健脾解毒方通过NF-κB/Nrf2/MRP2信号通路逆转大肠癌多药耐药的作用机制

Effects of Jianpi Jiedu Formula on NF-κB/Nrf2/MRP2 signaling pathway in multidrug resistance of colon cancer

目的:探讨健脾解毒方通过NF-κB/Nrf2/MRP2信号通路逆转大肠癌多药耐药的作用机制。方法:CCK-8法检测健脾解毒方逆转人肠癌HCT116/OXA细胞对奥沙利铂的耐药作用;细胞免疫荧光法检测健脾解毒方对NF-κB和Nrf2核转位情况;Western blot法检测健脾解毒方对NF-κB/Nrf2/MRP2信号通路的调控作用;利用RNAi干扰技术沉默Nrf2基因以及构建NF-κB慢病毒载体质粒,观察NF-κB对Nrf2/MRP2信号的调控作用。结果:与奥沙利铂组比较,健脾解毒方(12.5、25、50μg/mL)联合奥沙利铂(50μmol/L)后,细胞存活率从80.45%下降至43.15%(P<0.05,P<0.01);健脾解毒方抑制NF-κB及Nrf2的核转位及MRP2的表达;NF-κB过表达促进Nrf2核转位及MRP2的表达,增强细胞对奥沙利铂的耐药性(P<0.05,P<0.01);Nrf2基因沉默抑制MRP2表达,但不影响NF-κB的核转位。结论:健脾解毒方通过抑制NF-κB/Nrf2/MRP2信号通路发挥逆转大肠癌细胞多药耐药的作用。

Objective:To investigate the molecular mechanism of Jianpi Jiedu Formula(JPJD)on reversing multidrug resistance of colorectal cancer through inhibiting Nrf2/MRP2 signaling pathway via NF-κB inhibition.Methods:The reversal effect of JPJD at non-toxic concentrations on human colorectal cancer oxaliplatin-resistant cell HCT116/OXA was determined by CCK-8 assay.The effects of JPJD on nuclear translocation of NF-κB and Nrf2 were determined by immunocytochemical analysis.Regulation of JPJD on NF-κB/Nrf2/MRP2 signaling pathway was measured by Western blot.Nrf2/MRP2 signaling pathway mediated by NF-κB were observed by established NF-κB recombinant lentivirus and Nrf2-siRNA.Results:Compared with OXA(50μmol/L)alone,the cell survival rate decreased from 80.45%to 43.15%after applying JPJD(12.5,25,50μg/mL)on HCT116/OXA cell lines(P<0.05,P<0.01).JPJD inhibits the nuclear translocation of NF-κB and Nrf2 as well as inhibiting the expression of downstream target protein MRP2.NF-κB overexpression could promote the expression of Nrf2 nuclear translocation and its downstream target protein MRP2,and further enhance the resistance of human colorectal cancer HCT116/OXA cells to oxaliplatin(P<0.05,P<0.01).Nrf2 gene silencing inhibits MRP2 expression,but not affect nuclear translocation and expression of NF-κB.Conclusion:JPJD could reverse the multidrug resistance of colorectal cancer cells through NF-κB mediated Nrf2/MRP2 signaling parhway.