The invitro effect of lipopolysaccharide on proliferation, inflammatory factors and antioxidant enzyme activity in bovine mammary epithelial cells.

Abstract

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.