Porcine β-defensin Gene Expression And Its Regulation By Vitamin A

Defensin is one kind of antibiotic peptide, which has the extensive effects of antibiosis, anti-virus, anti-parasitic and immune regulation, and defensin is an important component of immune system. As a kind of immune modulator, vitamin A has significant effect on the ability of anti-infection, cytokines and related gene expression of animals. But, the gene expression regularity of porcineβ-defensin and the regulative action of vitamin A on porcineβ-defensin have not been reported yet. This research intended to study the difference of porcineβ-defensin expressed in species and tissues, and the effect of vitamin on the expression, to investigate the expression trait of porcineβ-defensin and the regulative effect of vitamin A. This research firstly studied the difference of porcineβ-defensin1,2,3 genes expression in two kinds of pig species with different disease resistance, and the effects of different vitamin supplementation on the expression ofβ-defensin1,2,3 genes through cell culture experiment in vitro, focused on the effect of vitamin A on porcineβ-defensin1,2,3 genes expression in animal test, initially investigated the mechanism of signal transduction of porcineβ-defensin1,2,3 genes expression which influenced by vitamin A. This study includes the following four experiments.Experiment 1:Research on the difference of pBD-1,2,3 genes expression of DLY and Meishan pigsThe expression difference ofβ-defensin gene in two pig species was compared through the study onβ-defensin expression level in different tissues of DLY and Meishan pigs. Six Meishan pigs and six DLY (Duroc×Landrace x Large) crossbred pigs all at 7-day-old were selected randomly, half male and half female. The mucous membrane of small intestine, heart, liver, spleen, lung, kidney, brain, skin, muscle, oral mucous membrane, respiratory epithelium, tongue, thymus, reproductive tract epithelium, and testis (male) or ovaries (female) were all selected immediately after pigs were slaughtered, and saved in liquid nitrogen for the extraction of total RNA to detect the levels of pBD-1,2,3 genes expression by real-time quantitative PCR.The results showed that, the pBD gene mRNA was detected in all tissues. The expression of pBD-1,2,3 genes in the tongue and oral mucosa were the highest for Meishan pigs. The expression of pBD-1,3 genes in the tongue and oral mucosa were the highest for DLY crossbred pigs, and also for the expression of pBD-2 genes in the kidney and liver. For the expression of pBD gene in most tissues of Meishan pigs was higher than that of the DLY crossbred pigs. The expression level of pBD-1,2,3 genes in the small intestinal mucosa, tongue, oral mucosa and genital mucosa which contacting with the outside world of Meishan pigs was higher than DLY crossbred pigs. So, it illustrated that the apparent species difference and tissue specificity existed for porcine pBD gene expression, the expression level of pBD-1,2,3 genes in most tissues of Meishan pigs were higher than DLY crossbred pigs.Experiment 2:The effect of VA, VD and VE on the expression of pBD genes and protein in cell line IPEC-J2To study the effect of VA, VD and VE on the expression of pBD-1,2,3 genes mRNA and protein, porcine small intestinal cell line IPEC-J2 was used and cultured in serum free DMEM/F12 medium with different levels of vitamin A, D, E. Total RNA and protein were extracted after 24h. The expression of pBD-1,2,3 gene mRNA were determined by Real-time PCR, and the protein content of pBD-1,2,3 were measured by ELISA, to compare the effect of different level of vitamin on the expression of pBD gene and protein.Results showed that, Vitamin A, D, E could induce the expression of pBD gene in porcine small intestinal epithelium and the secretion of pBD protein. Vitamin A at 0.5-20μmol/L improved the mRNA and protein expression level of pBD-1,2 and/or pBD-3 significantly. Vitamin D at 5-50nmol/L improved the mRNA or protein expression level of pBD-1, pBD2 or pBD-3 significantly. Vitamin E at 20 and 50μg/ml improved the mRNA or protein expression level of pBD-1 and pBD-3 significantly. Results illustrated that vitamin A, D, E could improve the mRNA and protein expression of pBD1,2,3 genes in this present condition, and vitamin A would be better for improving the expression of pBD1,2,3 genes.Experiment 3:VA influenced the expression of pBDs gene in DLY and Rongchang piglets24 Rongchang piglets (initially 3.5-4kg BW and 21-day-weaned) were randomly allotted to 4 treatment groups:group1,2,3 and 4 by weight; 30 DLY piglets (initially 6.5-7kg BW and 21-day-weaned) were randomly allotted to 5 treatment groups:group 5,6, 7,8 and 9 by weight, each treatment had 6 replicates with 1 piglets per replicate. The trial