Human Intestinal Epithelial Cells Research Articles

Transcriptional responses of human intestinal epithelial HT-29 cells to spore-displayed p40 derived from Lacticaseibacillus rhamnosus GG

BackgroundsThe aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells.Resultsp40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action.ConclusionsThis study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40.

Epithelial-derived galectin-9 containing exosomes contribute to the immunomodulatory effects promoted by 2'-fucosyllactose and short-chain galacto- and long-chain fructo-oligosaccharides.

Early life exposure to non-digestible oligosaccharides (NDO) or microbial components is known to affect immune development. NDO in combination with a TLR9 agonist mimicking bacterial triggers (CpG) promoted the secretion of galectins through unknown pathways. We aimed to study the contribution of exosomes in epithelial galectin secretion and subsequent immunoregulation upon exposure to a mixture of NDO by inhibiting exosome biogenesis. Human intestinal epithelial cells (IEC) (FHs 74 Int or HT-29) were apically exposed to 2'-fucosyllactose (2'FL) and short-chain galacto- and long-chain fructo-oligosaccharides (GF), alone or with CpG. Basolaterally, non-activated or αCD3/CD28-activated peripheral blood mononuclear cells (PBMC) were added. After 24h incubation, IEC were washed and incubated in fresh medium to analyze epithelial-derived galectin secretion. Additionally, before exposure to NDO and CpG, IEC were exposed to GW4869 to inhibit exosome biogenesis. After 24h of incubation, IEC were washed and incubated for additional 24h in the presence of GW4869, after which epithelial-derived galectin secretion was studied. Also, epithelial-derived exosomes were isolated to study the presence of galectins within the exosomes. Compared to CpG alone, exposure to 2'FL/GF mixture and CpG, significantly enhanced Th1-type IFNγ, and regulatory IEC-derived galectin-9 secretion in the HT-29/PBMC model. Similarly, in the FHs 74 Int/PBMC co-culture, 2'FL/GF induced immunomodulatory effects in the absence of CpG. Interestingly, galectin-9 and -4 were present in CD63-expressing exosomes isolated from HT-29 supernatants after IEC/PBMC co-culture. Exposure to GW4869 suppressed 2'FL/GF and CpG induced epithelial-derived galectin-9 secretion, which subsequently prevented the rise in IL-10 and reduction in IL-13 secretion observed in the HT-29/PBMC co-culture model upon exposure to 2'FL/GF and CpG. Exposure to 2'FL/GF and CpG or 2'FL/GF promoted Th1-type regulatory effects in HT-29/PBMC or FHs 74 Int/PBMC co-culture respectively, while Th2-type IL-13 was reduced in association with increased galectin-9 release. Galectin-9 and -4 were present in exosomes from HT-29 and the inhibition of exosome biogenesis inhibited epithelial-derived galectin secretion. This, also affected immunomodulatory effects in IEC/PBMC co-culture suggesting a key role of galectin expressing IEC-derived exosomes in the mucosal immune regulation induced by NDO.

Culture media from hypoxia conditioned mast cells aggravates hypoxia and reoxygenation injury of human intestinal cells

Intestinal ischemia-reperfusion (II/R) injury is a common clinical and pathological change; however, its underlying mechanisms remain unclear. Previous studies have shown that the inflammatory response induced by mast cell degranulation may be involved in the mechanism underlying II/R injury in rats. In this study, we established a human intestinal epithelial adenocarcinoma cell (Caco-2) hypoxia/reoxygenation (H/R) model and transwell system to investigate the effects of culture media (CM) from hypoxia conditioned human mast cell (HMC-1) and HMC-1 H/R on hypoxia/reoxygenation injury in Caco-2 under H/R conditions. Moreover, we assessed the barrier function of Caco-2 by measuring the 4-kDa fluorescein isothiocyanate (FITC)-dextran (FD4) flux and the tight junction protein expression. The results concluded that Caco-2 exposed to H/R insult showed an increase in lactate dehydrogenase (LDH) release, cell apoptosis index, cell permeability, Bax expression, phosphorylation of c-Jun N-terminal protein kinase (JNK) and p38, and a decrease in cell viability and expression of Bcl-2, ZO1, and occludin (all P < 0.05). Notably, preincubating Caco-2 with HMC-1CM resulted in an increase in cell injury (increased LDH levels and cell permeability, decreased cell viability), apoptosis index, p-JNK, and p-38 expression and a decrease in ZO1 and occludin expression by co-culture system (all P < 0.05). In conclusion, our results show that HMC-1 hypoxic and reoxygenated CM aggravates hypoxic and reoxygenated injury in Caco-2 by increasing the phosphorylation of JNK and p38 in vitro.

