Cis-Nerolidol Inhibits MAP Kinase and NF-κB Signaling Pathways and Prevents Epithelial Tight Junction Dysfunction in Colon Inflammation: In Vivo and In Vitro Studies.

Inflammation of the GI tract leads to compromised epithelial barrier integrity, which increases intestine permeability. A compromised intestinal barrier is a critical event that leads to microbe entry and promotes inflammatory responses. Inflammatory bowel diseases that comprise Crohn's disease (CD) and ulcerative colitis (UC) show an increase in intestinal permeability. Nerolidol (NED), a naturally occurring sesquiterpene alcohol, has potent anti-inflammatory properties in preclinical models of colon inflammation. In this study, we investigated the effect of NED on MAPKs, NF-κB signaling pathways, and intestine epithelial tight junction physiology using in vivo and in vitro models. The effect of NED on proinflammatory cytokine release and MAPK and NF-κB signaling pathways were evaluated using lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. Subsequently, the role of NED on MAPKs, NF-κB signaling, and the intestine tight junction integrity were assessed using DSS-induced colitis and LPS-stimulated Caco-2 cell culture models. Our result indicates that NED pre-treatment significantly inhibited proinflammatory cytokine release, expression of proteins involved in MAP kinase, and NF-κB signaling pathways in LPS-stimulated RAW macrophages and DSS-induced colitis. Furthermore, NED treatment significantly decreased FITC-dextran permeability in DSS-induced colitis. NED treatment enhanced tight junction protein expression (claudin-1, 3, 7, and occludin). Time-dependent increases in transepithelial electrical resistance (TEER) measurements reflect the formation of healthy tight junctions in the Caco-2 monolayer. LPS-stimulated Caco-2 showed a significant decrease in TEER. However, NED pre-treatment significantly prevented the fall in TEER measurements, indicating its protective role. In conclusion, NED significantly decreased MAPK and NF-κB signaling pathways and decreased tight junction permeability by enhancing epithelial tight junction protein expression.

Molecular Docking Identifies 1,8-Cineole (Eucalyptol) as A Novel PPARγ Agonist That Alleviates Colon Inflammation.

Inflammatory bowel disease, comprising Crohn's disease (CD) and ulcerative colitis (UC), is often debilitating. The disease etiology is multifactorial, involving genetic susceptibility, microbial dysregulation, abnormal immune activation, and environmental factors. Currently, available drug therapies are associated with adverse effects when used long-term. Therefore, the search for new drug candidates to treat IBD is imperative. The peroxisome proliferator-activated receptor-γ (PPARγ) is highly expressed in the colon. PPARγ plays a vital role in regulating colonic inflammation. 1,8-cineole, also known as eucalyptol, is a monoterpene oxide present in various aromatic plants which possess potent anti-inflammatory activity. Molecular docking and dynamics studies revealed that 1,8-cineole binds to PPARγ and if it were an agonist, that would explain the anti-inflammatory effects of 1,8-cineole. Therefore, we investigated the role of 1,8-cineole in colonic inflammation, using both in vivo and in vitro experimental approaches. Dextran sodium sulfate (DSS)-induced colitis was used as the in vivo model, and tumor necrosis factor-α (TNFα)-stimulated HT-29 cells as the in vitro model. 1,8-cineole treatment significantly decreased the inflammatory response in DSS-induced colitis mice. 1,8-cineole treatment also increased nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into the nucleus to induce potent antioxidant effects. 1,8-cineole also increased colonic PPARγ protein expression. Similarly, 1,8-cineole decreased proinflammatory chemokine production and increased PPARγ protein expression in TNFα-stimulated HT-29 cells. 1,8-cineole also increased PPARγ promoter activity time-dependently. Because of its potent anti-inflammatory effects, 1,8-cineole may be valuable in treating IBD.

Salmonella Typhimurium Infection Reduces the Ascorbic Acid Uptake in the Intestine.

