Inhibition of Transglutaminase 2 as a Therapeutic Strategy in Celiac Disease-In Vitro Studies in Intestinal Cells and Duodenal Biopsies.

Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a key mechanism in the pathogenesis of celiac disease (CD) and represents a potential therapeutic target. Recently, we have identified the small oxidative molecule PX-12 as an effective inhibitor of TG2 in vitro. In this study, we further investigated the effect of PX-12 and the established active-site directed inhibitor ERW1041 on TG2 activity and epithelial transport of gliadin peptides. We analyzed TG2 activity using immobilized TG2, Caco-2 cell lysates, confluent Caco-2 cell monolayers and duodenal biopsies from CD patients. TG2-mediated cross-linking of pepsin-/trypsin-digested gliadin (PTG) and 5BP (5-biotinamidopentylamine) was quantified by colorimetry, fluorometry and confocal microscopy. Cell viability was tested with a resazurin-based fluorometric assay. Epithelial transport of promofluor-conjugated gliadin peptides P31-43 and P56-88 was analyzed by fluorometry and confocal microscopy. PX-12 reduced TG2-mediated cross-linking of PTG and was significantly more effective than ERW1041 (10 µM, 15 ± 3 vs. 48 ± 8%, p < 0.001). In addition, PX-12 inhibited TG2 in cell lysates obtained from Caco-2 cells more than ERW1041 (10 µM; 12 ± 7% vs. 45 ± 19%, p < 0.05). Both substances inhibited TG2 comparably in the intestinal lamina propria of duodenal biopsies (100 µM, 25 ± 13% vs. 22 ± 11%). However, PX-12 did not inhibit TG2 in confluent Caco-2 cells, whereas ERW1041 showed a dose-dependent effect. Similarly, epithelial transport of P56-88 was inhibited by ERW1041, but not by PX-12. Cell viability was not negatively affected by either substance at concentrations up to 100 µM. PX-12 did not reduce TG2 activity or gliadin peptide transport in confluent Caco-2 cells. This could be caused by rapid inactivation or degradation of the substance in the Caco-2 cell culture. Still, our in vitro data underline the potential of the oxidative inhibition of TG2. The fact that the TG2-specific inhibitor ERW1041 reduced the epithelial uptake of P56-88 in Caco-2 cells further strengthens the therapeutic potential of TG2 inhibitors in CD.

Effects of Various Marine Toxins on the Mouse Intestine Organoid Model.

Because of their trace existence, exquisite structure and unique role, highly toxic marine biotoxins have always led to the development of natural product identification, structure and function research, chemistry and biosynthesis, and there are still many deficiencies in the injury and protection of highly toxic organisms, toxin biosynthesis, rapid detection, poisoning and diagnosis and treatment. In this study, a mouse intestine organoid (MIO) model was constructed to explore the effects of the marine toxins okadaic acid (OA) and conotoxin (CgTx) on MIO. The results showed that the cell mortality caused by the two toxins at middle and high concentrations was significantly higher than the cell mortality of the control group, the ATPase activity in each group exposed to OA was significantly lower than the ATPase activity of the control group, all the CgTx groups were significantly higher than that of the control group, and the number of apoptotic cells was not significantly higher than the number of apoptotic cells of the control group. Through RNA-Seq differential genes, Gene Ontology (GO) and pathway analysis, and Gene Set Enrichment Analysis (GSEA) experimental results, it was demonstrated that OA reduced cell metabolism and energy production by affecting cell transcription in MIO. Ultimately, cell death resulted. In contrast, CgTx upregulated the intracellular hormone metabolism pathway by affecting the nuclear receptor pathway of MIO, which resulted in cell death and the generation of energy in large amounts.

Different Effects of Lard and Vegetable Blend Oil on Intestinal Microorganisms, Enzyme Activity and Blood Routine in Mice.