Functional Properties of Dunaliella salina and Its Positive Effect on Probiotics.

The unicellular green microalga Dunaliella is a potential source of a wide range of nutritionally important compounds applicable to the food industry. The aim of this study was to assess the effect of Dunaliella salina dried biomass on the growth and adherence of 10 strains of Lactobacillus, Lacticaseibacillus, and Bifidobacterium. The immunomodulatory, antioxidant, and cytotoxic effects of D. salina on human peripheral mononuclear cells and simulated intestinal epithelial cell lines Caco-2 and HT-29 were evaluated. Furthermore, the hypocholesterolemic effects of the microalgae on lipid metabolism in rats fed a high-fat diet were analyzed. The addition of D. salina biomass had a positive effect on the growth of nine out of 10 probiotics and promoted the adherence of three bifidobacteria strains to human cell lines. The antioxidant and immunomodulatory properties of D. salina were concentration-dependent. The inflammatory cytokines (TNF-α and IL-6) were significantly increased following Dunaliella stimulation at the lowest concentration (0.5% w/v). Eight week supplementation of D. salina to the diet of hypercholesteromic rats significantly decreased the serum concentrations of LDL-C, VLDL, IDL-B, and IDL-C. D. salina is not cytotoxic in intestinal cell models; it promotes adherence of selected bifidobacteria, it affords immunomodulatory and antioxidant effects, and its addition to diets may help decrease atherosclerosis risk factors.

Label-free relative quantitative proteomics reveals extracellular vesicles as a vehicle for Salmonella effector protein delivery.

Extracellular vesicles are small vesicles with a diameter of 30-150 nm that are actively secreted by eukaryotic cells and play important roles in intercellular communication, immune responses, and tumorigenesis. Previous studies have shown that extracellular vesicles are involved in the process of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. However, changes in the protein content of extracellular vesicles elicited by S. Typhimurium infection have not been determined. Here, we extracted the extracellular vesicles with high purity from S. Typhimurium-infected Henle-407 cells, a kind of human intestinal epithelial cells, by ultracentrifugation combined with an extracellular vesicles purification kit, and analyzed their protein composition using label-free relative quantitative proteomics. The extracted extracellular vesicles exhibited an oval vesicular structure under electron microscopy, with a mean diameter of 140.4 ± 32.4 nm. The exosomal marker proteins CD9, CD63, and HSP70 were specifically detected. Compared with the uninfected group, nearly 1,234 specifically loaded proteins were uncovered in S. Typhimurium-infected Henle-407 cells. Among them were 409 S. Typhimurium-derived specific proteins, indicating a significant alteration in protein composition of extracellular vesicles by S. Typhimurium infection. Notably, these proteins included 75 secretory proteins and over 300 non-secretory proteins of S. Typhimurium, implicating novel pathways for bacterial protein delivery, although it remains unclear if their loading into extracellular vesicles is active or passive. To investigate the roles of these extracellular proteins, we exemplified the function of SopB, a well-known T3SS effector protein, and showed that the extracellular SopB could be taken up by RAW264.7 macrophages, activating the phosphorylation of Akt. This study provides new insights into the mechanism of Salmonella infection through extracellular vesicles that transport virulence proteins to uninfected neighboring cells to facilitate further infection.

Natural statin derivatives as potential therapy to reduce intestinal fluid loss in cholera.

