中文摘要

 

第一部分 川崎病血浆外泌体来源的蛋白质组学分析

 

目的：川崎病（Kawasaki disease，KD）是一种累及全身多器官的儿童血管炎性综合征，冠状动脉瘤（Coronary artery aneurysm，CAA）是KD最常见的并发症，也是目前儿童获得性心脏病的主要病因。目前，KD合并CAA的临床诊断主要依靠超声影像，而缺乏特异的诊断标志物。本研究探索外泌体来源的蛋白质作为KD合并CAA的早期诊断标志物，为临床决策提供依据。

 

方法：本研究为病例对照研究。病例组为2019年1月至2024年1月在首都儿科研究所附属儿童医院心血管内科住院治疗的初次发病KD患儿；健康对照组（Healthy control，HC）为同期于我院保健科体检的年龄-性别匹配儿童；发热对照组（Febrile control，FC）为同期于我院呼吸内科住院治疗的年龄-性别匹配明确病原的发热患儿。为了探索与冠脉病变严重程度相关的蛋白表达情况，KD组患儿根据冠脉病变程度分为3个亚组：KD不伴CAA患儿、KD伴小型CAA（Small coronary artery aneurysm，SCAA）和中型CAA（Medium coronary artery aneurysm，MCAA）患儿以及KD伴巨大CAA（Giant coronary artery aneurysm，GCAA）患儿。

采用超速离心法提取外泌体，通过电镜形态观察和免疫印迹（Western blotting，WB）检测外泌体标志物表达共同验证血浆来源的外泌体。采用液相色谱-质谱合并TMT6标记法分析KD组、HC组及FC组的外泌体蛋白表达谱，并进行生物信息学功能分析，绘制差异表达蛋白（Differentially expressed proteins，DEPs）富集KEGG、Reactome、Wiki通路图谱和GO功能富集图谱以及蛋白互作网络图，结合KD的病理机制筛选出特异表达蛋白并WB进行验证。

 

结果：

本研究共纳入60例儿童，其中40例（每组n=8）用于外泌体蛋白提取及生物信息学分析；20例（每组n=4）用于验证筛选蛋白的表达水平。电镜下形态观察和WB对外泌体标志物的验证提示所提取蛋白为外泌体来源。

初步筛选出具有显著差异的DEPs共995种（P＜0.05）。进行表达差异倍数为1.3的筛选后，与HC组相比，无论合并CAA与否，KD组共同表达DEPs共332种。与HC和KD组相比， FC组特异性表达DEPs共374种；与HC和FC组相比， KD组中特异性表达DEPs共104种；FC和KD两组相比，共同表达DEPs共228种。FC组特异性表达的374种DEPs中，富集到了胆固醇和脂质代谢相关通路。

在FC组和KD组，即发热性疾病共同表达的228种DEPs中，富集到了细胞结构和功能相关通路；趋势分析显示：在FC和KD亚组共24种DEPs随病情严重程度呈现显著下降趋势（P＜0.05）。进一步对这24种DEPs分析，富集到了感染相关通路，并筛选出在KD组表达显著下降的S100A9等蛋白进行后续验证。

对KD组特异性表达的104种DEPs通路分析显示：这些DEPs集中在补体相关通路、血小板功能激活通路等。对上述104种DEPs的趋势分析显示：54种显著改变的DEPs随KD并发症严重程度增加而下降，这些DEPs富集于G蛋白功能通路、胆汁酸受体相关的S1PR转导通路。综合DEPs富集的通路和表达趋势，我们筛选了补体通路中的C3、SERPNA1和G蛋白功能通路的GNAI2等蛋白进行后续验证。

对KD是否合并CAA亚组分析显示：91种DEPs在KD合并CAA患儿中特异性表达，且富集于补体相关通路和中性粒细胞体外诱捕网通路等。16种DEPs在KD不伴CAA患儿中特异性表达，且富集于病毒感染和免疫相关通路。对KD合并不同严重程度CAA亚组分析显示：共102种DEPs在KD合并SCAA/MCAA患儿中特异性表达，且富集于血小板激活相关通路等。34种DEPs在KD合并GCAA患儿中特异性表达，且富集于细胞功能相关通路等。

