Study On Molecular Mechanism Of The Interaction Between Clostridium Butyricum And Intestinal Epithelial Cells

Oral administration of Clostridium butyricum as probiotics is increasingly gaining importance in the treatment of intestinal inflammations and improvement of animal performance. However, the mechanisms of host cell receptor recognition of C. butyricum and the downstream immune signaling pathways leading to these benefits remain unclear. The aim of the study was to explain the beneficial properties of C. butyricum.Trial1. The HT-29cells were stimulated by C. butyricum to investigate its capability to influence the innate immune response of HT-29cells. The results showed that the C. butyricum was able to stimulate TLR (toll-like receptor)2production at mRNA level, however, TLR4, TLR5, TLR9, MyD88(myeloid differentiation primary response protein88) transcription levels were not up-regulated. The NF-κB (nuclear factor κB), IL (interleukin)-8and TNF-a (tumor necrosis factor alpha) levels in response to C. butyricum were significantly increased, indicating that HT-29cells were sensitised by C. butyricum. However, compared with Escherichia coli, the levels of NF-κB, IL-8and TNF-a induced by C. butyricum were far lower. Furthermore, we observed that the C. butyricum induced a significant increase in the levels of both IL-10and Hsp70(heat shock protein70), which may be associated with the beneficial properties of C. butyricum.Trial2. TLR2and MyD88specific siRNA (small interfering RNA) were used to silence expression of TLR2and MyD88. Knockdown of MyD88expression using siRNA in this manner did not affect C. butyricum-induced elevated levels of NF-κB, IL-8, IL-6and TNF-α, suggesting a MyD88-independent route to TLR signaling transduction. However, a significant reduction in the levels of NF-κB, IL-8, IL-6and TNF-α was evident in the absence of TLR2expression, implying the need for TLR2in C. butyricum recognition. Hence, C. butyricum activates TLR2-mediated MyD88-independent signaling pathway in human epithelial cells.Trial3. The TLR2is a key receptor for recognizing C. butyricum. We next analyzed the structure of TLR2and indentified its functional domain. Based on the X-ray crystal structure of mouse TLR2, a homology model of human TLR2was constructed. The human TLR2ligand agonist LTA (lipoteichoic acid) was docked into the optimized model, and the critical amino acid residues for binding were identified, which was very important for further site-directed mutagenesis study.Trial4. HT-29cells were treated with anti-IL-10(IL-10antibody) or siIL-10(specific siRNA) to disrupt IL-10. In both cases, the effects of C. butyri cum-induced NF-κB activation and IL-8expression were enhanced. We also found that neutralization or knockdown of IL-10could induce apoptosis and necrosis of HT-29cells treated with C. butyricum compared with control cells. These findings show that IL-10serves an important role in C. butyricum-mediated immune protection, and in host recognition of C. butyricum.Trial5. We investigated the effects of C. butyricum and its SCS (spent culture supernatants) on EHEC (Escherichia coli) growth and adherence to CEICs (chicken embryo intestinal cells). Indeed, the C. butyricum and its SCS exhibited significant inhibitory activity. On the other hand, we evaluated the potential of C. butyricum to inhibit EHEC-induced apoptosis in CEICs. Also, the C. butyricum showed significant inhibitory effect on the EHEC-induced apoptosis by modulating the expression of XIAP (X-linked inhibitor of apoptosis protein), BclXL (B-cell lymphoma-extra large), FAS, Bcl2(B-cell leukaemia/lymphoma-2), BAX (Bcl-2-associated X protein), P53(Tumor protein53) and via inhibition of caspase-9and caspase-3activation. Altogether, these results indicate that the C. butyricum possesses the ability to prevent the EHEC-induced intestinal disorders both directly through inhibiting EHEC viability and indirectly via medicating EHEC-induced apoptosis, which could help to explain the beneficial properties of C. butyricum. Furthermore, this data is novel in the case of poultry and the manner in which C. butyricum prevents the EHEC-induced apoptosis provides supportive information for the treatment of colibacilliosis.