Effect of hydroxylated and methylated flavonoids on cytochrome P450 activity in porcine intestinal epithelial cells.

Cytochrome P450 (CYP) oxidases are among the main metabolizing enzymes that are responsible for the transformation of xenobiotics, including clinically important drugs. Their activity can be influenced by several compounds leading to decreased efficacy or increased toxicity of co-administered medicines. Flavonoids exert various beneficial effects on human and animal health; therefore they are used as food and feed supplements. However, they are also well-known for their CYP modulating potential. Since the amount of CYP enzymes is highest in the liver, interaction studies are mainly conducted in hepatocytes, however, CYP activity in the gastrointestinal tract is also remarkable. In this study, effects of apigenin (API), quercetin (QUE) and their methylated derivatives trimethylapigenin (TM-API), 3-O-methylquercetin (3M-QUE) and 3',7-di-O-methylquercetin (3'7DM-QUE) on the CYP enzyme activity was examined in IPEC-J2 porcine intestinal epithelial cells. Potential food-drug interactions were studied using flavonoid treatment in combination with inducer and inhibitor compounds. API, TM-API, QUE and 3M-QUE significantly inhibited the CYP3A29 enzyme, while 3'7DM-QUE did not alter its activity. Enzyme inhibition has also been observed in case of some food-drug combinations. Our results support previous findings about CYP modulating effects of flavonoids and highlights the possibility of interactions when flavonoid-containing supplements are consumed during drug treatments.

Impact of heat-inactivated Lactobacillus on inflammatory response in endotoxin- and chemotherapeutic-treated porcine enterocytes.

Several factors such as pathogen bacteria, or oral chemotherapy disturb the intestinal integrity, leading to several undesirable effects. Inactivated probiotics may be beneficial in safely redress the physiological functions of the intestinal epithelium. Our aim is to determine the effect of tyndallized Lactobacillus on LPS- and 5-fluorouracil-treated porcine jejunal cells. IPEC-J2 cells derived from porcine jejunal epithelium were used as the in vitro model. The enterocyte cell cultures were treated with 109Lactobacillus reuteri cells/ml or 10 µg/ml lipopolysaccharides (LPS) or 100 µM 5-fluorouracil separately and simultaneously. We determined the alterations in mRNA levels of inflammatory mediators IL6, CXCL8/IL8, TNF. Furthermore, the protein level of IL-6 and IL-8 were measured. The fluorouracil treatment upregulated the IL6 gene expression, the endotoxin treatment upregulated the IL8 and TNF level. The heat-inactivated Lactobacillus increased the IL-8 level both at the gene expression and protein level. The co-administration of the non-viable probiotic with the 5-fluorouracil and the LPS resulted in decrease of IL6, IL8, and TNF level. The immune-modulator effect of tyndallized probiotic product is demonstrated in porcine jejunal cells. The inactivated Lactobacillus was able to prevent the accumulation of the selected inflammatory mediators in LPS- or 5-fluorouracil-exposed enterocytes.

In vitro study of chlorine dioxide on porcine intestinal epithelial cell gene markers.

BACKGROUND: Chlorine dioxide (ClO2 ) is an inorganic, potent biocide and is available in highly purified aqueous solution. It can be administered as an oral antiseptic in this form. OBJECTIVES: Our aim is to determine the level of inflammatory markers and cytochrome genes expressed by enterocytes exposed to different concentrations of hyperpure chlorine dioxide solution. METHODS: Porcine jejunal enterocyte cell (IPEC-J2) cultures were treated with the aqueous solution of hyper-pure chlorine dioxide of various concentrations. We determined the alterations in mRNA levels of inflammatory mediators, such as IL6, CXCL8/IL8, TNF, HSPA6 (Hsp70), CAT and PTGS2 (COX2); furthermore, the expression of three cytochrome genes (CYP1A1, CYP1A2, CYP3A29) were analysed by quantitative PCR method. RESULTS: The highest applied ClO2 concentration reduced the expression of all three investigated CYP genes. The gene expression of PTGS2 and CAT were not altered by most concentrations of ClO2 . The expression of IL8 gene was reduced by all applied concentrations of ClO2 . TNF mRNA level was also decreased by most ClO2 concentrations used. CONCLUSIONS: Different concentrations of chlorine dioxide exhibited immunomodulatory activity and caused altered transcription of CYP450 genes in porcine enterocytes. Further studies are needed to determine the appropriate ClO2 concentration for oral use in animals.

Alteration of avian hepatic cytochrome P450 gene expression and activity by certain feed additives.

