葛根素调节二氧化硅暴露肺泡巨噬细胞活化的可能机制

第1章前言1.1研究背景矽肺是由于长期吸入结晶型二氧化硅（SiO₂）粉尘引起的肺纤维化疾病，其病理特征为肺泡巨噬细胞异常活化驱动的持续性炎症反应与不可逆性肺组织纤维化。根据《2023年我国卫生健康事业发展统计公报》，尘肺病新发病例中矽肺占比高达66.61%，且随着新兴行业（如人造石材加工、珠宝抛光等）的快速发展，非传统职业群体的矽尘暴露风险显著增加，已成为全球性公共卫生问题[1]。尽管矽肺发病率呈下降趋势，但其病理进程不可逆转且缺乏有效治疗手段，患者常因呼吸衰竭或并发症过早死亡，对个人健康和社会经济造成沉重负担。因此，深入解析矽肺的分子机制并探索新型干预策略具有迫切意义。1.2国内外研究现状1.2.1矽肺纤维化病理机制矽肺纤维化的核心病理机制在于肺泡巨噬细胞的功能异常及代谢失衡。可吸入性二氧化硅颗粒在肺泡腔内沉积后，巨噬细胞通过模式识别受体启动吞噬过程，将颗粒内化。PlotegherN、PihánP等发现矽尘的理化特性（疏水性和晶体结构）可破坏溶酶体膜的完整性，促使水解酶（如组织蛋白酶）向胞质逸散，诱发线粒体能量代谢障碍及内质网应激反应ADDINZOTERO_ITEMCSL_CITATION{"citationID":"oGiwd3RF","properties":{"formattedCitation":"\\super[9,10]\\nosupersub{}","plainCitation":"[9,10]","noteIndex":0},"citationItems":[{"id":9197,"uris":["/users/11912958/items/8TPTG5PF"],"itemData":{"id":9197,"type":"article-journal","abstract":"Lysosomalstoragedisorders(LSDs)arerareinheriteddebilitatingandoftenfataldisorders.Causedbymutationsaffectinglysosomalproteins,LSDsarecharacterizedbytheaccumulationofundegradedmaterialinlysosomesandbylysosomaldysfunction.AlthoughLSDsaremultisystemicdiseases,themajoritydisplayneurologicsymptomsandneurodegeneration.OnlyrecentlyhasaroleemergedformitochondrialdysfunctioninthepathophysiologyofLSDs,suggestinganimpactoflysosomaldysfunctiononmitochondria.Moreover,mitochondrialdamagemayalsocauselysosomaldysfunction,furthersupportingtheactivityofcommonsignalingpathwaysandcrosstalkbetweenthetwoorganelles.InthisreviewweexplorethemechanismslinkinglysosomalandmitochondrialdysfunctiontoassesswhetherspecificmitochondrialpathwaysrepresentanewtherapeuticfrontierinthemanagementofLSDs.","container-title":"TrendsinMolecularMedicine","DOI":"10.1016/j.molmed.2016.12.003","ISSN":"1471-499X","issue":"2","journalAbbreviation":"TrendsMolMed","language":"eng","note":"PMID:28111024","page":"116-134","source":"PubMed","title":"MitochondrialDysfunctionandNeurodegenerationinLysosomalStorageDisorders","volume":"23","author":[{"family":"Plotegher","given":"Nicoletta"},{"family":"Duchen","given":"MichaelR."}],"issued":{"date-parts":[["2017",2]]}}},{"id":9194,"uris":["/users/11912958/items/AWS5QUG6"],"itemData":{"id":9194,"type":"article-journal","container-title":"NeuralRegenerationResearch","DOI":"10.4103/1673-5374.339487","ISSN":"1673-5374","issue":"12","journalAbbreviation":"NeuralRegenRes","note":"PMID:35662214\nPMCID:PMC9165380","page":"2695-2696","source":"PubMedCentral","title":"ProapoptoticRECS1:arequisitegatewaytolysosomaldysfunctionanddeath","title-short":"ProapoptoticRECS1","volume":"17","author":[{"family":"Pihán","given":"Philippe"},{"family":"Milani","given":"Mateus"},{"family":"Hetz","given":"Claudio"}],"issued":{"date-parts":[["2022",4,29]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}[2,3]。HuangL、LeungCC等发现此类亚细胞器损伤通过触发RIPK3-MLKL通路或NLRP3炎性小体相关的细胞焦亡机制，最终导致巨噬细胞程序性坏死或炎性消亡[4,5]。细胞崩解后释放的矽尘可被周围巨噬细胞再次吞噬，形成"吞噬-死亡-再吞噬"的恶性循环。ZhaoY等发现在此病理进程中，受损巨噬细胞通过线粒体呼吸链及NADPH氧化酶系统促使活性氧（ROS）大量生成，同时释放TNF-α、IL-1β等炎症介质和TGF-β1、PDGF等纤维化诱导因子。这些效应分子不仅直接损害肺泡上皮细胞，破坏肺泡-毛细血管屏障，还能刺激成纤维细胞分化为肌成纤维细胞，驱动细胞外基质的异常积聚。值得注意的是，慢性氧化应激状态可诱发肺泡上皮细胞发生上皮-间质转化（EMT），加剧肺组织结构的重塑。上述多环节的级联反应共同推动肺间质纤维化进程，形成典型矽结节病灶，并伴随肺顺应性下降和气体交换功能障碍[6-8]。这种多因素参与的病理改变构成了矽肺不可逆性呼吸功能损害的组织学基础。