As a leading cause of death in children under 5 years old, secretory diarrheas including cholera are characterized by excessive intestinal fluid secretion driven by enterotoxin-induced cAMP-dependent intestinal chloride transport. This study aimed to identify fungal bioactive metabolites possessing anti-secretory effects against cAMP-dependent chloride secretion in intestinal epithelial cells. Using electrophysiological analyses in human intestinal epithelial (T84) cells, five fungus-derived statin derivatives including α,β-dehydrolovastatin (DHLV), α,β-dehydrodihydromonacolin K, lovastatin, mevastatin and simvastatin were found to inhibit the cAMP-dependent chloride secretion with IC50 values of 1.8, 8.9, 11.9, 11.4 and 5 μM, respectively. Being the most potent statin derivatives, DHLV was evaluated for its pharmacological properties including cellular toxicity, mechanism of action, target specificity and in vivo efficacy. DHLV at concentrations up to 20 μM did not affect cell viability and barrier integrity of T84 cells. Electrophysiological analyses indicated that DHLV inhibited cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent apical chloride channel, via mechanisms not involving alteration of intracellular cAMP levels or its negative regulators including AMP-activated protein kinases and protein phosphatases. DHLV had no effect on Na+-K+ ATPase activities but inhibited Ca2+-dependent chloride secretion without affecting intracellular Ca2+ levels. Importantly, intraperitoneal (2 mg/kg) and intraluminal (20 μM) injections of DHLV reduced cholera toxin-induced intestinal fluid secretion in mice by 59% and 65%, respectively without affecting baseline intestinal fluid transport. This study identifies natural statin derivatives as novel natural product-derived CFTR inhibitors, which may be beneficial in the treatment of enterotoxin-induced secretory diarrheas including cholera.

Magnolia officinalis Bark Extract Prevents Enterocyte Death in a Colitis Mouse Model by Inhibiting ROS-Mediated Necroptosis.

Necroptosis is a form of programmed cell death with features of necrosis and apoptosis that occurs in the intestinal epithelium of patients with inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease. In addition, necroptosis has also been observed in enterocytes in animal models of dextran sulfate sodium (DSS)-induced colitis. Thus, the discovery of natural products for regulating necroptosis may represent an important therapeutic strategy for improving IBD. We found that Magnolia officinalis bark extract (MBE) prevented weight loss and suppressed the activation of the proinflammatory cytokine IL6 in DSS-induced colitis. Furthermore, MBE restored the length of the damaged colon and decreased the expression of necroptosis markers in mice with DSS-induced colitis. In vitro, necroptosis-induced reactive oxygen species (ROS) production was reduced by MBE, and the expression of COX2, a target protein of ROS, was simultaneously suppressed. Both magnolol and honokiol, the two major bioactive compounds in MBE, inhibited necroptosis in human primary intestinal epithelial cells and colorectal adenocarcinoma cells. Our findings highlight the effectiveness of MBE in modulating enterocyte necroptosis and suggest that MBE may be developed as a natural, disease-targeting drug for the treatment of colitis.

Green/Roasted Coffee and Silverskin Extracts Inhibit Sugar Absorption by Human Intestinal Epithelial (Caco-2) Cells by Decreasing GLUT2 Gene Expression.

Moderate coffee ingestion has been associated with a decrease in type 2 diabetes risk, mainly due to its richness in chlorogenic acids (CGA). To explore this, extracts of green beans, roasted beans, and silverskin were prepared by aqueous ultrasound-assisted extraction and characterized by a reversed-phase high-performance liquid chromatography-photodiode array detector (RP-HPLC-DAD). The effects on the uptake of glucose and fructose by human intestinal epithelial (Caco-2) cells and the influence on the expression of sugar transporter genes (by RT-qPCR) were investigated and compared. The uptake of 3H-deoxy-D-glucose and 14C-fructose by Caco-2 cells was significantly reduced by all the extracts, with green coffee (which also contained higher amounts of CGA) achieving the highest efficiency. Although silverskin presented the lowest amounts of CGA and caffeine, it promoted an inhibitory effect similar to the effects of green/roasted beans. In the case of glucose uptake, the effect was even higher than for roasted coffee. This activity is explained by the ability of the extracts to markedly decrease GLUT2, but not GLUT5 gene expression. In addition, a decrease in SGLT1 gene expression was also found for all extracts, although not at a statistically significant rate for silverskin. This study also revealed a synergistic inhibitory effect of caffeine and 5-CQA on the uptake of sugars. Thus, silverskin appears as an interesting alternative to coffee, since the valorization of this by-product also contributes to the sustainability of the coffee chain.

