Relationships between Circulating Sclerostin, Bone Marrow Adiposity, Other Adipose Deposits and Lean Mass in Post-Menopausal Women.

Sclerostin is a Wnt signaling pathway inhibitor that negatively regulates bone formation. Bone-marrow-derived stromal cell (BMSC) differentiation is influenced by the Wnt pathway, leading to the hypothesis that higher levels of sclerostin might be associated with an increase in bone marrow adiposity (BMA). The main purpose of this study was to determine whether a relationship exists between circulating sclerostin and BMA in post-menopausal women with and without fragility fractures. The relationships between circulating sclerostin and body composition parameters were then examined. The outcomes measures included vertebral and hip proton density fat fraction (PDFF) using the water fat imaging (WFI) MRI method; DXA scans; and laboratory measurements, including serum sclerostin. In 199 participants, no significant correlations were found between serum sclerostin and PDFF. In both groups, serum sclerostin was correlated positively with bone mineral density (R = 0.27 to 0.56) and negatively with renal function (R = -0.22 to -0.29). Serum sclerostin correlated negatively with visceral adiposity in both groups (R = -0.24 to -0.32). Serum sclerostin correlated negatively with total body fat (R = -0.47) and appendicular lean mass (R = -0.26) in the fracture group, but not in the controls. No evidence of a relationship between serum sclerostin and BMA was found. However, serum sclerostin was negatively correlated with body composition components, such as visceral adiposity, total body fat and appendicular lean mass.

Raman microspectroscopy reveals unsaturation heterogeneity at the lipid droplet level and validates an in vitro model of bone marrow adipocyte subtypes.

Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier in life and are more resilient to environmental and metabolic disruptions. In vivo, rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thus in vitro models remain the current standard to study nuances of adipocyte differentiation. No in vitro model has yet been described for the study of rBMAds/cBMAds. Here, we present an in vitro model of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression of Scd1 and Fads2 differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesis in vitro in a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 model in vitro system and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step.

Osteoporosis: A role for lipids.

An inverse relationship between bone marrow (BM) adiposity and bone mass has been described in different physiological and pathological conditions, including osteoporosis (OP). In osteoporotic patients, lower bone mass density is indeed associated with higher BM fat content, suggesting a potential role for bone lipids in the OP pathogenesis. Nevertheless, some questions remain. Is that BM adiposity a cause or a consequence of the bone loss? What kinds of lipids are involved? Human data are somehow contradictories regarding bone lipid signature related to OP, and animal data are needed to support on one or another way the human observations. Bone lipid signature associated to OP needs to be clarified if we want to understand better their roles in OP. In that context, by using an ovariectomy-induced OP murine model and looking at lipids in two bone compartments: BM and mineralized tissue (MT), our first challenge was to identify local lipid changes in relation to OP, in view to explore later the mechanisms by which those compounds could alter bone quality, particularly during the mineralization process. As the most striking data, long-term OP resulted in an accumulation of triglycerides, reduced levels of arachidonic and docosahexaenoic acids, an increase of stearoyl-CoA desaturase indices and a reduction of sphingomyelin in the MT, and potential consequences on bone properties and cell activities are discussed. The reader will appreciate that we are at an early stage of understanding the roles of lipids in the OP development and more investigations will be necessary.

Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur.

