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. 2025 Nov 25;57(6):1042–1050. [Article in Chinese] doi: 10.19723/j.issn.1671-167X.2025.06.005

非靶向代谢组学揭示原发性干燥综合征血小板减少患者血清差异代谢物及代谢通路

Untargeted metabolomics reveals differential serum metabolites and metabolic pathways in patients with primary Sjögren syndrome and thrombocytopenia

Zhao XIANG 1, Li YANG 2, Jing YANG 2,*
PMCID: PMC12711397  PMID: 41399064

Abstract

Objective

To systematically compare serum metabolome differences between patients with thrombocytopenia in primary Sjögren syndrome (pSS) and those with normal platelet count using non- targeted metabolomics technology, so as to identify differential metabolites, analyze the relationship between the relative quantification of these metabolites and platelet counts, and screen metabolic pathways associated with platelet counts in pSS patients with thrombocytopenia.

Methods

The patients with pSS were selected and grouped according to the presence or absence of thrombocytopenia. Serum samples were collected from the study subjects and analyzed by liquid chromatography-mass spectrometry (LC-MS). The samples were analysed by human metabolome database (HMDB), lipid metabolites and pathways strategy (LIPID MAPS) and other databases for classification and annotation. The samples were analyzed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) for multi-variate statistical analysis to screen the differential metabolites between the groups, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was made to study the functions and metabolic pathways of the metabolites. Correlation analysis was performed between the abundance of serum differential metabolites and platelet counts of pSS patients with thrombocytopenia.

Results

This study included 62 patients with pSS, of whom 32 had thrombocytopenia and 30 had normal platelet counts. A total of 137 differentially expressed metabolites, enriched in 54 metabolic pathways, were found in the serum of patients with thrombocytopenia compared with those without thrombocytopenia. Among them, the expression of desoxycorticosterone, hydrocortisone, and taurine was positively correlated with platelet count, and the expression of neopterin was negatively correlated with platelet count. Enrichment analysis showed that desoxycorticosterone and hydrocortisone were enriched in the steroid hormone biosynthesis pathway, taurine was enriched in the metabolic pathway of taurine and taurine, and neopterin was enriched in the folate metabolic pathway.

Conclusion

Thrombocytopenia in pSS patients may be related to the reduced activity of steroid hormone biosynthesis pathway and the metabolic pathway of taurine and taurine, and the increased activity of the pathway of folate metabolism.

Keywords: Sjögren syndrome, Thrombocytopenia, Metabolomics, Serum, Biomarkers

原发性干燥综合征(primary Sjögren syndrome，pSS)是一种全身性自身免疫病，约1/3的pSS患者表现为血液系统异常[1-3]，约5%~13%的患者存在血小板减少[4]，pSS伴严重的血小板减少会引起危及生命的脏器出血[5]。

pSS患者的血小板减少是一种继发的免疫性血小板减少(immune thrombocytopenia，ITP)[6]，其机制主要是由于IgG自身抗体与血小板结合，通过增强Fc受体介导的吞噬作用和脾脏破坏作用导致血小板破坏[7]。近年来，继发性ITP逐渐被认识，例如系统性红斑狼疮自身免疫紊乱产生大量自身抗体，进而导致血小板破坏增加或血小板生成减少[8-9]。有文献报道糖蛋白Ⅱb/Ⅲa抗体、糖蛋白Ⅰb-Ⅸ-Ⅴ复合物抗体、抗P-选择素抗体等自身抗体可能与pSS伴血小板减少有关[10]。pSS伴血小板减少的治疗方法包括糖皮质激素[11]、丙种球蛋白[12]、免疫抑制剂及生物制剂[13]等，但仍有部分患者的治疗效果不佳。因此，亟需运用新的研究方法探索其潜在发病机制，以助力研究新疗法。