Cytotoxicity of a Novel Compound Produced in Foods via the Reaction of Amino Acids with Acrolein along with Formaldehyde.

Acrolein (ACR) and formaldehyde (FA) are toxic aldehydes co-produced in foods. This work found that amino acids, the nucleophiles ubiquitously existing in foods, can react simultaneously with them. Six amino acids, including γ-aminobutyric acid (GABA), glycine, alanine, serine, threonine, and glutamine, can scavenge ACR and FA at 37, 85, and 160 °C. GABA had the highest scavenging capacity for ACR and FA, by 79 and 13% at 37 °C for 2 h, and 99 and 48% at 160 °C for 30 min, respectively. Moreover, a new type of compound with a basic structure of 5-formyl-3-methylene-3,6-dihydropyridin was identified in all reactions and formed by 1 molecule of FA and amino acid and 2 molecules of ACR. The content of this compound was higher than that of free ACR in typical thermally processed foods. Moreover, the compounds produced from different amino acids showed different cytotoxicity values. In gastric epithelial and human intestinal epithelial cell lines, the cytotoxicity values of serine-sourced and threonine-sourced products were lower than that of ACR but higher than that of FA, whereas others had less toxicity compared with the two aldehydes. Considering that the content of serine-sourced products was the highest in almost all tested foods, their safety needs to be evaluated.

The flavonoids extract from Okra flowers protects against DSS-induced colitis via regulating NF-κB signaling pathway and gut microbiota

Okra flower is an untapped flavonoid-rich resource, and flavonoids from it (AFE) have been demonstrated intestinal protective potential due to anti-colorectal cancer properties, but other intestinal-related effects remain to be explored. Here, we found that AFE, as a safe natural product, effectively alleviate 2.5% dextran sulfate sodium (DSS)-induced intestinal inflammation in C57BL/6N and DSS-induced injury of human intestinal epithelial cells NCM460. AFE maintained colon integrity by promoting the levels of tight junction proteins, ZO-1 and Occludin, and modulating the NF-κB signaling pathway to prevent further tissue damage. Meanwhile, gut microbiota was involved in the protective effect of AFE in vivo, with Akkermansia making a major contribution. Moreover, AFE exhibited similar inhibitory effects on the NF-κB pathway in vitro and exerted antioxidant effects as a ROS scavenger by regulating the Nrf2 signaling pathway. In summary, this study demonstrated the potential of AFE in restoring intestinal health in vitro and in vivo.

Transient receptor potential melastatin 7 aggravates necrotizing enterocolitis by promoting an inflammatory response in children.

As a rare disease in children, necrotizing enterocolitis (NEC) leads to high morbidity and mortality. However, its pathophysiology is largely unclear. Transient receptor potential melastatin 7 (TRPM7) is a membrane protein, which plays key roles in the inflammatory response. This study sought to examine the promoting effect of TRPM7 on NEC in children and explore the therapeutic effect of a TRPM7 inhibitor NS8593. First, we detected TRPM7 and NLR family pyrin domain containing 3 (NLRP3) expression and the state of inflammation in children with NEC through quantitative real-time polymerase chain reaction (RT-PCR), Western blot, and enzyme-linked immunosorbent assays. Next, Human intestinal epithelial cell lines were induced to NEC by lipopolysaccharides (LPSs). The level of cytokines and reactive oxygen species (ROS) were tested by RT-PCR and flow cytometry. The TRPM7 mediated calcium flux were determined by fluorescence. In addition, we used the TRPM7 inhibitor NS8593 to treat the in vivo rat model. The mRNA and protein expression were determined by real-time PCR and Elisa analysis, respectively. TRPM7 and NLRP3 expression was more increased in the samples from children with NEC compared to the control samples. Additionally, the elevated secretion of interleukin-1β, interleukin-6, and tumor necrosis factor alpha was also detected in the serum of children with NEC. These results showed that TRPM7 had a promoting effect on NEC development, possibly via the activation of NLRP3. To test our hypothesis, the TRPM7 inhibitor NS8593 was used to treat the LPS-stimulated IEC-6 cells. We found that the TRPM7 inhibitor NS8593 inhibited LPS-induced cytokine production and exhibited an anti-inflammatory effect by alleviating TRPM7-mediated NLRP3 inflammasome activation. Through in-vivo experiments, we found that TRPM7 was involved in the occurrence of NEC, and its inhibitor NS8593 played a certain therapeutic role in the rat model. Our study revealed TRPM7 inhibitors attenuated LPS-induced ROS and reduced the release of pro-inflammatory cytokines. It also exhibited protective effects on the NEC model.