Osteoporosis is characterized by a bone loss associated to an increased bone marrow adiposity; however, it is still unclear what kind of lipids are involved. Therefore, the main purpose of this study was to see if there is any local bone lipid changes related to osteoporosis, by using the ovariectomy-induced osteoporosis (OVX) rat model. Female SD rats (operated at 6 months of age for skeletal maturity) were divided in control SHAM and OVX groups (n = 6/group) and maintained for 9 month post-surgery. Lipids were analyzed in two compartments of femoral diaphyses: bone marrow (BM) and mineralized tissue (MT), by chromatographic methods. As expected, osteoporotic femurs had a larger BM mass associated with a two-fold increase of lipid content. The MT had a similar lipid enrichment, indicating that adiposity affected the mineral part as well. The main lipids concerned were triglycerides, sphingomyelin, phosphatidylcholine and phosphatidylserine in BM, and triglycerides and cholesterol esters in MT. The increase of both energy-storage and membrane-associated lipids in BM suggested that cell number and/or size was enhanced to allow more triglyceride storage. Interestingly, in MT of osteoporotic femurs, sphingomyelin was decreased, suggesting that its catabolism could be linked to osteoporosis. In both femoral compartments, fatty acid profiles were enriched in 14:0 and 16:1, lowered in 18:0 and 20:4 n-6, and two-fold higher stearoyl-CoA desaturase indexes (16:1/16:0 and 18:1/18:0 ratios), suggesting an increased de novo lipogenesis in osteoporotic femurs. Thus, the present study is first to report local changes of individual lipids in rat osteoporotic femurs and suggests that osteoporosis is a pathologic condition associated with an enhanced de novo lipogenesis. Further studies will be needed to better understand the consequences of these lipid changes in osteoporotic bones.

Lipid determination in bone marrow and mineralized bone tissue: From sample preparation to improved high-performance thin-layer and liquid chromatographic approaches.

In view of their key roles in the bone physiology (e.g., in the biomineralization process) and their potential implication in bone pathologies, an approach to study lipids in situ is needed. The aim of the present paper is to propose an original procedure to characterize lipids in both bone marrow (BM) and mineralized tissue (MT) compartments, taking into consideration sample preparation, lipid extraction and analytical issues, when using small sample size (≤ 0.5g of rat femurs). The potential contamination of the MT by marrow lipids and the poor accessibility of certain lipids from the MT - two major issues in bone handling - were taking care, respectively by performing two cleaning steps after BM removal and by adding a demineralization step to the overall lipid extraction protocol. For lipid analyses, a multi-one-dimensional HP-TLC method was developed to analyze the major neutral and polar lipids at once and showed an excellent resolution (for 15 standards) and a good precision (inter-day RSD<13%). When subjected to the entire "lipid extraction-HP-TLC" protocol, spike recoveries of the standards ranged between 76 and 122%. This HP-TLC method was suitable for lipid determination in both BM and MT [e.g., the MT had 5-times lesser lipids and a lower TG/phospholipid ratio than the BM (P <0.05)], and was quite reliable in term of lipid quantification. The demineralization step allowed to extract additional phosphatidylserine and esterified cholesterol from the MT, suggesting that these two species were associated to the mineralized matrix possibly in relation to their physiological role in the bone. Moreover, a reverse phase HPLC method for fatty acid determination as naphthacyl esters was set up to study fatty acids in bone samples and was used to validate the HP-TLC data. The fatty acid profile of the MT exhibited lower linoleic acid (18:2 n-6) and linolenic acid (18:3 n-3+n-6) levels and higher arachidonic acid (20:4 n-6) and docosahexaenoic acid (22:6 n-3) levels (P<0.05, compared to BM), suggesting that the MT is more metabolically active than the BM in term of long chain fatty acid production. In sum, the present work should contribute to facilitate future studies in the bone lipid field in view to understand better their implication in the marrow fat expansion-associated bone pathologies, such as osteoporosis.

Doses effects of zoledronic acid on mineral apatite and collagen quality of newly-formed bone in the rat's calvaria defect.