The intake of moderate oils and fats is necessary to maintain the body's energy balance, and the fatty acid composition of different oils and fats varies in their nutrition and function. The study aimed to investigate the effects of lard and vegetable blend oil on gut microbiota, intestinal enzyme activities, and blood routine. Kunming mice were assigned to the three groups: (1) Control group (CK) was gavage administration with distilled water, (2) Plant oil group (ZWY) was gavage administration with edible vegetable blend oil, (3) Lard group (DWY) was gavage administration with lard. After 42 days, microbiological, digestive enzymes, and blood routine were performed. Compared with the CK group, Escherichia coli, Lactobacilli, and Bifidobacteria were significantly decreased (p < 0.05), the activities of protease, cellulase, amylase, and xylanase were markedly reduced (p < 0.05), the hemoglobin was significantly increased (p < 0.05) in the ZWY group and DWY groups, and the hematocrit was increased in the ZWY group (p < 0.05), while other routine blood indices were increased (p > 0.05). Compared to the ZWY group, the activity of cellulase and amylase were significantly increased (p < 0.05), the intestinal microorganism and the routine blood indexes had no significant difference in the DWY group. Lard and vegetable blend oil diet affected the composition of the intestinal microorganisms, and the functions of digestive enzymes. Meanwhile, the levels of digestive enzymes may be correlated with the intestinal microbiota.

Microcystin-leucine arginine exposure induced intestinal lipid accumulation and MC-LR efflux disorder in Lithobates catesbeianus tadpoles.

Few studies exist on the toxic effects of chronic exposure to microcystins (MCs) on amphibian intestines, and the toxicity mechanisms are unclear. Here, we evaluated the impact of subchronic exposure (30 days) to environmentally realistic microcystin-leucine arginine (MC-LR) concentrations (0 µg/L, 0.5 µg/L and 2 µg/L) on tadpole (Lithobates catesbeianus) intestines by analyzing the histopathological and subcellular microstructural damage, the antioxidative and oxidative enzyme activities, and the transcriptome levels. Histopathological results showed severe damage accompanied by inflammation to the intestinal tissues as the MC-LR exposure concentration increased from 0.5 µg/L to 2 µg/L. RNA-sequencing analysis identified 634 and 1,147 differentially expressed genes (DEGs) after exposure to 0.5 µg/L and 2 µg/L MC-LR, respectively, compared with those of the control group (0 µg/L). Biosynthesis of unsaturated fatty acids and the peroxisome proliferator-activated receptor (PPAR) signaling pathway were upregulated in the intestinal tissues of the exposed groups, with many lipid droplets being observed on transmission electron microscopy, implying that MC-LR may induce lipid accumulation in frog intestines. Moreover, 2 µg/L of MC-LR exposure inhibited the xenobiotic and toxicant biodegradation related to detoxification, implying that the tadpoles' intestinal detoxification ability was weakened after exposure to 2 µg/L MC-LR, which may aggravate intestinal toxicity. Lipid accumulation and toxin efflux disorder may be caused by MC-LR-induced endoplasmic reticular stress. This study presents new evidence that MC-LR harms amphibians by impairing intestinal lipid metabolism and toxin efflux, providing a theoretical basis for evaluating the health risks of MC-LR to amphibians.

Phospholipase Cγ1 represses colorectal cancer growth by inhibiting the Wnt/ß-catenin signaling axis.

As essential phospholipid signaling regulators, phospholipase C (PLC)s are activated by various extracellular ligands and mediate intracellular signal transduction. PLCγ1 is involved in regulating various cancer cell functions. However, the precise in vivo link between PLCγ1 and cancer behavior remains undefined. To investigate the role of PLCγ1 in colorectal carcinogenesis, we generated an intestinal tissue-specific Plcg1 knock out (KO) in adenomatous polyposis coli (Apc) Min/+ mice. Plcg1 deficiency in ApcMin/+ mice showed earlier death, with a higher colorectal tumor incidence in both number and size than in wild-type mice. Mechanistically, inhibition of PLCγ1 increased the levels of its substrate phosphoinositol 4,5-bisphosphate (PIP2) at the plasma membrane and promoted the activation of Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3ß (GSK3ß) to enhance ß-catenin signaling. Enhanced cell proliferation and Wnt/ß-catenin signaling were observed in colon tumors from Plcg1 KO mice. Furthermore, low PLCγ1 expression was associated with a poor prognosis of colon cancer patients. Collectively, we demonstrated the role of PLCγ1 in vivo as a tumor suppressor relationship between the regulation of the PIP2 level and Wnt/ß-catenin-dependent intestinal tumor formation.

