Regulating Mechanism Of EPA And DHA On The Immunization Of Intestinal B Lymphocytes From Broiler Chickens

Large amount of studies showed that n-3 PUFA could modulate the immune function, maintain sound health, and consequently maximize the production performance of animals. B lymphocytes, bearing the ability of secreting IgA, were of crucial importance to the immune function of animals. The regulation of n-3 PUFA on mucosal immunity directly affected the growing performance of broilers, while most of the investigations, few of which referred to avians, were conducted in human and other laboratory animals. As the specific mechanism of n-3 PUFA on regulating the intestinal mucosal immune response was yet unclear, the nutritional supplementation of n-3 PUFA, to ease immunological stress and improve production performance of broilers, was usually in sightless dilemma. In this area, however, large numbers of recent articles focused on the immunomodulatory effcts of mixed n-3 PUFAs, other than the concentrations and ratios of two main ingredients - eicosapentaenoic acid （EPA） and docosahexaenoic acid （DHA）. To unravel the issues mentioned above, a series of trials were conducted as follows:1. The optimal working concentration inducing the maximal stimulating responses in vitro was determined by adding different concentrations （0, 5, 10, 15, 20μg/mL） of LPS to the B lymphocytes isolated from broiler chickens. B cells, isolated by nylon wool, were identified by flow cytometry, after which the cell morphology and viability were measured to establish the best isolating and cultivating method. The results showed that the purity and viability of B cells isolated by collagenase-1 digesting method were 96.46% and 97%, respectively, and LPS could induce the immune stimulation of B lymphocytes in vitro, with a maximal stimulating concentration at10μg / mL.2. A 5×5 factorial arrangement was used to study the effects of different concentrations （0, 20, 40, 60, 80μg/mL） and ratios （0:1, 1:0, 1:1, 1:2, 2:1） of EPA to DHA on the proliferation, differentiation and IgA secretion of B lymphocytes under stimulation in vitro, as well as the expression of CD5 and CD79a expression on B cells, to seek the optimal ratio and concentration of EPA and DHA regulating the response of intestinal B lymphocytes to immune stress. The resulsts were as follows: different ratios and concentrations of EPA and DHA inhibited the in vitro proliferation of lipopolysaccharide （LPS）-stimulated B lymphocytes; EPA and DHA significantly suppressed the ability of LPS-stimulated B cells to secrete IgA with ratios of 1:1 at 20μg/mL and 2:1 at 20μg/mL and 40μg/mL; CD5 and CD79a were maximally expressed, compared with those of control groups, on LPS-stimulated B cells when the EPA:DHA ratio was 2:1 at 20μg/mL. On basis of this, we concluded that in the present study the desired suppressing EPA: DHA ratio was 2:1 at 20μg/mL and 40μg/mL to down-regulate the immune response of intestinal B lymphocyte from young broilers.3. With the established immune stress moedel of B lymphocytes, this study sought to examine the effects of EPA and DHA （2:1, 20μg/mL）, in comparation with those of separately added EPA or DHA, on the membrane fatty acid composition and intracellular Ca²⁺, PGE-2 and COX-2 concentration of B lymphocytes, to clarify the immunomodulatory pathway of Ca²⁺ and eicosanoids. The results showed that either adding the EPA and DHA alone or together could significantly affect the membrane fatty acid composition, intracellular Ca²⁺, PGE-2 and COX-2 concentration of B lymphocytes.4. The expression of cAMP, cGMP, PLC and IP3 was determined using the immune stress moedel of B lymphocytes to unravel the regulating effects of EPA and DHA on the second messenger pathway and phosphatidylinositol signaling pathway of B cells isolated from broiler chickens. The results showed that either adding the EPA and DHA alone or together （2:1, 20μg/mL） could significantly affect the expression of cAMP, cGMP, PLC and IP3 of B lymphocytes, with a much more significant effects, nevertheless, when added together.Through the above-mentioned experiments, the in vivo immune stress moedel of B lymphocytes from broiler chickens was successfully established, by which we proved that EPA and DHA significantly suppressed the proliferation of LPS-stimulated B cells and then down-regulated the secretion of IgA, as well as the expression of CD5 and CD79a, with ratios of 2:1 at 20μg/mL. More specifically, n-3 PUFA may exert its immunomodulatory effects via changing the membrane fatty acid composition, down-regulating the concentration of intracellular Ca²⁺ and the expression of PGE-2, suppressing the expression of PLC and IP3 in phosphorylated inositol signaling pathway and altering the expression of cAMP and cGMP in the second messenger signaling pathway. The present study provided a complementary theoretical basis for the regulation of immunity and improving the gut healty of broiler chickens.