We investigated the effect of four feed additives, namely ß-glucan, a drinking water acidifier (DWA), a sanguinarine-containing product (SN) and fulvic acid, on hepatic cytochrome P450 (CYP) mRNA expression and CYP enzyme activity in chickens. The test substances were given to the chickens in the recommended dose or in tenfold dose. The administration of 5 mg/kg body weight (bw) ß-glucan and 0.1 ml/kg bw DWA for five days decreased the relative gene expression of CYP1A4 and CYP2C23a. The dosing of 50 mg/kg bw ß-glucan, 5 and 50 mg/kg bw SN, 1 ml/kg bw DWA and 250 mg/kg bw fulvic acid doubled the hepatic CYP1A4 activity. The activity of CYP2C and CYP3A remained unchanged. Avoidance of CYP1A-mediated feed-drug interactions requires accurate dosing of ß-glucan, DWA and fulvic acid. According to our results, no treatment resulted in excessive or less CYP2C and CYP3A protein formation, which reduces the risk of potential feed additive-drug interactions in chickens. However, the administration of feed additive SN containing a plant alkaloid should be avoided concomitantly with CYP1A-metabolised medicines.

Alteration in Inflammatory Responses and Cytochrome P450 Expression of Porcine Jejunal Cells by Drinking Water Supplements.

The intestinal epithelium is the first determining barrier to the drugs administered per os. Cytochrome P450 (CYP) enzymes are substantial in the initial step of xenobiotic metabolism; therefore, intestinal CYP enzyme activities could be an important influencing factor of the oral utilization of xenobiotic substances. In this study, the effect of four drinking water supplements on CYP mRNA levels of porcine intestinal epithelial cells was examined. Further goal of the study is to describe the effect of these feed additives on the proinflammatory response of the LPS-treated enterocytes. The nontransformed porcine intestinal epithelial cells (IPEC-J2) were grown on six-well polyester membrane inserts. Cell cultures were treated with LPS (10 µg/ml), ß-glucan (5 and 50 µg/ml), sanguinarine-containing additive (5 and 50 µg/ml), drinking water acidifier (0.1 and 1 µl/ml), and fulvic acid (25 and 250 µg/ml) for 1 hour. Cells were washed with culture medium and incubated for additional 1 h before total RNA isolation. IL-6, IL-8, TNF-α, HSP70, CYP1A1, CYP1A2, and CYP3A29 mRNA levels were measured. The LPS treatment upregulated the gene expression of IL-8 and TNF-α. The relative gene expression of IL-6 remained unchanged and TNF-α and HSP70 were downregulated after the treatment with each feed additive. CYP1A1 and CYP1A2 expressions increased after sanguinarine-containing solution, fulvic acid, and drinking water acidifier treatment. None of the treatments changed the gene expression of CYP3A29, responsible for the metabolism of the majority of drug substances used in swine industry. The feed additive substances inhibited the expression of proinflammatory mediators HSP70 and TNF-α; however, ß-glucan and fulvic acid elevated the production of the chemokine IL-8 mRNA in endotoxin-treated enterocytes. All acidic supplements increased the expression of CYP1A1 gene; their constituents may serve as a ligand of CYP1A1 nuclear receptors.

Chlorogenic Acid Combined with Lactobacillus plantarum 2142 Reduced LPS-Induced Intestinal Inflammation and Oxidative Stress in IPEC-J2 Cells.

This study was carried out to investigate protective effect of chlorogenic acid against lipopolysaccharide-induced inflammation and oxidative stress in intestinal epithelial cells. As a marker of inflammatory response, IL-6, IL-8, TNF-α mRNA and protein levels, furthermore, COX-2 mRNA level were followed up. Intracellular redox status and extracellular H2O2 level were also monitored by two fluorescent assays (DCFH-DA, Amplex Red). Moreover, the effect of gut microbiota metabolites in the above mentioned processes was taken into account in our model using Lactobacillus plantarum 2142 bacterial strain. Our data revealed that chlorogenic acid had significant lowering effect on the inflammatory response. Treatment with chlorogenic acid (25-50 µM) significantly decreased gene expression and concentration of proinflammatory cytokines IL-6 and IL-8 compared to LPS-treated cells. COX-2 and TNF-α mRNA levels were also reduced. Furthermore, chlorogenic acid reduced the level of reactive oxygen species in IPEC-J2 cells. Simultaneous application of chlorogenic acid and Lactobacillus plantarum 2142 supernatant resulted protective effect against LPS-induced inflammation and oxidative stress as well.

Polymethoxyflavone Apigenin-Trimethylether Suppresses LPS-Induced Inflammatory Response in Nontransformed Porcine Intestinal Cell Line IPEC-J2.