近年来，免疫学家认为免疫细胞的活化、增殖、凋亡和功能与特异性细胞内代谢途径的活化密切相关并依赖于其活化[14]。在pSS伴血小板减少患者中，免疫细胞的活动可能会引起诸多代谢途径的变化。代谢组学可用于鉴定可能改变细胞或生物体表型的代谢物[15]，代谢物不仅反映了组织的代谢活性，还可以影响临床表型[16]。目前，代谢组学已被广泛应用于pSS的研究。Lu等[17]对230例pSS患者和240例健康对照者的血清样本进行代谢组学分析，筛选潜在的脂质生物标志物，结果提示pSS代谢紊乱可能与氧化脂质、脂肪酸氧化和能量代谢有关。Fineide等[18]对pSS患者和健康人的唾液、眼泪及唇腺样本进行脂质组学分析，结果发现pSS患者唾液和眼泪的脂质谱与健康对照组有显著差异。Fernández-Ochoa等[19]通过对43例pSS患者和52名健康对照的血清和尿液进行代谢组学分析，发现pSS患者存在不饱和脂肪酸、磷脂酰肌醇、酰基甘氨酸、溶血磷脂酰胆碱、酰基肉碱及氨基酸的代谢异常。此外，还有研究对ITP患者的血浆进行了代谢组学研究，如张紫妍[20]研究发现，ITP患者存在柠檬酸循环、苯丙氨酸代谢、乙醛酸和二羧酸盐代谢以及苯丙氨酸、酪氨酸和色氨酸生物合成等异常；Xiong等[21]运用基于液相色谱-质谱联用(liquid chromatography-mass spectrometry，LC-MS)的血清代谢组学发现代谢物L-色氨酸、溶血磷脂酰胆碱(17 ∶ 0)和D-二氢鞘氨醇可作为辐射诱导的血小板减少症的生物标志物。这些研究发现提示pSS以及血小板减少的发生可能与机体代谢异常存在潜在的联系。

因此，本研究运用基于LC-MS的非靶向代谢组学方法对pSS伴和不伴血小板减少患者的血清样本进行代谢组学分析，筛选出差异代谢物，挖掘与pSS血小板减少有关的代谢通路，为研究pSS血小板减少的发病机制提供新方向。

1. 资料与方法

1.1. 研究对象

本研究为基于横断面调查的病例对照研究，选取自2020年7月至2022年7月于绵阳市中心医院就诊的pSS患者为研究对象。pSS诊断符合2016美国风湿病学会/欧洲抗风湿病联盟pSS分类标准[22]，血小板减少的诊断定义为血小板计数 <100× 109/L[23]。排除标准：(1)有高血压、糖尿病、肾功能损害等影响机体代谢的疾病；(2)原发性ITP以及再生障碍性贫血、肿瘤、肝硬化等引起的血小板减少；(3)合并类风湿性关节炎、系统性红斑狼疮等其他自身免疫性疾病；(4)合并感染及肿瘤；(5)有头颈部放疗史、肺间质纤维化；(6)近3个月内应用糖皮质激素、抗乙酰胆碱药(如阿托品、莨菪碱、溴丙胺太林、颠茄等)、免疫抑制剂等。所有入选对象均签署知情同意书，本研究获得绵阳市中心医院生物医学伦理委员批准(审批号：S20220342-01)。

1.2. 血清样本的处理

收集pSS患者治疗前的新鲜血液标本，于2 h内离心(10 000×g，10 min)，取血清分装于2个5 mL EP管中，并进行分组和编号，冻存于-80 ℃冰箱，用于备份和代谢组学研究。取100 μL血清样本置于EP管中，加入400 μL 80%(体积分数)甲醇水溶液，经涡旋震荡后在冰水中静置5 min，在4 ℃下以15 000×g离心20 min，取一定量上清液加入质谱级水稀释至甲醇浓度为53%，再于4 ℃以15 000× g离心20 min，收集上清液注入LC-MS系统进行非靶向代谢组学分析。从每个样品中取出等量的样品并混合作为质量控制样品。

