【作者】 王璐；

【导师】 孙鲜策；

【作者基本信息】 大连医科大学 ， 劳动卫生与环境卫生学， 2023， 硕士

【摘要】 目的:铁死亡是铁依赖的、由脂质过氧化引起的细胞死亡过程,铁在胞质中的蓄积与铁死亡的发生发展有着密不可分的联系。铁死亡在亚砷酸钠（Sodium arsenite,Na As O₂）所诱导的非酒精性脂肪性肝炎（Nonalcoholic steatohepatitis,NASH）中发挥重要作用,但是Na As O₂诱导铁死亡发生的具体分子机制仍不明确。铁硫簇家族蛋白2（CDGSH Iron Sulfur Domain 2,CISD2）对调控和维持线粒体铁稳态起着重要作用。然而,在Na As O₂诱导的NASH中,CISD2对胞质中铁的调控机制尚不清楚,CISD2是否对铁死亡有调控作用,以及与线粒体功能障碍之间的联系仍有待研究。因此,本研究的目的是探讨CISD2与Na As O₂致NASH中铁死亡与线粒体功能障碍之间的关系及其具体调控机制,为治疗NASH提供更多可能的干预靶点。方法:本研究以10周龄雄性Sprague-Dawley（SD）大鼠为体内研究对象,以人正常肝细胞系L-02细胞为体外研究对象。将雄性SD大鼠随机分为对照组与Na As O₂处理组,处理方法是以浓度分别为0 mg/kg BW和5 mg/kg BW灌胃,12 h/次,处理时间为9个月。使用实时荧光定量聚合酶链式反应（Realtime quantitative PCR,RT-q PCR）法检测NEET蛋白家族CISD1、CISD2、CISD3的基因表达水平;使用免疫蛋白印迹法（Western blot）检测CISD2、细胞色素C（Cytochrome c,Cyt C）以及线粒体呼吸链复合物的蛋白表达水平。L-02细胞以4μM Na As O₂进行处理,培养24 h后用RT-q PCR法检测NEET蛋白家族的基因表达水平;通过Western blot法检测L-02细胞内CISD2的蛋白表达水平。用CISD2过表达质粒转染L-02细胞24 h以及4μM Na As O₂处理24 h后,通过Western blot法和油红O染色法（oil red O）检测炎症因子肿瘤坏死因子α（TNFα）、白介素6（IL-6）、白介素1β（IL-1β）的蛋白表达水平和脂质沉积;通过Western blot法检测谷胱甘肽过氧化物酶4（Glutathione peroxidase 4,GPX4）和酰基辅酶A合成酶长链家族成员4（Acyl-Co A synthetase long chain family member 4,ACSL4）的蛋白表达水平,通过荧光探针检测脂质过氧化物（Lipid peroxides）的水平,比色法检测还原型谷胱甘肽（Glutathione,GSH）、丙二醛（Malondialdehyde,MDA）、总超氧化物歧化酶（Total superoxide dismutase,T-SOD）的含量;通过Western blot法检测粒体呼吸链复合物的蛋白表达水平,使用比色法检测三磷酸腺苷（Adenosine 5’-triphosphate,ATP）的含量,应用荧光探针检测线粒体膜电位（Mitochondrial membrane potential,MMP）的改变以及线粒体活性氧（Mitochondrial reactive oxygen species,Mt ROS）的生成,使用荧光探针检测细胞胞质与线粒体内铁的含量变化。结果:在大鼠肝脏与L-02细胞中,与对照组相比,RT-q PCR与Western blot结果表明CISD2的表达水平降低,因此对L-02细胞进行CISD2过表达质粒转染。Western blot结果表明,Na As O₂处理后TNFα、IL-6、IL-1β的蛋白表达水平表达上调,油红O结果显示脂滴蓄积,过表达CISD2后,与Na As O₂处理组相比,炎症因子表达水平降低,脂质堆积减少,提示CISD2能够缓解Na As O₂诱导的NASH。与对照组相比,Na As O₂处理后GPX4下调,ACSL4上调,脂质ROS增多,GSH、MDA含量增加,T-SOD活力降低,过表达CISD2后,与Na As O₂处理组相比GPX4上调,ACSL4下调,脂质ROS生成减少,GSH、MDA含量减少,T-SOD活力增加,提示CISD2可能阻止Na As O₂诱导的铁死亡。在大鼠肝脏中,线粒体中Cyt C减少外溢至胞浆中,线粒体呼吸链复合物的蛋白表达水平降低,提示大鼠肝脏发生线粒体功能障碍。在L-02细胞中,与对照组相比,Na As O₂处理后线粒体呼吸链复合物的蛋白表达水平降低,ATP含量减少、MMP降低以及Mt ROS生成增加,线粒体铁含量减少,胞质铁含量增加,过表达CISD2后,与Na As O₂处理组相比,这些指标均有所恢复,提示CISD2通过调控线粒体与胞质中的铁离子来缓解Na As O₂诱导的线粒体功能障碍。结论:Na As O₂暴露抑制CISD2的表达,CISD2能够促进铁离子在胞质内的蓄积,引起线粒体功能障碍,进而诱导铁死亡的发生,最终导致NASH。更多还原