Whole transcriptome analysis of HCT-8 cells infected by Cryptosporidium parvum

BackgroundCryptosporidium species are zoonotic protozoans that are important causes of diarrhoeal disease in both humans and animals. Non-coding RNAs (ncRNAs) play an important role in the innate immune defense against Cryptosporidium infection, but the underlying molecular mechanisms in the interaction between human ileocecal adenocarcinoma (HCT-8) cells and Cryptosporidium species have not been entirely revealed.MethodsThe expression profiles of messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) in the early phase of infection of HCT-8 cells with Cryptosporidium parvum and at 3 and 12 h post infection were analyzed using the RNA-sequencing technique. The biological functions of differentially expressed RNAs (dif-RNAs) were discovered through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The targeting relationships between three ncRNAs and mRNAs were analyzed using bioinformatics methods, followed by building a competing endogenous RNA (ceRNA) regulatory network centered on miRNAs.ResultsAfter strictly filtering the raw data, our analysis revealed 393 dif-lncRNAs, 69 dif-miRNAs and 115 dif-mRNAs at 3 hpi, and 450 dif-lncRNAs, 129 dif-miRNAs, 117 dif-mRNAs and one dif-circRNA at 12 hpi. Of these, 94 dif-lncRNAs, 24 dif-miRNAs and 22 dif-mRNAs were detected at both post-infection time points. Eleven dif-lncRNAs, seven dif-miRNAs, eight dif-mRNAs and one circRNA were randomly selected and confirmed using the quantitative real-time PCR. Bioinformatics analyses showed that the dif-mRNAs were significantly enriched in nutritional absorption, metabolic processes and metabolism-related pathways, while the dif-lncRNAs were mainly involved in the pathways related to the infection and pathogenicity of C. parvum (e.g. tight junction protein) and immune-related pathways (e.g. cell adhesion molecules). In contrast, dif-miRNAs and dif-circRNA were significantly enriched in apoptosis and apoptosis-related pathways. Among the downregulated RNAs, the miRNAs has-miR-324-3p and hsa-miR-3127-5p appear to be crucial miRNAs which could negatively regulate circRNA, lncRNA and mRNA.ConclusionsThe whole transcriptome profiles of HCT-8 cells infected with C. parvum were obtained in this study. The results of the GO and KEGG pathway analyses suggest significant roles for these dif-RNAs during the course of C. parvum infection. A ceRNA regulation network containing miRNA at its center was constructed for the first time, with hsa-miR-324-3p and hsa-miR-3127-5p being the crucial miRNAs. These findings provide novel insights into the responses of human intestinal epithelial cells to C. parvum infection.Graphical

Short-chain fatty acids ameliorate necrotizing enterocolitis-like intestinal injury through enhancing Notch1-mediated single immunoglobulin interleukin-1-related receptor, toll-interacting protein, and A20 induction.

