| Ovine rumen,the first stomach of ruminants,is constantly challenged by a variety of pathogenic microorganisms.The ruminal mucosal epithelium plays an important role in innate immunity,and can also secrete a series of innate immune molecules to prevent microbial attack.Defensins are cationic peptides that are produced by the mucosal epithelium and have broad-spectrum antimicrobial activity,and are considered potential replacements for antibiotics.Our group has demonstrated that the whole cell wall of Saccharomyces cerevisiae can up-regulate the expression of Sheep p-defensin-1(SBD-1)in ovine ruminal epithelial cells(ORECs).In order to further determine the active constituents of Saccharomyces cerevisiae cell wall-induced SBD-1 expression,the?-glucan derived from the cell wall of Saccharomyces cerevisiae was used to stimulate ORECs to observe its effect on the expression of SBD-1,and to further investigate the signaling pathways involved in this induction process.In a controlled environment,explant culture has the characteristics of retaining similar histological features in vivo.Therefore,explant culture is closer to the vivo environment than cells.This study explored the feasibility and optimal conditions of ovine rumen explants(OREs),and studied the effect of ?-glucan on the expression of antibacterial peptide SBD-1,proinflammatory cytokine IL-6 and anti-inflammatory cytokine IL-10 in OREs.The specific research contents and results are as follows;(1)After stimulating ORECs with different concentrations(0?5,10,20,50 and 100?g/mL)of ?-glucan for 8 h,qPCR and ELISA were used to detect the expression changes of SBD-1,thereby screening out the optimal P-glucan concentration that induces the expression of SBD-1.Then this concentration was used to stimulate the ORECs at different times(0,2,4,8,12,and 24 h),and the optimal stimulation time for induction of SBD-1 expression was also screened by qPCR and ELISA.The results showed that the level of SBD-1 mRNA and protein expression was highest when the ORECs were separately stimulated at 2 and 4 h with the concentration of 10 ?g/mL P-glucan.(2)The signaling pathway of ?-glucan-induced SBD-1 upregulation was detected by Western blot,RT-PCR,immunohistochemistry,immunocytochemistry,qPCR and ELISA.The results showed that Dendritic-cell-associated C-type lectin 1(Dectin-1)and Spleen tyrosine kinase(Syk)were expressed in ovine ruminal mucosa tissue and ORECs,and siRNAs interference or inhibitor inhibition the expression of Dectin-1,Syk,TLR-2 and MyD88 attenuated ?-glucan-induced SBD-1 expression.Treatment with ?-glucan resulted in significantly increased the expression of p38,JNK,ERK1/2 and NF-?B,while inhibition of MAPK and NF-?B pathways by inhibitors significantly reduced ?-glucan-induced SBD-1 expression,and the inhibitory effect of NF-?B was the most obvious.(3)OREs were cultured,and the structural integrity and cell viability of OREs were detected by H.E staining,qPCR and immunohistochemistry.H.E staining results showed that the OREs tissue structure is more complete in the medium without serum.qPCR and immunohistochemistry results showed that the expression of OREs E-cadherin within 24 h of culture was not significantly different from that of uncultured tissues,and there was a positive signal of CK-18 and Ki-67.(4)After co-cultivation of OREs with different concentrations(0,10,50,100,200 and 400 ?g/mL)of ?-glucan for 8 h,qPCR and ELISA were used to detect the expression of SBD-1,IL-6 and IL-10,thereby screening out the optimal ?-glucan concentration that induces the expression of SBD-1,IL-6 and IL-10.Then the OREs were stimulated with the optimal concentration of P-glucan for different time(0,2,4,8,12 and 24 h),and the optimal stimulation time for induction of SBD-1,IL-6 and IL-10 expression was also screened by qPCR and ELISA.The results showed that 50 ?g/mL ?-glucan stimulated the highest mRNA expression of SBD-1,IL-6 and IL-10 at 2,8 and 12 h.The expression of SBD-1 protein was peaked at 8 h after stimulation of OREs with 100 ?g/mL ?-glucan,while the expression of IL-6 and IL-10 was maximal at 12 h after stimulation of OREs with 50 and 200?g/mL,respectively.The results of this study indicate that p-glucan can increase the expression of SBD-1 in ORECs,and this process is mediated by Dectin-l-Syk/TLR-2-MyD88-MAPK/NF-KB signaling pathway,but may be mainly through Dectin-l-Syk/TLR-2-MyD88-NF-KB signaling pathway.In addition,the study also identified the feasibility of and optimal conditions of in vitro culture of OREs and demonstrated that P-glucan can activate the secretion of SBD-1,IL-6,and IL-10 in OREs to promote mucosal immunity. |
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