| Despite the widespread use of the oxygen-ozone in pain clinic,there is currently no consensus on its mechanisms of action and nearly no report for its action on nerve cells.Accordingly,the present studies were designed to assess the effects of oxygen-ozone on astrocytes of rats.Astrocytes were cultured in vitro through methods of trypsinization,different-speed cultivation,passaging to purify in accordance with McCarthy and Dvellis',and identified by glial fibrillary acidic protein(GFAP)immunohistochemistry staining. Then cells were seeded into 24 well plates and divided into 7 groups(n=7):blank group(C group),without intervention;oxygen group(O2 group),oxygen-ozone10 (O2-O310)group,O2-O320 group,O2-O340 group,O2-O360 group,O2-O380 group:added 400ul complete medium(CM)after effects of oxygen,or 10μg/mL,20μg/mL, 40μg/mL,60μg/mL,80μg/mL oxygen-ozone.After incubation for 2h or 4h,we observed cell morphous under inverted phase contrast microscope and detected endocellular superoxide dismutase(SOD)by XOD method,endocellular malondialdehyde(MDA)by TBA method,lactate dehydrogenase(LDH)leaking ratio by simple method of Qingtao Hong,dead cells' percentage by trypan blue staining.Our data revealed that(1)cells in O2-O360 group and O2-O380 group shew damaging:cell body turning hypertrophy and swelling,intracytoplasm having many degenerative grains and vacuoles,dead cells multiplying;(2)endocellular SOD increased(p＜0.05 or 0.01)except O2 group and O2-O310 group 4h,compared with C group;O2-O340 group increased(p＜0.05)compared with O2-O310 group and O2-O320 group;compared with O2 group,results were identifical with those of C group except O2-O310 group 2h;compared with 2h of ozone's action,SOD activity of ozone group except O2-O310 group all decreased(p＜0.05 or 0.01).(3)MDA in O2-O340 group 2h, O2-O360 group and O2-O380 group all increased(p＜0.05 or 0.01),while O2-O320 group 4h and O2-O340 group 4h decreased(p＜0.05)compared with C group;compared with O2 group,results were identifical with those of compared with C group;compared with 2h of ozone's action,MDA of O2-O320 group,O2-O360 group and O2-O380 group all decreased after ozone's action for 4h(p＜0.05 or 0.01).(4)LDH leaking ratio decreased(p＜0.05)in O2-O320 group 2h and O2-O340 group,and increased(p＜0.01)in O2-O360 group 4h and O2-O380 group compared with C group;compared with O2 group, results were identifical;compared with 2h of ozone's action,LDH leaking ratio increased in O2-O360 group and O2-O380 group after ozone's action for 4h(p＜0.05); (5)dead cells' percentage in O2-O360 group and O2-O380 group increased(p＜0.05 or 0.01)compared with C group;compared with O2 group,results were identifical; compared with 2h of ozone's action,O2-O360 group decreased(p＜0.05)and O2-O380 group increased after ozone's action for 4h(p＜0.01).We conclude that short time(2h,4h)after oxygen-ozone's intervention, oxygen-ozone showed a protective role at the concentration of 20μg/mL and 40μg/mL on astrocytes,validated by SOD increasing and LDH leaking ratio decreasing,while a damaging role at the concentration of 60μg/mL and 80μg/mL,validated by increase of MDA,LDH leaking ratio,dead cells' percentage and cells showing damaged.
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