Due to their inhibitory effects on resorption, bisphosphonates are widely used in the treatment of diseases associated to an extensive bone loss. Yet, little is known about bisphosphonates effects on newly-formed bone quality. In the present study, adult male Sprague-Dawley rats (n=80) with a bone defect calvaria area were used and short-term effects of zoledronic acid (ZA) were studied on the healing bone area. Three ZA treatments were tested by using either: 1°) a low single dose (120µgZA/kg, n=10; equivalent to human osteoporosis treatment), 2°) a low fractionated doses (20µgZA/kg daily for 6days either a total of 120µg/kg, n=15), and 3°) a high fractionated doses, (100µgZA/kg weekly for 6weeks, n=15; equivalent to 6months of human bone metastasis treatment). For each treatment, a control "vehicle" treatment was performed (with an identical number of rats). After ZA administration, the intrinsic bone material properties were evaluated by quantitative backscattered electron imaging (qBEI) and Raman microspectroscopy. Neither single nor fractionated low ZA doses modify the intrinsic bone material properties of the newly-formed bone compared to their respective control animals. On the opposite, the high ZA treatment resulted in a significant decrease of the crystallinity (-25%, P< 0.05) and of the hydroxyproline-to-proline ratio (-30%, P<0.05) in newly-formed bones. Moreover, with the high ZA treatment, the crystallinity was positively correlated with the hydroxyproline-to-proline ratio (ρ=0.78, P<0.0001). The present data highlight new properties for ZA on bone formation in a craniofacial defect model. As such, ZA at high doses disrupted the apatite crystal organization. In addition, we report here for the first time that high ZA doses decreased the hydroxyproline-to-proline ratio suggesting that ZA may affect the early collagen organization during the bone healing.

Bone Samples Extracted from Embalmed Subjects Are Not Appropriate for the Assessment of Bone Quality at the Molecular Level Using Raman Spectroscopy.

Bone samples extracted from embalmed cadavers are commonly used as controls in the study of bone. The effects of embalmment on the molecular composition of bone are unknown. The objective of this study was to determine the effect of embalmment on the molecular composition and structure of bone, as evaluated by Raman spectroscopy. Bone samples of femoral heads from five embalmed donors and five fresh-frozen donors were compared using Raman microspectroscopy with DuoScan technology. Physicochemical parameters simultaneously describing the organic and mineral phases of bone were compared using the Mann-Whitney U test. Partial least squares discriminant analysis (PLS-DA) was used to determine specific Raman spectral features of each group. Study of the mineral phase showed a 15% reduction of the mineral-to-matrix ratio (p < 0.001), an 8% decrease of type B carbonate substitution (p < 0.001), and a 2% increase in crystallinity (p < 0.001) in the embalmed donors group compared to those of the fresh donors group. Regarding the organic phase of bone, the hydroxyproline-to-proline ratio was increased by 18% in the embalmed group (p < 0.001), with no variation in both the relative proteoglycan content (GAG/CH3) (p = 0.08) and collagen maturity (p = 0.57). PLS-DA showed that the embalmed group was characterized mainly by peaks assigned to hydroxyproline, lipids, and collagen. Embalmment induces significant modifications of the molecular composition of bone. Bone samples from embalmed subjects should be avoided as controls for Raman spectroscopy studies. Preservation procedures performed prior to bone sampling should be reported in studies using human cadaver samples.

Understanding the local actions of lipids in bone physiology.

The adult skeleton is a metabolically active organ system that undergoes continuous remodeling to remove old and/or stressed bone (resorption) and replace it with new bone (formation) in order to maintain a constant bone mass and preserve bone strength from micro-damage accumulation. In that remodeling process, cellular balances--adipocytogenesis/osteoblastogenesis and osteoblastogenesis/osteoclastogenesis--are critical and tightly controlled by many factors, including lipids as discussed in the present review. Interest in the bone lipid area has increased as a result of in vivo evidences indicating a reciprocal relationship between bone mass and marrow adiposity. Lipids in bones are usually assumed to be present only in the bone marrow. However, the mineralized bone tissue itself also contains small amounts of lipids which might play an important role in bone physiology. Fatty acids, cholesterol, phospholipids and several endogenous metabolites (i.e., prostaglandins, oxysterols) have been purported to act on bone cell survival and functions, the bone mineralization process, and critical signaling pathways. Thus, they can be regarded as regulatory molecules important in bone health. Recently, several specific lipids derived from membrane phospholipids (i.e., sphingosine-1-phosphate, lysophosphatidic acid and different fatty acid amides) have emerged as important mediators in bone physiology and the number of such molecules will probably increase in the near future. The present paper reviews the current knowledge about: (1°) bone lipid composition in both bone marrow and mineralized tissue compartments, and (2°) local actions of lipids on bone physiology in relation to their metabolism. Understanding the roles of lipids in bone is essential to knowing how an imbalance in their signaling pathways might contribute to bone pathologies, such as osteoporosis.