Deficiency of AMPKα1 Exacerbates Intestinal Injury and Remote Acute Lung Injury in Mesenteric Ischemia and Reperfusion in Mice.

Mesenteric ischemia and reperfusion (I/R) injury can ensue from a variety of vascular diseases and represents a major cause of morbidity and mortality in intensive care units. It causes an inflammatory response associated with local gut dysfunction and remote organ injury. Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of metabolic homeostasis. The catalytic α1 subunit is highly expressed in the intestine and vascular system. In loss-of-function studies, we investigated the biological role of AMPKα1 in affecting the gastrointestinal barrier function. Male knock-out (KO) mice with a systemic deficiency of AMPKα1 and wild-type (WT) mice were subjected to a 30 min occlusion of the superior mesenteric artery. Four hours after reperfusion, AMPKα1 KO mice exhibited exaggerated histological gut injury and impairment of intestinal permeability associated with marked tissue lipid peroxidation and a lower apical expression of the junction proteins occludin and E-cadherin when compared to WT mice. Lung injury with neutrophil sequestration was higher in AMPKα1 KO mice than WT mice and paralleled with higher plasma levels of syndecan-1, a biomarker of endothelial injury. Thus, the data demonstrate that AMPKα1 is an important requisite for epithelial and endothelial integrity and has a protective role in remote organ injury after acute ischemic events.

Assessment of ameliorative effect of Trigonella foenum-graecum against CuO-NPs induced toxicity in Oreochromis mossambicus.

The current study assessed the ameliorative effect of Trigonella foenum graceum extract against copper oxide nanoparticles (CuO-NPs) induced toxicity in Oreochromis mossambicus. For this purpose 100 healthy fish weighing 20±2.34g were randomly divided into five different groups in duplicates and designated as control (C) no treatment, positive control (G*) treated with 0.12mg/L of CuO-NPs, experimental co-treated groups G1, G2 and G3 were treated with Trigonella foenum-graecum extract @ 18, 26 and 52mg/L along with 0.12 mg/L of CuO-NPs, respectively. In this study significant (P<0.05) changes were observed in the antioxidant activity of enzymes and histological alterations in the liver and intestine of fish in G*, G1 and G2 groups while a good ameliorative response of Trigonella foenum-graecum was observed in G3. Dose dependent alterations in glutathione, lipid peroxides, catalase, and malondialdehyde as well as histological architecture of liver and intestine were observed in treated groups, where more alterations were observed in positive control and low dose treated groups of Trigonella foenum-graecum. Moreover, more ameliorative effect was observed in high dose of Trigonella foenum-graecum treated group (G3). This study is novel as no previous data is available on the amelioration of Trigonella foenum-graecum extract against CuO-NPs induced toxicity in fish.

Molecular Mechanism of Stimulation of Na-K-ATPase by Leukotriene D4 in Intestinal Epithelial Cells.

Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and ß1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and ß1 subunits, mediated via the Ca-activated PKC pathway.

Coping strategies in response to different levels of elevated water hardness in channel catfish (Ictalurus punctatus): Insight into ion-regulatory and histopathological modulations.

