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TDK conference 2015

Investigations on the anti-inflammatory action of terpinen-4-ol and sodium n-butyrate on hepatocyte - Kupffer cell co-cultures
Orbán Kata - year 4
SzIU, Faculty of Veterinary Science, Department of Physiology and Biochemistry
Supervisors: Dr. Gábor Mátis, Dr. Zsuzsanna Neogrády


Nowadays, the application of alternative growth promoters is getting more abundant in animal production, so more and more studies are performed regarding the anti-inflammatory effects of biologically active substances of plants and those of bacterial metabolites. In the present study, the anti-inflammatory effects of terpinen-4-ol and sodium n-butyrate were investigated on primary porcine hepatocyte – Kupffer cell co-culture and hepatocyte mono-culture systems stimulated with bacterial lipopolysaccharids (LPS), serving as in vitro inflammatory models. Since the ratio of cell types involved in hepatic inflammation is changing during the inflammatory process, two types of the co-culture system have been previously established and characterized, containing hepatocytes and Kupffer cells in the ratio of 2:1 and 6:1, mimicking different states of inflammatory response.

Isolation of hepatocytes and Kupffer cells was performed by multi-step perfusion of processus caudatus of the liver, derived from male pigs of the Hungarian Large White breed, weighing 15 kg. At first, Kupffer cells were seeded on culture plates coated by collagen, which was followed by the seeding of hepatocytes. Confluent cell cultures, gained after 24 hours incubation, were concomitantly treated with LPS and terpinen-4-ol or sodium n-butyrate for 1 hour. Following additional 24 hours of incubation time, the concentration of IL-8 was determined from culture medium by using ELISA method. For confirmation of presence of the adjusted ratios of cell types in confluent cell cultures immunohistochemistry (detection of macrophage specific CD-68 antigen) was applied.

Based on immunohistochemical studies, the ratio of hepatocytes and Kupffer cells in co-culture systems was in accordance to the values set previously upon seeding. The concentration of IL-8 determined from culture medium of hepatocyte mono-cultures was significantly decreased either after terpinen-4-ol or sodium n-butyrate treatment compared to cells treated with LPS only. In contrast, in case of co-cultures the IL-8 concentration did not decrease either after incubation with terpinen-4-ol or sodium n-butyrate (compared to co-cultures treated with LPS only), although both of these compounds have remarkable anti-inflammatory effects based on literature data. A possible explanation can be that the ratio of Kupffer cells is significantly higher in the case of co-culture systems used as inflammatory models than in physiological conditions, thus the production of IL-8 increased so significantly after LPS treatment that the applied compounds could not ameliorate it sufficiently at the used concentrations. Our results justify the essential role of interleukin-producing Kupffer cells in the investigations of the effects of anti-inflammatory substances on in vitro models, mimicking the in vivo conditions more accurately, so the utilized co-culture systems can be proper tools for such examinations as well.

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