Selection And Verification Of Difference Proteins In Chicks Intestinal Mucosa With Selenium Deficiency And Research Of The Signal Pathway

Selenium(Se) is one of the important trace elements of human and animals. Se-deficiency can cause diarrhea in the young animal, which might be closely related to the intestinal mucosal immunity. However, whether selenium could regulates host intestinal mucosal immunity still remains elusive. In order to investigate the molecular mechanism that Se-deficiency impacting chick intestinal mucosal immunity, we attempted to reduce antioxidant activity of the chick duodenal mucosa(DM) via feeding the diet Se deficiency to increase inhibitor of nuclear factor kappa-B kinase subunit alpha(IKKα), activate the NF-κB pathway, and weaken the function of immunity.SPF chicks were firstly used to establish Se-deficiency chick model. The difference proteins between DM in Se-deficiency and normal chicks were confirmed by iTRAQ coupled with LC-MS/MS technology. The enrichment pathways were analyzed in different metabolic pathways using bioinformatics methods. Western blot was performed to validate difference proteins among DNA topoisomerase(TOPIIIβ), protein tyrosine kinases(PTK), protein serine/threonine kinase(AKT2), p50, IKKα and dual-specificity mitogen-activated protein kinase kinase 2(MEK2), and analyze NF-κB activation in DM. The RT-Q-PCR method was applied to analyze the mRNA levels of difference proteins TOPIIIβ、AKT2、PTK, and so on. Enzyme-linked immunosorbnent assay was performed to measure the level of secretion-type immunoglobulin A(SIgA) in each section, the activities of GSSG, GSH, GPx, the levels of p65, p65 subunits DNA-binding, p-IκB and IκB, and the activities of the related cytokines in chicks DM.The results included follows: 1. Se-deficiency caused Se concentration decreasing significantly in blood and DM compared with the control groups at day 7(p < 0.05). Se concentration decreased significantly at days 21 and 35(p < 0.01). The clinical symptoms of Se-deficiency chicks expressed depression, weight loss and diarrhea, and pathological examination exhibited pale muscle, duodena bleeding and exudative quality disease, which indicated that Se-deficiency chick model was successfully established. 2. The difference proteins were determined by iTRAQ in combination of quantitative analysis with software Mascot 2.2 and Proteome Discoverer1.4(thermo) library. Mascot screening found 67, 127, 119 kinds of difference proteins involved into the Se-deficiency chicks at days 7, 21 and 35 according to the score > 1.2, or value < 0.83(p < 0.05). There were 25 difference proteins had statistical significance with the consistency. Among the 25 identified proteins, 13 difference proteins were up-regulated, but 12 difference proteins were down-regulated. There were 18 KEGG pathways of the 25 difference protein enrichment in the “Ras signal pathway”, and so on(p < 0.05). 3. The results of Western Blot showed that expressions of TOPIIIβ in Se-deficiency groups were 0.37, 0.36 and 0.34 times than those of the control groups at days 7,21 and 35, respectively. The expressions of AKT2 in Se-deficiency groups were 0.76, 0.33 and 0.55 times than those of the control groups, respectively. The expressions of PTK in Se-deficiency groups were 1.12, 0.89 and 2.16 times than those of the control groups, respectively. These data were consistent with appraisal results of proteomic using iTRAQ. The expressions of IKKα in Se-deficiency groups were 2.16, 2.10 and 2.31 times than those of the control groups at the 3 time points, respectively. The expressions of MEK2 in Se-deficiency groups were 0.83 and 0.86 times than those of the control groups at days 7 and 21, respectively. However, the expression of MEK2 was 1.60 times in Se-deficiency groups than those of the control groups at day 35. At the 3 timepoints, the expressions of NF-κB(p50) increased significantly in Se-deficiency group(p < 0.05). The proteins involved in NF-κB signaling pathways were consistent with appraisal results of proteomic using iTRAQ. 4. The results of RT-Q-PCR found that the mRNA levels of PTK increased significantly in Se-deficiency groups compared with the control groups at days 7, 21 and 35, but the mRNA levels of TOPIIIβ, AKT2 and GPx1 obviously decreased. The levels of MEK2 mRNA increased in Se-deficiency groups at day 35(p < 0.05). These above results were consistent with the analysis of proteomic. 5. Our results showed that Se-deficiency caused chicks DM secretion of SIgA decreased(p <0.05) at days 7, 21 and 35. Se-deficiency decreased significantly secretion of SIgA in the chick jejunum, ileum, cecum and rectum(p < 0.05) at 21 and 35 day. Additionally, the Se deficiency elevated oxidized glutathione activity, and decreased glutathione peroxidase and glutathione activities(p < 0.05). These results showed that Se-deficiency elevated the activities of p65 and p65 subunit DNA-binding, and the p-IκBα, but decreased the level of IκBα(p < 0.01) in the DM. At the same time, the Se-deficiency increased the production of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, interferon gamma, and IL-17 A. In contrast, the expressions of anti-inflammatory cytokines, transforming growth factor-β1 and IL-10 were suppressed. These results implied that Se-deficiency activated NF-κB signaling pathway in chicks DM, and finally weakened the function of immunity.To sum up, on the basis of Se-deficiency proteomics, our results suggested that the Se-deficiency decreased the antioxidant activity and secretion of SIgA in chicks DM, then increased the expression of IKKα and p50, and activated the NF-κB pathway, which finally attenuated the function of duodenal mucosal immune and promoted inflammatory response.