The in vitro anti-inflammatory effect of apigenin and its trimethylated analogue (apigenin-trimethylether) has been investigated in order to evaluate whether these flavonoids could attenuate LPS-induced inflammation in IPEC-J2 non-transformed intestinal epithelial cells. Levels of IL-6, IL-8, TNF-α, and COX-2 mRNA were measured as a marker of inflammatory response. The extracellular H2O2 level in IPEC-J2 cells was also monitored by Amplex Red assay. Our data revealed that both compounds had significant lowering effect on the inflammatory response. Apigenin (at 25 µM) significantly decreased gene expression of IL-6 in LPS-treated cells, while apigenin-trimethylether in the same concentration did not influence IL-6 mRNA level. Both apigenin and apigenin-trimethylether reduced IL-8 gene expression significantly. TNF-α mRNA level was decreased by apigenin-trimethylether, which was not influenced by apigenin. Treatment with both flavonoids caused significant reduction in the mRNA level of COX-2, but the anti-inflammatory effect of the methylated analogue was more effective than the unmethylated one. Furthermore, both flavonoids reduced significantly the level of extracellular H2O2 compared to the control cells. In conclusion, the methylated apigenin analogue could avoid LPS-induced intestinal inflammation and it could be applied in the future as an effective anti-inflammatory compound.

Alternative treatment of serious and mild Pasteurella multocida infection in New Zealand White rabbits.

BACKGROUND: Pasteurella multocida causes numerous economically relevant diseases in livestock including rabbits. Immunisation is only variably effective. Prophylactic antibiotics are used in some species but are contra-indicated in rabbits, due to their adverse effects on the rabbit microbiota. There is therefore a substantial need for alternative forms of infection control in rabbits; we investigated the effect of oral ß-glucan on P. multocida infection in this species. RESULTS: Thirthy-five New Zealand White rabbits were randomly divided into five groups of seven animals. Three groups were inoculated with Pasteurella multocida intranasally (in.), a physiologically appropriate challenge which reproduces naturally acquired infection, and received either (1-3), (1-6) ß-glucans or placebo. Four other groups were inoculated both in. and intramuscularly (im.), representing a supra-physiological challenge, and received either (1-3), (1-6) ß-glucans, antibiotic or placebo. ß-glucans given prophylactically were highly effective in protecting against physiological (in.) bacterial challenge. They were less effective in protecting against supra-physiological bacterial challenge (in. and im.), although they extended survival times. This latter finding has practical relevance to breeders as it extends the window in which heavily infected and symptomatic animals can be salvaged with antibiotics. CONCLUSIONS: In our study, (1-3), (1-6) ß-glucans were highly effective in protecting against a model of naturally acquired P. multocida infection and extended survival times in the supra-physiological model. Enrofloxacin was effective in protecting against supra-physiological infection. We are currently reviewing the use of combined prophylaxis.

The effects of intestinal LPS exposure on inflammatory responses in a porcine enterohepatic co-culture system.

A porcine enterohepatic co-culture system, with primary hepatocytes as bottom layer and IPEC-J2 epithelial cells as upper layer, was developed to study the effects of lipopolysaccharides (LPS) on the gene expression profile of pro-inflammatory cytokines (interleukin-8 (IL-8) and tumor necrosis factor-α) and CYP enzymes (CYP1A1, CYP1A2, CYP3A29). The barrier integrity of IPEC-J2 cells was investigated by transepithelial electrical resistance measurements and by fluorescein isothiocyanate-dextran-based test. Basolateral IL-8 production was significantly elevated in LPS-treated IPEC-J2 and primary hepatocyte mono-cultures as well as in the co-culture system, in a dose-independent manner. The LPS-induced changes in the expression of the CYP1A2 and CYP3A29 genes in hepatocyte mono-cultures differed from those in co-culture after LPS treatment on the apical side of the IPEC-J2 cell layer. CYP1A2 was downregulated by the LPS treatment in mono-cultures but upregulated at 10 µg/ml LPS in co-culture; gene expression of CYP3A29 showed no significant LPS-induced change in the hepatocyte mono-culture but was significantly downregulated in co-culture. The newly established co-culture system capable of mimicking enterohepatic interplay in LPS-induced inflammatory responses in vitro can be used in the future for reliable screening of potential anti-inflammatory compounds.

Metabolites of Lactobacillus plantarum 2142 prevent oxidative stress-induced overexpression of proinflammatory cytokines in IPEC-J2 cell line.

Probiotics have already proven beneficial effects in the treatment of several intestinal infections, but the underlying mechanisms how the probiotics can affect responses of porcine IPEC-J2 enterocytes to oxidative stress remained to be elucidated. The immunomodulatory effect of five bacterial strains (Lactobacillus plantarum 2142, Lactobacillus casei Shirota, Bifidobacterium animalis subsp. lactis BB-12, Bacillus amyloliquefaciens CECT 5940 and Enterococcus faecium CECT 4515) on 1 mM peroxide-triggered upregulation of interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α) level was screened by q RT-PCR. Our data revealed that spent culture supernatant (SCS) of L. plantarum 2142 had significant lowering effect on IL-8 and TNF-α level with concomitant promoting activity on protective Hsp70 gene expression. According to our results, lactic acid (racemic, D: - and L: -lactic acid) and acetic acid produced by lactobacilli had no protective effect in quenching upregulation of proinflammatory cytokines. Furthermore, L. plantarum 2142-specific supernatant peptides were detected by gel electrophoresis and capillary zone electrophoresis.