Golden thistle (Scolymus hispanicus L.) hydromethanolic extracts ameliorated glucose absorption and inflammatory markers in vitro.

Golden thistle (GT, Scolymus hispanicus L.) is an edible plant native to the Mediterranean. Several activities have been reported for the GT, as it is used for traditional medicinal purposes in some cultures. In this study, we aimed to investigate the effects of GT crude extract on phenolic bioavailability, antidiabetic, and anti-inflammatory activities by using colonic epithelium (CaCo-2) and murine macrophage (RAW 264.7) cell lines. The CaCo-2 cells were grown on the bicameral membrane system for intestinal bioavailability and glucose efflux. Lipopolysaccharide (LPS, 0.5 µg/mL) was used to induce systemic inflammation on RAW 264.7. The inflammatory medium of RAW 264.7 cells was given to Caco-2 cells to mimic colonic inflammation. Our results showed that 5-o-caffeoylquinic acid had an apparent permeability of (1.82 ± 0.07) × 10-6 cm/s after 6 h. The extract lowered the glucose efflux by 39.4%-42.6%, in addition to the reductions in relative GLUT2 mRNA expressions by 49%-66% in pre- and co-treatments (p < .05). Decreases in systemic inflammation markers of nitric oxide, tumor necrosis factor-alpha, and interleukin-6 (IL-6) were also detected in 30%-45% range after pre-treatments with the GT extract (p < .05). Lastly, colonic inflammation markers of IL-6 and IL-8 were reduced by 8.7%-19.5% as a result of GT pre-treatments (p < .05). Thus, an in vitro investigation of GT extract revealed promising results on antidiabetic and anti-inflammatory activities.

Formulation of Gluten-Free Cookies Utilizing Chickpea, Carob, and Hazelnut Flours through Mixture Design.

Legume flours, which offer high nutritional quality, present viable options for gluten-free bakery products. However, they may have an objectionable flavor and taste for some consumers. In this study, it was aimed to improve the gluten-free cookie formulation by incorporating carob and hazelnut flours to pre-cooked chickpea flour and to investigate the techno-functional properties of the formulated cookies. The flours used in the formulations were assessed for their chemical and physical properties. This study employed a mixture design (simplex-centroid) to obtain the proportions of the flours to be used in the cookie formulations. The rheological characteristics of the doughs and the technological attributes of the baked cookies were determined. The addition of the hazelnut and carob flours had the overall effect of reducing the rheological characteristics of the cookie doughs. Furthermore, the textural attribute of the hardness of the baked cookies decreased as the ratio of hazelnut flour in the formulations was raised. The analysed results and sensory evaluation pointed to a formulation consisting of 30% pre-cooked chickpea/30% carob/30% hazelnut flours, which exhibited improved taste and overall acceptability scores. A total of 16.82 g/100 g of rapidly digestible starch, 5.36 g/100 g of slowly digestible starch, and 8.30 g/100 g of resistant starch exist in this particular cookie. As a result, combinations of chickpea, hazelnut, and carob flours hold promise as good alternatives for gluten-free cookie ingredients and warrant further exploration in the development of similar products.

Techno-Functional and In Vitro Digestibility Properties of Gluten-Free Cookies Made from Raw, Pre-Cooked, and Germinated Chickpea Flours.

Chickpea flour, which is produced in various forms, has high protein and fiber content; therefore, it can be a good ingredient for gluten-free cookies. The objective of this study was to investigate and compare the properties of cookies formulated using raw (RCF), cooked (CCF), and germinated (GCF) chickpea flours. The techno-functional properties of these flours were determined, and scanning electron microscope images and mid-infrared spectra were obtained. The rheological properties of cookie doughs were measured along with their mid-infrared spectra. Baked cookies were analyzed for their technological properties as well as their in vitro digestion properties. Sensory analysis was also performed for all the cookies. The most significant difference among the flours was observed in their water retention capacity, and CCF had 119.7% higher water retention capacity compared to RCF. The dough made with CCF had quite different rheological properties from the others. The cookies baked with GCF had the highest baking loss and spread ratio. The CCF-containing cookies had the hardest structure. The cookies made from RCF had a higher resistant starch content followed by the cookies with GCF. All the cookies had similar scores in all aspects tested in the sensory analysis. The use of three different forms of chickpea flour in cookie formulations resulted in products with very different properties; however, their overall acceptability levels were close.

