茵陈蒿汤对自身免疫性肝炎小鼠肝细胞铁死亡的抑制作用及其机制分析.docx

自身免疫性肝病DOI:10.12449/JCH240311茵陈蒿汤对自身免疫性肝炎小鼠肝细胞铁死亡的抑制作用及其机制分析钮蓉，陈晨，郭地，吕思学，武文，杨娜，刘杨山西中医药大学基础医学院，山西晋中030619通信作者：刘杨，Iiuyangl980SXtCm.educn(ORCID:0000-0002-6627-5002)摘要：目的探讨茵陈蒿汤对自身免疫性肝炎小鼠肝细胞铁死亡的抑制作用及机制。方法选取SPF级雌性C57BU6小鼠18只，采用随机数字表法分为正常组、模型组、治疗组，每组6只.模型组和治疗组采用刀豆蛋白A(CA)尾静脉注M制备自身免疫性肝炎小鼠模型，正常组注射生理盐水治疗组于造模前14天，给予茵陈蒿汤(4.68g生药kg)预防性灌胃治疗，末次灌胃给药后注射ConA。潮舱测ALT、AST、IFN-丫、TNF。、铁离子、GSH.ROS.ATPxMDA水平，计算肝fl螳数、脾脏指数，并观察GPX4、SLC7A11的表达；比较各组小鼠肝脏病理组织变化。正态分布的计量资料3组间比较采用单因素方差分析，进/两两比较采用LSD-检验.结果与正常组比,模型组肝蚂酸、脾蛇酸、ALT、AST、IFNYTNF-,铁离子、ROSxMDA水平均升高(Q值均0.05),GSH、ATP含量及GPX4、SLC7A11蛋白表达水平降低(户值均0.05).与睡组比较，治疗组酸、脾蛇黝、ALT、AST.IFN-,TNFa铁离子、ROS.MDA水平均降低(户值均005),GSH、ATP含量及GPX4、SLC7A11蛋白表达水平升高(户值均0.01)HE染色结果显示，与正常组匕瞰，模型组小鼠肝脏出现大麟肝细胞变性、坏死、门管区炎细胞聚集，治疗组处理的小鼠肝脏坏死和炎性浸润程度较模型组有所减轻。结论COnA诱导肝组织损伤可能与铁死亡有关。茵陈蒿汤可以增加SLC7A11/GPX4蛋白的表达水平进而抵抗ConA诱导的肝细胞铁死亡。关键词：肝炎，自身免疫性；茵陈蒿汤；铁死亡；谷胱甘肽过氧化酶；小鼠,近交C57BL基金项目：山西省科学技术厅山西省重点国别科技合作项目(202104041101013)；山西中医药大学科技创新团队项目(2022TD2003)；中西医结合防治风湿免疫病山西省科技创新人才重点团队(202204051002033)；山西中医药大学学科建设项目(2023XKJS-03)；山西省自然科学基金(202203021222272)RoleandmechanismofactionofYinchenhaoDecoctionininhibitingferroptosisofhepatocytesinmicewithautoimmunehepatitis1.IZhurong,CHENChen,GUODi,LYUSixue,WUJiawen,YANGNa,LIUYang.(SchoolofBasicMedicalScienceslShanxiUniversityofChineseMedicine,Jinzhong,Shanxi030619,China)Correspondingauthor:LIUYang,liuyang1980(ORCID:0000-0002-6627-5002)Abstract:ObjectiveToinvestigatetheroleandmechanismofactionofYinchenhaoDectionininhibitingferroptosisofhepatytesinmicewithautoimmunehepatitis.MethodsAtotalof18specificpathogen-freefemaleC57BL6micewereselectedanddividedintonormalgroup,modelgroup,andtreatmentgroupusingarandomnumbertable,with6miceineachgroup.ThemiceinthemodelgroupandthetreatmentgroupwereinjectedwithConcanavalinA(ConA)viathecaudalveintoestablishamousemodelofautoimmunehepatitis,andthoseinthenormalgroupwereinjectedwithnormalsaline.ThemiceinthetreatmentgroupweregivenprophylactictreatmentwithYinchenhaoDecoction(4.68gcrudedrug/kg)bygavageat14daysbeforemodeling,andConAwasinjectedafterthelastgavage.Thelevelsofalanineaminotransferase(ALT),aspartateamitransferase(AST),interferongamma(IFN-),tumornecrosisfactor-(TNF-a),ironion,glutathione(GSH),reactiveoxygenspecies(ROS)radenosinetriphosphate(ATP)fandmalondialdehyde(MDA)weremeasured;liverindexandspleenindexwerecallated;theexpressbnlevelsofGPX4andSLC7A11weremeasured;liverhistopathologicalchangeswerecomparedbetweengroups.Aonewayanalysisofvariancewasusedformparisonofnormallydistributedntinuousdatabetweenthreegroups,andtheleastsignificantdifferenceMestwasusedforfurthercomparisonbetweentwogroups.ResultsComparedwiththenormalgroup,themodelgrouphadsignificantincreasesinliverindex,spleenindexfALT,AST,IFN-,TNF-,ironion,ROSandMDA(allP<0.05)andsignificantreductionsinthententofGSHandATPandtheproteinexpressionlevelsofGPX4andSLC7A11(allP0.05).Comparedwiththemodelgroup,thetreatmentgrouphadsignificantreductionsinliverindex,spleenindex,ALT,AST,IFN-,TNFy,ironion,ROSandMDA(allP<0.05)andsignificantincreasesinthecontentofGSHandATPandtheproteinexpressionlevelsofGPX4andSLC7A11(allP<0.05).HEstainingshowedthatmparedwiththermalgroup,themodelgroupshowedmassivehepatocytedegenerationandnecrosisandinflammatorycellaggregationattheportalarea,andcomparedwiththemodelgroup,thetreatmentgrouphadalleviationoflivernecrosisandinflammatoryinfiltration.ConclusionLiverinjuryinducedbyConAmaybeassociatedwithfeoptosis.YinchenhaoDecoctioncanincreasetheproteinexpressionlevelsofSLC7A11andGPX4proteinandthusinhibitferroptosisofhepatocytesinducedbyConA.Keywords:Hepatitis,Autoimmune;YinchenhaoDection;Ferroptosis;GlutathionePeroxidase;Mice,InbredC57BLResearchfunding:ShanxiProvincialKeyCountryScienceandTechnologyCooperationProjectShanxiProvincialScienceandTechnologyDepartment(202104041101013);ScienceandTechnologyInnovationTeamProjectofShanxiUniversityofTraditionalChineseMedicine(2022TD2(X)3);KeyTeamofScienceandTechlogyInnovationTalentsofShanxiProvinceforPreventionandTreatmentofRheumatologicalandImmunologicaldiseasesbyIntegratedChineseandWesternMedicine(202204051002033);DisciplineConstructionProjectofShanxiUniversityofTraditionalChineseMedicine(2023XKJS-03);NaturalScienceFoundationProjectofShanxiProvin(202203021222272)自身免疫性肝炎（AIH）是一种由自身抗体和自身反应性T淋巴细胞诱导的肝脏炎症，其特点是循环自身抗体升高、高丙种球蛋白血症和界面性肝炎，肝细胞免疫耐受性丧失，可进展为肝硬化甚至肝衰竭l1-4½AIH主要发病人群为青少年及中老年女性O研究显示该病的发病率逐年上升AIH在全世界的年发病率为1.37/10万人，其中亚洲人群的年发病率为1.31/10万人15）。目前AIH的病因和发病机制尚不明确（6。现行治疗AIH的策略是单独使用糖皮质激素或与硫嗖瞟吟、免疫抑制剂组合。同时，为了保护肝脏、防止疾病的进展和肝功能的恶化，常规的治疗策略还包括保肝降酶等0然而，长期使用这些药物易使患者出现药物依赖性、耐药性及停药后复发率较高等问题，因此，亟需寻找安全有效的治疗方式m。茵陈蒿汤出自医圣张仲景所作伤寒杂病论，该方由茵陈、桅子、大黄三味药组成，具有清热利湿、利胆退黄的功效。现代医学研究表明，茵陈蒿汤用于治疗AlH具有良好的临床疗效l8-9jo但有关茵陈蒿汤治疗AIH的基础实验研究较少，其生物学机制尚未完全阐释清楚。有研究表明，铁死亡参与多种疾病的发生发展过程。铁死亡是一种由铁依赖的脂质过氧化诱导的调节性细胞死亡形式。过量的铁会引发体内脂质过氧化，从而导致铁死亡。胱氨酸-还原型谷胱甘肽（GSH）-谷胱甘肽过氧化物酶4（GPX4）是经典的铁死亡抑制系统。在正常生理条件下，胱氨酸通过抗转运蛋白SLC7A11进入细胞参与GSH的合成。合成的GSH可以被GPX4消耗，进而将活性氧（ROS）转化为脂质醇抑制铁死亡，保护细胞免受氧化应激，维持膜脂质双分子稳态f1°-11l）,已有研究表明ROS诱导的氧化应激是多种肝脏疾病的潜在病理生理机制i,211.那么，茵陈蒿汤是否通过抑制铁死亡及其介导的氧化应激