高脂饮食诱导的小鼠NAFLD模型肝组织中m6A甲基化修饰表达谱分析.pdf

Vol.43 No.10 Oct.2023上海交通大学学报（医学版）JOURNAL OF SHANGHAI JIAO TONG UNIVERSITY(MEDICAL SCIENCE)http:/上海交通大学学报（医学版），2023，43（10）高脂饮食诱导的小鼠 NAFLD 模型肝组织中 m6A 甲基化修饰表达谱分析刘君君1，2，逯素梅2，张炳杨2，李永清2，马万山1，21.山东大学临床医学院，济南 250014；2.山东第一医科大学第一附属医院（山东省千佛山医院）检验医学科，山东省医药卫生临床检验诊断学重点实验室，济南 250014摘要 目的利用微阵列芯片技术检测高脂饮食诱导的小鼠非酒精性脂肪性肝病（non-alcoholic fatty liver disease，NAFLD）模型中肝组织mRNA的m6A甲基化修饰和基因表达的变化。方法以68周龄雄性C57BL/6J小鼠为实验动物，高脂饲料喂养16周诱导建立NAFLD模型（高脂组，n=10）；另设基础组（n=10）为对照，给予含10%脂肪的基础饲料喂养。苏木精-伊红（hematoxylin-eosin，H-E）染色评估小鼠肝组织病理改变，判断NAFLD模型是否构建成功。运用甲基化RNA免疫共沉淀技术（methylated RNA immunoprecipitation，MeRIP）和微阵列测序技术（microarray表达谱分析）检测2组小鼠肝组织中mRNA的m6A甲基化和表达水平变化。结果基础组小鼠肝脏呈鲜红色，少见脂肪沉积；高脂组小鼠肝脏边界黄润，H-E染色可见肝细胞中脂滴弥漫浸润且相互融合，提示高脂饲料诱导的NAFLD模型构建成功。MeRIP-微阵列芯片检测结果显示，与基础组相比，高脂组小鼠肝脏中共有320个基因m6A甲基化修饰水平变化显著（P1.5），其中有108个上调基因和212个下调基因。将组间m6A甲基化水平差异显著的基因与mRNA差异表达基因取交集，发现有163个基因m6A甲基化水平和mRNA表达水平均差异显著。结论高脂饮食诱导的小鼠NAFLD模型中肝组织mRNA的m6A修饰变化显著，且该变化与mRNA的基因表达有关。关键词非酒精性脂肪性肝病；m6A甲基化修饰；mRNA；表观转录组学DOI10.3969/j.issn.1674-8115.2023.10.002 中图分类号R446 文献标志码AAnalysis of m6A methylation expression profiles in liver tissue of high-fat diet-induced mouse models of NAFLDLIU Junjun1,2,LU Sumei2,ZHANG Bingyang2,LI Yongqing2,MA Wanshan1,21.School of Clinical Medicine,Shandong University,Jinan 250014,China;2.Department of Clinical Laboratory Medicine,The First Affiliated Hospital of Shandong First Medical University/Shandong Provincial Qianfoshan Hospital,Shandong Medicine and Health Key Laboratory of Laboratory Medicine,Jinan 250014,ChinaAbstract Objective To detect the differences in m6A methylation modification and gene expression of liver tissue mRNA in high-fat diet-induced mouse models of non-alcoholic fatty liver disease(NAFLD)using microarray technology.Methods The NAFLD models were established in 6-8 weeks old male C57BL/6J mice fed with high-fat chow for 16 weeks(high-fat group,n=10).The basal group(n=10)was given 10%fat diet.Hematoxylin-eosin(H-E)staining was used to assess the histopathological changes in liver tissue and to determine the success of the NAFLD models.The changes of mRNA m6A methylation and expression levels in the liver tissues of the two groups were detected by using methylated RNA immunoprecipitation(MeRIP)and microarray expression profiling.Results The livers of the mice in the basal group were bright red with few fat deposits,while the livers of the mice in the high-fat group were yellowish with diffuse infiltration and fusion of lipid droplets in the hepatocytes by H-E