Intestinal Caco-2 Cells Research Articles

HMGB1 impacts the intestinal epithelial barrier by initiating NETs to regulate macrophage polarization.

High mobility group box 1 (HMGB1) has the capability of activating the immune response and taking part in macrophage polarization. Despite this, there is significant scope for exploration into how HMGB1 regulates macrophage polarization phenotype and influences intestinal epithelial barrier function. Investigating the role of HMGB1 in the creation of neutrophil extracellular traps (NETs) and the mechanism of its impact on macrophages could provide novel insights into intervening in intestinal inflammation and barrier damage. Therefore, the research examined the relationship between the macrophage polarization phenotype and HMGB1. Additionally, we analyzed how cell proliferation and cytokines changed in CaCo-2 cells following co-culture with HMGB1-influenced macrophages and intestinal epithelial CaCo-2 cells. We discovered that up-regulation of HMGB1 expression enhanced the creation of NETs, whereas inhibition of NETs formation led macrophages to switch from the anti-inflammatory M2 phenotype to the pro-inflammatory M1 phenotype. Additionally, we observed that macrophages induced by NETs containing HMGB1 can prompt CaCo-2 cell apoptosis and exacerbate the inflammatory response. HMGB1-containing NETs hinder tight junction protein expression in CaCo-2 cells by inducing macrophage M1 polarization, thereby impairing intestinal epithelial barrier function. Therefore, our findings indicate that by inhibiting the expression of HMGB1, the formation of NETs can be inhibited. This, in turn, mediates macrophage polarization and offers potential new therapies for intestinal diseases.

Molecular Mechanisms of Skatole-Induced Inflammatory Responses in Intestinal Epithelial Caco-2 Cells: Implications for Colorectal Cancer and Inflammatory Bowel Disease.

Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in intestinal epithelial cells significantly contribute to inflammatory bowel disease (IBD) and colorectal cancer (CRC). Given our previous findings that TNF-α is upregulated in intestinal epithelial Caco-2 cells induced by skatole, a tryptophan-derived gut microbiota metabolite, the present study aimed to explore the relationship between skatole and IL-6, alongside TNF-α. Skatole elevated the promoter activity of IL-6 as well as TNF-α, and increased IL-6 mRNA expression and protein secretion. In addition to activating NF-κB, the NF-κB inhibitor BAY 11-7082 reduced skatole-induced cell survival and the mRNA expression of IL-6 and TNF-α. NF-κB activation was attenuated by the extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 and the p38 inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. U126 and SB203580 also decreased the skatole-induced increase in IL-6 expression. When skatole-induced AhR activation was inhibited by CH223191, in addition to promoting NF-κB activation, IL-6 expression was enhanced in a manner similar to that previously reported for TNF-α. Taken together, these results suggest that skatole-elicited NF-κB activation induces IL-6 and TNF-α expression, although AhR activation partially suppresses this process. The ability of skatole to increase the expression of IL-6 and TNF-α may significantly affect the development and progression of these diseases. Moreover, the balance between NF-κB and AhR activation appears to govern the skatole-induced increases in IL-6 and TNF-α expression. Therefore, the present findings provide new insights into the mechanisms linking tryptophan-derived gut microbiota metabolites with colorectal disease.

Involvement of everolimus‑induced ABCB1 downregulation in drug‑drug interactions.

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 (IC50) 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 IC50 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.

Micronization of wholewheat flour increases iron bioavailability from hydrothermally processed wheat flour dough

Cereal products contribute significantly to dietary intake of essential minerals. In wheat, iron and zinc are stored in specific grain structures including the aleurone, scutellum and embryo. Wheat cell walls are resistant to digestion in the human gastrointestinal tract and therefore this study investigated the hypothesis that physical disruption of the cell walls would increase the bioaccessibility and bioavailability of iron and zinc from wheat-based foods. Flour was micronized using a combination of roller milling and a micro-mill and this reduced median particle size by two-thirds. Hydrothermally processed wheat flour doughs were subjected to in vitro digestion to determine mineral bioaccessibility. Mineral bioavailability from food digests was measured using human intestinal Caco-2 cells. Iron (but not zinc) bioavailability from wheat foods made using the micronized flour (2.5 ± 0.5 nmol/mg cell protein) was increased significantly compared with foods produced from standard milled flour (1.3 ± 0.1 nmol/mg cell protein; P = 0.031). Micronization of wheat flour has the potential to increase the absorption of the endogenous iron present in cereal foods and this might have health benefits for population groups with poor iron status.