Salmonella Typhimurium infection of the gastrointestinal tract leads to damage that compromises the integrity of the intestinal epithelium and results in enterocolitis and inflammation. Salmonella infection promotes the expression of inflammasome NLRP3, leading to activation and release of proinflammatory cytokines such as IL-1ß, and the infected host often displays altered nutrient levels. To date, the effect of Salmonella infection and proinflammatory cytokine IL-1ß on the intestinal uptake of ascorbic acid (AA) is unknown. Our results revealed a marked decrease in the rate of AA uptake in mouse jejunum infected with Salmonella wild type (WT). However, the nonpathogenic mutant (Δ invA Δ spiB) strain did not affect AA uptake. The decrease in AA uptake due to Salmonella WT infection is accompanied by significantly lower expression of mouse (m)SVCT1 protein, mRNA, and hnRNA levels. NLRP3 and IL-1ß expression levels were markedly increased in Salmonella-infected mouse jejunum. IL-1ß-exposed Caco-2 cells displayed marked inhibition in AA uptake and significantly decreased hSVCT1 expression at both protein and mRNA levels. Furthermore, the activity of the SLC23A1 promoter was significantly inhibited by IL-1ß exposure. In addition, GRHPR (a known SVCT1 interactor) protein and mRNA expression levels were significantly reduced in Salmonella-infected mouse jejunum. These results indicate that Salmonella infection inhibits AA absorption in mouse jejunum and IL-1ß-exposed Caco-2 cells. The observed inhibitory effect may partially be mediated through transcriptional mechanisms.

ß-Myrcene Mitigates Colon Inflammation by Inhibiting MAP Kinase and NF-κB Signaling Pathways.

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders that include Crohn's disease (CD) and ulcerative colitis (UC). The incidence of IBD is rising globally. However, the etiology of IBD is complex and governed by multiple factors. The current clinical treatment for IBD mainly includes steroids, biological agents and need-based surgery, based on the severity of the disease. Current drug therapy is often associated with adverse effects, which limits its use. Therefore, it necessitates the search for new drug candidates. In this pursuit, phytochemicals take the lead in the search for drug candidates to benefit from IBD treatment. ß-myrcene is a natural phytochemical compound present in various plant species which possesses potent anti-inflammatory activity. Here we investigated the role of ß-myrcene on colon inflammation to explore its molecular targets. We used 2% DSS colitis and TNF-α challenged HT-29 adenocarcinoma cells as in vivo and in vitro models. Our result indicated that the administration of ß-myrcene in dextran sodium sulfate (DSS)-treated mice restored colon length, decreased disease activity index (DAI), myeloperoxidase (MPO) enzyme activity and suppressed proinflammatory mediators. ß-myrcene administration suppressed mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways to limit inflammation. ß-myrcene also suppressed mRNA expression of proinflammatory chemokines in tumor necrosis factor-α (TNF-α) challenged HT-29 adenocarcinoma cells. In conclusion, ß-myrcene administration suppresses colon inflammation by inhibiting MAP kinases and NF-κB pathways.

Health Benefits, Pharmacological Effects, Molecular Mechanisms, and Therapeutic Potential of α-Bisabolol.

α-Bisabolol is one of the important monocyclic sesquiterpenes, derived naturally from essential oils of many edible and ornamental plants. It was first obtained from Matricaria chamomilla, commonly known as chamomile or German chamomile. The available literature indicates that this plant along with other α-Bisabolol containing plants is popularly used in traditional medicine for potential health benefits and general wellbeing. Nutritional studies are indicative of the health benefits of α-Bisabolol. Numerous experimental studies demonstrated pharmacological properties of α-Bisabolol including anticancer, antinociceptive, neuroprotective, cardioprotective, and antimicrobial. This review aims to collectively present different pharmacological activities based on both in vitro and in vivo studies. In the present review using synoptic tables and figures, we comprehensively present that α-Bisabolol possesses therapeutic and protective activities, therefore, it can be used for potential health benefits based on pharmacological effects, underlying molecular mechanism, and favorable pharmaceutical properties. Based on the studies mostly performed on cell lines or animal models, it is evident that α-Bisabolol may be a promising nutraceutical and phytomedicine to target aberrant biological mechanisms which result in altered physiological processes and various ailments. Given the polypharmacological effects and pleiotropic properties, along with favorable pharmacokinetics, and dietary availability and safety, α-Bisabolol can be used as a dietary agent, nutraceutical or phytopharmaceutical agent or as an adjuvant with currently available modern medicines. The regulatory approval of this molecule for use as food additives, and in cosmetics and fragrance industry is also supportive of its human usage. Moreover, further studies are necessary to address pharmaceutical, pharmacological, and toxicological aspects before clinical or nutritional usage in humans. The biological actions and health benefits open opportunities for pharmaceutical development with pharmacological basis of its use in future therapeutics.

