【作者】 刘敏；

【导师】 邵静；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2023， 博士

【副题名】基于神经细胞脱碘酶（Dio2和Dio3）的研究

【摘要】 目的:多溴联苯醚(Polybrominated Diphenyl Ethers,PBDEs)是一类广泛应用于工业和家庭产品中的溴代阻燃剂。由于PBDEs易从产品中释放入环境,且在动物体和人体中不断蓄积,而成为全球性有机污染物。PBDEs的同系物有多种,其中最具代表性的为十溴联苯醚(BDE209,高溴联苯醚)和四溴联苯醚(BDE47,低溴联苯醚),前者仍在大量使用,而后者毒性最大。因此,本课题选取BDE209和BDE47为研究对象探究PBDEs毒性的作用机制。目前,PBDEs的毒性主要体现在神经毒性,内分泌干扰毒性,生殖毒性以及致癌性四个方面,其中以前两个尤为重要。有观点认为PBDEs的神经毒性与机体甲状腺激素(Thyroid Hormones,TH)代谢紊乱密切相关,因此研究PBDEs与TH代谢的关系具有重要意义。TH具有维持机体正常生理功能的作用,并且不同组织细胞对TH需求不同,其在机体的稳态受脱碘酶(Deiodinase,Dios)的严格调控。Dios在神经系统的分布、表达及功能具有独特性,在不同脑区、不同发育阶段的表达也不同。在中枢神经系统分布的脱碘酶主要为Dio2和Dio3,而两者的组织分布与分子功能具有显著不同。Dio2主要分布于神经胶质细胞,催化T4生成活性产物T3,激活TH功能;Dio3主要分布于神经元细胞膜,催化T3生成无活性T2或催化T4转变为无活性rT3,而灭活TH功能。因此,基于Dios探究PBDEs对TH代谢的影响,进而揭示PBDEs介导神经细胞毒性的分子机制具有重要意义。神经胶质细胞和神经元细胞的细胞株有很多种,其中值得注意的是H4细胞和SK-N-AS细胞。H4细胞为几乎只表达Dio2不表达Dio3的神经胶质细胞,而SK-N-AS为几乎只表达Dio3不表达Dio2的神经元细胞。这两株独特的细胞系为研究脱碘酶在局部TH代谢紊乱造成神经损伤的作用提供了理想的体外细胞模型。细胞共培养体系是可以在体外模拟复杂的体内环境,实现细胞间信息的相互沟通的实验方法,目前已被广泛应用于现代细胞研究中。鉴于神经系统TH调节的复杂性,体内(动物)研究解决TH调节的反馈通路和机制较为困难,因此,细胞共培养体系为研究神经局部TH代谢紊乱的机制提供了合理的研究模型。本课题分为三个章节,以H4细胞和SK-N-AS细胞为研究模型,系统阐明PBDEs对神经系统局部TH代谢干扰的表型:即甲状腺功能减退。进一步机制研究,揭示了 PBDEs对神经系统局部TH干扰造成神经毒性的作用靶点:神经胶质细胞的Dio2以及神经元细胞的Dio3。方法:第一部分PBDEs对神经胶质细胞脱碘酶(Dio2)的影响和机制以及在局部TH水平和功能调节中的作用。1.采用神经胶质细胞H4细胞为体外研究模型,通过CCK-8法、细胞克隆形成实验以及实时无标记细胞增值检测方法,检测BDE209、BDE47对H4细胞的细胞毒性。2.通过LC/MS/MS方法和ELISA方法检测BDE209、BDE47对H4细胞TH代谢的干扰作用。3.运用Western、qPCR和siRNA干扰方法,确定BDE209诱导神经胶质细胞毒性的作用靶点为Dio2。4.通过Western、Co-IP、激光共聚焦以及分子模拟对接方法,揭示BDE209通过自噬溶酶体途径降低Dio2稳定性,诱导其降解的分子机制。第二部分PBDEs对神经元脱碘酶(Dio3)的影响以及在局部TH水平和功能调节中的作用。1.采用神经元细胞SK-N-AS细胞为体外研究模型,通过CCK-8法和细胞克隆形成实验,检测BDE47、BDE209对SK-N-AS细胞的细胞毒性。2.通过ELISA方法检测BDE47对SK-N-AS细胞TH代谢的干扰作用。3.运用Western、qPCR和siRNA干扰方法,确定BDE47诱导神经元细胞毒性的作用靶点为Dio3。4.运用生物信息学分析、基因编辑、荧光素酶报告基因以及qPCR方法预测并验证了调控Dio3表达microRNA——miR-24-3p。5.运用siRNA干扰、western、qPCR以及ELISA方法,明确BDE47以miR-24-3p依赖的方式抑制Dio3的表达。第三部分基于细胞共培养方法,探讨PBDEs通过对胶质细胞Dio2/神经元Dio3影响导致神经系统局部TH水平和功能紊乱。1.以神经胶质细胞H4和神经元细胞SK-N-AS为体外研究模型,采用插入式细胞共培养方法以及单细胞培养方法,通过CCK-8方法检测共培养体系下,PBDEs对神经胶质细胞和神经元细胞的毒性,并比较两种培养体系下的差别。2.通过ELISA和Western方法检测共培养体系下,PBDEs对胶质细胞Dio2及神经元细胞Dio3表达与活性的影响,并比较两种培养体系下的差别。结果:第一部分PBDEs对神经胶质细胞脱碘酶(Dio2)的影响和机制以及在局部TH水平和功能调节中的作用。1.PBDEs对神经胶质细胞H4的细胞毒性:(1)CCK-8,细胞克隆形成以及实时无标记细胞增殖实验显示,BDE209对H4细胞无急性细胞毒作用,但可缓慢抑制H4细胞增殖,说明BDE209对神经胶质细胞的细胞毒是慢性的,而非急性的。(2)BDE47对神经神经胶质细胞的增殖无显著抑制作用。2.PBDEs通过影响神经胶质细胞Dio2导致局部TH紊乱:(1)PBDEs造成神经胶质细胞局部TH紊乱,导致甲状腺功能减退:LC/MS/MS实验显示,BDE209可有效降低H4细胞培养基中内源性T3、T4的含量,造成局部甲状腺功能减退;而BDE47对H4细胞培养液中内源性T3和T4的含量无显著影响。因此,我们选取BDE209为研究对象,探究其造成神经毒性的分子机制。(2)BDE209对脱碘酶Dio2和Dio3表达的影响:选取H4细胞(几乎只表达Dio2)和SK-N-AS细胞(几乎只表达Dio3)为体外研究模型研究发现,BDE209对神经胶质细胞Dio2的抑制作用并非在于转录表达调控,而可能在于降低蛋白的稳定性;BDE209对神经元细胞Dio3蛋白无显著抑制作用。(3)BDE209对H4细胞TH转运蛋白的影响:BDE209不影响H4细胞中OATP14蛋白水平,但显著增加了 MCT8的蛋白含量。(4)BDE209对H4细胞T3核受体基因表达和核受体依赖性基因表达的影响:BDE209显著降低核受体下游依赖性基因RC3,UCP2,ENPP2的表达,而对H4细胞中THRA,THRB的表达无显著影响。(5)BDE209染毒H4细胞对外源性T4的调节能力:BDE209显著抑制H4细胞外源性T4向T3的转变。