NDUFA13与CRIP1调控慢阻肺B细胞损伤并具备潜在诊断价值

doi:10.12122/j.issn.1673-4254.2026.06.15

摘要/Abstract

摘要：

目的基于生物信息学方法筛选慢性阻塞性肺疾病（COPD）中与B细胞相关的生物标志物，并探究其诊断和治疗价值。方法基于基因表达综合数据库（GEO），通过差异表达分析、加权基因共表达网络分析（WGCNA）、单细胞RNA测序（scRNA-seq）、受试者工作特征曲线（ROC）分析及孟德尔随机化（MR）研究，鉴定COPD中B细胞相关生物标志物，构建诊断列线图并进行富集分析，运用伪时间分析和细胞通讯分析探索B细胞在COPD中的潜在作用。将20只ICR小鼠分为对照组与COPD组，每组10只，采用脂多糖（LPS）联合香烟烟雾诱导法构建COPD小鼠模型并验证生物标志物表达；其中COPD组经三溴乙醇麻醉后，于第1、14天气管滴注含20 μg LPS的30 μL生理盐水，直立静置15 s，其余时间每日置于密闭烟箱内进行香烟烟雾熏吸造模，测量相关指标。结果鉴定出两种COPD保护因子NDUFA13［优势比（OR）=0.2598，95%置信区间（CI）：0.2048~0.3297，P<0.0001］和CRIP1（OR=0.2690，95% CI：0.1460~0.4970，P<0.0001）作为生物标志物。这两种基因在COPD样本中均显著下调（P<0.05），且均参与氧化磷酸化通路；在COPD诊断中表现出良好效能，基于二者构建的诊断列线图的曲线下面积（AUC）为0.79。在单细胞水平上，B细胞表现出异质性；在COPD环境中，B细胞在发育初始及终末阶段的密度均升高（P<0.05）。在COPD背景下，B细胞与其他细胞（尤其是肺泡细胞）之间的相互作用及受体-配体配对显著增强（P<0.05）。基础实验结果显示，生物标志物在COPD样本的转录组和蛋白水平均下调（P<0.05），与公共数据库中的表达趋势一致。结论本研究证实NDUFA13和CRIP1可作为COPD中与B细胞相关的生物标志物，表明二者具备诊断潜力及降低COPD风险的能力。

关键词: 慢性阻塞性肺疾病, B细胞, 转录组学, 单细胞RNA测序, 孟德尔随机化, 生物标志物

Abstract:

Objective To identify B cell-associated biomarkers in chronic obstructive pulmonary disease (COPD) using bioinformatics and explore their diagnostic and therapeutic potential. Methods Based on the GEO database, differential expression analysis, WGCNA, scRNA-seq, ROC curve analysis, and Mendelian randomization analysis were performed to screen B cell-related biomarkers. Twenty ICR mice were divided equally into control and COPD groups, and COPD model was established using lipopolysaccharide (LPS) combined with cigarette smoke exposure to verify biomarker expression. Cultured murine B lymphoma CH12.LX cells were stimulated with 1 μg/mL LPS and transfected with plasmids overexpressing CRIP1 or NDUFA13 or with a negative control plasmid, and the changes in protein expressions of NDUFB8, MTCO1, ATP5A and ACTB were examined with Western blotting. CCK-8 assay and flow cytometry were used to analyze the viability and apoptosis of murine lung epithelial cells co-cultured with the treated CH12.LX cells, respectively. Results Two protective factors, namely NDUFA13 (OR=0.2598) and CRIP1 (OR=0.2690; P<0.0001), were identified as the key B cell-associated biomarkers in COPD. In the mouse models of COPD, the expressions of NDUFA13 and CRIP1 were significantly downregulated in the lung tissues at both the transcriptional and protein levels. In CH12.LX cells, stimulation with LPS significantly lowered the protein expressions of NDUFA13, CRIP1, NDUFB8, MTCO1 and ATP5A. Overexpression of CRIP1 or NDUFA13 effectively inhibited LPS-induced inflammatory responses and oxidative stress in CH12.LX cells, and promoted the viability and suppressed apoptosis of lung epithelial cells co-cultured with CH12.LX cells. Conclusion NDUFA13 and CRIP1 are B cell-associated protective biomarkers in COPD with potential diagnostic values for COPD.

