【作者】 刘娜；

【导师】 唐萌；

【作者基本信息】 东南大学 ， 卫生毒理学， 2023， 博士

【摘要】 研究目的:本研究旨在探讨生物成像材料碲化镉量子点(cadmium telluride quantum dots,CdTe QDs)在小鼠体内和体外巨噬细胞中引起炎性因子异常升高的机制,探讨细胞巨自噬和选择性自噬在该炎性反应中的参与作用并确定其中的关键蛋白和暴露剂量,为评估CdTe QDs的应用价值与毒作用提供参考。研究方法:1.采用电化学法合成3-巯基丙酸(3-mercaptopropionic acid,MPA)修饰的CdTe QDs(MPA-CdTe QDs),并对其进行表征。通过透射电子显微镜(transmission electron microscope,TEM)观察其形貌,用马尔文粒度仪分别测定其分散于超纯水和DMEM培养基中的水合粒径及zeta电位,用荧光分光光度计测定其激发和发射光谱以确定最大激发波长和最大发射波长。2.将雄性C57BL/6小鼠通过单次尾静脉注射急性暴露于MPA-CdTe QDs(1.25 μmol/Kg bw和12.5μmol/Kg bw)。24 h后,通过电感耦合等离子体质谱(inductively coupled plasma-mass spectrometry,ICP-MS)检测脏器中的 Cd 含量以反映 MPA-CdTe QDs 在小鼠体内的分布和排泄,通过HE染色观察肝、肾和肺的病理损伤,通过免疫荧光标记F4/80和CD86分子以指示巨噬细胞在肝、肾和肺内的浸润侵袭情况,通过酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)检测血清和脏器中促炎性细胞因子IL-1β和IL-6的水平,通过蛋白质免疫印迹(westernblot,WB)测定炎性反应相关蛋白的变化。持续观察上述指标至28 d以反映小鼠急性暴露于MPA-CdTe QDs后的恢复情况。3.以RAW264.7细胞为模型,多角度检测MPA-CdTe QDs对巨噬细胞的毒作用和巨噬细胞的炎性改变。通过MTT实验筛选巨噬细胞暴露于MPA-CdTe QDs的浓度。选定暴露剂量后,通过ICP-MS检测细胞暴露于0.1、0.5、1.0μmol/L MPA-CdTe QDs后细胞内的Cd含量,检测细胞培养液中的LDH以反映细胞膜的损伤,使用FITC-Annexin-V和PI检测巨噬细胞的坏死率和凋亡率。通过转录组测序概览MPA-CdTe QDs对RAW264.7细胞基因水平的影响,使用中性红检测巨噬细胞的吞噬能力,通过标记CD86和CD206检测M1型和M2型巨噬细胞的比例以及通过实时荧光定量PCR(real time quantitative PCR,qPCR)和WB分别测定各种炎性因子在转录水平和蛋白水平的变化。4.将 RAW264.7 细胞暴露于 0.1、0.5、1.0μmol/L MPA-CdTe QDs 24h后通过TEM观察巨噬细胞内自噬体和自噬溶酶体的数量,通过WB测定自噬相关蛋白ULK1、Beclin1、LC3B和p62的水平,通过LC3 turnover实验和双荧光LC3慢病毒感染的巨噬细胞检测自噬流的完整性,使用DCFH-DA和DAF-AM荧光探针分别检测细胞内活性氧(reactive oxygen species,ROS)和一氧化氮的含量。使用试剂盒检测细胞的总抗氧化能力、氧化与抗氧化物质的含量和抗氧化酶活性,包括:谷胱甘肽(reduced glutathione,GSH)、谷胱甘肽过氧化物酶(glutathione peroxidase,Gpx)、超氧化物歧化酶(superoxide dismutase,SOD)、还原型辅酶Ⅱ(nicotinamide adenine dinucleotide phosphate,NADPH)和 NADPH 氧化酶(NADPH oxidase,NOX)。分别使用抗氧化剂Trolox、NRF2 抑制剂 ML385 和 siRNA 确定 ROS、NRF2、ERK1/2 和 AMPK 在 MPA-CdTe QDs激活的RAW264.7细胞巨自噬中的参与作用,以及巨自噬对IL-1β和IL-6异常增多的介导作用。然后测定MPA-CdTe QDs处理后的巨噬细胞中溶酶体关联膜蛋白(LAMP1和LAMP2)和组织蛋白酶(CTSB和CTSD)的水平,使用AO染料检测溶酶体膜的完整性,使用pH依赖性荧光探针LysoSensorTM Green DND-189和Lyso-Tracker Red DND-99指示溶酶体pH的变化,用bafilomycin A1抑制溶酶体ATP酶(ATPase)后验证MPA-CdTe QDs是否通过溶酶体酸化造成细胞内ROS含量增多。5.接下来,研究0.1、0.5、1.0μmol/L MPA-CdTe QDs对巨噬细胞线粒体的结构、功能、数量、形态及线粒体质量控制过程的影响。通过TEM观察MPA-CdTe QDs对RAW264.7细胞线粒体结构的影响,使用JC-1探针检测线粒体膜电位,用MitoTracker标记线粒体的网络形态和数量,用MitoSOX Red检测线粒体超氧化物含量,用Fluo-3 AM检测细胞内钙离子含量,使用试剂盒检测细胞内ATP含量。通过WB检测线粒体质量控制相关蛋白的变化,包括:线粒体生物发生相关蛋白(PGC-1α、NRF-1和TFAM)、线粒体分裂-融合相关蛋白(DRP1、p-DRP1-Ser616、FIS1和MFF;OPA1、MFN1和MFN2)和线粒体自噬相关蛋白(PINK1和Parkin)。其中,着重关注线粒体自噬的发生,通过线粒体靶向超氧化物歧化酶MitoTEMPO、NRF2抑制剂ML385、针对ERK1/2的siRNA以及慢病毒敲减PINK1分别确定线粒体活性氧(mitochondrial ROS,mitoROS)、NRF2 和 ERK1/2 在 MPA-CdTe QDs 激活线粒体自噬中的参与作用,以及进而验证mitoROS和NRF2-ERK1/2通路参与的线粒体自噬对MPA-CdTe QDs引起的IL-1β和IL-6异常升高的介导作用。6.最后,使用试剂盒检测0.1、0.5、1.0μmol/LMPA-CdTe QDs 处理 24h 后的 RAW264.7细胞中的脂质过氧化产物丙二醛(malonicdialdehyde,MDA)含量,用Liperfluo荧光探针检测细胞内脂质过氧化物(lipid hydroperoxide,LPO)的含量,使用FerroOrange探针和Mito-FerroGreen探针分别检测细胞质和线粒体内游离二价铁离子(Fe2+)含量,然后使用各种细胞死亡方式抑制剂(铁死亡抑制剂ferrostatin-1和liproxstatin-1、铁离子螯合剂deferoxamine mesylate、自噬抑制剂3-methyladenine、溶酶体抑制剂chloroquine和bafilomycin A1、凋亡抑制剂Z-VAD-FMK、坏死性凋亡抑制剂necrostatin-1以及焦亡抑制剂VX-765)验证巨噬细胞中铁死亡是否发生,测定与铁死亡相关的GSH稳态调节蛋白和脂代谢调节蛋白的变化。