Hesperidin increases intestinal ß,ß-carotene 15-15' mono-oxygenase 1 (BCMO1) activity in Mongolian gerbils (Meriones unguiculatus) fed with ß-carotene-free diet.

ß,ß-Carotene 15-15' mono-oxygenase 1 (BCMO1) is a key enzyme in vitamin A (VitA) metabolism in mammals. Dietary compounds, such as carotenoids and polyphenols, were reported to influence BCMO1 activity. The aim of this study was to evaluate the effect of hesperidin (Hes), on the VitA bioefficacy of ß-carotene (Bc) from orange-fleshed sweet potato, using Mongolian gerbils, focussing on BCMO1 activity. Gerbils (n=50) depleted in VitA were divided into five groups fed with basal diet containing 3% white- or orange-fleshed sweet potatoes supplemented or not with Hes. Liver BCMO1 activity was low, with no significant differences between groups. Interestingly, intestinal mucosal BCMO1 activity was significantly higher in the gerbils fed without Bc or VitA than those fed with a VitA/Bc-supplemented diet. Finally, our results show that, under a low VitA status, Hes dramatically stimulated intestinal BCMO1 activity, an effect that could possibly be related to its action as an agonist of PPARγ.

The O-methylation of chrysin markedly improves its intestinal anti-inflammatory properties: Structure-activity relationships of flavones.

The aim of this study was to investigate whether methoxylated flavones versus their unmethylated analogs can modulate the intestinal inflammatory response. Flavone effects were assessed on soluble pro-inflammatory mediator (IL-8, IL-6, macrophage chemoattractant protein-1 (MCP-1), and cyclooxygenase-2 (COX-2)-derived PGE2) production and on nuclear factor (NF)-κB activation in 3d-confluent and 21d-differentiated Caco-2 cells stimulated with interleukin (IL)-1ß. Chrysin (CHRY) showed anti-inflammatory properties by decreasing COX-2-derived PGE2 and reducing NF-κB activation. Compared to CHRY, the dimethoxylated form (CHRY-DM) significantly reduced the secretion of all pro-inflammatory mediators, except IL-8, at both cellular stages (P<0.05); these effects being dose-dependent in 3d-cells. The reduction of NF-κB activation was significantly more pronounced with CHRY-DM. By evaluating other flavones, it was established that several structural dispositions of flavones seemed to be determinant in order to attenuate the intestinal inflammatory response, such as methoxylation of the 5- and 7-hydroxyl groups on the A-ring, non-methoxylation of the 3'-hydroxyl groups on the B-ring, and methoxylation of the 3-hydroxyl group on the C-ring. Of all flavones examined, CHRY-DM exhibited the strongest anti-inflammatory activity. These data indicate that, in the Caco-2 cell model, methoxylation of CHRY greatly improves its anti-inflammatory properties, probably through a more pronounced inhibition of the NF-κB signaling pathway. Nevertheless, methoxylation of other flavones was not systematically beneficial.

Among plant lignans, pinoresinol has the strongest antiinflammatory properties in human intestinal Caco-2 cells.

