Phytosterols are structurally similar to cholesterol but they are much less absorbed (<2%) than cholesterol (>50%) in the intestine. We hypothesize that phytosterols are poor substrates of intestinal acyl-CoA: cholesterol acyltransferase 2 (ACAT2), and thus minimal phytosterol esters are formed and packed into chylomicrons, leading to their low absorption. Two isotope tracing models, including a radioactive hamster microsomal ACAT2 reaction model and a differentiated Caco-2 cell model, were established to examine the specificity of ACAT2 to various sterols, including cholesterol, sitosterol, stigmasterol, and campesterol. Both models consistently demonstrated that only cholesterol but not phytosterols could be efficiently esterified by ACAT2 in a time- and dose-dependent manner. Molecular docking further suggested that unfavorable interactions existed between ACAT2 and phytosterols. In conclusion, phytosterols are poor substrates of ACAT2 and thus minimally absorbed. This work provides a theoretical basis for the use of phytosterol-based supplements in treating dyslipidemia and preventing heart diseases.

The main bioavailable phenolics from of Gongju (GJ) and their mechanism for hepato-protection remain unclear. To select the GJ phenolics with high bioavailability, chrysanthemum digestion and Caco-2 cells were used and their hepato-protective potential were examined by using AML-12 cells. The digestive recovery and small intestinal transit rate of the main phenolic compounds ranged from 28.52 to 69.53% and 6.57% ∼ 15.50%, respectively. Among them, chlorogenic acid, 3,5-dicaffeoylquinic acid, and 1,5-dicaffeoylquinic acid, showed higher small intestinal transit rates and digestive recoveries. Furthermore, we found that by increasing intracellular Catalase (CAT) and Superoxide dismutase (SOD) viability and lowering Malondialdehyde (MDA) level (P < 0.05), 3,5-dicaffeoylquinic acid significantly mitigated the oxidative damage of AML-12 liver cells more than the other two phenolics. Our results demonstrated that 3,5-dicaffeoylquninic acid was the primary phenolic compounds in GJ that effectively reduced liver damage, providing a theoretical basis for the development of GJ as a potentially useful resource for hepatoprotective diet.

This study examined the impact of Semen raphani on the absorption of ginsenosides from Panax ginseng C.A. Meyer (ginseng) using a Caco-2 cell model and Ultra-High-Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (UPLC-ESI-MS). Six primary ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rd) were quantified. Results showed that Semen Raphani increased the efflux rate of ginsenosides, particularly at higher concentrations, suggesting it inhibits their absorption. The research elucidates the intestinal absorption process of ginsenosides and the antagonistic mechanism of Semen Raphani against ginseng.

UNASSIGNED: Previous research has partially revealed distinct gut microbiota in ankylosing spondylitis (AS). In this study, we performed non-targeted fecal metabolomics in AS in order to discover the microbiome-metabolome interface in AS. Based on prospective cohort studies, we further explored the impact of the tumor necrosis factor inhibitor (TNFi) on the gut microbiota and metabolites in AS.
UNASSIGNED: To further understand the gut microbiota and metabolites in AS, along with the influence of TNFi, we initiated a prospective cohort study. Fecal samples were collected from 29 patients with AS before and after TNFi therapy and 31 healthy controls. Metagenomic and metabolomic experiments were performed on the fecal samples; moreover, validation experiments were conducted based on the association between the microbiota and metabolites.
UNASSIGNED: A total of 7,703 species were annotated using the metagenomic sequencing system and by profiling the microbial community taxonomic composition, while 50,046 metabolites were identified using metabolite profiling. Differential microbials and metabolites were discovered between patients with AS and healthy controls. Moreover, TNFi was confirmed to partially restore the gut microbiota and the metabolites. Multi-omics analysis of the microbiota and metabolites was performed to determine the associations between the differential microbes and metabolites, identifying compounds such as oxypurinol and biotin, which were correlated with the inhibition of the pathogenic bacteria Ruminococcus gnavus and the promotion of the probiotic bacteria Bacteroides uniformis. Through experimental studies, the relationship between microbes and metabolites was further confirmed, and the impact of these two types of microbes on the enterocytes and the inflammatory cytokine interleukin-18 (IL-18) was explored.
UNASSIGNED: In summary, multi-omics exploration elucidated the impact of TNFi on the gut microbiota and metabolites and proposed a novel therapeutic perspective: supplementation of compounds to inhibit potential pathogenic bacteria and to promote potential probiotics, therefore controlling inflammation in AS.