staining,suggesting that the high-fat diet-induced NAFLD models were successfully constructed.The results of the MeRIP-microarray showed that the m6A methylation levels of 320 genes in the livers of mice in the high-fat group were significantly altered compared with those in the basal group(P1.5),of which 108 genes were up-regulated and 212 genes were down-regulated.Genes with significant differences in m6A methylation levels between the two groups were intersected with those with differentially expressed mRNAs,and 163 genes were found to have significant differences in both m6A methylation level and mRNA expression level.论著 基础研究基金项目 山东省自然科学基金（ZR2021MH187）；山东省千佛山医院国家自然科学基金培育基金（QYPY2020NSFC1004，QYPY2021NSFC0804）。作者简介 刘君君（1992）女，主管技师，硕士生；电子信箱：。通信作者 马万山，电子信箱：。Funding Information Natural Science Foundation of Shandong Province(ZR2021MH187);Shandong Provincial Qianfoshan Hospital,National Natural Science Foundation of China Cultivation Fund(QYPY2020NSFC1004,QYPY2021NSFC0804).Corresponding Author MA Wanshan,E-mail:.12272023,43（10）上海交通大学学报（医学版）Vol.43 No.10 Oct.2023JOURNAL OF SHANGHAI JIAO TONG UNIVERSITY(MEDICAL SCIENCE)Conclusion The change in m6A modification of liver tissue mRNA in the high-fat diet-induced mouse models of NAFLD is significant and the change is associated with the gene expression of mRNA.Key words non-alcoholic fatty liver disease(NAFLD);m6A methylation modification;mRNA;epitranscriptomics随着社会经济的进步和人们物质生活的改善，非酒精性脂肪性肝病（non-alcoholic fatty liver disease，NAFLD）的发病率激增，已成为我国最常见的慢性肝病。有数据显示我国成年人 NAFLD 发病率为12%15%1，预计到 2030 年我国 NAFLD 患病人数将达到3.14亿2。NAFLD主要表现为肝细胞脂质代谢异常，肝细胞受损后引起炎症、胰岛素抵抗、纤维化等反应3。既往研究4-7表明，肥胖、2 型糖尿病、高脂血等代谢综合征与NAFLD的发生和发展关系密切，两者之间互为因果，并行发展8-9。NAFLD可引起失代偿期肝硬化、肝细胞癌，还参与2型糖尿病和动脉粥样硬化的发病10-12。NAFLD使患者生活质量降低，寿命缩短，还加重了国家的卫生经济负担13。基因在表达过程中会受到不同水平的调控。已有研究14-15表明聚焦 RNA 修饰的表观转录组学在NAFLD的发生和发展中起关键调控作用，而N6-甲基腺 嘌 呤（N6-methyladenosine，m6A）是 真 核 生 物mRNA中最普遍且动态可逆的修饰方式。m6A修饰可沉积在RNA的转录本上，调节RNA转运、剪接、降解及翻译等过程。本研究借助高脂饲料喂养构建小鼠 NAFLD 模型，利用甲基化 RNA 免疫共沉淀技术（methylated RNA immunoprecipitation，MeRIP）和微阵列测序技术，检测差异m6A甲基化修饰和差异基因表达，对数据进行联合分析，为NAFLD发病的基因调控机制研究提供理论依据。1对象与方法1.1实验动物SPF级C57B/6J雄性小鼠，68周龄，体质量1820 g，购自北京维通利华实验动物技术有限公司。动物生产许可证号为 SCXK（京）2016-0006，使用许可证号为SYXK（鲁）2018-0009。饲养环境采用12 h明和12 h暗交替，温度为2026、日温差1，相对湿度达40%70%，新鲜空气换气次数15次/h，气流速度0.18 m/s，压差25 Pa，氨浓度15 mg/m3，噪音1.5）和统计学显著性（P0.05），筛选2组之间差异m6A甲基化基因和差异表达基因。2结果2.1小鼠NAFLD肝组织中m6A修饰水平变化高脂饲料喂养16周后，基础组和高脂组的小鼠体质量出现明显差异，高脂组小鼠平均体质量为（455）g，而基础组小鼠为（255）g。高脂组的小鼠肝脏边界黄润，表面有颗粒感，且有明显的弥漫性肿大（图1A）。