Dietary lignans show some promising health benefits, but little is known about their fate and activities in the small intestine. The purpose of this study was thus to investigate whether plant lignans are taken up by intestinal cells and modulate the intestinal inflammatory response using the Caco-2 cell model. Six lignan standards [secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), pinoresinol (PINO), lariciresinol, matairesinol (MAT), and hydroxymatairesinol] and their colonic metabolites [enterolactone (ENL) and enterodiol] were studied. First, differentiated cells were exposed to SDG, SECO, PINO, or ENL at increasing concentrations for 4 h, and their cellular contents (before and after deconjugation) were determined by HPLC. Second, in IL-1ß-stimulated confluent and/or differentiated cells, lignan effects were tested on different soluble proinflammatory mediators quantified by enzyme immunoassays and on the NF-κB activation pathway by using cells transiently transfected. SECO, PINO, and ENL, but not SDG, were taken up and partly conjugated by cells, which is a saturable conjugation process. PINO was the most efficiently conjugated (75% of total in cells). In inflamed cells, PINO significantly reduced IL-6 by 65% and 30% in confluent and differentiated cells, respectively, and cyclooxygenase (COX)-2-derived prostaglandin E(2) by 62% in confluent cells. In contrast, MAT increased significantly COX-2-derived prostaglandin E(2) in confluent cells. Moreover, PINO dose-dependently decreased IL-6 and macrophage chemoattractant protein-1 secretions and NF-κB activity. Our findings suggest that plant lignans can be absorbed and metabolized in the small intestine and, among the plant lignans tested, PINO exhibited the strongest antiinflammatory properties by acting on the NF-κB signaling pathway, possibly in relation to its furofuran structure and/or its intestinal metabolism.

Anti-inflammatory effects of pomegranate (Punica granatum L.) husk ellagitannins in Caco-2 cells, an in vitro model of human intestine.

This study aimed at evaluating the anti-inflammatory properties of a pomegranate fruit husk (PomH) polyphenolic extract, rich in punicalagin, using Caco-2 cells, an in vitro model of human intestinal epithelium. Differentiated cells in bicameral inserts were pretreated or not with a PomH extract or punicalagin, as reference, at the apical side, representing the intestinal lumen. Inflammation was then induced with a cocktail of cytokines (Il-1ß, TNFα and IFNγ) and LPS. After 24 h incubation, 3 pro-inflammatory markers, i.e., interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1, were assayed both at their gene transcription (qRT-PCR) and secretion (ELISA) levels. As previously described, the pro-inflammatory cocktail significantly stimulated these 3 markers, at the gene transcript and secretion levels. In inflamed cells, a significant down-regulation of the transcription of the genes encoding IL-6 and MCP-1 was observed in the presence of the PomH extract or punicalagin, while IL-8 transcription was unaffected. Both treatments also decreased the amounts of the 3 proteins with dose-response effects, but only in the apical compartment. A lowered ELISA response was also observed when either IL-6, IL-8 or MCP-1 were mixed with punicalagin in a cell-free culture medium, indicating a direct molecular interaction. In conclusion, the punicalagin-rich PomH extract tested showed anti-inflammatory properties in the Caco-2 in vitro intestinal model. It acted both on the pro-inflammatory gene transcription and protein levels, the later phenomenon being possibly due to a direct molecular trapping. These data suggest that pomegranate husk could be an interesting natural source contributing to prevent intestinal chronic inflammation.

Dimethyl sulfoxide (DMSO) attenuates the inflammatory response in the in vitro intestinal Caco-2 cell model.

