Protective effects of ozone oxidative preconditioning on NRK-52E against ischemia and reperfusion injury in vitro

Objective To investigate the effects of ozone oxidative preconditioning (OzoneOP) on NRK-52E cells against ischemia and reperfusion (I/R) injury.Methods After all the cells were synchronized for 24 h,they were divided into three groups:the normal group,I/R group and OzoneOP group.In normal group,control buffer (1 ml) was added,and 3 h later,2 ml complete medium was given for culture of 24 h.In I/R group,the ischemic buffer (1 ml) was added and the cells were cultured under ischemic condition for 3 h,then 2 ml complete medium was given for culture of 24 h.In OzoneOP group,before the simulated ischemia,2 ml ozonated medium was given and cells were incubated for 2 h,and the rest were the same as I/R group.Flow cytometry was performed to detect cell apoptosis rate.The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were tested.The expression of B cell lymphoma/leukemia-2 (bcl-2) and bcl-2 associated X protein (bax) was detected by Western blotting.Results The cell apoptosis rate in normal group,I/R group and OzoneOP group was (4.58 ± 0.35) ％,(32.16 ± 3.27)％ and (18.73 ± 1.52)％,respectively.The SOD activities in normal group,L/R group and OzoneOP group were (14.31 ± 1.31),(7.03 ±3.94) and (10.45 ±2.35) U/mg protein,respectively.The MDA contents in normal group,I/R group and OzoneOP group were (0.48 ± 0.12),(2.13 ± 1.44) and (1.36 ±0.59) nmol/mg protein,respectively.The ratio of bcl-2/bax expression was downregulated in I/R group as compared with normal group.And OzoneOP could increase this ratio.Conclusion OzoneOP has protective effects on NRK-52E against I/R injury probably by regulating the ratio of the bcl-2/bax expression. Key words: Ozone oxidative preconditioning;  Ischemia and reperfusion injury;  Renal tubular epithelial cell