Water hardness above the optimal level can incite toxic effects in fish, which are often species specific. Hence, we aimed at obtaining insights on the potential effects of elevated water hardness as well as coping strategies in channel catfish (Ictalurus punctatus). First, a toxicity assay was performed where the 96 h-LC50 was calculated as 4939 mg/L CaCO3. Thereafter, to gain knowledge on the underlying adaptive strategies to high water hardness, fish were exposed to seven hardness levels (150, 600, 1000, 1500, 2000, 3000 and 4000 mg/L CaCO3 at pH 8.15) for 15 days. Results showed that branchial activities of Ca2+-ATPase and Na+/K+-ATPase, which facilitate Ca2+ uptake, reduced starting respectively from 1000 mg/L and 1500 mg/L CaCO3. Nevertheless, Ca2+ burden in plasma and tissue (gills, liver and intestine) remained elevated. Hardness exposure also disturbed cations (Na+, K+, Mg2+) and minerals (iron and phosphorus) homeostasis in a tissue-specific and dose-dependent manner. Both hemoglobin content and hematocrit dropped significantly at 3000-4000 mg/L CaCO3, with a parallel decline in iron content in plasma and gills. Muscle water content rose dramatically at 4000 mg/L CaCO3, indicating an osmo-regulation disruption. Higher hardness of 3000-4000 mg/L CaCO3 also incited a series of histopathological modifications in gills, liver and intestine; most likely due to excess Ca2+ accumulation. Overall, these data suggest that channel catfish can adapt to a wide range of elevated hardness by modulating Ca2+ regulatory pathways and histomorphological alterations, however, 1500 mg/L CaCO3 and above can impair the performance of this species.

Berberine and/or zinc protect against methotrexate-induced intestinal damage: Role of GSK-3ß/NRF2 and JAK1/STAT-3 signaling pathways.

AIM: The present study was undertaken to elucidate the potential protective mechanism of berberine (BBR) and/or zinc (Zn) against methotrexate (MTX)-induced intestinal injury. METHODS: Five groups of rats were assigned; normal group (received vehicle), MTX group (20 mg/kg; i.p. single dose), and the other three groups received a single daily oral dose of BBR (50 mg/kg), Zn (5 mg/kg), and BBR plus Zn respectively, for 5 days before MTX and 5 days after. RESULTS: Our results emphasized the toxic effect of MTX on rat's intestine as shown by disturbance of oxidant/antioxidant status, down-regulation of NRF2, SIRT1, FOXO-3, Akt, and mTOR expressions, along with up-regulation of GSK-3ß, JAK1, and STAT-3 expressions. Besides, severe intestinal histopathological changes were also observed. On the contrary, BBR and/or Zn produced marked protection against MTX-induced intestinal toxicity via amelioration of oxidative stress, improving NRF2, SIRT1, FOXO-3, GSK-3ß, Akt, mTOR, JAK1, and STAT-3 alterations. Moreover, our treatments significantly restored histopathological abnormalities. Interestingly, combination therapy of BBR plus Zn exhibited higher effectiveness than mono-therapy. SIGNIFICANCE: BBR plus Zn could be used as a novel therapy for the treatment of MTX-induced intestinal damage through modulation of GSK-3ß/NRF2, Akt/mTOR, JAK1/STAT-3, and SIRT1/FOXO-3 signaling pathways.

Expression Profiles of Digestive Genes in the Gut and Salivary Glands of Tarnished Plant Bug (Hemiptera: Miridae).

Host plant preference of agricultural pests may shift throughout the growing season, allowing the pests to persist on wild hosts when crops are not available. Lygus Hahn (Hemiptera: Miridae) bugs are severe pests of cotton during flowering and fruiting stages, but can persist on alternative crops, or on weed species. Diversity of digestive enzymes produced by salivary glands and gut tissues play a pivotal role in an organism's ability to utilize various food sources. Polyphagous insects produce an array of enzymes that can process carbohydrates, lipids, and proteins. In this study, the digestive enzyme repertoire of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was identified by high-throughput sequencing followed by cDNA cloning and sequencing. This study identified 87 digestive genes, including 30 polygalacturonases (PG), one ß-galactosidase, three α-glucosidases, six ß-glucosidases, 28 trypsin-like proteases, three serine proteases, one apyrase-like protease, one cysteine protease, 12 lipases, and two transcripts with low similarity to a xylanase A-like genes. RNA-Seq expression profiles of these digestive genes in adult tarnished plant bugs revealed that 57 and 12 genes were differentially expressed in the salivary gland and gut (≥5-fold, P ≤ 0.01), respectively. All polygalacturonase genes, most proteases, and two xylanase-like genes were differentially expressed in salivary glands, while most of the carbohydrate and lipid processing enzymes were differentially expressed in the gut. Seven of the proteases (KF208689, KF208697, KF208698, KF208699, KF208700, KF208701, and KF208702) were not detected in either the gut or salivary glands.

