Observation on the effect of medical ozone gas bath on the culture of normal human epidermal cells in vitro

Abstract

Objective To observe the effect of medical ozone gas on human epidermal cells in vitro by simulating ozone gas bath treatment. Methods 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. Results (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). 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). Conclusion 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. Key words: Epidermis; Cells; Ozone; Glutathione; Enzyme biochemistry