【作者】 徐淼；

【导师】 张立实； 杨辉； 贾旭东； 陈锦瑶；

【作者基本信息】 四川大学 ， 营养与食品卫生学， 2021， 博士

【摘要】 邻苯二甲酸酯类(Phthalate esters,PAEs)被广泛用作工业增塑剂,其健康危害越来越受重视。邻苯二甲酸二乙基己酯(Di(2-ethylhexyl)phthalate,DEHP)是PAEs中最常用者。由于PAEs常以非共价键结合于塑料,导致其极易从塑料中迁移至环境介质,因此人类可通过各种途径暴露于PAEs。此外,由于塑料被广泛用作食品包装材料,经口途径也成为一般人群暴露于DEHP的主要方式。DEHP和其他一些邻苯二甲酸酯类均具有内分泌干扰作用,可干扰生物体的代谢稳态并导致非酒精性脂肪肝(Nonalcoholic fatty liver disease,NAFLD)等各种不良健康后果。虽然DEHP对人类健康的影响已被广泛研究,但其对肝脏能量代谢的毒效应及作用机制仍存在争议。既往研究主要关注DEHP对肝细胞本身的作用及机制,认为肝细胞的过氧化物酶体增殖剂激活受体 α(Peroxisome proliferators-activated receptor α,PPARα)是主要靶点,但肝细胞PPARα激活不能解释所有DEHP诱发的脂肪肝。造成这种复杂性的原因可能是:在机体能量代谢紊乱导致疾病发生发展中,除主体的基质细胞外,免疫细胞也起到重要作用。因此,本研究从细胞之间相互作用角度,使用细胞特异性基因敲除的小鼠模型、人源性细胞模型,结合体内/外试验、计算生物学试验及转录组学、代谢组学分析等技术,探究在DEHP诱发的脂肪肝中,细胞特异性过氧化物酶体增殖剂激活受体γ(Peroxisome proliferators-activated receptor γ,PPARγ,基因名称 PPARG(人)/Pparg(小鼠))对肝脏脂质代谢紊乱的作用,阐明巨噬细胞对肝细胞能量代谢的影响。基于上述研究基础建立人源巨噬细胞模型,为快速筛选具有DEHP类似效应和机制的PAEs提供技术方法。第一部分巨噬细胞对邻苯二甲酸酯类诱发脂肪肝的调控作用及机制研究目的:阐明巨噬细胞分化、极化在邻苯二甲酸酯类诱发肝脏脂质代谢紊乱及脂肪肝中的作用及其机制方法:1、建立DEHP诱发小鼠脂肪肝模型:雄性C57BL/6J小鼠,6-8周龄,随机分为4组,每组10只,即溶剂对照组和DEHP低、中、高剂量组。DEHP各剂量组分别灌胃625、1250、2500 mg/kg bw的DEHP,溶剂对照组(DEHP 0 mg/kg bw)灌胃 0.5%(wt/vol)羧甲基纤维素(Carboxymethyl,CMC),连续28天。试验终点采集全血并处死小鼠,采用生化分析仪检测血浆甘油三酯(Triglycerides,TG)、胆固醇(Cholesterol,CHO)、胆碱酯酶(Cholinesterase,CHE)、高密度脂蛋白胆固醇(High-density lipoprotein cholesterol,HDL-C)、低密度脂蛋白胆固醇(Low-density lipoprotein cholesterol,LDL-C)、碱性磷酸酶(Alkaline phosphatase,ALP)、谷草转氨酶(Aspartate aminotransferase,AST)等;采用油红O染色检测肝脏脂质含量;采用RNA测序分析及实时荧光定量PCR(RT-qPCR)检测肝脏组织转录组学改变。2、DEHP诱发肝细胞特异性Pparg基因敲除小鼠脂肪肝模型:雄性6-8周龄C57BL/6J野生型(Wild type,WT)及肝细胞特异性Pparg敲除(Hep-KO)小鼠分别随机分组:野生型对照组(WT-CT,n=8)、野生型处理组(WT-DEHP,n=8)、肝细胞特异性Pparg敲除对照组(Hep-KO-CT,n=5)、肝细胞特异性Pparg 敲除处理组(Hep-KO-DEHP,n=5),共 4 个组。WT-DEHP 及Hep-KO-DEHP 组灌胃 2500 mg/kg bw 的 DEHP,WT-CT 及 Hep-KO-CT 灌胃0.5%(wt/vol)CMC,连续28天。试验终点时采集全血并处死小鼠,采用前述方法检测血浆TG、CHO、CHE、HDL-C、LDL-C、ALP等;检测肝脏脂质积累及转录组学改变;采用免疫组化(Immunohistochemistry,IHC)及多色免疫荧光(Multiplex Immunohistochemistry,mIF)检测肝脏 PPARγ、F4/80、CLEC4F蛋白表达。3、DEHP诱发巨噬细胞特异性Pparg基因敲除小鼠脂肪肝模型:雄性6-8周龄C57BL/6J WT小鼠及单核/巨噬细胞特异性Pparg敲除(Mac-KO)小鼠分别随机分组:WT-CT(n=8)、WT-DEHP(n=8)、单核/巨噬细胞特异性Pparg敲除对照组(Mac-KO-CT,n=5)、单核/巨噬细胞特异性Pparg敲除处理组(Mac-KO-DEHP,n=5),共 4 个组。WT-DEHP 及 Mac-KO-DEHP 组灌胃 2500 mg/kg bw 的 DEHP,WT-CT 及 Mac-KO-CT 灌胃 0.5%(wt/vol)CMC,连续28天。试验终点时采集全血并处死小鼠,同上述方法检测血浆TG、CHO、CHE、HDL-C、LDL-C、ALP等;检测肝脏脂质积累及转录组学改变;检测肝脏CLEC4F、IL-1RA、COX7A2L蛋白表达。此外采用流式细胞术检测肝脏组织中肝细胞、单核细胞、巨噬细胞的变化,采用非靶向脂质代谢组学检测肝脏脂质代谢水平的变化,采用基于磁珠的多重细胞因子试剂盒检测肝脏细胞因子水平。4、体外细胞试验:人单核细胞THP-1诱导分化为人源巨噬细胞,在诱导人源巨噬细胞极化过程中给予DEHP、邻苯二甲酸单(2-乙基己基)酯(Mono-(2-ethylhexyl)phthalate,MEHP)和(或)过氧化物酶体增殖因子活化受体(Peroxisome proliferator-activated receptors,PPARs)激动剂、拮抗剂、抑制剂处理,采用活细胞高内涵成像(Live-cell high content imaging,HCI)分析CD209、CD36表达;收集RNA进行常规转录组学测序分析;收集巨噬细胞上清液检测细胞因子改变。取自WT和Mac-KO小鼠骨髓的单核细胞诱导分化为骨髓源巨噬细胞(Bone marrow derived macrophages,BMDM),在BMDM极化过程给予DEHP或MEHP处理,采用HCI分析CD206、CD69蛋白表达。