Inhibition of α-glucosidases by tea polyphenols in rat intestinal extract and Caco-2 cells grown on Transwell.

Inhibition of maltase, sucrase, isomaltase and glucoamylase activity by acarbose, epigallocatechin gallate, epicatechin gallate and four polyphenol-rich tea extract from white, green, oolong, black tea, were investigated by using rat intestinal enzymes and human Caco-2 cells. Regarding rat intestinal enzyme mixture, all four tea extracts were very effective in inhibiting maltase and glucoamylase activity, but only white tea extract inhibited sucrase and isomaltase activity and the inhibition was limited. Mixed-type inhibition on rat maltase activity was observed. Tea extracts in combination with acarbose, produced a synergistic inhibitory effect on rat maltase activity. Caco-2 cells experiments were conducted in Transwells. Green tea extract and epigallocatechin gallate show dose-dependent inhibition on human sucrase activity, but no inhibition on rat sucrase activity. The opposite was observed on maltase activity. The results highlighted the different response in the two investigated model systems and show that tea polyphenols are good inhibitors for α-glucosidase activity.

The influence of coconut oil on the growth, immune, and antioxidative responses and the intestinal digestive enzymes and histomorphometry features of Nile tilapia (Oreochromis niloticus).

The trials of finding non-conventional and alternative aquafeed ingredients are increasing. In this sense, this study evaluated the influence of coconut oil on the growth, feed utilization, immune, and antioxidative responses of Nile tilapia. Five test diets were formulated by mixing coconut oil with the other ingredients at 0, 1, 2, 3, and 4% of the total ration and presented for tilapia for 60 successive days. The final weight, SGR, weight gain (WG), and feed intake were superior in fish delivered 2% of coconut oil (P < 0.05). Concurrently, fish that received 2% coconut oil had lower FCR and higher PER than fish of the control and 4% groups (P < 0.05). Higher lipase activity was observed in fish of 2% and 3% levels than the remaining groups (P < 0.05). Besides, the amylase and protease activities of fish in 1%, 2%, and 3% groups were higher than the 0% level (P < 0.05). The total blood cholesterol, RBCs, and PCV showed higher values in Nile tilapia fed 2% and 3% coconut oil (P < 0.05). The lysozyme and phagocytic activities were higher in fish fed 2% and 3% levels than the control (P < 0.05), while the phagocytic index in 2% and 3% levels was higher than 0% and 4% levels. Furthermore, SOD and CAT were higher in fish fed 1%, 2%, and 3% than fish fed 0% and 4% levels while GSH was higher in fish of 1%, 2%, and 3% than fish fed 0% level (P < 0.05). However, the MDA level was markedly lower in fish fed 25, 3%, and 4% coconut oil than the 0% level (P < 0.05). The intestine's histological structure in all groups appeared normal, forming of intestinal villi projecting from the intestinal wall. Also, the structure of the hepatopancreas had a normal architecture in all groups. To sum up, the inclusion of coconut oil at 2 to 3% is recommended as a replacer for fish oil in Nile tilapia diets.

Dietary Lysine Levels Improved Antioxidant Capacity and Immunity via the TOR and p38 MAPK Signaling Pathways in Grass Carp, Ctenopharyngodon idellus Fry.

