Effects and mechanism of sodium hydrosulfide on rat epidermal cells intervened with serum from burned rat

Abstract

Objective: To investigate the effects and mechanism of sodium hydrosulfide on rat epidermal cells intervened with serum from burned rat (hereinafter referred to as burn serum). Methods: The experimental research method was used. Ten male Sprague-Dawley (SD) rats aged eight months were taken to prepare normal rat serum (hereinafter referred to as normal serum), 30 male SD rats aged eight months were taken to prepare burn serum after full-thickness burn, and epidermal cells (the third passage)isolated from 10 SD rats born one day were used for the experiments. The cells were divided into normal serum group treated with normal serum and burn serum group treated with burn serum. Cell counting kit 8 method was used to detect cell survival rate after 1, 2, 4, 6, and 8 h of culture, respectively, to screen the subsequent intervention time of burn serum. The cells were divided into burn serum control group treated only with burn serum and 50, 100, 150, 200, 250 μmol/L sodium hydrosulfide groups treated with burn serum+ sodium hydrosulfide at corresponding final molarity. After 30 min of culture following the burn serum intervention, the cell survival rate was detected as above to screen the subsequent intervention concentration of sodium hydrosulfide. The cells were divided into burn serum control group treated with burn serum only and sodium hydrosulfide only group, glibenclamide only group, and sodium hydrosulfide+ glibenclamide group treated with burn serum+ corresponding reagents. After 5, 10, 15 min of culture following the burn serum intervention, the cell survival rate was detected as above to screen the subsequent intervention time of glibenclamide. The cells were divided into burn serum control group treated with burn serum and sodium hydrosulfide only group, glibenclamide only group, and sodium hydrosulfide+ glibenclamide group treated with burn serum+ corresponding reagents. After completing corresponding culture time of each reagent, the mitochondria were extracted to detect cytochrome c oxidase (CCO) activity using a spectrophotometer, and the protein expression level of adenosine triphosphate (ATP)-sensitive potassium channel was detected by Western blotting. Except for the number of samples for ATP-sensitive potassium channel protein detection, which was 3, the number of samples for the other indicators was 10. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference (LSD)-t test, LSD test, and Bonferroni correction. Results: Compared with that of normal serum group, the cell survival rate was significantly decreased in burn serum group after only 4 and 6 h of culture (t=4.02, 6.42, P<0.05). An overall comparison showed statistically significant differences in cell survival rate among the time points within normal serum group and burn serum group (F=19.74, 4.48, P<0.05 or P<0.01). Four hours of culture was selected as the subsequent intervention time of burn serum. After 30 min of culture following the burn serum intervention, compared with that of burn serum control group, only 150, 200, 250 μmol/L sodium hydrosulfide groups had a significantly higher cell survival rate (P<0.01), thus 150 μmol/L was selected as the subsequent intervention concentration of sodium hydrosulfide. Compared with that of burn serum control group, the cell survival rate decreased significantly in glibenclamide only group after 5 and 15 min of culture following burn serum intervention (P<0.05) and increased significantly in glibenclamide only group after 10 min of culture following the burn serum intervention and sodium hydrosulfide only group at each time point (P<0.05 or P<0.01). The cell survival rate in sodium hydrosulfide+ glibenclamide group was significantly lower than that of sodium hydrosulfide only group at each time point (P<0.05). The difference in cell survival rate was statistically significant among the time points within glibenclamide only group (F=11.81, P<0.01). Five minutes of culture was selected as the subsequent intervention time of glibenclamide. After 35 min of culture following the burn serum intervention, compared with (1.62±0.08) nmol·min(-1)·mg(-1) and 0.682±0.063 in burn serum control group, the CCO activity of cells and the protein expression level of ATP-sensitive potassium channel were significantly increased in sodium hydrosulfide only group ((1.99±0.09) nmol·min(-1)·mg(-1) and 0.932±0.014, P<0.01) and significantly decreased in glibenclamide only group ((1.44±0.09) nmol·min(-1)·mg(-1) and 0.600±0.012, P<0.01); the CCO activity of cells and the protein expression level of ATP-sensitive potassium channel in sodium hydrosulfide+ glibenclamide group ((1.79±0.06) nmol·min(-1)·mg(-1) and 0.744±0.071) was significantly lower than those of sodium hydrosulfide only group (P<0.05 or P<0.01). Conclusions: Sodium hydrosulfide can improve the survival rate of rat epidermal cells after burn serum intervention, by a mechanism which is related to the alleviation of epidermal cell mitochondrial damage and mediated by ATP-sensitive potassium channel.