基于上述生信分析结果，我们筛选了与补体相关、免疫相关、细胞结构和功能相关的DEPs进行WB蛋白表达水平验证，其中5种蛋白在不同组别中出现了显著差异：

与FC组相比，S100A9蛋白在KD组中表达水平显著降低（P＜0.05），并与质谱分析结果一致。

与HC组相比，补体C3蛋白在KD无CAA患儿组表达水平显著升高（P＜0.05）；与FC组相比，补体C3蛋白在KD无CAA患儿组表达水平显著升高（P＜0.01）；并与质谱分析结果一致。

与KD无CAA患儿相比，补体SERPINA1蛋白和G蛋白通路GNAI2蛋白在KD合并CAA患儿中表达水平显著下降（P＜0.05），并与质谱分析结果一致。

与KD合并SCAA/MCAA患儿相比，Rap1通路蛋白Rap1b 在KD合并GCAA患儿中表达水平显著下降（P＜0.05），并与质谱分析结果一致。

 

结论：本研究提供了KD及KD合并CAA患儿血浆外泌体蛋白图谱，提示SERPINA1和GNAI2蛋白可能作为KD合并CAA的潜在诊断标志物；Rap1b蛋白可能作为KD合并GCAA的潜在诊断标志物。

 

中文摘要

 

第二部分 川崎病血浆代谢组学分析

 

目的：川崎病（Kawasaki disease，KD）是一种全身免疫性血管炎，病程中伴随着机体多种代谢紊乱。基于前一部分外泌体蛋白质组学分析，发现KD患儿胆汁酸前体胆固醇及其相关脂质代谢通路改变。本研究利用代谢组学分析KD患儿外周血浆的差异代谢产物水平，主要聚焦于KD外周血浆胆碱/肉碱衍生物相关通路及胆汁酸代谢相关通路代谢物的变化。

 

方法：本研究为病例对照研究，病例组为2022年1月至2024年6月在首都儿科研究所附属儿童医院心血管内科住院治疗的初次发病KD患儿；健康对照组（Healthy control，HC）为同期于我院保健科体检年龄-性别匹配的健康儿童；发热对照组（Febrile control，FC）为同期于我院呼吸内科住院治疗的年龄-性别匹配且明确病原的发热患儿。首先，通过非靶向代谢组学检测及生信分析，探索KD患儿外周血浆代谢谱，并对筛选的代谢物进行靶向代谢检测及分析。

 

结果：

1. 本研究共纳入220例儿童，其中40例（KD组与HC组，每组n=20）用于KD组与HC组外周血浆非靶向代谢物检测及生物信息学分析；20例（KD组与HC组，每组n=10）用于KD组与HC组胆碱/肉碱代谢通路代谢物水平靶向检测；70例（HC组及FC组，每组n=20；KD组n=30）用于胆碱/肉碱的代谢终产物三甲胺-氮-氧化物（Trimethylamine-N-oxide，TMAO）水平的靶向检测；90例（每组n=30）用于靶向验证HC、FC及KD组胆汁酸水平。

2. 与HC组相比，具有显著差异且差异倍数为2的差异代谢产物（P＜0.05）共892种，其中在KD患儿中上调的差异代谢产物共430种，下调的差异代谢产物共462种；这些差异代谢产物富集于胆碱/肉碱衍生物及其代谢产物下游调控的胆汁酸代谢通路。

3. 对KD患儿靶向胆碱/肉碱通路代谢物分析显示：相比于HC组，甜菜碱在KD患儿中显著升高（4036.91±800.93 比2821.21±414.00ng/ml，P＜0.001），胆碱、肉碱及甜菜碱的终末代谢产物、同时也是胆汁酸的上游调控物TMAO水平在KD患儿中显著降低[5.03(3.34，57.57)VS 44.36（28.27，106.62）ng/ml，P＜0.05]。扩大样本量验证后发现，相比于HC和FC儿童，KD患儿外周血浆TMAO水平显著降低，组间相比：KD 比HC：[28.14(10.00，76.58)VS 163.34(84.83，298.32) ng/ml，P＜0.001]；KD 比FC：[28.14(10，76.58)VS 62.30(25.39，109.51) ng/ml，P＜0.05]。

