苯并(a)芘和邻苯二甲酸二丁酯联合染毒对L02细胞STAT3及p-STAT3水平的影响

Effects of BaP and DBP in combination on expression level of STAT3 and p-STAT3 in L02 cells

目的探讨苯并(a)芘[benzo(a)pyrene,BaP]和邻苯二甲酸二丁酯(dibutyl phthalate,DBP)体外染毒对人正常肝细胞(L02)细胞信号传导与转录激活因子3(signal transducers and activators of transcription 3,STAT3)的表达及其活化(p-STAT3)的影响。方法将体外培养的L02细胞分为对照组(0.1%DMSO)、BaP低、中、高剂量单独染毒组(12.5、25.0、50.0μmol/L BaP)、DBP低、中、高剂量单独染毒组(25.0、50.0、100.0μmol/L DBP)和BaP、DBP低、中、高剂量联合染毒组(12.5μmol/L BaP+25.0μmol/L DBP、25.0μmol/L BaP+50.0μmol/L DBP、50.0μmol/L BaP+100.0μmol/L DBP)。染毒72 h后,用倒置显微镜观察细胞形态,应用实时荧光定量聚合酶链反应(reverse transcription quantitative real-time polymerase chain reaction,qRT-PCR)法检测各染毒组STAT3 mRNA的表达水平,免疫印迹(Western blot)方法观察STAT3和p-STAT3蛋白的表达水平。结果细胞染毒72 h后,12.5μmol/L BaP、25.0μmol/L DBP和12.5μmol/L BaP+25.0μmol/L DBP低剂量染毒组的细胞形态改变不明显,仅少量细胞的细胞壁模糊;25.0μmol/L BaP、50.0μmol/L DBP和25.0μmol/L BaP+50.0μmol/L DBP中剂量染毒组细胞开始出现模糊,部分细胞由不规则形变为椭圆形,细胞间隙变大;50.0μmol/L BaP、100.0μmol/L DBP和50.0μmol/L BaP+100.0μmol/L DBP高剂量染毒组贴壁细胞数量减少,漂浮细胞增多,胞体缩小变圆,细胞间隙明显变大,细胞核增大;BaP、DBP联合染毒组较其对应的单独染毒组细胞形态学改变明显。与对照组相比,除12.5μmol/L BaP、25.0μmol/L BaP、25.0μmol/L DBP和12.5μmol/L BaP+25.0μmol/L DBP组外,其余各染毒组的STAT3 mRNA表达水平皆明显降低,差异有统计学意义(P<0.05)。BaP、DBP联合染毒组中,中剂量组(25.0μmol/L BaP+50.0μmol/L DBP)的STAT3 mRNA表达水平明显低于BaP中剂量单独染毒组(P<0.05),高剂量组(50.0μmol/L BaP+100.0μmol/L DBP)的STAT3 mRNA表达水平明显低于BaP、DBP高剂量单独染毒组(P<0.05)。与对照组相比,除12.5μmol/L BaP、25.0μmol/L BaP、25.0μmol/L DBP和12.5μmol/L BaP+25.0μmol/L DBP组外,其余各染毒组STAT3、p-STAT3蛋白的表达水平皆明显降低,差异有统计学意义(P<0.05)。BaP、DBP联合染毒组中,除低剂量组(12.5μmol/L BaP+25.0μmol/L DBP)外,中(25.0μmol/L BaP+50.0μmol/L DBP)、高(50.0μmol/L BaP+100.0μmol/L DBP)剂量组的STAT3、p-STAT3蛋白的表达水平皆明显低于各BaP、DBP中、高剂量单独染毒组,差异有统计学意义(P<0.05)。结论BaP、DBP染毒可下调L02细胞的STAT3转录表达水平,抑制STAT3的活化(p-STAT3)程度,尤以BaP、DBP联合染毒时抑制作用更为明显。此可能是BaP、DBP致肝细胞凋亡损伤的重要环节。

Objective To understand the expression levels of signal transducers and activators of transcription 3(STAT3)and p-STAT3 in human normal hepatocytes(L02)exposed to benzo(a)pyrene(BaP)and dibutyl phthalate(DBP)in combination.Methods L02 cells cultured in vitro were divided into control group(0.1%DMSO),BaP low,moderate and high doses groups(12.5,25.0 and 50.0μmol/L BaP),DBP low,moderate and high doses groups(25.0,50.0 and 100.0μmol/L DBP),BaP and DBP low,moderate and high doses co-exposure groups(12.5μmol/L BaP+25.0μmol/L DBP,25.0μmol/L BaP+50.0μmol/L DBP and 50.0μmol/L BaP+100.0μmol/L DBP).After 72 hours of exposure,the morphology of cells was observed by microscope,the expression of STAT3 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR),and the protein expression levels of STAT3 and p-STAT3 were observed by Western blot.Results After 72 hours of exposure,in 12.5μmol/L BaP,25.0μmol/L DBP and 12.5μmol/L BaP+25.0μmol/L DBP groups,no obvious changes in the cells were observed,except a small number of cells with blurred cell walls.In 25.0μmol/L BaP,50.0μmol/L DBP and 25.0μmol/L BaP+50.0μmol/L DBP groups,the shape of cells became obscure and ovalized,and the intercellular spaces became larger.In 50.0μmol/L Ba P,100.0μmol/L DBP and 50.0μmol/L BaP+100.0μmol/L DBP groups,there were more floating cells and larger intercellular space,and the cells exhibited shrank and round and the nucleus became enlarged;Also,in the combined exposure group,more obvious morphological changes were found than those in the exposed groups of BaP or DBP alone.Except for 12.5μmol/L BaP,25.0μmol/L BaP,25.0μmol/L DBP and 12.5μmol/L BaP+25.0μmol/L DBP groups,the expression levels of STAT3 mRNA in the other exposed groups were significantly lower than that in the control group(P<0.05).In the groups of Ba P and DBP in combination,the expression levels of STAT3 mRNA in moderate dose group was significantly lower than that in 25.0μmol/L BaP group(P<0.05);The expression levels of STAT3 mRNA in high dose group were significantly lower than that in 50.0μmol/L BaP or 100.0μmol/L DBP groups(P<0.05).Except 12.5μmol/L BaP,25.0μmol/L BaP,25.0μmol/L DBP and 12.5μmol/L BaP+25.0μmol/L DBP groups,the expression levels of STAT3 and p-STAT3 protein in the other exposed groups were significantly lower than those in the control group(P<0.05).In the groups of BaP and DBP in conbination,except 12.5μmol/L BaP+25.0μmol/L DBP group,the expression levels of STAT3 and p-STAT3 protein in the25.0μmol/L BaP+50.0μmol/L DBP and 50.0μmol/L BaP+100.0μmol/L DBP groups were significantly lower than those in the each corresponding individual exposure group(P<0.05).Conclusions BaP and DBP can reduce the expression levels of STAT3 and inhibit the activation of STAT3(p-STAT3)in L02 cells,especially in exposure to BaP and DBP in combination,the inhibitory effect is more obvious.This may be an important link in the apoptosis of hepatocytes induced by BaP and DBP.