HepG2细胞在MEHP处理条件下与人源M2型巨噬细胞共培养,或直接给予不同剂量IL-1RA与MEHP共处理,采用荧光探针四甲基罗丹明乙酯(Tetramethylrhodamine,ethyl ester,TMRE)、线粒体超氧化物指示剂(mitoSOX)和BODIPY493/503分别检测线粒体膜电位、线粒体活性氧及中性脂滴(Lipid droplet,LD)的改变;收集RNA进行常规转录组学测序分析。5、计算生物学实验:采用表面等离子共振(Surface plasmon resonance,SPR)分析PPARα、PPARδ及PPARγ分别与DEHP、MEHP的相互作用;采用分子对接及分子动力学模拟研究PPARα和PPARγ与MEHP的结合力。结果:1、DEHP诱发小鼠脂肪肝模型:DEHP处理使小鼠肝重、肝重体重比显著增加;血浆TG显著降低,而CHO、ALP、HDLC、LDLC显著升高;各剂量组亦见肝脏脂质堆积。DEHP在转录水平上显著激活PPAR信号通路及脂质代谢相关通路,使Pparg、Cd36、Fabp1等基因的表达显著增加。2、DEHP诱发肝细胞特异性Pparg敲除小鼠脂肪肝模型:DEHP处理使WT小鼠的肝重、肝重体重比、血浆CHE、CHO、ALP、LDLC、HDLC显著上调,血浆TG显著下调,而Hep-KO小鼠具有类似效应。在转录组水平,DEHP处理导致的WT与Hep-KO小鼠的基因改变相似,均显著激活PPAR信号通路。两种基因型动物在DEHP处理后PPARγ表达均增加,且非肝细胞(如:巨噬细胞)高于肝细胞。DEHP处理还可使肝枯否细胞(Kupffer cells,KCs)耗竭。3、DEHP诱发巨噬细胞特异性Pparg敲除小鼠脂肪肝模型:与WT小鼠相比,DEHP处理后,Mac-KO小鼠的肝重体重比及血浆TG、ALP、CHE、CHO、LDLC、HDLC均未显著改变,亦未见肝脏脂质蓄积。DEHP处理引起Mac-KO小鼠肝脏巨噬细胞表型转换,M2型巨噬细胞增加,枯否细胞增加;炎性单核细胞减少而巡逻单核细胞增加。此外DEHP处理还使Mac-KO小鼠的氧化磷酸化(Oxidative phosphorylation,OXPHOS)、花生四烯酸(Arachidonic acid,AA)代谢等通路显著增强。4、体外细胞试验:DEHP/MEHP处理可激活巨噬细胞PPARα和PPARγ,抑制人源、骨髓源巨噬细胞的M2极化。DEHP/MEHP处理引起HepG2细胞LD蓄积、线粒体氧化代谢(TMRE、mitoSOX)增强、脂质代谢及PPAR信号通路激活;M2型巨噬细胞共处理显著降低LD、抑制线粒体氧化代谢及上述通路的激活。PPAR激动剂、拮抗剂、抑制剂处理使人源M2型巨噬细胞的IL-1RA显著改变,而IL-1RA显著抑制DEHP诱导的HepG2细胞LD、mitoSOX、TMRE 增加。5、计算生物学实验:DEHP未能与PPARα、PPARδ、PPARγ结合。MEHP与PPARα和PPARγ均能结合,且与PPARγ亲和力更强,但不与PPARδ结合。结论:巨噬细胞特异性PPARα和PPARγ协同激活通过抑制细胞内脂质代谢(如:AA代谢),抑制M2极化,促进DEHP诱发的肝脏脂质代谢紊乱及脂肪肝形成。巨噬细胞特异性Pparg敲除引起巨噬细胞PPARα激活,通过增强AA代谢及促进M2型巨噬细胞极化而改善DEHP诱发的肝脏脂质代谢紊乱,阻滞脂肪肝形成。第二部分 花生四烯酸代谢调控巨噬细胞极化的机制研究目的:阐明AA代谢在调控巨噬细胞M2极化过程中的作用,并探究M2型肿瘤相关巨噬细胞与AA代谢的相关性。方法:人THP-1细胞诱导分化为M0型巨噬细胞,并诱导极化为M1、M2型巨噬细胞。采用转录组学及代谢组学分析M0、M1、M2型巨噬细胞的能量代谢相关通路的特征,采用HCI检测参与AA代谢的关键酶的表达。在M2极化过程中给予特异性抑制剂、AA及其代谢物等处理,检测M2型巨噬细胞表面标志物CD209及特征性细胞因子(IL-4、TARC、IL-1RA等)的改变。利用WT和Mac-KO小鼠骨髓来源的单核细胞诱导分化为BMDM并诱导M2极化,检测M2型巨噬细胞表面标志物CD206的表达,以探究PPARγ在AA代谢调控巨噬细胞极化中的作用。收集小鼠食管鳞状细胞癌(Esophageal squamous cell carcinoma,ESCC)组织进行测序,获得转录组学数据,结合癌症基因组图谱(The Cancer Genome Atlas,TCGA)中人ESCC的转录组学数据,分析ESCC中M2型肿瘤相关巨噬细胞与AA代谢的相关性。结果:转录组学分析显示脂质代谢与巨噬细胞极化密切相关,脂肪酸合成(Fatty acid biosynthesis,FAS)、OXPHOS等脂质代谢相关通路的关键酶抑制可直接影响M2型巨噬细胞极化。AA代谢在人源M0、M1、M2型巨噬细胞中显著不同;与M1型巨噬细胞相比,该代谢通路在M2型巨噬细胞显著上调;抑制此代谢通路可阻滞M2极化。在极化过程中给予AA处理显著抑制M2型巨噬细胞标志物(CD209、CD206、IL-4、TARC等)的表达,而前列腺素E2(Prostaglandin E2,PGE2)处理则显著促进上述标志物的表达。PGE2处理在基因和蛋白水平上均显著增强OXPHOS通路,同时PGE2处理激活 PPAR 反应元件(PPAR response element,PPRE),促进 CD36 表达,抑制PPARγ的表达和活性。此外,在人和小鼠ESCC中AA代谢与巨噬细胞M2极化都有一定的相关性。结论:AA代谢可通过生成PGE2而促进巨噬细胞M2极化,该过程可能涉及PPAR信号通路及OXPHOS通路。PGE2可激活PPAR信号通路,增强OXPHOS 通路。第三部分 人源PPRE巨噬细胞模型在邻苯二甲酸酯类危害识别中的应用研究目的:建立并验证含PPRE的人源巨噬细胞模型,利用此模型识别具有调控巨噬细胞M2极化作用的PAEs,并探讨该效应与PPAR信号通路激活的相关性。方法:通过基因或蛋白表达检测巨噬细胞、肝癌细胞HepG2、食管癌细胞TE-1对PPARy配体的响应。将PPRE promoter-H2B-eGFP序列稳定转染THP-1细胞,通过RNA测序及RT-qPCR对其进行鉴定。利用成功构建的PPRE-eGFP-THP1细胞诱导M0巨噬细胞(PPRE-MO),通过HCI测试PPARα和PPARγ的特异性激动剂、抑制剂、拮抗剂及DEHP、MEHP对PPRE的作用,同时通过HCI检测PPARs的靶基因CD36的蛋白表达;在巨噬细胞M2极化过程给予DEHP、MEHP处理,通过检测CD209蛋白表达检验DEHP、MEHP对M2极化的作用。