1.3. 仪器和分析条件

本研究使用的仪器有：质谱仪Q ExactiveTM HF-X(赛默飞，德国)、水色谱仪Vanquish UHPLC(赛默飞，德国)、色谱柱Hypesil Gold column(100×2.1 mm，1.9 μm，赛默飞，美国)、低温离心机D3024R(赛洛捷克，美国)。色谱柱条件：HypesilGoldcolumn(C18)，柱温40 ℃，流速0.2 mL/min。正离子模式：流动相A为0.1%(体积分数)甲酸(CAS-64-18-6)，流动相B为甲醇(CAS-67-56-1)。负离子模式：流动相A为5 mmol醋酸铵pH 9.0(CAS-631-61-8)，流动相B为甲醇(CAS-67-56-1)。质谱条件：扫描范围选择质荷比(m/z)100~1 500；电喷雾离子源设置：喷雾电压3.2 kV，鞘气流速40 arb，辅助气体流量10 arb，离子传输管温度320 ℃。极性：正离子模式、负离子模式；MS/MS二级扫描为数据依赖性采集。

1.4. 数据处理

将光谱分析生成的原始数据文件导入Compound Discoverer 3.1(CD3.1，赛默飞世尔)进行处理，对所有代谢物的保留时间、质荷比等参数进行简单筛选。设置0.2 min的保留时间偏差和5×10-6的质量偏差对不同样品进行峰对齐，以使鉴定更准确。随后，设置质量偏差5×10-6、信号强度偏差30%、信噪比3、最小信号强度、加和离子等信息进行峰值提取。同时，对峰面积进行定量，再对目标离子进行整合。通过分子离子峰和碎片离子进行分子式的预测，并与mzCloud、mzVault和Masslist数据库进行比对，用blank样本去除背景离子，对原始定量结果进行标准化处理。最后，得到代谢物的鉴定和相对定量结果。数据处理基于R 3.4.3、Python 2.7.6和CentOS 6.6进行。

1.5. 统计学方法

利用MetaX对检测得到的海量代谢物数据进行转换，然后执行主成分分析(principal component analysis，PCA)和偏最小二乘判别分析(partial least squares discriminant analysis，PLS-DA)，以此完成多元统计分析。PCA用于观察两组样本总体分布趋势，而PLS-DA是代谢物表达量与样品类别之间的关系模型，可预测样品类别，是一种有监督的判别分析统计学方法。采用t检验分析两组间每种代谢物表达差异，并计算两组间代谢物水平的倍数变化(fold change，FC)，通过R语言中cor. mtest函数进行显著性检验。运用Graphpad prism 8.0.1，采用D’Agostino-Pearson检验方法对差异代谢物表达量进行正态性检验，然后对血清差异代谢物表达量和血小板计数分别进行Pearson和Spearman相关性分析。采用错误发现率(false discovery rate，FDR)校正方法对P值进行处理，以控制总体Ⅰ类错误概率。 P < 0.05为差异具有统计学意义。

1.6. 代谢物鉴定和代谢通路分析

使用京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)数据库(https://www.genome.jp/kegg/pathway.html)、人类代谢组学数据库(human metabolome database，HMDB, https://hmdb.ca/metabolites)和脂质代谢途径研究计划(lipid metabolites and pathways strategy，LIPID MAPS) 数据库(http://www.lipidmaps.org/) 对鉴定到的代谢物进行注释，使用ggplot2绘制火山图和气泡图。

本研究将同时满足以下各条件的化合物保留为差异代谢物：(1)VIP>1；(2) P < 0.05且FDR＜0.05；(3)FC> 1.2或FC <0.833[24-26]。其中VIP(variable importance in the projection)是指PLS-DA模型第一主成分的变量投影重要度[24]，其数值大小表示检测到的代谢物对分组的贡献程度；FC代表每个代谢物在比较组中所有生物重复定量值的均值的比值；P值表示差异的统计显著性，并采用FDR对P值进行进一步校正(q＜0.05)。

本研究基于KEGG数据库研究代谢物的功能和代谢途径，当x/n>y/N时(其中N代表全部检测到的代谢物中参与KEGG代谢通路的代谢物数目，n为N中差异积累代谢物的数目，y为注释到某个KEGG通路的代谢物数目，x为富集到某个KEGG通路的差异积累代谢物数目)，认为该代谢途径富集，当代谢途径的 P < 0.05时，则该代谢途径显著富集。

2. 结果

2.1. 患者的一般情况

本研究共招募了278例pSS患者，根据纳入排除标准，最终纳入62例pSS患者，其中32例伴有血小板减少，30例血小板正常，研究对象的临床和人口统计学特征见表 1。

表 1.