H-E染色见高脂组小鼠肝组织细胞质中ControlHigh-fatAControlHigh-fatBCControlHigh-fat50 m50 m200 ng.L-1200 ng.L-1100 ng.L-1100 ng.L-150 ng.L-150 ng.L-1m6AMethylene blueRNA concentrationNote：A.General observation of the livers.B.H-E staining showed the cytoplasm of liver tissue in the high-fat group was filled with lipid droplets.C.Dot-blot showed the increase of m6A methylation levels in the high-fat group.图1高脂饮食诱导的小鼠NAFLD肝组织中m6A修饰水平变化Fig 1Changes of m6A modification levels in high-fat diet mouse livers12292023,43（10）上海交通大学学报（医学版）Vol.43 No.10 Oct.2023JOURNAL OF SHANGHAI JIAO TONG UNIVERSITY(MEDICAL SCIENCE)充满脂滴，且脂滴相互融合导致细胞核偏位（图1B）。以上结果均提示高脂饮食诱导的小鼠 NAFLD模型构建成功。Dot-blot实验结果显示，与基础组小鼠相比，高脂组小鼠肝组织内总 m6A 修饰水平升高（图 1C）。2.2基因微阵列芯片实验流程的质量控制基础组 3 只小鼠（编号为 7、8、9），高脂组 3只小鼠（编号为 10、11、12），经 PCA 分析后可知2 组样本之间的相似性低，符合基因芯片分析的要求（图 2A）。使用 Nano drop 对高脂组和基础组小鼠肝组织 RNA 进行定量及质量检测，检测结果合格的前提下，进行 MeRIP-qPCR 质量检测。结果显示，阴性对照%（MeRIP/Input）70，MeRIP 质 量 检 测 合 格（图 2B）。2.3小鼠肝脏mRNA差异m6A修饰基因与差异表达基因的联合分析2 组小鼠肝组织基因芯片差异甲基化分析发现，与基础组相比，高脂组有 320 个 mRNA 发生m6A 甲基化差异修饰（differentially methylated m6A site，DMMS）（变化倍数1.5，P1.5，且P0.05）取交集。将高甲基化高表达的基因和m6A甲基化修饰显著上调的基因取交集后，筛选出19个基因（图4A）；将高甲基化低表达的基因和m6AControlPC2(21.84%)PC1(30.75%)High-fatNegative ControlPositive ControlControlHigh-fatAB-7.55.02.50-2.5-5.0-2.52.55.07.50987111012-01220406080100%(MeRIP/Input)Note：A.PCA-plot.B.MeRIP-qPCR quality inspection bar chart showed negative and positive control.图2样品质控和MeRIP-qPCR质量检测Fig 2Sample quality control and MeRIP-qPCR quality testing1230刘君君，等高脂饮食诱导的小鼠NAFLD模型肝组织中m6A甲基化修饰表达谱分析http:/上海交通大学学报（医学版），2023，43（10）Hyper-up(25)Hyper-down (46)Hypo-up(58)Hypo-down(34)ABC-log10(P value)log2(Fold change)High-fat vs Controllog2(Gene expression fold change)High-fat vs Control-6543210-3-2-11230Micalc1Cyp2b10Ksr2Ybx1Paqr7Casc5Ubr3Dpm3Cd36Tsc22d1EsdS1c22a26Slc46a3Inpp5dHoxd9Romo1LitafMdh2Sycp3Folr1Not differentially methylated(34 073)Hypo-methylated(212)Methylated(108)12-high fat11-high fat10-high fat9-control8-control7-controlExpression-110log2(Methylation fold change)High-fat vs Control-6420-2-4-6-6-4-224680Note：A.Volcano plot of differential m6A methylation mRNAs.B.Cluster diagram showed top 10 differentially m6A-methylated mRNAs.C.Four quadrant diagrams showed differential methylation associated with gene expression in the high-fat group and the control group.