Study On The Effect And Mechanism Of Selenium-Enriched Probiotics On Porcine Cellular Immunity

Selenium (Se) is an essential trace element for animals and humans. Se has many biological functions including regulating metabolism, enhancing the antioxidative ability, immune function, reproductive performance, and reducing the risk of diabetes, cardiovascular disease, certain types of cancer and resisting to detoxification from some heavy metals. In many countries, supplementation of selenium in animal diet is a routine step on animal husbandry. Se is incorporated into selenoproteins as selenocysteine and exerts its biological functions. Probiotics, which are reported to exert a myriad of beneficial effects including balance of colonic microbiota, prevention and treatment of certain diarrhoeal diseases, improvement of growth performance, enhancement of immune response, reduction of serum cholesterol and inhibition of cancer, are diffusely served as feedstuff additive in stockbreeding. Adding sodium selenite as inorganic selenium in animal diet has been set limit in some countries because inorganic selenium has disadvantages of lower bioavailability, higher toxicity and potential environmental pollution. Precious studies have suggested that organic forms of Se (e.g., Se-enriched yeast, which contains selenomethionine) are less toxic than inorganic forms of Se such as sodium selenite, and the bioavailability of organic Se is relatively high. The preparation of Se-enriched Probiotics was made in our lab and used for nutritional supplementation in diets, which can exert dual effects of organic Se and probiotics at the same time. Invention patent for the preparation was awarded by State Intellectual Property Office of The P.R.C. The present study was conducted to evaluate the effects of supplementary Se-enriched probiotics on transform of Se, immune function, antioxidative ability and major selenoproteins mRNA levels of lymphocytes in piglets, and to explore its mechanism at the cellular and molecular levels, so that to provide a theoretical basis for application of Se-enriched probiotics in pig husbandry.Experiment1. Development of the method for determination of GPx1, GPx4, and TR1mRNA levels in pig by real-time PCRAccording to the published mRNA sequences of gene of P-actin, GPx1, GPx4, and TR1, four pairs of specific primers were designed by biological software and synthesized. Oligo dTs were used as primer and first strand cDNAs were synthesised from total RNA, which was isolated from bovine hepatocytes. Using the synthesised cDNAs as template, four target fragments were amplified by polymerase chain reaction (PCR), respectively. The PCR products were electrophoresed on2%agarose gels and extracted. The extracted PCR products were cloned into pMD18-T Vector and sequenced. The copies of extracted plasmids were measured and serially diluted by10-folds. The diluted plasmids were used as standard templates and real-time PCRs were performed. In addition, the contents and annealing temperatures of four pairs of specific primers were optimized, respectively. The results showed that one target band was amplified for each specific primer pair and consistent with the fact. The optimal contents and annealing temperatures of four primer pairs were10μmol/L0.4μL and60℃, respectively. All the R2of four standard curve for gene of β-actin, GPx1, GPx4, and TR1were higher than0.99. The melting curve analysis showed only one peak for each PCR product. These results indicate that the method for determination GPx1, GPx4and D1mRNA levels in pig by real-time PCR is successfully developed.Experiment2. Effect of supplementary selenium-enriched probiotics on growth performance and immune function in piglets48health piglets were divided into four groups in a randomized complete block design, of which3were assigned for test groups, and one served as control. The control group(C) received a basal diet. Both selenium-enriched probiotics (SP) and sodium selenite (SS) were added into basal diet at0.3mg/kg of Se to make two experiment diets. Probiotics was added into basal diet to make probiotics group (P). Whole feeding experiment lasted for42days. Blood samples were collected for analysis of serum IL-2and TNF-a concentrations on d0,14,28and42. On the d14and42of the experiment, the serum antibody level of swine fever in3test groups were significantly higher than the control group respectively (P <0.05); The T cell transformation of pig peripheral blood mononuclear cell (PBMC) were significantly higher in The SP, SS and P group than did the control group (P<0.05), and the SP group were significangly higher than the SS group; The serum concentrations of IL-2and TNF-a in3test groups were markedly higher than the control group on d42(P<0.05); The results testified that there is a better effect of supplementation SP than those of supplementation SS or probiotics in boosts immune responses. Experiment3. Effect of supplementary selenium-enriched probiotics on absorption and transform of selenium and antioxidative ability of pigletsExperiment design is the same as the experiment1. Whole feeding experiment lasted for42days. Blood samples were collected for analysis of whole blood selenium (Se) concentrations, erythrocyte glutathione peroxidase