Exposure of human intestinal epithelial cells and primary human hepatocytes to trypsin-like serine protease inhibitors with potential antiviral effect.

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 µM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 µM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 µM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.

The impact of quercetin on a porcine intestinal epithelial cell line exposed to deoxynivalenol.

Quercetin (Que) is present in many vegetables and fruits as a secondary antioxidant metabolite. Deoxynivalenol (DON) produced by various Fusarium mould species can induce cytotoxicity and oxidative stress in the gastrointestinal tracts of humans and farm animals. The aim of this study was to investigate the effects of Que on DON-induced oxidative stress in a non-tumourigenic porcine IPEC-J2 cell line. Two experimental designs were used in our experiments as follows: (a) pretreatment with 20 µmol/L Que for 24 h followed by 1-h 1 µmol/L DON treatment and (b) simultaneous application of 20 µmol/L Que and 1 µmol/L DON for 1 h. Cell cytotoxicity, transepithelial electrical resistance (TER) of cell monolayers and extracellular/intracellular redox status were studied. It was found that DON significantly decreased TER and triggered oxidative stress, while Que pretreatments were beneficial in maintaining the integrity of the monolayers and alleviated oxidative stress. However, co-treatment with Que was unable to preserve the integrity and redox balance of the cells exposed to DON. These results indicate that only the 24-h preincubation of cells with 20 µmol/L Que was beneficial in compensating for the disruption caused by DON in extracellular oxidative status.

Beneficial Effects of Rosmarinic Acid on IPEC-J2 Cells Exposed to the Combination of Deoxynivalenol and T-2 Toxin.

Mycotoxin contamination in feedstuffs is a worldwide problem that causes serious health issues both in humans and animals, and it contributes to serious economic losses. Deoxynivalenol (DON) and T-2 toxin (T-2) are major trichothecene mycotoxins and are known to challenge mainly intestinal barrier functions. Polyphenolic rosmarinic acid (RA) appeared to have antioxidant and anti-inflammatory properties in vitro. The aim of this study was to investigate protective effects of RA against DON and T-2 or combined mycotoxin-induced intestinal damage in nontumorigenic porcine cell line, IPEC-J2. It was ascertained that simultaneous treatment of DON and T-2 (DT2: 1 µmol/L DON + 5 nmol/L T - 2) for 48 h and 72 h reduced transepithelial electrical resistance of cell monolayer, which was restored by 50 µmol/L RA application. It was also found that DT2 for 48 h and 72 h could induce oxidative stress and elevate interleukin-6 (IL-6) and interleukin-8 (IL-8) levels significantly, which were alleviated by the administration of RA. DT2 administration contributed to the redistribution of claudin-1; however, occludin membranous localization was not altered by combined mycotoxin treatment. In conclusion, beneficial effect of RA was exerted on DT2-deteriorated cell monolayer integrity and on the perturbated redox status of IPEC-J2 cells.

Investigation of sphingosin-1-phosphate-triggered matriptase activation using a rat primary hepatocyte model.

Sphingosine-1-phosphate (S1P) has been reported as a matriptase activator. The aim of this study was to reveal if S1P can influence hepcidin production. Furthermore, we investigated how S1P can affect the viability and the redox status of primary hepatocytes. Rat primary hepatocytes were cultivated for 72 h and were treated with 50, 200, 1000 ng/ml S1P. Cell-free supernatants were collected every 24 h. Cell viability was tested by a colorimetric method using tetrazolium compound (MTS). The hepcidin levels in the cell-free supernatants were examined with hepcidin sandwich ELISA to determine the effect of S1P on the hepcidin-modulating ability of matriptase. In order to estimate the extent of S1P-generated oxidative stress, extracellular H2O2 measurements were performed by the use of fluorescent dye. Based on the findings, S1P treatment did not cause cell death for 72 h at concentrations up to 1000 ng/ml. S1P did not influence the extracellular H2O2 production for 72 h. The hepcidin levels were significantly suppressed in hepatocytes exposed to S1P treatment. Further studies would be needed to explore the exact mechanism of action of S1P.