3.BDE209对H4细胞Dio2影响的可能机制:(1)BDE209导致Dio2蛋白表达水平降低与UPS(泛素-蛋白水解酶途径)和自噬途径-溶酶体途径的关系:BDE209通过一条非蛋白酶体途径,即自噬溶酶体途径诱导Dio2泛素化降解。(2)BDE209通过促进WBS-1(泛素化酶)与Dio2的结合,抑制USP33(去泛素化酶)与Dio2的结合提高Dio2泛素化水平,促进p62/Dio2复合物的生成,降低Dio2蛋白水平。(3)BDE209通过与T4竞争Dio2活性空腔,从而抑制Dio2催化活性。第二部分PBDEs对神经元脱碘酶(Dio3)的影响以及在局部TH水平和功能调节中的作用。1.PBDEs对神经元细胞SK-N-AS的细胞毒性:CCK-8和细胞克隆形成实验显示,BDE47明显抑制SK-N-AS细胞的增殖能力,而BDE209对神经元细胞无显著影响。2.BDE47通过影响神经元细胞Dio3导致局部TH紊乱:(1)BDE47介导神经元细胞局部TH紊乱:ELISA方法显示,BDE47显著降低SK-N-AS细胞培养基中内源性rT3和T3的生成,而BDE209对此无显著抑制作用。因此,本部分我们以BDE47为研究对象,探究其诱导神经元局部TH代谢紊乱的分子机制。(2)BDE47对神经元细胞SK-N-AS脱碘酶Dio3表达的影响:siRNA干扰以及Western实验显示,Dio3 mRNA水平和蛋白水平受BDE47显著抑制,并且是BDE47介导神经元细胞毒性的关键靶点。(3)BDE47对SK-N-AS细胞TH转运蛋白的影响:BDE47可造成SK-N-AS细胞MCT8的蛋白水平显著升高。(4)BDE47不影响SK-N-AS细胞中THRA,THRB的表达,但可显著降低核受体下游依赖性基因RC3,UCP2,ENPP2的表达。(5)BDE47通过抑制Dio3活性抑制SK-N-AS细胞对外源性T4调节能力。3.BDE47对SK-N-AS细胞Dio3表达影响的可能机制:(1)miR-24-3p是调控Dio3表达的关键因子:生物信息学分析,基因编辑以及荧光素酶报告基因实验证实了 miR-24-3p通过与Dio3的3’-UTR结合,抑制其表达。(2)qPCR、荧光素酶报告基因以及ELISA实验显示BDE47通过上调miR-24-3p的表达来抑制Dio3的表达。(3)BDE47对自噬溶酶体途径的影响:BDE47介导神经元细胞Dio3蛋白水平的降低与自噬溶酶体途径无关。第三部分基于细胞共培养方法,探讨PBDEs通过对胶质细胞Dio2/神经元Dio3影响导致神经系统局部TH水平和功能紊乱:1.BDE209对共培养体系甲状腺功能的影响:(1)BDE209可造成共培养体系甲减,但甲减的程度较单细胞培养体系较轻。(2)BDE209对单细胞培养体系和共培养体系的神经胶质细胞均具有毒性作用;而对单细胞培养体系神经元细胞活力无抑制作用,对共培养体系神经元细胞活力显著抑制。(3)BDE209对单细胞培养体系和共培养体系的神经胶质细胞Dio2的表达与活性均具有抑制作用,但对共培养体系的抑制作用稍弱于单细胞培养体系。(4)BDE209对单细胞培养体系神经元细胞Dio3的表达与活性均无抑制作用,但对共培养体系有轻微抑制作用。2.BDE47对共培养体系甲状腺功能的影响:(1)BDE47可造成共培养体系甲减,但甲减的程度较单细胞培养体系较轻。(2)BDE47对单细胞培养体系和共培养体系的神经元细胞均具有毒性作用,对神经胶质细胞均无显著抑制作用。(3)BDE47对单细胞培养体系神经胶质细胞Dio2的表达与活性无显著影响,但会造成共培养体系神经胶质细胞Dio2活性轻微升高。(4)BDE47对单细胞培养体系和共培养体系的神经元细胞Dio3的表达与活性均具有抑制作用。结论:综上所述,本课题探究了环境有机污染物PBDEs介导神经细胞毒性的分子机制。不同类型的PBDEs介导神经细胞毒性的机制不同:高溴联苯醚BDE209通过诱导神经胶质细胞自噬,促进Dio2泛素化和降解,从而导致局部TH代谢紊乱(甲减),产生神经毒性;低溴联苯醚BDE47通过提高miR-24-3p表达,靶向抑制神经元细胞中Dio3表达,导致神经元细胞中TH代谢紊乱(甲减),产生神经毒性。此外,PBDEs在细胞共培养体系中对神经胶质细胞和神经元细胞的局部TH代谢紊乱和细胞毒性仍然存在,但由于神经胶质Dio2——神经元Dio3对局部TH协同调节的作用,对神经毒性作用有轻微减轻。更多还原

【Abstract】 Objective:Polybrominated diphenyl ethers(PBDEs)are a class of brominated flame retardants widely used in industrial and household products.PBDEs can freely release from products into the environment,accumulate in animals including human beings and become global organic pollutants.Among 209 PBDE congeners,decabromodiphenyl ether(BDE209),the highest bromodiphenyl ether,is still in heavy use,while tetrabromodiphenyl ether(BDE47),the lower bromodiphenyl ether,is considered the most toxic.Thus,we selected BDE209 and BDE47 as model compounds to explore the mechanism of PBDEs neurotoxicity.The toxicity of PBDEs is classified as neurotoxicity,endocrine disruption toxicity,reproductive toxicity and tumorigenicity,among which the first two are particularly important.It is believed that neurotoxicity of PBDEs is closely related to the disorder of thyroid hormone(TH)metabolism,and it is of great significance to clarify the relationship between PBDEs and TH metabolism.TH plays a crucial role in maintaining normal physiological