基于Nrf2/ARE信号通路探讨海南五指山裸花紫珠对鼻咽癌细胞顺铂诱导凋亡敏感性的作用及其机制

Effect of Wuzhishan Callicarpa nudiflora in Hainan Province on Cisplatin-induced Apoptosis of Nasopharyngeal Carcinoma Cells and Mechanism:Based on Nrf2/ARE Pathway

目的:探讨海南五指山裸花紫珠(LHZZ)对鼻咽癌(NPC)细胞顺铂(DDP)诱导凋亡敏感性的影响,并探究其作用机制。方法:细胞增殖与活性检测(CCK-8)法检测不同浓度DDP(0,2,4,8,16,32 mg·L^(-1))和不同浓度海南五指山裸花紫珠(0,25,50,75,100 mg·L^(-1))处理后的细胞存活率。实验分组为空白组(正常HNE1细胞),DDP组(8 mg·L^(-1),24 h),LHZZ组(50 mg·L^(-1),24 h),DDP+LHZZ组(8 mg·L^(-1)DDP+50 mg·L^(-1)LHZZ,24 h),DDP+LHZZ+核因子E2相关因子2(Nrf2)激活剂莱菔硫烷(SFN)组(8 mg·L^(-1)DDP+50 mg·L^(-1)LHZZ,24 h;然后10μmol·L^(-1)SFN,24 h),CCK-8法检测细胞存活率,集落克隆形成实验检测细胞集落形成能力,流式细胞术和原位末端标记法(TUNEL)染色检测细胞凋亡情况,荧光探针2',7'-二氯荧光黄双乙酸盐(DCFH-DA)检测培养细胞活性氧(ROS)水平,蛋白免疫印迹法(Western blot)检测细胞内凋亡相关蛋白B细胞淋巴瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax)和半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3)表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞内Nrf2,抗氧化反应元件(ARE)mRNA表达。结果:与空白组比较,经过不同浓度DDP和不同浓度LHZZ处理后,细胞存活率均明显下降(P<0.05);与DDP组和LHZZ组比较,DDP+LHZZ组细胞存活率下降,细胞集落克隆形成数目减少,细胞凋亡水平升高,Bcl-2蛋白表达水平下调,Bax和Caspase-3蛋白表达水平则上调,差异具有统计学意义(P<0.05);而在DDP+LHZZ组中加入SFN激活Nrf2/ARE信号通路,上述效果均受到抑制(P<0.05)。此外,与空白组比较,LHZZ组细胞内ROS水平下调(P<0.05);与DDP组比较,DDP+LHZZ组ROS水平明显下调(P<0.05);与DDP+LHZZ组比较,DDP+LHZZ+SFN组细胞ROS水平明显上调(P<0.05)。结论:LHZZ可增加DDP诱导的鼻咽癌细胞凋亡的敏感性,其机制可能与阻断Nrf2/ARE信号通路并抑制ROS水平相关。

Objective:To explore the effect of Wuzhishan Callicarpa nudiflora(LHZZ)on the sensitivity of nasopharyngeal carcinoma(NPC)cells to cisplatin(DDP)and the mechanism.Method:Cell counting kit-8(CCK-8)assay was used to detect the survival rate of NPC HNE1 cells after treatment with different concentration of DDP(0,2,4,8,16,32 mg·L^(-1))and different concentration of LHZZ(0,25,50,75,100 mg·L^(-1)).The following groups were designed:control group(normal HNE1 cells),DDP group(8 mg·L^(-1)DDP,24 h),LHZZ group(50 mg·L^(-1)LHZZ,24 h),DDP+LHZZ group(8 mg·L^(-1)DDP+50 mg·L^(-1)LHZZ,24 h),DDP+LHZZ+nuclear factor erythroid 2-related factor 2(Nrf2)activator sulforaphane(SFN)group(8 mg·L^(-1)DDP+50 mg·L^(-1)LHZZ,24 h,10μmol·L^(-1)SFN,24 h).Then CCK-8 assay was employed to examine cell survival rate,colony formation test the colony-forming ability,flow cytometry and in situ terminal end-labeling(TUNEL)staining cell apoptosis,fluorescent probe 2',7'-dichlorofluorescin diacetate(DCFH-DA)the level of reactive oxygen species(ROS)in cultured cells,Western blot the expression of apoptosis-related proteins in cells,such as B-cell lymphoma/leukemia-2(Bcl-2),Bcl-2 associated X protein(Bax),and cysteinyl aspartate specific proteinase-3(Caspase-3),and Real-time quantitative polymerase chain reaction(Real-time PCR)the expression of Nrf2 and antioxidant response element(ARE)mRNA in cells.Result:The survival rates of cells treated with different concentration of DDP and different concentration of LHZZ decreased compared with that in the control group(P<0.05).Compared with the DDP group and the LHZZ group,DDP+LHZZ group demonstrated decrease in cell survival rate,number of cell colonies,and Bcl-2 level,and increase in the apoptosis level and the expression of Bax and Caspase-3(P<0.05).However,after the addition of SFN,the Nrf2/ARE signaling pathway was activated and the above variation was inhibited(P<0.05).In addition,the level of intracellular ROS in the LHZZ group was lower than that in the control group(P<0.05)and the level in the DDP+LHZZ group was lower than that in the DDP group(P<0.05).Moreover,the ROS level in the DDP+LHZZ+SFN group was higher than that in the DDP+LHZZ group(P<0.05).Conclusion:LHZZ can enhance the sensitivity of DDP-induced NPC apoptosis,possibly by blocking the Nrf2/ARE signaling pathway and inhibiting the level of ROS.