Investigation of the inflammatory and oxidative stress-inducing effects of deoxynivalenol and T-2 toxin exposure in non-tumorigenic human intestinal cell model.

Fungi in the Fusarium genus produce trichothecene mycotoxins including deoxynivalenol (DON) and T-2 toxin which may elicit their damaging effects on the gastrointestinal tract following the consumption of contaminated cereal-based foods. The aim of our study was to evaluate the effects of these commonly occurring fusarotoxins alone and in combination using the human, non-cancerous intestinal epithelial cell line HIEC-6. Based on our experimental data, 24 h after treatment with fusarotoxins, hydrogen peroxide levels, intracellular oxidative stress and the amounts of inflammatory interleukins IL-6 and IL-8 significantly increased. Cell membrane localization of the tight junction protein claudin-1 decreased, whereas distribution of occludin remained unchanged. Taken together, the HIEC-6 cell line appears to be a suitable experimental model for monitoring the combined effects of mycotoxins at the cellular level including changes in the redox states of cells.

The Impact of Fermented Wheat Germ Extract on Porcine Epithelial Cell Line Exposed to Deoxynivalenol and T-2 Mycotoxins.

The effect of fermented wheat germ extract (FWGE) (Immunovet®) was evaluated with cotreatments with deoxynivalenol (DON) and T-2 toxin (T-2). These mycotoxins are produced by Fusarium mold species. The effects of FWGE on IPEC-J2 with DON and T-2 have not been studied until now. The IPEC-J2 porcine, nontumorigenic cell line was selected to investigate the outcome of the individually and simultaneously added compounds, as it has in vivo-like properties. The cells were treated for 24 h with the selected solutions; then, the IPEC-J2 cells were allowed to regenerate in a culture medium for an additional 24 h. In our results, DON and T-2 significantly increased the adverse impacts on cell viability and integrity of the cell monolayer. To elucidate the extent of oxidative stress, extracellular H2O2 concentrations and intracellular reactive oxygen species (ROS) were measured. FWGE appeared to be beneficial to IPEC-J2 cells given the separately and significantly decreased ROS levels. 1% and 2% FWGE could significantly reduce mycotoxin-induced oxidative stress. In conclusion, the results demonstrate that FWGE exerted protective effects to counteract the oxidative stress-provoking properties of applied fusariotoxins in the nontumorigenic IPEC-J2 cell line.

The Impact of Acute Matriptase Inhibition in Hepatic Inflammatory Models.

Purpose. Dysfunction of matriptase-2 can be involved in iron regulatory disorder via downregulation of hepcidin expression. In the present study, we investigated the effects of 3-amidinophenylalanine-derived matriptase inhibitors on porcine hepatic inflammatory cell models. Methods. Hepatocyte-Kupffer cell cocultures (ratio of 2 : 1 and 6 : 1) were treated with four structurally related matriptase inhibitors at 50 µM. Cell cytotoxicity and relative expressions of IL-6 and IL-8 and the levels of hepcidin were determined by MTS and porcine-specific ELISA. The extracellular H2O2 contents were analyzed by Amplex Red method. Results. Matriptase inhibitors at 50 µM for 24 h did not increase cell death rate. The elevated ROS production observed after short-term application of inhibitor MI-441 could be correlated with lowered hepcidin expression. MI-460 could significantly enhance hepcidin levels in the supernatants of cocultures (by 62.21 ± 26.8% in hepatocyte-Kupffer cell, 2 : 1, and by 42.6 ± 14.3% in hepatocyte-Kupffer cell, 6 : 1, cocultures, resp.). No significant changes were found in IL-6 and IL-8 levels in cocultures exposed to matriptase inhibitors. Conclusions. Based on in vitro findings, administration of MI-460 via modulation of hepcidin expression without cytotoxic and oxidative stress inducing properties might be a reliable alternative to treat iron overload in human and veterinary clinical practice.