进而采用上述方法从邻苯二甲酸丁苄酯(Butyl benzylphthalate,BBP)、邻苯二甲酸二丁酯(Di-n-butylphthalate,DBP)、邻苯二甲酸二甲酯(Di-methyl phthalate,DMP)、邻苯二甲酸二异癸酯(Diisodecyl phthalate,DIDP)、邻苯二甲酸二异壬酯(Diisononyl phthalate,DINP)、邻苯二甲酸二异丁酯(Diisobutyl phthalate,DIBP)、邻苯二甲酸二环己酯(Dicyclohexyl phthalate,DCHP)和邻苯二甲酸二乙酯(Diethyl phthalate,DEP)中筛选具有调控巨噬细胞M2极化作用的PAEs,同时监测PPAR信号通路的激活情况。结果:巨噬细胞对PPARγ配体的响应强于HepG2或TE-1细胞。在PPRE-M0,PPARα/γ的激动剂及DEHP、MEHP均能激活PPRE并促进CD36的表达,PPARα/γ的拮抗剂或抑制剂具有相反效应;DEHP、MEHP对PPRE的激活作用及CD36表达的促进作用可被PPARα/γ的抑制剂、拮抗剂阻滞。此外,在诱导M2极化过程中,DEHP、MEHP能够抑制M2型巨噬细胞的CD209、CD36表达及LD水平。检测的8种PAEs,除DCHP外,均在M2极化过程中显著抑制CD209表达;同时,DIBP、BBP和DIDP激活PPRE并促进CD36表达。结论:PPRE-eGFP-THP1细胞诱导的巨噬细胞具有识别PPAR信号通路激活作用及巨噬细胞M2极化作用的应用潜力。DIBP、BBP、DIDP、DINP、DEP、DMP及DBP均显著抑制巨噬细胞M2极化,其中DIBP、BBP和DIDP可能通过激活PPAR信号通路发挥该效应。全文总结在DEHP诱发小鼠脂肪肝形成过程中,代谢物MEHP除激活肝细胞PPARα导致脂质动员加速以外,还激活巨噬细胞特异性PPARγ,抑制巨噬细胞内AA代谢,从而抑制M2极化。巨噬细胞特异性Pparg敲除后,MEHP激活巨噬细胞PPARα,通过增强AA代谢,促进PGE2生成,从而促进M2极化。M2型巨噬细胞通过细胞因子(如:IL-1RA)以旁分泌的方式抑制肝细胞OXPHOS,从而减少肝细胞脂质蓄积,抑制脂肪肝形成。与DEHP类似,DIBP、BBP和DIDP可能通过激活PPAR信号通路抑制巨噬细胞M2极化,具有诱导肝脏脂肪肝形成的潜力。更多还原

【Abstract】 Phthalates(PAEs)are widely used as industrial plasticizers,and their health hazards have caused increasing attentions.Diethylhexyl phthalate(DEHP)is the most common member of PAEs.Due to non-covalently bond to plastics,PAEs can easily migrate from plastics to environmental media.Therefore,humans can be exposed to PAEs via various manners.In addition,because plastics are widely used in food packaging materials,the oral route has become the main way for the general population to be exposed to DEHP.DEHP and some other phthalates have shown endocrine disrupting effects,which may interfere with the body’s metabolic homeostasis and cause various adverse health consequences such as non-alcoholic fatty liver(NAFLD).Although the effects of DEHP on human health have been studied for half a century,its toxic effect and mechanism on liver energy metabolism are still controversial.Previous studies mainly focused on the mechanism of DEHP on hepatocytes.It is believed that the peroxisome proliferators-activated receptor α(PPARα)of hepatocytes is the main target,but the activation of hepatocytes’ PPARα cannot fully explain DEHP-induced fatty liver.The reason for this complexity may be that in the occurrence and development of metabolic diseases indicated by system energy metabolism disorders,immune cells also play an important role in addition to stromal cells.Therefore,this research intends to use in vivolin vitro experiments,computational biology experiments,transcriptomics and metabolomics analysis,etc.to investigated cell-cell interactions based on cell-specific gene knockout mouse models and human-derived cell models.We explore the effect of cell-specific peroxisome proliferators-activated receptor γ(PPARγ,encoded by PPARG(human)/Pparg(mouse))on DEHP induced liver lipids