4. 对KD患儿胆汁酸相关代谢物靶向分析显示：KD组有11种胆汁酸水平与HC组相比显著改变（P＜0.05）；其中牛磺石胆酸、牛磺胆酸等8种胆汁酸水平在KD患儿中明显升高；别胆酸、熊牛磺去氧胆酸和熊胆酸等3种胆汁酸在KD患儿中明显降低。

 

结论：本研究揭示了KD患儿血浆胆碱/肉碱代谢物TMAO及其下游胆汁酸水平的变化。提示TMAO-胆汁酸代谢通路可能参与KD的代谢紊乱；TMAO可能作为基于代谢紊乱的KD早期诊断标记物。

 

中文摘要

 

第三部分 TMAO作为川崎病早期诊断标志物的机制研究

 

目的：基于在川崎病（Kawasaki disease，KD）患儿人群中发现的胆碱代谢终产物三甲胺-氮-氧化物（Trimethylamine-N-oxide，TMAO）显著降低，在KD血管炎小鼠模型上通过代谢组学分析其血浆差异代谢产物的变化；通过单细胞组学分析TMAO相关基因在小鼠心脏组织表达变化；并采用TMAO干预血管炎小鼠，探索其对心脏炎症和下游胆汁酸代谢的影响。以期通过研究，阐明TMAO在KD血管炎小鼠模型中的作用及其机制，为其作为KD基于机制的诊断标志物提供依据。

 

方法：本研究通过腹腔注射干酪乳杆菌细胞壁提取物（Lactobacillus casei cell wall extract，LCWE）构建KD血管炎小鼠模型；采用液相色谱-质谱法检测小鼠血浆代谢物水平并进行生物信息学功能分析；采用单细胞测序分析KD血管炎和对照组小鼠心脏组织细胞水平的TMAO及胆汁酸相关基因；TMAO干预KD血管炎小鼠进行冠状动脉炎症评分及外周血胆汁酸水平检测。

 

结果：

对小鼠血浆的非靶向代谢组学分析显示：在KD血管炎小鼠血浆中共检测出1314种代谢物，这些代谢物主要是脂质及有机酸的衍生物，KEGG分析富集于胆碱代谢的胆汁分泌等代谢通路。与对照组小鼠相比，筛选出159种P＜0.05且VIP大于1的差异代谢物，这些差异代谢产物富集于蛋白质吸收降解、氨基酸代谢和胆汁酸代谢相关通路等；此外，另有30种代谢物在KD小鼠中特异性表达，这些代谢物富集于VEGF信号通路和趋化因子信号通路等。

对小鼠心脏组织单细胞分析显示：KD和健康小鼠心脏组织中存在巨噬细胞、中性粒细胞、内皮细胞、成纤维细胞和平滑肌细胞共5种主要细胞类型。与健康对照小鼠相比，KD小鼠心脏组织中不同巨噬细胞亚群的比例发生显著变化：Macro_Clqc亚群显著增加，而Macro_Isg15、Macro_Spp1和Macro_Il1b亚群以及单核细胞数量均减少。进一步的基因表达分析显示，KD小鼠巨噬细胞中炎症相关基因Il-1β、Nlrp3、Icam1等表达显著上调 (P < 0.01)。TMAO通路下游的关键基因Sirt2、Mapk1和Akt1的表达显著改变：在M2巨噬细胞中，KD血管炎组的Sirt2表达显著升高；在M1巨噬细胞中，KD血管炎组的Mapk1表达显著降低；在M1、M2巨噬细胞和前巨噬细胞中，KD血管炎组的Akt1表达均显著降低 (P < 0.001)。此外，胆汁酸合成的关键酶基因Cyp7a1在KD小鼠的Macro_Isg15、Macro_Spp1细胞和单核细胞中表达有升高趋势，但在Macro_Clqc细胞中表达降低；胆汁酸下游受体基因Fxr1在Macro_Clqc细胞中表达有升高趋势，但在Macro_Clqc、Macro_Cd209a、Macro_Il1b和单核细胞中表达降低；而Fxr2在Macro_Isg15和Macro_Spp1细胞中表达有升高趋势；S1pr2在KD小鼠的Macro_Spp1细胞中表达有升高趋势。

