【作者】 陈健；

【导师】 陆荣柱；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2024， 硕士

【摘要】 背景和目的:锰（manganese,Mn）是人体必需的金属元素,在人体的许多生理功能,尤其是神经发育中发挥重要作用。但是,锰也是一种公认的职业毒物和环境污染物。研究表明过量的锰暴露会导致神经损伤,但是其具体的机制尚未阐明,也缺乏有效的临床治疗手段。因此,探索锰的神经毒性机制有助于开发治疗锰中毒患者的新技术。铁死亡（ferroptosis,FPT）是由脂质过氧化（lipid peroxidation,LPO）驱动同时伴随着亚铁离子(Fe²⁺)积聚,区别于其他细胞死亡方式的一种调控性细胞死亡。许多研究表明,FPT与多种重金属毒性以及神经退行性疾病密切相关。因此,本研究采用ICR小鼠和多巴胺能（dopaminergic,DAergic）神经元样细胞（PC12）作为模型,通过体内外实验联合探索锰的DAergic神经毒性机制,为解释锰暴露诱导DAergic神经损伤提供新的视角以及为锰中毒治疗提供新的靶点。方法:1.体外模型构建及毒性实验:PC12细胞常作为DAergic神经细胞用于体外研究。我们先用四甲基偶氮唑蓝（methyl thiazolyl tetrazolium,MTT）筛选氯化锰（manganese chloride,Mn Cl₂）对PC12细胞的毒性浓度,然后以不同浓度（100μM、200μM、400μM和800μM）的Mn Cl₂处理PC12细胞24 h。MTT检测细胞存活率,显微镜白光下观察细胞形态,钙黄绿素/碘化丙啶（Calcein/PI）染色区分活细胞和死细胞,DCFH-DA探针检测细胞内活性氧（Reactive Oxygen Species,ROS）生成,JC-1探针检测线粒体膜电位（mitochondrial membrane potential,MMP）变化。2.体外实验FPT指标的检测以及FPT抑制剂铁抑素-1（ferrostatin-1,Fer-1）以及去铁胺（deferoxamine,DFO）验证:按照上述Mn Cl₂浓度处理PC12细胞,Ferro Orange探针检测细胞内Fe²⁺,BODIPY?581/591 C11探针检测细胞内脂质ROS,透射电镜（transmission electron microscope,TEM）观察细胞内线粒体形态变化,微孔板法检测细胞内GSH,WB检测GPX4、SLC7A11、p53以及转铁蛋白受体（Transferrin Receptor,Tf R）的表达,同时检测HIF-1α以及脯氨酸羟化酶2（prolyl hydroxylase 2,PHD2）。使用Fer-1和DFO进行验证,先用MTT筛选Fer-1和DFO的有效浓度,随后用Fer-1（10μM）或DFO（100μM）预处理PC12细胞3 h,接着用400μM Mn Cl₂与Fer-1或DFO共处理24 h后,检测FPT相关指标。3.体内模型构建以及实验:选用40只18-22 g的雄性ICR小鼠,随机分成4组[生理盐水对照组、Mn Cl₂组、Fer-1组和Fer-1+Mn Cl₂组],使其在受控环境中自适应至少一周,各组处理如下,（1）对照组:先连续三天腹腔注射0.9%的生理盐水,在接下来七天里每三天分别进行一次腹腔注射以及皮下注射0.9%生理盐水（两次操作间隔2 h内）;（2）Fer-1组:先连续三天腹腔注射1 mg/kg Fer-1,在接下来的七天里每三天腹腔注射一次1 mg/kg Fer-1,然后在两小时内皮下注射0.9%生理盐水;（3）Mn Cl₂组:先连续三天腹腔注射0.9%的生理盐水,在接下来的七天里每三天腹腔注射一次0.9%生理盐水,并在两小时内皮下注射100 mg/kg Mn Cl₂;（4）Fer-1+Mn Cl₂组:先连续三天腹腔注射1 mg/kg Fer-1,在接下来的七天里每三天腹腔注射一次1 mg/kg Fer-1,并在两小时内皮下注射100 mg/kg Mn Cl₂;最后一次处理后的第二天,所有小鼠进行行为学测试（旷场实验、平衡木实验以及爬杆实验,所有实验均未进行预训练）并记录数据。一天后,每组前三只小鼠心脏灌注,全脑组织横断位切片进行苏木精-伊红（hematoxylin-eosin,HE）染色,电感耦合等离子体质谱法（inductively coupled plasma-mass spectrometry,ICP-MS）检测小鼠脑组织内锰和铁含量,微孔板法检测血清中谷胱甘肽（glutathione,GSH）,免疫印迹法（Western blotting,WB）检测小鼠左脑纹状体区域的FPT相关蛋白溶质载体家族7成员11（solute carrier family 7 member 11,SLC7A11）,谷胱甘肽过氧化物酶4（glutathione peroxidase4,GPX4）以及肿瘤抑制蛋白53（tumor suppressor protein 53,p53）,同时检测缺氧诱导因子-1 alpha（hypoxia-inducible factor-1 alpha,HIF-1α）表达。4.HIF-1α激动剂二甲基草酸甘氨酸（dimethyloxalylglycine,DMOG）,抑制剂LW6干预验证HIF-1α在锰诱导的PC12细胞损伤中的作用以及p53小干扰核糖核酸（small interfering Ribonucleic Acid,siRNA）进行HIF-1α/p53通路验证:DMOG（1 m M）预处理PC12细胞3 h,接着DMOG和Mn Cl₂（400μM）共处理24h后,MTT检测细胞存活率,微孔板法检测细胞内GSH,BODIPY?581/591 C11探针检测细胞内脂质ROS,WB检测GPX4、SLC7A11、HIF-1α、p53表达。接着用LW6反向验证HIF-1α在锰诱导的PC12细胞损伤中的作用,LW6（10μM）预处理3 h后,400μM Mn Cl₂单独处理24 h,WB检测PC12细胞中HIF-1α的表达,MTT检测细胞存活率。p53-siRNAs干预进一步验证HIF-1α/p53通路,PCR检测HIF-1α、p53和SLC7A11的转录水平,从四个片段中挑选出敲减效率达到70%以上的片段进行后续实验,MTT检测细胞活性,WB检测细胞内HIF-1α、p53和SLC7A11表达。结果:1.锰暴露对PC12细胞的毒性效应:与对照组相比,Mn Cl₂组的PC12细胞存活率浓度依赖性地下降。同时,锰暴露后,显微镜下观察到PC12细胞密度下降,活死细胞染色结果也表明死细胞数量增多,细胞的MMP降低,ROS和脂质ROS生成增多,Fe²⁺升高。WB结果表明,p53和Tf R表达升高,SLC7A11和GPX4表达降低,HIF-1α出现了下降同时促进其降解的蛋白PHD2升高。2.Fer-1和DFO对锰暴露后PC12细胞的影响:MTT结果表明,高浓度的Fer-1（20和40μM）干预能部分缓解锰导致的PC12细胞活性降低。与Mn Cl₂组相比,Fer-1干预后,PC12细胞GSH回升,MMP上升,Fe²⁺蓄积减弱,ROS和脂质ROS生成下降。与体内结果相似,FPT相关蛋白以及HIF-1α表达也出现了回复。