Key words: chronic obstructive pulmonary disease, B cell, transcriptomics, single-cell RNA sequencing, Mendelian randomization, biomarkers

王淼, 卢必超, 孟昊阳, 周姝含, 毛宁峰, 张洋, 吕文亮. NDUFA13与CRIP1调控慢阻肺B细胞损伤并具备潜在诊断价值[J]. 南方医科大学学报, 2026, 46(6): 1349-1364.

Miao WANG, Bichao LU, Haoyang MENG, Shuhan ZHOU, Ningfeng MAO, Yang ZHANG, Wenliang LÜ. Overexpressing NDUFA13 and CRIP1 improves B cell injury and lung Lesions in COPD mouse models[J]. Journal of Southern Medical University, 2026, 46(6): 1349-1364.

图/表 12

表1 RT-PCR引物序列

Tab.1 Primer sequences for RT-PCR

Primer	Sequences (5'—3')
CRIP1	F1:CTACTTCCTTCTAGCTGCAG
	R1:ATGATTGCAGTAGGGCTTGC
NDUFA13	F1:CTACTTCCTTCTAG CTGCAG
	R1:TTTTCCCGGAGAATCTGCAG
SAMHD1	F1:ACTTTGCACAGCTTTACCAGG
	R1:GCATTTATGACCCCATAGTGG

图 1 差异表达及加权基因共表达网络分析

Fig.1 Differential expression and weighted gene co-expression network analyses. A, B: Volcano plot and heatmap of the differentially expressed genes (DEGs) in COPD relative to control samples. C: Hierarchical clustering dendrogram used in WGCNA. D: Determination of the optimal soft-thresholding power (β) to ensure the network conforms to a scale-free topology. E: Dendrogram of different gene modules. F: Heatmap showing the correlation between modules and COPD/control samples.

图 2 细胞类型注释及B细胞相关DEGs筛选

Fig.2 Cell type annotation and B cell-related DEG screening. A: UMAP visualization of 11 cell clusters derived from clustering analysis. B: UMAP visualization depicting 6 annotated cell types. C: Bubble plot displaying marker genes associated with the annotated cell types. D: Volcano plot illustrating DEGs within B cells between COPD patients and controls.

图 3 COPD候选B细胞相关生物标志物的筛选

Fig.3 Screening of candidate B cell-related biomarkers in COPD. A: Venn diagram showing the process of filtering for B-cell associated DEGs in COPD. B, C: Receiver operating characteristic (ROC) curves for B-cell specific DEGs in the training cohort and GSE38974 (validation cohort). D, E: Expression analysis of B-cell specific DEGs between COPD patients and controls in the training and the validation cohorts. *P<0.05, **P<0.01, ****P<0.0001.

表2 NDUFA13和CRIP1与COPD间的潜在关联

Tab.2 Association of NDUFA13 and CRIP1 with COPD

Outcome	Exposure	Gene	Method	SNP	P	OR	OR_LCI95	OR_UCI95
Chronic obstructive pulmonary disease (finn-b-COPD_ OPPORTUNIST_INFECTIONS)	eqtl-a-ENSG00000186010	NDUFA13	MR Egger	44	0.0011	0.2876	0.1430	0.5783
			Weighted median	44	0.0000	0.3021	0.2183	0.4181
			Inverse variance weighted (fixed effects)	44	0.0000	0.2598	0.2048	0.3297
			Simple mode	44	0.0000	0.2903	0.1737	0.4854
			Weighted mode	44	0.0000	0.3012	0.1942	0.467
	eqtl-a-ENSG00000213145	CRIP1	MR Egger	16	0.0460	0.0350	0.0020	0.7030
			Weighted median	16	0.0000	0.2170	0.0940	0.5010
			Inverse variance weighted (fixed effects)	16	0.0000	0.2690	0.1460	0.4970
			Simple mode	16	0.0200	0.1890	0.0540	0.6640
			Weighted mode	16	0.0080	0.1970	0.0690	0.5630