Sucrose-induced hyperglycemia dysregulates intestinal zinc metabolism and integrity: risk factors for chronic diseases.

Objective: Zinc is an essential micronutrient that is critical for many physiological processes, including glucose metabolism, regulation of inflammation, and intestinal barrier function. Further, zinc dysregulation is associated with an increased risk of chronic inflammatory diseases such as type II diabetes, obesity, and inflammatory bowel disease. However, whether altered zinc status is a symptom or cause of disease onset remains unclear. Common symptoms of these three chronic diseases include the onset of increased intestinal permeability and zinc dyshomeostasis. The specific focus of this work is to investigate how dietary sources of intestinal permeability, such as high sucrose consumption, impact transporter-mediated zinc homeostasis and subsequent zinc-dependent physiology contributing to disease development. Method: We used in vivo subchronic sucrose treatment, ex vivo intestinal organoid culture, and in vitro cell systems. We analyze the alterations in zinc metabolism and intestinal permeability and metabolic outcomes. Results: We found that subchronic sucrose treatment resulted in systemic changes in steady-state zinc distribution and increased 65Zn transport (blood-to-intestine) along with greater ZIP14 expression at the basolateral membrane of the intestine. Further, sucrose treatment enhanced cell survival of intestinal epithelial cells, activation of the EGFR-AKT-STAT3 pathway, and intestinal permeability. Conclusion: Our work suggests that subchronic high sucrose consumption alters systemic and intestinal zinc homeostasis linking diet-induced changes in zinc homeostasis to the intestinal permeability and onset of precursors for chronic disease.

Natural and synthetic nanovectors for cancer therapy.

Nanomaterials have been extensively studied in cancer therapy as vectors that may improve drug delivery. Such vectors not only bring numerous advantages such as stability, biocompatibility, and cellular uptake but have also been shown to overcome some cancer-related resistances. Nanocarrier can deliver the drug more precisely to the specific organ while improving its pharmacokinetics, thereby avoiding secondary adverse effects on the not target tissue. Between these nanovectors, diverse material types can be discerned, such as liposomes, dendrimers, carbon nanostructures, nanoparticles, nanowires, etc., each of which offers different opportunities for cancer therapy. In this review, a broad spectrum of nanovectors is analyzed for application in multimodal cancer therapy and diagnostics in terms of mode of action and pharmacokinetics. Advantages and inconveniences of promising nanovectors, including gold nanostructures, SPIONs, semiconducting quantum dots, various nanostructures, phospholipid-based liposomes, dendrimers, polymeric micelles, extracellular and exome vesicles are summarized. The article is concluded with a future outlook on this promising field.

Comparison of apical and basolateral Cu treatment for iron-related gene regulation during deferoxamine induced iron deficiency.

BACKGROUND: Intestinal copper transporter (Atp7a) mutant-brindled mice with systemic Cu deficiency had elevated Cu levels in enterocyte cells without any perturbation of iron-regulating genes, suggesting that blood Cu level might be important for intestinal iron homeostasis during iron deficiency (ID). We hypothesized that the blood Cu level and polarization (apical and basolateral) of enterocyte cells might be important regulators for the compensatory response on the regulation of genes in enterocyte cells during iron deficiency. METHODS: We grew Caco-2 cells on a bicameral cell culture plate to mimic the human intestine system and on a regular tissue culture plate. Iron deficiency was induced by deferoxamine (DFO). The cells were treated with Cu and Cu with Fe following mRNA expressions of DMT1, FPN, TFR, and ANKRD37 were analyzed. RESULTS: Our main finding was that basolateral treatment of Cu significantly reduced mRNA expressions of iron-regulated genes, including DMT1, FPN, TFR, and ANKRD37, compared to DFO-treated and DFO with apical Cu-treated groups in both bicameral and regular tissue culture plates. CONCLUSIONS: Cu level in the basolateral side of Caco-2 cells significantly influenced the intracellular gene regulation in DFO-induced iron-deficient condition, and polarization of the cells might be important factor gene regulation in enterocyte cells.