DFO干预后虽缓解了PC12细胞内Fe²⁺蓄积,但却加重了锰诱导的细胞活性下降。3.锰暴露对ICR小鼠的毒性效应以及Fer-1的保护作用:体内实验结果表明,四组小鼠的体重变化无明显差异,与对照组相比,Mn Cl₂组脑中锰浓度升高且Fer-1干预并不能缓解这种变化。同时,Mn Cl₂组出现运动障碍和脑病理改变,Fer-1干预后运动迟缓现象以及脑病理变化减弱。此外,相比于对照组,Mn Cl₂组的脑器官系数下降,脑中总铁浓度升高,纹状体组织中SLC7A11、GPX4下降,p53升高以及HIF-1α下降。Fer-1干预后,上述指标（除了p53）都得到部分程度的缓解。血清中GSH含量检测结果表明,Mn Cl₂组GSH含量升高,Fer-1干预后其浓度进一步升高。4.HIF-1α激动剂DMOG和抑制剂LW6对锰暴露后PC12细胞的影响:与Mn Cl₂组相比,DMOG干预后PC12细胞活性升高,GSH含量升高,脂质ROS水平下降,与Fer-1干预结果相似,逆转了p53的过表达,FPT相关蛋白（SLC7A11和GPX4）以及HIF-1α表达也出现了回复。而LW6预处理进一步降低了PC12细胞内HIF-1α表达,同时降低了其细胞活性。5.p53-siRNA对锰暴露后PC12细胞的作用以及通路验证:p53-siRNA2对p53的抑制率达到70%且对HIF-1α和SLC7A11无明显影响,因此选择其进行后续实验。总共分为六组[Control组、siRNA组、阴性对照（negative control,NC）组、Mn Cl₂组、Mn Cl₂+siRNA组和Mn Cl₂+NC组],与Mn Cl₂+NC组相比,Mn Cl₂+siRNA组在不改变HIF-1α表达的情况下,PC12细胞存活率升高,p53下降,SLC7A11升高。结论:1.锰暴露可诱导PC12细胞损伤且这种损伤与FPT有关;2.尽管DFO可降低细胞内Fe²⁺蓄积,但却加重细胞损伤,提示螯合铁并不能直接调控锰诱导的PC12细胞FPT样损伤;3.锰暴露会引起ICR小鼠的运动平衡功能障碍,大脑中总铁水平升高,纹状体组织病理变化以及FPT相关通路蛋白变化,且Fer-1能部分拮抗这种损伤,提示锰暴露可能会诱导DAergic神经FPT;4.HIF-1α激动剂DMOG抑制了锰暴露引起的p53过表达,使SLC7A11以及GPX4表达回复,从而拮抗锰诱导的PC12细胞FPT,而HIF-1α抑制剂LW6加重了锰诱导的PC12细胞损伤,提示HIF-1α对锰暴露诱导的PC12细胞FPT有一定的保护作用;5.p53-siRNA下调了细胞内的p53表达,使下游蛋白SLC7A11表达回升,从而抑制锰诱导的PC12细胞FPT。但是p53调控并不会影响锰暴露引起的HIF-1α抑制,而HIF-1α上调却能抑制锰暴露引起的p53过表达,提示在锰诱导的PC12细胞FPT通路中,HIF-1α是p53的上游蛋白。更多还原

【Abstract】 Background and Objective:Manganese（Mn）functions as an essential metal element in human body and plays a crucial role in carrying out various physiological functions,especially in maintaining neural development.However,Mn is a well-known occupational poison and environmental contaminant simultaneously.Studies have shown that excessive Mn exposure can cause neurological damage,while the specific mechanism underlying it has not been elucidated.Besides,neurological damage induced by excessive Mn exposure still lacks an effective clinical treatment.Therefore,the study of the neurotoxicity mechanism of Mn is helpful to provide theoretical support for the treatment of manganism in clinical practice.Ferroptosis（FPT）is a regulated cell death driven by lipid peroxidation（LPO）and accompanied by intracellular Fe²⁺accumulation,which is distinct from other cell death modes.Many studies have indicated that FPT is closely related to the toxicity of many heavy metals and a variety of neurodegenerative diseases.Therefore,in this study,ICR mice and dopaminergic neuron-like cells（PC12）were used as models to explore the mechanism of Mn neurotoxicity through in vitro and in vivo experiments,so as to provide a new perspective for explaining the damage of dopaminergic neuron induced by Mn exposure and provide a new target for the treatment of manganism.Methods:1.The construction and tests of in vitro modelsMethyl thiazolyl tetrazolium（MTT）was used to screen the toxic concentrations of Mn chloride（Mn Cl₂）,and then PC12 cells were treated with various concentrations of Mn Cl₂（100μM,200μM,400μM and 800μM）for 24 h.MTT was used to assay cell viability.The cell morphology was observed under a microscope.Live cells and dead cells were distinguished by Calcein/PI staining.Intracellular Reactive Oxygen Species（ROS）production was detected with DCFH-DA probe.The