activity (RBC GPx), plasma GPx3activity and plasma Malondialdehyde (MDA) concentrations on d0,14,28and42. The Se-Pro group and the Na2SeO3group had significantly higher Se concentrations in whole blood than did the control group and the Pro group (P<0.05) d14,28and42, respectively, and the Se concentrations in whole blood of SP group were significantly higher than SS group; Se concentrations in liver, kidney, muscle and spleen in the SP group and the Na2SeO3group were significantly higher than the control group and the Pro group (P <0.05), and the Se concentrations in the SP group were significantly higher than SS group(P<0.05); The Se-Pro group and the Na2SeO3group had significantly higher RBC GPx activities than did the control group and the Pro group (P<0.05) on d14,28and42, respectively, while there were no differences between Pro group and control group (P>0.05). The plasma GPx3activities were not different between the treatment groups and the control group in the whole experiment period (P>0.05). The plasma MDA concentrations in Se-Pro group and Na2SeO3group were significantly lower than that in control group and Pro group (P<0.05) on d14,28and42, respectively. Furthermore, the plasma MDA concentrations in SP group were significantly lower than that in Na2SeO3group (P<0.05) on d14,28and42, respectively. The results suggest that SP is more effective than Na2SeO3for increasing Se concentrations in whole blood and tissue, antioxidative ability and decreasing plasma MDA concentrations in piglets.Experiment4. Effect of supplementary selenium-enriched probiotics on GPxl, GPx4and TR1mRNA levels in PBMC of pigletsExperiment design is the same as the experiment1. Whole feeding experiment lasted for42days. Blood samples were collected for analysis of GPxl, GPx4and TR1mRNA levels on d0,14,28and42. The Se-Pro group and the Na2SeO3group had significantly higher GPxl and TR1mRNA levels than did the control group and the Pro group in the whole experiment period (P<0.05), while there were no differences between Pro group and control group (P>0.05). But the GPx4mRNA levels were not different between the Se-treatment groups and the control group in the whole experiment period (P>0.05). The results suggest that regulation of GPx1, TR1and GPx4mRNA level by Se are different, Se is more effective in improved GPx1mRNA levels than TR1and GPx4mRNA levels, and the GPx4mRNA levels were not affected by Se status.Experiment5. Regulation of different mitogens induced T-cell activation by selenium in primary cultured pig splenocytesIn this study, the effects of Se on T-cell proliferation and IL-2production were studied in primary porcine splenocytes. Splenocytes were treated with different mitogens in the presence of0.5-4μmol/L sodium selenite. Se significantly promoted T-cell receptor (TCR) or concanavalin A (ConA)-induced T-cell proliferation and IL-2production but failed to regulate T-cell response to phytohemagglutinin (PHA). In addition, Se significantly increased the levels of cytosolic glutathione peroxidase (GPx1) and thioredoxin reductase1(TR1) mRNA, the activity of GPx1and the concentration of reduced glutathione (GSH) in the unstimulated, or activated splenocytes.These results indicated that Se improved the redox status in all splenocytes, including unstimulated, TCR, ConA and PHA-stimulated, but only TCR and ConA-induced T-cell activation was affected by the redox status. N-acetylcysteine (NAC), a pharmacological antioxidant, increased T-cell proliferation and IL-2production by TCR and ConA stimulated splenocytes but had no effect on the response to PHA in primary porcine splenocytes confirming that PHA-induced T-cell activation is insensitive to the redox status. We conclude that Se promotes GPx1and TR1expression and increases antioxidative capacity in porcine splenocytes, which enhances TCR or ConA-induced T-cell activation but not PHA-induced T-cell activation. The different susceptibilities to Se between the TCR, ConA and PHA-induced T-cell activation may help to explain the controversy in the literature over whether or not Se boosts immune responses.Experiment6. Effects of different forms and concentrations of selenium on ConA induced T-cell proliferation in primary cultured pig splenocytesPrimary cultured pig splenocytes were incubated with0(control) and vary concentrations of Se as DL-selenomethionine (Se-Met), or sodium selenite (Na2SeO3) for48h in the presence of ConA. Compared to controls, significantly higher T-cell proliferation was observed at0.5-32μmol/L Se-Met, Significant increases of T-cell proliferation were obtained in Na2SeO3treated splenocytes at0.5-4μmol/L, with maximal effects at2μmol/L, and inhibited T-cell proliferation in a dose-dependent manner at8-32μmol/L. Intracellular reduced glutathione (GSH) contents and GPx activity in all Se-treated splenocytes were significantly higher than controls. An increases of GPx1mRNA level were obtained in all Se-treated splenocytes, but GPx4mRNA level was not affected by Se-treatment. And the MS, the inhibitor of the GPx1, significantly inhibited the proliferation improved by Se. We conclude that (i) regulation of ConA induced T-cell proliferation by different Se forms are different in primary cultured pig splenocytes, and GPxl maybe the key enzyme.