图3小鼠肝脏mRNA差异m6A修饰基因与差异表达基因的联合分析Fig 3Conjoint analysis of differential m6A methylation mRNAs and differentially expressed genes in the livers of mice12312023,43（10）上海交通大学学报（医学版）Vol.43 No.10 Oct.2023JOURNAL OF SHANGHAI JIAO TONG UNIVERSITY(MEDICAL SCIENCE)表1前10位m6A差异修饰的mRNA信息Tab 1Top 10 differentially m6A-methylated mRNAsGeneUp-regulated geneMicalc1Cyp2b10Ksr2Ybx1Paqr7Casc5Ubr3Dpm3Cd36Tsc22d1Down-regulated geneEsdSlc22a26Folr1Slc46a3Inpp5dHoxd9Romo1LitafMdh2Sycp3mRNA_IDENSMUST00000033033ENSMUST00000005477ENSMUST00000180430ENSMUST00000079644ENSMUST00000095074ENSMUST00000099542ENSMUST00000131553ENSMUST00000107462ENSMUST00000082367ENSMUST00000142683ENSMUST00000176957ENSMUST00000120522NM_001252553ENSMUST00000138244ENSMUST00000167032ENSMUST00000059272ENSMUST00000088610ENSMUST00000117360ENSMUST00000019323ENSMUST00000125612Locuschr7:112374345-112395355:+chr7:25897676-25926559:+chr5:117414000-117775003:+chr4:119277981-119294604:chr4:134497004-134510235:+chr2:119047119-119104121:+chr2:69897303-69938659:+chr3:89259358-89267077:+chr5:17782016-17835696:chr14:76487759-76506998:+chr14:74732384-74749848:+chr19:7781041-7802578:chr7:101858331-101870788:chr5:147893881-147894815:chr1:87676239-87720502:+chr2:74697727-74700208:+chr2:156144039-156145797:+chr16:10960824-10975579:chr5:135778480-135790391:+chr10:88459664-88473236:+RNA length/nt2 2591 82213 1251 6283 5506 5251 8018273 0167971 1172 8541 3373884 1252 1365697631 4561 169Fold change2.8402.7212.4842.3592.2742.2702.1131.9781.9651.9280.3560.4210.4370.4370.4620.4710.4840.4920.5090.513P value0.0040.0110.0010.0230.0020.0000.0050.0250.0210.0280.0100.0100.0000.0010.0100.0120.0050.0140.0320.044甲基化修饰显著上调的基因取交集后，筛选出18个基因（图4B）；将低甲基化高表达的基因和m6A甲基化修饰显著下调的基因取交集后，筛选出33个基因（图4C）；将低甲基化低表达的基因和m6A甲基化修饰显著下调的基因取交集后，筛选出 21 个基因（图4D）。1232刘君君，等高脂饮食诱导的小鼠NAFLD模型肝组织中m6A甲基化修饰表达谱分析http:/上海交通大学学报（医学版），2023，43（10）GeneAC168051.1Hacd2SpopGnai1Ube2uCes1gBtcGstm3Dyrk1aCyp2b10MtdhYbx1RP24-242P4.2RP23-158M23.2Ypel2TgfaPex3Cd36CideamRNA_IDENSMUST00000147525ENSMUST00000061156ENSMUST00000107722ENSMUST00000074694ENSMUST00000133493ENSMUST00000044602ENSMUST00000121044ENSMUST00000004136ENSMUST00000023614ENSMUST00000005477ENSMUST00000022865ENSMUST00000079644ENSMUST00000036819ENSMUST00000178224ENSMUST00000018571ENSMUST00000032066ENSMUST00000019945ENSMUST00000082367ENSMUST00000025404GeneCtsfMen1RP24-290O13.4PtafrSlc2a4Ly6gMicalc1Fam124bDnajb4Casc5RP24-284L24.1Anapc15Olfr1111Mid1ip1Slc3a2Zfp37Dpm3Nr2c2mRNA_IDENSMUST00000119694ENSMUST00000056391ENSMUST00000049476ENSMUST00000070690ENSMUST00000018710ENSMUST00000190262ENSMUST00000033033ENSMUST00000058748NM_001356364ENSMUST00000099542ENSMUST00000187643ENSMUST00000210984ENSMUST00000216378ENSMUST00000008179ENSMUST00000205538ENSMUST00000068822ENSMUST00000107462ENSMUST00000133133GeneMgat1Rbm26EsdLy6dMecrBpnt1Slc43a2Ltc4sPdgfrbNucb1Trpv2Zfp78Rasgef1bGhdcPde6gGlp2rArhgdibSlamf7Nkg7Hoxd9Zic4Gpx4ArtnSkp1aTbc1d7Phactr1Zfp239CfdIl15raLgals1Tmem176bRP23-342N3.3AgermRNA_IDENSMUST00000129588ENSMUST00000100327ENSMUST00000176957ENSMUST00000040404ENSMUST00000030742ENSMUST00000110965ENSMUST00000169547ENSMUST00000101265NM_008809ENSMUST00000209436uc007jjj.1ENSMUST00000108559ENSMUST00000031276ENSMUST00000139341ENSMUST00000026452ENSMUST00000051765NM_001301308ENSMUST00000192024ENSMUST00000070518ENSMUST00000059272ENSMUST00000172646ENSMUST00000183037NM_001284192ENSMUST00000109072ENSMUST00000220787ENSMUST00000128646ENSMUST00000172088ENSMUST00000061653ENSMUST00000138349ENSMUST00000089377ENSMUST00000203355ENSMUST00000058868ENSMUST00000015596GeneTff3AcppSh2d1aPolr2jTmc8Spin1VimEmc10Rnase4Slc46a3Cib3DnttBdkrb1AC101869.3Folr1RP23-422L16.28Phf21bSema4cFam46aZfp644Olfr532mRNA_IDENSMUST00000024827ENSMUST00000112590NM_001313688ENSMUST00000111129ENSMUST00000127080NM_001283029ENSMUST00000141365ENSMUST00000118808ENSMUST00000169895ENSMUST00000031655ENSMUST00000098630ENSMUST00000051806ENSMUST00000041229ENSMUST00000148020NM_001252553ENSMUST00000210714ENSMUST00000159939ENSMUST00000195620ENSMUST00000034802ENSMUST00000122980ENSMUST00000213801AHyper-upHyper-diffmethylatedBHyper-downHyper-diffmethylatedCHypo-upHypo-diffmethylatedDHypo-downHypo-diffmethylated6198826188912175332421187Note:A to DVenn diagrams for high/low m6A methylation and high/low expression genes.图4小鼠肝组织差异显著的m6A修饰且差异表达的基因信息Fig 4Differential m6A modification and differentially expressed genes in mouse liver tissue12332023,43（10）上海交通大学学报（医学版）Vol.43 No.10 Oct.2023JOURNAL OF SHANGHAI JIAO TONG UNIVERSITY(MEDICAL SCIENCE)3讨论NAFLD的病因及发病机制比较复杂，目前仍未完全阐明。现阶段NAFLD的治疗主要通过改善生活方式以稳定机体调节机制，积极寻找并去除可能的病因和诱因，以及减肥等，尚未发现可以根治该病的药物 16。因此，NAFLD的治疗也是全球面临的一大难题。m6A修饰是一种新型的表观转录后调控基因表达的重要方式，在多种疾病中的作用备受关注。已有研究17证实m6A在人类生命过程中的各个阶段起关键作用，并参与NAFLD、肥胖和2型糖尿病等疾病的发生和发展。PENG等18研究发现，mRNA的m6A修饰与脂质代谢关系密切，高脂饮食诱导脂肪肝的m6A修饰发生改变，影响肝脏的胰岛素敏感性。m6A甲基化作为新型生物标志物，为代谢性疾病的治疗提供了潜在的新靶点。因此，建立高脂饮食诱导下肝组织m6A修饰差异表达谱具有重要意义。近年来，微阵列芯片作为一种快速、并行、高效的分子生物学研究工具，对探究基因表达的调控机制有重要的辅助作用。本研究通过高脂饲料喂养小鼠，构建了稳定可靠的小鼠 NAFLD 模型。研究发现NAFLD小鼠总m6A甲基化水平升高，在RNA样本质检合格的前提下，进一步进行真核生物转录组基因m6A微阵列芯片表达谱分析，基于检测结果建立了高脂饮食诱导下肝组织m6A修饰差异表达谱。其中，有320个基因m6A甲基化修饰水平变化显著（P1.5），且发现有 163个基因 m6A甲基化水平和 mRNA表达水平均差异显著。该结果提示 m6A甲基化水平可能影响转录组基因表达的变化，且与肝脏脂质代谢的调控密切相关，这 163 个差异基因在NAFLD发生和发展中可能发挥重要的调控作用。现有文献证实，筛选出的部分差异表达基因与NAFLD的发生和发展有不同程度的相关性。例如，有研究19 表明小鼠肝脏组织Hacd2缺失可通过减少血清中长链脂肪酸的生成增加以产热为主的能量消耗，改善高脂饮食诱导的NAFLD等。