This study aimed to investigate dose effects of dimethyl sulfoxide (DMSO) (0.05-1%) on the intestinal inflammatory response in confluent- and differentiated-Caco-2 cells stimulated with interleukin (IL)-1ß or a pro-inflammatory cocktail for 24 h. Cyclooxygenase-2 (COX-2) activity was assayed by incubating inflamed cells with arachidonic acid and then measuring prostaglandin-E(2) (PGE(2)) produced. Soluble mediators (IL-8, IL-6, macrophage chemoattractant protein-1 (MCP-1), and COX-2-derived PGE(2)) were quantified by enzyme immunoassays and mRNA expression of 33 proteins by high throughput TaqMan Low Density Array. Data showed that DMSO decreased induced IL-6 and MCP-1 secretions in a dose-dependent manner (P<0.05), but not IL-8; these effects were cell development- and stimulus- independent. Moreover, in IL-1ß-stimulated confluent-cells, DMSO dose-dependently reduced COX-2-derived PGE(2) (P<0.05). DMSO at 0.5% decreased significantly mRNA levels of 14 proteins involved in the inflammatory response (including IL-6, IL-1α, IL-1ß, and COX-2). Thus, DMSO at low concentrations (0.1-0.5%) exhibits anti-inflammatory properties in the in vitro intestinal Caco-2 cell model. This point is important to be taken into account when assessing anti-inflammatory properties of bioactive compounds requiring DMSO as vehicle, such as phenolic compounds, in order to avoid miss-interpretation of the results.

Physio-pathological parameters affect the activation of inflammatory pathways by deoxynivalenol in Caco-2 cells.

The intake of deoxynivalenol (DON), a mycotoxin contaminating cereal food items, causes gastro-intestinal illness in human and animal. This study investigated whether intracellular inflammatory cascades (MAPKs and NF-κB), cell maturity (proliferating vs. differentiated), cell state (control vs. inflamed) and exposure duration (chronic vs. acute) affect IL-8 secretion and PGE-2 synthesis in Caco-2 cells exposed to plausible intestinal concentrations (50, 500 and 5000 ng/ml) of DON. IL-8 secretion and PGE-2 synthesizing capacity were dose-dependently upregulated in differentiated Caco-2 cells exposed to DON during 24h, reaching an increase of ∼25 and 1.7-fold respectively, whereas transcript level of IL-8 and COX-2 were increased by ∼40 and 17-fold. Similar results were obtained with proliferating cells. The upregulation decreased upon simultaneous incubation with inhibitors of MAPKs ERK1/2 or p38 or of transcription factor NF-κB. IL-8 secretion and PGE-2 synthesizing capacity increased respectively by ∼15 and 2-fold after chronic 21 day incubation with DON (50 ng/ml). IL-8 production was exacerbated (∼510-fold versus negative control) upon simultaneous exposure to inflammatory stimuli. These results suggest activation of inflammatory pathways in intestinal epithelial cells exposed chronically or acutely to DON. The sensitivity to DON, whereas not affected by cell differentiation, is exacerbated by the presence of additional stimuli.

Dietary polyphenols can modulate the intestinal inflammatory response.

Inflammatory bowel diseases (IBD) arise from multiple causes, including environmental factors, gut microflora, immunity, and genetic predispositions. In the course of IBD, immune homeostasis and intestinal mucosa barrier integrity are impaired. Among natural preventive treatments that have been identified to date, polyphenols appear as promising candidates. They have been shown to protect against several diseases, including cardiovascular diseases and cancers, and they have anti-inflammatory properties in non-intestinal models. This paper will review the literature that has described to date some effects of polyphenols on intestinal inflammation. Studies, conducted using in vivo and in vitro models, provide evidence that pure polyphenolic compounds and natural polyphenolic plant extracts can modulate intestinal inflammation.

Inhibition of inflammatory mediators by polyphenolic plant extracts in human intestinal Caco-2 cells.