对KD血管炎小鼠补充TMAO后，小鼠冠脉周围炎细胞浸润程度减轻，相比于KD小鼠炎症评分显著降低（1.30±0.43 vs 2.57±0.61，P＜0.001）。与KD小鼠相比，补充TMAO后KD小鼠外周血共有11种胆汁酸水平明显升高，包括脱甲胆酸、脱氧胆酸、甘氨胆酸和牛磺石胆酸等（P＜0.05）。

 

结论：本研究在KD小鼠血管炎心脏组织中发现TMAO及胆汁酸相关基因变化与单核-巨噬细胞亚群相关；补充TMAO后，小鼠心脏炎症减轻，血浆脱甲胆酸、脱氧胆酸、甘氨胆酸和牛磺石胆酸等胆汁酸水平升高，提示TAMO可能通过影响胆汁酸代谢参与KD的炎症反应。

 

ABSTRACT

PART 1  Proteomic Profiling of Plasma Exosomes in Kawasaki Disease

 

Objective: Kawasaki Disease (KD) is a systemic vasculitis syndrome which affects multiple organs. Coronary artery aneurysm (CAA) is the most common complication of KD and is currently the primary cause of acquired heart disease in children. At present, the clinical diagnosis of CAA in KD primarily relies on the echocardiography imaging, thus brings the need to explore specific diagnostic biomarkers. The aim of this study was to identifyexosomal candidate proteins for identifying KD with CAA in early stages, thereby providing a basis for clinical decision.

 

Methods: This study is a case-control study. The case group consisted of newly diagnosed KD patients hospitalized in the Cardiovascular Medicine Department of the Children’s Hospital Capital Institute of Pediatrics from January 2019 to January 2024. The age- and sex- matched healthy control (HC) children were recruited from the Medical Care Department and age- and sex- matched febrile control (FC) patients were recruited from the Respiratory Medicine Department of the same hospital during the same period. KD patients were further categorized into the following groups: those without CAA, those with small and medium CAA (SCAA/MCAA), and those with giant CAA (GCAA), to investigate protein expression related to CAA severity.

Exosomes were extracted by ultracentrifugation, and their presence was confirmed through morphological observation via electron microscopy and the detection of exosomal marker expression using Western blotting (WB) analysis. The exosomal protein expression profiles of the KD group, HC group, and FC group were analyzed using liquid chromatography-mass spectrometry (LC-MS) combined with TMT6 labeling. Based on the LC-MS results, we performed KEGG Pathway enrichment analysis, Reactome Pathway analysis, Wiki Pathway analysis, GO functional enrichment analysis, and protein-protein interaction (PPI) network analysis. Differentially expressed proteins (DEPs) were selected based on these analyses and the pathophysiology of KD. Additionally, Western blotting (WB) was performed to validate the expression levels of selected DEPs.

 

Results:

1. A total of 60 children were recruited in this study. Among those, 40 children (n=8 in each group) were used for exosomal protein extraction and bioinformatic analysis, 20 children (n=4 in each group) were used to validate the expression levels of the selected proteins. Morphological observations under electron microscopy and the verification of exosomal markers via WB analysis indicated that the extracted proteins were exosome-derived.