α-Bisabolol Mitigates Colon Inflammation by Stimulating Colon PPAR-γ Transcription Factor: In Vivo and In Vitro Study.

The incidence and prevalence of inflammatory bowel disease (IBD, Crohn's disease, and ulcerative colitis) are increasing worldwide. The etiology of IBD is multifactorial, including genetic predisposition, dysregulated immune response, microbial dysbiosis, and environmental factors. However, many of the existing therapies are associated with marked side effects. Therefore, the development of new drugs for IBD treatment is an important area of investigation. Here, we investigated the anti-inflammatory effects of α-bisabolol, a naturally occurring monocyclic sesquiterpene alcohol present in many aromatic plants, in colonic inflammation. To address this, we used molecular docking and dynamic studies to understand how α-bisabolol interacts with PPAR-γ, which is highly expressed in the colonic epithelium: in vivo (mice) and in vitro (RAW264.7 macrophages and HT-29 colonic adenocarcinoma cells) models. The molecular docking and dynamic analysis revealed that α-bisabolol interacts with PPAR-γ, a nuclear receptor protein that is highly expressed in the colon epithelium. Treatment with α-bisabolol in DSS-administered mice significantly reduced Disease Activity Index (DAI), myeloperoxidase (MPO) activity, and colonic length and protected the microarchitecture of the colon. α-Bisabolol treatment also reduced the expression of proinflammatory cytokines (IL-6, IL1ß, TNF-α, and IL-17A) at the protein and mRNA levels. The expression of COX-2 and iNOS inflammatory mediators were reduced along with tissue nitrite levels. Furthermore, α-bisabolol decreased the phosphorylation of activated mitogen-activated protein kinase (MAPK) signaling and nuclear factor kappa B (NFκB) proteins and enhanced colon epithelial PPAR-γ transcription factor expression. However, the PPAR-α and ß/δ expression was not altered, indicating α-bisabolol is a specific stimulator of PPAR-γ. α-Bisabolol also increased the PPAR-γ transcription factor expression but not PPAR-α and ß/δ in pretreated in LPS-stimulated RAW264.7 macrophages. α-Bisabolol significantly decreased the expression of proinflammatory chemokines (CXCL-1 and IL-8) mRNA in HT-29 cells treated with TNF-α and HT-29 PPAR-γ promoter activity. These results demonstrate that α-bisabolol mitigates colonic inflammation by inhibiting MAPK signaling and stimulating PPAR-γ expression.

Thymoquinone, a Dietary Bioactive Compound, Exerts Anti-Inflammatory Effects in Colitis by Stimulating Expression of the Colonic Epithelial PPAR-γ Transcription Factor.