The mitogen-activated protein kinases (MAPK) and nuclear factor kappaB (NF-kappaB) are involved in transduction cascades that play a key role in inflammatory response. We tested the ability of preselected natural polyphenolic extracts (grape seed, cocoa, sugar cane, oak, mangosteen and pomegranate) to modulate intestinal inflammation using human intestinal Caco-2 cells treated for 4h with these extracts and then stimulated by cytokines for 24 or 48h. The effect of polyphenolic extracts, at 50 micromol of gallic acid equivalent/l, was investigated on inflammation-related cellular events: (i) NF-kappaB activity (cells transfected with a NF-kappaB-luciferase construct), (ii) activation of Erk1/2 and JNK (western blotting), (iii) secretion of interleukin 8 (IL-8) (ELISA), (iv) secretion of prostaglandin (PG) E(2) (ELISA), (v) production of NO (Griess method). Results show that: (i) sugar cane, oak and pomegranate extracts inhibited NF-kappaB activity (from 1.6 to 1.9-fold) (P<0.001); (ii) pomegranate slightly inhibited Erk1/2 activation (1.3-fold) (P=0.008); (iii) oak and pomegranate decreased NO synthesis by 1.5-fold (P<0.001) and that of IL-8 by 10.3 and 6.7-fold respectively; (iv) pomegranate and cocoa decreased PGE(2) synthesis by 4.6 (P<0.0001) and 2.2-fold (P=0.001), respectively. We suggest that pomegranate extract could be particularly promising in dietary prevention of intestinal inflammation.

Modulation of signalling nuclear factor-kappaB activation pathway by polyphenols in human intestinal Caco-2 cells.

Recent studies support beneficial effects of polyphenols in various chronic inflammatory diseases, for example, the inflammatory bowel diseases. Inhibition of NF-kappaB activation by polyphenols could explain part of their anti-inflammatory properties, but few data are available on the intestine. The purpose of the present study was thus to investigate the effects of some polyphenols on NF-kappaB activation using human intestinal Caco-2 cells. Effects of standard polyphenols (50 mumol/l) were studied on different cellular events associated with NF-kappaB activation: (i) NF-kappaB activity using cells transiently transfected with a NF-kappaB-luciferase construct and stimulated by inflammatory agents (IL-1beta, TNF-alpha or lipopolysaccharides (LPS)); (ii) phosphorylation of the inhibitor of kappaB (IkappaB-alpha) analysed by Western blot; (iii) secretion of IL-8 quantified by ELISA assay. Results showed that chrysin and ellagic acid inhibited NF-kappaB activity, whereas genistein and resveratrol increased it. These effects were independent of the nature of the inducer, indicating that polyphenols may modulate NF-kappaB activation by acting on a common event to the cytokine- and LPS-mediated cascades. Chrysin strongly reduced (2.5-fold) IL-1beta-induced IkappaB-alpha phosphorylation, whereas ellagic acid increased it (1.7-fold). Ellagic acid, genistein and epigallocatechin gallate reduced (4- to 8-fold) IL-1beta-induced IL-8 secretion, while resveratrol promoted (1.7-fold) the secretion. Chrysin also diminished IL-8 secretion by 1.6-fold (but P>0.05). The data indicate that polyphenols can modulate the NF-kappaB activation pathway in the intestine. Chrysin could block NF-kappaB activation via the inhibition of IkappaB-alpha phosphorylation. The other molecular targets of the active polyphenols are still to be identified.

Xanthophylls are preferentially taken up compared with beta-carotene by retinal cells via a SRBI-dependent mechanism.

The purpose of this study was to investigate the mechanisms by which carotenoids [xanthophylls vs. beta-carotene(beta-C)] are taken up by retinal pigment epithelial (RPE) cells. The human RPE cell line, ARPE-19, was used. When ARPE-19 cells were fully differentiated (7-9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent 2-fold higher than beta-C (P < 0.05). At 9 weeks, cellular uptakes were 1.6, 2.5, and 3.2%, respectively, for beta-C, LUT, and ZEA. Similar extents were observed when carotenoids were delivered in either Tween 40 or "chylomicrons" produced by Caco-2 cells. Differentiated ARPE-19 cells did not exhibit any detectable beta-C 15,15'-oxygenase activity or convert exogenous beta-C into vitamin A. When using specific antibodies against the lipid transporters cluster determinant 36 (CD36) and scavenger receptor class B type I (SR-BI), cellular uptake of beta-C and ZEA were significantly decreased (40-60%) with anti-SR-BI but not with anti-CD36. Small interfering RNA transfection for SR-BI led to marked knockdown of SR-BI protein expression (approximately 90%), which resulted in decreased beta-C and ZEA uptakes by 51% and 87%, respectively. Thus, the present data show that RPE cells preferentially take up xanthophylls versus the carotene by a process that appears to be entirely SR-BI-dependent for ZEA and partly so for beta-C. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina.

