novel 3D cell culture models offer enormous potential for disease modeling and medication efficacy and safety assessment. However, the challenges provided by the inherent traits of Caco-2 cell variants and inter-laboratory methods have led to the development of irreducible data. Conclusion: These restrictions have an impact on the extrapolation of preclinical research results to clinical investigations on drug-drug and herbal-drug interactions.

Damage to the intestinal barrier causes intestinal inflammation, which could lead to the development of gastrointestinal diseases. Nut oils contain an abundance of unsaturated fatty acids, which are responsible for most of the bioactivities of nut oils. Here, the protective effect of four nut oils on the intestinal epithelial barrier was confirmed using Caco-2 cells damaged by tumor necrosis factor-α/interferon-γ treatment. Nut oils effectively increased transepithelial electronic resistance and lowered paracellular fluorescein isothiocyanate-dextran permeability in damaged Caco-2 cells. Furthermore, the nut oils inhibited the pro-inflammatory cytokine production of interleukin-6, which was induced by the inflammatory response. In particular, walnut oil effectively improved tight junction protein (occludin and zonula occludens-1) expression and distribution in damaged Caco-2 cells. In conclusion, our findings suggest that nut oils are promising candidates for impaired intestinal barrier protection.

7,8‐dihydroxyflavone (7,8‐DHF) is a biologically active flavone with various physiological activities, including neuroprotection, anti‐inflammation, and weight loss. Previous studies have found that the efflux protein P‐glycoprotein (P‐gp) significantly affects the transepithelial transport of 7,8‐DHF in the intestine, resulting in its low oral bioavailability. Based on this, in this study, a Caco‐2 monolayer cell model was used to investigate 14 dietary plant flavonoids as potential P‐gp inhibitors, and their effects on the transepithelial transport and in vitro digestion of 7,8‐DHF were explored. The results showed that among the 14 plant flavonoids, hesperetin, epigallocatechin gallate, fisetin, kaempferol, quercetin, and isoorientin increased and the apparent permeability coefficients (Papp) of 7,8‐DHF at AP → BL direction and lowered Papp value at BL → AP direction to varying degrees, reducing the efflux ratio of 7,8‐DHF less than 1.5. In particular, kaempferol and quercetin exhibited the best effect on promoting the transepithelial transport of 7,8‐DHF, especially when used at molar concentration ratios of 1:1 and 1:2 with 7,8‐DHF. This is beneficial for improving the oral bioavailability of 7,8‐DHF. Meanwhile, 7,8‐DHF was found to maintain structural stability in simulated saliva, gastric juice, and intestinal juice, and its stability was not affected by the coexistence of quercetin and kaempferol. Overall, this study provided a theoretical basis for seeking natural and safe P‐gp inhibitors to improve the oral absorption of natural products. [ABSTRACT FROM AUTHOR]

This work aimed to compare the performance of trans-ferulic acid-encapsulated nanostructured lipid carriers (NLCs) and solid lipid nanoparticles (SLNs) for transport by Caco-2 cells. The NLC particles (diameter: 102.6 nm) composed of Compritol® 888 ATO, ethyl oleate, Cremophor® EL, and Transcutol® P were larger than the SLNs (diameter: 86.0 nm) formed without liquid lipid (ethyl oleate), and the former had a higher encapsulation efficiency for trans-ferulic acid (p < 0.05). In vitro cultured Caco-2 cell transport was used to simulate intestinal absorption, and the cellular uptake of NLCs was higher than that of SLNs (p < 0.05). Compared to SLNs, NLCs greatly enhanced trans-ferulic acid permeation through the MillicellTM membrane (p < 0.05). This work confirms that NLCs have better properties than SLNs in terms of increasing drug transport by Caco-2 cells. This helps to comprehend the approach by which NLC-mediated oral bioavailability of trans-ferulic acid is better than that mediated by SLNs, as shown in our previous report.

SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.

In nonclinical studies, models that can predict the metabolism of drug candidates by cytochrome P450 (CYP), including Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) are helpful. CYP3A4-overexpressing human cells have been used universally to evaluate whether CYP3A4 metabolizes drug-candidate compounds. However, CYP3A4-overexpressing human cell lines are problematic because their activity levels are lower than that of in vivo human CYP3A4. Heme plays a paramount role in CYP activity. The rate-limiting step in heme biosynthesis is the generation of 5-aminolevulinic acid (5-ALA). In this study, we examined whether treatment with 5-ALA to CYP3A4-POR-UGT1A1-CES2 knockin and CES1 knockout (genome-edited) Caco-2 cells enhances CYP3A4 activity. A 7-day 5-ALA treatment increased intracellular heme levels in genome-edited Caco-2 cells without cytotoxicity. Moreover, consistent with the increase in intracellular heme content, 5-ALA treatment increased CYP3A4 activity in genome-edited Caco-2 cells. The results of this research are expected to be applied to pharmacokinetic studies using CYP-overexpressing human cells containing CYP3A4. • 5-ALA treatment increased intracellular heme levels in genome-edited Caco-2 cells. • Long-term 5-ALA treatment in genome-edited Caco-2 cells resulted in toxicity. • 5-ALA treatment increased CYP3A4 activity in genome-edited Caco-2 cells. [ABSTRACT FROM AUTHOR]

Resveratrol has anti-inflammatory, anti-cancer, and anti-aging pharmacological activities. There is currently a gap in academic research regarding the uptake, transport, and reduction of H2O2-induced oxidative damage of resveratrol in the Caco-2 cell model. This study investigated the role of resveratrol in the uptake, transport, and alleviation of H2O2-induced oxidative damage in Caco-2 cells. In the Caco-2 cell transport model, it was observed that the uptake and transport of resveratrol (10, 20, 40, and 80 μM) were time dependent and concentration dependent. Different temperatures (37 °C vs. 4 °C) could significantly affect the uptake and transportation of resveratrol. The apical to basolateral transport of resveratrol was markedly reduced by STF-31, a GLUT1 inhibitor, and siRNA intervention. Furthermore, resveratrol pretreatment (80 μM) improves the viability of Caco-2 cells induced by H2O2. In a cellular metabolite analysis combined with ultra-high performance liquid chromatography-tandem mass spectrometry, 21 metabolites were identified as differentials. These differential metabolites belong to the urea cycle, arginine and proline metabolism, glycine and serine metabolism, ammonia recycling, aspartate metabolism, glutathione metabolism, and other metabolic pathways. The transport, uptake, and metabolism of resveratrol suggest that oral resveratrol could prevent intestinal diseases caused by oxidative stress. [ABSTRACT FROM AUTHOR]

Angiopoietin-like protein 4 (ANGPTL4) is considered to be one of the important circulating mediators linking intestinal microorganisms and host lipid metabolism. The objective of this study was to assess the effects of peroxisome proliferator-activated receptor γ (PPARγ) on modulating ANGPTL4 synthesis in Caco-2 cells exposed to Clostridium butyricum. The viability of Caco-2 cells and the expression of PPARγ and ANGPTL4 in Caco-2 cells were detected after the Caco-2 cells were co-cultured with C. butyricum at the concentration of 1 × 106, 1 × 107 and 1 × 108 CFU/mL. The results showed that cell viability was enhanced by C. butyricum. Besides, PPARγ and ANGPTL4 expression and secretion in Caco-2 cells was significantly increased by 1 × 107 and 1 × 108 CFU/mL of C. butyricum. Furthermore, the effects of PPARγ on modulating ANGPTL4 synthesis in Caco-2 cells regulated by 1 × 108 CFU/mL of C. butyricum was also be expounded in PPARγ activation/inhibition model based on Caco-2 cells and via ChIP technique. It was found that C. butyricum promoted the binding of PPARγ to the PPAR binding site (chr19: 8362157-8362357, located upstream of the transcriptional start site of angptl4) of the angptl4 gene in Caco-2 cells. However, the PPARγ was not the only way for C. butyricum to stimulate ANGPTL4 production. Taken together, PPARγ played a role in the regulation of ANGPTL4 synthesis by C. butyricum in Caco-2 cells. [ABSTRACT FROM AUTHOR]

