Screening and validation of diagnostic biomarkers in metabolic dysfunction-associated fatty liver disease based on data-independent acquisition quantitative proteomics

Hongfei Song; Lijuan Dan; Xiuyan Li; Xiaojie You; Shengjie Wu; Haibo Yin; Wenping Liu; Qiao Li; Jie Mu

doi:10.12449/JCH260912

Volume 42 Issue 9

Sep. 2026

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Article Navigation > Journal of Clinical Hepatology > 2026 > 42(9): 2075-2085

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Screening and validation of diagnostic biomarkers in metabolic dysfunction-associated fatty liver disease based on data-independent acquisition quantitative proteomics

DOI: 10.12449/JCH260912

Hongfei Song¹,
Lijuan Dan²,
Xiuyan Li¹,
Xiaojie You¹,
Shengjie Wu²,
Haibo Yin¹,
Wenping Liu¹,
Qiao Li^{3
,
,
,},
Jie Mu^{1
,
,
,}

1.
School of Basic Medical Sciences，Chengdu University of Traditional Chinese Medicine，Chengdu 611137，China
2.
School of Clinical Medicine，Chengdu University of Traditional Chinese Medicine，Chengdu 610072，China
3.
Department of Rehabilitation Medicine，The Third Affiliated Hospital of Chengdu University of Traditional Chinese Medicine （West Branch），Chengdu 610000，China

Research funding:

National Natural Science Foundation of China (82505222);

Ministry of Education Humanities and Social Sciences Research Youth Foundation Project (23YJC870002);

Sichuan Provincial Natural Science Foundation (2025ZNSFSC1795)

More Information

Corresponding author: Li Qiao， liqiao37@outlook.com （ORCID： 0009-0002-3705-6131）; Mu Jie， 1041415560@qq.com （ORCID： 0000-0003-3466-238X）
Received Date: 2026-03-07
Accepted Date: 2026-04-17
Published Date: 2026-09-25

Abstract

Abstract

Objective To screen for and validate key differentially expressed proteins in serum of patients with metabolic dysfunction‑associated fatty liver disease （MAFLD）， and to provide a basis for identifying diagnostic biomarkers and developing intervention strategies for this disease. Methods A total of 20 patients who were diagnosed with MAFLD in the third affiliated hospital of Chengdu University of Traditional Chinese Medicine from December 2024 to January 2025 were enrolled as MAFLD group， and 20 healthy controls were enrolled as HC group. Demographic data and serum biochemical parameters were collected from both groups. Data-independent acquisition （DIA） quantitative proteomics was used to measure the relative expression levels of serum proteins， and related methods were used to obtain significantly differentially expressed proteins， including the gene ontology functional enrichment analysis， the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis， protein-protein interaction （PPI） network， Hub node interaction， and weighted gene co-expression network analysis （WGCNA）. Rats were given a high-fat diet to establish a model of MAFLD， and randomly assigned to the control group and model group. Serum biochemical parameters and inflammatory factors were measured， liver histopathological changes were observed， and the protein expression levels of MER proto-oncogene tyrosine kinase （MERTK） and matrix metalloproteinase-9 （MMP-9） in liver tissue were verified. The independent-samples t test was used for comparison of normally distributed continuous data between groups， while the Mann-Whitney U test was used for comparison of non-normally distributed continuous data or data with heterogeneity of variance between groups； the chi-square test or the Fisher’s exact test was used for comparison of categorical data between groups. Results Compared with the HC group， the MAFLD group had significant increases in the serum levels of aspartate aminotransferase （t=-7.348， P<0.05）， alanine aminotransferase （Z=5.088， P<0.05）， alkaline phosphatase （t=-3.943， P<0.05）， triglyceride （Z=2.070， P<0.05）， and high-density lipoprotein cholesterol （t=3.867， P<0.05）. A total of 3 559 proteins were identified by DIA serum proteomics， among which 160 differentially expressed proteins were mainly enriched in pathways including metabolic disorders， immune inflammatory response， and extracellular matrix remodeling. The PPI network and the Hub node analysis showed that serpin family E member 1， MMP-9， and intercellular cell adhesion molecule-1 were core regulatory proteins. WGCNA showed that the MEyellow module was significantly positively correlated with MAFLD phenotype （cor=0.55， P<0.001）， which was functionally enriched in the efferocytosis pathway. MERTK （P<0.001） and MMP-9 （P<0.001） showed the most significant differential expression in this module. A rat model of MAFLD was successfully established using a high-fat diet， and compared with the control group， the model group had enlarged， yellowish， and greasy liver and disordered structure of hepatic lobules， hepatocyte fatty degeneration， and inflammatory cell infiltration based on HE staining， as well as massive lipid droplet deposition based on oil red O staining， while no obvious fibrous cords were observed in Masson staining. Compared with the control group， the model group had significant increases in the serum levels of aspartate aminotransferase （t=-5.148， P<0.05）， alanine aminotransferase （t=-5.663， P<0.05）， total bilirubin （t=-13.061， P<0.05）， triglyceride （Z=-2.722， P<0.05）， total cholesterol （t=3.588， P<0.05）， and tumor necrosis factor-α （t=-16.741， P<0.05）， as well as significant increases in the protein expression levels of MERTK （t=-11.448， P<0.001） and MMP-9 （t=-10.875， P<0.001） in liver tissue. Conclusion MMP-9 and MERTK are abnormally expressed in MAFLD and may be used as potential diagnostic biomarkers for this disease.
- Metabolic Dysfunction‑Associated Fatty Liver Disease,
- Proteomics,
- Protein-Tyrosine Kinases,
- Matrix Metalloproteinase 9