Lutein and zeaxanthin protect photoreceptors from apoptosis induced by oxidative stress: relation with docosahexaenoic acid.

PURPOSE: Oxidative stress has been proposed as a major pathogenic factor in age-related macular degeneration (AMD), the leading cause of vision loss among elderly people of western European ancestry. Lutein (LUT) and zeaxanthin (ZEA), major components in macular pigment, are among the retinal antioxidants. Though xanthophyll intake may reduce the likelihood of having advanced AMD, direct evidence of neuroprotection is lacking. Prior work has shown that docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, delays apoptosis and promotes differentiation of photoreceptors. This study was conducted to investigate whether LUT, ZEA, and beta-carotene (BC), major dietary carotenoids protect photoreceptors from oxidative stress and whether this protection is synergistic with that of DHA. METHODS: Pure rat retinal neurons in culture, supplemented with LUT, ZEA, or BC, with or without DHA, were subjected to oxidative stress induced with paraquat and hydrogen peroxide. Apoptosis, preservation of mitochondrial membrane potential, cytochrome c translocation, and opsin expression were evaluated. RESULTS: Pretreatment with DHA, LUT, ZEA, and BC reduced oxidative stress-induced apoptosis in photoreceptors, preserved mitochondrial potential, and prevented cytochrome c release from mitochondria. ZEA and LUT also enhanced photoreceptor differentiation. In control cultures, photoreceptors failed to grow their characteristic outer segments; addition of DHA, ZEA, or LUT increased opsin expression and promoted the development of outer-segment-like processes. CONCLUSIONS: These results show for the first time the direct neuroprotection of photoreceptors by xanthophylls and suggest that ZEA and LUT, along with DHA, are important environmental influences that together promote photoreceptor survival and differentiation.

Mechanisms of provitamin A (carotenoid) and vitamin A (retinol) transport into and out of intestinal Caco-2 cells.

The purpose of this study was to compare the mechanisms of intestinal retinol (ROL) and carotenoid transport. When differentiated Caco-2 cells were incubated with ROL for varying times, cellular ROL plateaued within 2 h, whereas retinyl ester (RE) formation increased continuously. ROL and RE efflux into basolateral medium (BM) increased linearly with time, ROL in the nonlipoprotein fraction and REs in chylomicrons (CMs). In contrast to carotenoids, ROL uptake was proportional to ROL concentration (0.5-110 microM). ROL efflux into BM occurred via two processes: a) a saturable process at low concentrations (<10 microM) and b) a nonsaturable process at higher concentrations. When ROL-loaded cells were maintained on retinoid-free medium, free ROL, but not REs, was secreted into BM. Glyburide significantly reduced ROL efflux but not ROL uptake. Inhibition of ABCA1 protein expression by small interfering RNAs decreased ROL efflux but not carotenoid efflux. Scavenger receptor class B type I (SR-BI) inhibition did not affect ROL transport but decreased carotenoid uptake. The present data suggest that a) ROL enters intestinal cells by diffusion, b) ROL efflux is partly facilitated, probably by the basolateral transporter ABCA1, and c) newly synthesized REs, but not preformed esters, are incorporated into CM and secreted. In contrast to ROL transport, carotenoid uptake is mediated by the apical transporter SR-BI, and carotenoid efflux occurs exclusively via their secretion in CM.