然后通过WB确定巨噬细胞中与铁自噬相关的FTH1和NCOA4蛋白的时间变化趋势(0-48 h),并用ML385和siRNA 验证 NRF2 和 ERK1/2 对铁自噬的调节,最后用 ferrostatin-1、liproxstatin-1、deferoxamine mesylate、ML385 和 siRNA 确定 NRF2-ERK1/2-FTH1 通路介导的铁死亡在MPA-CdTe QDs引起的IL-1 β和IL-6异常升高中的参与作用。研究结果:1.MPA-CdTe QDs的表征。制备的MPA-CdTe QDs呈橘黄色,在超纯水中均匀分散,TEM观察呈圆形且具有明显的晶格结构,粒径为3.4±0.56 nm。在超纯水和DMEM培养基中的水合粒径分别为3.99±0.82 nm和5.42±1.84 nm,在超纯水中的zeta电位为-40.4±1.42 mV。最大激发波长是380 nm,380 nm激发波下的最大发射波长为603 nm。2.MPA-CdTe QDs的急性暴露在小鼠体内引起炎性因子异常升高及肝肾组织中巨噬细胞浸润,且持续至28d。小鼠通过尾静脉注射单次暴露于12.5μmol/Kgbw MPA-CdTe QDs后,21 d和28 d时体重增长被抑制,但是24 h、7 d、28 d时均没有观察到对各个脏器的重量和结构的影响。24h时,12.5μmol/Kgbw MPA-CdTe QDs在小鼠血清、肝脏和肾脏中引起白细胞升高、巨噬细胞浸润及IL-1β和IL-6的异常升高,并且7d和28 d时也观察到了同样的炎性改变。12.5μmol/Kg bw超出了小鼠的排泄能力且从暴露后24 h到28 d在小鼠体内进行了重新分布,最终蓄积在肾脏和肝脏。相较于12.5μmol/Kgbw,1.25μmol/Kgbw MPA-CdTe QDs 对小鼠没有明显毒性。3.MPA-CdTe QDs造成巨噬细胞的细胞毒性并引起IL-1β和IL-6显著升高。根据RAW264.7细胞暴露于MPA-CdTe QDs 24 h后的活性率,选择0.1(细胞活性率约为85%)、0.5、1.0μmol/L作为暴露剂量,选择24 h作为暴露时间。RAW264.7细胞对MPA-CdTe QDs的摄取和胞吐表现出暴露时间(3-24 h)和暴露剂量(0.1-1.0μmol/L)依赖性增多。1.0μmol/L MPA-CdTe QDs可以损伤RAW264.7细胞的胞膜,促进细胞发生凋亡,增强巨噬细胞的吞噬能力,使巨噬细胞中M1促炎型细胞的比例增加、M2抑炎型细胞的比例减少,并引起RAW264.7细胞中IL-1β和IL-6显著增多。此外,转录组测序结果显示1.0 μmol/L MPA-CdTe QDs导致巨噬细胞中177个基因发生差异表达,“iron ion transport”、“glutathione metabolic process”、“interleukin-1 beta production”、“ERK1 and ERK2 cascade”、“lysosome”、“ferroptosis”“mitochondrial depolarization”等基因集显著改变。4.ROS-NRF2-ERK1/2-AMPK 通路介导的巨自噬参与 MPA-CdTe QDs 升髙 RAW264.7细胞中IL-1β和IL-6。1.0μmol/L MPA-CdTe QDs激活RAW264.7细胞中巨自噬的发生且自噬通量增加,表现为TEM观察到自噬体和自噬溶酶体数量增加,ULK1、Beclin1及LC3BII蛋白增加,底物蛋白p62无明显增加。stubRFP-sensGFP-LC3双荧光慢病毒指示了 MPA-CdTe QDs在巨噬细胞内引起GFP蛋白的淬灭,加之溶酶体抑制剂bafilomycin A1和E64d抑制了 MPA-CdTe QDs处理的巨噬细胞内LC3BII和p62的水解(LC3 Turnover试验),表明了自噬通量的增加。此外,MPA-CdTe QDs造成细胞内氧化水平与抗氧化能力失衡,增加细胞内的ROS和一氧化氮,引起GSH消耗但增强细胞总抗氧化能力。MPA-CdTe QDs引起的ROS升高下调了 NRF2蛋白,而NRF2的降低进而增加ERK1/2和AMPK的磷酸化,激活巨自噬。而该ROS-NRF2-ERK1/2-AMPK通路介导的巨自噬也参与了 MPA-CdTe QDs引起的RAW264.7细胞过表达IL-1β和IL-6,该分子通路的作用被抗氧化剂Trolox、NRF2抑制剂ML385、针对 AMPK 和 ERK1/2 的 siRNA 验证。5.MPA-CdTe QDs造成巨噬细胞溶酶体结构和功能异常,并经此参与了部分ROS升高。转录组测序结果提示1.0μmol/L MPA-CdTe QDs造成巨噬细胞内关于溶酶体的“Lysosome”GO term 和 KEGG pathway 显著富集,溶酶体膜蛋白 LAMP1 和 LAMP2显著下降。MPA-CdTe QDs使巨噬细胞内溶酶体酸性增强,该溶酶体酸化可以被V-ATPase 抑制剂 bafilomycin A1 缓解。此外,1.0μmol/L MPA-CdTe QDs 使巨噬细胞中CTSB和CTSD蛋白水平显著下降,溶酶体膜通透性增加,而该溶酶体膜损伤依赖于溶酶体酸化,溶酶体酸化也造成胞质内ROS的含量升高,该反应由bafilomycin A1验证,bafilomycin A1也减轻了 MPA-CdTe QDs引起的IL-1β和IL-6的异常升高。6.mitoROS和NRF2-ERK1/2-PINK1介导的线粒体自噬参与了 MPA-CdTe QDs引起的RAW264.7 细胞中 IL-1β 和 IL-6 增多。结果表明,1.0μmol/L MPA-CdTe QDs 破坏RAW264.7细胞的线粒体嵴、使线粒体膜电位下降、增加mitoROS含量且抑制ATP合成。此外,MPA-CdTe QDs增加了巨噬细胞中线粒体的数量,使线粒体的网状结构向碎片化的点状和短棒状转变,这与MPA-CdTe QDs促进线粒体生物发生、促进线粒体分裂但抑制线粒体融合是一致的。而且,MPA-CdTe QDs引起RAW264.7细胞发生线粒体自噬:表现为PINK1和Parkin蛋白显著升高、线粒体和溶酶体的共定位增多及线粒体外膜受体TOM20蛋白和LC3B的共定位增多。MPA-CdTe QDs引起的mitoROS升高与NRF2下降导致的ERK1/2磷酸化协同激活了线粒体自噬,由线粒体靶向超氧化物歧化酶MitoTEMPO、NRF2抑制剂ML385、针对ERK1/2的siRNA验证,且PINK1介导的线粒体自噬参与了 MPA-CdTe QDs引起的RAW264.7细胞过表达IL-1β和IL-6,因为使用慢病毒敲减RAW264.7细胞中PINK1后IL-1β和IL-6的异常增多被缓解。7.