Cd36缺失的小鼠肝脏中Srebp1、Ppar和Fasn等成脂相关基因表达降低，在HepG2细胞中也证实CD36通过与INSIG2结合，促进肝脏SREBP1表达和脂质从头合成途径，从而促进NAFLD的发生和发展20。外周血PPAR和CIDEA的mRNA分子表达水平与血浆三酰甘油和总胆固醇水平呈显著负相关21。SANS等22 的研究表明：在肥胖患者中，肝脏CIDEA的表达与NAFLD活性评分和肝损伤密切相关；肝脏CIDEA的表达与肥胖程度呈正相关，且会加重NAFLD的进展。TANG等23 报道，高脂饮食小鼠肝脏中CIDEA的m6A甲基化水平升高与mRNA表达水平呈显著正相关，经实验证实mettl16介导 的 m6A 甲 基 化 通 过 上 调 CIDEA 表 达 水 平 促 进NAFLD进展。有研究24 显示，Ces1/Es-x在小鼠肝脏脂肪代谢中起调节作用，Ces1/Es-x基因敲除小鼠出现肥胖、脂肪肝、高胰岛素血症等NAFLD表现。高脂诱导的肥胖小鼠中，MECR基因的表达水平与2型糖尿病密切相关，ATP降低MECR蛋白水平可能参与胰岛素抵抗的发生25。高脂饮食可诱导小鼠肝脏发生严重的脂肪样变，而纤维化相关基因Pdgfrb、Tgfb在小鼠体内表达升高26。以上研究都提示，本次研究中筛选出的差异基因对NAFLD的发病机制研究有重要意义。本研究未对筛选出的差异基因进行进一步验证。后续我们将围绕差异基因进行验证，并通过干扰或过表达差异基因，开展进一步的功能分析，为NAFLD的预防和治疗提供新的靶点。综上所述，本研究通过比较正常小鼠与NAFLD模型小鼠中肝脏组织的m6A甲基化及mRNA表达差异，发现NAFLD小鼠肝组织中m6A修饰水平发生了显著变化，且 mRNA表达水平变化显著，表明 m6A甲基化可能通过影响mRNA的表达调控NAFLD的发生和发展。本研究建立了主要差异基因表达谱，为筛选更多的NAFLD治疗靶点提供了一定的理论基础。利益冲突声明/Conflict of Interests 所有作者声明不存在利益冲突。All authors disclose no relevant conflict of interests.伦理批准和动物权利声明/Ethics Approval and Animal Right本研究涉及的所有动物实验均已通过山东第一医科大学第一附属医院医学伦理委员会的审核批准，审批号为2022动伦审字（S081）号。所有实验过程均遵照实验动物福利伦理审查指南的条例进行。All experimental animal protocols in this study were reviewed and approved by Medical Ethics Committee of The First Affiliated Hospital of Shandong First Medical University(Approval Letter:2022 Animal Ethics Society No.S081),and all experimental animal protocols were carried out by following the guidelines of Guideline for Ethical Review of Animal Welfare.作者贡献/Authors Contributions马万山、逯素梅、刘君君参与了实验设计以及论文的写作和修改，1234刘君君，等高脂饮食诱导的小鼠NAFLD模型肝组织中m6A甲基化修饰表达谱分析http:/上海交通大学学报（医学版），2023，43（10）刘君君、张炳杨、李永清负责实验操作与数据分析。所有作者均阅读并同意了最终稿件的提交。The study was designed by MA Wanshan,LU Sumei and LIU Junjun.The manuscript was drafted and revised by MA Wanshan,LU Sumei and LIU Junjun.The experiments were performed by LIU Junjun,ZHANG Bingyang and LI Yongqing.The data were analyzed by LIU Junjun,ZHANG Bingyang and LI Yongqing.All the authors have read the last version of paper and consented for submission.Received:2023-04-14 Accepted:2023-09-12 Published online:2023-10-28 参考文献 1 祥蔚.IL-17A基因敲除减轻LPS诱导的脂肪肝小鼠炎症性肝损伤及其机制研究D.重庆:第三军医大学,2017.XIANG W.IL-17A deficiency alleviates LPS-induced steatotic liver injury in mice and the underlying mechanismD.Chongqing:Third Military Medical University of Chinese P.L.A.,2017.2 ESTES C,ANSTEE Q M,ARIAS-LOSTE M T,et al.Modeling NAFLD disease burden in China,France,Germany,Italy,Japan,Spain,United Kingdom,and United States for the period 20162030J.J Hepatol,2018,69(4):896-904.3 CHENG Y,HOU T,PING J,et al.Quantitative succinylome analysis in the liver