1.1.2溶酶体的功能溶酶体（lysosome）是真核细胞的核心降解代谢中心，其独特的酸性微环境（pH4.5-5.5）为各类水解酶提供了理想的反应条件ADDINZOTERO_ITEMCSL_CITATION{"citationID":"ATnTxxS5","properties":{"formattedCitation":"\\super[17]\\nosupersub{}","plainCitation":"[17]","noteIndex":0},"citationItems":[{"id":9200,"uris":["/users/11912958/items/6A3UXXEM"],"itemData":{"id":9200,"type":"article-journal","abstract":"TheacidicenvironmentwithinlysosomesismaintainedwithinanarrowpHrange(pH4.5-5.0)optimalfordigestingautophagiccargomacromoleculessothattheresultingbuildingblockmetabolitescanbereused.ThispHhomeostasisisaconsequenceofprotoninfluxproducedbyaV-typeH+-translocatingATPase(V-ATPase)andrapidprotoneffluxthroughanunidentified\"leak\"pathway.Byperformingacandidateexpressionscreening,wediscoveredthattheTMEM175geneencodesaproton-activated,proton-selectivechannel(LyPAP)thatisrequiredforlysosomalH+\"leak\"currents.TheactivityofLyPAPismostactivewhenlysosomesarehyper-acidified,andcellslackingTMEM175exhibitlysosomalhyper-acidificationandimpairedproteolyticdegradation,bothofwhichcanberestoredbyoptimizinglysosomalpHusingpharmacologicalagents.VariantsofTMEM175thatareassociatedwithsusceptibilitytoParkinsondisease(PD)causeareductioninTMEM175-dependentLyPAPcurrentsandlysosomalhyper-acidification.Hence,ourstudiesnotonlyrevealanessentialH+-dissipatingpathwayinlysosomes,butalsoprovideamoleculartargettoregulatepH-dependentlysosomalfunctionsandassociatedpathologies.","container-title":"Autophagy","DOI":"10.1080/15548627.2022.2125629","ISSN":"1554-8635","issue":"4","journalAbbreviation":"Autophagy","language":"eng","note":"PMID:36120744\nPMCID:PMC10012942","page":"1368-1370","source":"PubMed","title":"TheAcidGateintheLysosome","volume":"19","author":[{"family":"Hu","given":"Meiqin"},{"family":"Chen","given":"Jingzhi"},{"family":"Liu","given":"Siyu"},{"family":"Xu","given":"Haoxing"}],"issued":{"date-parts":[["2023",4]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}[9]。ChaoX等发现该细胞器通过膜结构形成的动态区室结构，利用蛋白酶、脂肪酶及糖苷酶等水解酶系的协同效应，实现对胞吞物质、损伤细胞器和大分子物质的高效降解与循环利用[10]。PrietoHuarcayaS等研究发现，增强溶酶体酶活性可有效分解异常折叠蛋白及脂质沉积等病理代谢产物，这一特性在神经退行性疾病及溶酶体贮积症的治疗策略中具有潜在价值ADDINZOTERO_ITEMCSL_CITATION{"citationID":"nnmeTNcW","properties":{"formattedCitation":"\\super[19]\\nosupersub{}","plainCitation":"[19]","noteIndex":0},"citationItems":[{"id":9203,"uris":["/users/11912958/items/AN3GDCGL"],"itemData":{"id":9203,"type":"article-journal","container-title":"NeuralRegenerationResearch","DOI":"10.4103/1673-5374.363181","ISSN":"1673-5374","issue":"8","journalAbbreviation":"NeuralRegenRes","language":"eng","note":"PMID:36751788\nPMCID:PMC10154497","page":"1713-1714","source":"PubMed","title":"Therapeuticpotentialoflysosomalcathepsinsforneurodegenerativediseases","volume":"18","author":[{"family":"PrietoHuarcaya","given":"Susy"},{"family":"Zunke","given":"Friederike"}],"issued":{"date-parts":[["2023",8]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}[11]。在众多水解酶中，组织蛋白酶家族因其核心蛋白分解功能而备受关注。TurkV等发现组织蛋白酶D（CathepsinD,CTSD）作为天冬氨酸蛋白酶家族成员，在酸性条件下特异性分解多种蛋白底物；而组织蛋白酶B（CathepsinB,CTSB）作为半胱氨酸蛋白酶代表，不仅具备内切酶活性，还表现独特的羧基端二肽酶特性，共同参与细胞外基质重塑及抗原提呈等重要生理活动[12]。最新研究还揭示二者在肿瘤转移与程序性死亡调控中的双重作用。溶酶体功能的精准调节还依赖其膜系统特殊蛋白组分。