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Home » Archive » 2013

TDK conference 2013

Examination of bacterial lipopolysaccharide-induced inflammation on primary cultures of porcine hepatocytes
Lipták Antónia - year 4
SzIU, Faculty of Veterinary Science, Department of Physiology and Biochemistry
Supervisors: Gábor Mátis DVM, Anna Kulcsár

Abstract:

Stress factors, such as release of lipopolysaccharides (LPS) of Gram-negative bacteria can lead to formation of acute and chronic inflammation. In this case, the inflammatory mediators produced by the cells under stress, first of all cytokines – such as interleukin 8 (IL-8) – are produced in larger quantities. The inflammatory mediators produced by the enterocytes and the absorbed LPS can easily enter the liver through the portal vein, where they can induce the activation of a new inflammatory cascade.

We examined the effects of bacterial LPS on the liver in a culture of pig liver cells, which served as an in vitro system. We were able to determine the viability of the liver cells based on their albumin production, and the state of the inflammatory processes by the concentration of IL-8 in the culture medium. We also examined the gene expression of the microsomal cytochrome P450 (CYP) enzymes of the hepatocytes, which have an important role in the biotransformation of the xenobiotics.

For the cell isolation, 15-kg castrated male pigs were used. The isolated tissue was perfused in multiple stages and digested by collagenase, as a result of which the liver cells were dissociated. The hepatocytes were cultured using a collagen-coated, six-well plate for 24 hours, after which the cell cultures were confluent in every case.

The samples were then exposed to LPS extracted from the cell membrane of Salmonella typhimurium, with differing concentrations (1 és 10 µg/ml), for one hour. After the treatment the ELISA method was used to determine the medium’s albumin and IL-8 content. The gene expression of CYP enzymes was also examined by using qRT-PCR.

Albumin concentration corresponding to intact hepatocytes was significantly reduced 24 h after LPS treatment with either of the two concentrations of LPS. IL-8 production of hepatocytes tended to be increased by the lower dose of LPS treatment. CYP1A1 and CYP1A2 gene expression of hepatocytes was significantly decreased after exposure to 1 µg/ml LPS, but CYP3A29 was not altered. No significant difference could be found between 10 µg/ml LPS-stimulated and control cells concerning the expression of hepatic CYP1A1 and CYP3A29 genes, however, CYP1A2 was down-regulated after treatment with the applied higher LPS concentration.

Based on our results, the cell culture we applied is an appropriate model for the study of various inflammatory processes. It was found in the present study that gene expression of various CYP enzymes can be reduced by bacterial LPS. However, this effect may vary depending on the applied LPS concentration and examined isoenzyme. In the future, this model is suitable to examine the effects of anti-inflammatory substances besides LPS, such as Lactobacillus plantarum and n-butyrate, on cells. Furthermore, the applied liver cell culture will serve as a good basis for the development of a novel enterohepatic co-culture model.



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