metabolism disorder/fatty liver and the role of macrophages in regulating liver cell energy metabolism.In addition,we planned to establish a cell model for quickly screening out PAEs with similar effects and mechanisms to DEHP.Part One The role and mechanism of macrophages in phthalates-induced fatty liverObjective:To clarify the role and mechanism of macrophage differentiation/polarization in phthalates-induced fatty liver.Method:1.DEHP-induced fatty liver mice model.Male C57BL/6J mice,6-8 weeks old,were randomly divided into 4 groups,namely solvent control group and DEHP low,medium and high dose groups,each with 10 mice.DEHP low,medium and high dose groups were given 625,1250,and 2500 mg/kg bw of DEHP by gavage,respectively,for 28 consecutive days.The solvent control group(DEHP 0 mg/kg bw)was given 0.5%(wt/vol)carboxymethyl(CMC)by gavage.Animal were sacrificed at the endpoint of experiment.Plasma triglycerides(TG),cholesterol(CHO),cholinesterase(CHE),high-density lipoprotein cholesterol(HDL-C),low Low-density lipoprotein cholesterol(LDL-C),alkaline phosphatase(ALP)and Aspartate aminotransferase(AST)were detected via a biochemical analyzer.Oil red O staining was used to detect liver lipids;RNA sequencing and real-time quantitative PCR(RT-qPCR)were used to detect transcriptomic changes.2.DEHP-induced fatty liver model of hepatocyte-specific Pparg knockout mice.Male 6-8 week old C57BL/6J wild-type(WT)mice and hepatocyte-specific PPARy knockout(Hep-KO)mice were respectively divided into two groups.A total of 4 groups were obtained:wild-type control group(WT-CT,n=8),wild-type treated group(WT-DEHP,n=8),Hep-KO control group(Hep-KO-CT,n=5),Hep-KO treated group(Hep-KO-DEHP,n=5).The WT-DEHP and Hep-KO-DEHP groups were given 2500 mg/kg bw of DEHP by gavage for 28 consecutive days.0.5%(wt/vol)CMC was given to WT-CT and Hep-KO-CT via gavage.Animal were sacrificed and whole blood and liver were collected at the end point.Plasma TG,CHO,CHE,HDL-C,LDL-C and ALP were detected with the above method.Liver lipid accumulation and transcriptomic changes were investigated as above described.Liver protein expression of PPARγ,F4/80 and CLEC4F were determined via immunohistochemistry(IHC)or multicolor immunofluorescence(mIF).3.DEHP-induced fatty liver model of macrophage-specific Pparg knockout mice.Male 6-8 weeks old C57BL/6J WT mice and monocyte/macrophage-specific PPARy knockout(Mac-KO)mice were respectively divided into two groups,and a total of 4 group including WT-CT(n=8),WT-DEHP(n=8),Mac-KO control group(Mac-KO-CT,n=5),Mac-KO treated group(Mac-KO-DEHP,n=5)were obtained.The WT-DEHP and Mac-KO-DEHP groups were given 2500 mg/kg bw of DEHP by gavage for 28 consecutive days.0.5%(wt/vol)CMC was given to WT-CT and Mac-KO-CT groups via gavage.Animal were sacrificed and whole blood and liver