Iron Absorption: Factors, Limitations, and Improvement Methods.

Iron is an essential element for human life since it participates in many functions in the human body, including oxygen transport, immunity, cell division and differentiation, and energy metabolism. Iron homeostasis is mainly controlled by intestinal absorption because iron does not have active excretory mechanisms for humans. Thus, efficient intestinal iron bioavailability is essential to reduce the risk of iron deficiency anemia. There are two forms of iron, heme and nonheme, found in foods. The average daily dietary iron intake is 10 to 15 mg in humans since only 1 to 2 mg is absorbed through the intestinal system. Nutrient-nutrient interactions may play a role in dietary intestinal iron absorption. Dietary inhibitors such as calcium, phytates, polyphenols and enhancers such as ascorbic acid and proteins mainly influence iron bioavailability. Numerous studies have been carried out for years to enhance iron bioavailability and combat iron deficiency. In addition to traditional methods, innovative techniques are being developed day by day to enhance iron bioavailability. This review will provide information about iron bioavailability, factors affecting absorption, iron deficiency, and recent studies on improving iron bioavailability.

Effects of Golden Thistle (Scolymus hispanicus L.) on Cytotoxic Activity: Cell Cycle Arrest and Apoptotic Properties on the CaCo-2 Cell Line.

Cancer is a global concern for many individuals with high mortality rates, with colon cancer being the third most common diagnosed cancer worldwide. A phytochemical-rich diet is often recommended in the prevention and during the treatment of cancer cases. Golden thistle (GT) plant (Scolymus hispanicus L.) is a wild edible plant widely consumed in the Mediterranean countries. In this study, we aimed to obtain a hydromethanolic extract from three parts of the GT plant and test its antiproliferative activity in the CaCo-2 human adenocarcinoma cell line. Concentrations of the golden thistle extract (GTE) were used to treat CaCo-2 cells and the most significant reduction was detected with 4 mg/mL GTE after 72 h, with 78.3% decrease in cell viability (P < .05). Additionally, 4 mg/mL GTE caused 7.8-fold higher release of lactate dehydrogenase enzyme, indicating cell death after treatment. Flow cytometric analyses concluded both 3.3-fold higher early and late apoptotic activity of the 4 mg/mL GTE compared with the nontreated control group (P < .05). Last, 4 mg/mL GTE showed 24.1% reduction in the G1 phase and 38.1% increase in the S phase of cell cycle distribution. The alteration of G1 and S phases in the cell cycle led to growth reduction of CaCo-2 cells and caused apoptosis.

The Effect of Ankaferd Blood Stopper on Colonic Inflammation: An In Vitro Study in RAW 264.7 and Caco-2 Cells.

Ankaferd Blood Stopper (ABS) is a medicinal plant extract that has anti-inflammatory effect. Inflammatory bowel disease is a pathological condition that directly affects colon health and increases the risk of colon cancer. Especially inflammation is an important factor in the formation and progression of this disease. The aim of the study was to investigate the protective effect of ABS on colonic inflammation. Caco-2 and RAW 264.7 cells were used as a model of in vitro colonic inflammation. RAW 264.7 cells were treated with lipopolysaccharide for 12 h to induce inflammation, and an inflammatory medium (IM) was obtained. Caco-2 cells were treated with 15 µL/mL ABS for 4 h, then incubated with IM. The cells also were incubated with 15 µL/mL ABS and IM together for 12 h. Tumor necrosis factor alpha (TNF-α) protein levels were targeted in testing inflammatory condition and cyclooxygenase-2 (COX-2) mRNA level was used as a marker gene to show the possible anti-inflammatory effect of ABS in Caco-2 cells. TNF-α level was 26.1-fold higher than the control group. IM caused 3.2-fold increase in COX-2 expression in Caco-2 cells. Pretreatment of Caco-2 cells with ABS resulted in 3.3-fold decrease in COX-2 mRNA levels relative to IM group. Furthermore, COX-2 mRNA level reduced 4.7-fold when ABS and conditional medium were given at the same time. ABS has suppressive effect on COX-2 mRNA expression in Caco-2 cells. These results suggest that ABS might have protective and therapeutic effect for colonic inflammation.

