基于高内涵筛选技术的吴茱萸次碱肝毒性研究  

Study on hepatoxic liver of rutecarpine based on high-content screening technology

作　　者：郭丽[1] 路青瑜 李娇[1] 杨付梅[1] 孙黔云[1] GUO Li;LU Qing-yu;LI Jiao;YANG Fu-mei;SUN Qian-yun(The Key Lab of Chemistry for Natural Products,Guizhou Province and Chinese Acadamy of Science,State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550014,China)

机构地区：[1]贵州省中国科学院天然产物化学重点实验室,贵州医科大学省部共建药用植物功效与利用国家重点实验室,贵州贵阳550014

出　　处：《中国药理学通报》2022年第10期1548-1558,共11页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No U1812403);贵州省科技计划及平台人才项目(黔科合人才[2016]4018号、黔科合平台人才[2016]5625号、黔科合支撑[2019]2753号、黔科合平台人才[2019]5702号、黔科合基础[2017]1115号)。

摘　　要：目的利用高内涵筛选技术研究吴茱萸次碱(ruteacarpine,RUT)的肝毒性及其可能机制。方法HepG2细胞暴露于不同浓度的RUT后作用不同时间,MTT法检测细胞活力,采用高内涵检测RUT对细胞存活、细胞核面积、线粒体膜电位(MMP)、ROS、钙离子内流、细胞膜完整性(DIR)和MAPK、NF-κB、JAKs-STATs信号通路活化水平的影响,流式细胞检测细胞凋亡。结果100μmol·L^(-1)RUT明显抑制了HepG2细胞的活力(P<0.01),表现为RUT作用24 h后,细胞核面积减少,核形态不均一,作用48 h后存活细胞数量明显减少(P<0.01),并伴随着细胞的早期凋亡(P<0.01);从6 h开始,100μmol·L^(-1)RUT组细胞内活性氧和钙离子水平明显升高(P<0.01),细胞膜完整性明显降低(P<0.01);100μmol·L^(-1)RUT作用24 h后,ERK1/2、JNK、STAT3和p38的磷酸化水平明显升高(P<0.01,P<0.05),总蛋白未见明显变化;在3 h可检测到c-Jun、c-Fos的表达上调,与对照组相比,差异具有显著性(P<0.01);在3 h时间点,可明显检测到p-NF-κB p65的表达上调(P<0.01),但NF-κB p65入核不明显。结论吴茱萸次碱在100μmol·L^(-1)的条件下对HepG2细胞表现出细胞毒性,其毒性机制主要与氧化应激和炎症反应导致的细胞损伤有关。Aim To investigate the hepatotoxicity of rutecarpine(RUT)by using high-content screening technology.Methods HepG2 cells were exposed to different concentrations of RUT for different time,then cell viability was detected by MTT method.Cell count,nucleus injury,mitochondrial membrane potential(MMP),reactive oxygen species(ROS),internal flow of calcium,cell membrane integrity(DIR)were measured by high-content screening technology.The activation of MAPK,NF-κB and JAKs-STATs was assayed by high-content screening technology.The apoptosis was detected by flow cytometry.Results The viability was significantly reduced by 100μmol·L^(-1)RUT(P<0.01)after HepG2 cell exposure to RUT for 24 h,the nuclear area decreased and the nuclear morphology was uneven,and after 48 h,the cell count was significantly reduced(P<0.01),the early apoptosis was detected(P<0.01).After HepG2 cell exposure to RUT for 6 h,the levels of ROS and internal flow of calcium significantly increased(P<0.01),and the cell membrane integrity was obviously damaged(P<0.01).After exposure to 100μmol·L^(-1)RUT for 24 h,the phosphorylation of ERK,JNK,STAT3 and p38 significantly increased(P<0.01,P<0.05),but there was no significant change in total protein level.The expression of c-Jun and c-Fos was up-regulated at 3 h(P<0.01),and at 3h time point,the phosphorylation of NF-κB p65 significantly increased(P<0.01),but nuclear translocation was not significant.Conclusions Under certain conditions,RUT shows cytotoxicity on HepG2 cells,and its toxic mechanism is mainly related to injury caused by oxidative stress and inflammatory response.

关 键 词：吴茱萸次碱 肝毒性 高内涵筛选技术 氧化应激 HEPG2 MAPK 

分 类 号：R284.1[医药卫生—中药学] R322.47[医药卫生—中医学] R345.57R349.1