Abstract
目的
探索类风湿关节炎(rheumatoid arthritis，RA)患者肌肉量减少的生物标志物。
方法
本研究为横断面研究，采用非标记液相色谱-串联质谱技术检测基线病情活动且2年随访期间保持肌肉量正常或肌肉量减少的RA患者血清蛋白组学特征，并通过生物信息学分析两组患者血清中的差异蛋白；进一步纳入102例RA患者(肌肉量减少组和肌肉量正常组各51例)及招募志愿者51例作为健康对照组(healthy controls，HC)进行差异蛋白的ELISA验证，并采用Logistic回归分析RA患者基线肌肉量减少的相关因素。
结果
10例肌肉量减少组与9例肌肉量正常组RA患者通过基线血清蛋白组学分析筛选出38个差异蛋白，其中间-α-胰蛋白酶抑制蛋白重链3(inter-alpha-trypsin inhibitor heavy chain H3，ITIH3)表达差异显著(log2FC=2.09)，并在所有血清样本中稳定表达。ELISA验证结果显示102例RA患者血清ITIH3水平显著高于HC组[(119.4±79.7) mg/L vs. (42.3±16.6) mg/L, P < 0.001]，且肌肉量减少组RA患者血清ITIH3显著高于肌肉量正常组[(148.1±94.7) mg/L vs. (90.8±46.6) mg/L, P < 0.001]。多因素Logistic回归分析结果显示，校正混杂因素后，血清ITIH3水平仍是RA患者肌肉量减少的独立危险因素(OR=1.024，95%CI：1.013~1.038，P < 0.001)。
结论
血清ITIH3水平是RA患者肌肉量减少的独立危险因素，提示ITIH3可能是RA肌肉量减少的潜在生物标志物。
Keywords: 类风湿关节炎, 肌肉量减少, 蛋白质组学, 生物标志物, 间-α-胰蛋白酶抑制蛋白重链3
Abstract
Objective
To explore the serum biomarkers of myopenia in patients with rheumatoid arthritis (RA) via serum proteomic profiling.
Methods
This cross-sectional study recruited active RA patients who either sustained non-myopenic or myopenia state over a 2-year follow-up period and unlabeled liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze the serum proteomic profiles. Bioinformatics analyses were then applied to identify differentially expressed proteins between the two groups. Further validation cohort enrolled 102 RA patients (including 51 cases of non-myopenia group and 51 cases of myopenia group) and 51 healthy controls (HC) with age- and gender- matched propensity score at a 1 ∶ 1 ∶ 1 ratio. Enzyme-linked immunosorbent assay (ELISA) was used to verify the expression levels of the candidate protein. Multivariate logistic regression analysis was performed to identify baseline factors associated with myopenia in the RA patients.
Results
Initial proteomic analysis of baseline serum samples from 9 non-myopenia RA patients and 10 myopenia RA patients identified 38 differentially expressed proteins. Among them, inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) showed a significant upregulation in the myopenia group (log2FC=2.09) and was consistently detected across all the samples. Subsequent ELISA validation confirmed that the serum ITIH3 level in 102 RA patients was significantly higher than that in 51 HC [(119.4±79.7) mg/L vs. (42.3±16.6) mg/L, P < 0.001], in which both myopenia group and non-myopenia group of the RA patients showed higher levels of serum ITIH3 than the HC group (both P < 0.001). Importantly, the serum ITIH3 level in the 51 patients with myopenia were significantly higher than that in the 51 patients without myopenia [(148.1±94.7) mg/L vs. (90.8±46.6) mg/L, P < 0.001]. After adjustment for confounding covariates including gender, age, disease duration, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), radiological joint destruction and previous treatment, the multivariate Logistic regression analysis showed that the baseline serum ITIH3 level was an independent risk factor for myopenia in the RA patients (OR=1.024, 95%CI: 1.013-1.038, P < 0.001).
Conclusion
This study identifies serum ITIH3 as a significant and independent risk factor for myopenia in patients with RA, which imply that ITIH3 might be a potential biomarker of myopenia. Further longitudinal large-sample multicenter validation is warranted to elucidate the precise role of ITIH3 in the pathophysiology of RA-associated myopenia and the clinical utility in risk stratification and management.