Physicochemical and Active Properties of Gelatine-Based Composite Gels Loaded with Lysozyme and Green Tea Polyphenols.

RESEARCH BACKGROUND: The use of gel-based systems as a novel method for the delivery of natural antimicrobial, antioxidant and bioactive compounds is a developing innovative solution for the food industry. This research aims to develop multifunctional active edible gels based on gelatine and its composites with improved mechanical properties. EXPERIMENTAL APPROACH: Antilisterial and bioactive composite gels showing different physical and active properties from classical gelatine gel were developed by loading lysozyme and green tea extract into gelatine/starch and gelatine/wax composite gels. Mechanical properties, swelling profiles, colour, release profiles, and antimicrobial and bioactive properties of the gels were characterised. RESULTS AND CONCLUSIONS: Gelatine/wax gels showed 1.3- to 2.1-fold higher firmness and cutting strength than gelatine and gelatine/starch composite gels that had similar firmness and cutting strengths. Work to shear of both composite gels was 1.4- to 1.9-fold higher than that of gelatine gel. The gelatine/starch gel showed the highest water absorption capacity. Green tea extract reduced soluble lysozyme in all gels, but composite gels contained higher amount of soluble lysozyme than gelatine gel. All the gels with lysozyme inhibited Listeria innocua growth in the broth media, while green tea extract showed antilisterial activity only in gelatine/wax gels. Gels with green tea extract showed antioxidant, antidiabetic (α-glucosidase and α-amylase inhibition), antihypertensive (angiotensin-converting enzyme inhibition) and antiproliferative activities (on Caco-2 human colon carcinoma cells). However, gelatine and gelatine/wax gels showed the highest antioxidant and antidiabetic activity. The gelatine/wax gels prevented phenolic browning, while green tea extract in other gels showed moderate or extensive browning. NOVELTY AND SCIENTIFIC CONTRIBUTION: This work clearly showed the possibility of improving mechanical properties and modifying water absorption and controlled release profiles of gelatine gels using gelatine/starch and gelatine/wax composites. The novel composite gels reduced browning of incorporated polyphenols and showed antilisterial and bioactive properties.

The development of lentil derived protein-iron complexes and their effects on iron deficiency anemia in vitro.

Iron deficiency anemia (IDA) is the most common nutrient-dependent health problem in the world and could be reversed by commercially available iron supplementation. The form of iron supplement is important due to its toxicity on the gastrointestinal system (GI), so the development of new dietary strategies might be important for the prevention of IDA. It has been shown that plant-based proteins bind to iron and might decrease the free form of iron before absorption and increase iron bioavailability. Thus, we aimed to form lentil derived protein-iron complexes and to test the functional properties of hydrolysed protein-iron complexes in anemic Caco-2 cell line. Our main findings were that (i) lentil derived proteins had the capacity to chelate iron minerals and (ii) hydrolysed protein-iron complexes significantly reduced the mRNA levels of iron regulated divalent metal transporter-1 (DMT1), transferrin receptor (TFR), and ankyrin repeat domain 37 (ANKRD37) marker genes that were induced by iron deficiency anemia. The current findings suggest that hydrolysed protein-iron complexes might have functional properties in iron deficiency anemia in vitro. Further in vivo studies are necessary to show lentil derived proteins and iron might be used as supplements or food additives to reduce the risk of iron deficiency anemia.