铁自噬介导的铁死亡参与了 MPA-CdTe QDs升高RAW264.7细胞中促炎因子IL-1β和IL-6的过程。1.0μmol/LMPA-CdTe QDs处理RAW264.7细胞24 h后,巨噬细胞发生铁死亡,表现为:GSH消耗、LPO增多、细胞质和线粒体内游离Fe2+增多、铁死亡抑制剂ferrostatin-1和liproxstatin-1及铁螯合剂deferoxamine mesylate可以降低细胞的死亡率、GSH稳态调节蛋白(SLC7A11、GPX4和AIFM2)异常改变及脂质代谢调节蛋白(SCD、ACSL4、NOX4和COX2)异常改变。MPA-CdTe QDs在巨噬细胞中激活铁自噬的发生,表现为处理后的巨噬细胞内FTH1和NCOA4异常降低、FTH1在溶酶体和过氧化物酶体中被水解(因为bafilomycin A1、E64d和MG-132可以抑制FTH1的水解)及NCOA4与LC3B的共定位程度增加。且铁自噬介导了 MPA-CdTe QDs引起的巨噬细胞铁死亡,因为bafilomycin A1、E64d和MG-132均不同程度地减轻了 MPA-CdTe QDs引起的ROS过量增多、LPO异常增多和游离Fe2+异常增多。MPA-CdTe QDs下调NRF2后促使ERK1/2磷酸化随后激活铁自噬,使FTH1水解后释放Fe2+。ML385和针对ERK1/2的siRNA分别加重和缓解了 MPA-CdTe QDs引起的FTH1水解,分别加重和缓解了 ROS、LPO及游离Fe2+的增多。最后,NRF2-ERK1/2-FTH1轴介导的铁死亡参与了 MPA-CdTe QDs升高巨噬细胞中促炎性细胞因子IL-1β和IL-6的过程。研究结论:1.MPA-CdTe QDs在体内和体外引起的促炎因子IL-1β和IL-6异常增多均表现出剂量依赖性:在小鼠体内,12.5μmol/Kg bw MPA-CdTe QDs引起明显的炎性反应;在体外RAW264.7细胞中,1.0μmol/LMPA-CdTe QDs引起显著的炎性反应。2.急性单次尾静脉注射1.25μmol/Kg bw MPA-CdTe QDs对小鼠没有明显毒作用。12.5μmol/Kg bw MPA-CdTe QDs导致小鼠肝脏、肾脏和血液中炎性因子IL-1β和IL-6异常升高,肝脏和肾脏中巨噬细胞浸润,持续至28 d,最终主要蓄积在肾脏和肝脏。3.首次观察到MPA-CdTe QDs在巨噬细胞中激活并增加的自噬通量表现为毒性作用,参与了巨噬细胞中促炎因子IL-1β和IL-6异常增多的过程。4.MPA-CdTe QDs在巨噬细胞中引发了选择性自噬——线粒体自噬和铁自噬,并且参与了细胞内炎性因子IL-1β和IL-6异常升高的过程。5.MPA-CdTe QDs在巨噬细胞中造成了细胞器损伤(线粒体和溶酶体)和多种细胞死亡形式(凋亡、铁死亡和自噬),从多个角度厘清预防MPA-CdTe QDs毒性产生的思路。6.MPA-CdTe QDs在巨噬细胞中激活的巨自噬和选择性自噬是由ROS-NRF2-ERK1/2通路参与的,它们是减轻MPA-CdTe QDs潜在毒作用的关键靶点。更多还原

【Abstract】 Aim:The aim of this study is to investigate the mechanism by which cadmium telluride quantum dots(CdTe QDs),a bioimaging material,cause abnormal elevation of inflammatory cytokines in mouse and macrophages,to investigate the involvement of macroautophagy and selective autophagy in this inflammatory response and to identify the key proteins and exposure doses.Finally,provide data to evaluate the application and toxic effects of CdTe QDs.Methods:1.3-mercaptopropionic acid(MPA)modified CdTe QDs(MPA-CdTe QDs)were synthesized by electrochemical method and characterized.Their morphology was observed by transmission electron microscopy(TEM),hydrodynamic size and zeta potential dispersed in ultrapure water and DMEM medium were measured by Malvern laser particle size analyzer,and the excitation and emission spectra were scanned by fluorescence spectrophotometer to determine the maximum excitation wavelength and maximum emission wavelength.2.Male C57BL/6 mice were acutely exposed to MPA-CdTe QDs(1.25 and 12.5μmol/Kg bw)by single tail vein injection.After 24 h,the Cd content in organs was measured by inductively coupled plasma-mass spectrometry(ICP-MS)to reflect the distribution and excretion of MPA-CdTe QDs in mice.The pathological damage of liver,kidney and lung were detected by HE staining.F4/80 and CD86 were marked through immunofluorescence to indicate macrophage infiltration in liver,kidney and lung.The levels of inflammatory cytokines IL1β and IL-6 in serum and organs were measured by enzyme linked immunosorbent assay(ELISA)and western blot(WB).The changes of inflammatory response-related proteins were measured by WB.And