was conducted for 28 days. Groups 1,3,5 and 7 were fed on vitamin A deficient diet, group 9 were fed on higher vitamin A diet (5*NRC) and other groups were fed on normal vitamin A diet. The trial period included 2 weeks preliminary experiment and 4 weeks formal experiment. In formal experiment day 14, the piglets of group 3,4,7,8 and 9 were injected PRRS modified live vaccine vaccination or PBS as a control.Results showed that,1. DLY piglets' ADG and ADFI were significantly higher than Rongchang piglets. After piglets vaccinated PRRS, the performance of both Rongchang and DLY piglets were declined. The effect of PRRS to DLY piglets performance was bigger than to Rongchang piglets.2. The retinol level in serum and liver of DLY piglets were significantly higher than Rongchang piglet. PRRS vaccination decreased the serum retinol level but improved the liver retinol level. Vitamin A deficient treatment significantly decreased the retinol level in serum and liver. High vitamin A diet improved both the liver and serum retinol level.3. IgA,IgG and IgM in serum increased significantly after PRRS vaccination. Vitamin A deficient treatment significantly decreased the serum IgA,IgG and IgM contents. The effect of PRRS to DLY piglets serum IgA and IgG was greater than to Rongchang piglets.4. IL-1a,IL-1βand IL-10 in serum increased significantly after PRRS vaccination. Vitamin A deficient treatment significantly increased the serum IL-la,IL-1βand decreased the serum IL-10 contents.5. PRRS vaccination showed little effect on the mRNA expression of pBD-1 and pBD-3, but improved significantly the mRNA expression of pBD-2 in lung, spleen, mesenteric nodes and inguinal lymph nodes. The level of vitamin A in diet had little effect on the mRNA expression of pBD-2 gene, and had significant effect on the mRNA expression of pBD-1 and pBD-3 in tongue, oral mucosa, respiratory mucosa and mucous membrane of small intestine.Results indicated that experimental animal'ADG, ADFI and serum retinol level were declined after PRRS vaccination, but the serum IgA,IgG,IgM increased significantly, the mRNA expression of pBD-2 in lung, spleen, mesenteric nodes and inguinal lymph nodes also increased. In comparison, the effect of PRRS to DLY piglets was greater than to Rongchang piglets. Vitamin A deficient treatment significantly decreased the serum retinol level and mRNA expression of pBD-1 and pBD-3 in some tissues, and this effect can be aggravated by PRRS vaccination.Experiment 4:The possible signal transduction channels of VA influenced the expression of pBDs geneIn order to preliminary reveal the signal transduction mechanisms of vitamin A promoting induction expression of pBD-1,2,3 gene, the experiment used IPEC-J2 cell line model for the study to investigate the effet of signaling pathway blockade on induction expression of VA in pBD-1,2,3 gene mRNA. We chose MG-132 and PD98059 for NF-κB and MEK-ERK signaling pathway inhibitor, respectively, and applied single factor design with six treatment groups, that was control group, the vitamin A group, MG-132 group, PD98059 group, vitamin A+MG-132 group and the vitamin A+PD98059 group.The results show that, MG-132 very significantly reduced the induction expression of pBD-2 gene of vitamin A, PD98059 significantly reduced the pBD-1 and pBD-3 gene induction of vitamin A. The results suggest that, NF-κB pathway may be one of the signal transduction pathway of vitamin A affection the expression of pBD-2 gene; MEK-ERK pathway may be one of the signaling pathways of vitamin A affection pBD-1,3 expression.In conclusion, data reported here demonstrated:1. The distribution and expression of pBD in Meishan and DLY pigs with different immunity were different, the expression of pBD1,2 and 3 in mucous membrane of small intestine, tongue, oral mucosa and mucous membrane of reproductive tract in Meishan pigs with higher immunity were all higher than DLY pigs.2. Vitamin A, D and E could all induce the expression of pBD-1,2 and 3 gene in small intestine epithelium, and motivate the secretion of its protein, this mode of action exhibited the dose dependent relation. The promotive effect of vitamin A was the most significant one.3. Under the situation of normal and PRRS vaccination, the content of vitamin A in diet showed the significant effect on the expression of pBD-1 and 3 genes in tongue, oral mucosa, Respiratory Mucosa and mucous membrane of small intestine, diet with higher vitamin A improved the expression of pBD gene in these tissues.4. This study demonstrated initially that MEK-ERK passageway was one of the signal transduction channels of vitamin A inducing the expression of pBD-1 and 3 genes, NF-κB passageway was one of the signal transduction channels of vitamin A inducing the expression of pBD-2 gene.