were collected at the end point.Plasma TG,CHO,CHE,HDL-C,LDL-C and ALP were detect with the above method.Liver lipid accumulation and transcriptomic changes were investigated as above described.Liver protein expression of CLEC4F,IL-1RA and COX7A2L were determined using above methods.In addition,changes in hepatocytes,monocytes and macrophages within mouse liver were detected via flow cytometry.Changes in liver lipid metabolomics were determined via untargeted lipid metabolomics analysis.Cytokines level within mouse liver were detected via a magnetic bead-based multiple cytokine detection kit.4.In vitro cell test.Human monocytic THP-1 cells were differentiated into MO macrophages,and mono-(2-ethylhexyl)phthalate(MEHP)and/or PPAR agonists,antagonists,inhibitors were treated during M1/M2 polarization.Live cell immunofluorescence combined with high-content imaging are used to analyze CD209 and CD36 expression.Total RNA were collected for routine transcriptomics sequencing analysis.Cell supernatant was collect to detect cytokines level.Monocytes taken from the bone marrow of WT and Mac-KO mice were differentiated into bone-marrow derived macrophages(BMDM)and polarized into M1/M2 type macrophages.DEHP or MEHP treatments were added during the polarization process.Live cell immunofluorescence and high-content imaging were used to analyze the proteins expression of CD206 and CD69.HepG2 cells were treated with MEHP in the presence or absence of M2 type macrophages,or directly treated with IL-1RA in co-culture with MEHP.Fluorescent probes(Tetramethylrhodamine,ethyl ester,TMRE),mitochondrial superoxide indicator(mitoSOX),BODIPY 493/503 were used to detect mitochondrial membrane potential,mitochondrial reactive oxygen species and neutral lipid droplets(LD),respectively.Total RNA was collected for routine transcriptomics sequencing analysis.5.Computational biology experiments.The interaction of PPAR subtypes with DEHP and MEHP were analyzed using surface plasmon resonance(SPR).Molecular docking and molecular dynamics simulation were used to study the binding force of PPARα/γ and MEHP.Results:1.DEHP-induced fatty liver model in mice,DEHP treatment significantly increased liver weight and liver index in mice.Plasma TG were significantly reduced,while CHO,ALP,HDLC,LDLC were significantly increased.Liver lipid accumulation was seen in each dose group.DEHP significantly activates the PPAR signaling pathway and lipid metabolism-related pathways at the transcription level,and the expression of Pparg,Cd36,Fabp1 and other genes are significantly increased.2.DEHP induced fatty liver model in hepatocyte-specific Pparg knockout mice,DEHP treatment makes the liver weight,liver index,plasma TG,CHE,CHO and other indexes of WT and Hep-KO mice change to a similar degree.At the transcriptome