基于高内涵分析技术研究山萘酚的人肾近曲小管细胞HK-2毒性及机制  

Nephrotoxicity and mechanism of kaempferol on HK-2 cells based on high-content analysis technology

作　　者：路青瑜 郭丽 李娇[1,2] 杨付梅 孙黔云[1,2] LU Qing-yu;GUO Li;LI Jiao;YANG Fu-mei;SUN Qian-yun(State Key Laboratory of Function and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550014,China;Key Laboratory of Chemistry for Natural Products,Guizhou Province and Chinese Academy of Sciences,Guiyang 550014,China)

机构地区：[1]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 [2]贵州省中国科学院天然产物化学重点实验室,贵州贵阳550014

出　　处：《中国药理学与毒理学杂志》2023年第2期121-129,共9页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(U1812403);贵州省科技计划项目(黔科合人才[2016]4018号);贵州省科技计划项目(黔科合平台人才[2016]5625号);贵州省科技计划项目(黔科合平台人才[2019]5702号)。

摘　　要：目的基于高内涵分析技术探究山萘酚的肾细胞毒性作用及机制。方法人肾近曲小管细胞HK-2分为细胞对照组(二甲亚砜终浓度<0.01%)、山萘酚5,10,20,50和100μmol·L^(-1)组及多柔比星(Dox)10μmol·L^(-1)组。Hoechst 33342染色和噻唑蓝(MTT)比色法分别检测活细胞数目和细胞存活率;荧光探针DCFH-DA,Mito-Tracker Red CMXRos和Fluo-4 AM分别检测活性氧(ROS)、线粒体膜电位(MMP)和Ca^(2+)内流水平;化学发光法检测细胞ATP含量;AnnexinⅤ/PI双染法检测细胞凋亡率;Hoechst 33342染色检测DNA含量;免疫荧光法检测NF-κB p65入核情况,以及丝裂原活化蛋白激酶(MAPK)通路蛋白〔p38 MAPK、磷酸化p38 MAPK(p-p38 MAPK)、c-Jun N端激酶(JNK)、p-JNK、细胞外信号调节激酶(ERK)和p-ERK〕和酪氨酸激酶2(JAK2)/信号转导与转录激活因子3(STAT3)通路蛋白(p-STAT3和STAT3)的表达。结果与细胞对照组相比,山萘酚5,10,20,50和100μmol·L^(-1)组活细胞数目明显减少(P<0.05,P<0.01);山萘酚10,20,50和100μmol·L^(-1)组细胞存活率显著下降(P<0.05,P<0.01);山萘酚10,20,50和100μmol·L^(-1)组细胞ROS水平升高(P<0.05,P<0.01);山萘酚5,10,20,50和100μmol·L^(-1)组细胞MMP下降(P<0.01);山萘酚50和100μmol·L^(-1)组细胞Ca^(2+)内流水平明显升高(P<0.05,P<0.01),ATP含量明显降低(P<0.05,P<0.01);山萘酚20,50和100μmol·L^(-1)组细胞凋亡率均明显升高(P<0.05,P<0.01);山萘酚各剂量组DNA含量均无明显变化。免疫荧光检测结果显示,山萘酚50和100μmol·L^(-1)使NF-κB p65发生磷酸化并入核;山萘酚上调MAPK通路p-p38 MAPK,p-ERK,p-JNK和JNK蛋白及JAK2/STAT3通路p-STAT3蛋白的表达(P<0.05,P<0.01)。结论山萘酚对HK-2细胞有明显毒性,机制与其促进ROS水平升高而介导氧化应激损伤和细胞凋亡有关。OBJECTIVE To investigate the toxic effect and mechnism of kaempferol on human kidney proximal tubular epithelial cells(HK-2)based on high content analysis technology.METHODS HK-2 cells were divided into the control group(final concentration of dimethylsulfoxide<0.01%),kaempferol 5,10,20,50 and 100μmol·L^(-1)and doxorubicin(Dox,final concentration 10μmol·L^(-1))groups.The count of living cells was detected via Hoechst 33342 staining,the cell viability was determined by MTT assay,the levels of reactive oxygen species(ROS),mitochondrial membrane potential(MMP)and the intracel⁃lular calcium ion were detected by fluorescence probes of DCFH-DA,Mito-Tracker Red CMXRos and Fluo-4 AM.The ATP content was measured with the chemiluminescence method,and apoptosis by flow cytometry.The DNA content was detected by Hoechst 33342 staining.The nuclear translocation of NF-κB p65,the expressions of p38 mitogen-activated protein kinases(p38 MAPK),p-p38 MAPK,extracellular signal regulated kinase(ERK),p-ERK and c-Jun N-terminalkinases(JNK),p-JNK protein in associated mitogen activated protein kinases(MAPK)signal pathway,and those of signal transducer and activator of transcription-3(STAT3),p-STAT3 protein in janus kinase-2(JAK2)/STAT3 signal signal pathway were detected by immunofluorescence assay.RESULTS Compared with the control group,the count of living cells in the kaempferol 5,10,20,50 and 100μmol·L^(-1)groups and the cell viability in the kaempferol 10,20,50 and 100μmol·L^(-1)groups were significantly deceased(P<0.05,P<0.01).The levels of ROS were significantly increased in the kaempferol 10,20,50 and 100μmol·L^(-1)groups(P<0.05,P<0.01),while those of MMP in 5,10,20,50 and 100μmol·L^(-1)groups were obviously decreased(P<0.01).The levels of calcium influx in kaempferol 50,100μmol·L^(-1)groups were increased significantly(P<0.05,P<0.01),while the ATP content in the kaempferol 50 and 100μmol·L^(-1)groups was obviously decreased(P<0.05,P<0.01).The apoptotic rates in kaempferol 20,50,100μmol·L^(-1)groups were up-r

关 键 词：高内涵分析技术 山萘酚 肾毒性 NF-ΚB 

分 类 号：R992[医药卫生—毒理学]