Effect of Jania rubens algal extract on an in vitro model of inflammatory bowel disease

Abstract

Inflammatory Bowel Diseases (IBD) is a detrimental disease that severely alters the quality of life for those experiencing it. It is characterized by chronic inflammation in the intestinal region as well as ulcerating lesions. IBD describes two main conditions which are Crohn’s Diseases (CD) and Ulcerative colitis (UC). This study aimed to establish an in vitro model of IBD by co-culturing immune cells, particularly monocytes from the THP-(acute monocytic leukemia) cell line with intestinal epithelial cells from the Caco-2 (colorectal adenocarcinoma) cell line and inducing inflammation by treatment with bacterial lipopolysaccharide (LPS). Moreover, the potential anti-inflammatory role of the DM Soxhlet extract of the Lebanese red algae Jania rubens was studied by treating these cultures with a fixed concentration (250μg/ml) of the extract over three different time points (6, 12, 24h). An MTT cytotoxicity and proliferation assay as well as a Trypan Blue dye exclusion assay were carried out to test the cytotoxicity of the DM Soxhlet extract. The proliferation rates of THP 1 and Caco-2 were also calculated in order to determine the number of cells to be seeded to ensure a 1:1 ratio during co-culture. RT-qPCR was carried out on kit-extracted RNA from these co-cultures and the gene expression of TLR4, IL-10, IL-6 and IL-8 under different conditions of treatment was studied. The results show that after 6h treatment with DM Soxhlet extract there was 18% dead Caco-2 cells and after 24h treatment there was 18% dead THP-1 cells. Proliferation of THP-1 increased after 6- and 12-hours treatment with DM Soxhlet extract. For the co-culture, Caco-2 cells were seeded one day before at a density of 10x105cells/well and THP-1 were seeded the next day with the treatments at a density of 15x105 cells/well. Gene expression analysis on the extracted RNA from the co-cultures exhibited decreased expression of pro-inflammatory markers IL-6, IL-8 and TLR4 and increased expression of the anti-inflammatory cytokine IL-10 at the 24-hour timepoint, suggesting that DM Soxhlet could exhibit anti-inflammatory properties, hence, could be used against a panel of inflammatory diseases such as IBD