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders with increasing incidence and prevalence worldwide. Here, we investigated thymoquinone (TQ), a naturally occurring phytochemical present in Nigella sativa, for anti-inflammatory effects in colonic inflammation. To address this, we used in vivo (mice) and in vitro (HT-29 cells) models in this investigation. Our results showed that TQ treatment significantly reduced the disease activity index (DAI), myeloperoxidase (MPO) activity, and protected colon microscopic architecture. In addition, TQ also reduced the expression of proinflammatory cytokines and mediators at both the mRNA and protein levels. Further, TQ decreased phosphorylation of the activated mitogen-activated protein kinase (MAPK) signaling pathway and nuclear factor kappa B (NF-κB) proteins and enhanced colon epithelial PPAR-γ transcription factor expression. TQ significantly decreased proinflammatory chemokines (CXCL-1 and IL-8), and mediator (COX-2) mRNA expression in HT-29 cells treated with TNF-α. TQ also increased HT-29 PPAR-γ mRNA, PPAR-γ protein expression, and PPAR-γ promoter activity. These results indicate that TQ inhibits MAPK and NF-κB signaling pathways and transcriptionally regulates PPAR-γ expression to induce potent anti-inflammatory activity in vivo and in vitro models of colon inflammation.

Nerolidol Mitigates Colonic Inflammation: An Experimental Study Using both In Vivo and In Vitro Models.

Nerolidol (NED) is a naturally occurring sesquiterpene alcohol present in various plants with potent anti-inflammatory effects. In the current study, we investigated NED as a putative anti-inflammatory compound in an experimental model of colonic inflammation. C57BL/6J male black mice (C57BL/6J) were administered 3% dextran sodium sulfate (DSS) in drinking water for 7 days to induce colitis. Six groups received either vehicle alone or DSS alone or DSS with oral NED (50, 100, and 150 mg/kg body weight/day by oral gavage) or DSS with sulfasalazine. Disease activity index (DAI), colonic histology, and biochemical parameters were measured. TNF-α-treated HT-29 cells were used as in vitro model of colonic inflammation to study NED (25 µM and 50 µM). NED significantly decreased the DAI and reduced the inflammation-associated changes in colon length as well as macroscopic and microscopic architecture of the colon. Changes in tissue Myeloperoxidase (MPO) concentrations, neutrophil and macrophage mRNA expression (CXCL2 and CCL2), and proinflammatory cytokine content (IL-1ß, IL-6, and TNF-α) both at the protein and mRNA level were significantly reduced by NED. The increase in content of the proinflammatory enzymes, COX-2 and iNOS induced by DSS were also significantly inhibited by NED along with tissue nitrate levels. NED promoted Nrf2 nuclear translocation dose dependently. NED significantly increased antioxidant enzymes activity (Superoxide dismutase (SOD) and Catalase (CAT)), Hemeoxygenase-1 (HO-1), and SOD3 mRNA levels. NED treatment in TNF-α-challenged HT-29 cells significantly decreased proinflammatory chemokines (CXCL1, IL-8, CCL2) and COX-2 mRNA levels. NED supplementation attenuates colon inflammation through its potent antioxidant and anti-inflammatory activity both in in vivo and in vitro models of colonic inflammation.

Phytochemical drug candidates for the modulation of peroxisome proliferator-activated receptor γ in inflammatory bowel diseases.

Plant-based compounds or phytochemicals such as alkaloids, glycosides, flavonoids, volatile oils, tannins, resins, and polyphenols have been used extensively in traditional medicine for centuries and more recently in Western alternative medicine. Extensive evidence suggests that consumption of dietary polyphenolic compounds lowers the risk of inflammatory diseases. The anti-inflammatory properties of several phytochemicals are mediated through ligand-inducible peroxisome proliferator-activated receptors (PPARs), particularly the PPARγ transcription factor. Inflammatory bowel disease (IBD) is represented by ulcerative colitis, which occurs in the mucosa of the colon and rectum, and Crohn's disease (CD) that can involve any segment of gastrointestinal tract. Because of the lack of cost-effective pharmaceutical treatment options, many IBD patients seek and use alternative and unconventional therapies to alleviate their symptoms. PPARγ plays a role in the inhibition of inflammatory cytokine expression and activation of anti-inflammatory immune cells. The phytochemicals reported here are ligands that activate PPARγ, which in turn modulates inflammatory responses. PPARγ is highly expressed in the gut making it a potential therapeutic target for IBDs. This review summarizes the effects of the currently published phytochemicals that modulate the PPARγ pathway and reduce or eliminate colonic inflammation.

