Porcine β-defensin Gene Expression And Its Regulation By Arginine

P-defensin is a direct effector molecule of anti-microbial immunity, which is critical for antibiosis, anti-virus, anti-parasitic and immune regulation. P-defensin is also a linkage between innate and adaptive immunity. As a kind of immune modulators, arginine is important for immune organ development, cellular and humoral immunity and animal stress relief. However, gene expression pattern of porcineβ-defensin (pBD) and the impact of arginine on pBD gene expression pattern have not been reported yet. This research intended to study the difference expression pattern of pBD in different swine breed and tissues, as well as the effect of arginine on pBD expression in order to understand the expression character of pBD and the regulative effect of arginine on pBD.Experiment 1 Study on the difference of pBD-1,2,3 genes expression in DLY and Tibetan pigsTo compare the difference of P-defensin gene expression between two swine breeds, Six 7-day-old Tibetan pigs and six 7-day-old DLY (Duroc x Landrace x Yorkshire) crossbred pigs were selected randomly and slaughtered by exsanguination. 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 collected immediately, and stored at-80℃for further extraction of total RNA. Then pBD-1,2,3 genes expression were determined using real-time quantitative PCR. The results showed that the pBD gene mRNA expression could be detected in all tissues. The pBD-1 and 3 were expressed at high levels in tongue and oral epithelium, but pBD-2 in kidney and liver. The expressions of pBD-1 and 3 of Tibetan pigs in most tissues were higher than those of crossbred pigs, but pBD-2 level was lower. The finding illustrated that there were obvious breed differences and tissue specificity of pBD expression.Experiment 2 The effects of arginine, isoleucine and Zn2+on the expression of pBD genes and protein in IPEC-J2 cellsTo study the effect of arginine (Arg), isoleucine (Ile) and Zn2+on the expression of pBD-1,2,3 mRNA and protein, porcine intestinal epithelial cell line (IPEC-J2) was used and cultured in serum free DMEM/F12 medium with different levels of Arg,Ile and Zn2+ Total RNA and protein were extracted after 24 h. The expression of pBD-1,2,3 mRNA were determined by real-time PCR, and the protein content of pBD-1,2,3 were measured by ELIS A. Data indicated that Arg, Ile and Zn2+could induce the expression of pBD gene and the secretion of pBD protein in porcine intestinal epithelial cell line IPEC-J2. All concentrations of Arg in this experiment significantly increased the mRNA and protein expression level of pBD-1,2,3 significantly increased the mRNA and protein expression level of pBD-3. The 100μg/mL was the optimal concentration of Arg to regulate the expression of the pBD. The optimal concentration of Ile and Zn2+to regulate the expression of the pBD were at 25 g/mL and 100μmol/mL, respectively. These results suggested that Arg, Ile, Zn2+could improve the mRNA and protein expression of pBD-1,2, 3 genes in the experimental concentration range and Arg would be better for improving the expression of pBD-1,2,3 genes.Experiment 3 The effect of Arg on endogenous defensin expression in DLY pigletIn order to investigate the effect of arginine on endogenous defensin expression in weanling piglets,36 DLY piglets (initially 6.5-7kg BW and 21-day-weaned) were randomly allotted to 6 treatment groups by weight according to a 2 x 3 factorial design. There were 6 replicates with 1 piglet per replicate in each treatment. During the whole experiment period, groups 1 and 4 were fed with basal diet, group 2 and 5 were fed with basal+0.5%arginine diet, and the other groups were fed with basal+1%arginine diet. In the morning of the eighth day after determining the fasting weight, group 4,5 and 6 totally 18 piglets were injected with 4 mL S.C500(Salmonlla Choleraesuis) for each piglet. The group 1,2 and 3 were injected saline as a control. On the tenth day after S.C500 vaccination, all piglets were slaughted and sampled. The results were shown as follows:1. Dietary arginine supplementation tended to increase the ADG as well as ADFI of piglets, and significantly declined F/G. of piglets. Vaccination of S.C500 decreased the performance. The ADG and ADFI of first week and the overall ADG were significantly reduced after S.C500 injection. After S.C500 vaccination, the F/G tended to be declined. The arginine supplementation improved the performance of piglets vaccinated S.C500. The 1% Arg group was better than other groups on improvement of piglets performance.2. Arginine supplementation in diet caused the decreasing trend of blood urea nitrogen levels. After immune stress, the blood urea nitrogen level tended to be increased. The blood urea nitrogen concentrations of 1% Arg group were lower than 0.5% Arg group.3. For un-vaccinated piglets, dietary arginine supplementation increased free arginine (Before or 1,3,7 and 10 days after stress), ornithine (Before or 1,3,7 and 10 days after stress), and citrulline (Before or 3 and 7 days after stress) concentrations and decreased threonine (1 