Single immunoglobulin interleukin-1-related receptor (SIGIRR), toll-interacting protein (TOLLIP), and A20 are major inhibitors of toll-like receptor (TLR) signaling induced postnatally in the neonatal intestine. Short-chain fatty acids (SCFAs), fermentation products of indigestible carbohydrates produced by symbiotic bacteria, inhibit intestinal inflammation. Herein, we investigated the mechanisms by which SCFAs regulate SIGIRR, A20, and TOLLIP expression and mitigate experimental necrotizing enterocolitis (NEC). Butyrate induced NOTCH activation by repressing sirtuin 1 (SIRT1)-mediated deacetylation of the Notch intracellular domain (NICD) in human intestinal epithelial cells (HIECs). Overexpression of NICD induced SIGIRR, A20, and TOLLIP expression. Chromatin immunoprecipitation revealed that butyrate-induced NICD binds to the SIGIRR, A20, and TOLLIP gene promoters. Notch1-shRNA suppressed butyrate-induced SIGIRR/A20 upregulation in mouse enteroids and HIEC. Flagellin (TLR5 agonist)-induced inflammation in HIEC was inhibited by butyrate in a SIGIRR-dependent manner. Neonatal mice fed butyrate had increased NICD, A20, SIGIRR, and TOLLIP expression in the ileal epithelium. Butyrate inhibited experimental NEC-induced intestinal apoptosis, cytokine expression, and histological injury. Our data suggest that SCFAs can regulate the expression of the major negative regulators of TLR signaling in the neonatal intestine through Notch1 and ameliorate experimental NEC. Enteral SCFAs supplementation in preterm infants provides a promising bacteria-free, therapeutic option for NEC.NEW & NOTEWORTHY Short-chain fatty acids (SCFAs), such as propionate and butyrate, metabolites produced by symbiotic gut bacteria are known to be anti-inflammatory, but the mechanisms by which they protect against NEC are not fully understood. In this study, we reveal that SCFAs regulate intestinal inflammation by inducing the key TLR and IL1R inhibitors, SIGIRR and A20, through activation of the pluripotent transcriptional factor NOTCH1. Butyrate-mediated SIGIRR and A20 induction represses experimental NEC in the neonatal intestine.

Milk-Derived miR-22-3p Promotes Proliferation of Human Intestinal Epithelial Cells (HIECs) by Regulating Gene Expression.

MicroRNA (miRNA) is small non-coding RNA involved in gene silencing and post-transcriptional regulation of gene expression. Milk exosomes are microvesicles containing microRNAs (miRNAs). miR-22-3p (miR-22) is plentiful in human milk exosomes and may contribute to intestinal development since milk exosomes and microRNAs are resistant to gastrointestinal digestion in infants. After miR-22 mimics were transfected to human intestinal crypt-like epithelial cells (HIECs) using Lipofectamine for 24 h, RNA was isolated for microarray assay. Microarray results show that miR-22 markedly regulates gene expression, and the roles of miR-22 include promotion of proliferation, regulation of immune functions, and inhibition of apoptosis. Based on the microarray results and miR-22 predicted target genes, CCAAT/enhancer-binding protein δ (C/EBPδ) may be an important direct target of miR-22. C/EBPδ is a transcription factor that regulates numerous biological processes including cell proliferation. In miR-22 transfected HIECs, expression of the C/EBPδ gene was significantly inhibited. Silencing of the C/EBPδ gene by siRNA resulted in increased proliferation of HIECs. A luciferase assay showed that miR-22 specifically binds to the 3'-untranslated region of C/EBPδ mRNA. In summary, milk-derived miR-22 promotes intestinal proliferation by modifying gene expression, and C/EBPδ may be an important target for miR-22 involved in this effect.

Modelling of p-tyramine transport across human intestinal epithelial cells predicts the presence of additional transporters.