2. A total of 995 DEPs were identified among all groups（P＜0.05）. Selected by a fold-change of 1.3, 332 DEPs were found to be commonly expressed in the KD groups compared to the HC group, regardless of the presence of CAA. Additionally, 374 DEPs were specifically expressed in the FC group compared to both the HC and KD groups. In contrast, 104 DEPs were specifically expressed in the KD group compared to the HC and FC groups. Additionally, 228 DEPs were commonly expressed between the FC and KD groups. Among the 374 DEPs specifically expressed in the FC group, pathways related to cholesterol and lipid metabolism were enriched.

3. Among the 228 DEPs commonly expressed in both the FC and KD groups, which were associated with febrile diseases, pathways related to cell structure and function were enriched. Trend analysis revealed that 24 DEPs showed a significant decreasing trend with increasing disease severity in the FC and KD subgroups (P < 0.05). Further analysis of these 24 DEPs identified enrichment in infection-related pathways. Proteins such as S100A9, which showed a significant decrease in expression in the KD group, were selected for subsequent validation.

4.Pathway enrichment analysis of the 104 DEPs specifically expressed in the KD group revealed that these proteins were enriched in complement-related pathways and platelet activation pathways. Trend analysis of these 104 DEPs showed that 54 DEPs showed a significant decreasing trend with increasing CAA severity. These DEPs were enriched in G protein signaling pathways and bile acid receptor-related S1PR transduction pathway. Based on the enrichment of pathways and expression trends of the DEPs, proteins such as C3 and SERPINA1 from the complement pathway, and GNAI2 from the G protein signaling pathway, were selected for further validation.

5.Subgroup analysis stratified by the presence of CAA in KD patients revealed that 91 DEPs were specifically expressed in KD patients with CAA. These DEPs were enriched in complement-related pathways and neutrophil extracellular trap pathways. In KD patients without CAA, 16 DEPs were specifically expressed and were enriched in pathways related to viral infection and immune response. Further analysis of KD subgroups with varying severity of CAA showed that 102 DEPs were specifically expressed in KD patients with SCAA/MCAA and were enriched in pathways related to platelet activation. In contrast, 34 DEPs were specifically expressed in KD patients with GCAA, and they were enriched in pathways related to cell function.

6.Based on the results of the aforementioned bioinformatics analysis, DEPs associated with complement, immune response, and cell structure/function were selected for WB validation of protein expression levels. Among these, five proteins showed significant differences across different groups:

1) Compared to the FC group, the expression level of the S100A9 protein was significantly reduced in the KD group (P < 0.05), which was consistent with the results from the mass spectrometry analysis.

2) Complement protein C3 expression was significantly increased in KD patients without CAA group compared to both the HC group (P < 0.05) and the FC group (P < 0.01), which was consistent with the results from the mass spectrometry analysis.

3) Compared to KD patients without CAA, the expression levels of SERPINA1 and GNAI2 were significantly decreased in KD patients with CAA (P < 0.05). which were consistent with the results from the mass spectrometry analysis.

4) Compared to KD patients with SCAA/MCAA, the expression level of Rap1b was significantly decreased in KD patients with GCAA (P < 0.05), which was consistent with the results from the mass spectrometry analysis.

 

Conclusion: This study provides a proteomic profile of plasma exosomes in KD patients with and without CAA. These findings suggest that the proteins SERPINA1 and GNAI2 may serve as potential diagnostic biomarkers for KD with CAA, while the Rap1b protein may be a potential diagnostic biomarker for KD with GCAA.

 

 

ABSTRACT

PART 2  Metabolomic Analysis of Plasma in Kawasaki Disease

 

Objective: Kawasaki disease (KD) is a systemic immune vasculitis which is accompanied by various metabolic disturbance throughout its course. Previous exosome proteomics analysis identified alterations in bile acid precursor cholesterol and related lipid metabolism pathways in KD patients. This study employed metabolomics analysis to investigate differentially expressed metabolites in the peripheral plasma of KD patients, primarily focusing on changes in choline/carnitine derivative-related pathways and bile acid metabolism-related pathways.