the above indicators were continuously observed until 28 d to reflect the recovery of mice after acute exposure to MPA-CdTe QDs.3.RAW264.7 macrophages were applied as in vitro model,the toxic effects of MPA-CdTe QDs on macrophages and the inflammatory changes in macrophages were examined from multiple angles.The concentrations of MPA-CdTe QDs being exposed to macrophages were screened by MTT assay.After that,the intracellular Cd concent after macrophages being exposed to 0.1,0.5 and 1.0μmol/L MPA-CdTe QDs were detected by ICP-MS.The level of LDH in cell culture medium was measured to reflect the damage of cell membranes,and FITC-Annexin-V and PI kit was used to detect the necrosis and apoptosis rates of macrophages.The genes changed by MPA-CdTe QDs in RAW264.7 cells were outlined by transcriptome sequencing.The phagocytic capacity of macrophages was detected using neutral red.The ratio of M1-and M2-macrophages was distinguished by labeling CD86 and CD206.Both real-time quantitative fluorescence PCR(qPCR)and WB were applied to determine the changes in various inflammatory cytokines at the transcriptional and protein levels,respectively.4.RAW264.7 cells were exposed to 0.1,0.5 and 1.0 μmol/LMPA-CdTe QDs for 24 h to observe the number of autophagosomes and autolysosomes by TEM.The levels of autophagy-related proteins ULK1,Beclinl,LC3B and p62 were measured by WB.LC3 turnover assay and dual fluorescence LC3 lentivirus-infected macrophages were used to detect the integrity of autophagic flow.Intracellular levels of reactive oxygen species(ROS)and nitric oxide was detected by fluorescent probes DCFH-DA and DAF-AM,respectively.The total antioxidant capacity of cells as well as the levels of oxidative and antioxidant substances or activity of antioxidant enzymes was detected by kits including glutathione(GSH),glutathione peroxidase(Gpx),superoxide dismutase(SOD),reduced coenzyme Ⅱ(nicotinamide adenine dinucleotide phosphate,NADPH)and NADPH oxidase(NOX).The involvement of ROS,NRF2,ERK1/2 and AMPK in MPA-CdTe QDs-induced macroautophagy in RAW264.7 cells and the mediating role of macroautophagy on IL-1β and IL-6 overexpression were verified using the antioxidant Trolox,NRF2 inhibitor ML385 and siRNA,respectively.The levels of lysosome-associated membrane proteins(LAMP1 and LAMP2)and cathepsins(CTSB and CTSD)were then measured in MPA-CdTe QDs-treated macrophages.The integrity of lysosomal membranes was examined using AO dye,changes in lysosomal pH were indicated using pH-dependent fluorescent probes LysoSensorTM Green DND-189 and Lyso-Tracker Red DND-99,and the inhibitor of lysosomal ATPase bafilomycin A1 verified that lysosomal acidification contributed to a partial increase in intracellular ROS.5.Next,the effects of 0.1,0.5 and 1.0μmol/L MPA-CdTe QDs on mitochondrial structure,function,number,morphology and mitochondrial quality control processes in macrophages were investigated.The effects of MPA-CdTe QDs on the mitochondrial