研究对象的临床和人口统计学特征

Clinical and demographic characteristics of the study population

Clinical and demographic characteristics pSS with thrombocytopenia (n=32) pSS without thrombocytopenia (n=30) P
pSS, primary Sjögren syndrome; ESSDAI, European Alliance of Associations for Rheumatology (EULAR) Sjögren syndrome disease activity index.
Age/years, x±s 52.84±13.33 47.9±11.96 0.140 0
Female, n(%) 31 (96.88) 29 (93.33) 0.999 9
Course of disease/years, x±s 5.73±5.15 1.65±2.77 0.000 2
ESSDAI, x±s 2.59±0.49 1.13±0.67 ＜0.000 1
Platelet count/(×109/L), x±s 39.72±28.31 181±55.23 ＜0.000 1
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2.2. pSS伴和不伴血小板减少患者的血清代谢组差异

PCA得分图显示，pSS伴和不伴血小板减少患者的血清代谢组有分离趋势(图 1A、1B)，为了更好地区分两组患者的血清代谢差异，本研究进行了PLS-DA分析(图 1C、1D)，结果显示pSS伴和不伴血小板减少患者的血清代谢物存在明显差异。

图 1.

PCA得分图(A、B)和PLS-DA得分图(C、D)

PCA score (A, B) and PLS-DA score (C, D)

A and B show the PCA score plots of metabolites in positive and negative ion modes; C and D show the PLS-DA score plots of metabolites in positive and negative ion modes. pSS_lowPLT, primary Sjögren syndrome (pSS) with thrombocytopenia group; pSS_normalPLT, pSS without thrombocytopenia group; PC, principal component; PCA, principal component analysis; PLS-DA, partial least squares discriminant analysis.

图 1

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2.3. pSS伴和不伴血小板减少患者的血清差异代谢物筛选

差异代谢物筛选结果显示，pSS伴血小板减少组与不伴血小板减少组相比，有137种差异代谢物，包括87种上调的代谢物和50种下调的代谢物(图 2)。

图 2.

差异代谢物的火山图

Volcano plot of differential metabolites

A and B show the volcano plots of positive and negative ion patterns in the primary Sjögren syndrome (pSS) with thrombocytopenia and pSS without thrombocytopenia groups. Points in the volcano plot indicate individual metabolites, with red dots representing significantly upregulated metabolites and green dots indicating significantly downregulated ones. The grey dots represent metabolites with no significant difference. The variable importance in the projection (VIP) value is indicated by the size of the points.

图 2

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2.4. 差异代谢物KEGG通路富集分析

KEGG富集分析结果显示，pSS伴和不伴血小板减少患者的137种差异代谢物主要富集于54条代谢途径，图 3的气泡图展示了两组间正负离子模式差异代谢物所富集的前20位代谢通路。

图 3.

KEGG富集通路气泡图

KEGG enrichment pathway bubble plot

A and B display the pathway enrichment results of differential metabolites between the primary Sjögren syndrome (pSS) with thrombocytopenia and pSS without thrombocytopenia groups in positive and negative ion modes. Point colors indicate P-values from hypergeometric tests, where smaller values signify more statistically significant results. Point sizes reflect the number of enriched differential metabolites within each pathway, with larger points indicating higher abundance. KEGG, Kyoto Encyclopedia of Genes and Genomes; TCA, tricarboxylic acid.

