Exploration of a mammary epithelial cell model for the study of epithelial inflammation and mechanisms of anti-inflammatory activity in medicinal plants

Failure to terminate the inflammatory response results in a state of chronic inflammation that might lead to cell transformation and cancer especially in epithelial cells. Hence, there is increased interest in the role of non immune epithelial cells in the regulation of inflammation to identify the link between chronic inflammation and cancer. The aim of this study was to investigate the regulation of inflammatory responses in non immune mammary epithelial cells and how this regulation is modulated by the cell microenvironment in order to establish an in vitro non immune cell model as a tool to understand inflammation in epithelial cells, and the mechanisms of action of potential anti-inflammatory agents in inflamed epithelia.; Bacterial endotoxin (ET) treated SCp2 cells showed sharp induction of both interleukin-6 (IL-6) secretion and nitric oxide (NO) production, but with unexpected delay in iNOS mRNA expression compared to that of IL-6 mRNA expression. ET also induced the activation of NFkappaB subunits p65 and p50 at 1 h after ET application (post-ET); however, p65 activation was transient, while that of p50 remained high throughout the experiment. Selective inhibition of NFkappaB activation pathway by inhibiting inhibitory kappa B kinase (IKK) alpha and IKKbeta by Wedelolactone reduced ET-induced IL-6 mRNA expression and protein secretion but not that of iNOS mRNA or NO production, suggesting that differences in regulation of ET-induced IL-6 and iNOS involve NFkappaB activation.; Interestingly, serum supplementation transiently up regulated cytokine protein secretion (IL-6) and mRNA expression (IL-6 and TNFalpha) in response to ET, but sharply reduced ET-induced iNOS mRNA expression and NO production, confirming the different modes of regulation of those inflammatory genes. Addition of exogenous extracellular matrix (EHS) to cultured SCp2 cells on plastic had no apparent effect on the temporal pattern of ET-induced IL-6 or iNOS mRNA expression.; Surprisingly, the coculture of SCp2 cells on a confluent monolayer of SCg6 mouse mammary myoepithelial cells induced a dramatic increase in IL-6 secretion to very high levels in the absence of ET exposure. Addition of ET to SCp2:SCg6 cell cocultures further increased IL-6 secretion to higher levels in sharp contrast to the meager induction of NO to maximum levels of ¼ to ½ that in SCp2 cells alone. These results showed that the microenvironment of the inflamed cell is important in the regulation of inflammation and for understanding the link between inflammation and cancer in epithelia.; Finally, the ET-induced inflammation in SCp2 cells was used to screen and identify the mechanism of action of anti-inflammatory fractions of methanol extracts of wild Lebanese Centaurea ainetensis, a plant used in Lebanese traditional folk medicine to treat inflammatory diseases. A partially purified fraction of C. ainetensis eluted by 60% methanol from solid phase extraction (SPE) columns followed by methanol gradient elution on reverse phase high liquid performance chromatography (RP-HPLC) strongly inhibited ET-induced IL-6 secretion with a drastic reduction of the cytotoxicity observed in the crude methanol extract. The partially purified fraction of C. ainetensis also reduced ET-induced NO production by SCp2 cells, and inhibited the expression of ET-induced iNOS mRNA without affecting that of ET-induced IL-6 mRNA, suggesting an inhibitory role on the MAPK pathway but not on the NFkappaB pathway of inflammation. Concentration and partial purification of anti-inflammatory bioactivity from crude extracts of C. ainetensis by SPE columns followed by RP-HPLC suggest feasibility of purification and characterization. Moreover the use of non immune mammary epithelial cells for investigation of anti-inflammatory drugs presents a potential model to further investigate the link between chronic inflammation and cancers of epithelia.