Effects of an Extract of Physalis Peruviana Linnaeus on the Expression of Inflammatory Markers in the Caco-2 Intestinal Epithelium-like Cell Line

Objective: Physalis Peruviana Linnaeus (PPL) is an herbaceous species characterized by a wide variety of bioactive compounds to which anti-inflammatory properties have been attributed. This makes this fruit a possible complementary therapy for diseases that involve chronic inflammation, such as inflammatory bowel diseases (IBD). In the present study, the effect of a PPL extract on the expression of inflammatory markers in the Caco-2 cell line was evaluated. Methods: An in vitro gastric digest (50 g PPL pulp) was performed, obtaining an extract that was used to challenge Caco-2 cells for 24 and 72 hours. This extract was characterized by LC-MS/MS. Then, the relative mRNA expression of NF-kB, TLR4, IL-18 and MCP-1 was determined through qRT-PCR and the protein levels of TNF-α, IL-6, IL-8, IL-18 and MCP-1 through Luminex Immunoassay. Results: From the characterization of the extract, compounds with bioactive potential such as isothiocyanates, indoles and coumarins were found. Treatment of Caco-2 cells with PPL extract (80 µg/ml), particularly for 72 hours, produced a reduction of IL-18 and MCP-1 mRNA expression (p < 0.01), in addition to IL-18 (p < 0.01), IL-8 (p < 0.0001) and MCP-1 (p < 0.01) protein levels, however, no effects on NF-kB p65 (p = 0.09) and TLR4 (p = 0.20) mRNA expression were observed. Conclusion: The results obtained in this study open the possibility that the regular consumption of 50 g of PPL could constitute a possible complementary therapy for the treatment of IBD, improving the quality of life of these patients.

Biological activity and chemical characteristics studies of new oligomannose produced by Erwinia gerundensis

Natural polymers have attracted considerable attention in recent decades among scientists due to their potential therapeutic uses, particularly as antimicrobial and antitumor agents. In this research, novel EPSs were extracted from garlic rhizosphere bacteria. The antibacterial and antitumor activities of the polymer were evaluated through biological assays. The antibacterial activity was tested against gram-positive microorganisms (such as Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus) and gram-negative organisms (such as Shigella sonnei and Escherichia coli). The most significant inhibition zone was observed with Listeria monocytogenes and S. typhi, measuring 35 mm, while the most miniature antibacterial effect was seen with Staphylococcus aureus at 23.67 mm.Furthermore, the inhibitory effect of the crude polymer was assessed using a broth medium with two strains of E. coli and Bacillus cereus. Electron microscope images displayed varying degrees of damage to bacterial cells in the treated broth. The antitumor activity was determined using the MTT test on colon carcinoma cells (HCT-116), hepatocellular carcinoma cells (HepG-2), and CaCO2 (intestinal carcinoma cells), with IC50 values of 188.86±6.17 µg/mL, 221.66±8.02 µg/mL, and 203.65±7.43 µg/mL, respectively, after 48 h. The bacteria responsible for polymer production were isolated from garlic plant rhizospheres and identified as Erwinia gerundensis CCASU-2024–69 through 16S rRNA sequencing. FTIR and NMR techniques determined the crude EPS's main components and functional groups, including carbonyl, carboxylic, methylene, and silanol. GC–MS analysis revealed 34 bioactive compounds, while HPLC analysis indicated that the EPS was a hetero-monosaccharide consisting of d-xylose, d-glucose, l-arabinose, ribose, and d-mannose. This research study represents the initial exploration into the exopolysaccharide derived from Erwinia gerundensis. To assess the interaction between the exopolysaccharide and the active sites of Bacillus cereus and E. coli, molecular docking experiments were conducted using five monosaccharides: d-xylose, d-glucose, l-arabinose, ribose, and d-mannose. The data obtained from the molecular docking analysis strongly correlates with the findings from biological studies.Furthermore, these highly active compounds exhibit a favorable proposed ADMET profile. This particular exopolysaccharide shows potential as a natural antibiotic and holds promise in treating gastrointestinal cancer. A comprehensive assessment of laboratory animals is essential before its potential use as a prebiotic in nutrition.