Keywords: Rheumatoid arthritis, Myopenia, Proteomics, Biomarker, Inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3)
随着全球人口老年化，肌少症已经引起越来越多的关注与重视。肌少症是一种与增龄相关、以肌肉量减少、肌肉力量下降和(或)躯体功能障碍为主要特征的老年综合征，可增加衰弱、跌倒、残疾甚至死亡等不良结局的发生风险，2016年作为独立疾病首次列入国际疾病分类(international classification of diseases, ICD)疾病编码管理。肌少症可根据病因分为原发性和继发性，其不仅见于老年群体，慢性疾病患者[如类风湿关节炎(rheumatoid arthritis, RA)]更是高发人群，且与原发病相互加重，使患者预后显著恶化[1]。我国2024版《中国肌少症诊疗指南》强调肌少症的规范化诊断，包括肌肉力量、肌肉量和躯体功能三大指标[1]。然而，值得注意的是，RA患者由于关节炎症导致的关节肿痛会干扰肌肉力量和躯体功能的评估，因此肌肉量减少的评估、监测及预测在RA肌少症的研究中尤为重要[2]。
肌肉量减少是一种具有临床意义的肌肉萎缩，可发生于任何疾病及各个年龄段[3]。本课题组的前瞻性队列研究表明，RA患者基线肌肉量减少不仅与高疾病活动度、躯体功能障碍加重有关[4]，还是随访一年的RA患者出现放射学关节破坏进展的独立预测因素[5]。RA肌肉量减少不仅导致患者躯体功能退化、关节破坏加速，还与心血管事件及心血管死亡风险升高有关[6]。目前临床缺乏肌肉量减少的早期筛查及预测指标，现有的肌肉量评估技术中，磁共振成像(magnetic resonance imaging，MRI)和计算机断层扫描(computed tomography，CT)因高成本、操作复杂及存在辐射风险等因素限制了其在临床的广泛使用，而生物电阻抗分析(bioelectrical impedance analysis，BIA)尽管操作简便、无辐射，但在临床普及度不高[7]。因此，临床亟需寻找新的指标辅助RA患者肌肉量减少的早期筛查及预测。本文旨在通过非标记的液相色谱-串联蛋白质谱(liquid chromatography-tandem mass spectrometry，LC-MS/MS)筛选出RA患者肌肉量减少的血清生物标志物。
1. 资料与方法
1.1. 研究对象的选择及分组
本研究为横断面研究，RA患者来自中山大学孙逸仙纪念医院风湿免疫科建立的RA前瞻性队列[4-5, 8-10]，RA诊断符合2010年美国风湿病协会(American College of Rheumatology, ACR)/欧洲抗风湿病联盟(European League Against Rheumatism, EULAR)联合修订的RA分类标准[11]。本研究纳入2015年8月至2020年8月入组的基线完成肌肉量测定并有基线血清结果、随访满2年并且至少有2次肌肉量测定数据，同时排除合并其他自身免疫病(如系统性红斑狼疮、系统性硬化症、皮肌炎等)、恶性肿瘤、严重肝肾疾病的RA患者。健康志愿者作为健康对照(healthy control, HC)组为2019年8月至2022年3月自愿参加本研究的中山大学孙逸仙纪念医院的职工或学生。本研究开始前已经中山大学孙逸仙纪念医院伦理委员会审查批准(批文号SYSEC-2009-06和SYSEC-KY-KS-2019-097)，所有参与本研究的受试者包括患者和健康志愿者均签署知情同意书。
1.2. 临床资料收集
收集RA患者的临床资料，包括年龄、性别、病程、吸烟情况、RA疾病活动度评估、功能评估、放射学评估，以及实验室检测类风湿因子(rheumatoid factors, RF)、抗环瓜氨酸肽(cyclic citrullinated peptide, CCP)抗体等检查结果。RA疾病活动度评估包括28个压痛关节数和28个肿胀关节数、患者总体疾病活动度评估和医生总体疾病活动度评估、疼痛视觉模拟评分(visual analogue scale, VAS)、红细胞沉降率(erythrocyte sedimentation rate, ESR)和C反应蛋白(C-reactive protein, CRP)；计算临床疾病活动指数(clinical disease activity index，CDAI)，CDAI>2.8为RA疾病活动期；采用Stanford健康评估问卷-残疾指数(health assessment questionnaire-disability index，HAQ-DI)评估患者躯体功能，HAQ-DI≥1定义为躯体功能障碍；采用改良Sharp评分法评估双手(包括腕关节)正位X线片，放射学关节破坏(radiological joint destruction，RJD)定义为改良Sharp评分总分(modified total Sharp score，mTSS)大于10[11]。
1.3. 肌肉量测量