Investigation of the influence of high glucose on molecular and genetic responses: an in vitro study using a human intestine model.

BACKGROUND: Dietary glucose consumption has increased worldwide. Long-term high glucose intake contributes to the development of obesity and type 2 diabetes mellitus (T2DM). Obese people tend to eat glucose-containing foods, which can lead to an addiction to glucose, increased glucose levels in the blood and intestine lumen, and exposure of intestinal enterocytes to high dietary glucose. Recent studies have documented a role for enterocytes in glucose sensing. However, the molecular and genetic relationship between high glucose levels and intestinal enterocytes has not been determined. We aimed to identify relevant target genes and molecular pathways regulated by high glucose in a well-established in vitro epithelial cell culture model of the human intestinal system (Caco-2 cells). METHODS: Cells were grown in a medium containing 5.5 and 25 mM glucose in a bicameral culture system for 21 days to mimic the human intestine. Transepithelial electrical resistance was used to control monolayer formation and polarization of the cells. Total RNA was isolated, and genome-wide mRNA expression profiles were determined. Molecular pathways were analyzed using the DAVID bioinformatics program. Gene expression levels were confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: Microarray gene expression data demonstrated that 679 genes (297 upregulated, 382 downregulated) were affected by high glucose treatment. Bioinformatics analysis indicated that intracellular protein export (p = 0.0069) and ubiquitin-mediated proteolysis (p = 0.024) pathways were induced, whereas glycolysis/gluconeogenesis (p < 0.0001), pentose phosphate (p = 0.0043), and fructose-mannose metabolism (p = 0.013) pathways were downregulated, in response to high glucose. Microarray analysis of gene expression showed that high glucose significantly induced mRNA expression levels of thioredoxin-interacting protein (TXNIP, p = 0.0001) and lipocalin 15 (LCN15, p = 0.0016) and reduced those of ATP-binding cassette, sub-family A member 1 (ABCA1, p = 0.0004), and iroquois homeobox 3 (IRX3, p = 0.0001). CONCLUSIONS: To our knowledge, this is the first investigation of high glucose-regulated molecular responses in an intestinal enterocyte model. Our findings identify new target genes that may be important in the intestinal glucose absorption and metabolism during high glucose consumption.

Ankaferd Influences mRNA Expression of Iron-Regulated Genes During Iron-Deficiency Anemia.

Ankaferd Blood Stopper (ABS) comprises a mixture of plants and stops bleeding via forming a protein network by erythroid aggregation. Bleeding causes reduction of iron levels in body. It has been indicated that ABS contains significant amount of iron. Thus, we investigated the biological activity of ABS-derived iron on iron-regulated genes during iron-deficiency anemia (IDA). IDA We selected Caco-2 and HepG2 cell lines as in vitro models of human intestine and liver, respectively. Iron deficiency anemia was induced by deferoxamine. The cells were treated with ferric ammonium citrate (FAC) and ABS. Messenger RNA levels of iron-regulated genes were analyzed by quantitative reverse transcription polymerase chain reaction to elucidate whether iron in ABS behaved similar to inorganic iron (FAC) during IDA. The results showed that ABS-derived iron influenced transcriptions of iron-regulated marker genes, including divalent metal transporter ( Dmt1), transferrin receptor ( TfR), ankyrin repeat domain 37 ( Ankrd37), and hepcidin ( Hamp) in IDA-induced Caco-2 and HepG2 cells. Our results suggest that when ABS is used to stop tissue bleeding, it might have an ability to reduce levels of IDA.

Intestinal hephaestin potentiates iron absorption in weanling, adult, and pregnant mice under physiological conditions.

