Lipopolysaccharide adsorption on activated rice shell in intestine epithelial cell culture

Akimbekov N.Sh.

Al-Farabi Kazakh National University, Almaty city, The Republic of Kazakhstan nur_akimbek@yahoo.com

This study confirmed the toxic effect of lipopolysaccharide (LPS) on intestine epithelial cell (IEC-6) and adsorption of LPS on an activated material based on rice shell (ARS). The source of LPS in organism is gram negative intestine microflora. Previous works show that lipopolysaccharide exert influences on IEC-6 and its ability to migrate. Cell migration is a central process in the development and maintenance. Tissue formation during wound healing and immune responses all require the orchestrated movement of cells in particular directions towards specific locations. In the present work the effects of LPS on IEC-6 migration was studied in vitro by assessing the ability of confluent cells to reconstitute a scraped wound. The objective of this work is endotoxin removal by the sorption on the activated sorbent in IEC-6 culture.

ARS is one of the most complex nanostructured solids and multifaceted molecule because of its extremely high surface area and micropore volume. It’s bimodal and rarely trimodal pore size distribution provides good access for sorbate molecules into the interior. The structure of activated carbon is complex. It is basically composed of an amorphous structure and a graphite-like microcrystalline structure. Of the two, the graphitic structure is important for its capacity to provide "space" in terms of slit-shaped channels accommodating molecules. ARS is believed to be promising material that might show efficient and so far unknown LPS adsorption properties.

IEC-6 culture was used in the experiment. The culture between passages 5-13 was filled with the appropriate volume of PromoCell Growth Medium (2 ml/well) in cell culture vessels (Nunclon Surface) and placed in an incubator (37°C, 5% CO₂). The next day, IEC-6 cells were scraped with a cell scraper. At the same time LPS was added at the concentration of 2µg/ml to the first cell culture; LPS (2µg/ml) and ARS (5µg/ml) to the second; ARS (5µg/ml) alone was added to the third and the fourth untreated culture was used as a control. The cell cultures were placed under a microscope equipped with a camera. Differential interference contrast images were obtained every 300 min. Digital images from LPS, LPS-ARS and ARS treated IEC-6 cell cultures were obtained at t= 0; 6 and 12 h, revealing inhibited wound closure as a result of impaired migration for the former. The experiments were repeated twice with triplicate samples.

In control cell cultures, the wound was completely closed by 12 h. The same was observed in cell cultures with ARS and LPS-ARS.

In contrast, LPS-treated cell cultures revealed a significant inhibition of migration rate and consistent inability to close the wound at any given time point. However, LPS had no effect on the cells viability. The results indicate that LPS significantly impairs IEC-6 migration in vitro.

The present work describes the adsorption process using ARS for the removal of LPS from medium with intestine epithelial cell cultures. It also shows that ARS does not have any adverse effect on IEC-6 migrating ability. These studies are very important for discovering new medicines for normalization of intestine microecology as well as for the treatment of intestine disorders.