采用生物电阻抗分析仪(InBody 230, Biospace Co, 中国上海)测量HC和RA患者四肢骨骼肌质量(appendicular skeletal muscle mass, ASM)，并计算四肢骨骼肌指数(appendicular skeletal muscle mass index, ASMI)=ASM(kg)/身高(m)2。肌肉量减少定义为ASMI男性 < 7.0 kg/m2、女性 < 5.7 kg/m2[1]。
1.4. RA患者血清蛋白质组学非标记液相色谱-串联质谱分析
收集基线病情活动(CDAI＞2.8)且两年随访期间肌肉量减少(n=10)或正常(n=9)的RA患者血清进行LC-MS/MS分析。取出在-80 ℃冻存的19例血清样本，经梯度复融后统一进行前处理。首先，使用ProteoMinerTM试剂盒去除血清中的高丰度蛋白(如白蛋白和免疫球蛋白)；随后，取200 ~300 μg蛋白加入胰酶溶液(0.1 g/L，10 mmol/L醋酸配制)和50 mmol/L NH4HCO3，于37 ℃进行酶解(≥12 h)，反应以三氟乙酸(trifluoroacetic acid，TFA)终体积分数为0.4%终止。酶解产物经C18除盐柱纯化：依次用甲醇、0.2%(体积分数) TFA/80%(体积分数)乙腈和0.2%TFA冲洗活化，上样后以0.2% TFA冲洗3次，再以80%乙腈洗脱。洗脱液经真空干燥后，用0.1%(体积分数)甲酸复溶。
复溶样品采用LC-MS/MS分析。酶解肽段使用毛细管填充柱(20 cm×75 μm)分离，流动相为溶剂A(含0.1%甲酸的2%乙腈水溶液)和溶剂B(含0.1%(体积分数)甲酸的98%乙腈溶液)，流速300 nL/min，梯度洗脱程序表示为时间(溶剂B起始浓度→溶剂B结束浓度)：0 min (2%→5%), 1~ 90 min (5%→25%), 91~120 min (25%→40%)。质谱分析采用Thermo FAIMS-Fusion高分辨质谱，全扫描范围质荷比300~1 800，分辨率设为60 000 (质荷比400)。采集模式为数据依赖型(data dependent acquisition，DDA)，每张MS全扫描图中选取10个最强的母离子进行碰撞诱导解离(collision-induced dissociation, CID)碎裂MS/MS分析。原始数据使用Proteome Discoverer 2.4软件处理，检索UniProt恒河猴数据库。检索参数包括：母离子质量容差(相对分子质量)0.001，碎片离子质量容差0.5，胰蛋白酶酶切(允许最多2个漏切位点)，固定修饰为半胱氨酸羧甲基化，可变修饰为甲硫氨酸氧化及蛋白质N端乙酰化。
1.5. 血清间-α-胰蛋白酶抑制蛋白重链3(inter-alpha-trypsin inhibitor heavy chain H3, ITIH3)检测
根据性别、年龄进行倾向性评分1 ∶ 1 ∶ 1匹配，分别纳入RA肌肉量减少组51例、RA肌肉量正常组51例和HC组51例作为血清ELISA验证队列。采用ELISA定量检测试剂盒(CSB-EL011896HU，CUSABIO公司，中国)检测RA患者和HC血清ITIH3水平。按照试剂盒说明进行操作，使用酶标仪(Multiskan FC，Thermo Fisher Scientific公司，美国)在450 nm下读取光密度(D)值。样品(质量)浓度通过绘制试剂盒提供的标准品标准曲线计算得出(单位: mg/L)。
1.6. 生物信息学和统计学分析
所有生物信息学和统计学分析均通过R(4.3.2版本)完成。使用R软件nonrandom包进行倾向评分匹配入组对象，limma包筛选LC-MS/MS差异表达蛋白(differentially expressed proteins, DEPs)，筛选阈值为log2 FC >1，P < 0.05；使用pheatmap包绘制热图。
符合正态分布的连续变量以 x±s表示，两组间比较采用t检验，多组比较采用方差分析，不符合正态分布的连续变量以M(Q1，Q3)表示，组间比较采用Mann-Whitney U检验，多组比较采用Friedman检验。分类变量以n(%)表示，多组间比较采用χ2检验或Fisher精确概率法，Bonferroni检验用于多组间的两两比较，校正后的显著性阈值为P < 0.016 7。采用单因素和多因素Logistic回归分析RA患者基线肌肉量减少的相关因素。所有显著性检验均为双侧检验，P < 0.05认为差异有统计学意义。
2. 结果
2.1. RA患者肌肉量减少相关的血清蛋白组学特征及生物信息学分析
收集19例基线疾病活动(CDAI > 2.8)且两年随访期间肌肉量保持正常或减少的RA患者基线血清, 进行LC-MS/MS分析，其中肌肉量正常组9例，平均年龄为(45.1±9.5)岁，女性占77.8%，中位病程8.0(5.5, 10.0)个月；肌肉量减少组10例，平均年龄为(40.0±10.0)岁，女性占80.0%，中位病程10.0(1.0, 12.5)个月。两组间年龄、性别、病程、主动吸烟比例及基线CDAI评分差异均无统计学意义(表 1)。
表 1.