图 3

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2.5. pSS伴血小板减少患者的血清差异代谢物的表达量与血小板计数的关系

为了研究血清代谢物的变化与pSS伴血小板减少之间的关联，本研究将pSS伴和不伴血小板减少患者之间的血清差异代谢物的表达量和pSS伴血小板减少患者的血小板计数进行了相关性分析。正态性检验结果显示，pSS伴血小板减少患者血小板计数与34种差异代谢物的表达量符合正态分布(图 4A、4B)，对这34种差异代谢物的表达量和血小板计数进行Pearson相关分析，对另外103种不符合正态分布的差异代谢物的表达量与血小板计数进行Spearman相关分析，结果显示总共有11种差异积累代谢物的表达量与血小板计数相关(表 2)。其中牛磺酸、四烯二酸、没药淄酮、磷脂酰胆碱(7 ∶ 0/8 ∶ 0)、去氧皮质酮、氢化可的松、10-硝基油盐酸、(11E, 15Z)-9, 10, 13-三羟基十八烷-11, 15-二烯酸的表达量与pSS伴血小板减少患者的血小板计数呈正相关，新蝶呤、咪唑烯酸、对甲苯磺酸的表达量与pSS伴血小板减少患者的血小板计数呈负相关(图 4C、4D)。

图 4.

差异代谢物表达量和血小板计数之间的关系

Relationship between differential metabolites abundance and platelet count

A, normal Q-Q plots of 34 differentially metabolized compounds; B, normal Q-Q plot of platelets, where the x-axis represents actual observed values and the y-axis indicates predicted values. C, Pearson linear correlation plot between 2 differentially metabolized compounds and platelets; D, Spearman linear correlation plot between 9 metabolites and platelets. LPE, lysophosphatidylethanolamine; GHB, γ-hydroxybutyric acid; AIMP, aminoacyl-tRNA synthetase-interacting multifunctional protein; PLT, platelet.

图 4

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表 2.

11种差异代谢物的表达量与血小板计数的关系

Relationship between expression of 11 differential metabolites and platelet count

Differential metabolite r P q Up/Down KEGG pathway
The r-values for taurine and neopterin were analyzed using Pearson correlation, while the remaining correlations were analyzed using Spearman correlation. KEGG, Kyoto Encyclopedia of Genes and Genomes.
Taurine 0.376 7 0.036 7 0.041 9 Down Aurine and hypotaurine metabolism
Neopterin -0.578 1 0.000 7 0.007 7 Down Folate biosynthesis
Imidazolelactic acid -0.391 5 0.026 7 0.041 9 Up
P-toluenesulfonic acid -0.404 1 0.021 8 0.041 9 Down
Tetradecanedioic acid 0.365 4 0.039 7 0.041 9 Up
Guggulsterone 0.361 8 0.041 9 0.041 9 Down
Phosphatidylcholine (7 ∶0/8 ∶0) 0.391 5 0.026 7 0.041 9 Down
Desoxycortone 0.425 0 0.015 3 0.041 9 Down Steroid hormone biosynthesis
Cortisol 0.380 6 0.031 6 0.041 9 Down Steroid hormone biosynthesis
10-nitrolinoleate 0.535 8 0.001 6 0.008 8 Up
(11E, 15Z)-9, 10, 13-trihydroxyoctadeca-11, 15-dienoic acid 0.403 7 0.021 9 0.041 9 Up
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根据KEGG富集分析结果，与pSS伴血小板减少患者的血小板计数相关的血清差异积累代谢物中的去氧皮质酮和氢化可的松与血小板计数呈正相关，富集于类固醇激素生物合成(steroid hormone biosynthesis)途径；牛磺酸的表达量与血小板计数呈正相关，富集于牛磺酸和亚牛磺酸的代谢(taurine and hypotaurine metabolism)途径；新蝶呤与血小板计数呈负相关，富集于叶酸生物合成(folate biosynthesis)途径；而其余8种差异积累代谢物未注释到对应的KEGG通路。上述结果表明，pSS伴血小板减少患者的血小板计数可能与类固醇激素生物合成、牛磺酸和亚牛磺酸的代谢及叶酸生物合成途径有关。