Neonatal Fc receptor is a functional receptor for classical human astrovirus.

Human astrovirus (HAstV) is a global cause of gastroenteritis in infants, the elderly, and the immunocompromised. However, the molecular mechanisms that control its susceptibility are not fully understood, as the functional receptor used by the virus has yet to be identified. Here, a genome-wide CRISPR-Cas9 library screen in Caco2 cells revealed that the neonatal Fc receptor (FcRn) can function as a receptor for classical HAstV (Mamastrovirus genotype 1). Deletion of FCGRT or B2M, which encode subunits of FcRn, rendered Caco2 cells and intestinal organoid cells resistant to HAstV infection. We also showed that human FcRn expression renders non-susceptible cells permissive to viral infection and that FcRn binds directly to the HAstV spike protein. Therefore, our findings provide insight into the entry mechanism of HAstV into susceptible cells. We anticipate that this information can be used to develop new therapies targeting human astroviruses, providing new strategies to treat this global health issue.

Lack of effects of polystyrene micro- and nanoplastics on activity and expression of human drug transporters

Micro- and nanoplastics (MPs/NPs) constitute emerging and widely-distributed environmental contaminants to which humans are highly exposed. They possibly represent a threat for human health. In order to identify cellular/molecular targets for these plastic particles, we have analysed the effects of exposure to manufactured polystyrene (PS) MPs and NPs on in vitro activity and expression of human membrane drug transporters, known to interact with chemical pollutants. PS MPs and NPs, used at various concentrations (1, 10 or 100 µg/mL), failed to inhibit efflux activities of the ATP-binding cassette (ABC) transporters P-glycoprotein, MRPs and BCRP in ABC transporter-expressing cells. Furthermore, PS particles did not impair the transport of P-glycoprotein or BCRP substrates across intestinal Caco-2 cell monolayers. Uptake activities of solute carriers (SLCs) such as OCT1 and OCT2 (handling organic cations) or OATP1B1, OATP1B3, OATP2B1, OAT1 and OAT3 (handling organic anions) were additionally not altered by PS MPs/NPs in HEK-293 cells overexpressing these SLCs. mRNA expression of ABC transporters and of the SLCs OCT1 and OATP2B1 in Caco-2 cells and human hepatic HepaRG cells were finally not impaired by a 48-h exposure to MPs/NPs. Altogether, these data indicate that human drug transporters are unlikely to be direct and univocal targets for synthetic PS MPs/NPs.

Effect of ultrasound treatment on quality parameters and health promoting activity of fish protein hydrolysates extracted from side streams of Atlantic mackerel (Scomber scombrus).

Fish protein hydrolysates (FPH) obtained by enzymatic hydrolysis allows for smart valorization of fish side streams. However, further treatments are normally needed to enhance bioactive and functional properties of the obtained FPH. At present, the commonly used methods to improve functional properties of FPH include chemical and enzymatic modification. Chemical treatments often cause environmental problems, while the enzymatic modification method requires the use of quite expensive enzymes. In recent years, emerging technologies such as ultrasound treatment (US-treatment) have shown great potential in protein modification with high efficiency and safety, low energy consumption, and low nutritional destructiveness. In this study, high-power ultrasound treatments were applied to fish protein hydrolysates (FPH) extracted from Atlantic mackerel (Scomber scombrus) side streams to improve their quality parameters. The effect of three different treatments of 300 W, 450 W and 600 W at the operating frequency of 20 kHz for 10 min on the physicochemical, structural, and functional characteristics of FPH, were examined. The results have shown that with an increase in ultrasound power, the protein solubility of FPH increased linearly, and the changes were significant for all US-treated samples compared to control (untreated) samples. US-treatment significantly increased the degree of hydrolysis of FPH samples treated with 450 W and 600 W compared to control samples. The carbonyl content of FPH increased (significantly for 450 W and 600 W), while thiol groups decreased (significantly for 300 W and 450 W). This indicated that some US-treatments induced oxidation of FPH, however the values of the protein oxidation were low. Amino acid composition of FPH revealed that US-treatment increased the proportion of essential amino acids in the sample treated with 300 W and 450 W, but the increase was not significant. After the US-treatment, all FPH samples became lighter and less yellowish and reddish, which suggest potentially higher attractiveness to consumers. In addition, the in vitro antioxidant activity was assessed using the DPPH, FRAP, and ABTS assays and the cell-free dipeptidyl peptidase IV (DPP-IV) inhibitory activity was also measured. Moreover, these biological activities were measured at cellular level utilizing human intestinal Caco-2 cells. Specifically, the FPH capacity to lower H2O2-induced reactive oxygen species (ROS) and lipid peroxidation levels was used to measure its antioxidant activity. The findings suggest that Scomber scombrus hydrolysates could find use as ingredients for promoting health.