structure of RAW264.7 cells were observed by TEM,the mitochondrial membrane potential was detected using the JC-1 probe,the network morphology and number of mitochondria were labeled with MitoTracker,the mitochondrial superoxide content was detected with MitoSOX Red,and the intracellular calcium ion content was detected with Fluo-3 AM probe.The intracellular ATP content was detected using the kit.We also detected the change of mitochondrial quality control-related proteins by WB,including:mitochondrial biogenesisrelated proteins(PGC-1α,NRF-1 and TFAM),mitochondrial fission-fusion-related proteins(DRP1,p-DRP1-Ser616,FIS1 and MFF;OPA1,MFN1 and MFN2)and mitophagy-related proteins(PINK1 and Parkin).Among them,focusing on the occurrence of mitophagy,the mitochondria-targeted superoxide dismutase MitoTEMPO,the NRF2 inhibitor ML385,siRNAs targeting ERK1/2,and lentiviral knockdown PINK1 were used to make sure the role of mitochondrial reactive oxygen species(mitoROS)and NRF2-ERK1/2 pathway in the activation of mitophagy by MPA-CdTe QDs,respectively.And then validating if mitoROS and NRF2-ERK1/2 pathway was involved in elevating IL-1β and IL-6 induced by MPACdTe QDs through mitophagy.6.Finally,the levels of malonic dialdehyde(MDA),a lipid peroxidation product,were detected in RAW264.7 cells treated with 0.1,0.5 and 1.0μmol/L MPA-CdTe QDs for 24 h.The intracellular lipid peroxide(LPO)was detected with Liperfluo fluorescent probe,the content of free divalent iron ion(Fe2+)in cytoplasm and mitochondria was detected using FerroOrange probe and Mito-FerroGreen probe,respectively.Various inhibitors of cell death(ferrostatin-1 and liproxstatin-1,inhibitors of ferroptosis;deferoxamine mesylate,iron ion chelator;3-methyladenine,autophagy inhibitor;chloroquine and bafilomycin A1,lysosomal inhibitors;Z-VAD-FMK,apoptosis inhibitor;necrostatin-1,necroptosis inhibitor;and VX765,pyroptosis inhibitor)were applied to verify that MPA-CdTe QDs caused ferroptosis in macrophages.And to determine the changes in GSH homeostasis regulatory proteins and lipid metabolism regulatory proteins associated with ferroptosis.Then the temporal trends(0-48 h)of FTH1 and NCOA4 proteins associated with ferritinophagy in macrophages were determined by WB.If NRF2-ERK1/2 pathway regulated ferritinophagy was verified by ML385 and siRNA.Finally,the participation of NRF2-ERK1/2-FTH1 pathway-mediated ferroptosis in the abnormal elevation of IL-1β and IL-6 induced by MPA-CdTe QDs was determined by ferrostatin-1,liproxstatin-1,deferoxamine mesylate,ML385 and siRNA.Results:1.Characterization of MPA-CdTe QDs.The prepared MPA-CdTe QDs were orange in color,homogeneously dispersed in ultrapure water.The TEM observation showed a round shape and distinct lattice structure with the particle size of 3.4±0.56 nm.The hydrodynamic size of MPA-CdTe QDs in ultrapure water and DMEM medium were 3.99±0.82 nm and 5.42 ±1.84 nm,respectively.The zeta potential in ultrapure water was-40.4±1.42 