The Molecular Mechanism Of Clostridium Butyricum And Clostridium Butyricum Teichoic Acid Acting On Intestinal Epithelial Cells

This study was conducted to investigate the molecular mechanism of Clostridium butyricum and Clostridium butyricum teichoic acid acting on intestinal epithelial cells. The colon adenocarcinoma cells,called HT-29cells were to act as guinea pigs for our tests which were seeded in six-well plates. Teichoic acid was extracted from Clostridium butyricum by adopting TX114and the purity was detected by H nuclear magnetic resonance (NMR)and infrared spectrum (IR). The FITC labeled concentration for109CFU/mL of Clostridium butyricum was added to the cells which were treated for24h with concentration respectively for0ug/mL、50ug/mL.80ug/mL of teichoic acid.The adhesion of bacteria were observed using Inverted fluorescence microscope after2h.Total cellular RNA was abstracted from HT-29cells which were treated for12h with concentration respectively for0.10CFU/mL103CFU/mL.105CFU/mL.107CFU/mL.109CFU/mL of Clostridium butyricum and for0.10μg/mL.20ug/mL.50ug/mL.80μg/mL.100μg/mL of teichoic acid by adopting Trizol. Total RNA was reverse-transcribed to generate cDNA and genes of immune factors,IL-6. IL-8. IL-10. Hsp. TNF-α and NF-κB were obteined by PCR reaction Using specific primers for amplification.We observed the effects of Clostridium butyricum and teichoic acid on the mRNA expression of immune factors.Cell cultures were collected after the cells treated for24h with concentration respectively for0.10CFU/mL.103CFU/mL.105CFU/mL.107CFU/mL.109CFU/mL of Clostridium butyricum and for0.10μg/mL.20μg/mL.50μg/mL.80μg/mL.100μg/mL of teichoic acid.We determined the effects of Clostridium butyricum and teichoic acid on extracellular proteins,IL-6. IL-10. IL-12. TNF-α expression in HT-29cells.Experimental results show that:Clostridium butyricum teichoic acids extracted by TX114have not residues of nucleic acids and proteins,contain diagnostic radical and possess great purity and biological activity detected by H nuclear magnetic resonance (NMR)and infrared spectrum (IR).The amount of Clostridium butyricum adhering to HT-29cells declined significantly as the TAs-treated concentration increased when using Clostridium butyricum and teichoic acid treated cells at the same time.Among proinflammatory factors,the mRNA expression of IL-8.the mRNA expression of TNF-a decreased and the mRNA expression of IL-6has no change;Among inflammation inhibiting factors,the mRNA expression of IL-10decreased and the mRNA expression of Hsp70increased;The mRNA expression of signaling pathway protein,NF-KB,declined.The concentrations of immune factors,IL-6、IL-10、IL-12、 TNF-a were extremely low and undetectable.We can concluded from the above findings:Clostridium butyricum teichoic acids can be extracted and puried by adopting TX114.Clostridium butyricum teichoic acids play an important role in the process of Clostridium butyricum adhering to intestinal cells.Clostridium butyricum and teichoic acid are able to induce the inflammatory response in HT-29cells, nevertheless they can regulate the inflammation by means of increasing inflammation inhibitory factor secretion to avoid tissue damage,which incarnate the probiotic properties of Clostridium butyricum.On the other hand,the expression of IL-8was probably increased for the basic state of alert of cells,which in order to prevent the intrusion of foreign bodies.We also observed that the effect trends of Clostridium butyricum and teichoic acid were identical.So we speculated initially that:Clostridium butyricum adhere and colonize on intestinal cell surface and regulate the immune response by using teichoic acid.There is no doubt about that we need thorough studies to verification this conjecture.