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References

[1]	Teng M L, Ng C H, Huang D Q, et al. Global incidence and prevalence of nonalcoholic fatty liver disease[J]. Clin Mol Hepatol, 2023, 29( Suppl): S32- S42. DOI: 10.3350/cmh.2022.0365.
[2]	Riazi K, Azhari H, Charette J H, et al. The prevalence and incidence of NAFLD worldwide: A systematic review and meta-analysis[J]. Lancet Gastroenterol Hepatol, 2022, 7( 9): 851- 861. DOI: 10.1016/S2468-1253(22)00165-0.
[3]	Chinese Society of Hepatology, Chinese Medical Association. Guidelines for the prevention and treatment of metabolic dysfunction-associated(non-alcoholic) fatty liver disease(version 2024)[J]. J Pract Hepatol, 2024, 27( 4): 494- 510. DOI: 10.3760/cma.j.cn501113-20240327-00163. 中华医学会肝病学分会. 代谢相关(非酒精性)脂肪性肝病防治指南(2024年版)[J]. 实用肝脏病杂志, 2024, 27( 4): 494- 510. DOI: 10.3760/cma.j.cn501113-20240327-00163.
[4]	Nan Y M, An J H, Bao J F, et al. The Chinese Society of Hepatology position statement on the redefinition of fatty liver disease[J]. J Hepatol, 2021, 75( 2): 454- 461. DOI: 10.1016/j.jhep.2021.05.003.
[5]	Febbraio M A, Reibe S, Shalapour S, et al. Preclinical models for studying NASH-driven HCC: How useful are they?[J]. Cell Metab, 2019, 29( 1): 18- 26. DOI: 10.1016/j.cmet.2018.10.012.
[6]	Stevanović J, Beleza J, Coxito P, et al. Physical exercise and liver“fitness”: Role of mitochondrial function and epigenetics-related mechanisms in non-alcoholic fatty liver disease[J]. Mol Metab, 2020, 32: 1- 14. DOI: 10.1016/j.molmet.2019.11.015.
[7]	Fröhlich K, Fahrner M, Brombacher E, et al. Data-independent acquisition: A milestone and prospect in clinical mass spectrometry–based proteomics[J]. Mol Cell Proteom, 2024, 23( 8): 100800. DOI: 10.1016/j.mcpro.2024.100800.
[8]	Lei N, Song H F, Zeng L, et al. Persistent lipid accumulation leads to persistent exacerbation of endoplasmic reticulum stress and inflammation in progressive NASH via the IRE1α/TRAF2 complex[J]. Molecules, 2023, 28( 7): 3185. DOI: 10.3390/molecules28073185.
[9]	Dan Lijuan, Song Hongfei, Li Xiuyan, et al. Mechanism of Sini Power combined with Linggui Zhugan Decoction on NAFLD based on transcriptomics and metabolomics[J]. China J Chin Materia Med, 2025, 50( 23): 6685- 6693. DOI: 10.19540/j.cnki.cjcmm.20250708.706. 淡丽娟, 宋虹霏, 李岫滟, 等. 基于转录组学与代谢组学探讨四逆散联合苓桂术甘汤对NAFLD的作用机制[J]. 中国中药杂志, 2025, 50( 23): 6685- 6693. DOI: 10.19540/j.cnki.cjcmm.20250708.706.
[10]	Guo Z W, Wu D J, Mao R H, et al. Global burden of MAFLD, MAFLD related cirrhosis and MASH related liver cancer from 1990 to 2021[J]. Sci Rep, 2025, 15( 1): 7083. DOI: 10.1038/s41598-025-91312-5.
[11]	Quek J, Chan K E, Wong Z Y, et al. Global prevalence of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in the overweight and obese population: A systematic review and meta-analysis[J]. Lancet Gastroenterol Hepatol, 2023, 8( 1): 20- 30. DOI: 10.1016/S2468-1253(22)00317-X.
[12]	Horn P, Tacke F. Metabolic reprogramming in liver fibrosis[J]. Cell Metab, 2024, 36( 7): 1439- 1455. DOI: 10.1016/j.cmet.2024.05.003.
