锰对人源性脉络丛乳头状瘤细胞系合成GDF-15的影响及机制研究  

Exploring the influence and mechanism of manganese on GDF-15 in human-derived choroid plexus papilloma cell line

作　　者：张雨 张国艳 李子南[1,2] 田永吉[3] 宁钧宇[1,2] 洪昭雄 高珊[2] 谭壮生[2] 敬海明[1,2] 李国君[1,2] ZHANG Yu;ZHANG Guo-yan;LI Zi-nan;TIAN Yong-ji;NING Jun-yu;HONG Jau-Shyong;GAO Shan;TAN Zhuang-sheng;JING Hai-ming;LI Guo-jun(School of Public Health,Capital Medical University,Beijing 100069,China;Beijing Center for Disease Prevention and Control,Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning,Beijing 100013,China;Department of Pediatric Neurosurgery,Beijing Tiantan Hospital,Capiyal Medical University,Beijing 100070,China;Neuropharmacology Section,Neurobiology Laboratory,National Institute of Environmental Health,Sciences,P.O.Box 12233,Research Triangle Park,NC 27709,USA.)

机构地区：[1]首都医科大学公共卫生学院,北京100069 [2]北京市疾病预防控制中心,食物中毒诊断溯源技术北京市重点实验室,北京100013 [3]首都医科大学附属北京天坛医院小儿神经外科,北京100070 [4]美国国立卫生环境健康研究所神经生物学实验室,德汉姆北卡州27709

出　　处：《毒理学杂志》2024年第3期207-214,242,共9页Journal of Toxicology

基　　金：首都高层次公共卫生技术人才建设卫生毒理学科“领军人才”项目(#02-03)。

摘　　要：目的应用人源性脉络丛乳头状瘤细胞系(hCPP)探究锰对脑脉络丛上皮细胞中生长分化因子-15(growth differentiation factor-15,GDF-15)合成的影响及其调控机制。方法应用CCK-8试剂盒检测氯化锰(以下简称锰)处理后hCPP的细胞活力;不同浓度的锰(25、50、75、100、150和200μmol/L)处理hCPP细胞24、48和72 h,通过Western blot、Real-Time PCR检测GDF-15蛋白表达和Gdf-15 mRNA水平在锰作用下的剂量和时间-效应关系。给予hCPP细胞100μmol/L的锰处理24 h,通过Western blot检测p38 MAPK、NF-κB、AMPK、PI3K/AKT通路的激活情况,随后,给予相应的通路抑制剂后检测GDF-15的蛋白表达。100μmol/L的锰处理hCPP细胞0、6、12、24、36、48、72 h,检测GDF-15、p38 MAPK蛋白表达的时间-效应关系。使用荧光探针试剂盒检测hCPP锰处理(100μmol/L,24 h)后细胞活性氧(reactive oxygen species,ROS)的含量,给予ROS抑制剂N-acetylcysteine(NAC)后检测GDF-15、p38 MAPK蛋白表达。结果用200μmol/L以下的不同浓度的锰处理hCPP细胞24、48和72 h,可引起细胞活力降低、GDF-15蛋白合成增加、Gdf-15 mRNA水平增加,且随锰浓度的增高而变化。100μmol/L的锰处理hCPP细胞24 h,通过增加p38 MAPK、NF-κB p65和AMPKα的磷酸化水平激活p38 MAPK、NF-κB和AMPK信号通路,给予SB 202190(p38 MAPK通路抑制剂)可逆转锰处理hCPP引起的GDF-15合成增加效应。100μmol/L的锰处理hCPP细胞0、6、12、24、36、48和72 h,GDF-15蛋白水平和p38 MAPK磷酸化蛋白水平随着锰处理时间的增加而增高。100μmol/L的锰处理hCPP细胞15、30、60 min以及24 h,细胞ROS水平与对照组相比差异无统计学意义,给予NAC处理不能逆转锰引起的GDF-15合成增加及p38 MAPK通路的激活状态。结论本研究表明,锰处理可增加hCPP细胞中GDF-15的合成,这可能是p38 MAPK信号通路激活后介导的。这些发现为今后阐明GDF-15在锰所致中枢神经系统的毒性效应及分子机制提供了新的线�Objective This study aimed to investigate the effect and mechanism of manganese on Growth differentiation factor 15(GDF-15)in a newly established human-derived Choroid Plexus Papilloma Cell Line(hCPP).Methods The dose-dependent and time-related toxic effects of manganese on hCPP cell viability were assessed using the CCK-8 assay.The expression of GDF-15 protein and the level of Gdf-15 mRNA were examined through Western blot and Real-Time PCR,respectively.After treatment with 100μmol/L MnCl_2 for 24 hours,the phosphorylation levels of p38 MAPK,NF-κB p65,and AMPKαwere evaluated through Western blot,followed by the detection of GDF-15 protein expression after treatment with corresponding pathway inhibitors.The content of reactive oxygen species(ROS)following manganese exposure(100μmol/L,24 h)was measured using the fluorescent probe Dihydroethidium kit.Additionally,GDF-15 and p38 MAPK protein levels were assessed through Western blotting after treatment with N-acetylcysteine(NAC),a ROS inhibitor.Results Treatment of hCPP with different concentrations of manganese for 24 h,48 h,and 72 h led to time-and concentration-dependent decreases in cell viability,accompanied by an increase in GDF-15 protein synthesis and the level of Gdf-15 mRNA.Treatment with 100μmol/L manganese for 24 h activated several signaling pathways,evidenced by increased phosphorylation levels of p38 MAPK,NF-κB p65,and AMPKα.Furthermore,treatment with SB 202190(a p38 MAPK pathway inhibitor)reversed the effect of manganese stimulation on hCPP-induced increase in GDF-15 synthesis.Stimulation of hCPP with 100μmol/L manganese for various durations revealed time-dependent increases in GDF-15 protein and p38 MAPK protein phosphorylation levels.However,no significant difference in ROS levels was observed between the control group and cells treated with 100μmol/L manganese for 15 min,30 min,60 min,and 24 h.The lack of ROS involvement was further supported by the failure of acetylcysteine treatment to reverse the manganese-induced increase in GDF-15 s

关 键 词：生长分化因子-15(GDF-15) 氯化锰 脉络丛 人源性脉络丛乳头状瘤细胞系(hCPP) p38 MAPK信号通路 

分 类 号：R99[医药卫生—毒理学] R114[医药卫生—药学]