【Abstract】 Objective:Ferroptosis is a form of cell death that is iron-dependent and caused by lipid peroxidation,,iron accumulation in the cytoplasm is closely related to the occurrence and development of ferroptosis.Ferroptosis plays an important role in nonalcoholic steatohepatitis（NASH）caused by sodium arsenite（Na As O₂）.CISD2（CDGSH Iron Sulfur Domain 2）plays an important role in the regulation of iron and the maintenance of mitochondrial homeostasis.However,in NASH caused by Na As O₂,the regulatory mechanism of CISD2 on iron is unclear,and the specific link between the regulatory effect of CISD2 on ferroptosis and mitochondrial dysfunction remains to be studied.Therefore,this study aims to explore the relationship between ferroptosis and mitochondrial dysfunction in CISD2 and NASH which is caused by Na As O₂,as well as its specific regulatory mechanism,and to provide more possible intervention targets for the treatment of NASH.Methods:In this study,10-week-old male Sprague-Dawley rats were used in vivo and L-02 cells were used in vitro.Male Sprague-Dawley rats were divided into control and Na As O₂ treatment groups by gavage at 0 mg/kg BW and 5 mg/kg BW for 12 h/time for 9 months.The gene expression levels in CISD1,CISD2,and CISD3 were measured by real-time quantitative PCR（RT-q PCR）.The protein expression levels in CISD2,cytochrome C（Cyt C）,and mitochondrial respiratory chain complexes were measured by western blot.L-02 cells were treated with 4μM Na As O₂ for 24 h,and the gene expression levels of CISD1,CISD2 and CISD3 were detected by RT-q PCR.The protein expression level of CISD2 in L-02 cells was detected by western blot.After transfection of L-02cells with CISD2 overexpression plasmid for 24 h and 4μM Na As O₂ for 24 h,the protein expression levels and lipid deposition of inflammatory factors TNFα,IL-6 and IL-1βwere detected by western blot and oil red O staining.Protein expression levels of glutathione peroxidase 4（GPX4）and acyl-Co A synthetase long chain family member 4（ACSL4）were detected by western blot,levels of lipid peroxides were detected by fluorescent probes,and reduced glutathione（GSH）,malondialdehyde（MDA）,total superoxide dismutase（T-SOD）were detected by colorimetric detection content.The protein expression levels of the mitochondrial respiratory chain complex were detected by the western blot.Adenosine 5’-triphosphate（ATP）was detected by colorimetry and fluorescent probes were used to detect the changes in mitochondrial membrane potential（MMP）and the generation of mitochondrial reactive oxygen species（Mt ROS）.The changes in iron content in cytoplasm and mitochondria were detected using fluorescent probes.Results:In vivo and in vitro,RT-q PCR and western blot results showed that the expression level of CISD2 was significantly reduced compared to the control group,and L-02 cells were transfected with the CISD2 overexpression plasmid.The results of western blot showed that the protein expression levels of inflammatory factors TNFα,IL-6 and IL-1βwere upregulated after Na As O₂treatment,and the oil red O Indicated that lipid droplets accumulated significantly,and after overexpression of CISD2,the expression level of inflammatory factors was reduced and lipid accumulation was reduced.These results demonstrated that CISD2 could alleviate the NASH caused by Na As O₂.Compared with the control group,after Na As O₂ treatment,the expression of ferroptosis-related proteins GPX4 was down-regulated,ACSL4 expression was upregulated,lipid ROS was significantly increased,GSH and MDA content were significantly increased,T-SOD was significantly reduced,and after overexpression of CISD2,GPX4 was increased significantly compared with Na As O₂ treatment group,ACSL4 was decreased,lipid ROS was decreased,GSH and MDA content were decreased,and T-SOD content increased.CISD2 was shown to prevent the ferroptosis caused by Na As O₂.The expression of Cyt C in mitochondria was reduced and spilled into the cytoplasm.The protein expression levels of mitochondrial respiratory chain complexes were decreased significantly,indicating mitochondrial dysfunction in Na As O₂-treated rat liver.In L-02 cells,compared with the control group,the protein expression levels of mitochondrial respiratory chain complex were significantly reduced after Na As O₂ treatment,and ATP content was decreased,MMP was decreased,Mt ROS production was increased.Mitochondrial iron content was decreased and cytosolic iron content was increased,after overexpression of CISD2,compared with Na As O₂ treatment group,their changes have been reversed,shows that CISD2 improves Na As O₂-induced mitochondrial dysfunction by transporting iron in mitochondria and cytoplasm.Conclusion:Na As O2 exposure inhibits the expression of CISD2,which causes mitochondrial dysfunction by promoting cytoplasmic iron accumulation,which in turn induces the occurrence of ferroptosis and NASH.更多还原