Therapeutic Potential of Plants and Plant Derived Phytochemicals against Acetaminophen-Induced Liver Injury.

Acetaminophen (APAP), which is also known as paracetamol or N-acetyl-p-aminophenol is a safe and potent drug for fever, pain and inflammation when used at its normal therapeutic doses. It is available as over-the-counter drug and used by all the age groups. The overdose results in acute liver failure that often requires liver transplantation. Current clinical therapy for APAP-induced liver toxicity is the administration of N-acetyl-cysteine (NAC), a sulphydryl compound an approved drug which acts by replenishing cellular glutathione (GSH) stores in the liver. Over the past five decades, several studies indicate that the safety and efficacy of herbal extracts or plant derived compounds that are used either as monotherapy or as an adjunct therapy along with conventional medicines for hepatotoxicity have shown favorable responses. Phytochemicals mitigate necrotic cell death and protect against APAP-induced liver toxicityby restoring cellular antioxidant defense system, limiting oxidative stress and subsequently protecting mitochondrial dysfunction and inflammation. Recent experimental evidences indicat that these phytochemicals also regulate differential gene expression to modulate various cellular pathways that are implicated in cellular protection. Therefore, in this review, we highlight the role of the phytochemicals, which are shown to be efficacious in clinically relevant APAP-induced hepatotoxicity experimental models. In this review, we have made comprehensive attempt to delineate the molecular mechanism and the cellular targets that are modulated by the phytochemicals to mediate the cytoprotective effect against APAP-induced hepatotoxicity. In this review, we have also defined the challenges and scope of phytochemicals to be developed as drugs to target APAP-induced hepatotoxicity.

Frondanol, a Nutraceutical Extract from Cucumaria frondosa, Attenuates Colonic Inflammation in a DSS-Induced Colitis Model in Mice.

Frondanol is a nutraceutical lipid extract of the intestine of the edible Atlantic sea cucumber, Cucumaria frondosa, with potent anti-inflammatory effects. In the current study, we investigated Frondanol as a putative anti-inflammatory compound in an experimental model of colonic inflammation. C57BL/6J male black mice (C57BL/6J) were given 3% dextran sodium sulfate (DSS) in drinking water for 7 days to induce colitis. The colitis group received oral Frondanol (100 mg/kg body weight/per day by gavage) and were compared with a control group and the DSS group. Disease activity index (DAI) and colon histology were scored for macroscopic and microscopic changes. Colonic tissue length, myeloperoxidase (MPO) concentration, neutrophil and macrophage marker mRNA, pro-inflammatory cytokine proteins, and their respective mRNAs were measured using ELISA and real-time RT-PCR. The tissue content of leukotriene B4 (LTB4) was also measured using ELISA. Frondanol significantly decreased the DAI and reduced the inflammation-associated changes in colon length as well as macroscopic and microscopic architecture of the colon. Changes in tissue MPO concentrations, neutrophil and macrophage mRNA expression (F4/80 and MIP-2), and pro-inflammatory cytokine content (IL-1β, IL-6 and TNF-α) both at the protein and mRNA level were significantly reduced by Frondanol. The increase in content of the pro-inflammatory mediator leukotriene B4 (LTB4) induced by DSS was also significantly inhibited by Frondanol. It was thus found that Frondanol supplementation attenuates colon inflammation through its potent anti-inflammatory activity.

Protective effect of Lagerstroemia speciosa against dextran sulfate sodium induced ulcerative colitis in C57BL/6 mice.