Background: Disruption of intestinal barrier function and an imbalance in intestinal immunity are crucial for the occurrence and development of ulcerative colitis. Because of their important roles in regulating inflammation and immunity, exosomes (Exos) released from bone marrow mesenchymal stem cells (BMSCs) may be useful for treating ulcerative colitis. The EphB/EphrinB signaling pathway plays a crucial role in the inflammatory process and the development and function of immune cells, and can mediate long-distance intercellular communication through extracellular vesicles. This study was conducted to explore the effects of pre-modified BMSC-Exos expressing EphB2 (EphB2-Exos) on immunoregulation in vitro. Methods: We transfected a lentivirus vector encoding EphB2 into BMSCs and isolated EphB2-Exos from the culture supernatant. Inflammation and oxidative damage in the human colon adenocarcinoma cell line (Caco-2) were induced by dextran sulfate sodium/hydrogen peroxide. In addition, spleen CD4+ T lymphocytes of rats were sorted in vitro. We conducted a series of experiments to explore the biological functions of EphB2-Exos. Results: EphB2-Exos were successfully isolated and were found to significantly protect the activity, proliferation, and migration of Caco-2 cells that were inhibited by dextran sulfate sodium. EphB2-Exos alleviated inflammation and apoptosis and increased the activity of antioxidant enzymes while inhibiting oxidative stress in Caco-2 cells. EphB2-Exos restored intestinal barrier function by inhibiting the RhoA/ROCK pathway and regulated the polarization of CD4+T cells. Conclusion: EphB2-Exos enhanced intestinal barrier function and regulated the immune balance by inhibiting the RhoA/ROCK pathway in vitro. These findings suggest that EphB2-Exos can be applied as a cell-free therapy for ulcerative colitis. [ABSTRACT FROM AUTHOR]

Blastocystis hominis is an intestinal protozoan that is often neglected, despite causing abdominal pain and diarrhea. Previous research has demonstrated that lipids can be synthesized by B. hominis or can accumulate in growth medium, but their function and mechanisms in the pathogenesis of Blastocystis remain unclear. Our study found that lipid-rich Blastocystis ST7-B can increase inflammation and disrupt Caco-2 cells more than the same parasite without the lipovenoes supplement. Additionally, the cysteine protease of Blastocystis, a virulence factor, is upregulated and has higher activity in lipid-rich Blastocystis. In order to better understand the effects of lipids on Blastocystis pathogenesis, we treated lipid-lowering pravastatin during Blastocystis ST7-B culturing with a lipovenoes supplement, which decreased the lipid levels of the Blastocystis and reduced the Blastocystis-induced inflammation and cell disruption of Caco-2 cells. We also analyzed the fatty acid composition and possible synthesis pathway in Blastocystis ST7-B, finding significantly higher ratios of arachidonic acid, oleic acid, and palmitic acid than in the other lipid components in lipid-rich Blastocystis ST7-B. These results suggest that lipids play a significant role in the pathogenesis of Blastocystis and provide important information on the molecular mechanisms of and potential treatments for Blastocystis infection. [ABSTRACT FROM AUTHOR]

Abstract Background Lysiphyllum strychnifolium (Craib) A. Schmitz, a traditional Thai medicinal plant, is mainly composed of polyphenols and flavonoids and exhibits several pharmacological activities, including antioxidant, anticancer, antimicrobial, and antidiabetic activities. However, the mechanism by which pure compounds from L. strychnifolium inhibit glucose catalysis in the small intestine and their effect on the glucose transporter remain unknown. Methods The objectives of this research were to examine the effect of 3,5,7-trihydroxychromone-3-O-𝛼-L-rhamnopyranoside (compound 1) and 3,5,7,3’,5’-pentahydroxy-flavanonol-3-O-𝛼-L-rhamnopyranoside (compound 2) on the inhibition of α-amylase and α-glucosidase, as well as glucose transporters, such as sodium-glucose cotransporter 1 (SGLT1), glucose transporter 2 (GLUT2), and glucose transporter 5 (GLUT5), using Caco-2 cells as a model of human intestinal epithelial cells. Additionally, the binding affinity and interaction patterns of compounds against two receptor proteins (SGLT1 and GLUT2) were determined for the first time utilizing a molecular docking approach. Results In the α-amylase inhibition assay, a concentration-dependent inhibitory response was observed against the enzyme. The results indicated that compound 1 inhibited α-amylase activity in a manner similar to that of acarbose (which exhibit IC50 values of 3.32 ± 0.30 µg/mL and 2.86 ± 0.10 µg/mL, respectively) in addition to a moderate inhibitory effect for compound 2 (IC50 = 10.15 ± 0.53 µg/mL). Interestingly, compounds 1 and 2 significantly inhibited α-glucosidase and exhibited better inhibition than that of acarbose, with IC50 values of 5.35 ± 1.66 µg/mL, 510.15 ± 1.46 µg/mL, and 736.93 ± 7.02 µg/mL, respectively. Additionally, α-glucosidase activity in the supernatant of the Caco-2 cell monolayer was observed. In comparison to acarbose, compounds 1 and 2 inhibited α-glucosidase activity more effectively in Caco-2 cells without cytotoxicity at a concentration of 62.5 µg/mL. Furthermore, the glucose uptake pathways mediated by SGLT1, GLUT2, and GLUT5- were downregulated in Caco-2 cells treated with compounds 1 and 2. Additionally, molecular modeling studies revealed that compounds 1 and 2 presented high binding activity with SGLT1 and GLUT2. Conclusion In summary, our present study was the first to perform molecular docking with compounds present in L. strychnifolium extracts. Our findings indicated that compounds 1 and 2 reduced glucose uptake in Caco-2 cells by decreasing the expression of glucose transporter genes and inhibiting the binding sites of SGLT1 and GLUT2. Therefore, compounds 1 and 2 may be used as functional foods in dietary therapy for postprandial hyperglycemia modulation of type 2 diabetes.