Okadaic Acid, a type of diarrhetic shellfish poison, is widely distributed and harmful, causing symptoms such as diarrhea, vomiting, and more in humans. Recent studies have demonstrated that OA can lead to various toxicities such as cytotoxicity, neurotoxicity, embryotoxicity, and hepatotoxicity. In order to investigate the immunotoxicity of OA on intestinal cells, a transcriptome analysis was conducted to compare the differences in the Caco-2 cell transcriptional group before and after administration. The CCK-8 experiment demonstrated that OA had a detrimental effect on the activity of Caco-2 cells, with an IC50 value of 33.98 nM. Transcriptome data revealed changes in immune-related genes between the experimental and control groups, including inflammatory factors, heat shock proteins, and zinc finger proteins. The analysis of the results suggests that OA can induce the production of inflammatory factors and apoptosis in cells, and may also affect cell ferroptosis. These findings indicate that OA has a significant impact on intestinal immunity, providing valuable insights for the study of immune toxicity associated with OA.

Phenolics of mulberry (Morus alba L.) leaves (MLs) have potential anti-diabetic effects, but they may be chemically modified during gastrointestinal digestion so affect their biological activity. In this study, an in vitro digestion model coupled with Caco-2 monolayer and Caco-2/insulin-resistant HepG2 coculture model were used to study the transport and hypoglycemic effects of phenolics in raw MLs (U-MLs) and solid-fermented MLs (F-MLs). The results of LC-MS/MS analysis showed that the Papp (apparent permeability coefficient, 10-6cm/s) of phenolics in digested MLs ranged from 0.002 ± 0.00 (quercetin 3-O-glucoside) to 60.19 ± 0.67 (ferulic acid), indicating higher phenolic acids absorbability and poor flavonoids absorbability. The Papp values of phenolic extracts of F-MLs in Caco-2 monolayer were significantly higher (p > 0.05) than that of U-MLs. Digested phenolic extracts inhibited the activities of sucrase (60.13 ± 2.03 %) and maltase (82.35 ± 0.78 %) and decreased 9.28 ± 0.84 % of glucose uptake in Caco-2 monolayer. Furthermore, a decrease in the mRNA expression of glucose transporters SGLT1 (0.64 ± 0.18), GLUT2 (0.14 ± 0.02) and the sucrase-isomaltase (0.59 ± 0.00) was observed. In Caco-2/insulin-resistant HepG2 co-culture model, phenolic extracts regulated glucose metabolism by up-regulating the mRNA expressions of IRS1 (9.32-fold), Akt (17.07-fold) and GYS2 (1.5-fold), and down-regulating the GSK-3β (0.22-fold), PEPCK (0.49-fold) and FOXO1 (0.10-fold) mRNA levels. Both U-MLs and F-MLs could improve glucose metabolism, and the partial least squares (PLS) analysis showed that luteoforol and p-coumaric acid were the primary phenolics that strongly correlated with the hypoglycemic ability of MLs. Results suggested that phenolics of MLs can be used as dietary supplements to regulate glucose metabolism.

In the former report, the casein peptide TKLTEEEKNR (PfCN) exhibits strong thrombin inhibitory activity in vitro. Its absorption capabilities, however, are unclear. Therefore, we studied its absorption characteristics both in vivo and in vitro. PfCN was carried by cells from the apical chamber to the basolateral chamber via active translocation in Caco-2 cells. Meanwhile, it can also be transported by HUVECs. We found that PfCN can be taken up by HUVECs using confocal laser imaging. PfCN has been proven to have good absorption properties in in vivo experiments. After five minutes of oral treatment, PfCN was identified in the blood, peaking at 82.75 ± 36.52 ng/mL in 30 min. And PfCN vanished from the blood circulation after 120 min. According to in vivo experiments, excessive concentrations of PfCN will alter the permeability of HUVECs. As a result, there is a foundation for PfCN application in the food sector. Meanwhile, we also hope this article can give an idea to the researchers who studying the absorption of functional peptides.