The protective effect of methanolic extract of Lagerstroemia speciosaleaves (LS) was evaluated against dextran sulfate sodium (DSS) induced ulcerative colitis in C57BL/6 mice. The administration of DSS (2.5% in drinking water ad libitum) in C57BL/6 mice induced ulcerative colitis in 7 days. The LS was orally administered for 7 days at daily doses of 100 and 200 mg/kg. At the end of 7 days of treatment the animals were sacrificed, colonic tissues were removed and processed for further analysis of oxidative stress, and histopathology. In DSS treated mice the oxidative stress markers were elevated compared to controls. There was also significant reduction in the anti-oxidant defense levels marked by reduced cellular glutathione, catalase, and superoxide dismutase. The DSS-induced damage to the colon epithelium was evident from a significant increase in the lipid peroxidation. The histology of colon sections revealed inflammatory changes and marked impairment in the integrity of the mucosal lining with inflammatory changes. Both the doses of LS significantly prevented DSS-induced inflammatory and ulcerative damages of the colon, reduced lipid peroxidation and also restored the levels of innate antioxidants in the colon tissue. These findings indicate the protective effects of LS against the DSS-induced inflammatory and oxidative damage in the mouse colon. Further investigation involving bioactivity guided fractionation of the LS can yield potent constituent which may have a significant role in the treatment of inflammatory bowel disease and ulcerative colitis.

The location of pacemakers in the uteri of pregnant guinea pigs and rats.

The pregnant uterus is a smooth muscle organ whose pattern of contraction is dictated by the propagation of electrical impulses. Such electrical activity may originate from one or more pacemakers, but the location of these sites has not yet been determined. To detect the location of the pacemaker in the gravid uterus, two approaches were used: 1) determine the site from where the contraction started using isolated uteri from the pregnant guinea pig, and videotape their contractions; and 2) record, in isolated uteri from pregnant term rats, with 240 extracellular electrodes simultaneously, and determine where the electrical bursts started. In both the contractile and electrophysiological experiments, there was not a single, specific pacemaker area. However, most contractions (guinea pig 87%) and bursts (rat 76%) started close to the mesometrial border (mean 2.7 ± 4.0 mm SD in guinea pigs and 1.3 ± 1.4 mm in rats). In addition, in the rat, most sites of initiations were located closer to the ovarial end of the horn (mean distance from the ovarial end 6.0 ± 6.2 mm SD), whereas such an orientation was not seen in the guinea pig. In both guinea pig and rat uteri at term, there is not one specific pacemaker area. Rather, contractile and electrical activity may arise from any site, with the majority starting close to the mesometrial border. Furthermore, in the rat, most activities started at the ovarial end of the horn. This may suggest a slightly different pattern of contraction in both species.

Effect of Ethanol Exposure on Slow Wave Activity and Smooth Muscle Contraction in the Rat Small Intestine.

BACKGROUND: Ethanol ingestion causes a variety of gastrointestinal disturbances including motility alterations. Slow wave propagation coordinates gastrointestinal motility, and abnormal slow wave activity is thought to contribute to motility disorders. To date, however, little is known about the effect of acute ethanol on motility disturbances associated with slow wave activity. AIM: To investigate the effect of ethanol on small intestine slow wave activity. METHODS: Segments (3-5 cm long) were isolated from the rat duodenum, jejunum, and ileum and mounted in an organ bath superfused with a normal Tyrode solution or with 1, 3, or 5% ethanol containing Tyrode. The electrical activities were recorded using an array of 121 extracellular electrodes, and motility recordings were performed using a digital video camera. RESULTS: The frequency and amplitude of slow wave activity were not altered at 1, 3, or 5% ethanol concentrations, but a significant drop in velocity was found at 3 and 5% ethanol. Furthermore, inexcitable areas appeared in a dose-dependent manner. Slow wave was sometimes also seen to propagate in a circular fashion, thereby describing a reentrant loop. Finally, in all duodenal, jejunal, and ileal segments, ethanol inhibited contractions and became fully quiescent at 3-5%. CONCLUSIONS: These studies for the first time demonstrate that ethanol significantly inhibits slow wave and spike activity in a dose-dependent manner and could also initiate reentrant activities. Intestinal contractions were also inhibited in a dose-dependent manner. In conclusion, ethanol inhibits both slow wave activity and motor activity to cause ethanol-induced intestinal disturbances.

Chronic alcohol feeding inhibits physiological and molecular parameters of intestinal and renal riboflavin transport.

