益气解毒方通过诱导自噬促进鼻咽癌细胞凋亡的作用研究  

Qi-Boosting Toxin-Resolving Formula Promotes Apoptosis of Nasopharyngeal Carcinoma Cells by Inducing Autophagy

作　　者：蔺婷 罗晶婧 周芳亮[4] 范婧莹[3] 戴娜 胡梅[4] 何迎春 LIN Ting;LUO Jingjing;ZHOU Fangliang;FAN Jingying;DAI Na;HU Mei;HE Yingchun(Hunan Provincial Key Laboratory for Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Chinese Medicine,Changsha 410208 Hunan,China;Hunan Provincial Engineering and Technological Research Centerfor Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Chinese Medicine and Protecting Visual Function,Changsha 410208 Hunan,China;College of Integrated Traditional Chinese and Western Medicine of Hunan University of Chinese Medicine,Changsha 410208 Hunan,China;Medical School,Hunan University of Chinese Medicine School,Changsha 410208 Hunan,China;Graduate School of Hunan Universityof Chinese Medicine,Changsha 410208 Hunan,China)

机构地区：[1]中医药防治眼耳鼻咽喉疾病湖南省重点实验室,湖南长沙410208 [2]湖南省中医药防治眼耳鼻咽喉疾病与视功能保护工程技术研究中心,湖南长沙410208 [3]湖南中医药大学中西医结合学院,湖南长沙410208 [4]湖南中医药大学医学院,湖南长沙410208 [5]湖南中医药大学研究生院,湖南长沙410208

出　　处：《中药新药与临床药理》2019年第10期1149-1158,共10页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目（81573721、81874408）;湖南省自然科学基金项目（2016JJ6117、2017JJ3246）;湖南省教育厅科研基金项目（16C1212）;湖南中医药大学研究生创新课题项目（2017CX33）;中医药防治眼耳鼻咽喉疾病湖南省重点实验室开放基金项目（2018YZD02）

摘　　要：目的研究益气解毒方(Qi-boosting toxin-resolving Formula,QBTRF)对人鼻咽癌CNE-2Z细胞自噬和凋亡的影响,探讨CNE-2Z细胞自噬和凋亡之间的关系。方法CCK-8法检测不同浓度益气解毒方干预鼻咽癌CNE-2Z细胞24 h和48 h后的细胞存活率,并计算48 h的IC50;采用Annexin V-FITC/PI流式细胞术Western Blot和透射电镜等方法检测QBTRF干预鼻咽癌CNE-2Z细胞后的自噬和凋亡情况。结果CCK-8法结果显示,QBTRF能显著抑制CNE-2Z细胞增殖(P<0.01),48 h的IC50为0.5 mg·mL-1;AnnexinV-FITC/PI结果显示,与Control组比较,QBTRF干预不同时间后细胞凋亡率明显升高(P<0.01),与QBTRF组比较,3-MA(自噬抑制剂)+QBTRF组的细胞凋亡率显著下降(P<0.01)。MDC染色法结果显示,与Control组比较,0.5 mg·mL-1QBTRF处理CNE-2Z细胞不同时间后荧光增强增加(P<0.05,P<0.01),干预24 h时,荧光强度最大(P<0.01)。Western Blot结果显示,与Control组比较,QBTRF干预CNE-2Z细胞不同时间后cleaved Caspase-3、Bax、cleaved PARP、LC3-II、Beclin1蛋白表达上调(P<0.05,P<0.01),Bcl-2、PI3K、p-AKT和p-mTOR的蛋白表达量下降(P<0.01),AKT蛋白表达无变化(P>0.05);与0.5 mg·mL-1QBTRF组比较,3-MA+QBTRF组cleaved Caspase-3、cleaved PARP、Bax的蛋白表达量下降(P<0.01),Bcl-2、PI3K、p-AKT、和p-mTOR的蛋白表达量上调(P<0.05,P<0.01),AKT蛋白表达无变化(P>0.05),Z-VAD-fmk(凋亡抑制剂)+QBTRF组的LC3-II和Beclin1蛋白表达量下调(P<0.05)。透射电镜结果显示,QBTRF干预CNE-2Z细胞不同时间后细胞内自噬体和凋亡小体出现不同程度增加,24 h自噬最为显著,48 h凋亡最为明显。结论QBTRF可抑制人鼻咽癌CNE-2Z细胞增殖并诱导细胞凋亡和自噬;QBTRF可能通过诱导自噬促进鼻咽癌细胞凋亡。Objective To study the effect of Qi-boosting Toxin-resolving formula(QBTRF)on autophagy and apoptosis of CNE-2 Z cells of human nasopharyngeal carcinoma,and to explore the relationship between autophagy and apoptosis of CNE-2 Z cells.Methods CCK-8 assay was used to detect the cell viability of nasopharyngeal carcinoma CNE-2 Z cells treated with different concentrations of QBTRF for 24 h and 48 h,and IC50(Half Inhibitory Rate Concentration)was calculated at 48 h.Annexin V-FITC/PI,Western Blot and transmission electron microscope were used to detect the autophagy and apoptosis of nasopharyngeal carcinoma CNE-2 Z cells.Results CCK-8 results showed that QBTRF significantly inhibited the proliferation of CNE-2 Z cells(P<0.01),and IC50 was0.5 mg·mL-1 at 48 h.AnnexinV-FITC/PI results showed that the apoptosis rate of CNE-2 Z cells treated with QBTRF was significantly increased(P<0.01).Compared with the QBTRF group,the apoptotic rate of the 3-MA+QBTRF group was significantly decreased(P<0.01).MDC staining results showed that,compared with the Control group,the fluorescence intensity of CNE-2 Z cells increased after treatment with 0.5 mg·mL-1 QBTRF for different duration(P<0.05,P<0.01);and the fluorescence intensity was the highest at 24 h after intervention(P<0.01).Western Blot results showed that after QBTRF intervention,the expression levels of cleaved Caspase-3,Bax,cleaved PARP,LC3-II and Beclin1 were up-regulated(P<0.05,P<0.01),while the expression of Bcl-2,PI3 K,pAKT and p-mTOR protein were down-regulated(P<0.01);and the expression of AKT protein was unchanged(P>0.05).Compared with 0.5 mg·mL-1 QBTRF group,the expression of cleaved Caspase-3,cleaved PARP and Bax decreased in 3-MA+QBTRF group(P<0.01),while the expression of Bcl-2,PI3 K,p-AKT and p-mTOR increased(P<0.05,P<0.01),the expression of AKT protein remained unchanged(P>0.05).The expression of LC3-II and Beclin1 protein decreased in Z-VAD-fmk+QBTRF group(P<0.05).Transmission electron microscopy showed that QBTRF interfered with CNE-2 Z cells at different tim

关 键 词：益气解毒方 自噬 凋亡 鼻咽癌 CNE-2Z细胞 

分 类 号：R285[医药卫生—中药学]