The aim of this study was to evaluate the in situ insulinotropic effects of pea protein hydrolysates (PPHs) mediated by active glucagon-like peptide-17-36 (active GLP-1) using a 2D and dual-layered coculture cell model. Following this model, a mixed Caco-2 and NCI-H716 cell monolayer was differentiated on the apical side to study the effects of PPHs on active GLP-1 levels; meanwhile, the beta-TC-6 cells were seeded on the basolateral side to investigate the insulin responses induced by active GLP-1. The in situ DPP-4 half-maximal inhibitory concentration (IC50) of PPHs, PPHs-120G, and PPHs-120I was 2.94, 3.43, and 2.26 mg/mL, respectively. They directly stimulated active GLP-1 secretion in NCI-H716 cells by 3.03 ± 0.21, 1.99 ± 0.03, and 2.24 ± 0.02 times, respectively. Insulin release in beta-TC-6 cells was directly stimulated by PPHs but not by PPHs-120G and PPHs-120I. Interestingly, PPHs-120G and PPHs-120I indirectly stimulated insulin release in this coculture cell model by enhancing active GLP-1 concentrations. More importantly, PPHs, PPHs-120G, and PPHs-120I increase active GLP-1 levels by their dual function of stimulating active GLP-1 secretion and DPP-4 inhibition. This study suggests that the 2D and dual-layered coculture cell model supports a more comprehensive assessment of in situ insulinotropic effects of protein hydrolysates mediated by active GLP-1.

BACKGROUND: Oral bioavailability (F), which is evaluated by permeability and solubility, is one of the key parameters in drug discovery. Currently, Caco-2 and Ussing chamber are both used in the study of intestinal permeability of drugs at different stages of drug development. However, comparative research between the Ussing chamber and Caco-2 for predicting the intestinal availability data (Fa×Fg) in humans has not been reported.
METHODS: In the present study, we evaluated the permeability of 22 drugs in rat intestines by Ussing chamber and compared them with the reported permeability data from Caco-2. In addition, the active transport of gabapentin was evaluated by Ussing Chamber.
RESULTS: Intestine segments were selected by corresponding absorption site for Ussing chamber analysis. BCS Class I and II compounds were more absorbed in the duodenum and jejunum, and Class III and IV compounds were more absorbed in the ileum. Papp values in the Caco-2 model were moderately correlated with human Fa×Fg (R2=0.722), and the Papp of the rat in the Ussing chamber revealed a better correlation with human Fa×Fg (R2=0.952). In addition, we also used the Ussing chamber to identify the transporter of gabapentin, and the results showed that the active absorption of gabapentin was related to LAT1.
CONCLUSIONS: Ussing chamber combined with rat intestinal tissue would be a significant tool for predicting the intestinal absorption and metabolism of compounds with diverse physiochemical characteristics.

The intestinal epithelium barrier serves as a highly dynamic immunologic frontier in the defense against invading pathogenic bacteria and viruses. Hence, understanding of the complicated underlying relationship between enteric pathogens and the intestinal epithelium barrier is vital for developing strategies to improve the intestinal health of farm animals. To this end, Caco-2 cells were stimulated by 1 µg/ml lipopolysaccharide (LPS) for 24 h and 5 µg/ml polyinosinic-polycytidylic acid (ploy(I:C)) for 4 h to imitate bacterial and viral infection processes, respectively. The specific alterations in gene expression of Caco-2 cells after stimulation were characterized by transcriptome sequencing. Seventy differentially expressed genes (DEGs) were identified under LPS exposure, and 17 DEGs were observed under ploy(I:C) exposure. We found that most DEGs were specific, and only one common DEG SPAG7 was observed. Gene Ontology (GO) annotation analysis indicated that all DEGs identified in the different treatments were mainly derived from GO terms related to the maintenance of cellular homeostasis. Moreover, specific DEGs such as SLC39A10, MT2A, and MT1E regulated by LPS treatment, while IFIT2 and RUNX2 mediated by ploy(I:C) treatment, which are derived from immune function modulation related GO terms, were confirmed by both transcriptome sequencing and qRT-PCR. In addition, both transcriptome sequencing and qRT-PCR results verified that LPS specifically down-regulated the DEGs INHBE and ARF6, which are involved in inflammation responses related to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway including the TGF-beta signaling pathways and the Ras signaling pathway. Ploy(I:C) uniquely suppressed the DEGs GABARAP and LAMTOR3, which participated in viral replication-associated pathways including autophagy and mTOR signaling pathway.