溶酶体相关膜蛋白（Lysosome-AssociatedmembraneProteins，LAMPs）作为跨膜糖蛋白家族，不仅通过稳定溶酶体膜结构防止酶类泄漏，其LAMP-2a等亚型更参与分子伴侣介导的自噬过程。SaftigP等发现该家族蛋白还能与自噬体膜ATG8同源蛋白相互作用，调控巨自噬过程中自噬体-溶酶体融合的关键环节[13]。这些机制共同确立了溶酶体在细胞稳态调控中执行与调控的双重功能。然而，现有研究多聚焦于溶酶体损伤的病理后果，对靶向修复溶酶体膜完整性的干预策略仍缺乏系统性探索，尤其是天然药物成分如何通过调控LAMP1稳定性或TFEB核转位恢复溶酶体功能尚未阐明。1.1.3自噬的作用自噬是维持细胞稳态的核心调控系统。该过程通过调节胞内成分合成与分解的动态平衡，清除损伤细胞器及错误折叠蛋白ADDINZOTERO_ITEMCSL_CITATION{"citationID":"f976BPAs","properties":{"formattedCitation":"\\super[23]\\nosupersub{}","plainCitation":"[23]","noteIndex":0},"citationItems":[{"id":9208,"uris":["/users/11912958/items/G9UU3R3G"],"itemData":{"id":9208,"type":"article-journal","abstract":"Ionexchangebetweenintracellularandextracellularspacesisthebasicmechanismforcontrollingcellmetabolismandsignaltransduction.Thisprocessismediatedbyionchannelsandtransportersontheplasmamembrane,orintracellularmembranesthatsurroundvariousorganelles,inresponsetoenvironmentalstimuli.Macroautophagy(hereafterreferredtoasautophagy)isoneofthelysosomal-dependentdegradationpathwaysthatmaintainshomeostasisthroughthedegradationandrecyclingofcellularcomponents(e.g.,dysfunctionalproteinsanddamagedorganelles).Althoughautophagy-related(ATG)proteinsplayacentralroleinregulatingtheformationofautophagy-relatedmemberstructures(e.g.,phagophores,autophagosomes,andautolysosomes),theautophagicprocessalsoinvolveschangesinexpressionandfunctionofionchannelsandtransporters.Herewediscusscurrentknowledgeofthemechanismsthatregulateautophagyinmammaliancells,withspecialattentiontotheionchannelsandtransporters.Wealsohighlightprospectsforthedevelopmentofdrugstargetingionchannelsandtransportersinautophagy.","container-title":"Autophagy","DOI":"10.1080/15548627.2021.1885147","ISSN":"1554-8635","issue":"1","journalAbbreviation":"Autophagy","language":"eng","note":"PMID:33657975\nPMCID:PMC8865261","page":"4-23","source":"PubMed","title":"IonChannelsandTransportersinAutophagy","volume":"18","author":[{"family":"Zhang","given":"Ruoxi"},{"family":"Kang","given":"Rui"},{"family":"Klionsky","given":"DanielJ."},{"family":"Tang","given":"Daolin"}],"issued":{"date-parts":[["2022",1]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}[14]。其生物学流程可分为四个阶段：在营养缺失等应激状态下，mTOR（哺乳动物雷帕霉素靶蛋白）信号通路受抑触发自噬启动；ATG基因调控下形成具有双层膜结构的自噬体（autophagosome）；自噬体与溶酶体融合形成自噬溶酶体复合体（autolysosome）；最终在酸性微环境及水解酶系作用下，内容物被分解为可循环利用的基本单位[15]。溶酶体作为自噬过程的核心细胞器，不仅为自噬提供了必要的酸性环境，其含有的组织蛋白酶、酸性磷酸酶等水解酶系可协同分解蛋白聚集体、异常脂质等病理产物。各种致病因子通过多重机制破坏溶酶体稳态系统：一方面通过损害溶酶体膜稳定性引发组织蛋白酶的异常释放，另一方面干扰自噬体-溶酶体膜融合进程，造成自噬流阻断。更为重要的是，这些致病因子可显著干扰溶酶体腔室酸化状态，导致水解酶系活化受阻，从而全面损害自噬溶酶体的分解功能。这种级联性的细胞器功能障碍持续激活肺泡巨噬细胞的炎性应答，同时促进成纤维细胞的病理性分化，共同推动肺实质组织向不可逆性纤维化重构演变。SiO₂颗粒与肺泡巨噬细胞的相互作用主要发生在溶酶体这一关键细胞器。SydorMJ等研究发现，SiO₂颗粒与溶酶体膜磷脂头部基团发生特异性结合，引发膜相结构改变，这种异常不仅干扰溶酶体正常功能，还可导致自噬小体异常积累，最终诱发溶酶体功能失代偿[16]。自噬流阻滞被认为是矽肺纤维化的核心病理环节，对于自噬-溶酶体融合调控路径，TanS等研究表明，SiO₂暴露可导致自噬体（LC3-II阳性）与溶酶体（LAMP1阳性）共定位率下降，p62/SQSTM1蛋白异常积累，提示自噬体-溶酶体融合效率受损，SaftigP等研究发现通过激活TFEB（转录因子EB）促进溶酶体生物合成，或抑制mTORC1信号以增强自噬起始，已被证实可部分缓解矽肺纤维化[17]。然而，现有干预手段（如雷帕霉素）因副作用显著难以临床转化，且针对自噬流动态（如LC3-II/p62比值）的精准调控机制仍不明确。1.1.4葛根素的应用被誉为“亚洲人参”的葛根（PuerariaeLobataeRadix，Plr）是豆科藤本植物野葛的根，广泛分布于东亚及东南亚地区的丘陵地带。这种豆科藤本植物凭借其强适应性和高资源再生能力，兼具药用开发潜力与农业经济价值，在民间药膳文化中具有悠久的应用历史。葛根素（Puerarin，Pue）是葛属植物的化学分类学标志物。