图4 孟德尔随机化(MR)分析

Fig.4 Results of Mendelian randomization (MR) analysis. A-D: Scatter plots, forest plots, funnel plots, and Leave-One-Out (LOO) test in MR analysis.

图 5 NDUFA13和CRIP1在COPD中通路富集分析

Fig.5 Gene set enrichment analysis (GSEA) of NDUFA13 and CRIP1. A, B: Top 5 enriched KEGG pathways for NDUFA13 and CRIP1.

图 6 COPD环境中B细胞的发育轨迹和细胞通讯分析

Fig.6 Developmental trajectory and cell communication analyses of B cells in the COPD microenvironment. A, B: Pseudo-time trajectory analysis of B cells. C: Cell trajectories of B cells in COPD and controls. D: Distribution of biomarker expression levels along the developmental trajectory in B cells. E, F: Annotated cellular communication network illustrating the number and strength of interactions in controls (E) and COPD (F). G: Receptor-ligand pair interactions mediated by B cells in controls. H: Receptor-ligand pair interactions mediated by B cells in COPD.

图 6 COPD环境中B细胞的发育轨迹和细胞通讯分析

图 7 COPD小鼠模型中B细胞相关生物标志物的表达验证

Fig.7 Expression validation of B cell-related biomarkers in the COPD mouse model. A-C: Representative histopathological sections of mouse lungs showing infiltrating inflammatory cells (A), fluid infiltration within the alveolar cavity (red arrow) and sunken bronchial walls (green arrow) (B), structural destruction of the alveoli, and bleeding (C). D, E: Immunohistochemical analysis of CRIP1 (D) and NDUFA13 (E). F: Quantitative results of CRIP1 and NDUFA13 expressions in immunohistochemical analysis. **P<0.01. G, H: Relative expression levels of biomarkers in RT-PCR (G) and Western blotting (H). **P<0.01.

图8 过表达CRIP1和NDUFA13改善模型诱导的B细胞氧化磷酸化损伤

Fig.8 CRIP1 and NDUFA13 overexpression alleviates LPS-induced oxidative phosphorylation dysfunction in cultured murine B cells (n=3). A, B, H, I: Western blotting and quantitative detection of CRIP1 (A, B) and NDUFA13 (H, I) protein levels in B cells of each group. **P<0.01. C-F, J-M: Western blotting and quantitative determination of the expression levels of oxidative phosphorylation-related proteins NDUFB8, MTCO1 and ATP5A in B cells of each group. **P<0.01. G, N: Detection of ATP production in B cells of each group. **P<0.01.

图9 CRIP1和NDUFA13过表达抑制B细胞炎症及氧化应激,并减轻其对肺上皮细胞的损伤

Fig.9 Overexpression of CRIP1 and NDUFA13 suppresses inflammation and oxidative stress in B cells and attenuates their damaging effects on lung epithelial cells (n=3). A-C: Expression levels of IL-6, TNF-α and IL-10 in B cells of each group. **P<0.01. F-H: Secretion levels of IL-6, TNF-α and IL-10 in B cells of each group. **P<0.01. D, E, I, J: Mitochondrial ROS levels (MitoSOX fluorescence intensity) in B cells measured by flow cytometry with MitoSOX staining. **P<0.01. K, L: Cell viability of lung epithelial cells co-cultured with B cells from different treatment groups. **P<0.01. M-O: Apoptosis of lung epithelial cells co-cultured with B cells detected with flow cytometry using Annexin V-FITC/PI double staining. **P<0.01.

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