mitochondrial membrane potential（MMP）was detected by JC-1 probe.2.The detection of the FPT indicators in vitro and the FPT inhibitor ferrostatin-1（Fer-1）and deferoxamine（DFO）for validation of FPTPC12 cells were treated at the Mn Cl₂ concentrations described above.Intracellular Fe²⁺and lipid ROS were detected with Ferro Orange and BODIPY?581/591 C11 probes,respectively.The morphological changes of mitochondria were observed under a transmission electron microscope（TEM）.Intracellular GSH was detected by microplate method.The expressions of GPX4,SLC7A11,p53 and transferrin receptor（Tf R）were detected by Western blotting.HIF-1αand prolyl hydroxylase 2（PHD2）expressions were also detected.Fer-1 and DFO were applied for validation of FPT.The effective concentrations of Fer-1 and DFO were screened by MTT,and then PC12 cells were pretreated with Fer-1（10μM）and DFO（100μM）for 3 h,respectively,followed by co-treatment with 400μM Mn Cl₂ and Fer-1 or DFO for 24 h.After the treatment,FPT-related indicators were detected.3.The construction and experiments of In vivo models:Forty male ICR mice with the body weight（b.w）between 18-22 g were randomly divided into four groups[saline control group,Mn Cl₂ group,Fer-1 group and Fer-1+Mn Cl₂ group]to acclimate in a controlled environment for at least one week.Each group was treated as follows:（1）Control group:0.9%normal saline was injected intraperitoneally for three consecutive days,and 0.9%normal saline was injected intraperitoneally and subcutaneously every three days for the next seven days.The interval between two injections is limited to 2 h.（2）Fer-1 group:Fer-1 at the dosage of 1 mg/kg b.w was injected i.p.for three consecutive days,and then Fer-1 was injected intraperitoneally every three days for the next seven days,followed by a subcutaneous injection of 0.9%saline within 2 h.（3）Mn Cl₂ group:0.9%normal saline was intraperitoneally injected for three consecutive days,and 0.9%normal saline was intraperitoneally injected every three days for the next seven days,and Mn Cl₂ at the dosage of 100 mg/kg b.w was injected subcutaneously within 2 h.（4）Mn Cl₂+Fer-1 group:Fer-1 at the dosage of 1 mg/kg b.w was intraperitoneally injected for three consecutive days,and 1 mg/kg Fer-1 was intraperitoneally injected every three days for the next seven days,and Mn Cl₂ at the dosage of 100 mg/kg b.w was injected subcutaneously within 2 h.On the next day after the final treatment,all mice underwent behavioral tests（open field test,beam test and pole test,all without pre-training）and data were recorded.On the other day,the first three mice per group were cardiac perfused,and the whole brain tissue were cross-sectioned to slices and stained with hematoxylin-eosin（HE）.Inductively coupled plasma-mass spectrometry（ICP-MS）was used to detect Mn and iron content in the right brain of mice.Glutathione（GSH）in serum was detected by microplate method.Western blotting（WB）was used to detect FPT-related proteins in the left striatum region of mice,including solute carrier family 7 member 11（SLC7A11）,glutathione peroxidase 