In this study, an oral colonic delivery system is designed using octenyl succinic anhydride (OSA) starch grafted with folic acid (FA). OSA starch with different degrees of substitution (DS) (0.011, 0.027, and 0.039) is prepared by reacting OSA with waxy maize starch. To optimize the drug‐loading efficiency, the study choses OSA6 starch (DS = 0.027) as the substrate to prepare OSA starch grafted with FA (FA‐OSA6). Fourier transform infrared spectrum confirms the successful introduction of FA. The scanning electron micrograph indicates that the surface of the FA‐OSA starch granules is rough and uneven. Drug loading analysis shows that FA‐OSA6 starch efficiently loaded doxorubicin hydrochloride (DOX) up to 87.71%. In vitro release studies suggest that the drug‐loaded complex released only 15.8% DOX through the upper gastrointestinal tract. The cell viability assay shows that the drug‐loaded complex effectively inhibits the growth of colon cancer cells (Caco‐2). These results suggest that OSA6 starch grafted with FA has potential as an oral colonic delivery carrier for DOX. [ABSTRACT FROM AUTHOR]

Recently, we demonstrated that a Citrus flavanone mix (FM) shows antioxidant and anti-inflammatory activity, even after gastro-duodenal digestion (DFM). The aim of this study was to investigate the possible involvement of the cyclooxygenases (COXs) in the anti-inflammatory activity previously detected, using a human COX inhibitor screening assay, molecular modeling studies, and PGE2 release by Caco-2 cells stimulated with IL-1β and arachidonic acid. Furthermore, the ability to counteract pro-oxidative processes induced by IL-1β was evaluated by measuring four oxidative stress markers, namely, carbonylated proteins, thiobarbituric acid-reactive substances, reactive oxygen species, and reduced glutathione/oxidized glutathione ratio in Caco-2 cells. All flavonoids showed a strong inhibitory activity on COXs, confirmed by molecular modeling studies, with DFM, which showed the best and most synergistic activity on COX-2 (82.45% vs. 87.93% of nimesulide). These results were also corroborated by the cell-based assays. Indeed, DFM proves to be the most powerful anti-inflammatory and antioxidant agent reducing, synergistically and in a statistically significant manner (p < 0.05), PGE2 release than the oxidative stress markers, also with respect to the nimesulide and trolox used as reference compounds. This leads to the hypothesis that FM could be an excellent antioxidant and COX inhibitor candidate to counteract intestinal inflammation. [ABSTRACT FROM AUTHOR]

Drug degradation at low pH and rapid clearance from intestinal absorption sites are the main factors limiting the development of oral macromolecular delivery systems. Based on the pH responsiveness and mucosal adhesion of hyaluronic acid (HA) and poly[2-(dimethylamino)ethyl methacrylate] (PDM), we prepared three HA–PDM nano-delivery systems loaded with insulin (INS) using three different molecular weights (MW) of HA (L, M, H), respectively. The three types of nanoparticles (L/H/M-HA–PDM–INS) had uniform particle sizes and negatively charged surfaces. The optimal drug loadings of the L-HA–PDM–INS, M-HA–PDM–INS, H-HA–PDM–INS were 8.69 ± 0.94%, 9.11 ± 1.03%, and 10.61 ± 1.16% (w/w), respectively. The structural characteristics of HA–PDM–INS were determined using FT-IR, and the effect of the MW of HA on the properties of HA–PDM–INS was investigated. The release of INS from H-HA–PDM–INS was 22.01 ± 3.84% at pH 1.2 and 63.23 ± 4.10% at pH 7.4. The protective ability of HA–PDM–INS with different MW against INS was verified by circular dichroism spectroscopy and protease resistance experiments. H-HA–PDM–INS retained 45.67 ± 5.03% INS at pH 1.2 at 2 h. The biocompatibility of HA–PDM–INS, regardless of the MW of HA, was demonstrated using CCK-8 and live–dead cell staining. Compared with the INS solution, the transport efficiencies of L-HA–PDM–INS, M-HA–PDM–INS, and H-HA–PDM–INS increased 4.16, 3.81, and 3.10 times, respectively. In vivo pharmacodynamic and pharmacokinetic studies were performed in diabetic rats following oral administration. H-HA–PDM–INS exhibited an effective hypoglycemic effect over a long period, with relative bioavailability of 14.62%. In conclusion, these simple, environmentally friendly, pH-responsive, and mucoadhesive nanoparticles have the potential for industrial development. This study provides preliminary data support for oral INS delivery. [ABSTRACT FROM AUTHOR]

Nutritional supplementation with L-arginine has been shown to promote cell growth and exercise performance, and is helpful to various disorders. Astragalus membranaceus and Panax notoginseng are traditional herbal medicines that display a wide range of beneficial activities. To understand their effects on L-arginine absorption in human intestinal cells, in vitro and in vivo experiments were performed using a standardized mixture of Astragalus membranaceus and Panax notoginseng saponins (APS). Our studies indicated that APS increased the expression of cationic amino acid transporter 1 (CAT1) and L-arginine transport in human Caco-2 cells. In addition, APS benefits TNBS-induced-colitis rats by increasing food intake, body weight, intestinal epithelial integrity, CAT1 gene expression, and blood L-arginine level. APS also reduces intestinal inflammation associated with increased myeloperoxidase activity in colitis rats. Further studies indicated that the plasma L-arginine level significantly increases in human subjects administered APS. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the physicochemical properties of whey protein hydrolysate and determine changes in absorption rate due to enzymatic hydrolysis. The molecular weight distribution analysis of whey protein concentrate (WPC) and low-molecule whey protein hydrolysate (LMWPH) using the Superdex G-75 column revealed that LMWPH is composed of peptides smaller than those in WPC. Fourier-transform infrared spectroscopy indicated differences in peak positions between WPC and LMWPH, suggesting hydrolysis-mediated changes in secondary structures. Moreover, LMWPH exhibited higher thermal stability and faster intestinal permeation than WPC. Additionally, oral LMWPH administration increased serum protein content at 20 min, whereas WPC gradually increased serum protein content after 40 min. Although the total amount of WPC and LMWPH absorption was similar, LMWPH absorption rate was higher. Collectively, LMWPH, a hydrolysate of WPC, has distinct physicochemical properties and enhanced absorptive characteristics. Taken together, LMWPH is composed of low-molecular-weight peptides with low antigenicity and has improved absorption compared to WPC. Therefore, LMWPH can be used as a protein source with high bioavailability in the development of functional materials.

In this study, we analyzed the eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) lipid composition of fish oil obtained through enzymatic treatment, fractional distillation and silica gel column purification, and further assessed EPA/DHA bioavailability. Lipid subclass composition information was obtained through ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), and bioavailability tests were performed using the Caco-2 cell monolayer model. Results showed that enzymatic treatment improved the incorporation of EPA/DHA as diacylglycerol (DG) while silica gel column chromatography enriched the content of EPA/DHA as phosphatidylglycerol (PG) (12.58%) and phosphatidylethanolamine (PE) (4.99%). Furthermore, increasing the purity of EPA/DHA could improve its bioavailability and after 24 incubation, binding forms of triglyceride (TG) was superior to ethyl ester (EE) (p < 0.05) at the same purity level. Those findings are helpful to provide research basis for exploring the bioactivity of fish oil.

