Studies On Oxidative Stress And P38MAPK Mediated Apoptosis Of NRK-52e Cell Induced By Melamine

Objective To study the oxidative damage of renal tubular epithelial cells(NRK-52e cell line) induced by melamine and the cytoprotective effect of Trolox, a water-soluble antioxidant, to NRK-52e cells and the role of p38MAPK pathway on apoptosis induced by melamine.Methods Melamine was directly dissolved in DMEM medium and was diluted to 40mM,30mM,24mM,16mM,8mM, OmM. Half inhibition concentration (IC50) of melamine was test by MTT assay. According to the IC50, three doses were selected as experimental groups, as follows 24mM,16mM,8mM. At the same time, each group was set up to correspond to the protection group of Trolox, which the final concentration was 1 mM. In the experiment, trolox and melamine in protection groups were mixed to add to the medium. After 8 hours, the cell culture medium was collected for the Lactate dehydrogenase (LDH) detection. And the cells were cleaved by cell lysis solution and collected. For the cell lysate, total SOD activity was determinated by NBT staining method (NBT assay), GSH-Px activity was detected by UV colorimetric assay and MDA content in samples was test through thiobarbituric acid (TBA assay). In addition, the Lactate dehydrogenase activity in cell culture medium and the level of reactive oxygen species(ROS) in cell were measured by 2,4-dinitrophenylhydrazine assay and the fluorescent probe DCFH-DA. The expressions of p38 and p-p38 in intercellular were detected by cytoimmunochemistry. In additional, the quality expressions of p38 and p-p38 were detected using the western blot. The apoptosis rate of NRK-52e cells induced by melamine and treated with trolox or SB203580 were detected by fluid cytometry.Results The IC50 of melamine on NRK-52e cells was 28.22mM and the IC50 was 33.05mM after treated with trolox. In the detection of oxidative damage indicators in toxin groups, the activity of antioxidant enzymes of SOD and GSH-Px decreased with the concentration increasing of melamine. Comparing with the control group, the activity of SOD and GSH-Px in 8mM group decreased significantly (P<0.05), and the other two groups decreased very significantly(P<0.01). Meanwhile, MDA content in each group increased. MDA content of 8mM group was significantly higher (P<0.05), and the other groups of 16mM and 24mM was very significantly difference (P<0.01), compared with control group. In protection groups of Trolox, SOD activity of 16mM group was significantly difference than its toxin group (P<0.05). GSH-Px activity of 24mM group was significantly higher than its corresponding to toxin group (P<0.01), while its MDA content was significantly lower than its toxin group (P<0.05). In addition, LDH activity of 8mM group in culture medium was incresed with the rised concentration of melamine under the protection of trolox. And the reactive oxygen species (ROS) levels in 24mM and 16mM groups were significantly lower than the control group (P<0.01). And the apoptosis rate of NRK-52e cells exposured to melamine increased, corresponding to the concentration of melamine, and showed significant difference (P<0.05). And the apoptosis rate of protected-trolox groups reduced significantly (P<0.01). The p38 protein expressed in every group in immunocytochemistry detection. In treatment group of melamine, p-p38 expression showed that the apoptotic sign could induce the phosphate of p38 protein and performance the bio-function. Compared with melamine group, the expression of p-p38 reduced significantly after treating with SB203580 (P<0.05). Also, the apoptosis rate of NRK-52e cells reduced, compared to the melamine group. It meant that SB203580 could inhibit the phosphate of p38 protein to reduce the apoptosis rate.Conclusion The results showed that melamine decrease the activity of antioxidant enzymes to accelerate cell lipid peroxidation, destroy the cell membrane integrity. Meanwhile, the melamine can also promote the cells to produce excessive reactive oxygen species, which may also cause cell damage and induce cell apoptosis. In addition, the damage melamine-induced decrease by adding membrane protective angent trolox. It means that trolox can enhance the activity of antioxidant enzymes to eliminate oxygen free radicals to protect cells from cell apoptosis. In additional, this existed a relationship between the melamine-induced apoptosis of NRK-52e cells and the p38MAPK pathway. And the inhibition of p38MAPK pathway could stop the apoptosis induced by melamine.