An 8-week rearing trial was designed to appraise the dietary lysine levels on intestinal antioxidant capacity and immunity of grass carp fry. Six practical diets were prepared with graded levels of lysine (1.44, 1.79, 1.97, 2.44, 2.56 and 2.87% dry matter), and these diets were fed to grass carp fry. The results showed that the activities of intestinal antioxidant factors including catalase and glutathione peroxidase were markedly improved by the 2.44% dietary lysine compared with the control diet (1.44% dietary lysine) (P < 0.05). In terms of antioxidants, compared with the control diet, the 2.44% diet markedly upregulated the mRNA expression levels of target of rapamycin, S6 kinase1 and nuclear factor erythroid 2-related factor 2 pathway-related antioxidant genes, containing catalase and glutathione peroxidase 1α (P < 0.05) and downregulated the mRNA levels of Kelch-like ECH-associated protein 1 (P > 0.05). The mRNA levels of 4E-binding protein 2 showed the opposite trend compared with those of target of rapamycin, and the minimum value was observed in the group of 1.97% dietary lysine (P < 0.05). In terms of immunity, compared with the 1.44% diet, the 2.44% diet markedly suppressed the intestinal p38 mitogen-activated protein kinase and interferon γ2 mRNA levels (P < 0.05). Moreover, nuclear factor-kappa B p65, tumor necrosis factor α, interleukin 6, interleukin 8, and interleukin 15 mRNA levels all exhibited the same trend as p38 mitogen-activated protein kinase and interferon γ2; however, the difference among all the lysine treatments groups was not significant (P > 0.05). The anti-inflammatory cytokines transforming growth factor ß2 and interleukin 4/13B mRNA levels in the intestine were remarkably upregulated by high dietary lysine levels (2.56 and 2.87%) (P < 0.05), and when the dietary lysine level reached 2.44%, the interleukin 4/13A mRNA levels were strikingly increased (P < 0.05). Overall, the data suggested that 2.44% dietary lysine could strengthen the immune and antioxidant capacities of grass carp fry via activating the target of rapamycin (TOR) signaling pathway, and suppressing the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway, which then improve the survival rate.

Anatomical distribution and expression of CYP in humans: Neuropharmacological implications.

The cytochrome P450 (CYP450) superfamily is responsible for the metabolism of most xenobiotics and pharmacological treatments generally used in clinical settings. Genetic factors as well as environmental determinants acting through fine epigenetic mechanisms modulate the expression of CYP over the lifespan (fetal vs. infancy vs. adult phases) and in diverse organs. In addition, pathological processes might alter the expression of CYP. In this selective review, we sought to summarize the evidence on the expression of CYP focusing on three specific aspects: (a) the anatomical distribution of the expression in body districts relevant in terms of drug pharmacokinetics (liver, gut, and kidney) and pharmacodynamics, focusing for the latter on the brain, since this is the target organ of psychopharmacological agents; (b) the patterns of expression during developmental phases; and (c) the expression of CYP450 enzymes during pathological processes such as cancer. We showed that CYP isoforms show distinct patterns of expression depending on the body district and the specific developmental phases. Of particular relevance for neuropsychopharmacology is the complex regulatory mechanisms that significantly modulate the complexity of the pharmacokinetic regulation, including the concentration of specific CYP isoforms in distinct areas of the brain, where they could greatly affect local substrate and metabolite concentrations of drugs.

Evaluation of inhibitors of intestinal UDP-glucuronosyltransferases 1A8 and 1A10 using raloxifene as a substrate in Caco-2 cells: Studies with four flavonoids of Scutellaria baicalensis.