Soybean bioactivity is significantly enhanced during tempeh fermentation. This study aimed to evaluate the efficacy of tempeh on colorectal cancer cells in vitro and colon precancerous lesions (aberrant crypt foci, ACF) in vivo. In the in vitro assay, tempeh water extract (WET) could inhibit the proliferation of Caco-2 cells. In the animal assay using 1,2-dimethylhydrazine (DMH)-induced Sprague–Dawley (SD) rats, 12-weeks daily feeding of tempeh could decrease the level of Clostridium perfringens in cecum contents and reduce the number of large (≥4 foci) ACF in the colon of treated rats, compared to the DMH control. By the results of TOF-MS and Edman degradation, the isolated antioxidant dipeptide, tripeptides, and tetrapeptides from WET might contain methionine, proline, and lysine. The bioactive peptides in tempeh might inhibit colon cancer by suppressing the growth of C. perfringens in the intestinal tract. [ABSTRACT FROM AUTHOR]

Foods containing chestnuts (Castanea mollissima Blume) are relatively uncommon, despite the high nutrient and starch contents and purported health benefits. In this study, we examine the flavor-related metabolites, volatile compounds, and amino acids in a traditional glutinous rice fermented beverage supplemented with chestnuts as a fermentation substrate for lactic acid bacteria (LAB). Changes in antioxidant activity towards free radicals and effects on cellular oxidative stress are compared between beverages with or without chestnuts. The fermented chestnut-rice beverage (FCRB) has higher sensory scores and a wider range of volatiles and flavor-related compounds (74 vs. 38 species compounds), but lower amino acid contents, than the traditional fermented glutinous rice beverage (TFRB). In free radical scavenging assays, the FCRB exhibits higher activity than the TFRB in vitro. Furthermore, while neither beverage induces cytotoxity in Caco-2 cells at concentrations up to 2 mg/mL, pretreatment with the FCRB results in lower rates of apoptosis and necrosis and higher overall viability in cells with H2O2-induced oxidative stress compared to pretreatment with the TFRB. The enhanced reactive oxygen species neutralization in vitro and protection against oxidative damage in cells, coupled with increased diversity of volatiles and flavor-related metabolites of LAB, support the addition of chestnuts to enhance flavor profile and antioxidant properties of fermented functional foods. [ABSTRACT FROM AUTHOR]

Several therapeutically active molecules are poorly water-soluble, thereby creating a challenge for pharmaceutical scientists to develop an active solution for their oral drug delivery. This study aimed to investigate the potential for novel polymer-surfactant-based formulations (designated A and B) to improve the solubility and permeability of curcumin. A solubility study and characterization studies (FTIR, DSC and XRD) were conducted for the various formulations. The cytotoxicity of formulations and commercial comparators was tested via MTT and LDH assays, and their permeability by in vitro drug transport and cellular drug uptake was established using the Caco-2 cell model. The apparent permeability coefficients (Papp) are considered a good indicator of drug permeation. However, it can be argued that the magnitude of Papp, when used to reflect the permeability of the cells to the drug, can be influenced by the initial drug concentration (C0) in the donor chamber. Therefore, Papp (suspension) and Papp (solution) were calculated based on the different values of C0. It was clear that Papp (solution) can more accurately reflect drug permeation than Papp (suspension). Formulation A, containing Soluplus® and vitamin E TPGs, significantly increased the permeation and cellular uptake of curcumin compared to other samples, which is believed to be related to the increased aqueous solubility of the drug in this formulation. [ABSTRACT FROM AUTHOR]

The cell viability of intestinal epithelial Caco-2 cell could be determined by CCK-8 assay, and cell survival and apoptosis detected by Annexin V-FITC/ PI double-straining. The expressions of anti-apoptosis protein (Bcl-2), pro-apoptosis protein (Bax), and tight junction protein were determined by protein immunoblotting. This paper investigated the protective effects of Areca catechu L. polysaccharide (AP) on oxidative stress in Caco-2 cells. The results showed that AP could down-regulate the expression of Bax, up-regulate the expression of Bcl-2, and inhibit cell apoptosis caused by DMNQ, and improve endoplasmic stress, up-regulate the expression of Caco-2 tight junction protein Z0-1, Occludin and Claudin 1. AP also maintained the integrity of cells through improving the expression of tight junction protein. These results suggested that the AP could exhibit a protective effect to against DMNQ-induced oxidative stress in Caco-2 cells. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) as probiotic candidates have various beneficial functions, such as regulating gut microbiota, inhibiting intestinal pathogens, and improving gut immunity. The colonization of the intestine is a prerequisite for probiotic function. Therefore, it is necessary to screen the highly adherent LAB. In this study, the cell surface properties, such as hydrophobicity, auto-aggregation, co-aggregation, and adhesion abilities of the six chicken-derived LAB to Caco-2 cells were investigated. All six strains showed different hydrophobicity (21.18–95.27%), auto-aggregation (13.61–30.17%), co-aggregation with Escherichia coli ATCC 25922 (10.23–36.23%), and Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311 (11.71–39.35%), and adhesion to Caco-2 cells (8.57–26.37%). Pediococcus pentosaceus 2–5 and Lactobacillus reuteri L-3 were identified as the strains with strong adhesion abilities (26.37% and 21.57%, respectively). Moreover, these strains could survive in a gastric acid environment at pH 2, 3, and 4 for 3 h and in a bile salt environment at 0.1%, 0.2%, and 0.3% (w/v) concentration for 6 h. Furthermore, the cell-free supernatant of P. pentosaceus 2–5 and L. reuteri L-3 inhibited the growth of enteropathogenic bacteria and the strains inhibited the adhesion of these pathogens to Caco-2 cells. In this study, these results suggested that P. pentosaceus 2–5 and L. reuteri L-3, isolated from chicken intestines might be good probiotic candidates to be used as feed additives or delivery vehicles of biologically active substances. [ABSTRACT FROM AUTHOR]

Phycotoxins are a class of multiple natural metabolites produced by microalgae in marine and freshwater ecosystems that bioaccumulate in food webs, particularly in shellfish, having a great impact on human health. Phycotoxins are mainly leached and absorbed in the small intestine when human consumers accidentally ingest toxic aquatic products contaminated by them. To assess the intestinal uptake and damage of phycotoxins, a typical in vitro model was developed and widely applied using the human colorectal adenocarcinoma Caco-2 cell line. In this review, the application cases were summarized for multiple phycotoxins, including microcystins (MCs), cylindrospermopsins (CYNs), domoic acids (DAs), saxitoxins (STXs), palytoxins (PLTXs), okadaic acids (OAs), pectenotoxins (PTXs) and azaspiracids (AZAs). The results of the previous studies showed that each group of phycotoxins presented different cytotoxicity and mechanisms to Caco-2 cells, and significant discrepancies in the transport of phycotoxin across the Caco-2 cell monolayers. Therefore, this review describes the evaluation assays of the Caco-2 cell monolayer model, illustrates the principles of several primary cytotoxicity evaluation assays, and summarizes the cytotoxicity of each group of phycotoxins to Caco-2 cells line and their cellular transport, and finally proposes the development of multicellular intestinal models for future comprehensive studies on the toxicity and absorption of phycotoxins in the intestine. It will improve the understanding of Caco-2 cell monolayer models in the toxicology studies on phycotoxins and the potentially detrimental effects of microalgal toxins on the human intestine.