level,DEHP treatment caused similar genetic changes in WT and Hep-KO mice,and both significantly activated the PPAR signaling pathway.The expression of PPARy in animals of both genotypes increased after DEHP treatment,and non-hepatocytes(e.g.macrophages)were higher than hepatocytes.DEHP treatment also depletes liver Kupffer cells.3.DEHP induced fatty liver model in macrophage-specific Pparg knockout mice.Compared with WT,after DEHP treatment,the liver index,plasma TG,ALP,CHE and other indexes of Mac-KO mice did not significantly change.DEHP did not cause liver lipid accumulation.DEHP treatment caused the macrophages phenotype switch in Mac-KO mouse liver,and caused increase of M2 type macrophages.DEHP decreased inflammatory monocytes while increased patrolling monocytes and Kupffer cells.Oxidative phosphorylation(OXPHOS),arachidonic acid(AA)metabolism and other pathways were significant changed by DEHP in Mac-KO mice.4.In vitro cell test.DEHP/MEHP treatment activated macrophages PPARαand PPARy,and inhibited the M2 polarization of human and bone marrow-derived macrophages.DEHP/MEHP treatment caused the accumulation and enhancement of LD in HepG2 cells,and activated lipid metabolism as well as PPAR signaling pathway.M2 macrophage co-treatment significantly reduced LD,inhibited mitochondrial oxidative metabolism and activated the above pathways.PPAR ligand significantly changed the release of IL-1RA in human M2 macrophages.And IL-1RA significantly inhibits the increase of LD,mitoSOX,TMRE in HepG2 cells caused by DEHP.5.Computational biology experiment.DEHP did not bind to PPARα,PPARδ or PPARγ.MEHP can bind to both PPARα and PPARγ,and has a stronger affinity with PPARγ.MEHP did not bind to PPARδ.Conclusion:The synergistic activation of macrophage-specific PPARa and PPARy inhibits intracellular lipid metabolism including AA metabolism,inhibits M2 polarization,and promotes DEHP-induced liver lipid metabolism disorders and fatty liver formation.Macrophage-specific Pparg knockout causes PPARαactivation,which improves DEHP-induced liver lipid metabolism disorders by enhancing AA metabolism and promoting the polarization of M2 macrophages,therefore blocking the formation of fatty liver.Part Two:Mechanism of Arachidonic Acid Metabolism in Regulating Macrophage PolarizationObjective:Clarify the role of AA metabolism in regulating the M2 polarization of macrophages and explore the relationship between tumor associated M2 macrophages and AA metabolism.Methods:Human THP-1 cells were differentiated into M0 macrophages and polarized to M1,M2 type macrophages.Transcriptomics and metabolomics were used to analyze the characteristics of the energy metabolism-related pathways of human M0,M1,M2 type macrophages.And the protein expression of key enzymes in AA metabolism were detected