Lipopolysaccharide (LPS) was selected as a stimulus to investigate its effect on cell viability and oxidative stress in bovine mammary epithelial cells (BMEC) by detecting the cell relative growth rate (RGR), antioxidant indicators and inflammatory factors. This information was used to provide the theoretical basis for the establishment of a LPS-induced oxidative damage model. The experiment was divided into two parts. The first part used a two-factor experimental design to determine the appropriate incubation time of LPS by detecting the RGR. The third-passage BMEC were divided into 24 groups with six replicates in each group. The first factor was LPS concentration, which was 0 (control), 0.1, 1.0 and 10.0 μg/mL; the second factor was LPS incubation time (2, 4, 6, 8, 12 and 24 h). The optimum LPS incubation time was 6 h according to the results of the first part of the experiment. The second part of the experiment was conducted using a single-factor experimental design, and the third-passage cells were divided into four groups with six replicates in each group. The cells were incubated with culture medium containing different concentrations of LPS (0 [control], 0.1, 1.0 and 10.0 μg/mL) for 6 h to select the appropriate concentration of LPS to measure the antioxidant indicators and inflammatory factors. The results showed the RGR was significantly reduced as the concentration of LPS and the incubation time increased; the interaction between concentration and incubation time was also significant. The cells treated with 0.1 μg/mL of LPS for 6 h had no significant difference in the activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) (P > 0.05) compared with the cells in the control group. On the contrary, catalase (CAT) activity and malondialdehyde (MDA) content were markedly lower and higher, respectively, in the 0.1 μg/mL LPS-treated group for 6 h compared with the control group (P < 0.05). The activities of GPx, CAT and SOD in the BMEC treated with 1.0 or 10.0 μg/mL of LPS were significantly lower compared with the cells treated with 0.1 μg/mL of LPS and cells in the control group after 6 h of incubation; however, the opposite trend was detected in MDA content. There was no significant (P > 0.05) difference between the 10.0 and 1.0 μg/mL LPS-treated groups. Compared with the control group, interleukin-1, interleukin-6 and nitric oxide concentrations and the activity of inducible nitric oxide synthase in the 0.1 μg/mL LPS-treated group significantly increased (P < 0.0001), but the levels of tumour necrosis factor did not significantly change (P > 0.05). All of observed indicators were higher in the 1.0 and 10.0 μg/mL LPS-treated groups (P < 0.0001) compared with the other groups, but there was no significant (P > 0.05) difference between the 1.0 and 10.0 μg/mL LPS-treated groups. The results indicated that a concentration of 1.0 μg/mL of LPS and an incubation time of 6 h were the optimum conditions necessary to induce oxidative stress in the BMEC and establish a model for oxidative damage.

It is known that physiological overproduction of nitric oxide (NO) contributes to oxidative stress and inflammation. Our published studies indicated that vitamin A (VA) reduces NO-induced oxidative stress in bovine mammary epithelial cells (BMECs) by increasing antioxidant enzyme activities. However, the precise mechanism is unclear. The present study was conducted to examine the protective effects of VA on NO-induced damage to BMECs in vitro using diethylenetriamine nitric oxide (DETA-NO) as the NO donor and to explore the intracellular signaling mechanisms of VA that involve nuclear factor erythroid 2-related factor (Nrf2) and nuclear factor kappa-B (NF-κB). Subconfluent BMECs were divided into 10 treatment groups with 6 replicates per treatment and were cultured with dimethyl sulfoxide (DMSO, vehicle negative control) or 0, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, or 4 μg/mL of VA for 24 h and then incubated in the absence or presence of DETA-NO (1,000 μmol/liter) and VA for an additional 6 h. The results showed that exposure to DETA alone decreased cell proliferation compared with the negative control. Pretreatment with VA promoted the proliferation of BMECs, increased the activities of antioxidative enzymes including selenoprotein glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) and their gene and protein expression but decreased NO and interleukin 1 (IL-1) contents in a quadratic manner (P < 0.05). In addition, the expression of mRNA and protein of factors that are related to NF-κB or Nrf2 signaling pathways in BMECs were regulated by VA in a quadratic dose-dependent manner; VA at a concentration of 1 μg/mL exhibited the strongest effect. Together, these results suggest that VA promotes antioxidant functions of BMECs by regulating the synthesis of selenoproteins including GPx and TrxR and by reducing concentrations of IL-1 and NO in vitro by modulating Nrf2 and NF-κB signaling pathways.