Assessment of drug-drug interaction of dapagliflozin with LCZ696 based on an LC-MS/MS method.

The co-administration of dapagliflozin (DPF) and sacubitril/valsartan (LCZ696) has emerged as a promising therapeutic approach for managing heart failure. Given that DPF and LCZ696 are substrates for P-glycoprotein, there is a plausible potential for drug-drug interactions when administered concomitantly. To investigate the pharmacokinetic changes when these drugs are co-administered, we have established and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of simultaneously detecting DPF, LBQ657 (the active metabolite of sacubitril) and valsartan in rat plasma. This method has demonstrated selectivity, sensitivity, and accuracy. Drug-drug interactions were examined by the LC-MS/MS method. The mechanisms were investigated using everted intestinal sac models and Caco-2 cells. The results showed that DPF significantly increased the area under the curve (AUC(0-t)) (3,563.3 ± 651.7 vs. 7,146.5 ± 1,714.9h μg/L) of LBQ657 (the active metabolite of sacubitril) and the AUC(0-t) (24,022.4 ± 6,774.3 vs. 55,728.3 ± 32,446.3h μg/L) of valsartan after oral co-administration. Dapagliflozin significantly increased the amount of LBQ657 and valsartan in intestinal sacs by 1- and 1.25-fold at 2.25 h. Caco-2 cell uptake studies confirmed that P-glycoprotein is the transporter involved in this interaction. This finding enhances the understanding of drug-drug interactions in the treatment of heart failure and provides a guidence for clinical therapy.

P06-17 Assessment of genome-wide methylation changes caused by TiO2 nanoparticles on human intestinal caco-2 cells

P06-17 Assessment of genome-wide methylation changes caused by TiO2 nanoparticles on human intestinal caco-2 cells

Apoptosis and Oxidative Stress in Human Intestinal Epithelial Caco-2 Cells Caused by Marine Phycotoxin Azaspiracid-2.

When humans consume seafood contaminated by lipophilic polyether phycotoxins, such as azaspiracids (AZAs), the toxins are mainly leached and absorbed in the small intestine, potentially causing intestinal damage. In this study, human intestinal epithelial Caco-2 cells were used to investigate the adverse effects of azaspiracid-2 (AZA-2) on human intestinal epithelial cells. Cell viability, apoptosis, oxidative damage and mitochondrial ultrastructure were investigated, and ribonucleic acid sequence (RNA-seq) analysis was applied to explore the potential mechanisms of AZA-2 toxicity to Caco-2 cells. Results showed that AZA-2 significantly reduced the proliferation of Caco-2 cells in a concentration-dependent response, and the 48 h EC50 of AZA-2 was 12.65 nmol L-1. AZA-2 can induce apoptosis in Caco-2 cells in a dose-dependent manner. Visible mitochondrial swelling, cristae disintegration, membrane rupture and autophagy were observed in Caco-2 cells exposed to AZA-2. Reactive oxygen species (ROS) and malondialdehyde (MDA) content were significantly increased in Caco-2 cells after 48 h of exposure to 1 and 10 nmol L-1 of AZA-2. Transcriptome analysis showed that KEGG pathways related to cellular oxidative damage and lipid metabolism were affected, mainly including mitophagy, oxidative phosphorylation, cholesterol metabolism, vitamin digestion and absorption, bile secretion and the peroxisome proliferator-activated receptor signaling pathway. The cytotoxic effects of AZA-2 on Caco-2 cells may be associated with ROS-mediated autophagy and apoptosis in mitochondrial cells. Results of this study improve understanding of the cytotoxicity and molecular mechanisms of AZA-2 on Caco-2 cells, which is significant for protecting human health.