 

Methods: This case-control study included children with newly diagnosed KD patients hospitalized in the Cardiovascular Medicine Department of the Children’s Hospital Capital Institute of Pediatrics from January 2022 to June 2024. The age- and sex- matched healthy control (HC) children were recruited from the Medical Care Department and age- and sex- matched febrile control (FC) patients were recruited from the Respiratory Medicine Department of the same hospital during the same period. Initially, non-targeted metabolomics detection and bioinformatics analysis were conducted to explore the metabolic profile in the plasma of KD patients, focusing on changes in metabolites related to choline/carnitine and bile acid metabolism pathways. Subsequently, targeted metabolic detection and analysis were performed on the metabolites within these pathways.

 

Results:

1.A total of 220 children were included in this study. Among them, 40 children (n=20 in each group) were used for non-targeted metabolite detection and bioinformatics analysis of plasma between the KD and HC groups. Another 20 children (n=10 in each group) were used for targeted detection of metabolite levels in the choline/carnitine metabolism pathway between the KD and HC groups. A total of 70 children were included in the study (n=20 in the HC and FC groups, and n=30 in the KD group) for the targeted detection of trimethylamine-N-oxide (TMAO), the choline/carnitine metabolic end product. Additionally, 90 children (n=30 in each group) were used for the targeted validation of bile acid levels in the HC, FC, and KD groups.

2. Compared to the HC group, a total of 892 metabolites exhibited significant differences with a fold change of 2 (P < 0.05). Among these, 430 metabolites were upregulated and 462 were downregulated in KD patients. These differentially expressed metabolites were enriched in the bile acid metabolism pathway, which was regulated downstream by choline/carnitine derivatives and their metabolic product TMAO.

3. Targeted analysis of metabolites in the choline/carnitine pathway in KD patients revealed that betaine levels were significantly elevated compared to the HC group (4036.91 ± 800.93 vs. 2821.21 ± 414.00 ng/ml, P < 0.001). In contrast, the level of TMAO, which was the end metabolite of choline, carnitine, and betaine, and an upstream regulator of bile acids, were significantly reduced in KD patients [5.03 (3.34, 57.57) vs. 44.36 (28.27, 106.62) ng/ml, P < 0.05]. Upon validation with a larger cohort, plasma TMAO level was found to be significantly lower in KD patients compared to the HC group [28.14(10.00，76.58) vs. 163.34(84.83，298.32) ng/ml，P＜0.001], and it was found to be significantly lower in KD patients compared to the FC group[28.14(10，76.58) vs. 62.30(25.39，109.51) ng/ml，P＜0.05].

4. Targeted analysis of bile acid-related metabolites in KD patients showed that 11 bile acids had significantly different expression levels compared to the HC group (P < 0.05). Among these, eight bile acids, including taurolithocholic acid and taurocholic acid, were significantly elevated in KD patients. Conversely, three bile acids, including allocholic acid, taurodeoxycholic acid, and ursodeoxycholic acid, were significantly reduced in KD patients.

 

Conclusion: This study revealed changes in plasma choline/carnitine metabolite TMAO and its downstream bile acid levels in KD patients. It suggested that the TMAO-bile acid metabolic pathway may be involved in the metabolic disturbances associated with KD. TMAO could potentially serve as an early diagnostic marker for KD based on metabolic disturbances.

 

 

ABSTRACT

PART 3  Mechanism Investigating: TMAO as an Early Diagnostic Marker for Kawasaki Disease

 

Objective: Given the significant reduction of the choline metabolism end-product trimethylamine-N-oxide (TMAO) observed in children with Kawasaki disease (KD), this study aimed to analyze changes in plasma differentially expressed metabolites in KD vasculitis mouse models using metabolomics. Additionally, single-cell RNA sequencing was employed to examine the expression changes of TMAO-related genes in mouse cardiac tissues. Furthermore, TMAO intervention was implemented in the KD vasculitis mouse models to explore its effects on cardiac inflammation and downstream bile acid metabolism. Through this research, we aimed to elucidate the role of TMAO in the KD vasculitis mouse model, thereby providing a basis for its potential application as a mechanism-based diagnostic marker for KD.