Regulation of intestinal iron absorption is crucial to maintain body iron levels because humans have no regulated iron-excretory system. Elucidating molecular events that mediate intestinal iron transport is thus important for the development of therapeutic approaches to modify iron absorption in pathological states. The process of iron uptake into duodenal enterocytes is relatively well understood, but less is known about the functional coupling between the iron exporter ferroportin 1 and the basolateral membrane iron oxidase hephaestin (Heph). Initial characterization of intestine-specific Heph knockout (Hephint) mice demonstrated that adult male mice were mildly iron deficient; however, the specific role of intestinal Heph has not been determined in weanling mice, in female mice, or during physiological states which stimulate iron absorption. Furthermore, because ferroportin 1-mediated iron export from some tissues (eg, liver) is impaired in the absence of the Heph homolog, ceruloplasmin, we hypothesized that Heph is rate limiting for intestinal iron absorption, especially when iron demands increase. Our experimental approach was to assess various physiological parameters and iron (59Fe) absorption and tissue distribution in weanling, adult, and pregnant Hephint mice (and controls) under physiological conditions and in adult Hephint mice after dietary iron deprivation or acute hemolysis. Results demonstrate that intestinal Heph is essential for optimal iron transport in weanlings and adults of both sexes and during pregnancy, but not in adult mice with iron-deficiency or hemolytic anemia. Moreover, activation of unidentified, intestinal ferroxidases was noted, which may explain why intestinal Heph is not always required for optimal iron absorption.

Silencing of the Menkes copper-transporting ATPase (Atp7a) gene increases cyclin D1 protein expression and impairs proliferation of rat intestinal epithelial (IEC-6) cells.

The Menkes copper-transporting ATPase (Atp7a) has dual roles in mammalian enterocytes: pumping copper into the trans-Golgi network (to support cuproenzyme synthesis) and across the basolateral membrane (to deliver dietary copper to the blood). Atp7a is strongly induced in the rodent duodenum during iron deprivation, suggesting that copper influences iron homeostasis. To investigate this possibility, Atp7a was silenced in rat intestinal epithelial (IEC-6) cells. Irrespective of its influence on iron homeostasis, an unexpected observation was made in the Atp7a knockdown (KD) cells: the cells grew slower (∼40% fewer cells at 96h) and were larger than negative-control shRNA-transfected cells. Lack of Atp7a activity thus perturbed cell cycle control. To elucidate a possible molecular mechanism, expression of two important cell cycle control proteins was assessed. Cyclin D1 (CD1) protein expression increased in Atp7a KD cells whereas proliferating-cell nuclear antigen (PCNA) expression was unaltered. Increased CD1 expression is consistent with impaired cell cycle progression. Expression of additional cell proliferation marker genes (p21 and Ki67) was also investigated; p21 expression increased, whereas Ki67 decreased, both consistent with diminished cell growth. Further experiments were designed to determine whether increased cellular copper content was the trigger for the altered growth phenotype of the Atp7a KD cells. Copper loading, however, did not influence the expression patterns of CD1, p21 or Ki67. Overall, these findings demonstrate that Atp7a is required for normal proliferation of IEC-6 cells. How Atp7a influences cell growth is unclear, but the underlying mechanism could relate to its roles in intracellular copper distribution or cuproenzyme synthesis.

Mechanistic and regulatory aspects of intestinal iron absorption.

Iron is an essential trace mineral that plays a number of important physiological roles in humans, including oxygen transport, energy metabolism, and neurotransmitter synthesis. Iron absorption by the proximal small bowel is a critical checkpoint in the maintenance of whole-body iron levels since, unlike most other essential nutrients, no regulated excretory systems exist for iron in humans. Maintaining proper iron levels is critical to avoid the adverse physiological consequences of either low or high tissue iron concentrations, as commonly occurs in iron-deficiency anemia and hereditary hemochromatosis, respectively. Exquisite regulatory mechanisms have thus evolved to modulate how much iron is acquired from the diet. Systemic sensing of iron levels is accomplished by a network of molecules that regulate transcription of the HAMP gene in hepatocytes, thus modulating levels of the serum-borne, iron-regulatory hormone hepcidin. Hepcidin decreases intestinal iron absorption by binding to the iron exporter ferroportin 1 on the basolateral surface of duodenal enterocytes, causing its internalization and degradation. Mucosal regulation of iron transport also occurs during low-iron states, via transcriptional (by hypoxia-inducible factor 2α) and posttranscriptional (by the iron-sensing iron-regulatory protein/iron-responsive element system) mechanisms. Recent studies demonstrated that these regulatory loops function in tandem to control expression or activity of key modulators of iron homeostasis. In health, body iron levels are maintained at appropriate levels; however, in several inherited disorders and in other pathophysiological states, iron sensing is perturbed and intestinal iron absorption is dysregulated. The iron-related phenotypes of these diseases exemplify the necessity of precisely regulating iron absorption to meet body demands.

