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目的研究煤焦沥青烟提取物对BEAS-2B细胞Nrf2、Keap1、NQO1的基因及其蛋白表达的影响,以探讨煤焦沥青烟提取物在造成细胞氧化损伤过程中Nrf2-Keap1/ARE通路的作用机制。方法设立未处理对照组、0.1%DMSO溶剂对照组、5μg/ml B(a)P阳性对照组、6μmol/L全反式维甲酸组、6μmol/L全反式维甲酸预处理0.5 h后5μg/ml CTP染毒组和CTP染毒组(1、5、10和20μg/ml),染毒3、6、12和24 h后提取总RNA;RT-PCR检测Nrf2、Keap1、NQO1 mRNA的相对表达量;Western blot测定Nrf2、Keap1、NQO1蛋白的相对表达量。结果不同CTP染毒剂量作用后各个基因mRNA相对表达量差异无统计学意义(P>0.05),而不同时间点相对表达量差异有统计学意义(P<0.05)。Western blot结果显示,随着CTP烟提取物染毒浓度的增加,Keap1蛋白无明显变化,NQO1蛋白表达量逐渐上升;Nrf2蛋白在5μg/ml染毒浓度时表达最高,在5μg/ml CTP染毒浓度作用下,BEAS-2B细胞Nrf2蛋白表达量随时间逐渐升高,在染毒6 h后达到最高,之后表达量又呈下降趋势。结论煤焦沥青烟提取物作用于BEAS-2B细胞后,代谢酶NQO1表达上调,推测Keap1-Nrf2/ARE通路可能通过上调细胞保护性基因NQO1的表达而对抗煤焦沥青毒性作用,降低细胞氧化损伤。
Objective To investigate the effects of coal tar pitch smoke extract on gene and protein expression of Nrf2, Keap1 and NQO1 in BEAS-2B cells in order to investigate the role of coal tar pitch smoke extract in Nrf2-Keap1 / ARE pathway during oxidative injury mechanism. Methods The untreated control group, 0.1% DMSO solvent control group, 5μg / ml B (a) P positive control group, 6μmol / L all-trans retinoic acid group, 6μmol / L all- The total RNA was extracted after 3, 6, 12 and 24 h exposure to CTP and CTP (1, 5, 10 and 20 μg / ml), respectively. The relative mRNA levels of Nrf2, Keap1 and NQO1 The expression of Nrf2, Keap1, NQO1 protein was detected by Western blot. Results There was no significant difference in the relative expression levels of mRNA between different CTP doses (P> 0.05), while the relative expression levels at different time points had statistical significance (P <0.05). Western blot results showed that with the increase of CTP smoke extract concentration, Keap1 protein did not change significantly, NQO1 protein expression gradually increased; Nrf2 protein highest expression at 5μg / ml exposure concentration, 5μg / ml CTP exposure The concentration of Nrf2 protein in BEAS-2B cells gradually increased with time and reached the highest level at 6 h after exposure. The expression level of Nrf2 decreased afterwards. CONCLUSIONS: The expression of NQO1, a metabolic enzyme, was up-regulated after extract of coal tar pitch in BEAS-2B cells. It is speculated that Keap1-Nrf2 / ARE pathway may be able to counteract the toxic effects of coal tar pitch by up-regulating the expression of NQO1, .