虎七散诱导食管癌EC9706细胞线粒体凋亡和铁死亡的研究  

Study on Mitochondrial Apoptosis and Ferroptosis Induced by Huqi Powder(虎七散)in EC9706 Esophageal Cancer Cells

作　　者：张超男 李洪霖 刘素彤 代圣民 卢娟娟 马纯政[2] ZHANG Chaonan;LI Honglin;LIU Sutong;DAI Shengmin;LU Juanjuan;MA Chunzheng(The Second Clinical Medical College of Henan University of Chinese Medicine,Zhengzhou 450002;Henan Province Hospital of Tradi-tional Chinese Medicine,Zhengzhou 450002;First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000)

机构地区：[1]河南中医药大学第二临床医学院,郑州450002 [2]河南省中医院,郑州450002 [3]河南中医药大学第一附属医院,郑州450000

出　　处：《中药药理与临床》2024年第8期19-28,共10页Pharmacology and Clinics of Chinese Materia Medica

基　　金：河南省中医药科学研究专项课题(编号:2022ZY1089);河南省科学技术厅科技攻关课题(编号:212102311118);河南省中医管理局,河南省中医药科学研究专项课题(编号:2022ZY2018)。

摘　　要：目的:本研究旨在探讨虎七散能否通过调控氧化还原和线粒体稳态诱导线粒体凋亡和铁死亡,并进一步分析其对P53蛋白表达的影响。方法:线粒体凋亡试验分为空白对照组、虎七散2、4、8 mg/mL组;铁死亡试验分为空白对照组、铁抑素-110μmol/L组、联合给药组(铁抑素-110μmol/L+虎七散8 mg/mL)、虎七散8 mg/mL组。采用CCK-8法检测细胞存活率;采用试剂盒检测细胞内GSH、MDA含量,SOD活力;ROS荧光探针(DCFH-DA)、脂质过氧化荧光探针(BODIPYTM581/591C11)、线粒体膜电位探针JC-1及铁离子Phen green SK(PGSK)探针,分别测定ROS、脂质ROS、线粒体膜电位及铁离子的荧光强度;Transwell法检测细胞侵袭和迁移能力;TUNEL一步凋亡法检测细胞凋亡;Western blot法检测细胞线粒体凋亡蛋白(BCL-2、BAX、CytC、Active Caspase-3)、铁死亡蛋白(xCT、ACLS4、GPX4)及P53蛋白的表达。结果:通过线粒体凋亡试验,与空白对照组相比,虎七散8 mg/mL组明显抑制食管癌EC9706细胞增殖,细胞内GSH含量和SOD活力降低,ROS水平增加和线粒体膜电位单体的荧光强度增强,细胞的凋亡率升高,细胞的侵袭和迁移降低,BAX、CytC、Active Caspase-3和P53表达上调,BCL-2表达下调(P<0.05或P<0.01)。铁死亡实验结果显示,与空白对照组相比,虎七散8 mg/mL组的细胞内GSH含量减少,MDA含量增加,ROS水平增加以及线粒体膜电位单体荧光强度增强,铁离子荧光强度降低,ACSL4和P53表达上调,xCT和GPX4表达下调(P<0.05);与虎七散8 mg/mL组相比,铁抑素-110μmol/L+虎七散8 mg/mL组细胞内GSH含量增加,MDA含量降低,ROS水平及线粒体膜电位单体荧光强度均降低,铁离子荧光强度增强,ACSL4和P53蛋白表达下调,xCT和GPX4蛋白表达上调(P<0.05)。结论:虎七散有促氧化作用,能通过促进ROS和脂质ROS过度累积,调控氧化还原和线粒体稳态,诱导线粒体凋亡和铁死亡,上调P53表达;其靶向氧化还原、线粒体稳态及P53,协同线Objective:The purpose of this study was to investigate whether Huqi Powder(虎七散)could induce mitochondrial apoptosis and ferroptosis by regulating redox and mitochondrial homeostasis,and to further analyze its effect on p53 protein expression.Methods:In the mitochondrial apoptosis experiment,the esophageal cancer EC9706 cells were divided into control group,Huqi Powder 2 mg/mL,4 mg/mL and 8 mg/mL groups.In ferroptosis assay,the cells were divided into control group,ferrostatin-110μmol/L group,combination group(ferrostatin-110μmol/L+Huqi Powder 8 mg/mL),and Huqi Powder 8 mg/mL group.Cell viability was measured by CCK-8 assay.The levels of intracellular GSH and MDA,and SOD activity were measured by the assay kits.The fluorescence probes such as DCFH-DA,C11-BODIPY 581/591,JC-1,and Phen Green SK(PGSK)were used to measure the fluorescence intensity of reactive oxygen species(ROS),lipid peroxidation of ROS,mitochondrial membrane potential,and iron ions,respectively.Cell invasion and migration were measured by the Transwell invasion and migration assay.One step TUNEL apoptosis assay kit was used to detect apoptosis.Expression of mitochondrial apoptosis-related proteins(Bcl-2,Bax,CytC,active caspase-3),ferroptosis-related proteins(xCT,ACLS4,GPX4)and p53 protein were measured by Western Blot.Results:In mitochondrial apoptosis assays,compared with control group,8 mg/mL Huqi Powder effectively inhibited EC9706 cell proliferation,decreased intracellular GSH content and SOD activity,increased the fluorescence intensities of ROS and mitochondrial membrane potential,increased cell apoptosis,and inhibited cell invasion and migration,up regulated the expressions of Bax,CytC,active caspase-3 and p53,and down regulated the Bcl-2 expression(P<0.05 or P<0.01).Ferroptosis assays showed that compared with control group,8 mg/mL Huqi Powder reduced GSH level and increased MDA level,increased the fluorescence intensities of ROS,lipid peroxidation of ROS and mitochondrial membrane potential,decreased iron ion fluorescence,up regulat

关 键 词：虎七散 氧化还原 线粒体 线粒体凋亡 铁死亡 P53蛋白 

分 类 号：R285[医药卫生—中药学]