4（GPX4）,tumor suppressor protein53（p53）.The expression of hypoxia-inducible factor-1 alpha（HIF-1α）was also detected.4.HIF-1αagonist dimethyloxalylglycine（DMOG）and inhibitor LW6 for verifying the role of HIF-1αin Mn-induced PC12 cell damage,and p53 small interfering ribonucleic acid（siRNA）for verifying the HIF-1α/p53 pathwayPC12 cells were pretreated with DMOG（1 m M）for 3 h,followed by co-treatment with DMOG and Mn Cl₂（400μM）for 24 h.MTT was used to measure cell viability.Intracellular GSH was detected by microplate method.BODIPY?581/591 C11 probe was used to detect intracellular lipid ROS.Western blotting was used to detect the expressions of GPX4,SLC7A11,HIF-1α,and p53.Then LW6 was used to verify the role of HIF-1αin Mn-induced PC12 cell injury.After pretreatment with LW6（10μM）for 3 h,PC12 cells were treated with400μM Mn Cl₂alone for 24 h.The HIF-1αexpression and cell viability were detected.The HIF-1α/p53 pathway was further verified by p53-siRNAs intervention.The transcriptional levels of HIF-1α,p53 and SLC7A11 were detected by PCR.Then,the siRNA fragment with knockdown efficiency of more than 70%was selected from the four fragments for subsequent experiments.The cell viability and proteins expression（HIF-1α,p53,SLC7A11）were detected.Results:1.Toxic effects of Mn Cl₂ exposure on PC12 cells:Compared with the control group,the viability of PC12 cells in the Mn Cl₂ group decreased in a concentration-dependent manner.Meanwhile,after Mn exposure,a decrease in PC12 cell density was observed under the microscope.The results of Calcein/PI staining also showed that the number of dead cells increased.In addition,MMP decreased and morphological changes in mitochondria was also observed.Increased production of ROS and lipid ROS,and the level of Fe²⁺also occurred in PC12 cells of the Mn Cl₂ group.WB showed that the expressions of p53 and Tf R increased,and the expressions of SLC7A11 and GPX4decreased.Besides,HIF-1αwas decreased and PHD2,which promoted HIF-1αdegradation,was increased.2.Effects of Fer-1 and DFO on PC12 cells exposed to Mn:MTT assay showed that higher concentrations of Fer-1（20 and 40μM）could partially alleviate the Mn-induced reduction of PC12 cell viability.Compared with the Mn Cl₂ group,Fer-1 treatment increased GSH content and MMP in PC12 cells.Fer-1 treatment also alleviated the accumulation of Fe²⁺and decreased the production of ROS as well as lipid ROS.Consistent with the in vivo results,the expression of FPT-related proteins and HIF-1αwas also restored.Although DFO decreased the accumulation of Fe²⁺in PC12 cells,it aggravated the decline of cell viability induced by Mn.3.Toxic effects of Mn Cl₂ exposure and protective effects of Fer-1 in ICR mice:The results of in vivo experiments showed that there was no significant difference in body weight change among the four groups of mice.Compared with the control group,the Mn Cl₂group had an increased brain Mn concentration,and Fer-1 intervention could not alleviate this change.Meanwhile,Mn Cl₂ group showed movement disorders and brain pathological changes,and Fer-1 intervention