葛根素因其生物活性被广泛研究，具有抑制炎症、改善纤维化以及调节血压和血糖的作用ADDINZOTERO_ITEMCSL_CITATION{"citationID":"S7P58oun","properties":{"formattedCitation":"\\super[27\\uc0\\u8211{}31]\\nosupersub{}","plainCitation":"[27–31]","noteIndex":0},"citationItems":[{"id":189,"uris":["/users/11912958/items/NGXDCPY5"],"itemData":{"id":189,"type":"article-journal","abstract":"Inordertoevaluatetheanti-inflammatoryandantioxidantactivitiesofPuerarialobatarootsanditsactivecomponents,invitroinhibitoryactivitiesagainstlipopolysaccharide(LPS)-inducednitricoxide(NO)production,induciblenitricoxidesynthase(iNOS),cyclooxygenase-2(COX-2)proteinexpression,andtert-butylhydroperoxide(t-BHP)-inducedreactiveoxygenspecies(ROS)generationinRAW264.7cells,aswellasinvitroscavengingactivitiesagainst1,1-diphenyl-2-picrylhydrazyl(DPPH),peroxynitrite(ONOO−),nitricoxide(NO·),superoxideanion(·O2−)andtotalROS,andinhibitoryactivitiesagainstONOO−-mediatedtyrosinenitration,weredetermined.Repeatedcolumnchromatographywasperformedtoisolatefourknowncompoundsfromtheanti-inflammatoryandantioxidantEtOAcfraction:daidzein;genistein;puerarin;(+)-puerarolB-2-O-glucopyranoside;fourknowncompoundsfromtheanti-inflammatoryn-hexanefraction:lupenone;lupeol;puerarol;coumestrol;sevenknowncompoundsfromtheantioxidantn-BuOHfraction:allantoin;3′-hydroxypuerarin;daidzein8-C-apiosyl-(1→6)-glucoside;puerarin;genistin;3′-methoxypuerarin;daidzin.Amongthesecompounds,lupenoneandlupeolreducedNOproduction,aswellasiNOSandCOX-2proteinlevelsinLPS-stimulatedRAW264.7cells.Furthermore,lupeolshowedsignificantinhibitoryactivityagainstintracellularROSgenerationbyt-BHP.Meanwhile,3′-hydroxypuerarinshowedmarkedONOO−,NO·,totalROSscavengingactivities,andweak·O2−scavengingactivity,while3′-methoxypuerarinshowedONOO−scavengingactivityandweakNO·andO2−scavengingactivities,suggestingthatexistenceofthe3′-hydroxylgroupinpuerarinplaysanimportantroleinthescavengingofONOO−,NO·,andtotalROS,aswellasinhibitingtheONOO−-mediatedtyrosinenitrationmechanism.TheseresultsindicatethatP.lobatarootsanditsconstituentsmaybeausefultherapeuticandpreventiveapproachtovariousinflammatorydiseasesandoxidativestress-relateddisease.","container-title":"ArchivesofPharmacalResearch","DOI":"10.1007/s12272-012-0508-x","ISSN":"1976-3786","issue":"5","journalAbbreviation":"Arch.Pharm.Res.","language":"en","page":"823-837","source":"SpringerLink","title":"Anti-inflammatoryandantioxidantactivitiesofconstituentsisolatedfromPuerarialobataroots","volume":"35","author":[{"family":"Jin","given":"SeongEun"},{"family":"Son","given":"YouKyung"},{"family":"Min","given":"Byung-Sun"},{"family":"Jung","given":"HyunAh"},{"family":"Choi","given":"JaeSue"}],"issued":{"date-parts":[["2012",5,1]]}}},{"id":1627,"uris":["/users/11912958/items/5L7LG4JB"],"itemData":{"id":1627,"type":"article-journal","abstract":"BACKGROUND:Previousstudieshavereportedthatpuerarinpossessescardioprotective,vasodilatory,anti-inflammatory,anti-apoptotic,andhypoglycemicproperties.However,theimpactofpuerarinonsepsis-associatedencephalopathy(SAE)remainsunexplored.Inthisstudy,weexploredwhetherpuerarincanmodulatemicroglia-mediatedneuroinflammationforthetreatmentofSAEanddelvedintotheunderlyingmechanisms.\nMETHODS:WeestablishedamurinemodelofSAEthroughintraperitonealinjectionoflipopolysaccharide(LPS).Thepuerarintreatmentgroupreceivedpretreatmentwithpuerarin.Forinvitroexperiments,BV2cellswerepre-incubatedwithpuerarinfor2