19例进行血清LC-MS/MS检测的RA患者的基线临床特征比较
Comparisons of baseline characteristics of RA patients in serum LC-MS/MS analysis
| Items | Non-myopenia RA patients (n=9) | Myopenia RA patients (n=10) | Statistics | P |
| RA, rheumatoid arthritis; ASMI, appendicular skeletal muscle mass index; CDAI, clinical disease activity index. *showing 2-sided significancy of Fisher’ s exact test. | ||||
| Female, n (%) | 7(77.8) | 8(80.0) | χ2=0.000 | >0.999 |
| Age/years, x±s | 45.1±9.5 | 40.0±10.0 | t=1.139 | 0.271 |
| ASMI/(kg/m2), x±s | 6.50±0.55 | 5.17±0.62 | t=4.932 | ＜0.001 |
| Disease duration/month, M (Q1, Q3) | 8.0(5.5, 10.0) | 10.0(1.0, 12.5) | Z=-0.369 | 0.712 |
| Active smoking, n (%) | 0(0.0) | 1(10.0) | - | >0.999* |
| CDAI, M (Q1, Q3) | 19.0(14.5, 29.0) | 22.5(11.8, 27.0) | Z=-0.286 | 0.775 |
LC-MS/MS检测结果共鉴定出506个蛋白质，主成分分析(principal component analysis，PCA)显示，肌肉量减少组与肌肉量正常组之间存在较大重叠，但仍表现出一定程度的分离(图 1A)。以log2 FC >1、P < 0.05为阈值，筛选出38个差异表达蛋白，其中上调蛋白26个，下调蛋白12个(图 1B)。进一步对两组不同样本的蛋白表达进行可视化分析，热图定量分析显示，两组间蛋白表达水平存在显著差异(图 1C)。在差异倍数排名前八的差异表达蛋白中，ITIH3在所有血清样本中均可稳定测出，且在肌肉量减少组的表达量为肌肉量正常组的7.49倍(log2FC=2.09，图 1D)。
图 1.
肌肉量减少组和肌肉量正常组RA患者血清LC-MS/MS检测及生物信息学分析
Serum LC-MS/MS and bioinformatics analysis of myopenia and non-mypoenia RA patients
Serum protein profile analysis showing principal component analysis plot (A), DEPs volcano plot (B), DEPs heatmap (C), DEPs sample distribution plot (D) of myopenia and non-mypoenia RA patients. DEPs, differentially expressed proteins; PC, principal component; FC, fold change.
2.2. ELISA法检测RA患者基线血清ITIH3水平
进一步纳入102例RA患者和51例HC志愿者进行血清ITIH3的ELISA验证，RA患者组平均年龄为(49.1±11.5)岁，女性占79.4%，中位病程41(10, 120)个月，85.3%的患者为疾病活动期，28.4%的患者有躯体功能障碍，46.1%的患者有放射学关节破坏。
HC组平均年龄(46.9±8.8)岁，女性占86.3%。与肌肉量正常组RA患者相比，肌肉量减少组RA患者病程更长[65 (14, 133) 个月vs. 39 (6, 111)个月，P=0.028]，且ESR水平更高[49 (24, 77) mm/h vs. 32 (19, 50) mm/h，P=0.011，表 2]。
表 2.
健康对照组与RA患者组的基线临床特征比较
Comparisons of baseline characteristics between RA patients and healthy controls
| Items | HC(n=51) | Non-myopenia RA patients (n=51) | Myopenia RA patients (n=51) | All RA patients(n=102) | Statistics | P |
| HC, healthy controls; RA, rheumatoid arthritis; ASMI, appendicular skeletal muscle mass index; RF, rheumatoid factor; ACPA, anti-cyclic citrullinated protein antibodies; 28TJC, 28 tender joint counts; 28SJC, 28 swollen joint counts; PtGA, patient global assessment of disease activity; PrGA, physician global assessment of disease activity; VAS, visual analogue scale; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; CDAI, clinical disease activity index; HAQ-DI, health assessment questionnaire-disability index; mTSS, modified total Sharp score. a, comparison among HC, non-myopenia RA patients and myopenia RA patients; b, comparison of HC to all RA patients; c, comparison of non-myopenia RA patients to myopenia RA patients. * showing 2-sided significancy of Fisher’ s exact test between HC and all RA patients. | ||||||
| Female, n (%) | 44 (86.3) | 39(76.5) | 42(82.4) | 81 (79.4) | χ2 =2.753a | 0.252 |