p-Tyramine (TYR) is an endogenous trace amine, which can also be synthesized by intestinal microbiota, and is present in commonly consumed diets. TYR is an agonist for the intracellular trace amine-associated receptor 1, which has been implicated in psychiatric, metabolic, and immune-related disorders. We have previously demonstrated TYR readily diffuses across lipid bilayers, while transport across Caco-2 cell membranes involves Organic Cation Transporter 2 (OCT2) and a Na+-dependent active transporter. Here we developed mathematical models to determine whether known kinetics for these processes are sufficient to explain observed transcellular TYR passage. Ordinary differential equations were developed for known TYR transport processes to predict concentration-time relationships. Michaelis-Menten kinetics were assumed for all transporter-mediated processes and a one phase exponential function used for simple diffusion. Modelled concentration-time plots were compared to published experimental results. Additional transporter functions were sequentially added to models to improve consistency, and a least squares error minimization approach utilized to determine added transporter kinetics. Finally, possible TYR compartmentalization was also modelled. Following apical loading, transport across the apical, but not the basolateral, membrane was modelled without additional transporters, suggesting a basolateral transporter was missing. Consistent with this, models of basolateral compartment loading did not match experimental observations, indicating missing basolateral transporters were bidirectional. Addition of a transporter with the kinetic characteristics of OCT2 did not improve models. Varying the kinetic parameters of the added transporter improved models of basolateral, but worsened apical, loading models, suggesting the need for either a directional preference in transporters, or intracellular TYR compartmentalization. Experimental parameters were recapitulated by introducing asymmetry into the apical OCT2 (Kt_OCT2_apicaltocell = 110.4 nM, Kt_OCT2_celltoapical = 1,227.9nM), and a symmetric basolateral facilitated diffusion transporter (Vmax = 6.0nM/s, Kt = 628.3nM). The apparent directionality of OCT2 may reflect altered TYR ionization due to known pH differences between compartments. Models for asymmetry and compartmentalization were compared by root mean square deviation from experimental data, and it was found that TYR compartmentalization could only partially replace the need for asymmetry of OCT2. In conclusion, modelling indicates that known TYR transport processes are insufficient to explain experimental concentration-time profiles and that asymmetry of the apical membrane OCT2 combined with additional, low affinity, basolateral membrane facilitated diffusion transporters are required.

Acute cytomegalovirus infection modulates the intestinal microbiota and targets intestinal epithelial cells.

Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive - in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to assess the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes coincided with high-titer MCMV replication in the colon, crypt hyperplasia, increased colonic pro-inflammatory cytokine levels, and a transient increase in the expression of the antimicrobial protein Regenerating islet-derived protein 3 gamma (Reg3γ). Further analyses revealed that murine and human intestinal epithelial cell lines, as well as primary intestinal crypt cells and organoids represent direct targets of CMV infection causing increased cell death. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV transiently induces colitis in immunocompetent hosts by altering the intestinal homeostasis.

PGC-1α in mediating mitochondrial biogenesis and intestinal epithelial differentiation promoted by purple potato extract

• Purple potato polyphenolic extract (PPE) promotes gut epithelial differentiation. • PPE upregulates PGC-1α and enhances mitochondria biogenesis in Caco-2 cells. • PPE promotes intracellular oxidative metabolism. • PGC-1α is indispensable in PPE-induced differentiation & mitochondrial biogenesis. Intestinal epithelial differentiation plays an important role in maintaining gut health and is impaired due to many diseases. Purple potatoes are rich in polyphenols that promote the differentiation of intestinal epithelial cells. In this study, we found that purple potato (PP) extract up-regulated the level of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, the expression of mitochondrial-related enzymes, the content of mitochondrial DNA, and the levels of tricarboxylic acid cycle metabolites in human intestinal epithelial Caco-2 cells. Inhibition of PGC-1α by either PGC-1α inhibitor SR-18292 or siRNA-mediated knockdown eliminated the enhancing effects of PP extract on the expression of differentiation markers, mitochondrial metabolite contents and mitochondrial DNA copy number, as well as the expression of mitochondrial DNA-encoded enzymes. In summary, PP extract promoted mitochondrial biogenesis and the differentiation of intestinal epithelial cells, and these promotion effects are dependent on the activation of PGC-1α.

Physicochemical and biopharmaceutical characterization of new sulfonamide derivatives of gallic acid

Gallic acid (GA) is known for its antioxidant activitywhichis restricted due to its low oral permeability. In this project the carboxylic group of (GA) was substitutedwith sulfonamide group and hydroxyl groups were methylated which resulted insignificantly (p&amp;lt;0.01) increased permeability of 3,4,5-trimethoxybenzenesulfonamide (TMBS) and 3,4,5-trihydroxybenzenesulfonamide (THBS) over GA, inParallel Artificial Membrane Permeability Assay studies with simulatedgastrointestinal fluids and Human intestinal epithelial cells HIEC-6 cells.Biochemical studies confirmed TMBS was O-demethylated by CYP2D6. THBS and GAhad increased antioxidant activity with increased concentration in DPPH assaywhile TMBS indicated lower activity at all tested concentration. Theantioxidant activity of TMBS was greater than GA in HIEC-6 cells which mainlyrelated to its O-demethylation by CYP2D6.&amp;nbsp;

In Vitro Evaluation of Anti-Rotaviral Activity and Intestinal Toxicity of a Phytotherapeutic Prototype of Achyrocline bogotensis (Kunth) DC.

