| This thesis is divided into five experiments to investigate the effect and underlying mechanism of selenium(Se) on antioxidant function of mammary gland in dairy cows. The experiment 1 was conducted as the single factor completely randomized design, and twenty-one healthy Chinese Holstein cows with the similar body weight, parity and milk yield were randomly divided into three groups with seven cows in each group. The cows in the three groups were fed with 0.3(recommended dose), 0.6 and 0.9mg/kg Se from Se yeast, respectively, and to explore the effect of Se supplementation higher than the recommended dose on milk performance, antioxidant and immune function in dairy cows. The method of cell cultivation in vitro was used in the next four experiments. The experiment 2 was conducted as the single factor completely randomized design to explore the effect of different levels of Se supplementation(0, 10, 20, 50 and 100nmol/L) on selenoproteins synthesis and antioxidant function of bovine mammary epithelial cells(BMEC), and to select the appropriate Se supplemental dose ensuring the better antioxidant function. Based on the results in experiment 2, H2O2 was selected as a stressor and BMEC were randomly divided into four groups(control group, Se treatment group, H2O2 damage group and Se prevention group) to investigate the underlying protective mechanism of Se from H2O2-induced oxidative damage in experiment 3. In experiment 4, 2,4-dinitrochlorobenzene(DNCB) was selected as the inhibitor of thioredoxin reductase(Trx R) activity to induce oxidative damage of BMEC. The oxidative damage model was established by detecting cell viability, Trx R activity and antioxidant indicators in BMEC. According to the concentration and action time of DNCB selected in that model, BMEC in the experiment 5 were randomly divided into four groups as control group, Se treatment group, DNCB inhibition group and Se prevention group. The mechanism of Se regulating antioxidant function of mammary gland by Trx R-mitogen activated protein kinase(MAPK) pathway-arachidonic acid(ARA) pathway was investigated further.The results of the present study showed as followed:(1) Compared with the commended dose of 0.3mg/kg diet, Se supplementation of 0.6-0.9mg/kg diet had no significant effect on milk performance. But Se supplementation of 0.6mg/kg diet significantly increased the activities of glutathion peroxidase(GPX), superoxide dismutase(SOD) and catalase(CAT), and total antioxidant capacity(T-AOC), as well as the content of selenoprotein P(Sel P) in serum. Se supplementation of 0.6mg/kg diet also significantly decreased 5-lipoxygenase(5-LOX) activity and reactive oxygen species(ROS) level in serum. Se supplementation of 0.9mg/kg diet had no significant effect on the antioxidant function.(2) Se supplementation of 0.6mg/kg diet significantly promoted the immune function of dairy cows, and the contents of interleukin(IL) 2, tumor necrosis factor(TNF-a), immunoglobulin(Ig) M and G in serum were markedly increased. However, the positive effect of Se tended to be suppressed when Se was increased to 0.9mg/kg diet compared with that of 0.6mg/kg diet,and the contents of IL-2, TNF-a and Ig M in serum were decreased markedly.(3) The results indicated that adding appropriate Se to 0.6mg/kg in diet of dairy cows above the commended dose could improve the antioxidant function and immune function. It is probably disadvantageous to the antioxidant function and immune function when the supplemented dose was increased to 0.9mg/kg diet.(4) Se supplementation increased cell proliferation, the activities of GPX and SOD, T-AOC and Sel P content, and decreased ROS and malondialdehyde(MDA) levels in a dose-dependent manner, and Se supplementation of 50-100nmol/L had the better effect. Se supplementation also increased Trx R activity in a dose-dependent manner, and Se supplementation of 20-50nmol/L had the better promoting effect. The dose-dependent response between m RNA and protein expression of GPX1 and Trx R1, as well as Sel P m RNA expression and Se supplementation were also observed in this experiment. The m RNA and protein expression of GPX1 were up-regulated with the addition of 50-100nmol/L Se, and the m RNA expression of Trx R1 and Sel P were up-regulated with the addition of 20-100nmol/L Se. Taking all these parameters into consideration, Se supplementation of 50nmol/L had the better promoting effect on the antioxidant function of BMEC.(5) Compared with control group, the activities of GPX, Trx R, CAT and SOD, T-AOC and Sel P content, the m RNA expression of GPX1, Trx R1 and Sel P, and the protein expression of GPX1 and Trx R1 in H2O2 damage group were decreased significantly. The MDA and ROS levels were increased markedly. However, Se pretreatment markedly suppressed the corresponding changes of those parameters induced by H2O2, and indicated that Se had significant protective effect on H2O2-induced cell damage.(6) H2O2-induced cell oxidative injury leaded to an significant increase in the phosphorylation levels of p38 MAPK and c-Jun N-terminal kinase(JNK), the m RNA expression and activities of cytosolic phospholipase A2(c PLA2), 5-LOX and cyclooxygenase-2(COX-2), and the contents of ARA and 15-liydroperoxyeicosatetraenoic acid(15-HPETE). However, the above parameters in Se prevention group were decreased markedly compared with those in H2O2 damage group. These results implied that the protection of Se from oxidative damage induced by H2O2 may be associated with the decrease of ARA concentration resulting from the increase of Trx R activity.(7) Cell oxidative injury induced by 300 μmol/L DNCB for 2h was observed, and the cell proliferation and Trx R activity were decreased to 73% and 56%, respectivly, and the activities of GPX, SOD and CAT were supressed, and the MDA level in cells was increased. When DNCB concentration was increased to 300 μmol/L and its reaction time lasted for 2h, a significant oxidative stress was produced in BMEC, which is used to establish a standard model of cell oxidative damage.(8) Compared with the control, DNCB treatment decreased significantly cell proliferation, Trx R activity and its m RNA and protein expression, and increased markedly c PLA2 activity and its m RNA expression, which resulted in an increase of ARA content. However, Se pretreament markedly attenuated those changes of the above parameters induced by DNCB, and resulted in an increase of Trx R activity and a decrease of ARA content.(9) DNCB treatment resulted in a significant increase of the apoptosis signaling kinase 1(ASK-1) activity, a marked enhancement in the phosphorylation levels of p38 MAPK and JNK, the m RNA expression and activities of c PLA2 and 5-LOX, as well as the contents of ARA and 15-HPETE. However, those parameters in Se prevention group had a reverse change compared with DNCB inhibition group.(10) These results revealed that the antioxidant mechanism of Se via the Trx R-MAPK-ARA pathway, that is to say, the protective role of Se to cell oxidative damage was related to the inhibition of MAPK signaling pathway and then the decrease of ARA content resulting from the increase of Trx R activity.Taking all these results into consideration, Se improved mammary antioxidant function of dairy cows, and Se protected cells from oxidative damage mainly by increasing the activity of Trx R, inhibiting the activation of MAPK signaling pathway, and then decreasing the contents of ARA and its related metabolic parameters. |
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