 

Methods: In this study, KD vasculitis mouse models were established through intraperitoneal injection of Lactobacillus casei cell wall extract (LCWE). Liquid chromatography-mass spectrometry (LC-MS) was employed to detect plasma metabolite levels in the mice, followed by bioinformatic functional analysis. Single-cell sequencing was used to analyze the expression of TMAO and bile acid-related genes at the cellular level in cardiac tissues of KD vasculitis and control group mice. Additionally, TMAO intervention was conducted on KD vasculitis mice to assess coronary artery inflammation scores and peripheral plasma bile acid levels.

 

Results:

1. Untargeted metabolomic analysis identified 1,314 metabolites in the plasma of KD vasculitis mice. These metabolites were primarily derivatives of lipids and organic acids. KEGG pathway analysis indicated enrichment in metabolic pathways such as choline metabolism and bile secretion. Using criteria of P < 0.05 and VIP > 1, 159 differentially expressed metabolites were identified in KD mice compared to controls. These differentially expressed metabolites were enriched in pathways related to protein absorption and degradation, amino acid metabolism, and bile acid metabolism. Additionally, 30 metabolites were specifically expressed in KD mice, with enrichment in pathways such as the VEGF signaling pathway and chemokine signaling pathway.

2. Single-cell analysis of mouse cardiac tissue revealed the presence of five major cell types: macrophages, neutrophils, endothelial cells, fibroblasts, and smooth muscle cells. Compared to healthy control mice, the proportions of different macrophage subpopulations in the cardiac tissue of KD mice showed changes: the Macro_Clqc subpopulation was significantly increased, while the Macro_Isg15, Macro_Spp1, and Macro_Il1b subpopulations, as well as monocyte numbers, were decreased. Further gene expression analysis indicated a significant upregulation of inflammation-related genes such as Il-1β, Nlrp3, and Icam1 in the macrophages of KD mice (P < 0.01). Key genes of the TMAO pathway downstream, including Sirt2, Mapk1, and Akt1, exhibited significant expression changes in KD vasculitis mice: Sirt2 expression was significantly increased in M2 macrophages; Mapk1 expression was significantly reduced in M1 macrophages; and Akt1 expression was significantly decreased in M1, M2 macrophages, and precursor macrophages (P < 0.001). Additionally, the expression of the key enzyme gene for bile acid synthesis, Cyp7a1, showed an increasing trend in Macro_Isg15, Macro_Spp1 cells, and monocytes of KD mice, but decreased in Macro_Clqc cells. The expression of the downstream bile acid receptor gene Fxr1 showed an increasing trend in Macro_Clqc cells but decreased in Macro_Clqc, Macro_Cd209a, Macro_Il1b, and monocytes. Fxr2 expression showed an increasing trend in Macro_Isg15 and Macro_Spp1 cells, while S1pr2 expression showed an increasing trend in Macro_Spp1 cells of KD mice.

3. After administering TMAO to KD vasculitis mice, there was a reduction in the degree of inflammatory cell infiltration around the coronary arteries. The inflammation score was significantly lower compared to KD mice without TMAO supplementation (1.30±0.43 vs 2.57±0.61, P < 0.001). Additionally, in comparison to untreated KD mice, the peripheral plasma bile acid levels in TMAO-supplemented KD mice showed a significant increase in 11 different bile acids, including lithocholic acid, deoxycholic acid, glycocholic acid, and taurolithocholic acid (P < 0.05).

 

Conclusion: This study identified that changes in TMAO and bile acid-related genes are associated with monocyte-macrophage subpopulations in the cardiac tissue of KD vasculitis mice. Following TMAO supplementation, there was a reduction in cardiac inflammation and an increase in plasma levels of bile acids such as lithocholic acid, deoxycholic acid, glycocholic acid, and taurolithocholic acid. These findings suggest that TMAO may participate in the inflammatory response of KD by influencing bile acid metabolism.