Viruses represent the primary etiologic agents (70-80%) of acute diarrheal disease (ADD), and rotavirus (RV) is the most relevant one. Currently, four rotavirus vaccines are available. However, these vaccines do not protect against emerging viral strains or are not available in low-income countries. To date, there are no approved drugs available against rotavirus infection. In this study, we evaluated the in vitro anti-rotaviral activity and intestinal toxicity of a phytotherapeutic prototype obtained from Achyrocline bogotensis (Kunth) DC. (PPAb); medicinal plant that contains compounds that inhibit the rotavirus replication cycle. Virucidal and viral yield reduction effects exerted by the PPAb were evaluated by immunocytochemistry and flow cytometry. Furthermore, the toxic impact of the PPAb was evaluated in polarized human intestinal epithelial C2BBe1 cells in terms of cytotoxicity, loss of cytoplasmic membrane asymmetry, and DNA fragmentation by MTT and fluorometry. PPAb concentrations under 0.49 mg/mL exerted significant virucidal and viral yield reduction activities, and concentrations under 16 mg/mL neither reduced cell viability, produced DNA fragmentation, nor compromised the C2BBe1cell membrane stability after 24-h incubation. Based on these results, the evaluated phytotherapeutic prototype of Achyrocline bogotensis might be considered as a promising alternative to treat ADD caused by rotavirus.

Ginsenoside Rc attenuates DSS-induced ulcerative colitis, intestinal inflammatory, and barrier function by activating the farnesoid X receptor.

Objectives: Farnesoid X receptor (FXR) activation is involved in ameliorating inflammatory bowel disease (IBD), such as ulcerative colitis (UC), and inflammatory regulation may be involved in its mechanism. Ginsenoside Rc (Rc) is a major component of Panax ginseng, and it plays an excellent role in the anti-inflammatory processes. Our aim is to explore the alleviative effect of Rc on dextran sulfate sodium (DSS)-induced inflammation and deficiencies in barrier function based on FXR signaling. Materials and Methods: In vitro, we treated human intestinal epithelial cell lines (LS174T) with LPS to explore the anti-inflammatory effect of Rc supplementation. In vivo, a DSS-induced IBD mice model was established, and the changes in inflammatory and barrier function in colons after Rc treatment were measured using the disease activity index (DAI), hematoxylin and eosin (H&E) staining, immunofluorescence, ELISA, and qPCR. Molecular docking analysis, luciferase reporter gene assay, and qPCR were then used to analyze the binding targets of Rc. DSS-induced FXR-knockout (FXR-/-) mice were used for further validation. Results: Rc significantly recovered the abnormal levels of inflammation indexes (TNF-α, IL-6, IL-1β, and NF-KB) induced by LPS in LS174T. DSS-induced C57BL/6 mice exhibited a significantly decreased body weight and elevated DAI, as well as a decrease in colon weight and length. Increased inflammatory markers (TNF-α, IL-6, IL-1β, ICAM1, NF-KB, F4/80, and CD11b displayed an increased expression) and damaged barrier function (Claudin-1, occludin, and ZO-1 displayed a decreased expression) were observed in DSS-induced C57BL/6 mice. Nevertheless, supplementation with Rc mitigated the increased inflammatory and damaged barrier function associated with DSS. Further evaluation revealed an activation of FXR signaling in Rc-treated LS174T, with FXR, BSEP, and SHP found to be upregulated. Furthermore, molecular docking indicated that there is a clear interaction between Rc and FXR, while Rc activated transcriptional expression of FXR in luciferase reporter gene assay. However, these reversal abilities of Rc were not observed in DSS-induced FXR-/- mice. Conclusion: Our findings suggest that Rc may ameliorate inflammation and barrier function in the intestine, which in turn leads to the attenuation of DSS-induced UC, in which Rc may potentially activate FXR signaling to protect the intestines from DSS-induced injury.