via live cell immunofluorescence and high content imaging system(HCIS).Then by using specific inhibitors,AA and its metabolites during the M2 polarization process,followed by detection of macrophages markers(CD209,IL-4,TARC,etc.),the polarization effects of these compounds were investigated.Subsequently,using Mac-KO mouse bone marrow-derived monocytes to obtain BMDM and explore whether PPARy are involved in the regulation of polarization by AA metabolism.Finally,mouse esophageal squamous cell carcinoma(ESCC)tissues were collected for RNA sequencing.The obtained mice ESCC transcriptomics data,combined with the transcriptomics data of human ESCC from The Cancer Genome Atlas(TCGA)were used for analyzing the correlation between M2 type tumor-associated macrophages and AA metabolism.Results:Transcriptomics analysis showed that lipid metabolism is closely related to macrophage polarization,and inhibition of lipid metabolism-related pathways such as fatty acid biosynthesis(FAS)and OXPHOS can directly affect M2 macrophage polarization.AA metabolism is significantly different in human-derived M0,M1,and M2 macrophages.Compared with M1 macrophages,this metabolic pathway is significantly up-regulated in M2 macrophages and inhibition of this metabolic pathway can block M2 polarization.During the polarization process,AA treatment significantly inhibited the expression of M2 type macrophage markers(CD209,CD206,IL-4,TARC,etc.),while prostaglandin E2(PGE2)treatment significantly promoted the above-mentioned markers expression.PGE2 treatment significantly enhanced the OXPHOS pathway at both gene and protein levels,and PGE2 treatment could inhibit the expression and activity of PPARy.In addition,there is a correlation between AA metabolism and M2 polarization in human and mouse ESCC.Conclusion:AA metabolism can directly promote the M2 polarization of macrophages through PGE2 biosynthesis,which involves the PPAR signaling pathway and OXPHOS.PGE2 activates PPAR signaling pathway and enhance OXPHOS.Part Three:Application of human-derived PPRE-macrophage model in the identification of phthalates hazardsObjective:Establish and verify a human-derived macrophage model containing PPAR response element(PPRE).Use this model to identify PAEs that regulate the M2 polarization of macrophages,and explore the potential mechanism of PPAR signaling pathway activation.Methods:Human macrophages,liver cancer cell line HepG2 cells and esophageal cancer cell line TE-1 cells were used to compare the differential response of PPARy ligands by determining gene or protein expression.The PPRE promoter-H2B-eGFP sequence was stably transferred into THP-1 cells to construct PPRE-eGFP-THP1 cells.And the obtained cells were validated by RNA sequencing and RT-qPCR.Using the successfully constructed PPRE-eGFP-THP1 