| Antimicrobial peptides are important effector molecules of innate immune, which play a critical role in the host against invasion of pathogenic microorganisms. In chicken, two main classes of antimicrobial peptides have been described which respectively are P-defensins and cathelicidins, with P-defensins is primary.1,25-(OH)2-D3have been found to be highly potent in elevating the expression of human antimicrobial peptides which mediated by VDR in present. The results suggested that VDR may play an important role in regulating the expression of avian β-defensins as a kind of transcription factors. However, it remains to know whether vitamin D3 can up-regulate the expression of chicken P-defensins by inducing the expression of VDR and how does it works in vivo. The objective of this study was to evaluate the effect of vitamin D3 and LPS on regulating chicken β-defensins.In this study, Taihe Silky Fowl was adopted as the experimental materials. At first, fourteen kinds of chicken P-defensins gene were analyzed by means of bioinformatics analysis and predicted weather exist VDRE’s typical structure. At 84 d, LPS or vitamin D3 was injected subcutaneously. Small intestines were harvested for RNA isolation at 4,24, and 48 h postinjection. Relative levels of RNA expression were measured for VDR gene and the chicken P-defensins genes with putative VDREs in their promoters (GAL-3,-4,-5,-6,-7,-9 and-10) by qPCR. The results were as follows:1) The published fourteen kinds of gallinacin genes cDNA sequence (National Center for Biotechnology Information [GenBank]) containing a 2000-bp 5’flanking sequences were investigated for the putative transcription factor binding sites of VDR using NHR-Scan. Results showed that seven kinds of gallinacin genes have DR3 or ER6 VDREs, that is, GAL-3,-4,-5,-6,-7,-9 and -10, respectively.2) Real-time PCR analyses reveal that injection of vitamin D3 showed significantly up-regulated the expression of VDR, GAL-4,-6,10 in small intestine(P<0.05). At the same time, expression of GAL-3,-5,-7 was up-regulated in small intestine. These results suggested that expression of VDR and GALs seems to be induced by vitamin D3 and was concluded the tissues expressing VDR responds to vitamin D3 and in turn up-regulate this tissues cellular functions to synthesize GALs.3) The correlation analysis of the expression level of VDR and GALs in VD3-injected chickens revealed that the mRNA expression of VDR has a highly significant positive association with the mRNA expression of GAL-5 (P<0.01) and a significant positive association with the mRNA expression of GAL-10 (P<0.05).The results suggested that vitamin D3 can mediated the expression of part β-defensins in small intestine by activating VDR.4) According to the change of VDR and GALs mRNA expression level in VD3-injected chickens at 4,24 and 48h postinjection, we found that collection time was affected VDR and GALs expression level significantly. VDR had higher level of RNA expression at 4 and 24 h (P<0.05). The difference is significant of GAL-3, GAL-4, GAL-6 and GAL-10 expression at 4h with 24h or 48h (P<0.05),and no differential expression of these four genes at 24h with 48h (P>0.05). Therefore, we speculate that VDR and GALs expressed a rise-decline pattern as vitamin D3 metabolism in vivo.5) At 4 and 24 h postinjection, the expression of VDR, GAL-4,GAL-5,GAL-6 and GAL-10 gene was up-regulated significantly in the injection of LPS and vitamin D3 compared to LPS-injected (P<0.05). These results suggested that pathogenic microorganism invasion could induce the expression of β-defensins genes, and LPS and vitamin D3 worked synergistically in up-regulating the expression of β-defensins to improve the body disease resistance thereby. |
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