mV.The maximum excitation wavelength was 380 nm and the maximum emission wavelength was 603 nm at 380 nm excitation wave.2.Acute exposure to MPA-CdTe QDs induced abnormal elevation of inflammatory cytokines and macrophage infiltration in liver and kidney tissues in mice,and persisted until 28 d.After single exposure to 12.5μmol/Kg bw MPA-CdTe QDs by tail vein injection for 24 h,the growth of body weight was inhibited at 7 d and 28 d,but no effects on the weight and structure of organs were observed at 24 h,7 d and 28 d.At 24 h,12.5μmol/Kg bw MPACdTe QDs caused leukocytes elevation,macrophages infiltration,and abnormal elevation of IL-1β and IL-6 in the serum,liver and kidney of mice,and the same inflammatory changes were observed at 7 and 28 d.12.5μmol/Kg bw MPA-CdTe QDs exceeded the excretory capacity of the mice and redistributed in mice from 24 h to 28 d after exposure,with kidney and liver being the most important accumulation organs at 28 d.Compared with 12.5μmol/Kg bw,1.25μmol/Kg bw MPA-CdTe QDs were not significantly toxic to mice.3.MPA-CdTe QDs caused toxic effects on macrophages and abnormally increased IL-1βand IL-6.Based on the cell activity rate of RAW264.7 cells after 24 h exposure to MPACdTe QDs,0.1(cell activity rate of about 85%),0.5 and 1.0μmol/L were selected as exposure dose and 24 h were selected as exposure time.The uptake and excretion of MPA-CdTe QDs by RAW264.7 cells showed time-dependent(3-24 h)and dose-dependent(0.1-1.0μmol/L).1.0μmol/L MPA-CdTe QDs damaged cell membrane,promoted cell apoptosis,enhanced phagocytosis of macrophages,increased the proportion of M1 pro-inflammatory macrophages and decreased the proportion of M2 anti-inflammatory macrophages,and caused overexpression of IL-1β and IL-6 in RAW264.7 cells.In addition,the transcriptome sequencing showed that 1.0μmol/L MPA-CdTe QDs caused 177 differentially expressed genes(DEGs)in RAW264.7 cells,some gene sets were significantly altered,including "iron ion transport","glutathione metabolic process","interleukin-1 beta production","ERK1 and ERK2 cascade",and "lysosome","feroptosis","mitochondrial depolarization",etc.4.ROS-NRF2-ERK1/2-AMPK pathway-mediated macroautophagy was involved in MPA-CdTe QDs-induced overexpression of IL-1β and IL-6 in RAW264.7 cells.1.0μmol/L MPA-CdTe QDs activated macroautophagy and increased autophagic flux in RAW264.7 cells,as evidenced by an increase in the number of autophagosomes and autolysosomes observed by TEM,and an increase in ULK1,Beclin-1,and LC3BII proteins,with no significant increase in p62.The stubRFP-sensGFP-LC3 dual fluorescent lentivirus indicated that MPA-CdTe QDs caused quenching of GFP protein in macrophages,coupled with the inhibition of LC3BII and p62 hydrolysis in MPA-CdTe QDs-treated macrophages by the lysosomal inhibitors bafilomycin A1 and E64d(LC3 turnover assay),indicating the autophagic flux was unobstructed.In addition,MPA-CdTe QDs caused an imbalance between oxidation level and