Background: Lysiphyllum strychnifolium (Craib) A. Schmitz, a traditional Thai medicinal plant, is mainly composed of polyphenols and flavonoids and exhibits several pharmacological activities, including antioxidant, anticancer, antimicrobial, and antidiabetic activities. However, the mechanism by which pure compounds from L. strychnifolium inhibit glucose catalysis in the small intestine and their effect on the glucose transporter remain unknown. Methods: The objectives of this research were to examine the effect of 3,5,7-trihydroxychromone-3-O-훼-L-rhamnopyranoside (compound 1) and 3,5,7,3',5'-pentahydroxy-flavanonol-3-O-훼-L-rhamnopyranoside (compound 2) on the inhibition of α-amylase and α-glucosidase, as well as glucose transporters, such as sodium-glucose cotransporter 1 (SGLT1), glucose transporter 2 (GLUT2), and glucose transporter 5 (GLUT5), using Caco-2 cells as a model of human intestinal epithelial cells. Additionally, the binding affinity and interaction patterns of compounds against two receptor proteins (SGLT1 and GLUT2) were determined for the first time utilizing a molecular docking approach. Results: In the α-amylase inhibition assay, a concentration-dependent inhibitory response was observed against the enzyme. The results indicated that compound 1 inhibited α-amylase activity in a manner similar to that of acarbose (which exhibit IC50 values of 3.32 ± 0.30 µg/mL and 2.86 ± 0.10 µg/mL, respectively) in addition to a moderate inhibitory effect for compound 2 (IC50 = 10.15 ± 0.53 µg/mL). Interestingly, compounds 1 and 2 significantly inhibited α-glucosidase and exhibited better inhibition than that of acarbose, with IC50 values of 5.35 ± 1.66 µg/mL, 510.15 ± 1.46 µg/mL, and 736.93 ± 7.02 µg/mL, respectively. Additionally, α-glucosidase activity in the supernatant of the Caco-2 cell monolayer was observed. In comparison to acarbose, compounds 1 and 2 inhibited α-glucosidase activity more effectively in Caco-2 cells without cytotoxicity at a concentration of 62.5 µg/mL. Furthermore, the glucose uptake pathways mediated by SGLT1, GLUT2, and GLUT5- were downregulated in Caco-2 cells treated with compounds 1 and 2. Additionally, molecular modeling studies revealed that compounds 1 and 2 presented high binding activity with SGLT1 and GLUT2. Conclusion: In summary, our present study was the first to perform molecular docking with compounds present in L. strychnifolium extracts. Our findings indicated that compounds 1 and 2 reduced glucose uptake in Caco-2 cells by decreasing the expression of glucose transporter genes and inhibiting the binding sites of SGLT1 and GLUT2. Therefore, compounds 1 and 2 may be used as functional foods in dietary therapy for postprandial hyperglycemia modulation of type 2 diabetes. [ABSTRACT FROM AUTHOR]

Introduction: Genistin, as a kind of natural isoflavone glycoside, has good biological activity, and its weak absorption makes it closely related to intestinal flora. However, the role of the intestinal flora is still unclear and whether the metabolites produced by the intestinal flora are absorbed systemically is also variable., Methods: Genistin was fermented for 24 h based on fecal bacteria fermentation technology. The components were qualitatively and quantitatively analyzed by HPLC and UHPLC-Q-Exactive Orbitrap Mass spectrometry. The composition of intestinal flora in fermentation samples from fecal bacteria was detected by 16S rRNA sequencing. Five representative probiotics were cultured in vitro and fermented with genistin to determine similarities and differences in genistin metabolites by different bacteria at different times. Finally, the absorption results of metabolites by fermentation were verified by a Caco-2 cell monolayer., Results: The HPLC results of fecal fermentation showed that genistein levels increased from 0.0139 ± 0.0057 mg/mL to 0.0426 ± 0.0251 mg/mL and two new metabolites were produced. A total of 46 metabolites following fecal fermentation were identified, resulting from various biotransformation reaction products, such as decarbonylation, hydroxylation, and methylation. Simultaneously, the 16S rRNA results showed that the intestinal flora changed significantly before and after fermentation and that the intestinal microorganisms in the control (Con) group and the fermentation (Fer) group showed a significant separation trend. Five genera, Lactobacillus , Bifidobacterium , Parabacteroides , Sutterella , and Dorea , were considered the dominant flora for genistin fermentation. The qualitative results of fermentation of genistin by five probiotics at different times showed that there were significant differences in small molecule metabolites by fermentation of different bacteria. Meanwhile, most metabolites could be identified following fecal bacteria fermentation, which verified the importance of the dominant bacteria in the feces for the biotransformation of components. Finally, the absorption results of the metabolites based on the Caco-2 cell monolayer showed that 14 metabolites could be absorbed into the circulation in vivo through the mesentery., Discussion: The small molecule metabolites of genistin by fermentation of fecal bacteria can be well absorbed systemically by the body. These studies provide a reference value for explaining the transformation and absorption of flavonoid glycosides in the intestine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Wang, Wang, Wu, Zhao, Gong, Jiang and Xia.)

Everolimus is an oral mammalian target of rapamycin (mTOR) inhibitor used in cancer chemotherapy and transplantation. Due to its therapeutic properties, everolimus has been used long-term in clinical practice. Drug interactions with everolimus during gastrointestinal absorption can alter the oral bioavailability of everolimus and/or concomitant drugs. However, the effects of everolimus on gastrointestinal absorption remain unknown. The present study assessed the impact of continuous exposure to everolimus on expression and function of the ATP-binding cassette (ABC) transporter ABCB1 and ABCG2 using a Caco-2 intestinal cell model. Caco-2 subline, Caco/EV, was established by continuously exposing Caco-2 cells to 1 µM everolimus. Cell viability was evaluated using WST-1 assay. mRNA levels were measured by reverse transcription-quantitative PCR. Transport activity of ABCB1 was evaluated through the cellular accumulation of Rhodamin 123, a substrate for ABCB1. The half-maximal inhibitory concentration (IC 50 ) values for everolimus in Caco-2 and Caco/EV cells were 0.31 and 4.33 µM, respectively, indicating 14-fold resistance in Caco/EV cells. Sensitivity to paclitaxel and 7-ethyl-10-hydroxycamptothecin, which are substrates for ABCB1 and ABCG2, respectively, was enhanced in Caco/EV, but not in Caco-2 cells. The IC 50 values of cisplatin were comparable in both cell lines. Furthermore, mRNA expression levels of ABCB1 and ABCG2 were lower in Caco/EV cells than in Caco-2 cells, and the cellular accumulation of Rhodamine 123 was significantly higher in Caco/EV cells. These findings demonstrated that continuous exposure to everolimus suppressed the expression and function of ABCB1 and ABCG2, suggesting potential drug-drug interactions via the suppression of ABCB1 and ABCG2 in the intestinal tract., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Nakayama et al.)

Based on in vitro digestion, micellar synthesis, and Caco-2 cell model, this study investigated the effects of typical flavonoids in citrus (naringenin, naringin, hesperetin, hesperidin, quercetin, and rutin) at different doses on the micellization and cellular uptake of β -carotene. In in vitro digestion, low-dose flavonoids enhanced β -carotene bioaccesssibility by regulating the stability and dispersibility of the intestinal medium, particularly quercetin, which significantly increased the bioaccessibility by 44.6% ( p < 0.05). Furthermore, naringenin, hesperetin, hesperidin, and quercetin enhanced the micellar incorporation rate of β -carotene; however, naringin and rutin exhibited an opposite effect, particularly naringin, which significantly reduced it by 71.3% ( p < 0.05). This phenomenon could be attributed to the high solubility of naringin and rutin in micelles, resulting in a competitive inhibitory effect on β -carotene. Besides, all treatments significantly enhanced β -carotene cellular uptake ( p < 0.05) by promoting the expression of scavenger receptor class B type I and Niemann-Pick C1-Like 1.

A monolayer Caco-2 cell model was established to explore the effects of sea bass sausage digestive juice containing phosphate on calcium ion transport. Differential proteins of Caco-2 cells treated with fish sausage juice were detected and analyzed by gene ontology (GO) functional annotation and kyoto encyclopedia of genes and genomes (KEGG) pathway analyses. Results revealed that after treatment with 0.23 mg/mL digestive juice of perch sausage in vitro, Caco-2 cell viability was the highest at 72 h (99.84%). Additionally, 0.23 mg/mL digestive juice of perch sausage in vitro significantly increased calcium ion transport. The transfer volume was 1.396 μg/well. Fish sausages containing phosphate significantly affected the protein expression levels of Caco-2 cells. Two hundred one differential proteins were detected, including 114 up-regulated and 87 down-regulated proteins. The main differential proteins included P02795, Q9P0W0, Q96PU5, Q9GZT9 and Q5EBL8. The adjustment ratios of the fish sausage group were 0.7485, 1.373, 1.2535, 0.6775, and 0.809, respectively. The pathway analysis showed that phosphate affected calcium ion absorption and transport through the P02795 enrichment pathway. The fish sausage group showed that the immune-related functions of cells were affected. This study expounds the effects of water-retaining agents on the nutritional quality of aquatic products and provides theoretical support for the research and application of surimi products.

Blastocystis hominis is an intestinal protozoan that is often neglected, despite causing abdominal pain and diarrhea. Previous research has demonstrated that lipids can be synthesized by B. hominis or can accumulate in growth medium, but their function and mechanisms in the pathogenesis of Blastocystis remain unclear. Our study found that lipid-rich Blastocystis ST7-B can increase inflammation and disrupt Caco-2 cells more than the same parasite without the lipovenoes supplement. Additionally, the cysteine protease of Blastocystis, a virulence factor, is upregulated and has higher activity in lipid-rich Blastocystis. In order to better understand the effects of lipids on Blastocystis pathogenesis, we treated lipid-lowering pravastatin during Blastocystis ST7-B culturing with a lipovenoes supplement, which decreased the lipid levels of the Blastocystis and reduced the Blastocystis-induced inflammation and cell disruption of Caco-2 cells. We also analyzed the fatty acid composition and possible synthesis pathway in Blastocystis ST7-B, finding significantly higher ratios of arachidonic acid, oleic acid, and palmitic acid than in the other lipid components in lipid-rich Blastocystis ST7-B. These results suggest that lipids play a significant role in the pathogenesis of Blastocystis and provide important information on the molecular mechanisms of and potential treatments for Blastocystis infection.

Resveratrol has anti-inflammatory, anti-cancer, and anti-aging pharmacological activities. There is currently a gap in academic research regarding the uptake, transport, and reduction of H2O2-induced oxidative damage of resveratrol in the Caco-2 cell model. This study investigated the role of resveratrol in the uptake, transport, and alleviation of H2O2-induced oxidative damage in Caco-2 cells. In the Caco-2 cell transport model, it was observed that the uptake and transport of resveratrol (10, 20, 40, and 80 μM) were time dependent and concentration dependent. Different temperatures (37 °C vs. 4 °C) could significantly affect the uptake and transportation of resveratrol. The apical to basolateral transport of resveratrol was markedly reduced by STF-31, a GLUT1 inhibitor, and siRNA intervention. Furthermore, resveratrol pretreatment (80 μM) improves the viability of Caco-2 cells induced by H2O2. In a cellular metabolite analysis combined with ultra-high performance liquid chromatography-tandem mass spectrometry, 21 metabolites were identified as differentials. These differential metabolites belong to the urea cycle, arginine and proline metabolism, glycine and serine metabolism, ammonia recycling, aspartate metabolism, glutathione metabolism, and other metabolic pathways. The transport, uptake, and metabolism of resveratrol suggest that oral resveratrol could prevent intestinal diseases caused by oxidative stress.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a broad host range. With its housekeeping sigma factor and four alternative ones (namely SigB, SigC, SigH, and SigL), L. monocytogenes can express genes in response to changing environments. However, the roles of these sigma factors in intracellular survival are still unclear. The objectives of this study were to characterize the role of each alternative σ factor on L. monocytogenes invasion and growth inside human epithelial colorectal adenocarcinoma Caco-2 cells. We used L. monocytogenes 10403S wild type and its 15 alternative sigma factor deletion mutants at a multiplicity of infection of 100 and 1 in invasion and intracellular growth assays in the Caco-2 cells, respectively. At 1.5, 2, 4, 6, 8, 10, and 12 h post-infection, Caco-2 cells were lysed, and intracellular L. monocytogenes were enumerated on brain-heart infusion agar. Colony-forming and growth rates were compared among strains. The results from phenotypic characterization confirmed that (i) SigB is the key factor for L. monocytogenes invasion and (ii) having only SigA (ΔsigBCHL strain) is sufficient to invade and multiply in the host cell at similar levels as the wild type. Our previous study suggested the negative role of SigL in bile stress response. In this study, we have shown that additional deletion of the rpoN (or sigL) gene to ΔsigB, ΔsigC, or ΔsigH could restore the impaired invasion efficiencies of the single mutant, suggesting the absence of SigL could enhance host invasion. Therefore, we further investigated the role of SigL during extracellular and intracellular life cycles. Using RNA sequencing, we identified 118 and 16 SigL-dependent genes during the extracellular and intracellular life cycles, respectively. The sigL gene itself was induced by fivefolds prior to the invasion, and 5.3 folds during Caco-2 infection, further suggesting the role of SigL in intracellular growth. [ABSTRACT FROM AUTHOR]

In this study, Shiitake mushroom-derived extracellular nanovesicles (SMDENVs) were isolated from fresh Shiitake mushrooms by ultracentrifugation and sucrose gradient ultracentrifugation. The morphological characteristics of SMDENVs were investigated via Transmission Electron Microscopy and Laser Scanning Confocal Microscopy. SMDENVs were spherical, hollow, and uniform in size, with an average diameter of 177.6 ± 51.4 nm. Based on the analysis of lipidomics and proteomics, 383 lipids species and 1290 proteins were identified in SMDENVs. Compared with the conventional liposomes, SMDENVs demonstrated higher stability in different environmental conditions. Furthermore, we observed that SMDENVs were cytocompatible and inhibited the proliferation of Caco-2 cells. SMDENVs could be phagocytized by Caco-2 cells in a time-dependent manner. Further, SMDENVs also inhibited the proliferation of Caco-2 cells in a dose-dependent manner, and the half-maximal inhibitory concentration (IC 50) was 236.2 ± 3.2 μg/mL. Additionally, SMDENVs induced cellular apoptosis by increasing the levels of reactive oxygen species and decreasing the mitochondrial membrane potential. [Display omitted] • The nanovesicles were isolated from shiitake mushrooms (SMDENVs). • SMDENVs were purified by sucrose gradient ultracentrifugation. • The morphology, structure, and composition of SMDENVs were characterized. • SMDENVs showed higher stability compared with conventional liposomes. • SMDENVs could be phagocytosed by Caco-2 cells and induce cellular apoptosis. [ABSTRACT FROM AUTHOR]