The present study was conducted to examine the effects of retinoic acid (RA) on the synthesis of selenoprotein and the antioxidative indices of bovine mammary epithelial cells (BMEC) in vitro and to explore the antioxidative mechanisms of RA in the BMEC. The subconfluenced BMEC were divided into six treatments with six replicates per treatment and cultured in a Dulbecco's Modified Eagle's Medium/F12 media (10% fetal bovine serum, 5 µg/ml ovine prolactin, 10 ng/ml epidermal growth factor, 1 g/ml hydrocortisone, 0.5% insulin-transferrin-selenium) containing different levels of RA (0 (control), 0.05, 0.1, 0.2, 1 or 2 mg/ml) for 24 h. Addition of RA promoted the proliferation of BMEC, increased the activities of catalase, superoxide dismutase, total antioxidant capacity, glutathione peroxidase (GPX), thioredoxin reductase (TRXR), and the content of selenoprotein P (SELP) in a dose-dependent manner (P < 0.05). The optimal RA dose was 1 μg/ml. However, positive effect of RA tended to be suppressed when RA was increased to 2 μg/ml. The expressions of mRNA and protein of GPX in BMEC were up-regulated by RA in a quadratic dose-response relationship (P < 0.01), and the addition of 1 μg/ml RA showed the best effect. The mRNA expressions of TRXR1 and SELP as well as the protein expression of TRXR1 were higher at 1-2 μg/ml RA. These results suggested that RA promoted antioxidant function of BMEC by regulating the synthesis of selenoprotein including GPX, TRXR, and SELP in vitro. Keywords: vitamin A; antioxidant function; dairy cowsList of abbreviations: BMEC = bovine mammary epithelial cells, NO = nitric oxide, VA = vitamin A, GPX = glutathione peroxidase, TRXR = thioredoxin reductase, ROS = reactive oxygen species, MDA = malondialdehyde, SELP = selenoprotein P, RA = retinoic acid, ID = iodothyronine deiodinases, BW = body weight, DMEM = Dulbecco's Modified Eagle's Medium, DMSO = dimethyl sulfoxide, MTT = methyl thiazolyl tetrazolium, DTNB = dithio-bis-nitrobenzoic acid, GAPDH = glyceraldehyde phosphate dehydrogenase, SOD = superoxide dismutase, CAT = catalase, T-AOC = total antioxidant capacity, iNOS = inducible nitric oxide synthase, MAPK = mitogen-activated protein kinase, RT = reverse transcriptase, RT-PCR = real-time polymerase chain reaction, PBS = phosphate buffered solution

ABSTRACT:The considerable increase in oxygen requirements due to the high metabolic rate of the bovine mammary epithelial cells (BMEC) during lactation results in an augmented production of reactive nitrogen species (RNS), such as nitric oxide (NO), which may expose cows to increased oxidative stress. Vitamin A (VA) has been shown in several studies to enhance the antioxidant defence system against oxidative stress, but whether the reason is related to a reduced NO production remains unclear. Diethylene triamine-nitric oxide polymer (NOp) is a type of NO-generating compound, which is safe, efficacious, and releases NO over a long period. The current study was conducted to investigate the effect of VA on the antioxidant function in BMEC and the underlying mechanism by discussing the protection of VA on NO-induced oxidative stress of BMEC. The experiment was conducted using a single-factor completely randomized arrangement. Primary BMEC were isolated from the mammary glands of Holstein dairy cows. The third generation cells were randomly divided into four equal groups with six replicates each. Each group received different combinations of VA and NOp treatment as follows: controls (without VA and NOp), NOp treatment alone, VA treatment alone, and VA and NOp treatment together. The lysates were collected to evaluate the activities of glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) and the contents of reactive oxygen species (ROS) and malondialdehyde (MDA), and the cell-free supernatants were collected to analyze selenoprotein P (SelP) content, inducible nitric oxide synthase (iNOS) activities and nitric oxide (NO), interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) contents. The results suggested that compared to the control, the cell proliferation, the activity of the antioxidants GPx and TrxR, the content of SelP and the antioxidant gene expressions of GPx1, GPx4, and TrxR1 were significantly decreased (P < 0.05), and the contents of ROS and MDA, the activity of iNOS, the contents of NO and IL-1, IL-6, TNF-α, and their mRNA expressions were increased dramatically in the NOp treatment alone group (P < 0.05), but the opposite changes were observed in the VA treatment alone group. Compared to the NOp treatment alone, the VA and NOp treatment together significantly improved cell proliferation, the activities of the antioxidants GPx and TrxR, and the gene expressions of GPx1 and TrxR1, and dramatically decreased the contents of ROS and MDA, the activity of iNOS, the contents of NO and IL-1, IL-6, TNF-α and their mRNA expression levels (P < 0.05). The present research suggests that VA can improve the antioxidant function of BMEC and protect the cells from experiencing the NOp-induced oxidative stress by regulating antioxidant gene expression. The probable mechanism is that VA can reduce the activity of iNOS and its mRNA expression by down-regulating of the expression of IL-1, IL-6, and TNF-α to reduce NO production. However, the exact mechanism warrants future exploration. Keyword...

Nitinol exhibits unique (thermo)mechanical properties that make it central to the design of many medical devices. However, nitinol nominally contains 50 atomic percent nickel, which if released in sufficient quantities, can lead to adverse health effects. While nickel release from nitinol devices is typically characterized using in vitro immersion tests, these evaluations require lengthy time periods. We have explored elevated temperature as a potential method to expedite this testing. Nickel release was characterized in nitinol materials with surface oxide thickness ranging from 12 to 1564 nm at four different temperatures from 310 to 360 K. We found that for three of the materials with relatively thin oxide layers, ≤ 87 nm nickel release exhibited Arrhenius behavior over the entire temperature range with activation energies of 80 to 85 kJ/mol. Conversely, the fourth ''black-oxide'' material, with a much thicker, complex oxide layer, was not well characterized by an Arrhenius relationship. Power law release profiles were observed in all four materials; however, the exponent from the thin oxide materials was approximately 1/4 compared with 3/4 for the black-oxide material. To illustrate the potential benefit of using elevated temperature to abbreviate nickel release testing, we demonstrated that a > 50 day 310 K release profile could be accurately recovered by testing for less than 1 week at 340 K. However, because the materials explored in this study were limited, additional testing and mechanistic insight are needed to establish a protective temperature scaling that can be applied to all nitinol medical device components.