antioxidation capacity,increasing intracellular ROS and nitric oxide,depleting GSH but enhancing total cellular antioxidant capacity.The abnormal elevation of ROS caused by MPA-CdTe QDs downregulated NRF2 protein,which in turn increased the phosphorylation of ERK1/2 and AMPK which activated macroautophagy.In turn,this ROS-NRF2-ERK1/2-AMPK pathway-mediated macroautophagy was also participated in MPA-CdTe QDs-induced overexpression of IL-1β and IL-6 in RAW264.7 cells,and the role of this molecular pathway was validated by the antioxidant Trolox,the NRF2 inhibitor ML385,and siRNAs targeting AMPK and ERK1/2.5.MPA-CdTe QDs caused structural and functional abnormalities in macrophage lysosomes,which partially contributed to ROS overproduction.The transcriptome sequencing results suggested that 1.0μmol/L MPA-CdTe QDs caused significant enrichment of the "Lysosome" GO term and KEGG pathway in macrophages,and a significant decrease of lysosomal membrane proteins LAMP 1 and LAMP2.After treatment,lysosomal acidity was strengthened,and this lysosomal acidification was alleviated by the V-ATPase inhibitor bafilomycin A1.In addition,1.0μmol/L MPA-CdTe QDs caused a significant decrease in CTSB and CTSD proteins,and increased lysosomal membrane permeability in macrophages,which was dependent on lysosomal acidification.And lysosomal acidification also caused abnormal elevation of ROS,this was verified by bafilomycin A1,which also attenuated the abnormal elevation of IL-1β and IL-6 induced by MPA-CdTe QDs.6.mitoROS-and NRF2-ERKl/2-PINK1-mediated mitophagy contributed to the abnormal increase of IL-1β and IL-6 in RAW264.7 cells treated by MPA-CdTe QDs.The results showed that 1.0μmol/L MPA-CdTe QDs disrupted the mitochondrial cristae,decreased the mitochondrial membrane potential,increased mitoROS,and inhibited ATP synthesis in RAW264.7 cells.In addition,MPA-CdTe QDs increased the number of mitochondria in macrophages and shifted the mitochondrial network to a fragmented punctate,which was consistent with MPA-CdTe QDs promoting mitochondrial biogenesis,facilitating mitochondrial fission,and inhibiting mitochondrial fusion.Moreover,MPA-CdTe QDs activated mitophagy in RAW264.7 cells,as evidenced by significantly elevated PINK1 and Parkin proteins,increased co-localization of mitochondria and lysosomes,and increased co-localization of mitochondrial outer membrane receptor TOM20 and LC3B.MPA-CdTe QDs-induced elevated mitoROS synergistically activated mitophagy with phosphorylation of ERK1/2 regulated by decreased NRF2,verified by mitochondria-targeted superoxide dismutase MitoTEMPO,NRF2 inhibitor ML385,siRNA against ERK1/2.And PINK1mediated mitophagy was involved in MPA-CdTe QDs-induced overexpression of IL-1β and IL-6 in RAW264.7 cells,as the overexpression of IL-1β and IL-6 were alleviated after knockdown of PINK1 in RAW264.7 cells using lentivirus.7.Ferritinophagy-mediated ferroptosis was engaged in the overexpression of IL-1β and IL-6 