UDP glucuronosyltransferases (UGTs) of the gastrointestinal tract play a crucial role in protection against the toxic effects of xenobiotics in the environment. UGTs such as UGT1A8 and UGT1A10 are predominantly expressed in gastrointestinal tissues. In this study, we examined the phase II metabolism of raloxifene in differentiated Caco-2 monolayers by inducing UGT1A8 and UGT1A10 expression in these cells. The present study evaluated the following four flavonoids of Scutellaria baicalensis as model herbal compounds: scutellarein, salvigenin, baicalein, and wogonin. All test compounds, endpoint substrates, and their metabolites were quantified using liquid chromatography and high-resolution mass spectrometry. The transepithelial electrical resistance values for the individual compounds were comparable regardless of whether they were measured individually. Salvigenin significantly inhibited UGT1A8 and UGT1A10 activities in a concentration-dependent manner. All individual compounds except scutellarein inhibited UGT1A8 and UGT1A10 activity at a concentration of 100 µM. In addition, all individual flavonoids at 100 µM, except wogonin, significantly increased the amount of raloxifene in the basolateral chambers. The positive control, canagliflozin, significantly inhibited both UGT1A8 and UGT1A10 activities. These findings suggest that the Caco-2 assay can be utilized for identifying UGT1A8 and UGT1A10 inhibitors and indicate the potential of salvigenin for enhancing the pharmacological effects of UGT substrate drugs.

Evaluation of Tissue Stem Cell-Derived Human Intestinal Organoids, a Physiologically Relevant Model to Evaluate Cytochrome P450 Induction in Gut.

Induction of cytochrome P450 can cause drug-drug interactions and efficacy failure. Induction risk in liver and gut is typically inferred from experiments with plated hepatocytes. Organoids are physiologically relevant, multicellular structures originating from stem cells. Intestinal stem cell-derived organoids retain traits of normal gut physiology, such as an epithelial barrier and cellular diversity. Matched human enteroid and colonoid lines, generated from ileal and colon biopsies from two donors, were cultured in extracellular matrix for 3 days, followed by a single 48-hour treatment with rifampin, omeprazole, CITCO, and phenytoin at concentrations that induce target genes in hepatocytes. After treatment, mRNA was analyzed for induction of target genes. Rifampin induced CYP3A4; estimated EC50 and maximal fold induction were 3.75 µM and 8.96-fold, respectively, for ileal organoids and 1.40 µM and 11.3-fold, respectively, for colon organoids. Ileal, but not colon, organoids exhibited nifedipine oxidase activity, which was induced by rifampin up to 14-fold. The test compounds did not increase mRNA expression of CYP1A2, CYP2B6, multidrug resistance transporter 1 (P-glycoprotein), breast cancer resistance protein, and UDP-glucuronosyltransferase 1A1 in ileal organoids. Whereas omeprazole induced CYP3A4 (up to 5.3-fold, geometric mean, n = 4 experiments), constitutive androstane receptor activators phenytoin and CITCO did not. Omeprazole failed to induce CYP1A2 mRNA but did induce CYP1A1 mRNA (up to 7.7-fold and 15-fold in ileal and colon organoids, respectively, n = 4 experiments). Despite relatively high intra- and interexperimental variability, data suggest that the model yields induction responses that are distinct from hepatocytes and holds promise to enable evaluation of CYP1A1 and CYP3A4 induction in gut. SIGNIFICANCE STATEMENT: An adult intestinal stem cell-derived organoid model to test P450 induction in gut was evaluated. Testing several prototypical inducers for mRNA induction of P450 isoforms, UDP-glucuronosyltransferase 1A1, P-glycoprotein, and breast cancer resistance protein with both human colon and ileal organoids resulted in a range of responses, often distinct from those found in hepatocytes, indicating the potential for further development of this model as a physiologically relevant gut induction test system.