cells to induce macrophages,i.e.PPRE-M0 cells and high-content imaging analysis,the PPRE response of PPARs specific activators,inhibitors,antagonists and DEHP,MEHP was determined.In parallel,live cell immunofluorescence was used to detect the protein level of CD36(PPARs target).DEHP and MEHP were added to macrophages during the M2 polarization process and their polarization effects were determined by CD209 protein expression.Furthermore,butyl benzyl phthalate(BBP),di-n-butyl phthalate(DBP),di-methyl phthalate(DMP),diisodecyl phthalate(DIDP),diisononyl phthalate(DINP),diisobutyl phthalate(DIBP),dicyclohexyl phthalate(DCHP)and diethyl phthalate(DEP)were applied on the above cell model for identifying the polarization of M2 macrophages and exploring potential PPAR signaling pathway activation.Results:The response of macrophages to PPARy ligands was much stronger than that of HepG2 or TE-1 cells.In macrophages induced by PPRE-eGFP-THP1 cells,PPARα/γ agonists and DEHP and MEHP can activate PPRE and promote the expression of CD36.PPARα/γ antagonists or inhibitors have the opposite effect.The activation of PPRE and the promotion of CD36 expression induced by DEHP and MEHP can be blocked by PPARα/γ inhibitors and antagonists.In addition,in the process of inducing M2 polarization,DEHP and MEHP can inhibit the expression of CD209,CD36 and LD level of M2 type macrophages.The tested PAEs(DBP,DEP,DMP,DINP,DIDP,DIBP and BBP)significantly inhibited the expression of CD209 during M2 polarization while DCHP did not significantly impact this process.Among them,DIBP,BBP and DIDP activated PPRE and promoted the expression of CD36.Conclusion:The macrophages induced by PPRE-eGFP-THP1 cells have the potential to recognize PPAR signaling pathway activation and the M2 polarization of macrophages.The tested PAEs significantly inhibited the M2 polarization of macrophages except for DCHP.DIBP,BBP and DIDP may function by activating the PPAR signaling pathway.SummaryDuring the formation of DEHP induced fatty liver in mice,MEHP not only activates hepatocytes’ PPARα,leading to accelerated lipid mobilization,but also activates macrophage-specific PPARy,inhibits macrophages intracellular AA metabolism,thereby inhibiting M2 polarization.After macrophage-specific Pparg is knocked out,MEHP activates macrophages PPARα,and promotes the production of PGE2 by enhancing AA metabolism,thereby promoting M2 polarization.M2 type macrophages inhibit hepatocytes’ OXPHOS in a paracrine manner through cytokines(such as IL-1RA),thereby reducing lipid accumulation in hepatocytes and inhibiting the formation of fatty liver.Similar to DEHP,DIBP,BBP and DIDP may inhibit the M2 polarization of macrophages by activating the PPAR signaling pathway,and have the potential to induce the formation of liver fatty liver.更多还原