基于JAK2/STAT3信号通路探讨雷公藤甲素诱导宫颈癌耐药细胞自噬性死亡的机制  

Mechanism of Triptolide-Induced Autophagic Death of Drug-Resistant Cervical Cancer Cells Based on JAK2/STAT3 Signaling Pathway

作　　者：刘秀玲 王茹娜 符小春 张海龙 王培培 赵广彩 LIU Xiu-ling;WANG Ru-na;FU Xiao-chun;ZHANG Hai-long;WANG Pei-pei;ZHAO Guang-cai(Department of Gynecological Oncology,Zhumadian Central Hospital,Zhumadian 463000,China;Department of Pharmacy,Zhumadian Central Hospital,Zhumadian 463000,China;Department of Obstetrics,Qilu Hospital of Shandong University,Qingdao 266003,China)

机构地区：[1]驻马店市中心医院肿瘤妇科,河南驻马店463000 [2]驻马店市中心医院药学部,河南驻马店463000 [3]山东大学齐鲁医院,山东青岛266003

出　　处：《中国药学杂志》2022年第24期2099-2107,共9页Chinese Pharmaceutical Journal

摘　　要：目的探讨雷公藤甲素(triptolide,TPL)增敏顺铂(cisplatin,DDP)促进宫颈癌耐药细胞自噬性死亡的作用机制。方法体外构建人宫颈癌DDP耐药细胞株HeLa/DR,分为空白组、DDP(10μg·mL^(-1))组、TPL组(50 nmol·L^(-1))、DDP+TPL组用于药效学研究;分为TPL组、N-乙酰-L-半胱氨酸(NAC,5 mmol·L^(-1))组、TPL+NAC组、Janus激酶2/信号转导与转录激活因子3(JAK2/STAT3)抑制剂AG490(8μg·mL^(-1))组、TPL+JAK2/转录信号传导子和激活子3(STAT3)激活剂RO8191(20μmol·L^(-1))组用于机制研究。采用CCK-8法检测细胞增殖率,计算半数抑制浓度(IC50)和药物敏感度指数(SI),采用中位药效法(chou-talalay)计算DDP和TPL联合用药指数(CI)。采用流式细胞术检测细胞凋亡和细胞周期分布;采用MitoTracker green和DQ BSA red染色观察溶酶体数量和吞噬功能;采用GFP-LC3和MitroTracker共定位染色法观察线粒体自噬体数量。Western blot法检测相关蛋白表达。结果TPL剂量≤200 nmol·L^(-1)时,CI<1,与DDP具有协同作用;50 nmol·L^(-1)TPL与DDP的协同作用最显著,可增加HeLa/DR细胞对DDP、5-氟尿嘧啶和紫杉醇的敏感性(SI=8.05±0.28,3.17±0.14,4.05±0.12)。与DDP组相比,TPL+DDP组细胞凋亡率和G0/G1期细胞比例明显升高(P<0.05)。此外,虽然LC3-Mitro共定位绿色荧光增多且线粒体总蛋白中LC3-II/LC3-I比值和Beclin1蛋白表达升高(P<0.05),但是DQ BSA red/Lysotracker green荧光比值降低(P<0.05)。与空白组相比,TPL组HeLa/DR细胞的2′,7′-二氯荧光素(DCF)荧光强度明显升高,而加入NAC后可显著降低活性氧(ROS)生成量,同时Beclin蛋白相对表达量和LC3-II/LC3-I蛋白比值降低。网络药理学揭示了TPL-宫颈癌-JAK2/STAT3通路之间的关系。Western blot结果显示,与空白组相比,TPL组、AG490组p-JAK2/JAK2、p-STAT3/STAT3蛋白表达比值明显降低,但是TPL+RO8191组和TPL+NAC组p-JAK2/JAK2、p-STAT3/STAT3蛋白表达比值则较TPL组升高(P<0.001)。结论TPL通过诱导ROS产生�OBJECTIVE To investigate the effect and mechanism of triptolide(TPL)-induced autophagic death of cisplatin(DDP)-resistant cervical cancer cells.METHODS DDP-resistant cervical cancer cell lines HeLa/DR was cultured in vitro,and divided into blank group,DDP(10μg·mL^(-1))group,TPL(50 nmol·L^(-1))group,DDP+TPL group for pharmacodynamic research and into TPL group,N-acetyl-L-cysteine(NAC,5 mmol·L^(-1))group,TPL+NAC group,inhibitor of Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)AG490(8μg·mL^(-1))group,TPL+activator of JAK2/STAT3(RO8191,20μmol·L^(-1))group for mechanism research.CCK-8 method was used to detect the cell proliferation,the IC50and drug sensitivity index(SI)were calculated,and the combined drug use index(CI)of DDP and apatinib was calculated by median pharmacodynamics(chou-talalay)method.The apoptosis and cell cycle were detected by Flow cytometry.The number of lysosomes and phagocytic function were observed by Mitotracker Green and DQ BSA red staining,and the number of mitochondrial autophagosomes was observed by GFP-LC3 and Mitrotracker co-localization staining.Western blot was used to detect the expression of the related proteins.RESULTS When the dosage of TPL was less than 200 nmol·L^(-1),the CI was<1,indicating that TPL had synergistic effect with DDP,and when the dosage of TPL was 50 nmol·L^(-1),the synergistic effect of TPL and DDP was most significant.Besides,50 nmol·L^(-1)of TPL could increase the sensitivity of HeLa/DR cells to DDP,5-fluorouracil,and paclitaxel(SI=8.05±0.28,3.17±0.14,4.05±0.12).Compared with DDP group,the apoptosis rate and the ratio of G0/G1phase cells were significantly increased in DDP+TPL group(P<0.05).Besides,although co-localization of LC3-Mitro green fluorescence and LC3-II/LC3-I ratio and Beclin1 expression in total mitochondrial proteins were increased(P<0.05),the fluorescence ratio of BSA DQ red/Lysotracker green decreased(P<0.05).Compared with the blank group,the DCF fluorescence intensity of HeLa/DR cells in TPL group was

关 键 词：宫颈癌 雷公藤甲素 Janus激酶2/信号转导与转录激活因子3通路 顺铂耐药 线粒体-溶酶体交互作用 

分 类 号：R965[医药卫生—药理学]