hbeforeLPSexposure.Weemployednetworkpharmacology,theMorrisWaterMaze(MWM)test,NovelObjectRecognition(NOR)test,immunofluorescencestaining,enzyme-linkedimmunosorbentassay(ELISA),Westernblotting,andquantitativereal-timePCR(qRT-PCR)toelucidatethemolecularmechanismofunderlyingpuerarin'seffectsinSAEtreatment.\nRESULTS:Ourfindingsdemonstratethatpuerarinsignificantlyreducedtheproductionofinflammatorycytokines(TNF-αandIL-6)intheperipheralbloodofLPS-treatedmice.Moreover,puerarintreatmentmarkedlyamelioratedsepsis-associatedcognitiveimpairment.PuerarinalsoexhibitedinhibitoryeffectsonthereleaseofTNF-αandIL-6frommicroglia,therebypreventinghippocampalneuronalcelldeath.NetworkpharmacologyanalysisidentifiedAKT1asapotentialtherapeutictargetforpuerarininSAEtreatment.Subsequently,wevalidatedtheseresultsinbothinvitroandinvitroexperiments.OurstudyconclusivelydemonstratedthatpuerarinreducedLPS-inducedphosphorylationofAKT1,withtheAKTactivatorSC79reversingpuerarin'santi-inflammatoryeffectsthroughtheactivationoftheAKT1signalingpathway.\nCONCLUSION:Puerarinexertsananti-neuroinflammatoryeffectagainstSAEbymodulatingtheAKT1pathwayinmicroglia.","container-title":"Phytomedicine:InternationalJournalofPhytotherapyandPhytopharmacology","DOI":"10.1016/j.phymed.2023.155119","ISSN":"1618-095X","journalAbbreviation":"Phytomedicine","language":"en","note":"PMID:37801894","page":"155119","source":"PubMed","title":"Puerarinpreventssepsis-associatedencephalopathybyregulatingtheAKT1pathwayinmicroglia","volume":"121","author":[{"family":"Lin","given":"Shao-Peng"},{"family":"Zhu","given":"Lidong"},{"family":"Shi","given":"Hongjian"},{"family":"Ye","given":"Shan"},{"family":"Li","given":"Qi"},{"family":"Yin","given":"Xiaofang"},{"family":"Xie","given":"Qiangda"},{"family":"Xu","given":"Qizhong"},{"family":"Wei","given":"Jue-Xian"},{"family":"Mei","given":"Fen"},{"family":"Zhu","given":"Yongcheng"},{"family":"Lin","given":"Pei-Yi"},{"family":"Chen","given":"Xiao-Hui"}],"issued":{"date-parts":[["2023",12]]}}},{"id":1836,"uris":["/users/11912958/items/I65YDHCC"],"itemData":{"id":1836,"type":"article-journal","abstract":"Puerarialobata(knownasGegen)isanedibleandmedicinalherbthatisanutritiousmedicinefoodhomologyplantinChina.PreviousstudiesindicatedthatP.lobataplaysanessentialroleincontrollingcytokines.However,theexactmechanismoftheinflammationresponseisstillunknown.Inthisstudy,weobservedtheuptakeofP.lobata-derivedexosomes(Exos)inisolatedmousemacrophages.OurresultsshowthatP.lobata-derivedExosshiftM1macrophagestowardtheM2.ThesedatapresentthatP.lobataandpuerarinmightexertandenhanceanti-inflammatoryeffectsthroughtheactivationofexosomesandshiftsinmacrophagepolarization,providingstrongevidencefortheapplicationofP.lobataasnovelananti-inflammatorytherapeuticbiomaterial.","container-title":"Molecules","DOI":"10.3390/molecules27238184","ISSN":"1420-3049","issue":"23","journalAbbreviation":"Molecules","language":"en","page