Preliminary Studies On Expression And Signaling Pathway Of β-Defensin-1 Induced By Lactobacillus In Sheep Rumen Epithelia Cells

Lactobacillus exists in the mucosa of mammalian body and can resist against other pathogens in mouth and digestive tract. Defensins were widely found in animals, plants and insects, which composed the first defensive line against pathogen in a host. Recent years, numerous studies concluded that defensins could be induced by Lactobacillus. There are two β-defensins in sheep, namely sheep β-defensin-1 (SBD-1) and SBD-2, moreover SBD-1 expressed in the entire digestive tract and respiratory, while the SBD-2 only expressed in the tongue and the distal ileum, therefore SBD-1 may be an important part of gastrointestinal innate immune response in sheep. A mature and stable culture system for sheep rumen epithelia cells (RECs) were established firstly in vitro, then detected the expression of SBD-1 induced by lactobacilli, and investigated the signal transduction mechanism of SBD-1 expression induced by the dominant strain Lactobacillus plantarum. The research contents and results are as follows:1. We used the method either clipped the rumen papillae or isolated bluntly the rumen tissue, then digested with 0.25% trypsin, respectively. The results showed that relatively pure cells can be obtained by trypsin digestion after rumen blunt dissection. We selected three kinds of common cell culture media to culture cells, the results showed that DMEM/F12 was more conducive to growth of RECs. RECs of primary culture was purified through trypsin with different time, then identified by immunocytochemical (ICC) staining. The growth curve of cultured cells was plotted and indicted that the growth law was in agreement with the biological characteristics of cells. Thus, the culture system of sheep RECs was established successfully in vitro.2. Two lactobacilli, Lactobacillus casei Zhang (L. casei Zhang) and Lactobacillus plantarum P-8 (L. plantarum P-8), were selectedto stimulate RECs at different concentrations for 2h, then changed the medium and culturedcells continue to induce the expression of SBD-1 at different times. Real-time quantitative PCR (RT-qPCR) method was used to detect the expression of SBD-1 mRNA.The results showed that the expression of SBD-1 mRNA had a time and dose-dependent with lactobacilli and had a very higher level than control when the concentration of lactobacilli was 107 CFU/mL and cells were induced for 8 h, furthermore L. plantarum P-8 was superior to L. casei Zhang.3. RECs were induced for 8 h by viable, heat-inactivated and supernatant of 107 CFU/mL L. plantarum P-8, respectively. Then RT-qPCR and enzyme-linked immunosorbent assay (ELISA) were taken to detect the SBD-1 expression of mRNA and protein. The results showed that inactived L. plantarum P-8 can also significantly induced SBD-1 gene and protein expression, but weaker than live, while supernatant had no effect on SBD-1 expression. The implication is that lactobacilli can induce expression of SBD-1 differentially in RCEs, and when RECs were induced by L. plantarum P-8 at 107 CFU/mL for 8 h, SBD-1 expression was the highest.4. RECs were induced by L. plantarum P-8 at 107 CFU/mL for 8 h, RT-qPCR and Western Blot were applied to detect the expression of TLR2 and MyD88, we found that the gene and protein levels of TLR2 and MyD88 were significantly higher than control. The expression of SBD-1 induced by L. plantarum P-8 was inhibited after TLR2 blocking, indicating that TLR2 is involved in the process that cells recognized and transferred the signal of L. plantarum P-8.5. RECs were induced by L. plantarum P-8 at 107 CFU/mL for 8 h, RT-qPCR was used to detect the gene transcription levels of NF-κB1 and NF-κB2, which were significantly higher than control. The protein expression of phospho-IκBα was also higher than control detected by Western Blot. SBD-1 induction by L. plantarum P-8 were suppressed with NF-κB signaling pathway inhibitor (PDTC), suggesting that the signaling pathway of NF-κB was involved in L. plantarum P-8 inductionon SBD-1 expression.6. RECs were induced by L. plantarum P-8 at 107 CFU/mL for 8 h, confirmed that gene expression levels of ERK and p38 increased significantly detected by RT-qPCR, and the levels of phospho-ERK1/2 and phospho-p38 were also significantly higher than control detected by Western Blot; then the cells were pretreated by the signaling pathway inhibitors of ERK1/2 and p38 (PD98059 and SB202190), respectively, the results showed L. plantarum P-8 can not induce the expression of SBD-1. The conclusion is that L. plantarum P-8 inducing the expression of SBD-1 was regulated by both ERK 1/2 signaling pathway and p38 signaling pathway.Our findings indicated that lactobacilli could induce SBD-1 expression differentially in the RECs, and viable L. plantarum P-8 induced the expression of SBD-1 was involved in the regulation by multiple signaling pathways, probably by TLR2-NF-κB/MAPKs (ERK1/2 and p38) pathways.