Microwave radiation affects the phosphorylation of nuclear factor erythroid-2-related factor-2 and activity of protein kinase C in vascular endothelial cells

Min ZHONG; Yang CHEN; Guangbin ZHANG; Zhenping YU

ABSTRACT

BACKGROUND:

Nuclear factor erythroid-2-related factor-2 (NF-E2-related factor-2) is an important transcription factor to regulate anti-oxidative stress reaction. Some researches indicate that NF-E2-related factor-2 can be phosphorylated by numerous members of protein kinase C family. In order to investigate generant mechanism of microwave radiation on oxidative stress injury, whether microwave radiation can influence on anti-oxidative regulating system through NF-E2-related factor-2 or not should be further studied.OBJ ECTIVE To analyze the effect of microwave radiation on phosphorylation of NF-E2-related factor-2 and activity of protein kinase C in vascular endothelial cells.

DESIGN:

Observational-contrast study.

SETTING:

Department of Labor Hygiene, the Third Military Medical University of Chinese PLA.MATERIALS Vascular endothelial cell strain; H332PO4; Protein-A Sepharose (Sigma Company); mono-antibody of NF-E2-related factor-2 (H-300, Santa Cruz); α-mono-antibody of protein kinase C (Santa Cruz); glass microfiber filters(Whatman Company); gel scanning system (Gel Doc 2000, Bio-Rad); liquid scintillation spectrometer (LKB-117, Sweden).

METHODS:

The experiment was carried out in Laboratory of Electromagnetic radiation and Biological Effect, Department of Labor Hygiene, the Third Military Medical University of Chinese PLA from March to July 2003. ① Analysis of phosphorylation of NF-E2-related factor-2 Vascular endothelial cells were cultured with DMEM medium till the period of productive growth and incubated with 32Pi for 2 hours. And then, cultured bottle was maintained in water bath at 37℃ and performed with microwave radiation in dark chamber, whose reflectivity was about zero. It was regarded as radiation group, and the average power density of radiation was 30 mW/cm2; in addition, the duration of radiation was 30 minutes.Cells did not deal with microwave radiation were regarded as control group. Phosphorylation level of NF-E2-related factor-2 was measured at 2, 4, 8 and 24 hours after radiation with immune coprecipitation-autoradiography technique and dealt with semi-quantitative analysis with gel scanning system. Cells in the control group were analyzed directly. ②Active analysis and expressional measurement of protein kinase C Cells in the radiation group and the control group were dealt with the same cultured method, condition, radiation styles, dosage and environment as mentioned above. At 2, 4, 8 and 24 hours after radiation, cells were split to extract plasma and membrane protein. Furthermore, activity of protein kinase C was measured with r-32P-ATP labeled liquid scintillation spectrometer; gray value of protein strap was dealt with semi-quantitative analysis with gel scanning system; staining degree of plasma was observed after immunocytochemical staining of protein kinase C. In addition, cells in the control group were measured and observed directly.MAIN OUTCOME

MEASURES:

① Phosphorylation level of NF-E2-related factor-2 in radiation group and control group; ②Results of active analysis and expressional measurement of protein kinase C in radiation group and control group.

RESULTS:

① Phosphorylation level of NF-E2-related factor-2 in radiation group and control group Gray value of NF-E2-related factor-2 was higher in radiation group than that in control group at 2, 4 and 8 hours after radiation.Phosphorylation level of NF-E2-related factor-2 reached the peak at four hours after radiation. In addition, results of semi-quantitative scanning analysis showed that, at 2, 4 and 8 hours after radiation, phosphorylation level of NF-E2-related factor-2 was increased 33%, 261% and 141% in radiation group as compared with that in control group,respectively (t = 2.974, 4.209, 4.047, P ＜ 0.05), and then, fallen down to normal value 24 hours later. ② Results of active analysis and expressional measurement of protein kinase C in radiation group and control group At 2, 4 and 8 hours after microwave radiation, expression of protein kinase C in radiation group was higher than that in control group,especially at the 4 hour. In addition, at 24 hours after radiation, expression of protein kinase C recovered the normal value. Results of immunocytochemical staining showed that staining of plasma was deeper in radiation group than that in control group at 4 hours after radiation. Moreover, results of r-32P-ATP labeled liquid scintillation spectrometer also suggested that, at 2, 4 and 8 hours after radiation, activity of protein kinase C was increased 36%, 93% and 47% in radiation group as compared with that in control group, respectively (t =2.801, 3.654, 3.035, P ＜ 0.05). And then, activity of protein kinase C was decreased after 24 hours, otherwise, activity of protein kinase C reached the peak at 4 hours after radiation.

CONCLUSION:

Microwave radiation can strengthen the phosphorylation level of NF-E2-related factor-2 in vascular endothelial cells during a special period; meanwhile, it can also cause the increase of expression of protein kinase C.Time effect of activity of protein kinase C is coincidence with phosphorylation level of NF-E2-related factor-2.