":"8184","source":"DOI.org(Crossref)","title":"EdiblePuerarialobata-DerivedExosomesPromoteM2MacrophagePolarization","volume":"27","author":[{"family":"Wu","given":"Jiaqi"},{"family":"Ma","given":"Xiaoyu"},{"family":"Lu","given":"Yu"},{"family":"Zhang","given":"Tao"},{"family":"Du","given":"Zuoqin"},{"family":"Xu","given":"Jin"},{"family":"You","given":"Jingcan"},{"family":"Chen","given":"Ni"},{"family":"Deng","given":"Xin"},{"family":"Wu","given":"Jianbo"}],"issued":{"date-parts":[["2022",11,24]]}}},{"id":1609,"uris":["/users/11912958/items/6WY4779T"],"itemData":{"id":1609,"type":"article-journal","abstract":"Dysbiosisofgutmicrobiotaandmetabolicpathwaydisordersarecloselyrelatedtotheulcerativecolitis.Throughnetworkpharmacology,wefoundthatpuerarinisapotentialingredientthatcanimprovethecryptdeformationandinflammatoryinfiltrationinmice,anddecreasethelevelsofIL-1β,IL-6andTNF-αsignificantly.Listeria,AlistipesandP.coprigraduallybecamedominantbacteriainUCmice,whichwerepositivelycorrelatedwithinflammatoryfactors.PuerarineffectivelyimproveddysbiosisbyreducingtheabundanceofAlistipes,P.copriandVeillonella,andincreasingthelevelofDesulfovibrionacea.Correlationnetworkandmetabolicfunctionpredictionanalysisofthemicrobiotashowedthattheyformedatightlyconnectednetworkandwerewidelyinvolvedincarbohydratemetabolismandaminoacidmetabolism.Specifically,weobservedsignificantchangesinthetryptophanmetabolismpathwayinDSSmice,withanincreaseintheabundanceofBacteroidetesandEnterobacteriaceaeinvolvedintryptophanmetabolism.However,thismetabolicdisorderwasalleviatedafterpuerarintreatment,includingthereversalof3-HAAlevelsandanincreaseintheabundanceofRhodobacteraceaeandHalomonadaceaeinvolvedinkynureninemetabolism,aswellasasignificantincreaseinthepurinemetaboliteguanosine.Inconclusion,ourstudysuggeststhatpuerarinhasagoodtherapeuticeffectonUC,whichispartiallyachievedbyrestoringthecompositionandabundanceofgutmicrobiotaandtheirmetabolism.","container-title":"FrontiersinMicrobiology","DOI":"10.3389/fmicb.2023.1279029","ISSN":"1664-302X","journalAbbreviation":"FrontMicrobiol","language":"en","note":"PMID:37908541\nPMCID:PMC10614640","page":"1279029","source":"PubMed","title":"Puerarinalleviatesinflammationandpathologicaldamageincolitismicebyregulatingmetabolismandgutmicrobiota","volume":"14","author":[{"family":"Zou","given":"Yixin"},{"family":"Ding","given":"Wenjiao"},{"family":"Wu","given":"You"},{"family":"Chen","given":"Tingting"},{"family":"Ruan","given":"Zheng"}],"issued":{"date-parts":[["2023"]]}}},{"id":1625,"uris":["/users/11912958/items/VRA7HQLA"],"itemData":{"id":1625,"type":"article-journal","abstract":"Maleinfertilityisaglobalconcern,withanoticeableincreaseinthedeclineofspermatogenesisandspermquality.However,therearelimitedclinicallyeffectivetreatmentsavailable.Thisstudyaimedtoinvestigatethepotentialeffectivenessofpuerarinintreatingmaleinfertility,whichleadstogonadalchanges.TheresultsobtainedfromvariousanalysessuchasCASA,immunofluorescence,DIFF-Quick,hematoxylinandeosin(H&E),andperiodicacid-Schiff(PAS)stainingdemonstratedthatpuerarinsupplementationsignificantlyalleviatedthebusulfan-inducedreductioninspermatogenesisandspermqualityinbothyoungandadultmice.Furthermore,puerarinexhibitedamarkedimprovementinthedamagecausedbybusulfantothearchitectureofseminiferoustubules,causalepididymis,blood-testicularbarrier(BTB),aswellasspermatogoniaandSertolicells.Similarly,puerarinsignificantlyreducedthelevelsoftotalantioxidantcapacity(T-AOC),malondialdehyde(MDA),andcaspase-3inthetestesofbusulfan-inducedmice,asdeterminedbymicroplatereaderanalysis.Additionally,RNA-seqdata,RT-qPCR,andwesternblottingrevealedthatpuerarinrestoredtheabnormalgeneexpressionsinducedbybusulfantonearlyhealthylevels.Notably,puerarinsignificantlyreversedtheimpactofbusulfanontheexpressionofmarkergenesinvolvedinspermatogenesisandoxidativestress.Moreover,puerarinsuppressedthephosphorylationofp38,ERK1/2,andJNKinthetestes,asobservedthroughtesticularanalysis.Consequently,thisstudyconcludesthatpuerarinmayserveasapotentialalternativefortreatingbusulfan-induceddamagetomalefertilitybyinactivatingthetesticularMAPKpathways.Thesefindingsmaypavethewayfortheuseofpuerarininaddressingchemotherapy-orotherfactors-inducedmaleinfertilityinhumans.","container-title":"Biomedicine&Pharmacotherapy=Biomedecine&Pharmacotherapie","DOI":"10.1016/j.biopha.2023.115231","ISSN":"1950-6007","journalAbbreviation":"BiomedPharmacother","language":"en","note":"PMID:37516022","page":"115231","source":"PubMed","title":"Puerarinimprovesbusulfan-induceddisruptionofspermatogenesisbyinhibitingMAPKpathways","volume":"165","author":[{"family":"Li","given":"Hai-Tao"},{"family":"Zhong","given":"Kun"},{"family":"Xia","given":"Yun-Fei"},{"family":"Song","given":"Jian"},{"family":"Chen","given":"Xiao-Qing"},{"family":"Zhao","given":"Wei"},{"family":"Zeng","given":"Xu-Hui"},{"family":"Chen","given":"Tian-Xing"}],"issued":{"date-parts":[["2023",9]]}}}],"schema":"/citation-style-language/schema/raw/master/csl-citation.json"}[18–22]。WangLY等研究发现，该化合物还能通过促进自噬体-溶酶体融合机制，改善镉暴露所致的自噬流阻断现象[23]。现有研究主要聚焦于其在糖尿病、心肌炎等领域的治疗机制，而针对矽肺纤维化进程的调节作用尚未见系统性研究报道。阐明葛根素如何通过改善自噬溶酶体功能障碍，进而缓解矽肺尤为重要。1.3研究目的探讨Pue缓解SiO2暴露诱导的肺泡巨噬细胞异常活化及炎症反应的分子机制，为缓解矽肺提供科学依据。通过细胞实验（MH-S肺泡巨噬细胞），明确Pue是否通过恢复溶酶体功能（如LAMP1表达、CTSB/CTSD活性）和自噬溶酶体融合，缓解SiO₂暴露引起的自噬流阻滞及炎症反应。验证Pue对关键分子（TFEB、LC3II/p62）的调控作用，揭示其通过促进TFEB核转位改善溶酶体生物合成。利用分子对接技术，分析Pue与炎症因子、自噬标志物及溶酶体蛋白的结合能力，验证其通过多靶点协同抑制矽肺进展的分子基础。1.4研究意义矽肺的核心病理机制涉及SiO2诱导的肺泡巨噬细胞异常活化，但其分子调控网络尚未完全阐明。本研究聚焦Pue对巨噬细胞自噬溶酶体功能的调控作用。通过解析Pue对溶酶体膜蛋白（LAMP1）、组织蛋白酶（CTSD/CTSB）及转录因子TFEB的调控用，进一步探索了中药活性成分通过靶向溶酶体稳态缓解矽肺的机制，为职业性肺病的分子病理学研究提供了新视角。结合分子对接技术，深入探讨Pue与炎症因子、自噬标志物和溶酶体相关指标的相互作用，为阐明其多靶点调控网络奠定基础，进一步拓展了中医药活性成分的分子药理学研究维度。聚焦SiO₂暴露引发的溶酶体膜通透性改变、自噬流阻滞及炎症反应，揭示溶酶体功能障碍在矽肺病理进程中的核心地位。研究结果可为矽肺的早期诊断标志物（如溶酶体碱化、Gal3/LAMP1共定位减少）筛选提供理论依据，同时为其他尘肺病（如煤工尘肺、石棉肺）的靶向治疗提供参考。针对矽肺缺乏有效治疗手段的现状，Pue对矽肺治疗的积极作用可显著降低患者对氧疗或肺移植的依赖，减轻医疗经济负担。研究结果可为公共卫生部门制定职业健康防护政策提供科学支持，例如将Pue进一步开发为膳食补充剂功能成分或预防性药物制剂，用于采矿、建材等职业暴露人群的长期防护，进而在一定程度上降低矽肺发病率。第2章材料与方法2.1主要仪器与试剂实验所用主要仪器设备见表2.1，主要试剂见表2.2。表2.1仪器设备仪器名称生产厂家二氧化碳恒温培养箱德国ThermoScientific公司倒置光学显微镜日本OLYMPUS公司高压灭菌器日本SANYO公司多功能酶标仪美国MolecularDevices公司金属浴Kylin-Bell有限公司高速冷冻离心机德国ThermoScientific公司制冰机冰山松洋生物科技（大连）有限公司全自动PCR分析系统中国上海宏石医疗科技有限公司PCR扩增仪美国BIO-RAD公司-80℃超低温冰箱中国海尔有限公司离心机长沙湘智离心机仪器有限公司涡旋器中国佑宁有限公司续表2.1仪器名称生产厂家低温摇床上海丙林电子科技有限公司超纯水机四川优普超纯科技有限公司组织破碎仪上海净信有限公司PR224ZH/E电子天平中国奥豪斯仪器有限公司垂直电泳槽美国Bio-Rad有限公司MiniTrans-Blot转膜槽美国Bio-Rad有限公司NEMO®MicroCT平生医疗科技有限公司迷你小型离心机中国佑宁有限公司化学发光成像系统上海勤翔科学仪器有限公司超声破碎机冠森生物科技有限公化学发光成像系统上海勤翔科学仪器有限公司表2.2主要试剂