Molecular mediators governing iron-copper interactions.

Given their similar physiochemical properties, it is a logical postulate that iron and copper metabolism are intertwined. Indeed, iron-copper interactions were first documented over a century ago, but the homeostatic effects of one on the other has not been elucidated at a molecular level to date. Recent experimental work has, however, begun to provide mechanistic insight into how copper influences iron metabolism. During iron deficiency, elevated copper levels are observed in the intestinal mucosa, liver, and blood. Copper accumulation and/or redistribution within enterocytes may influence iron transport, and high hepatic copper may enhance biosynthesis of a circulating ferroxidase, which potentiates iron release from stores. Moreover, emerging evidence has documented direct effects of copper on the expression and activity of the iron-regulatory hormone hepcidin. This review summarizes current experimental work in this field, with a focus on molecular aspects of iron-copper interplay and how these interactions relate to various disease states.

Silencing the Menkes copper-transporting ATPase (Atp7a) gene in rat intestinal epithelial (IEC-6) cells increases iron flux via transcriptional induction of ferroportin 1 (Fpn1).

The Menkes copper-transporting ATPase (Atp7a) gene is induced in rat duodenum during iron deficiency, consistent with copper accumulation in the intestinal mucosa and liver. To test the hypothesis that ATP7A influences intestinal iron metabolism, the Atp7a gene was silenced in rat intestinal epithelial (IEC-6) cells using short hairpin RNA (shRNA) technology. Perturbations in intracellular copper homeostasis were noted in knockdown cells, consistent with the dual roles of ATP7A in pumping copper into the trans-Golgi (for cuproenzyme synthesis) and exporting copper from cells. Intracellular iron concentrations were unaffected by Atp7a knockdown. Unexpectedly, however, vectorial iron ((59)Fe) transport increased (∼33%) in knockdown cells grown in bicameral inserts and increased further (∼70%) by iron deprivation (compared with negative control shRNA-transfected cells). Additional experiments were designed to elucidate the molecular mechanism of increased transepithelial iron flux. Enhanced iron uptake by knockdown cells was associated with increased expression of a ferrireductase (duodenal cytochrome b) and activity of a cell-surface ferrireductase. Increased iron efflux from knockdown cells was likely mediated via transcriptional activation of the ferroportin 1 gene (by an unknown mechanism). Moreover, Atp7a knockdown significantly attenuated expression of an iron oxidase [hephaestin (HEPH); by ∼80%] and membrane ferroxidase activity (by ∼50%). Cytosolic ferroxidase activity, however, was retained in knockdown cells (75% of control cells), perhaps compensating for diminished HEPH activity. This investigation has thus documented alterations in iron homeostasis associated with Atp7a knockdown in enterocyte-like cells. Alterations in copper transport, trafficking, or distribution may underlie the increase in transepithelial iron flux noted when ATP7A activity is diminished.

Investigation of iron metabolism in mice expressing a mutant Menke's copper transporting ATPase (Atp7a) protein with diminished activity (Brindled; Mo (Br) (/y) ).