functions especially during neurodevelopment.Different tissue cells have different requirements for TH,and its homeostasis in local areas is strictly regulated by Deiodinase(Dios).Distribution,expression,and function of Dios in the central nervous system(CNS)are unique,and expression patterns are modulated across different brain regions and trimmed along different developmental stages.Dio2 and Dio3 are the major deiodinases in the CNS.Dio2 is mainly distributed in glial cells,catalyzing T4 to generate active product T3 as functional TH.Dio3 is mainly expressed in neuronal cell membrane,catalyzing formation of inactive T2 from T3 or conversion of T4 to inactive rT3 for optimizing TH levels.In the present study,we explored the effect of PBDEs on Dio2 and Dio3 and their potential roles in PBDEs mediated TH disorders and neurotoxicity using two in vitro models,a glial cell line,notably H4 cells,and a neuronal cell line,so called SK-N-AS cells.H4 cells are known to solely express Dio2,and SK-N-AS cells mainly express Dio3,making them the unique and ideal in vitro cell models for fulfilling our research objectives.This study was categorized into three chapters to systematically clarify the role of Dio2 and Dio3 and thus mechanistic basis underlying PBDEs interference of TH metabolism in glial cells and neuronal cells.The co-culture system of H4 and SK-N-AS cells was to simulate the internal environment by building the communication between two cell types.Due to the complexity of nervous system regulation,it would be very difficult to conduct in vivo(animal)studies for feedback pathway and mechanism of TH regulation.Cell co-culture system provided a reasonable research model for studying the mechanism of local TH metabolism disorder.Methods:Chapter 1:The mechanism of PBDEs on deiodinase 2(Dio2)and the effect of PBDEs in the interference of local TH level and function in glial cells.1.Glial H4 cells were utilized as the in vitro model,and the cytotoxicity of BDE209 and BDE47 on H4 cells was assessed using the CCK-8 method,cell cloning and formation experiments,and real-time marker-free cell proliferation detection.2.Interference of BDE209 and BDE47 on TH metabolism in H4 cells was evaluated through LC/MS/MS and ELISA methods.3.Dio2 was identified as the target of BDE209 induced glial cytotoxicity via Western blotting,qPCR analysis,and siRNA interference techniques.4.The molecular mechanism underlying BDE209-induced degradation of Dio2 via autophagic lysosome pathway was elucidated using Western blotting,Co-IP,confocal microscopy,and molecular simulation docking methods.Chapter 2:The mechanism of PBDEs on deiodinase 3(Dio3)and the effect of PBDEs in the interference of local TH level and function regulation in neuronal cells.1.Using SK-N-AS cells as an in vitro model,the cytotoxicity of BDE47 and BDE209 was assessed via clone formation and CCK-8 