ROS/PERK介导的内质网应激在白花蛇舌草总黄酮诱导CNE1鼻咽癌细胞周期阻滞和凋亡中的作用  

Effect of ROS/PERK Mediated Endoplasmic Reticulum Stress on Cycle Arrest and Apoptosis of CNE1 Nasopharyngeal Carcinoma Cells Induced by Total Flavonoids of Scleromitrion diffusum

作　　者：王慧敏[1] 蔡纪堂[1] 吴紫陆 柴峰[1] WANG Hui-min;CAI Ji-tang;WU Zi-lu;CHAI Feng(Henan Province Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China;Second Clinical Medical College,Henan University of Chinese medicine,Zhengzhou 450002,China)

机构地区：[1]河南省中医院,河南郑州450002 [2]河南中医药大学第二临床医学院,河南郑州450002

出　　处：《中药材》2024年第3期705-712,共8页Journal of Chinese Medicinal Materials

基　　金：2023年度河南省中医药科学研究专项课题(2023ZYZD08);2022年度河南省中医药科学研究专项普通课题(2022ZY1075)。

摘　　要：目的:探究白花蛇舌草总黄酮对鼻咽癌细胞CNE1周期和凋亡的影响及可能的分子作用机制。方法:①为研究白花蛇舌草总黄酮对CNE1细胞周期和凋亡的影响,在考察白花蛇舌草总黄酮对鼻咽癌细胞CNE1和鼻咽上皮细胞NP69细胞活力的影响后,将CNE1细胞分为对照组、白花蛇舌草总黄酮(25、50、100μg/mL)组;四甲基偶氮唑蓝法(MTT)检测细胞活力;平板克隆检测白花蛇舌草总黄酮对CNE1细胞克隆形成能力的影响;JC-1荧光探针检测线粒体膜电位;Hoechst 33258染色检测细胞凋亡形态学变化;流式细胞术检测细胞周期和细胞凋亡率;②为研究白花蛇舌草总黄酮抑制CNE1细胞增殖可能的分子机制,将CNE1细胞分为对照组、白花蛇舌草总黄酮(100μg/mL)组,采用Western Blot检测p-PERX、ATF4、CHOP蛋白表达,并利用转录组测序技术获取基因表达,对差异表达基因进行生物学分析;③为研究类PKR的内质网激酶(PERK)在白花蛇舌草总黄酮诱导CNE1细胞周期阻滞和凋亡中的作用,采用小干扰核糖核酸(siRNA)进行干预;④为研究活性氧(ROS)在调控PERK信号中的作用,在白花蛇舌草总黄酮处理过程中孵育N-乙酰半胱氨酸(NAC)。结果:与对照组比较,白花蛇舌草总黄酮组CNE1细胞的存活率显著降低(P<0.05),其作用呈剂量及时间依赖性。白花蛇舌草总黄酮处理引起的细胞周期阻滞于G_(0)/G_(1)期并诱导CNE1细胞的凋亡(P<0.05),其作用呈浓度依赖性。转录组测序结果表明,白花蛇舌草总黄酮处理后细胞中PERK信号通路变化显著。与对照组比较,白花蛇舌草总黄酮处理引起p-PERK、ATF4、CHOP蛋白表达显著升高(P<0.05)。与NC siRNA+白花蛇舌草总黄酮组比较,PERK siRNA组和PERK siRNA+白花蛇舌草总黄酮组细胞凋亡率、G_(0)/G_(1)期比例及p-PERK、ATF4、CHOP蛋白表达显著降低(P<0.05)。与白花蛇舌草总黄酮组比较,NAC+白花蛇舌草总黄酮组CNE1细胞ROS水平和p-PERK蛋白表�Objective:To investigate the effects of total flavonoids of Scleromitrion diffusum on CNE1 cell cycle and apoptosis of nasopharyngeal carcinoma and its possible molecular mechanism.Methods:For the study of the effect of Scleromitrion diffusum flavonoids on CNE1 cell cycle and apoptosis,the effect of total flavonoids of Scleromitrion diffusum on the viability of nasopharyngeal carcinoma cell CNE1 and nasopharyngeal epithelial cell NP69 were investigated,then CNE1 cells were divided into control group and Scleromitrion diffusum total flavonoids(25,50,100μg/mL)groups.The cell viability was detected by tetramethyl azazole blue(MTT)method.The effect of total flavonoids of Scleromitrion diffusum on the clonal formation of CNE1 cells was detected by plate cloning.JC-1 fluorescent probe was used to detect mitochondrial membrane potential.The apoptotic morphological changes were detected by Hoechst 33258 staining.The cell cycle and apoptosis rate were detected by flow cytometry.In order to investigate the possible molecular mechanism of inhibiting proliferation of CNE1 cells by total flavonoids of Scleromitrion diffusum,CNE1 cells were divided into control group and total flavonoids of Scleromitrion diffusum(100μg/mL)group.The protein expressions of p-PERK,ATF4,CHOP were detected by Western Blot.Transcriptome sequencing technology was used to obtain gene expression,and the differentially expressed genes were analyzed.In order to study the effect of PKR-like endoplasmic reticulum kinase(PERK)on CNE1 cell cycle arrest and apoptosis induced by total flavonoids of Scleromitrion diffusum,small interfering ribonucleic acid(siRNA)was used.For the study of the role of reactive oxygen species(ROS)in the regulation of PERK signaling,N-acetylcysteine(NAC)was cultured during the treatment of total flavonoids of Scleromitrion diffusum.Results:Compared with the control group,the survival rate of CNE1 cells in the Scleromitrion diffusum total flavonoids group was significantly decreased in a dose-dependent manner(P<0.05).Scleromitrion

关 键 词：白花蛇舌草总黄酮 鼻咽癌 内质网应激 细胞凋亡 细胞周期 

分 类 号：R285.5[医药卫生—中药学]