Soup is an important presence in diet, but its absorption and transport mechanism by the human body remains unclear. In this study, Caco-2 intact cell and monolayer cell models were constructed to simulate small intestine absorption on colloidal nanoparticles (CNPs) isolated from lamb soup. The intracellular localization of CNPs was viewed by laser confocal microscopy (LSCM). CNPs uptake and release pathways were explored by differences in CNPs concentrations in 5 endocytosis inhibitor models and 4 exocytosis inhibitor models. Results indicated that CNPs endocytosis by Caco-2 cells was restrained by Nystatin and Cytochalasin D, with exocytosis being inhibited by Nocodazole and Monensin. Therefore, the major absorption pathways for CNPs were caveolin-dependent endocytosis, macropinocytosis and phagocytosis. The major transport pathways were microtubule-vesicle-mediated protein transport to the membrane and transportation between the Golgi apparatus and membrane. This study may provide theoretical support for the transport mechanism of soup products in the small intestine. • Laser confocal microscopy was used to visualize the soup products' CNPs transport in Caco-2 cell. • CNPs absorption pathways were caveolin-dependent, macropinocytosis and phagocytosis. • Microtubules and Golgi apparatus were essential in CNPs exocytosis. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a broad host range. With its housekeeping sigma factor and four alternative ones (namely SigB, SigC, SigH, and SigL), L. monocytogenes can express genes in response to changing environments. However, the roles of these sigma factors in intracellular survival are still unclear. The objectives of this study were to characterize the role of each alternative σ factor on L. monocytogenes invasion and growth inside human epithelial colorectal adenocarcinoma Caco-2 cells. We used L. monocytogenes 10403S wild type and its 15 alternative sigma factor deletion mutants at a multiplicity of infection of 100 and 1 in invasion and intracellular growth assays in the Caco-2 cells, respectively. At 1.5, 2, 4, 6, 8, 10, and 12 h post-infection, Caco-2 cells were lysed, and intracellular L. monocytogenes were enumerated on brain-heart infusion agar. Colony-forming and growth rates were compared among strains. The results from phenotypic characterization confirmed that (i) SigB is the key factor for L. monocytogenes invasion and (ii) having only SigA (ΔsigBCHL strain) is sufficient to invade and multiply in the host cell at similar levels as the wild type. Our previous study suggested the negative role of SigL in bile stress response. In this study, we have shown that additional deletion of the rpoN (or sigL) gene to ΔsigB, ΔsigC, or ΔsigH could restore the impaired invasion efficiencies of the single mutant, suggesting the absence of SigL could enhance host invasion. Therefore, we further investigated the role of SigL during extracellular and intracellular life cycles. Using RNA sequencing, we identified 118 and 16 SigL-dependent genes during the extracellular and intracellular life cycles, respectively. The sigL gene itself was induced by fivefolds prior to the invasion, and 5.3 folds during Caco-2 infection, further suggesting the role of SigL in intracellular growth.

[Display omitted] • A Caco-2 monolayer model was developed using medium customization and sodium valerate. • The abbreviated model showed improved transporter activity and monolayer integrity. • The model demonstrated consistent and reliable performance over multiple days. • The model has economic and practical advantages for drug permeability assessment. The human colorectal adenocarcinoma cell line Caco-2, widely used for studying intestinal drug permeability, is typically grown on permeable filter supports and matures in 21 days with frequent media changes. The process is labor-intensive, prone to contamination, and has low throughput, contributing to the overall high utilization cost. Efforts to establish a low-cost, high-throughput, and short-duration model have encountered obstacles, such as weaker tight junctions causing monolayer leaks, incomplete differentiation resulting in low transporter expression, intricate and challenging protocols, and cytotoxicity, limiting the usability. Hence, this study aimed to develop a low-cost, efficient, and short-duration model by addressing the aforementioned concerns by customizing the media and finding a safe differentiation inducer. We generated a new rapid model using sodium valerate, which demonstrated sufficient transporter activity, improved monolayer integrity, and higher levels of differentiation markers than the 21-day model. Furthermore, this model exhibited consistent and reliable results when used to evaluate drug permeability over multiple days of repeated use. This study demonstrates the potential of a sodium valerate-assisted abbreviated model for drug permeability assessment with economic and practical advantages. [ABSTRACT FROM AUTHOR]

The frequency presence of emamectin benzoate in agricultural production highlights the need for studying their toxicity against human intestinal epithelial barrier (IEB). Herein, we combined a Caco-2 cell model with transcriptome analysis to assess the intestinal toxicity of emamectin benzoate and its disease-causing potential. Results showed that the half maximal inhibitory concentration (IC 50) of emamectin benzoate on Caco-2 cell viability after 24, 48, and 72 h of exposure were 18.1, 9.9, and 8.3 μM, respectively. Emamectin benzoate exposure enhanced the Caco-2 monolayer paracellular permeability, damaged the IEB, and increased cellular apoptosis. Key driver gene analysis of 42 apoptosis - related DEGs, identified 10 genes (XIAP , KRAS , MCL1 , NRAS , PIK3CA , CYCS , MAPK8 , CASP3 , FADD , and TNFRSF10B) with the strongest correlation with emamectin benzoate - induced apoptosis. Transcriptomics identified 326 differentially expressed genes (DEGs, 204 upregulated and 122 downregulated). The functional terms of neurodegeneration - multiple diseases was enriched with the most number of DEGs, and the Parkinson disease pathway had the highest enrichment degree. Our findings provided support for environmental toxicology studies and the health risk assessment of emamectin benzoate. [Display omitted] [ABSTRACT FROM AUTHOR]

As a natural emulsifier in coconut milk, coconut globulin (CG) is a potential plant-based protein source to replace animal protein. However, due to its poor solubility and poor digestive properties, CG is not sufficiently utilized. The aim of this study was to improve the digestibility of CG by employing atmospheric cold plasma (ACP) technology. The influence of ACP treatment on the digestive properties of coconut globulin was evaluated by measurements of digestibility, degree of hydrolysis, free amino acids (FAAs), and polypeptide distribution after gastrointestinal digestion. These results implied that application of ACP treatment facilitated the digestion of CG, resulting in the hydrolysis of the compound into numerous smaller peptides (<1000 Da) and FAAs. The quartz crystal microbalance with dissipation technique was utilized to monitor the dynamic digestion process of CG. The results observed that ACP-treated CG had increased affinity for pepsin and trypsin, especially at 60 kV (the adsorbed mass was 7.60 μg/cm2). Various analytical techniques, including circular dichroism spectroscopy, Fourier transform infrared spectroscopy, fluorescence spectroscopy, and particle size distribution, demonstrated that ACP treatment facilitated the structural unfold of CG, resulting in increased exposure of enzyme binding sites and hydrolysis into smaller fragments. In addition, in vitro and cell-based antioxidant experiments provided evidence of the significant antioxidant activity of the hydrolysate derived from ACP-treated CG. Hence, the utilization of ACP treatment at 60 kV emerged as the optimal approach for enhancing the digestibility of CG (from 71.65% to 78.65%). This finding provides a theoretical basis for the use of ACP treatment in protein-rich foods, thereby providing insights for expanding the application of ACP treatment in the field of food processing. [Display omitted] • ACP-treated CG facilitated the augmentation of enzyme binding sites. • ACP stimulated the hydrolysis of CG to small peptides and free amino acids. • Hydrolysate of ACP-treated CG exhibited substantial antioxidant activity. [ABSTRACT FROM AUTHOR]

Docosahexaenoic acid (DHA) in the form of triacylglycerols (TAG) are widely recognized for their health benefits. As naturally occurring DHA in the form of TAG are limited, this work developed and optimized a Candida rugosa lipase-catalyzed selective hydrolysis process to enrich DHA in tuna oil. The DHA-enriched tuna oil produced at optimal conditions (lipase concentration of 4 %, ratio of water to tuna oil of 1:3, reaction temperature of 55°C) contained 46.12 % of DHA, in which 55.24 % of DHA was located in sn -2 position of the glycerides. In-vitro bioavailability test shows, the DHA-enriched tuna oil (7.89 %) had significantly higher cellular uptake of DHA as compared to unprocessed oil. The DHA-enriched oil demonstrated increased intracellular oxidation and mitochondrial damage of Caco-2 cell. • Candida rugosa lipase - catalyzed process to produce DHA-enriched tuna oil was optimized. • DHA-enriched tuna oil contained 46.12 % of DHA and 55.24 % of DHA in sn-2 of TAG. • DHA-enriched oil had better DHA uptake than the unprocessed oil. [ABSTRACT FROM AUTHOR]

