Abstract: 【Objective】The regulatory effect of probiotic Saccharomyces cerevisiae on the β-defensin-1 (Sheep Beta-Defensin-1, SBD-1) expression was explored in cultured ruminal epithelial cells of sheep, which would provide a theoretical foundation and basis for the regulation mechanism of probiotics to defensins at the molecular level. 【Method】 Firstly, the primary culture of the sheep ruminal epithelial cells were carried out after treating the sheep ruminal tissue with PBS and antibiotic. When the number of cells in primary cell culture bottles grew to more than 85%, the cells were passaged to 12-well plates for cell stimulation test, meanwhile, a parallel plate count test was conducted for five times after the activated and purified S. cerevisiae were cultured at 25℃, 180 r/min aerobically for 48 h. A concentration of 5.2 × 10⁸ CFU·mL^-1 S. cerevisiae was obtained according to processing method of plate count, then the concentration was further diluted to the concentrations of 5.2×10⁷, 5.2×10⁶, 5.2×10⁵ and 5.2×10⁴ CFU·mL^-1 by a serial dilution. The cultured sheep ruminal epithelial cells were stimulated for different times (2, 4, 8, 12 and 24 h) by different concentrations of S. cerevisiae (5.2×10⁴ - 5.2×10⁸ CFU·mL^-1), and a control group was set up with DMEM/F12 medium instead of S. cerevisiae. Secondly, the total RNA was extracted from the tested cells and reversely transcripted into cDNA, then different expressions of SBD-1 were examined by real time fluorescence quantitative PCR and enzyme-linked immunosorbent assay (ELISA) tests. Finally, the related differences analysis of data was done by SAS 9.2 software. 【Result】The realtime fluorescence quantitative PCR results showed that the expression of SBD-1 mRNA in each time group increased and then decreased following the increase of concentrations, the expression of SBD-1 mRNA was the highest when the cells were stimulated with the concentration of 5.2 × 10⁷ CFU·mL^-1, and significantly higher than the control group and the other concentration groups (P＜0.01). When the concentration was constant, the expression of SBD-1 mRNA increased with the extension of stimulating time, which the expression of SBD-1 mRNA was the highest at 12 h, then downward. Therefore, the expression of SBD-1 was the highest at the concentration of 5.2 × 10⁷ CFU·mL^-1 and 12 h stimulating, compared with the other time groups at this concentration was significantly different (P＜0.01 ). The results of ELISA test showed that the expression trend of SBD-1 protein was consistent with the SBD-1 mRNA. After the sheep ruminal epithelial cells were stimulated with different concentrations of S. cerevisiae for 2, 4, 8, 12 and 24 h, the expression of SBD-1 protein in the co-culture supernatants increased, and there was a significant difference compared with the control group (P＜0.01 ). The expression of SBD-1 protein reached the highest level at concentration of 5.2 × 10⁷ CFU·mL^-1 and 12 h stimulating, the difference was significant compared with the each time group under this concentration (P＜0.01).【Conclusion】The results of this study indicated that S. cerevisiae could improve the expression of SBD-1 in ruminal epithelial cells of sheep. And the level of SBD-1 expression was the highest when the ruminal epithelial cells were stimulated for 12 h with the concentration of 5.2 × 10⁷ CFU·mL^-1.

Key words:  β-defensin-1, ruminal epithelial cells, Saccharomyces cerevisiae, realtime fluorescence quantitative PCR, ELISA, sheep