摘要
目的:探讨黄芩苷(Baicalin)对弥漫大B细胞淋巴瘤细胞株DB增殖、焦亡的影响及其作用机制。方法:用不同浓度(0、5、10、20、40μmol/L)的黄芩苷处理DB细胞,通过CCK-8法检测细胞增殖活性并计算半数抑制浓度(IC50)值;倒置显微镜下观察焦亡形态,LDH含量释放检测验证细胞膜完整性,实时荧光定量PCR和Western blot检测细胞焦亡相关mRNA及蛋白(NLRP3、GSDMD、GSDME、N-GSDMD、N-GSDME)表达。为进一步阐明黄芩苷诱导DB细胞焦亡与ROS生成之间的关联,将细胞分为对照组、黄芩苷组、NAC组及NAC联合黄芩苷组,NAC组用ROS抑制剂N-乙酰半胱氨酸(NAC)2 mmol/L预处理DB细胞2 h;联用组经预处理后加入黄芩苷,培养48 h,用DCFH-DA法检测细胞中活性氧(ROS)含量的变化。结果:黄芩苷剂量依赖性抑制DB细胞的增殖活性(r=-0.99),作用48 h的IC50为20.56μmol/L,倒置显微镜观察到药物组DB细胞焦亡形态学改变。与对照组相比,黄芩苷组细胞释放LDH明显升高(P<0.01),表明细胞膜完整性丧失;实时荧光定量PCR以及Western blot检测结果表明,黄芩苷剂量依赖的增加焦亡通路中NLRP3、N-GSDMD和N-GSDME基因和蛋白的表达水平(P<0.05)。ROS检测显示,与对照组相比,黄芩苷组细胞内ROS水平显著升高(P<0.05),NAC组ROS含量显著降低(P<0.05);与NAC组比较,NAC联合黄芩苷组ROS含量增加,提示黄芩苷显著减弱了NAC对ROS生成的抑制作用(P<0.05)。同样地,Western blot检测结果显示,与对照组比较,黄芩苷组焦亡相关蛋白表达量增加(P<0.05)。NAC组抑制NLRP3表达并减少了N-GSDMD和N-GSDME的切割(P<0.05);与NAC组比较,NAC联合黄芩苷组焦亡相关蛋白表达升高。表明,黄芩苷能有效诱导DB细胞焦亡,并逆转NAC对ROS生成的抑制作用。结论:黄芩苷能抑制DLBCL细胞株DB增殖,其机制可能通过调控ROS生成来影响焦亡通路实现的。
Objective:To investigate the effect of Baicalin on the proliferation and pyroptosis of diffuse large B-cell lymphoma cell line DB and its mechanism.Methods:DB cells were treated with baicalin at different concentrations(0,5,10,20,40 μmol/L).Cell proliferation was detected by CCK-8assay and half maximal inhibitory concentration(IC50)was calculated.The morphology of pyroptosis was observed under an inverted microscope,the integrity of the cell membrane was verified by LDH content release assay,and the expressions of pyroptosis-related mRNA and protein(NLRP3,GSDMD,GSDME,N-GSDMD,N-GSDME)were detected by real-time fluorescence quantitative PCR and Western blot.In order to further clarify the relationship between baicalin-induced pyroptosis and ROS production in DB cells,DB cells were divided into control group,baicalin group,NAC group and NAC combined with baicalin group.DB cells in the NAC group were pretreated with ROS inhibitor N-acetylcysteine(NAC) 2mmol/L for2h.Baicalin was added to the combined treatment group after pretreatment,and the content of reactive oxygen species(ROS)in the cells was detected by DCFH-DA method after48hours of culture.Results:Baicalin inhibited the proliferation of DB cells in a dose-dependent manner(r=-0.99),and the IC50was20.56 μmol/L at48h.The morphological changes of pyroptosis in DB cells were observed under inverted microscope.Compared with the control group,the release of LDH in the baicalin group was significantly increased(P<0.01),indicating the loss of cell membrane integrity.Baicalin dose-dependently increased the expression levels of NLRP3,N-GSDMD,and N-GSDME mRNA and protein in the pyroptosis pathway(P<0.05).Compared with the control group,the level of ROS in the baicalin group was significantly increased(P<0.05),and the content of ROS in the NAC group was significantly decreased(P<0.05).Compared with the NAC group,the content of ROS in the NAC+baicalin group was increased.Baicalin significantly attenuated the inhibitory effect of NAC on ROS production(P<0.05).Similarly,Wes
作者
卢铭
何春玲
吴振天
吕垚
段晓晖
王冰璇
王世雄
王健红
梁蓉
LU Ming;HE Chun-Ling;WU Zhen-Tian;LYU Yao;DUAN Xiao-Hui;WANG Bing-Xuan;WANG Shi-Xiong;WANG Jian-Hong;LIANG Rong(The Second Clinical Medical College of Shaanxi University of Chinese Medicine,Xi’an 712046,Shanxi Province,China;Department of Hematology,The First Affiliated Hospital of Air Force Military Medical University,Xi’an 710032,Shaanxi Province,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2023年第6期1706-1713,共8页
Journal of Experimental Hematology
基金
西京医院学科助推计划项目(XJZT21CZ07)
陕西省自然科学基金(2023-JC-YB-651)。