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.

Expression of the locus of enterocyte effacement genes during the invasion process of the atypical enteropathogenic Escherichia coli 1711-4 strain of serotype O51:H40.

Atypical enteropathogenic Escherichia coli (aEPEC) is a significant cause of diarrhea in low- and middle-income countries. Certain aEPEC strains, including the Brazilian representative strain of serotype O51:H40 called aEPEC 1711-4, can use flagella to attach to, invade, and persist in T84 and Caco-2 intestinal cells. It can also translocate from the gut to extraintestinal sites in a rat model. Although various aspects of the virulence of this strain were studied and the requirement of a type III secretion system for the efficiency of the invasion process was demonstrated, the expression of the locus of enterocyte effacement (LEE) genes during the invasion and intracellular persistence remains unclear. To address this question, the expression of flagella and the different LEE operons was evaluated during kinetic experiments of the interaction of aEPEC 1711-4 with enterocytes in vitro. The genome of the strain was also sequenced. The results showed that flagella expression remained unchanged, but the expression of eae and escJ increased during the early interaction and invasion of aEPEC 1711-4 into Caco-2 cells, and there was no change 24 h post-infection during the persistence period. The number of actin accumulation foci formed on HeLa cells also increased during the 6-h analysis. No known gene related to the invasion process was identified in the genome of aEPEC 1711-4, which was shown to belong to the global EPEC lineage 10. These findings suggest that the LEE components and the intimate adherence promoted by intimin are necessary for the invasion and persistence of aEPEC 1711-4, but the detailed mechanism needs further study.IMPORTANCEAtypical enteropathogenic Escherichia coli (aEPEC) is a major cause of diarrhea, especially in low- and middle-income countries, like Brazil. However, due to the genome heterogeneity of each clonal group, it is difficult to comprehend the pathogenicity of this strain fully. Among aEPEC strains, 1711-4 can invade eukaryotic cells in vitro, cross the gut barrier, and reach extraintestinal sites in animal models. By studying how different known aEPEC virulence factors are expressed during the invasion process, we can gain insight into the commonalities of this phenotype among other aEPEC strains. This will help in developing preventive measures to control infections caused by invasive strains. No known virulence-encoding genes linked to the invasion process were found. Nevertheless, additional studies are still necessary to evaluate the role of other factors in this phenotype.

Effect of docosahexaenoic acid as an anti-inflammatory for Caco-2 cells and modulating agent for gut microbiota in children with obesity (the DAMOCLE study).

Docosahexaenoic acid (DHA) is a long-chain omega-3 polyunsaturated fatty acid. We investigated the dual health ability of DHA to modulate gut microbiota in children with obesity and to exert anti-inflammatory activity on human intestinal Caco-2 cells. In a pilot study involving 18 obese children (8-14years), participants received a daily DHA supplement (500mg/day) and dietary intervention from baseline (T0) to 4months (T1), followed by dietary intervention alone from 4months (T1) to 8months (T2). Fecal samples, anthropometry, biochemicals and dietary assessment were collected at each timepoint. At preclinical level, we evaluated DHA's antioxidant and anti-inflammatory effects on Caco-2 cells stimulated with Hydrogen peroxide (H2O2) and Lipopolysaccharides (LPS), by measuring also Inducible nitric oxide synthase(iNOS) levels and cytokines, respectively. Ten children were included in final analysis. No major changes were observed for anthropometric and biochemical parameters, and participants showed a low dietary compliance at T1 and T2. DHA supplementation restored the Firmicutes/Bacteroidetes ratio that was conserved also after the DHA discontinuation at T2. DHA supplementation drove a depletion in Ruminococcaceae and Dialisteraceae, and enrichment in Bacteroidaceae, Oscillospiraceae, and Akkermansiaceae. At genus level, Allisonella was the most decreased by DHA supplementation. In Caco-2 cells, DHA decreased H2O2-induced reactive oxygen species (ROS) and nitric oxide (NO) production via iNOS pathway modulation. Additionally, DHA modulated proinflammatory (IL-1β, IL-6, IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokine production in LPS-stimulated Caco-2 cells. An improvement in gut dysbiosis of children with obesity seems to be triggered by DHA and to continue after discontinuation. The ability to modulate gut microbiota, matches also with an anti-inflammatory effect of DHA on Caco-2 cells.

