To observe the effect of medical ozone gas on human epidermal cells in vitro by simulating ozone gas bath treatment.

Normal skin tissue was taken, and the primary culture of epidermal cells were digested and separated. The third generation cells were used for experimental intervention in 50 mg/L ozone environment. (1) Observe the primary and inherited cells, and observe cell morphological and apoptosis changes by Hoechst 33258 staining; (2) Cell viability assay was divided into oxygen group and ozone group (n=5), the cell counting kit-8 (CCK-8) method and enzymatic marke were employed to detect the cell viability trend of cells directly interfered with cells in the absence of medium in short period of time (0, 5, 10, 15, 20, 25, 30 s); (3)Enzyme biochemical detection was divided into air group, oxygen group and ozone group (n=5), the activity level of superoxide dismutase (SOD), malondialdehyde content, LDH leakage rate detection, and the indicators of enzyme biochemical after intervention 10, 30, 60 min were detected respectively. The levels of intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured according to the intervention time of 10, 30, 60 min, and GSH/GSSG value was calculated. The overall comparison of cell viability was analyzed by repeated measures analysis of variance. The LSD-t test was used to compare the two groups and multiple time points in the same group of enzyme biochemical.

(1) The original cultured epidermal cells showed oval or multi-angle adherent walls, and showed typical of "paving stones" after stabilization. After fluorescence staining of Hoechst 33258, the normal nucleus showed diffuse and uniform pale blue fluorescence. And after ozone interference, with the prolonged of the duration, the dense bright blue particles appeared, epidermal cells gradually undergo nuclear condensation, chromatin condensation and apoptotic bodies, and partial epidermal cells appeared to fall off after 60 min of intervention. (2) Ozone directly interfered with the epidermal cells, and the cell activity showed a significant decline with the increase of time. Intervened by Ozone for 30 s, the cell absorbance value in the ozone group was (98.72±1.20)%, and intervened by oxygen for 30 s, the cell absorbance value in the oxygen group was(22.70±3.78)%, the difference was statistically significant (t=48.758, P<0.05). (3) Biochemical effects of ozone on cell enzymes: there were no significant differences in SOD, malondialdehyde, and LDH values between the oxygen group and the air group intervention for 60 min (with P values below 0.05). Intervened by ozone for 10 min, the values of SOD, malondialdehyde and LDH were (153.63±8.41) U/mg prot, (52.41±6.30) nmol/mg prot and (186.19±20.20) U/g prot, intervened by Ozone for 30 min, the values of SOD, malondialdehyde and LDH were (84.31±7.23) U/mg prot, (79.09±6.98) nmol/mg prot and (221.22±20.79) U/g prot, the differences was statistically significant(t=13.972, -6.343, -2.703; with P values below 0.05). Intervened by Ozone for 60 min, the values of SOD, malondialdehyde and LDH were (30.31±2.79) U/mg prot, (97.5±7.35) nmol/mg prot and (280.76±20.06) U/g prot, compared with the intervention for 30 min, the difference was statistically significant (t=15.569, -4.059, -4.608; with P value below 0.01). Comparison between ozone group and oxygen group, the difference of the SOD, malondialdehyde and LDH values were statistically significant (with P values below 0.05). There were no significant differences in GSH, GSSG, GSH/GSSG values between the air group and the oxygen group (with P values above 0.05). Between ozone group and oxygen group, there were no significant differences of the GSH value between the ozone group intervention for 10 min and the oxygen group intervention for 60 min (t=1.811, P>0.05). The differences of GSH, GSSG and GSH/GSSG values in the other times intervened by ozone were statistically significant (with P values below 0.05). Intervened by ozone for 10 min, the values of GSH, GSSG, GSH/GSSG were (11.67±1.37) μmol/L, (1.83±0.18) μmol/L, 6.48±1.28, intervened by ozone for 30 min, the values of GSH, GSSG, GSH/GSSG were (9.37±0.75) μmol/L, (1.59±0.2) μmol/L and 6.00±1.23. The value of GSH decreased obviously, the difference was statistically significant (t=3.295, P=0.011), and the difference of the values of GSSG and GSH/GSSG was no statistically significant (t=1.98, 0.605; with P values above 0.05). Intervened by ozone for 60 min, the values of GSH, GSSG, GSH/GSSG were (8.34±1.16) μmol/L, (2.02±0.24) μmol/L and 4.13±0.44, compared with the intervention for 30 min, the difference was statistically significant, the difference of the values of GSH and GSSG was no statistically significant (t=1.673, -3.08; with P values above 0.05), and the value of GSH/GSSG decreased significantly, the difference was statistically significant (t=3.216, P<0.05).

Human epidermal cells cultrued in vitro are sensitive to ozone gas, and clinical application of ozone gas bath therapy to reat skin wounds should be considered to control treatment time.