Effects And Underlying Mechanism Of Vitamin A On Synthesis Of Selenoprotein And Antioxidative Function Of Mammary Glands In Dairy Cows

The present study was divided into five parts to examine the effects and underlying mechanism of vitamin A (VA) on selenoprotein synthesis and antioxidative function of mammary glands of dairy cows. The experiment1was conducted using a single factor randomized arrangement, and twenty-eight Holstein cows with similar body weight, parity, milk production were divided into two groups with14replicates in each group. The only independent factor in this experiment was the concentration of supplemental VA provided in the cows’diet (control:110IU/kg BW or high dose VA supplementation:220IU/kg BW) in order to explore the effect of high dose of VA on the milk performance, antioxidant and immune function of dairy cows. The next four experiments were done in vitro. The experiment2was also conducted as a single factor randomized arrangement to examine the effects of different concentrations of VA (0,0.05,0.1,0.2,1and2μg/mL) on cell viability and synthesis of selenoprotein, as well as the antioxidative function in bovine mammary epithelial cells (BMEC), to select the appropriate dose of VA ensuring better antioxidative function and to explore the mechanism for VA to enhance antioxidant function basing on selenoprotein synthesis, gene transcription and protein expression. The experiment3examined the effect of hydrogen peroxide (H2O2) on cell viability and antioxidant paramaters to establish the oxidative damage model of BMEC. On the basis of the experiment3, the experiment4was divided into control, H2O2treatment, VA treatment and VA prevention group, to further discuss the protective effect of VA on the BMEC oxidative damage induced by H2O2. The experiment5was divided into control, DNCB treatment resulting in suppressed thioredoxin reductase (TrxR) activity, VA group, VA+DNCB group, and to further explore the mechanism by which VA improved antioxidant function via the TrxR-MAPK-ARA pathways.The results of this study showed as follows:(1) Compared with110IU/kg BW VA, supplementation of the diet with220IU of VA/kg of BW exceeding the NRC recommended dose did not affect milk performance but enhanced antioxidantive function, and significantly increased the activties of glutathione peroxidase (GPx), TrxR, superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) as well as selenoprotein P (SelP) content, reduced the concentrations of malondialdehyde (MDA) and reactive oxygen species (ROS) in the serum of dairy cows.(2) Supplementation with220IU of VA/kg of BW significantly enhanced the immune function of dairy cows, and increased the concentratons of immunoglobulin M, immunoglobulin G, immunoglobulin A, interleukin-1, tumor necrosis factor, VA, soluble CD4content and soluble CD4/soluble CD8in the serum, but reduced somatic cells in the milk and soluble CD8content in the serum.(3) Addition of VA could promote the proliferation of BMEC, increase the activities of CAT, SOD, T-AOC, GPx, TrxR and content of SelP in a dose-dependent manner. The optimal VA dose was1μg/mL. However, positive effect of VA tended to be suppressed when VA was increased to2μg/mL. The expressions of mRNA and protein of GPx in BMEC were up-regulated by VA in a dose-dependent manner, and the addition of1μg/mL VA had the best effect. The mRNA expression of TrxRl and SelP as well as the protein expression of TrxRl were greater at1-2μg/mL VA. Taking all these factors into consideration,1μg/mL VA gave the best effect.(4) The BMEC survival significantly reduced to79.79%when the cells were induced by600μmol/L H2O2for6h. Cell oxidative injury induced by600μmol/L H2O2for6h also significantly reduced the activities of GPx, SOD and CAT, significantly increased MDA content in BMEC.When H2O2concentration was increased to600μmol/L and its reaction time lasted6h, a significant oxidative stress was produced in BMEC, which was used to establish a standard model of oxidative stress.(5) Cell oxidative injury induced by H2O2significantly decreased the cell survival, GPx and TrxR activities, SelP content, the activities of CAT, SOD and T-AOC, gene expressions of GPx1, TrxRland SelP as well as protein expressions of GPxl and TrxRl, and increased the contents of MDA and ROS. The addition of VA prevented the corresponding changes of those paramaters and protected the BMEC from oxidative stress.(6) Cell oxidative injury induced by H2O2significantly increased the arachidonic acid (ARA) concentration, the gene expression and activity of cytosolic phospholipase A2(cPLA2) and5-lipoxygenase (5-LOX), as well as the contents of15-hydroxy twenty-four arachidonic acid (15-HETE) and hydroxy peroxide tetracosenic arachidonic acid (15-HPETE). VA pretreatment prevented the corresponding increases of those paramaters and increased the activity of TrxR. Moreover, adding VA prevented the oxidative stress which can lead to increase of phosphorylation levels of p38mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK), and effectively regulated ARA content. These results implied that the protection of VA on oxidative damage induced by H2O2may be associated with lower ARA concentration resulting from the increasing TrxR activity.(7) DNCB group significantly inhibited cell viability and the activity, gene and protein expressions of TrxR, and increased the cPLA2activity and gene expression as well as ARA content. VA pretreatment prevented the corresponding changes of TrxR activity and ARA content.(8) DNCB group significantly increased the cell signaling kinase activity, and the phosphorylation levels of JNK and p38MAPK, ARA content as well as5-LOX activity, LTB4and15-HPETE contents related to LOX metabolisms. Addition of VA significantly suppressed the changes of these paramaters, and increased the activity of TrxR.(9) These results revealed that the antioxidation mechanism of VA via the TrxR-MAPK-ARA pathway, that is to say, VA protected the BMEC from oxidative stress associated with elevating the TrxR activity that inhibited the MAPK signaling pathway, which futher leaded to the decreased concentration of ARA.Taking all these results into consideration, VA might promote antioxidant function of mammary glands of dairy cows, and its mechanism was that VA could regulate the synthesis of selenoprotein including Gpx, TrxR and SelP, and that VA protected the BMEC from oxidative stress by regulation of TrxR activity which caused a decrease in the concentration of ARA resulting from the inhibition of MAPK signaling pathway.