| Antimicrobial peptides (AMPs) are short peptides coded by genes, play a vital role in resisting pathogen invading as effcter of host defense. AMPs have broad spectrum inhibitory activity to bacteria, fungi, virus and mycoplasm, and show a different expression pattern which are inducible expression and inherent expression. A variety of factors have been shown positive regulation effect on AMPs, LPS and VD are the best examples of the immune stimulus factors and the nutrition factors, respectively. Chicken have two main AMPs, which are 14 P-defensins and 4 cathelicidins.1,25(OH)2D3 have been found to be highly potent in regulating the expression of HDPs in human, mouse and bovine studies, which indicates that we can use this mechanism to up regulate domestic animals’ AMP expression to resist pathogen with better image to reduce using antibiotics in animal production. However, whether VD can induce the expression of chicken β-defensins remains unknown. Intestine is an organ mainly function in digestion and absorption, it is an ideal model to investigate the effect of VD on expression of chicken β-defensins with or without LPS stimulation.In this study, we optimized the culture method of chicken intestinal epithelial cell for further use. The optimization results showed that using the intestine of 15 d chicken embryo is better for IECs culture than using the intestine of 192 d laying hens and 1d chick; more intact crypts were harvested when we increased the number of digestion without total digestion time change; compared with differential adhension technique, time used for cell culture were greatly saved and higher purity of cells were obtained by using cell strainer to separate the crypts and single cells. Setting adhension completed as the starting point, IECs grew slowly in the first 12 h, showed a logarithmic growth phase between 24-48 h, after 72 h, cells began to apoptosis with boundary blurred.To find the optimal concentration of LPS (0,50,100,200,400,800 μg/mL) and VD (0,0.2,2,20,200 ng/mL) for further study, IECs were treated by a series of VD and LPS concentration gradient, respectively. After 12 h, cells were harvested for real time qPCR to detect the expression of the genes involved in immune activation. IECs were found could be activated by 800 μg/mL LPS treated, with TLR4, IL-1 and IL-6 significantly been upregulated; IECs showed no response to single VD treatments, while VDR and inflammatory factors(IL-1 and IL-6) was respectively up regulated and down regulated by VD treatments when IECs were immune activated by 800 μg/mL LPS, and this effect was proportional to the concentration of VD 800 μg/mL LPS and 20 ng/mL VD were selected for further study.To investigate the effct of VD and LPS on chicken β-defensins, IECs were placed into a 2×2 factorial randomized block design of VD x LPS. The results showed that LPS had positive effect in up regulating the expression of GAL-1, GAL-3. GAL-4, GAL-5, GAL-6, GAL-9, GAL-10, GAL-12 and GAL-13; VD had a significant effect in manipulating the expression of GAL-3, GAL-4, GAL-5, GAL-6 and GAL-9, but it only occurred when IECs had been activated by LPS.7 of total 14 chicken (3-defensins were found have VDREs in their promoter, and 6 of them can be induced by VD.In conclusion, VD can induce the expression of chicken β-defensins under immune challenge, and this induction may rely on VDRE in the promoter of β-defensin genes. However, further studies are needed to explain how it works. |
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