煤焦沥青烟提取物对BEAS-2B细胞Nrf2-Keap1/ARE通路的影响

Effect of Coal Tar Pitch Extract on Nrf2-Keap1/ARE Pathway in BEAS-2B Cells

[目的]研究煤焦沥青烟提取物对BEAS-2B细胞Nrf2、Keap1、NQO1 mRNA和Nrf2蛋白表达的影响,以期探讨对抗煤焦沥青致细胞氧化损伤的分子靶标和通路。[方法]GC-MS检测煤焦沥青烟提取物的主要成分;采用细胞急性毒性实验,根据文献以及以往的实验将煤焦沥青烟提取物分为0.00、1.25、2.50、5.00、10.00 mg/L五个染毒组,以BEAS-2B细胞为染毒对象进行实验;MTT法检测染毒BEAS-2B细胞生长增殖情况;RT-PCR检测Nrf2、Keap1、NQO1 mRNA的表达量;Western blot测定Nrf2蛋白的表达量。[结果]煤焦沥青烟提取物主要成分中86.02%为多环芳烃类化合物;浓度为1.25 mg/L时促进BEAS-2B细胞增殖,2.5～10 mg/L抑制细胞增殖;染毒组Nrf2 mRNA及蛋白表达量低于对照组,而Keap1 mRNA表达量高于对照组,差别均有统计学意义(P<0.05);在1.25～5.00 mg/L浓度范围内,随着染毒浓度的增加,Nrf2 mRNA和蛋白表达呈递增趋势,而染毒浓度为10.00 mg/L时,Nrf2 mRNA和蛋白的表达量均有所减少(P<0.05);NQO1 mRNA在2.5～5.00 mg/L浓度范围内随染毒浓度的增加表达水平逐渐升高。[结论]煤焦沥青烟提取物刺激BEAS-2B细胞后,可能通过Nrf2-Keap1/ARE通路来调节细胞抗氧化应激能力。

[Objective] To study the effect of coal tar pitch extract on the expressions of Nrf2,Keap1,NQO1 mRNA and Nrf2 protein in BEAS-2B cells,and to explore the target genes and pathway in defense against cell oxidative damage caused by coal tar pitch.[Methods] GC-MS was applied to analyze the ingredients of coal tar pitch（CTP）extract.According to literature and previous experiments,BEAS-2B cells were exposed separately to different concentration of CTP extract,namely 0.00,1.25,2.50,5.00,and 10.00 mg/L in the acute cell toxicity test.The proliferation of BEAS-2B exposed to the extract of CTP was analyzed by MTT.The reverse transcription polymerase chain reaction（RT-PCR）was applied to measure the expressions of Nrf2,Keap1 and NQO1 mRNA,and Western blot was used to determine the level of Nrf2 protein.[Results] The main ingredients of coal tar pitch extract were polycyclic aromatic hydrocarbon compounds,accounted for 86.02%,which promoted cell proliferation in the 1.25 mg/L concentration and restrained cell proliferation in the concentrations 2.5-10 mg/L.The expressions of Nrf2 mRNA and protein in all test groups were lower than those in the control group while the expressions of Keap1 mRNA was higher than control with significant statistical difference（P 0.05）.The expressions of Nrf2 increased with increasing concentration of CTP extract,however,it decreased in the 10.00 mg/L group.The expression level of NQO1 mRNA increased gradually with increasing concentration of CTP extract among the concentrations 2.5-10 mg/L.[Conclusion] The ability against antioxidant stress might be enhanced through the Nrf2-Keap1/ARE pathway when CTP extract stimulated the BEAS-2B cells.