[Display omitted] • Caco-2 cell monolayer model used to investigate Se species transport and uptake. • SeCys 2 and MeSeCys had high translocation rates in Cardamine violifolia leaves. • Translocations of SeMet, Se(IV), and Se(VI) were passive diffusional transport. • Organic Se bioavailability was higher than inorganic Se. • The bioavailability of Se species was affected by the food composition. Inadequate Se intake can enhance vulnerability to certain health risks, with supplementation lessening these risks. This study investigated the bioavailability of Se and Se species in five Se compounds and in Se-rich Cardamine violifolia using in vitro digestion coupled with a Caco-2 cell monolayer model, which enabled the study of Se transport and uptake. Translocation results showed that SeCys 2 and MeSeCys had high translocation rates in C. violifolia leaves (CVLs). The uptake rate of organic Se increased with time, and MeSeCys exhibited a higher uptake rate than that for SeCys 2 and SeMet. The translocation mechanisms of SeMet, Se(IV), and Se(VI) were passive transport, whereas those of SeCys 2 and MeSeCys were active transport. The bioavailability of organic Se was higher than that of inorganic Se, with a total Se bioavailability in CVLs of 49.11 %. This study would provide a theoretical basis for the application of C. violifolia in the functional food. [ABSTRACT FROM AUTHOR]

Drug degradation at low pH and rapid clearance from intestinal absorption sites are the main factors limiting the development of oral macromolecular delivery systems. Based on the pH responsiveness and mucosal adhesion of hyaluronic acid (HA) and poly[2-(dimethylamino)ethyl methacrylate] (PDM), we prepared three HA–PDM nano-delivery systems loaded with insulin (INS) using three different molecular weights (MW) of HA (L, M, H), respectively. The three types of nanoparticles (L/H/M-HA–PDM–INS) had uniform particle sizes and negatively charged surfaces. The optimal drug loadings of the L-HA–PDM–INS, M-HA–PDM–INS, H-HA–PDM–INS were 8.69 ± 0.94%, 9.11 ± 1.03%, and 10.61 ± 1.16% (w/w), respectively. The structural characteristics of HA–PDM–INS were determined using FT-IR, and the effect of the MW of HA on the properties of HA–PDM–INS was investigated. The release of INS from H-HA–PDM–INS was 22.01 ± 3.84% at pH 1.2 and 63.23 ± 4.10% at pH 7.4. The protective ability of HA–PDM–INS with different MW against INS was verified by circular dichroism spectroscopy and protease resistance experiments. H-HA–PDM–INS retained 45.67 ± 5.03% INS at pH 1.2 at 2 h. The biocompatibility of HA–PDM–INS, regardless of the MW of HA, was demonstrated using CCK-8 and live–dead cell staining. Compared with the INS solution, the transport efficiencies of L-HA–PDM–INS, M-HA–PDM–INS, and H-HA–PDM–INS increased 4.16, 3.81, and 3.10 times, respectively. In vivo pharmacodynamic and pharmacokinetic studies were performed in diabetic rats following oral administration. H-HA–PDM–INS exhibited an effective hypoglycemic effect over a long period, with relative bioavailability of 14.62%. In conclusion, these simple, environmentally friendly, pH-responsive, and mucoadhesive nanoparticles have the potential for industrial development. This study provides preliminary data support for oral INS delivery.

Polysaccharides are abundant in natural resources and perform numerous physiological functions. Polysaccharide structures often lack chromophore groups; thus, current analytical methods cannot distinguish polysaccharide metabolites in the body or polysaccharide prototypes in biological samples. Thus, the measurement of polysaccharides in blood, bodily fluid, and cell-culture medium is difficult. Our early-stage research resulted in the isolation of two homogeneous polysaccharides from Pseudostellaria heterophylla, PHP0.5MSC-F and PHPH-1-2, which have anti-hyperglycemia and insulin resistance improvement effects for type 2 diabetes. In this study, the reducing terminal sugars of PHP0.5MSC-F and PHPH-1-2 were labeled with 2-aminobenzamide (2-AB) to prepare novel fluorescent probes for HPLC-coupled fluorescence detection (HPLC-FLD). Quantitative analysis was performed in reference to T40, and the detection limit for PHP0.5MSC-F was found to be 8.84 μg/mL with a linear range of 29.45–683.28 μg/mL. In reference to T70, the detection limit for PHPH-1-2 was found to be 13.89 μg/mL with a linear range of 46.29–462.76 μg/mL. This method was used to measure the bidirectional transport of polysaccharides across caco-2 cells from apical to basolateral (AP→BL) or from basolateral to apical (BL→AP) directions and to evaluate the polysaccharide bioavailability. The drug absorption capacity was determined based on the apparent permeability coefficient (Papp), and the Papp values for the two polysaccharides were found to be greater than 1 × 10−6 cm/s, which suggests easy absorption. Regarding bidirectional transport, the AP→BL Papp values were greater than the BL→AP values; thus, PHP0.5MSC-F and PHPH-1-2 mainly underwent passive transference. The two membrane permeable polysaccharides were not P-gp efflux transporter substrates. The absorption mechanism of PHP0.5MSC-F complies with passive diffusion under a concentration gradient, whereas PHPH-1-2 mainly utilizes a clathrin-mediated endocytic pathway to enter caco-2 cells. This innovative HPLC-FLD method can help to track polysaccharide internalization in vitro and in vivo to facilitate cellular uptake and biodistribution exploration. [ABSTRACT FROM AUTHOR]

Diarrheagenic Escherichia coli (DEC) can cause epidemic diarrhea worldwide. The pathogenic potential of different strains is diverse and the continuous emergence of pathogenic strains has brought serious harm to public health. Accurately distinguishing and identifying DEC with different virulence is necessary for epidemiological surveillance and investigation. Clustered regularly interspaced short palindromic repeats (CRISPR) typing is a new molecular method that can distinguish pathogenic bacteria excellently and has shown great promise in DEC typing. The purpose of this study was to investigate the discrimination of CRISPR typing method for DEC and explore the pathogenicity potential of DEC based on CRISPR types (CT). The whole genome sequences of 789 DEC strains downloaded from the database were applied CRISPR typing and serotyping. The D value (Simpson's index) with 0.9709 determined that CRISPR typing had a higher discrimination. Moreover, the same H antigen strains with different O seemed to share more identical spacers. Further analyzing the strains CRISPR types and the number of virulence genes, it was found that there was a significant correlation between the CRISPR types and the number of virulence genes (p < 0.01). The strains with the largest number of virulence genes concentrated in CT25 and CT56 and the number of virulence genes in CT264 was the least, indicating that the pathway potential of different CRISPR types was variable. Combined with the Caco-2 cell assay of the laboratory strains, the invasion capacity of STEC strains of different CRISPR types was different and there was no significant difference in the invasion rate between different CRISPR type strains (p > 0.05). In the future, with the increase of the number of strains that can be studied experimentally, the relationship between CRISPR types and adhesion and invasion capacities will be further clarified. [ABSTRACT FROM AUTHOR]