Effect of pasteurization and storage on the bioavailability of milk proteins during in vitro intestinal digestion and Caco-2 cell transport

This study fills a specific knowledge gap by comprehensively investigating the intestinal digestive dynamics and transport kinetics of pasteurized milk proteins during cold storage, utilizing dynamic in vitro digestion models, human colorectal adenocarcinoma cell line (Caco-2) transport simulations, peptideomics and amino acid analysis. The content of free amino acids during intestinal digestion and their transport capacity (excluding lysine) were highest in pasteurized milk stored for 7 days (PAST-D7), intermediate in pasteurized milk stored for 0 days (PAST-D0) and lowest in raw milk (RM). Conversely, the quantity and intensity of bioactive peptides during digestion decreased with pasteurization and storage. Additionally, pasteurization and storage reduced the intensity of bioactive peptides transported through Caco-2 cells and the types and intensity of potential allergenic peptides. These findings indicate that while pasteurization and storage may diminish some physiological benefits of milk, they may also enhance protein bioavailability and reduce allergenicity.

Identification of COL3A1 as a candidate protein involved in the crosstalk between obesity and diarrhea using quantitative proteomics and machine learning

BackgroundIncreasing epidemiologic studies have shown a positive correlation between obesity and chronic diarrhea. Nevertheless, the precise etiology remains uncertain. MethodsWe performed a comprehensive proteomics analysis utilizing the data-independent acquisition (DIA) technique on jejunal tissues from patients with obesity and chronic diarrhea (OD, n = 33), obese patients (OB, n = 10), and healthy controls (n = 8). Differentially expressed proteins (DEPs) in OD vs. control and OD vs. OB comparisons were subjected to pathway enrichment and protein-protein interaction (PPI) network analysis. Machine learning algorithms were adopted on overlapping DEPs in both comparisons. The candidate protein was further validated using Western blot, immunohistochemistry (IHC), and in vitro experiments. ResultsWe identified 189 and 228 DEPs in OD vs. control and OD vs. OB comparisons, respectively. DEPs in both comparisons were co-enriched in extracellular matrix (ECM) organization. Downregulated DEPs were associated with tight junction and ECM-receptor interaction in OD vs. control and OD vs. OB comparisons, respectively. Machine learning algorithms selected 3 proteins from 14 overlapping DEPs in both comparisons, among which collagen alpha-1(III) chain (COL3A1) was identified as a core protein in PPI networks. Western blot and IHC verified the expression of COL3A1. Moreover, the tight junction-related proteins decreased after the knockdown of COL3A1 in Caco2 intestinal cells upon PA challenge, consistent with the proteomics results. ConclusionsWe generated in-depth profiling of a proteomic dataset from samples of OD patients and provided unique insights into disease pathogenesis. COL3A1 was involved in the crosstalk between obesity and intestinal homeostasis via the ECM-receptor interaction pathway.

Identification of Antioxidant Methyl Derivatives of Ortho-Carbonyl Hydroquinones That Reduce Caco-2 Cell Energetic Metabolism and Alpha-Glucosidase Activity.

