The Protective Effect Of Metabolites Of Lactic Acid Bacteria Isolated From Deep Seawater On The Tight Junctions Of Caco-2 Cells

The intestine barrier is in correlation with human health.Intestinal epithelium is an important part of intestinal mucosa layer which can not only absorb useful substances and nutrients but also provide a barrier against harmful ones.The epithelium is composed by several kinds of intestinal epithelial cells(IECs),between which there exist some contiguous,relatively impermeable and dynamic structures which called tight junctions(TJs).TJs consisting of several functional proteins protect the host from intrusion of harmful molecules,bacteria,virus and other pathogens by regulating the paracellular permeability.Caco-2 cells,which are one kind of IECs,become differentiated and polarized under specific culturing conditions to form a confluent monolayer on the cell culture insert filter.In this case,Caco-2 cells are used to evaluate structural integrity and the function of TJs.And the ability of the epithelial barrier constructed by Caco-2 cells is measured by the trans-epithelial electrical resistance(TEER).There were only a few reports on the efficacy of lactic acid bacteria(LABs)isolated from marine environment,although it was well known that LABs isolated from the terrestrial including Lactobacillus plantarum were useful for human health.The bacteria which can survive in the extreme environment such as deep-sea may have developed corresponding characteristics superior than the ones from the terrestrial source.The present study aims to investigate the effects of LABs isolated from marine environment on TJs by the intestinal epithelial model of Caco-2 cells.In recent years,many people are suffering from various gastrointestinal diseases due to unhealthy excessive drinking and western diet with energy-dense food.That's why this study haspractical value to guarantee the intestinal health by the diet containing the LABs and their metabolites.L.plantarum strain BF1-13 isolated from deep seawater(800m)of Izu-Akazawa,Shizuoka prefecture was used in this study.The intestinal epithelial model constructed by Caco-2 cells were prepared and optimized for investigating the efficacy of the culture supernatant of L.plantarum strain BE1-13 on the barrier function.Caco-2 cells were incubated in the culture inserts(Millicell)until the confluence-stage to form the monolayer.TEER values which were measured by Millicell-ERS(Merck)were analysed as the temperature-corrected TEER(tc TEER)value by temperature correcting mathematic method.First,the effect of methanol and H2O2 as the damage inducer separately(DI)on the model was studied.Then,the effects of the culture supernatants of the sample along with quercetin(QU)as the positive control(PC)were also investigated by this evaluation system.Following results were obtained in the present study.1)the model was completed after 5-11 days incubation which is much shorter than the traditional Caco-2 model(21days);2)MRS showed no significant influences on the tc TEER values;3)100?M quercetin was used as the PC which increase about 15% of the original tc TEER value 2hours after the addition;4)methanol which changed the pore size of membrane of culture-insert was not suitable as the DI in this study;5)1m M H2O2 was suitable for the DI,which decreased about 30% of the original tc TEER value 2 hours after the addition;6)oxidative-induced damage by H2O2 on intestinal barrier function was released by the supernatant of L.plantarum strain BF1-13,which may even be inhibited by the high concentration at 10%.