During iron deficiency, perturbations in copper homeostasis have frequently been documented. Previous studies in iron-deprived rats demonstrated that enterocyte and hepatic copper levels increase and a copper transporter (the Menkes Copper ATPase; Atp7a) is induced in the duodenal epithelium in parallel to iron transport-related genes (e.g. Dmt1, Dcytb, Fpn1). Moreover, two ferroxidase proteins involved in iron homeostasis, hephaestin expressed in enterocytes and ceruloplasmin, produced and secreted into blood by the liver, are copper-dependent enzymes. We thus aimed to test the hypothesis that Atp7a function is important for the copper-related compensatory response of the intestinal epithelium to iron deficiency. Accordingly, iron homeostasis was studied for the first time in mice expressing a mutant Atp7a protein with minimal activity (Brindled [Mo (Br) (/y) ]). Mutant mice were rescued by perinatal copper injections, and, after a 7-8 week recovery period, were deprived of dietary iron for 3 weeks (along with WT littermates). Adult Mo (Br) (/y) mice displayed copper-deficiency anemia but had normal iron status; in contrast, iron-deprived Mo (Br) (/y) mice were iron deficient and more severely anemic with partial amelioration of the copper-deficient phenotype. Intestinal iron absorption in both genotypes (WT and Mo (Br) (/y) ) increased ∼3-fold when mice consumed a low-iron diet and ∼6-fold when mice were concurrently bled. WT mice exhibited no alterations in copper homeostasis in response to iron deprivation or phlebotomy. Conversely, upregulation of iron absorption was associated with increased enterocyte and liver copper levels and serum ferroxidase (ceruloplasmin) activity in Mo (Br) (/y) mice, typifying the response to iron deprivation in many mammalian species. We thus speculate that a copper threshold exists that is necessary to allow appropriate regulate of iron absorption. In summary, Mo (Br) (/y) mice were able to adequately regulate iron absorption, but unlike in WT mice, concurrent increases in enterocyte and liver copper levels and serum ferroxidase activity may have contributed to maintenance of iron homeostasis.

The Dual Diverse Dynamic Reversible Effects of Ankaferd Blood Stopper on EPCR and PAI-1 Inside Vascular Endothelial Cells With and Without LPS Challenge.

OBJECTIVE: Ankaferd blood stopper (ABS) is comprised of a mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. ABS is used as a topical hemostatic agent due to its antihemorrhagic effect, yet its hemostatic mechanism of action remains to be investigated. ABS does not affect the levels of coagulation factors II, V, VII, VIII, IX, X, XI and XII. The aim of this study was to investigate the effects of ABS on endothelium and immune response. As such, we evaluated changes in endothelial cell protein C receptor (EPCR) and plasminogen activator inhibitor type-1 (PAI-1) expression inside human umbilical vein endothelial cells (HUVECs) in the presence and absence of lipopolysaccharides (LPSs). MATERIAL AND METHODS: We exposed HUVECs to 10 µL and 100 µL of ABS for 5 min, 25 min, 50 min, 6 h, and 24 h. Additionally, 10 µg mL-1 of LPS was administered for 1 h to observe the effects of LPS challenge on HUVECs, and then the cells were treated with ABS for 5 min, 25 min, 50 min, and 6 h to observe the effects of ABS on HUVECs. Total RNA was isolated from HUVECs and then the level of expression of EPCR and PAI-1 mRNA was measured. RESULTS: Cells were microscopically observed to arise from the surface and adhere to each other following the administration of ABS to HUVECs. Additionally, after 24 h the cells had normal growth and physiology, which suggests that the adhesive cellular effects of ABS might be reversible. ABS had a negative effect on EPCR and PAI-1 expression; the effect in response to 100 µL was greater than that to 10 µL. EPCR and PAI-1 expression increased over time in response to LPS and 10 µL of ABS. EPCR and PAI-1 expression was very low during the first hour of exposure to LPS and 100 µL of ABS, but after 6 h increased to levels similar to those observed in response to LPS and 10 µL of ABS. CONCLUSION: It was observed that ABS had dual diverse dynamic reversible effects on EPCR and PAI-1 expression in HUVECs, which were dependent on dose and concentration. ABS might play a role in numerous cellular mechanisms, in addition to having hemostatic effects. CONFLICT OF INTEREST: None declared.