3. 讨论

本研究采用基于LC-MS的检测技术对pSS伴和不伴血小板减少患者的血清样本进行非靶向代谢组学分析，发现两者之间存在137种差异代谢物，富集于54条代谢途径。进一步的相关性分析发现，牛磺酸、四烯二酸、没药淄酮、磷脂酰胆碱、去氧皮质酮、氢化可的松、10-硝基油盐酸、(11E, 15Z)-9, 10, 13-三羟基十八烷-11, 15-二烯酸的表达量与pSS伴血小板减少患者的血小板计数呈正相关，新蝶呤、咪唑烯酸、对甲苯磺酸的表达量与pSS伴血小板减少患者的血小板计数呈负相关。KEGG富集分析结果表明，pSS伴血小板减少患者的血小板计数可能与类固醇激素生物合成、牛磺酸和亚牛磺酸的代谢及叶酸生物合成途径有关，这些代谢途径为研究pSS伴血小板减少的发生提供了可能的线索。

目前，已有研究表明代谢物谱的改变与pSS、ITP有关，但尚无研究关注pSS伴血小板减少患者的代谢特征。本研究的代谢组学分析表明，pSS伴血小板减少患者存在血清代谢异常。Bengtsson等[27]观察到pSS与系统性红斑狼疮患者相比，血清中的富马酸等有机酸增加，而本研究发现，与pSS不伴血小板减少患者相比，pSS伴血小板减少患者血清中富马酸表达量增加。Li等[28]发现，与健康对照组相比，pSS患者血清中氢化可的松水平高于健康组，本研究显示，与pSS不伴血小板减少患者相比，pSS伴血小板减少患者血清中的氢化可的松水平降低。在生物标记物方面，Lu等[17]基于非靶向脂质代谢组学分析，将磷脂酰胆碱(18 ∶ 0/22 ∶ 5)、甘油三酯(16 ∶ 0/18 ∶ 0/18 ∶ 1)和酰基肉碱(12 ∶ 0)确定为pSS的特定生物标志物。而本研究则不尽相同，本研究结果显示腺苷和硫酸睾酮有较高的诊断价值。张紫妍[20]研究发现ITP患者存在柠檬酸循环代谢异常，本研究中pSS伴血小板减少患者也存在柠檬酸循环代谢异常，说明pSS血小板减少与ITP的发生机制可能存在相似之处。

类固醇激素生物合成与血小板可能存在联系。本研究发现，与pSS不伴血小板减少患者相比，pSS伴血小板减少患者血清中去氧皮质酮、氢化可的松的表达量下调，而去氧皮质酮和氢化可的松富集于类固醇激素生物合成途径，表明pSS伴血小板减少患者的类固醇激素生物合成可能是减弱的。此外，本研究还观察到pSS伴血小板减少患者血清中的雌二醇代谢产物——雌三醇的水平是下调的。由此推断，与pSS不伴血小板减少患者相比，pSS伴血小板减少患者血清中的雌二醇水平也应该是降低的。至于血小板的产生，目前普遍认为是造血干细胞使巨核细胞成熟，形成血小板前体细胞，血小板前体细胞形成珠状前体血小板并释放到血流中，然后再分裂为成熟的血小板[29]。据报道，雌二醇会通过刺激前血小板来促进血小板生成[30]。虽然雌激素分泌和年龄有关，但本研究中pSS伴和不伴血小板减少患者的年龄组成并无显著差异。因此，我们认为pSS伴血小板减少的发生可能与雌二醇等类固醇激素的生物合成减少有关。但本研究并未直接观察到雌二醇水平的变化，可能与采用的代谢组学方法局限性有关，可进一步进行针对类固醇激素的靶向代谢组学检测来证实pSS伴血小板减少患者血清中雌二醇以及其他可能与pSS伴血小板减少有关的代谢物的变化。