试剂名称生产厂家RPMI-1640培养基中国逍鹏生物科技有限公司双抗（Pen-StrepSolution）中国逍鹏生物科技有限公司胎牛血清中国瑞帕特生物科技有限公司β-巯基乙醇中国碧云天生物技术有限公CCK8细胞活力检测试剂盒中国瑞帕特生物科技有限公司PBS中国瑞帕特生物科技有限公司DMSO美国SIGMA公司葛根素北京百灵威科技有限公司氯仿替代物中国瑞帕特生物科技有限公司异丙醇天津市津东天正精细化学试剂厂无水乙醇上海泰坦科技股份有限公司DEPC水北京聚合美生物科技有限公司反转录试剂盒中国聚合美有限公司RT-qPCR试剂盒中国聚合美有限公司蛋白酶抑制剂上海雅酶生物科技有限公司磷酸酶抑制剂中国瑞帕特生物科技有限公司RIPA裂解液中国瑞帕特生物科技有限公司BCA蛋白定量试剂盒中国瑞帕特生物科技有限公司续表2.2核质分离试剂盒中国碧云天生物技术有限公司快速SDS凝胶制备试剂盒中国北京庄盟生物科技有限公司甘氨酸（Glycine）北京博奥拓达科技有限公司十二烷基硫酸钠（SDS）北京博奥拓达科技有限公司三羟甲基氨基甲烷（Tris）北京博奥拓达科技有限公司脱脂奶粉上海雅酶生物医药科技有限公司5X蛋白上样缓冲液中国瑞帕特生物科技有限公司丽春红S染液中国碧云天生物技术有限公司通用型抗体稀释液中国北京庄盟生物科技有限公司吐温-20中国瑞帕特生物科技有限公司预染蛋白Marker中国中实同创科技有限公司LysoSensorBlue荧光探针美国Invitrogen生命技术公司不含DAPI抗淬灭封片剂中国瑞帕特生物科技有限公司抗荧光衰减封片剂（含DAPI）中国瑞帕特生物科技有限公司IL-6中国瑞帕特生物科技有限公司IL-1β武汉爱博泰克生物科技有限公司IL-4武汉爱博泰克生物科技有限公司IL-10江苏亲科生物研究中心有限公司TGF-β中国Arigobio生物α-SMA杭州华安生物技术有限公司P62日本MBL公司LC3日本MBL公司LAMP1武汉爱博泰克生物科技有限公司CTSD杭州华安生物技术有限公司CTSB武汉爱博泰克生物科技有限公司TFEB武汉爱博泰克生物科技有限公司α-Tubulin中国瑞帕特生物科技有限公司β-actin美国Proteintech公司HistoneH3杭州华安生物技术有限公司荧光二抗中国瑞帕特生物科技有限公司SiO2粉尘美国Sigma公司 2.2实验对象及分组选择小鼠肺泡巨噬细胞系MH-S进行体外细胞学实验。完全培养液：10%胎牛血清+1%双抗+0.05mMβ-巯基乙醇+89%RMPI-1640。实验分组:将MH-S细胞分为对照组（Con）、SiO2（50μg/cm²）暴露组、Pue（10μM）+SiO2（50μg/cm²）联合处理组、Pue（25μM）+SiO2（50μg/cm²）联合处理组、Pue（50μM）+SiO2（50μg/cm²）联合处理组，时间方案为Pue预处理2h后加入SiO2共同处理6h/24h。2.3主要溶液配置2.3.1SiO2悬液配置在超净台中将消毒完毕的SiO2粉尘置于洁净无菌的15mL离心管内，再用移液器小心加入RMPI-1640培养液，拧紧离心管盖子，充分混匀，配置成浓度为10mg/mL的SiO2悬液，备用。使用时现用现配，使用前使用超声机以100%功率超声30min。2.3.2Pue溶液配制使用DMSO充分溶解Pue粉末，使其浓度为400mM进行保存，使用时用空白培养液稀释成需要的浓度。2.4分子对接利用Pubchem数据库下载Pue的3D结构图，以SDF格式保存。使用OpenBabelGUL软件将SDF格式转换为PDB格式。从PDB数据库（/）[24]下载相应蛋白的三维结构图，并将其保存为PDB格式，然后将它们导入AutoDock软件进行分子对接。使用PyMol软件将对接结果进行可视化。2.5CCK8细胞活力检测2.5.1实验分组将MH-S细胞分为对照组（Con）、DMSO对照组、Pue10μM组、Pue25μM组、Pue50μM组、Pue100μM组和Pue200μM组。2.5.2实验步骤经不同浓度Pue预处理26h后，在每孔中加入10μlCCK8溶液，于恒温37℃，5%CO2细胞培养箱培养孵育3h。于450nm波长酶标仪检测吸光度值。2.6MH-S细胞基因转录活性测定2.6.1Tiozol法提取细胞mRNA首先进行样品预处理；然后在样品中加入Trizol使样品的核酸蛋白复合物分离；之后加入氯仿替代物，使液体分层后离心；接下来收集上层液体并加入等体积异丙醇，混匀并静置后离心得到总RNA沉淀；使用70%酒精漂洗总RNA两次后，将残留酒精烘干，加入10μl无酶水溶解总RNA。2.6.2mRNA逆转录使用聚合美（MF-166）反转录试剂盒将mRNA反转录为cDNA，该部分加样时需在冰上进行，详细用法见表2.3。加入样品和反应试剂后轻轻混匀并短暂离心，经37℃孵育15min；85℃加热5s，反应后置于冰上恢复室温，进行后续实验或冷冻保存。表2.3PCR扩增步骤试剂名称使用体积10×gDNAplusremovermix1μlRNA模板500ng/c（mRNA浓度）DEPC-ddH2O补足至10μl反应液轻轻混匀，短暂离心，42℃温育2min，然后置于冰上冷却。5×M5RTSuperPlusMix4μlDEPC-ddH2O6μl总体积20μl2.6.3PCR检测PCR检测反应体系见表2.4，反应条件见表2.5，引物序列见表2.6。将反应体系加入八连管后，盖上盖子震荡后离心，进行上机反应。表2.4PCR反应体系试剂名称使用量模板DNA2μl2×M5HiperRealtimePCRSupermixwithLowROX10μlPrimer1(10μM)0.5μlPrimer2(10μM)0.5μl表2.5PCR反应时间反应温度（℃）反应时间（s）9560951540cycles551572609515Meltcurves606095306015表2.6引物序列基因名称前引物（5’~3’）后引物（5’~3’）Il-6GCAGGATACACCACTCCTCAAAAAGACGAATCAGCAGCACAAIl-1βTTCAGGCAGGCAGTATCACTCGAAGGTCCACGGGAAAGACACIl-4GGTCTCAACCCCCAGCTAGTGCCGATGATCTCTCTCAAGTGATIl-10CTTACTGACTGGCATGAGGATCAGCAGCTCTAGGAGCATGTGGTgf-βCTCCCGTGGCTTCTAGTGCGCCTTAGTTTGGACAGGATCTGTnf-αCTGGACTCTACGACTTCACAATGAGTTGGCGATCACTGACAGTTGapdhTGTGTCCGTCGTGGATCTGATTGCTGTTGAAGTCGCAGGAG2.7MH-S细胞蛋白表达水平测定2.7.1细胞蛋白提取总蛋白的提取使用RIPA蛋白酶裂解液与蛋白酶抑制剂以100：1比例混合的混合液提取细胞总蛋白。细胞核质分离蛋白的提取使用核质分离试剂盒按照说明书操作提取MH-S细胞核蛋白和胞浆