attenuated the phenomenon of bradykinesia and brain pathological changes.In addition,compared with the control group,the organ coefficient of brain in the Mn Cl₂ group was decreased and the total iron concentration in brain was increased.Besides,in the Mn Cl₂ group,SLC7A11 and GPX4 were decreased,while p53 was increased.The level of HIF-1αwas also decreased.After Fer-1 intervention,all the above indicators（except p53）were partially alleviated.The detection of GSH content in serum showed that GSH in Mn Cl₂ group was increased,and its concentration was further increased after the intervention of Fer-1.4.Effects of HIF-1αagonist DMOG and inhibitor LW6 on PC12 cells treated with Mn:Compared with the Mn Cl₂ group,PC12 cell viability and GSH content increased after DMOG intervention.Besides,lipid ROS levels decreased.Similar with the results of Fer-1intervention,the overexpression of p53 was reversed.In addition,the expressions of FPT-related proteins（SLC7A11 and GPX4）and HIF-1αwere also recovered.However,LW6pretreatment further reduced the expression of HIF-1αand cell viability in PC12 cells.5.Effect of p53-siRNA on PC12 cells after Mn exposure and pathway verification:The inhibition rate of p53 by p53-siRNA2 reached 70%and had no significant effect on HIF-1αand SLC7A11,so p53-siRNA2 was selected for subsequent experiments.PC12 cells were divided into six groups[Control group,siRNA group,negative control（NC）group,Mn Cl₂ group,Mn Cl₂+siRNA group and Mn Cl₂+NC group].Compared with the Mn Cl₂+NC group,the viability of Mn Cl₂+siRNA group increased.p53-siRNA intervention decreased the level of p53,and increased the level of SLC7A11 without changing the expression of HIF-1α.Conclusion:1.Mn exposure can cause PC12 cells damage.Meanwhile,it causes changes in FPT-related indicators in PC12 cells,and Fer-1 can partially antagonize this damage,suggesting that exposure to Mn induces FPT in PC12 cells.2.DFO can alleviate the accumulation of intracellular Fe²⁺,but still aggravate the Mn damage,indicating that the regulation of Mn-induced FPT-like damage in PC12 cells cannot depend on directly chelation of iron.3.Mn exposure can cause motor dysfunction,pathological changes of brain and the changes of FPT-related indicators in ICR mice,and Fer-1 can partially antagonize these damages,implying that exposure to Mn induces DAergic FPT.4.HIF-1αagonist DMOG can antagonize Mn-induced FPT in PC12 cells through reversing the overexpression of p53 and restoring the expression of SLC7A11 and GPX4,while HIF-1αinhibitor LW6 can aggravate Mn-induced injury in PC12 cells,implying that HIF-1αhas protective effects on Mn-induced FPT in PC12 cells.5.p53-siRNA can downregulate the expression of p53 and upregulate the expression of SLC7A11,thereby inhibiting the Mn-induced FPT in PC12 cells.However,p53 regulation does not affect the inhibition of HIF-1αinduced by Mn exposure,while up-regulation of HIF-1αreversed the overexpression of p53 induced by Mn exposure,suggesting that HIF-1αis an upstream protein of p53 in the FPT pathway induced by Mn exposure in PC12 cells.更多还原