assays.2.The impact of BDE47 on TH metabolism in SK-N-AS cells was evaluated using ELISA.3.Dio3 was identified as a target for inducing neuronal cytotoxicity through Western,qPCR and siRNA interference methods.4.Bioinformatics analysis,gene editing,luciferase assays and qPCR were employed to predict and validate the involvement of miRNAs in regulating Dio3 expression,specifically miR-24-3p.5.Combination of siRNA interference,Western,qPCR and ELISA methods revealed that BDE47 inhibited Dio3 expression in a miR-24-3p dependent manner.Chapter 3:Establishment of H4/SK-N-AS co-culture and verification on role of Dio2 and Dio3 in PBDEs induced local TH level and neurotoxicity in the CNS.1.Glial cell H4 and neuronal cell SK-N-AS co-culture system was established to simulate internal interaction of glial cells and neuronal cells.2.The impact of PBDEs on expression and activity of glial cell Dio2 and neuron cell Dio3 in a co-culture system was assessed using ELISA and Western blotting techniques,with subsequent comparison to discern differences between the two culture systems.Results:Chapter 1:The mechanism of PBDEs on deiodinase 2(Dio2)and the effect of PBDEs in the interference of local TH level and function in glial cells.1.Cytotoxicity of PBDEs in H4 glial cells:CCK-8 assay,cell cloning assay and realtime unlabeled cell proliferation assay demonstrated that BDE209 did not exert acute cytotoxicity on H4 cells,but could gradually impede the proliferation of H4 cells,indicating that the toxic effect of BDE209 in glial cells was chronic rather than acute.On the other hand,BDE47 had no significant inhibitory effect on neuroglial cell proliferation.2.PBDEs can cause local TH disorders by affecting Dio2 in H4 glial cells:(1)PBDEs can induce local thyroid hormone disorders in glial cells,leading to hypothyroidism.LC/MS/MS experiments have demonstrated that BDE209 effectively reduced endogenous T3 and T4 content in H4 cell medium,resulting in localized hypothyroidism.In contrast,BDE47 has no significant effect on endogenous T3 and T4 content in H4 cell culture medium.Therefore,we selected BDE209 as our model compound to explore its molecular mechanism of neurotoxicity in glial cells.(2)Effects of BDE209 on Dio2 and Dio3 expression:H4 cells,which mainly expressed Dio2,and SK-N-AS cells,which mainly expressed Dio3,were further certified as our in vitro study models.It was observed that the inhibitory effect of BDE209 on glial cell Dio2 was not due to transcriptional regulation but rather attributed to protein destabilization.Conversely,BDE209 did not significantly inhibit the expression of Dio3 protein in neuronal cells.(3)Effects of BDE209 on TH transporter in H4 cells:No significant impact was observed on OATP14 protein by exposure to BDE209.