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Home » Archive » 2022

TDK conference 2022

Luteolin and chrysin could prevent lipopolysaccharide-ochratoxin combination-caused inflammation and oxidative stress in vitro
Wohlert, Annelie - year 4
University of Veterinary Medicine Budapest, Department of Pharmacology and Toxicology
Supervisor: Erzsebet Pasztine Gere

Abstract:

Ochratoxin A (OTA) produced by Aspergillus and Penicillium spp. can contaminate cereal-based foods and feedstuffs and their oral consumption can cause severe gastrointestinal, kidney and liver disorders both in humans and in the livestock. Gram-negative bacterial lipopolysaccharide (LPS) is also reported to cause chronic inflammation of the gut and is capable of stimulating liver injury. However, plant-derived flavonoids such as chrysin (CHR) and luteolin (LUT) display many positive aspects, such as anti-inflammatory and antioxidant properties.

In this study, not cancerous IPEC-J2 cells from neonatal piglets as well as minipig hepatocytes were used to study on the 24 hrs lasting impact of OTA/LPS and for screening beneficial effects of the applied flavonoids. The cells were treated with OTA (1 µM, 5 µM and 20 µM), LPS (10 µg/ml), CHR (1 µM) and LUT (2.5 µg/ml) alone and in combinations in IPEC-J2. The minipig hepatocytes were exposed to OTA (20 µM) in the absence or in the presence of CHR (100 nM, 500 nM, 1000 nM). To determine the cell viabilities MTS assay (in IPEC-J2 cells) and CCK-8 test (minipig hepatocytes) were performed and to evaluate extracellular (EC) hydrogen-peroxide (H2O2) Amplex red assay was applied. Intracellular (IC) reactive oxygen species (ROS) were assessed using 2’-7’dichlorodihydrofluorescein diacetate (DCFH-DA) method. ELISA kit assay was used to assess IL-6 and IL-8 secretion in IPEC-J2 cells.

It was found that OTA decreased cell viabilities both in IPEC-J2 cells and in minipig hepatocytes (p<0.001), while LPS reduced viability in IPEC-J2 cells (p<0.01). This effect could not be alleviated by LUT or CHR (p>0.05), however EC H2O2 was successfully suppressed by LUT in IPEC-J2 cells (p<0.001) and CHR in minipig hepatocytes (p<0.01). OTA in combination with LPS would significantly elevate the IC ROS which was successfully counteracted by CHR and LUT (p<0.001). Inflammatory cytokines IL-6 and IL-8 secretion was elevated by LPS (p<0.01) and LPS+ OTA (p<0.001) and could be successfully quenched by LUT (IL-6 p<0.01; IL-8 p<0.001).

Based on our results flavonoids (CHR and LUT) exert beneficial effects on IC ROS levels as well as on cytokine secretion (LUT) in vitro. These flavonoids might be used as dietary and feed supplements in the future to avoid OTA and LPS- related health risks in humans and in farm animals after further in vivo safety studies.



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