| Age/years, x±s | 46.9±8.8 | 48.7±9.8 | 49.5±13.0 | 49.1±11.5 | F=0.811a | 0.446 |
| ASMI/(kg/m2), x±s | 6.31±0.73 | 6.81±0.67 | 5.23±0.63 | 6.0±1.0 | F=72.335a | < 0.001 |
| Myopenia, n (%) | 10 (19.6) | 0(0.0) | 51(100.0) | 51 (50.0) | - | < 0.001b* |
| Disease duration/month, M(Q1, Q3) | - | 39 (6, 111) | 65 (14, 133) | 41 (10, 120) | Z=-2.202c | 0.028 |
| Active smoking, n (%) | - | 7(13.7) | 6(11.8) | 13 (14.6) | χ2=0.088c | 0.767 |
| Positive RF, n (%) | - | 38(74.5) | 42(82.4) | 80 (78.4) | χ2=0.927c | 0.336 |
| Positive ACPA, n (%) | - | 37(72.5) | 41(80.4) | 78 (76.5) | χ2=0.872c | 0.350 |
| 28TJC, M (Q1, Q3) | - | 3 (1, 8) | 4 (1, 7) | 3 (1, 7) | Z=-0.199c | 0.842 |
| 28SJC, M (Q1, Q3) | - | 2 (1, 6) | 2 (0, 5) | 2 (1, 5) | Z=-0.149c | 0.882 |
| PtGA/cm, M (Q1, Q3) | - | 4 (2, 7) | 4 (3, 6) | 4 (2, 6) | Z=-0.236c | 0.814 |
| PrGA/cm, M (Q1, Q3) | - | 3 (2, 7) | 4 (3, 6) | 4 (2, 6) | Z=-0.508c | 0.611 |
| Pain VAS/cm, M (Q1, Q3) | - | 3 (2, 7) | 4 (2, 5) | 4 (2, 5) | Z=-0.784c | 0.433 |
| ESR/(mm/h), M (Q1, Q3) | - | 32 (19, 50) | 49 (24, 77) | 37 (21, 64) | Z=-2.534c | 0.011 |
| CRP/(mg/L), M (Q1, Q3) | - | 5.9 (3.3, 10.6) | 10.2 (3.3, 25.5) | 6.8 (3.3, 17.9) | Z=-1.581c | 0.114 |
| CDAI, M (Q1, Q3) | - | 13 (6, 28) | 16 (9, 23) | 14 (8, 24) | Z=-0.378c | 0.705 |
| Active disease, n (%) | 44 (86.3) | 43(84.3) | 87 (85.3) | χ2=0.078c | 0.780 | |
| HAQ-DI, M (Q1, Q3) | - | 0.25 (0.00, 1.00) | 0.50 (0.13, 1.25) | 0.38 (0.00, 1.06) | Z=-0.892c | 0.372 |
| Physical dysfunction, n (%) | 14 (27.5) | 15(29.4) | 29(28.4) | χ2=0.048c | 0.826 | |
| mTSS, M (Q1, Q3) | - | 5 (0, 32) | 11 (3, 46) | 8 (0, 36) | Z=-1.564c | 0.118 |
| Radiological joint destruction, n (%) | 19 (37.3) | 27 (52.9) | 46 (45.1) | χ2=2.534c | 0.111 | |
ELISA检测基线血清ITIH3水平的结果显示，RA患者的总体血清ITIH3水平显著高于HC组[(119.4± 79.7) mg/L vs. (42.3±16.6) mg/L, P < 0.001，图 2A]。102例RA患者中，肌肉量减少组和肌肉量正常组RA患者的血清ITIH3水平均显著高于HC组[(148.1±94.7) mg/L vs. (42.3±16.6) mg/L, (90.8±46.6) mg/L vs. (42.3±16.6) mg/L, P均 < 0.001，图 2B]，并且肌肉量减少组RA患者血清ITIH3水平显著高于肌肉量正常组[(148.1±94.7) mg/L vs. (90.8±46.6) mg/L, P < 0.001，图 2B]。
图 2.
健康对照组与RA患者组基线血清ITIH3水平的比较
Comparisons of baseline serum ITIH3 levels of RA patients and healthy controls
A, the serum ITIH3 levels of RA patients and healthy controls were detected by ELISA; B, comparison of serum ITIH3 levels among myopenia/non-mypoenia RA patients and healthy controls. RA, rheumatoid arthritis; HC, healthy controls; ELISA, enzyme- linked immunosorbent assay; ITIH3, inter- alpha-trypsin inhibitor heavy chain 3.
2.3. 基线血清ITIH3水平升高是RA患者基线肌肉量减少的独立危险因素
采用Logistic回归分析RA患者基线肌肉量减少的相关因素，单因素分析结果显示，基线血清ITIH3水平(OR=1.012，95%CI：1.005~1.019，P < 0.001)、ESR(OR=1.016，95%CI：1.003~1.031，P=0.013)与RA患者基线肌肉量减少呈正相关(表 3)。进一步将单因素分析中有显著意义及P值相对较小的自变量(血清ITIH3水平、病程、ESR、CRP、放射学关节破坏)纳入多因素Logistic回归分析，结果显示血清ITIH3水平仍是RA患者基线肌肉量减少的独立危险因素(OR=1.018，95%CI：1.009~1.029，P < 0.001)。考虑到可能存在的潜在混杂因素的影响，在上述多因素Logistic回归分析中进一步纳入性别、年龄、既往用药情况进行校正，结果显示血清ITIH3水平仍是RA患者基线肌肉量减少的独立危险因素(OR=1.024，95%CI：1.013~1.038，P < 0.001)，并且RA患者血清水平ITIH3水平每升高1 mg/L，肌肉量减少事件发生的风险比平均增加2.4%。
表 3.