(4)The impact of BDE209 on T3 nuclear receptor gene expression and downstream nuclear receptordependent gene expression in H4 cells were investigated.Results showed that BDE209 significantly decreased the expressions of RC3,UCP2,and ENPP2 genes,while no significant effect was observed on THRA and THRB expressions.(5)The regulatory capacity of BDE209-infected H4 cells on exogenous T3 and T4:BDE209 significantly impeded conversion of exogenous T4 to T3 in H4 cells.3.The possible mechanism of BDE209 induced Dio2 downregulation in H4 cells:(1)The relationship between decreased level of Dio2 protein induced by BDE209 and UPS(ubiquitin-proteolytic enzyme pathway)and the autophagy-ly sosome pathway:BDE209 induced ubiquitination-mediated degradation of Dio2 through the autophagy-lysosome pathway,rather than the proteasome pathway.(2)By promoting the binding of WBS-1(ubiquitination enzyme)to Dio2,BDE209 inhibited binding of USP33(deubiquitination enzyme)to Dio2 to improve the ubiquitinati on level of Dio2,promote the formation of p62/Dio2 complex,and reduce the level of Dio2 protein.(3)BDE209 inhibited the catalytic activity of Dio2 by competing with T4 in the Dio2 active cavity.Chapter 2:The mechanism of PBDEs on deiodinase 3(Dio3)and the effect of PBDEs in the interference of local TH level and function regulation in neuronal cells.1.The cytotoxicity of PBDEs on SK-N-AS neuronal cells was evaluated through CCK-8 assays and cell cloning and formation experiments.Results indicated that BDE47 significantly inhibited the proliferation of SK-N-AS cells,while BDE209 had no significant effect on neuronal cells.2.BDE47 induces local disturbance of thyroid hormone(TH)by affecting neuron Dio3:(1)BDE47 mediated TH disruption in neuronal cells:ELISA analysis revealed that BDE47 significantly decreased the production of endogenous rT3 and T3 in SK-N-AS cell medium,while BDE209 had no significant inhibitory effect on this process.Therefore,we focused on investigating the molecular mechanism underlying the induction of local TH metabolism disorder in neurons by using BDE47 as a model compound.(2)The impact of BDE47 on Dio3 expression in SK-N-AS neuronal cells:siRNA interference and Western experiments demonstrated that both mRNA and protein levels of BDE3 are significantly suppressed by BDE47,which was the key target responsible for BDE47-mediated neuronal cytotoxicity.(3)Effects of BDE47 on TH transporter in SK-N-AS cells:BDE47 significantly upregulated the protein expression of MCT8 in SK-N-AS cells.This increase was a result of negative feedback due to local hypothyroidism caused by BDE47,indicating that Dio3 played a crucial role in the disruption of TH metabolism induced by BDE47.(4)BDE47 had no effect on the expression of THRA and THRB in SK-N-AS cells,but significantly reduced the downstream dependent genes RC3 of nuclear receptor,UCP2,and ENPP2.