day after stress), methionine (1,3,7 and 10 days after stress), valine (1 day after stress) and histidine (7 day after stress) concentrations in sera. After S.C500 challenge serum lysine (1,3,7 and 10 days after stress), methionine (1,3, and 7 days after stress), valine (1,7 and 10 days after stress) and phenylalanine (1,3,7 and 10 days after stress) concentrations tended to be increased, and arginine (3 days after stress), citrulline (1 and 3 days after stress) and threonine (1,3 and 10 days after stress) concentrations tended to be decreased. The interaction between Arg supplementation and S. C500 vaccination impacted methionine (3 and 7 days after stress), leucine (7 days after stress), phenylalanine (7 days after stress) and citrulline (7 days after stress) concentrations in sera.4. Dietary supplementation of arginine increased the NO concentration (Before stress) and elevated the activity of TNOS (7 days after stress) and iNOS (1,3 and 7 days after stress). But there was no impact on cNOS activity in serum. Vaccination of S.C500 also enhanced the NO concentration (10 days after stress) and elevated the activity of TNOS (7 days after stress) and iNOS (3 and 7 days after stress). There was no effect of Arg supplementation and S.C 500 vaccination interaction on serum NO concentration and abilities of TNOS and iNOS.5. For un-vaccinated piglet, dietary arginine supplementation increased serum IgA (3, 7, and 10 days after stress), IgG (1,3,7 and 10 days after stress) and IgM (3 days after stress). IgA (3 and 10 days after stress), IgG (1,3 and 10 days after stress) and IgM (1,7 and 10 days after stress) in serum increased significantly after S.C500 vaccination. There was no effect of Arg supplementation and S.C 500 vaccination interaction on IgA^ IgG and IgM levels.6. Dietary arginine supplementation decreased serum IL-1β(Before or 1,3 and 7 days after stress), TNF-a (Before or 1,7 and 10 days after stress), and cortisol (Before or 1,3 and 10 days after stress), increased IL-2 (1,3 and 7 days after stress). The levels of IL-1β(1,7 and 10 days after stress), TNF-a(l,3,7 and 10 days after stress), and cortisol (1,3,7 and 10 days after stress) in serum were increased significantly and IL-2 (1,3,7 and 10 days after stress) level was decreased after S.C500 vaccination. There was no effect of Arg supplementation and S.C 500 vaccination interaction on serum IL-1β, TNF-α, cortisol and IL-2 levels.7. Arginine supplementation increased the expression of iNOS in liver and kidney, ARGⅠand ARGⅡin kidney and jejunum, decreased the expression of CAT-1,2,3 in jejunum. Challenge of S.C500 decreased the expression of eNOS in liver, ARG I and ARG II in jejunum, increased the expression of CAT-1,2 in jejunum. The interaction between arginine supplementation and S.C 500 vaccination significantly influenced the expression of CAT-3 in piglets. 8. Dietary supplementation of arginine increased the expression of pBD-1,2,3 in all tissues. S.C500 challenge didn't significantly change the expression of pBD-1,3, but significantly increased the pBD-2 expression in tongue, oral mucosa, respiratory mucosa, jejunum mucosa, ileum mucosa, mesenteric nodes and inguinal lymph nodes. These data suggested that S.C500 challenge increased the expression of pBD-2. Arginine supplementation could upregulate the expression of pBD-1,2,3 in all tissues.The results implicated that S.C500 challenge increased the expression of pBD-2. Dietary supplementation of arginine increased the expression of pBD-1,2,3 in all tissues. Supplementation of arginine to piglets under stress could alleviate the stress responseExperiment 4 The preliminary study of the possible signal transduction pathway by which arginine regulates the expression of pBDs geneIn order to preliminary reveal the signal transduction mechanism of pBD-1,2,3 gene expression induced by arginine, IPEC-J2 cell line was used as a model to investigate the effect of signal inhibitor on expression of pBD-1,2,3 gene induced by arginine. MG-132 and PD98059 were chosen as NF-κB and MEK-ERK signaling pathway inhibitor, respectively. A single factor design with six treatment groups was applied. Six groups included control group, the arginine group, MG-132 group, PD98059 group, arginine+ MG-132 group and the arginine+PD98059 group. The results showed that MG-132 significantly decreased the expression of pBD-2 gene induced by arginine; PD98059 significantly decreased the pBD-1 and pBD-3 gene induced by arginine.The results suggested that NF-κB pathway may be one of the signal transduction pathways by which arginine regulates the expression of pBD-2 gene; MEK-ERK pathway may be one of the signaling pathways by which arginine regulates the pBD-1,3 expression.In conclusion, there were obvious breed difference and tissue specificity of pBD gene expression in Tibetan pigs and DLY pigs. The cell culture experiment and animal experiment both showed that arginine could induce the expression of pBD-1,2,3. The NF-κB pathway and MEK-ERK pathway may be the signaling pathway involved in arginine induction of pBD-2 and pBD-1,3 gene expressions, respectively.