Logistic回归分析RA患者基线肌肉量减少的相关因素
Logistic regression analysis of factors related to baseline myopenia in RA patients
| Items | Univariate | Multivariate1 | Multivariate2 | |||||
| OR(95%CI) | P | OR(95%CI) | P | OR(95%CI) | P | |||
| ITIH3, inter-alpha trypsin inhibitor heavy chain 3; RA, rheumatoid arthritis; RF, rheumatoid factor; ACPA, anti-cyclic citrullinated protein antibodies; 28TJC, 28 tender joint counts; 28SJC, 28 swollen joint counts; PtGA, patient global assessment of disease activity; PrGA, physician global assessment of disease activity; VAS, visual analogue scale; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; CDAI, clinical disease activity index; HAQ-DI, health assessment questionnaire-disability index, mTSS, modified total Sharp score; csDMARDs, conventional synthetic disease-modifying antirheumatic drugs; bDAMRDs, biological disease-modifying antirheumatic drugs; tsDMARDs, target synthetic disease-modifying antirheumatic drugs. Multivariate1, including ITIH3, disease duration, ESR, CRP and radiological joint destruction. Multivariate2, including ITIH3, gender, age, disease duration, ESR, CRP, radiological joint destruction and previous treatment. | ||||||||
| ITIH3 | 1.012 (1.005-1.019) | < 0.001 | 1.018 (1.009-1.029) | < 0.001 | 1.024 (1.013-1.038) | < 0.001 | ||
| Female | 1.436 (0.548-3.875) | 0.462 | 1.137 (0.215-6.436) | 0.880 | ||||
| Age | 1.006 (0.972-1.042) | 0.721 | 1.002 (0.949-1.059) | 0.939 | ||||
| Disease duration | 1.004 (0.999-1.009) | 0.112 | 1.000 (0.993-1.007) | 0.977 | 1.000 (0.993-1.008) | 0.960 | ||
| Active smoking | 0.813 (0.241-2.667) | 0.731 | ||||||
| Positive RF | 1.596 (0.619-4.271) | 0.335 | ||||||
| Positive ACPA | 1.551 (0.619-4.003) | 0.350 | ||||||
| 28TJC | 0.990 (0.918-1.065) | 0.779 | ||||||
| 28SJC | 0.961 (0.865-1.063) | 0.442 | ||||||
| PtGA | 1.002 (0.874-1.150) | 0.972 | ||||||
| PrGA | 1.010 (0.877-1.165) | 0.886 | ||||||
| Pain VAS | 1.036 (0.885-1.215) | 0.660 | ||||||
| ESR | 1.016 (1.003-1.031) | 0.013 | 1.021 (1.000-1.045) | 0.056 | 1.024 (0.999-1.052) | 0.065 | ||
| CRP | 1.009 (0.995-1.026) | 0.213 | 0.986 (0.959-1.015) | 0.321 | 0.986 (0.954-1.022) | 0.403 | ||
| CDAI | 0.995 (0.966-1.026) | 0.761 | ||||||
| HAQ-DI | 1.039 (0.617-1.758) | 0.884 | ||||||
| Physical dysfunction | 1.112 (0.449-2.771) | 0.818 | ||||||
| mTSS | 1.005 (0.996-1.016) | 0.289 | ||||||
| Radiological joint destruction | 1.945 (0.851-4.531) | 0.115 | 1.435 (0.4595-4.463) | 0.530 | 1.833 (0.521-6.576) | 0.344 | ||
| Previous treatment | ||||||||
| Treatment naÏve | 0.804 (0.316-2.013) | 0.641 | 0.805 (0.052-16.06) | 0.881 | ||||
| Glucocorticoids | 1.314 (0.569-3.060) | 0.522 | 3.126 (0.874-13.37) | 0.097 | ||||
| csDMARDs | 1.458 (0.545-4.013) | 0.453 | 0.436 (0.040-5.478) | 0.494 | ||||
| bDAMRDs/tsDMARDs | 1.537 (0.243-12.12) | 0.644 | 1.022 (0.099-10.94) | 0.985 | ||||
3. 讨论
RA是一种免疫介导的以慢性疼痛性关节炎症为特征的全身性疾病。RA患病率占世界总人口的0.5%~1.0%，中国大陆地区患病率为0.42%(估测患病总人数达500万)[11]。约40%的RA患者可合并一种或多种关节外表现，包括累及肌肉系统，导致骨骼肌肌肉量减少、肌肉功能减退。在RA患者中，肌少症的患病率约为25%，不但增加RA患者关节炎症和关节损伤的负担，还与跌倒、骨折和残疾的风险增加有关，会显著降低患者生活质量，延长住院时间，甚至增加死亡风险[6]。RA患者肌肉量减少患病率由于诊断标准和种族的差异在不同研究中差异较大(17%~60%)[6]。本课题组前期真实世界RA队列研究表明，36.5%的RA患者有肌少症，69.0%有肌肉功能下降，57.8%有肌肉力量下降，37.1%有躯体功能障碍，而肌肉量减少的发生率为46.1%，明显高于普通人群(13.2%)[4, 8]。RA患者在病程早期(一年内)即可出现肌肉量减少[9]，42.1%的早期RA患者存在肌肉量减少[9]，而在老年RA患者中肌肉量减少的发生率高达54.5%[5]。