(5)By inhibiting Dio3,BDE47 impaired SK-NAS cells’ ability to regulate exogenous T4.3.The impact of possible mechanisms on Dio3 expression in SK-N-AS cells was investigated,revealing that:(1)MiR-24-3p was a crucial factor in regulating Dio3 expression:bioinformatics analysis,gene editing,and luciferase reporter gene experiments demonstrated that miR-24-3p was bound to the 3’-UTR of Dio3 and inhibited its expression.(2)qPCR,luciferase reporter gene,and ELISA experiments revealed that BDE47 upregulated expression of miR-24-3p,leading to downregulation of Dio3 expression.(3)Effect of BDE47 on the autophagic lysosome pathway:The reduction of Dio3 protein in neurons induced by BDE47 was independent of the autophagic lysosome pathway.Chapter 3:Establishment of H4/SK-N-AS co-culture and verification on role of Dio2 and Dio3 in PBDEs induced local TH level and neurotoxicity in the CNS.1.Effects of BDE209 on thyroid function in co-culture system:(1)BDE209 induced hypothyroidism in co-culture systems,albeit to a lesser extent than in single-cell culture systems.(2)BDE209 exhibited toxicity towards glial cells in both single-cell and coculture systems.While the activity of neuron cells remained unaffected in the former,it was significantly inhibited in the latter.(3)BDE209 suppressed the expression and activity of Dio2 in both single-cell and co-cultured glial cells,with a slightly weaker inhibitory effect observed in the co-culture system.(4)BDE209 did not affect the expression and activity of Dio3 in single-cultured neuron cells but exhibited a slight inhibitory effect on co-cultured neuron cells.2.Effects of BDE47 on thyroid function in co-culture system:(1)BDE47 can induce hypothyroidism in both co-culture and single-culture systems,with a milder effect observed in the former.(2)BDE47 exhibited neurotoxicity in both culture systems while not significantly inhibiting glial cells.(3)BDE47 did not significantly affect the expression and activity of Dio2 in glial cells cultured individually,but slightly increased the activity of Dio2 in co-cultured system.(4)BDE47 inhibited the expression and activity of Dio3 in both single-cell and co-culture systems.Conclusion:This study investigated the molecular mechanisms underlying neurotoxicity mediated by environmental organic pollutant PBDEs.Different types of PBDEs induced neurotoxicity through distinct pathways:BDE209,a highly brominated diphenyl ether,promoted ubiquitination and degradation of Dio2 via glial cell autophagy,leading to TH metabolism disorders and subsequent neurotoxicity;BDE47,a lower brominated diphenyl ether,inhibited Dio3 expression in neuronal cells by upregulating miR-24-3p.In addition,the local metabolism disorder of thyroid hormone and cytotoxicity of PBDEs on glial and neuronal cells persisted in the cell co-culture system.However,due to the synergistic regulation of local thyroid hormone by glial Dio2-neuron Dio3,neurotoxicity in co-culture system was slightly reduced.更多还原