Altered Intestinal ACE2 Levels Are Associated With Inflammation, Severe Disease, and Response to Anti-Cytokine Therapy in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The host receptor for severe acute respiratory syndrome coronavirus 2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small bowel (SB). Our aim was to identify factors influencing intestinal ACE2 expression in Crohn's disease (CD), ulcerative colitis (UC), and non-inflammatory bowel disease (IBD) controls. METHODS: Using bulk RNA sequencing or microarray transcriptomics from tissue samples (4 SB and 2 colonic cohorts; n = 495; n = 387 UC; n = 94 non-IBD), we analyzed the relationship between ACE2 with demographics and disease activity and prognosis. We examined the outcome of anti-tumor necrosis factor and anti-interleukin-12/interleukin-23 treatment on SB and colonic ACE2 expression in 3 clinical trials. Univariate and multivariate regression models were fitted. RESULTS: ACE2 levels were consistently reduced in SB CD and elevated in colonic UC compared with non-IBD controls. Elevated SB ACE2 was also associated with demographic features (age and elevated body mass index) associated with poor coronavirus disease 2019 outcomes. Within CD, SB ACE2 was reduced in patients subsequently developing complicated disease. Within UC, colonic ACE2 was elevated in active disease and in patients subsequently requiring anti-tumor necrosis factor rescue therapy. SB and colonic ACE2 expression in active CD and UC were restored by anti-cytokine therapy, most notably in responders. CONCLUSIONS: Reduced SB but elevated colonic ACE2 levels in IBD are associated with inflammation and severe disease, but normalized after anti-cytokine therapy, suggesting compartmentalization of ACE2-related biology in SB and colonic inflammation. The restoration of ACE2 expression with anti-cytokine therapy might be important in the context of severe acute respiratory syndrome coronavirus 2 infection and potentially explain reports of reduced morbidity from coronavirus disease 2019 in IBD patients treated with anti-cytokines.

The Angiotensin-(1-12)/Chymase axis as an alternate component of the tissue renin angiotensin system.

The identification of an alternate extended form of angiotensin I composed of the first twelve amino acids at the N-terminal of angiotensinogen has generated new knowledge of the importance of noncanonical mechanisms for renin independent generation of angiotensins. The human sequence of the dodecapeptide angiotensin-(1-12) [N-Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10-Val1-Ile12-COOH] is an endogenous substrate that in the rat has been documented to be present in multiple organs including the heart, brain, kidney, gut, adrenal gland, and the bone marrow. Newer studies have confirmed the existence of Ang-(1-12) as an Ang II-forming substrate in the blood and heart of normal and diseased patients. Studies to-date document that angiotensin II generation from angiotensin-(1-12) does not require renin participation while chymase rather than angiotensin converting enzyme shows high catalytic activity in converting this tissue substrate into angiotensin II directly.

Prediction of Cyclosporin-Mediated Drug Interaction Using Physiologically Based Pharmacokinetic Model Characterizing Interplay of Drug Transporters and Enzymes.

Uptake transporter organic anion transporting polypeptides (OATPs), efflux transporters (P-gp, BCRP and MRP2) and cytochrome P450 enzymes (CYP450s) are widely expressed in the liver, intestine or kidney. They coordinately work to control drug disposition, termed as "interplay of transporters and enzymes". Cyclosporine A (CsA) is an inhibitor of OATPs, P-gp, MRP2, BCRP and CYP3As. Drug-drug interaction (DDI) of CsA with victim drugs occurs via disordering interplay of transporters and enzymes. We aimed to establish a whole-body physiologically-based pharmacokinetic (PBPK) model which predicts disposition of CsA and nine victim drugs including atorvastatin, cerivastatin, pravastatin, rosuvastatin, fluvastatin, simvastatin, lovastatin, repaglinide and bosentan, as well as drug-drug interactions (DDIs) of CsA with nine victim drugs to investigate the integrated effect of enzymes and transporters in liver, intestinal and kidney on drug disposition. Predictions were compared with observations. Most of the predictions were within 0.5-2.0 folds of observations. Atorvastatin was represented to investigate individual contributions of transporters and CYP3As to atorvastatin disposition and their integrated effect. The contributions to atorvastatin disposition were hepatic OATPs >> hepatic CYP3A > intestinal CYP3As ≈ efflux transporters (P-gp/BCRP/MRP2). The results got the conclusion that the developed PBPK model characterizing the interplay of enzymes and transporters was successfully applied to predict the pharmacokinetics of 10 OATP substrates and DDIs of CsA with 9 victim drugs.