in RAW264.7 cells by MPA-CdTe QDs.After 1.0μmol/L MPA-CdTe QDs treated RAW264.7 cells for 24 h,ferroptosis occurred as evidenced by GSH depletion,increased LPO,increased free Fe2+in cytoplasm and mitochondria,the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 and the iron chelator deferoxamine mesylate reduced cellular mortality,abnormal alterations in GSH homeostasis regulatory proteins(SLC7A11,GPX4 and AIFM2)and lipid metabolism regulatory proteins(SCD,ACSL4,NOX4 and COX2).MPA-CdTe QDs activated ferritinophagy in macrophages,as evidenced by abnormal decrease of FTH1 and NCOA4,the hydrolysis of FTH1 was happened in both lysosomes and peroxisomes(because bafilomycin A1,E64d and MG-132 inhibited the hydrolysis of FTH1)and increased co-localization of NCOA4 and LC3B.In MPA-CdTe QDs-treated macrophages,ferritinophagy mediated ferroptosis,as bafilomycin A1,E64d and MG-132 all differentially attenuated abnormally increased ROS,LPO and free Fe2+.MPA-CdTe QDs downregulating NRF2 prompted ERK1/2 phosphorylation followed by activation of ferritinophagy,and then Fe2+release after FTH1 hydrolysis.ML385 and siRNA targeting ERK1/2 aggravated and alleviated MPA-CdTe QDs-induced FTH1 hydrolysis and increases in ROS,LPO and free Fe2+,respectively.Finally,NRF2-ERK1/2-FTH1 axis-mediated ferroptosis was involved in the elevation of pro-inflammatory cytokines IL-1β and IL-6 in macrophages induced by MPA-CdTe QDs.Conclusion:1.MPA-CdTe QDs induced abnormal increases of pro-inflammatory cytokines IL-1β and IL-6 both in vivo and in vitro with dose-dependence:in mice,12.5μmol/Kg bw MPA-CdTe QDs induced a significant inflammatory response;in RAW264.7 cells,1.0μmol/L MPA-CdTe QDs induced significant inflammatory responses.2.Acute single tail vein injection of 1.25μmol/Kg bw MPA-CdTe QDs was not significantly toxic to mice.12.5μmol/Kg bw MPA-CdTe QDs caused abnormal elevation of inflammatory cytokines IL-1β and IL-6 in the liver,kidney and blood,and macrophage infiltration in liver and kidney,which lasted until 28 d and eventually accumulated mainly in kidney and liver.3.MPA-CdTe QDs-increased autophagic flow exerting toxic effects in treated macrophages were observed for the first time,which were involved in the abnormal increase of proinflammatory cytokines IL-1β and IL-6.4.MPA-CdTe QDs triggered selective autophagy(mitophagy and ferroptosis)in macrophages and selective autophagy was involved in the overexpression of intracellular inflammatory cytokines IL-1β and IL-6.5.MPA-CdTe QDs damaged organelles(including mitochondria and lysosomes)and educed multiple types of cell death(including apoptosis,ferroptosis and autophagy)in macrophages,and clarified the idea of preventing the toxicity of MPA-CdTe QDs from multiple perspectives.6.Macroautophagy and selective autophagy activated by MPA-CdTe QDs in macrophages were involved by the ROS-NRF2-ERK1/2 pathway,and they were key targets to mitigate the potential toxic effects of MPA-CdTe QDs.更多还原