Vitamin B2 (riboflavin, RF) is essential for normal human health. Mammals obtain RF from exogenous sources via intestinal absorption and prevent its urinary loss by reabsorption in the kidneys. Both of these absorptive events are carrier-mediated and involve specific RF transporters (RFVTs). Chronic alcohol consumption in humans is associated with a high prevalence of RF deficiency and suboptimal levels, but little is known about the effect of chronic alcohol exposure on physiological and molecular parameters of the intestinal and renal RF transport events. We addressed these issues using rats chronically fed an alcohol liquid diet and pair-fed controls as a model. The results showed that chronic alcohol feeding significantly inhibits carrier-mediated RF transport across the intestinal brush-border and basolateral membrane domains of the polarized enterocytes. This inhibition was associated with a parallel reduction in the expression of the rat RFVT-1 and -3 at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels. Chronic alcohol feeding also caused a significant inhibition in RF uptake in the colon. Similarly, a significant inhibition in carrier-mediated RF transport across the renal brush-border and basolateral membrane domains was observed, which again was associated with a significant reduction in the level of expression of RFVT-1 and -3 at the protein, mRNA, and hnRNA levels. These findings demonstrate that chronic alcohol exposure impairs both intestinal absorption and renal reabsorption processes of RF and that these effects are, at least in part, mediated via transcriptional mechanism(s) involving the slc52a1 and slc52a3 genes.

Differentiation-dependent regulation of intestinal vitamin B(2) uptake: studies utilizing human-derived intestinal epithelial Caco-2 cells and native rat intestine.

Intestinal epithelial cells undergo differentiation as they move from the crypt to the villi, a process that is associated with up- and downregulation in expression of a variety of genes, including those involved in nutrient absorption. Whether the intestinal uptake process of vitamin B(2) [riboflavin (RF)] also undergoes differentiation-dependent regulation and the mechanism through which this occurs are not known. We used human-derived intestinal epithelial Caco-2 cells and native rat intestine as models to address these issues. Caco-2 cells showed a significantly higher carrier-mediated RF uptake in post- than preconfluent cells. This upregulation was associated with a significantly higher level of protein and mRNA expression of the RF transporters hRFVT-1 and hRFVT-3 in the post- than preconfluent cells; it was also accompanied with a significantly higher rate of transcription of the respective genes (SLC52A1 and SLC52A3), as indicated by the higher level of expression of heterogeneous nuclear RNA and higher promoter activity in post- than preconfluent cells. Studies with native rat intestine also showed a significantly higher RF uptake by epithelial cells of the villus tip than epithelial cells of the crypt; this again was accompanied by a significantly higher level of expression of the rat RFVT-1 and RFVT-3 at the protein, mRNA, and heterogeneous nuclear RNA levels. These findings show, for the first time, that the intestinal RF uptake process undergoes differentiation-dependent upregulation and suggest that this is mediated (at least in part) via transcriptional mechanisms.

Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: studies utilizing Caco-2 cells and Rab8a knockout mice.

BACKGROUND: Ascorbic acid (AA) is required for normal human health and development. Human intestine expresses two sodium-dependent vitamin C transporters (hSVCT-1 and -2) that mediate cellular AA transport, with hSVCT1 targeting to the apical membrane of polarized epithelia. Studies have shown a role for the Rab8a in the apical membrane targeting of transporters in intestinal cells. AIMS: The purpose of this study was to determine whether Rab8a impacts the function and/or targeting of hSVCT1, and intestinal AA uptake. METHODS: We used human intestinal cells and cells from a Rab8a knockout mouse. (14)C-AA uptake was performed to determine functionality. PCR and western blotting were performed to determine RNA and protein expression, respectively. Confocal imaging was performed to determine co-localization. RESULTS: We show that hSVCT1 co-localized with Rab8a in intestinal cells. Knockdown of Rab8a lead to a significant inhibition in AA uptake and cell surface biotinylation studies revealed a lower cell surface expression of hSVCT1 in Rab8a siRNA-treated cells. Similarly, in the small intestine of a Rab8a knockout mouse, AA uptake was significantly inhibited. This effect again resulted from a decreased expression level of mSVCT1 protein, even though mRNA expression of SVCT1 was similar in intestinal cells from Rab8a knockout and wild-type litter-mates. The latter data are suggestive of enhanced lysosomal degradation of hSVCT1 protein in Rab8a-deficient cells; indeed, confocal imaging of Rab8a siRNA-treated intestinal cells revealed a strong overlap between hSVCT1-YFP and LAMP1-RFP. CONCLUSIONS: These findings show a role for Rab8a in the physiological function of hSVCT1 in intestinal epithelia.

Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption.

The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.

Relative contribution of THTR-1 and THTR-2 in thiamin uptake by pancreatic acinar cells: studies utilizing Slc19a2 and Slc19a3 knockout mouse models.

Thiamin is essential for normal function of pancreatic acinar cells, and its deficiency leads to a reduction in pancreatic digestive enzymes. We have recently shown that thiamin uptake by rat pancreatic acinar cells is carrier-mediated and that both thiamin transporter (THTR)-1 and THTR-2 are expressed in these cells; little, however, is known about the relative contribution of these transporters toward total carrier-mediated thiamin uptake by these cells. We addressed this issue using a gene-specific silencing approach (siRNA) in mouse-derived pancreatic acinar 266-6 cells and Slc19a2 and Slc19a3 knockout mouse models. First we established that thiamin uptake by mouse pancreatic acinar cells is via a carrier-mediated process. We also established that these cells as well as native human pancreas express THTR-1 and THTR-2, with expression of the former (and activity of its promoter) being significantly higher than that of the latter. Using gene-specific siRNA against mouse THTR-1 and THTR-2, we observed a significant inhibition in carrier-mediated thiamin uptake by 266-6 cells in both cases. Similarly, thiamin uptake by freshly isolated primary pancreatic acinar cells of the Slc19a2 and Slc19a3 knockout mice was significantly lower than uptake by acinar cells of the respective littermates; the degree of inhibition observed in the former knockout model was greater than that of the latter. These findings demonstrate, for the first time, that both mTHTR-1 and mTHTR-2 are involved in carrier-mediated thiamin uptake by pancreatic acinar cells.

Thiamin uptake by pancreatic acinar cells: effect of chronic alcohol feeding/exposure.

Thiamin is important for normal function of pancreatic acinar cells, but little is known about its mechanism of uptake and about the effect of chronic alcohol use on the process. We addressed these issues using freshly isolated rat primary and rat-derived cultured AR42J pancreatic acinar cells as models. Results showed thiamin uptake by both primary and cultured AR42J pancreatic acinar cells to be via a specific carrier-mediated mechanism and that both of the thiamin transporters 1 and 2 (THTR-1 and THTR-2) are expressed in these cells. Chronic alcohol feeding of rats was found to lead to a significant inhibition of carrier-mediated thiamin uptake by pancreatic acinar cells and was associated with a significant reduction in level of expression of THTR-1 and THTR-2 at the protein and mRNA levels. Chronic exposure (96 h) of AR42J cells to alcohol also led to a significant decreased carrier-mediated thiamin uptake, an effect that was associated with a significant decrease in the activity of the human SLC19A2 and SLC19A3 promoters expressed in these cells. We also examined the effect of chronic alcohol feeding of rats on level of expression of key thiamin metabolizing enzymes (thiamin phosphokinase and thiamin pyrophosphatase) as well as on level of expression of the mitochondrial thiamin pyrophosphate transporter of pancreatic acinar cells and observed a significant inhibition in all these parameters. These results demonstrate for the first time that thiamin uptake by pancreatic acinar cells is via a carrier-mediated process and that both the THTR-1 as well as THTR-2 are expressed in these cells. Also, chronic alcohol feeding/exposure inhibits thiamin uptake process and the inhibition is, at least in part, being exerted at the transcriptional level. Furthermore, chronic alcohol feeding also negatively impacts intracellular parameters of thiamin metabolism in pancreatic acinar cells.