硫氢化钠对经烧伤大鼠血清干预的大鼠表皮细胞的影响及其机制

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

目的探讨硫氢化钠对经烧伤大鼠血清(下称烧伤血清)干预的大鼠表皮细胞的影响及其机制。方法采用实验研究方法。取10只8个月龄雄性SD大鼠制备正常大鼠血清(下称正常血清),取30只8个月龄雄性SD大鼠制成Ⅲ度烧伤后制备烧伤血清,取分离自10只1 d龄SD大鼠的表皮细胞(第3代)进行实验。将细胞分为用正常血清处理的正常血清组和烧伤血清处理的烧伤血清组,分别于培养1、2、4、6、8 h采用细胞计数试剂盒8法检测细胞存活率,筛选后续烧伤血清干预时间。将细胞分为仅用烧伤血清处理的烧伤血清对照组和用烧伤血清+相应终物质的量浓度硫氢化钠处理的50、100、150、200、250μmol/L硫氢化钠组,烧伤血清干预后培养30 min,同前检测细胞存活率筛选后续硫氢化钠干预浓度。将细胞分为仅用烧伤血清处理的烧伤血清对照组和用烧伤血清+相应试剂处理的单纯硫氢化钠组、单纯格列本脲组、硫氢化钠+格列本脲组,烧伤血清干预后培养5、10、15 min,同前检测细胞存活率筛选后续格列本脲干预时间。将细胞分为烧伤血清处理的烧伤血清对照组和用烧伤血清+相应试剂处理的单纯硫氢化钠组、单纯格列本脲组、硫氢化钠+格列本脲组,按相应试剂处理时间培养结束,提取线粒体采用分光光度计检测细胞色素c氧化酶(CCO)活性,采用蛋白质印迹法检测ATP敏感性钾离子通道蛋白表达水平。除ATP敏感性钾离子通道蛋白检测样本数为3外,其余指标样本数均为10。对数据行析因设计方差分析、单因素方差分析、LSD-t检验、LSD检验、Bonferroni校正。结果与正常血清组比较,烧伤血清组仅培养4、6 h细胞存活率明显降低(t=4.02、6.42,P<0.05);正常血清组、烧伤血清组组内细胞存活率各时间点总体比较,差异有统计学意义(F=19.74、4.48,P<0.05或P<0.01)。选取培养4 h作为后续烧伤血清干预时间。烧伤血清干预后培养30 min,与烧伤血清对照组比较,仅150、200、250μmol/L硫氢化钠组细胞存活率明显升高(P<0.01)。选取150μmol/L作为后续硫氢化钠干预浓度。与烧伤血清对照组比较,单纯格列本脲组烧伤血清干预后培养5、15 min细胞存活率明显降低(P<0.05),单纯格列本脲组烧伤血清干预后培养10 min及单纯硫氢化钠组各时间点细胞存活率明显升高(P<0.05或P<0.01);硫氢化钠+格列本脲组各时间点细胞存活率均明显低于单纯硫氢化钠组(P<0.05)。仅单纯格列本脲组组内细胞存活率各时间点总体比较,差异有统计学意义(F=11.81,P<0.01)。选取培养5 min作为后续格列本脲干预时间。烧伤血清干预后培养35 min,与烧伤血清对照组的(1.62±0.08)nmol·min^(-1)·mg^(-1)、0.682±0.063比较,单纯硫氢化钠组细胞CCO活性[(1.99±0.09)nmol·min^(-1)·mg^(-1)]和ATP敏感性钾离子通道蛋白表达水平(0.932±0.014)明显升高(P<0.01),单纯格列本脲组细胞CCO活性[(1.44±0.09)nmol·min^(-1)·mg^(-1)]和ATP敏感性钾离子通道蛋白表达水平(0.600±0.012)明显降低(P<0.01);硫氢化钠+格列本脲组细胞CCO活性[(1.79±0.06)nmol·min^(-1)·mg^(-1)]和ATP敏感性钾离子通道蛋白表达水平(0.744±0.071)明显低于单纯硫氢化钠组(P<0.05或P<0.01)。结论硫氢化钠能够提高经烧伤血清干预的大鼠表皮细胞的存活率,与减轻表皮细胞线粒体损伤有关,并且由ATP敏感性钾离子通道介导。

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 50,100,150,200,250μmol/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 150,200,250μmol/L sodium hydrosulfide groups had a significantly higher cell survival rate(P<0.01),thus 150μmol/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.