苯并[a]芘的细胞毒性及诱导IL-1β和IL-8表达作用研究

Cytotoxicity and expression-induction of IL-1β and IL-8 by benzo [a]pyrene in NCI-H292 cells

目的探讨不同浓度苯并[a]芘(BaP)对人肺癌(淋巴结转移)NCI-H292的细胞毒性及其对NCI-H292细胞炎性因子分泌的影响。方法取对数期生长的NCI-H292细胞,分别以不同剂量BaP(0、4、8、16和32μmol/L)染毒24、48和72 h后,采用实时无标记细胞分析系统(RTCA)法检测细胞存活率,ELISA法检测细胞内白介素-1β(IL-1β)和白介素-8(IL-8)水平。本实验按照5×3析因设计。结果与对照组相比,各剂量BaP染毒组在24、48和72 h时间点细胞存活率均显著降低(P<0.001),细胞存活率与染毒剂量之间呈高度负相关关系(r;=-0.986、-0.974和-0.993,P<0.01);与同剂量组24 h时比较,各实验组在染毒48 h和72 h细胞存活率均显著升高(P<0.001),细胞存活率与染毒时间之间呈高度正相关关系(r;=0.958、0.996、0.994、0.999和1.000,P<0.01)。NCI-H292细胞内IL-1β水平在染毒剂量和染毒时间上有交互效应(P<0.001),在48 h和72 h时BaP高剂量(8~32μmol/L)染毒组IL-1β水平均呈随着染毒剂量升高而降低(P<0.01),72 h时低剂量BaP染毒组IL-1β水平则呈随着染毒剂量升高而升高的剂量-效应关系(P<0.01);同时,各实验组细胞IL-1β水平在24、48和72 h均呈随着染毒时间增加而上升的时间-效应关系(P<0.01)。NCI-H292细胞内IL-8水平在染毒剂量和染毒时间上有交互效应(P<0.01):在染毒72 h,BaP染毒剂量为0~16μmol/L时IL-8水平呈随着染毒剂量升高而升高的剂量-效应关系(P<0.01);4、8μmol/L染毒组细胞IL-8水平在24、48和72 h呈随着染毒时间增加而上升的时间-效应关系(P<0.01)。结论BaP染毒可引起NCI-H292细胞的存活率下降,且随着染毒剂量增加毒性作用增强;BaP可刺激NCI-H292细胞分泌IL-1β和IL-8,其可能在BaP致肺肿瘤炎症过程中发挥重要作用。

Objective To study the toxicity of different concentrations of benzo[a]pyrene(BaP) on the cytotoxicity and their effect on secretion of inflammatory cytokines in NCI-H292 cells(human lung cancer cells with lymph node metastasis). Methods NCI-H292 cells in logarithmic growth phase were exposed to different concentrations of BaP(0,4,8,16 and 32 μmol/L) for 24,48 and 72 h, respectively, then the cell survival rate was determined by real time cell analysis(RTCA),the expression levels of interleukin-1β(IL-1β) and interleukin-8(IL-8) in cell supernatant were detected by ELISA.The experiment was designed according to 5×3 factor analysis.Results The results showed that compared with control group of the same exposure time point, the cell survival rate in all exposed groups at all time points were obviously decreased(P<0.001),and Pearson correlation coefficients between survival rate and BaP exposure dose were-0.986,-0.974 and-0.993,respectively(all P<0.001);compared with same dose group at 24 h time point, the cell survival rate in BaP exposed groups at 48 h and 72 h time point were obviously increased(P<0.001),and the Pearson correlation coefficient were 0.958,0.996,0.994,0.999 and 1.000,respectively(P<0.001),that means there was a highly positive correlation between cell survival rate and exposure time.There seemed some interaction effect between exposure doses and exposure time on IL-1β level in NCI-H292 cells(P<0.001): in higher BaP exposure groups(8~32 μmol/L) at 48 h and 72 h time point, the IL-1β decreased with increase of BaP exposure doses(both P<0.01),while in lower BaP exposure groups(0~8 μmol/L)at 72 h point, the IL-1β was increased with increase of BaP exposure doses(P<0.01). Additionally, there was also some interaction effect between exposure doses and exposure time on IL-8 level in NCL-H292 cells(P<0.01): the IL-1β levels in BaP dose groups of 0~16 μmol/L,presented a rise trend with the exposure dose of BaP(P<0.01) at the time point of 72 h, and the levels of IL-8 in 4 and 8 μmol/L groups were also presented a rise trend and with time-effect relationship at the time points of 24,48 and 72 h after exposure.Conclusion The results suggested that BaP might induce the decrease of the survival rate of NCI-H292 cells and the toxicity showed increased with the increase of exposure dose;meanwhile, BaP could also induce the secretion of inflammatory cytokines such as IL-1β and IL-8 in NCI-H292 cells, which suggested that these cytokines may play an importanrt role in the inflammation process mediated lung carcinogenesis induced by BaP.