RA肌肉量减少的产生机制涉及慢性炎症、蛋白质合成障碍及神经肌肉功能失调等。RA疾病过程中持续产生的肿瘤坏死因子-α、白介素-6等促炎细胞因子可以通过诱导泛素-蛋白酶体系统促进蛋白水解，并增加肌细胞自噬、线粒体功能障碍，从而导致肌肉量减少以及肌肉萎缩[6]。RA肌肉量减少与营养不良、恶病质等“低肌肉量综合征”高度重叠。本课题组既往研究发现，超半数(54.8%)RA患者有低肌肉量综合征且合并营养不良，3.2%同时合并恶病质[12]。现行的肌少症筛查工具(如SARC-F问卷)对RA患者肌肉量减少的敏感性不足，漏诊率高，血清标志物(如肌酐/胱抑素C比值)易受肾功能及RA炎症干扰，无法可靠反映肌肉损耗程度。血液样本一直被视为潜在生物标志物的宝库，然而，血浆中蛋白质的丰度跨度极大(超过十个数量级)，给定量分析带来了巨大挑战。基于质谱技术的蛋白质分析正是破解这一难题的有效方法，通过精准测定蛋白质的质量，结合生物信息学，蛋白质谱可鉴定上万种蛋白，分析其丰度、翻译后修饰(如磷酸化、乙酰化)及空间分布。本研究通过血清蛋白质组学特征分析筛选及ELISA验证RA肌肉量减少相关的潜在生物标志物，结果发现肌肉量减少的RA患者血清ITIH3水平显著高于肌肉量正常的RA患者和HC，并且基线血清ITIH3水平是RA患者基线肌肉量减少的独立危险因素。
ITIH3属于间-α-胰蛋白酶抑制剂家族，是一种具有内肽酶抑制活性的分泌蛋白。ITIH3可与细胞外基质主要成分透明质酸结合，进而参与维持细胞外基质稳定及组织修复过程[13]。另外，ITIH3还可通过抑制补体级联早期反应减轻组织损伤[13]。近期研究发现，因长期卧床而导致肌肉量减少的个体中，其血浆ITIH3水平明显升高，提示肌肉丢失可能与ITIH3调控相关[14]。ITIH3是与射血分数保留的心力衰竭发展相关的3种血清蛋白质之一，也可能是妊娠期高血压和子痫前期促血栓炎症的关键介质[15-16]。针对重症肌无力的研究则提示ITIH3可作为重症肌无力疾病活动性和治疗反应的潜在生物标志物[17]。本研究结果发现，在肌肉量减少的RA患者血清中ITIH3上调，其机制可能与ITIH3调控炎症和组织修复的功能相关。值得注意的是，RA患者总体的血清ITIH3水平显著高于健康对照组，同时肌肉量正常组RA患者血清ITIH3水平也较健康对照组显著升高，但较肌肉量减少组RA患者低，提示该组RA患者虽表现为肌肉量正常，但仍有进展为肌肉量减少的潜在风险。
本研究结果尽管具有启发性，但存在一定局限。首先，本研究样本量较小，可能影响结果的普适性，未来需在大规模、多中心队列中进行更高精度的血清蛋白组学筛选及多中心队列的验证；其次，未来临床研究仍需收集营养状态、体力活动水平等临床数据，充分考虑潜在的混杂因素，进一步分析ITIH3对肌肉量减少的影响；再次，仅对基线血清进行检测无法明确ITIH3与肌肉量减少之间的因果关系，需通过前瞻性的纵向研究探索基线ITIH3水平升高是否与肌肉量减少的进展相关。未来血清ITIH3在RA患者肌肉量减少的早期筛查及预测中的价值值得进一步探讨。
综上所述，本研究拓展了RA患者血清蛋白质组学研究的新方向，聚焦于RA合并肌肉量减少患者的血清蛋白质组学特征，发现ITIH3可作为诊断RA肌肉量减少的潜在生物学标志物，可能为RA患者肌肉量减少的筛查和监测提供一种更便捷、经济的方法。
Funding Statement
国家自然科学基金(82471832)、广州市科技市校(院)企联合资助专题(2024A03J0912)、广州市科技计划项目-逸仙优秀青年科学基金项目(2023A03J0709)、中山大学逸仙临床研究5010计划项目(SYS-5010-202407)
Supported by National Natural Science Foundation of China (82471832), Guangzhou Science and Technology Joint Funding Project for University-Enterprise Collaboration (2024A03J0912), Guangzhou Municipal Science and Technology Project and Yat-sen Excellent Yong Scientists Fund (2023A03J0709), and Yixian Clinical Research 5010 Program Project (SYS-5010-202407)
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 吴滔、林建子：设计研究方案，收集、分析、整理数据资料，撰写论文；朱亚峰：协助血清蛋白组学检测及分析数据；马剑达、贾霈雯、潘婕：协助临床资料整理和数据分析；杨莉娟：ELISA检测；邹耀威、杨迎、卢烨：协助临床资料收集；戴冽：提出研究思路，设计研究方案，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。
References
- 1.中华医学会老年医学分会, 国家老年疾病临床医学研究中心(湘雅医院) 中国肌肉减少症诊疗指南(2024版) 中华医学杂志. 2025;105(3):181–203. [Google Scholar]
- 2.Jin H, Wang G, Lu Q, et al. Pathophysiology of myopenia in rheumatoid arthritis. Bone Res. 2025;13(1):64. doi: 10.1038/s41413-025-00438-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Fearon K, Evans WJ, Anker SD. Myopenia: A new universal term for muscle wasting. J Cachexia Sarcopenia Muscle. 2011;2(1):1–3. doi: 10.1007/s13539-011-0025-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Lin JZ, Liang JJ, Ma JD, et al. Myopenia is associated with joint damage in rheumatoid arthritis: A cross-sectional study. J Cachexia Sarcopenia Muscle. 2019;10(2):355–367. doi: 10.1002/jcsm.12381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Ma JD, Chen CT, Lin JZ, et al. Muscle wasting, a neglected complication associated with physical dysfunction in elderly patients with rheumatoid arthritis: A cross-sectional observational study. Scand J Rheumatol. 2021;50(4):280–289. doi: 10.1080/03009742.2020.1842902. [DOI] [PubMed] [Google Scholar]