α-glucosidase, a pharmacological target for type 2 diabetes mellitus (T2DM), is present in the intestinal brush border membrane and catalyzes the hydrolysis of sugar linkages during carbohydrate digestion. Since α-glucosidase inhibitors (AGIs) modulate intestinal metabolism, they may influence oxidative stress and glycolysis inhibition, potentially addressing intestinal dysfunction associated with T2DM. Herein, we report on a study of an ortho-carbonyl substituted hydroquinone series, whose members differ only in the number and position of methyl groups on a common scaffold, on radical-scavenging activities (ORAC assay) and correlate them with some parameters obtained by density functional theory (DFT) analysis. These compounds' effect on enzymatic activity, their molecular modeling on α-glucosidase, and their impact on the mitochondrial respiration and glycolysis of the intestinal Caco-2 cell line were evaluated. Three groups of compounds, according their effects on the Caco-2 cells metabolism, were characterized: group A (compounds 2, 3, 5, 8, 9, and 10) reduces the glycolysis, group B (compounds 1 and 6) reduces the basal mitochondrial oxygen consumption rate (OCR) and increases the extracellular acidification rate (ECAR), suggesting that it induces a metabolic remodeling toward glycolysis, and group C (compounds 4 and 7) increases the glycolysis lacking effect on OCR. Compounds 5 and 10 were more potent as α-glucosidase inhibitors (AGIs) than acarbose, a well-known AGI with clinical use. Moreover, compound 5 was an OCR/ECAR inhibitor, and compound 10 was a dual agent, increasing the proton leak-driven OCR and inhibiting the maximal electron transport flux. Additionally, menadione-induced ROS production was prevented by compound 5 in Caco-2 cells. These results reveal that slight structural variations in a hydroquinone scaffold led to diverse antioxidant capability, α-glucosidase inhibition, and the regulation of mitochondrial bioenergetics in Caco-2 cells, which may be useful in the design of new drugs for T2DM and metabolic syndrome.

Unlocking the nutraceutical potential of Corylus avellana L. shells: microwave-assisted extraction of phytochemicals with antiradical and anti-diabetic properties.

In recent years, the demand for high-quality natural extracts to be included in nutraceutical formulations has increased sharply. Hazelnut (Corylus avellana L.) shells (HZS) are underrated agricultural by-products that could be exploited as a source of active ingredients with pro-healthy properties. In the present study, a fully green microwave-assisted extraction (MAE) method was established for the first time aiming to recover bioactive constituents from HZS with significant nutraceutical value. Key MAE parameters, including ethanol in water concentration, microwave power, irradiation time and solvent-to-powder ratio, were optimized through response surface methodology utilizing a Box-Behnken design to achieve the highest total phenolic content and antioxidant/antiradical activities in the final extract. The optimal MAE conditions (28% v/v ethanol/water, 270 s, 670 W, and 37 mL g-1) yielded an extract with significant scavenging capacity against reactive oxygen species and remarkable inhibitory activity towards both α-amylase (IC50 = 7.73 μg mL-1) and α-glucosidase (IC50 = 49.44 μg mL-1), demonstrating stronger hypoglycaemic properties than the anti-diabetic drug acarbose. Additionally, fluorescence spectroscopy results highlighted the ability of the optimized extract from HZS (OHS-E) to counteract advanced glycation end-product formation throughout the glycation cascade in a dose-dependent manner. Liquid chromatography/electrospray ionization-tandem mass spectrometry profiling unveiled the presence of fatty acids and phenolic compounds, including lignans, flavonoids, gallic acid derivatives and diarylheptanoids. Lastly, the biocompatibility of OHS-E was attested on HT29-MTX and Caco-2 intestinal cells. Altogether, these findings encourage the potential application of OHS-E as an effective nutraceutical component against type 2 diabetes mellitus and oxidative stress. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Lipid-lowering effect and oral transport characteristics study of curculigoside.

The incidence of metabolic disorders during pregnancy is increasing year by year, with diseases including hypertension and hyperlipidemia. Statins are the primary drugs for treating hyperlipidemia or atherosclerosis, yet some patients remain unresponsive to them, and pregnant women are prohibited from taking statins. Curculigoside is the major biologically active natural product present in Curculigo orchioides. In this study, A high-fat mice model was developed to study the lipid-lowering effect of curculigoside. Using intestinal Caco-2 cell monolayer, the curculigoside transport properties at two temperatures and possible transporters were systemically studied. Curculigoside at concentrations used during the experiments have no toxic effect to Caco-2 cells. The curculigoside transfer from the apical to the basolateral side was strongly influenced by temperature. P-glycoprotein, breast cancer resistance protein, and efflux transporters are crucial components of the human intestinal cell line Caco-2. The curculigoside can significantly affect the contents of total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol in mice. The transport properties and potential mechanism of curculigoside offer valuable insights for the design of development of hypolipidemic drugs like anti-atherosclerotic drugs and also be helpful to the further study of the pharmacological activity of curculigoside.