牛磺酸代谢可能与外周血小板计数有关。本研究结果显示，与pSS不伴血小板减少患者相比，pSS伴血小板减少患者血清中牛磺酸水平也是降低的，牛磺酸富集于牛磺酸和亚牛磺酸代谢途径，表明牛磺酸和亚牛磺酸代谢活动可能也是减弱的。据报道，牛磺酸在血小板中的浓度比其他氨基酸高6倍[31]，根据Maupin[32]的数据，血小板-血浆牛磺酸浓度梯度为440 ∶ 1。Ahtee等[33]认为血小板基于主动转运蛋白从外周环境中蓄积牛磺酸，产生和维持高血小板-血浆牛磺酸浓度梯度。因此，从理论上来说，血小板数量减少，血小板从外周血中摄取的牛磺酸也会减少，外周血中的牛磺酸就会有所增加。然而，本研究却发现pSS伴血小板减少患者血清中的牛磺酸是减少的。因此，我们推测pSS伴血小板减少患者的血清中牛磺酸水平的降低可能是牛磺酸和亚牛磺酸代谢活动减弱的结果，牛磺酸和亚牛磺酸代谢途径的下调可能与pSS伴血小板减少的发生有关。

至于叶酸生物合成，此前已有研究表明叶酸在体外有抑制前列腺素E2产生[34]和在体内抑制环加氧酶活化的作用[35]。当机体的血细胞产生受损时，会发生髓外造血[36]，肝和脾是髓外造血的重要器官[37]。近期，Meng等[38]在研究暴露于炭黑(carbon black)的小鼠的血栓形成机制时发现，环加氧酶依赖性前列腺素E2产生的激活促进了脾巨噬细胞中白细胞介素6的表达，导致脾和血液循环中血小板计数增加，而叶酸通过抑制巨噬细胞内环加氧酶2和前列腺素合酶的表达，抑制小鼠脾髓外造血，从而使暴露于炭黑的小鼠血小板计数维持在正常水平。本研究显示，与pSS不伴血小板减少患者相比，pSS伴血小板减少患者血清中的新蝶呤表达量减少，且与血小板计数呈负相关，而新蝶呤富集于叶酸生物合成途径，表明pSS伴血小板减少患者的叶酸生物合成也是减弱的。当pSS伴血小板减少发生时，机体可能发生了髓外造血，叶酸生物合成的减弱促进了髓外造血。遗憾的是，本研究也并未直接观察到pSS伴血小板减少患者血清中叶酸表达量的变化。因此，叶酸生物合成途径的减弱是疾病本身引起的代谢扰动，还是继发于血小板减少的代偿性变化，仍有待进一步研究。

本研究仍存在一定局限性和不足：首先，样本量较小；其次，无法确保这一发现适用于不同生活方式和饮食的基因多样性人群；再次，未对pSS伴血小板减少与其他风湿免疫性疾病相关的血小板减少进行比较；此外，本研究虽然发现了可能与pSS伴血小板减少发生有关的代谢途径，但未能对差异代谢物进行额外的验证，比如未进行靶向代谢组学分析。因此，仍需要更进一步的研究。

综上所述，pSS伴血小板减少患者存在明显的血清代谢异常，与血小板正常的pSS患者相比，血小板减少患者的去氧皮质酮及氢化可的松、牛磺酸的表达量与其血小板计数呈正相关，而新蝶呤的表达量与其血小板计数呈负相关。根据KEGG富集分析结果，pSS患者血小板减少可能与类固醇激素生物合成途径以及牛磺酸和亚牛磺酸代谢途径活性减弱、叶酸生物合成途径活性增强有关。

Funding Statement

四川省科技厅重点研发项目(2017SZ0148)

Supported by Key Research and Development Project of Science and Technology Department of Sichuan Provincial (2017SZ0148)

Footnotes

利益冲突  所有作者均声明不存在利益冲突。

作者贡献声明  向钊：收集研究对象临床资料和血液样本，分析整理数据，研究设计并撰稿；杨莉：筛选研究对象，提供血液样本，管理数据；杨静：研究设计指导，提供资源，管理及监督项目进展，总体把关和审定论文。所有作者均参与论文修改，并对最终文稿进行审读和确认。

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