- 6.Bennett JL, Pratt AG, Dodds R, et al. Rheumatoid sarcopenia: Loss of skeletal muscle strength and mass in rheumatoid arthritis. Nat Rev Rheumatol. 2023;19(4):239–251. doi: 10.1038/s41584-023-00921-9. [DOI] [PubMed] [Google Scholar]
- 7.Tosato M, Marzetti E, Cesari M, et al. Measurement of muscle mass in sarcopenia: From imaging to biochemical markers. Aging Clin Exp Res. 2017;29(1):19–27. doi: 10.1007/s40520-016-0717-0. [DOI] [PubMed] [Google Scholar]
- 8.Jia PW, Lin JZ, Zou YW, et al. The characteristic of muscle function for sarcopenia in patients with rheumatoid arthritis: A large-scale real-world cross-sectional study. Medicina. 2025;61(4):551. doi: 10.3390/medicina61040551. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Pan J, Zou YW, Zhu YY, et al. Muscle mass loss is associated with physical dysfunction in patients with early rheumatoid arthritis. Front Nutr. 2022;9:1007184. doi: 10.3389/fnut.2022.1007184. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Lin JZ, Liu Y, Ma JD, et al. Reduced skeletal muscle inde-pendently predicts 1-year aggravated joint destruction in patients with rheumatoid arthritis. Ther Adv Musculoskelet Dis. 2020;12:1759720X20946220. doi: 10.1177/1759720X20946220. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.国家皮肤与免疫疾病临床医学研究中心(北京协和医院), 中国医师协会风湿免疫专科医师分会, 中国康复医学会风湿免疫病康复专业委员会, et al. 2024中国类风湿关节炎诊疗指南. 中华内科杂志. 2024;63(11):1059–1077. [Google Scholar]
- 12.贾 霈雯, 杨 迎, 邹 耀威, et al. 类风湿关节炎患者低肌肉量综合征的临床特征及其对躯体功能的影响. 北京大学学报(医学版) 2024;56(6):1009–1016. doi: 10.19723/j.issn.1671-167X.2024.06.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Adair JE, Stober V, Sobhany M, et al. Inter-α-trypsin inhibitor promotes bronchial epithelial repair after injury through vitronectin binding. J Biol Chem. 2009;284(25):16922–16930. doi: 10.1074/jbc.M808560200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Okroj M, Holmquist E, Sjölander J, et al. Heavy chains of inter alpha inhibitor (IαI) inhibit the human complement system at early stages of the cascade. J Biol Chem. 2012;287(24):20100–20110. doi: 10.1074/jbc.M111.324913. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Karuna N, Tonry C, Ledwidge M, et al. Proteomic-based bio-marker discovery reveals panels of diagnostic biomarkers for early identification of heart failure subtypes. J Transl Med. 2025;23(1):546. doi: 10.1186/s12967-025-06563-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.de Almeida LGN, Young D, Chow L, et al. Proteomics and metabolomics profiling of platelets and plasma mediators of thrombo-inflammation in gestational hypertension and preeclampsia. Cells. 2022;11(8):1256. doi: 10.3390/cells11081256. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Schroeter CB, Nelke C, Stascheit F, et al. Inter-alpha-trypsin inhibitor heavy chain H3 is a potential biomarker for disease activity in myasthenia gravis. Acta Neuropathol. 2024;147(1):102. doi: 10.1007/s00401-024-02754-6. [DOI] [PMC free article] [PubMed] [Google Scholar]