[13]	Li Xiuyan, Lei Na, Song Hongfei, et al. The role of endoplasmic reticulum stress in non-alcoholic fatty liver disease and related targeted therapies[J]. J Clin Hepatol, 2024, 40( 11): 2300- 2305. DOI: 10.12449/JCH241126. 李岫滟, 雷娜, 宋虹霏, 等. 内质网应激在非酒精性脂肪性肝病中的作用及相关靶向治疗[J]. 临床肝胆病杂志, 2024, 40( 11): 2300- 2305. DOI: 10.12449/JCH241126.
[14]	Wang Dan, Zhang Jinxue, Su Lining, et al. Mechanism of action of mitochondrial dysfunction in metabolic associated fatty liver disease and the current status of research on traditional Chinese medicine[J]. J Clin Hepatol, 2025, 41( 12): 2615- 2621. DOI: 10.12449/JCH251224. 王丹, 张金雪, 苏李宁, 等. 线粒体功能紊乱在代谢相关脂肪性肝病中的作用机制及中医药干预研究现状[J]. 临床肝胆病杂志, 2025, 41( 12): 2615- 2621. DOI: 10.12449/JCH251224.
[15]	Fromenty B, Roden M. Mitochondrial alterations in fatty liver diseases[J]. J Hepatol, 2023, 78( 2): 415- 429. DOI: 10.1016/j.jhep.2022.09.020.
[16]	Xu S J, Lu F, Gao J H, et al. Inflammation-mediated metabolic regulation in adipose tissue[J]. Obes Rev, 2024, 25( 6): e13724. DOI: 10.1111/obr.13724.
[17]	Lou R H, Shui W Q. Acquisition and analysis of DIA-based proteomic data: A comprehensive survey in 2023[J]. Mol Cell Proteom, 2024, 23( 2): 100712. DOI: 10.1016/j.mcpro.2024.100712.
[18]	Tian Z L, He W X, Tang J N, et al. Identification of important modules and biomarkers in breast cancer based on WGCNA[J]. Onco Targets Ther, 2020, 13: 6805- 6817. DOI: 10.2147/OTT.S258439.
[19]	Poon I K H, Ravichandran K S. Targeting efferocytosis in inflammaging[J]. Annu Rev Pharmacol Toxicol, 2024, 64: 339- 357. DOI: 10.1146/annurev-pharmtox-032723-110507.
[20]	Shi H X, Moore M P, Wang X B, et al. Efferocytosis in liver disease[J]. JHEP Rep, 2024, 6( 1): 100960. DOI: 10.1016/j.jhepr.2023.100960.
[21]	Wang X C, He Q F, Zhou C L, et al. Prolonged hypernutrition impairs TREM2-dependent efferocytosis to license chronic liver inflammation and NASH development[J]. Immunity, 2023, 56( 1): 58- 77.e11. DOI: 10.1016/j.immuni.2022.11.013.
[22]	Lee Y S, Olefsky J. Eating macrophages for a healthy anti-NASH meal[J]. Immunity, 2023, 56( 1): 3- 5. DOI: 10.1016/j.immuni.2022.12.011.
[23]	Petta S, Valenti L, Marra F, et al. MERTK rs4374383 polymorphism affects the severity of fibrosis in non-alcoholic fatty liver disease[J]. J Hepatol, 2016, 64( 3): 682- 690. DOI: 10.1016/j.jhep.2015.10.016.
[24]	Cai B S, Dongiovanni P, Corey K E, et al. Macrophage MerTK promotes liver fibrosis in nonalcoholic steatohepatitis[J]. Cell Metab, 2020, 31( 2): 406- 421. e 7. DOI: 10.1016/j.cmet.2019.11.013.
[25]	Bartneck M, Warzecha K T, Tacke F. Therapeutic targeting of liver inflammation and fibrosis by nanomedicine[J]. Hepatobiliary Surg Nutr, 2014, 3( 6): 364- 376. DOI: 10.3978/j.issn.2304-3881.2014.11.02.
[26]	Giannitrapani L, Soresi M, Bondì M L, et al. Nanotechnology applications for the therapy of liver fibrosis[J]. World J Gastroenterol, 2014, 20( 23): 7242- 7251. DOI: 10.3748/wjg.v20.i23.7242.
[27]	Verma R P, Hansch C. Matrix metalloproteinases(MMPs): Chemical-biological functions and(Q)SARs[J]. Bioorg Med Chem, 2007, 15( 6): 2223- 2268. DOI: 10.1016/j.bmc.2007.01.011.
[28]	Kurbatova I V, Topchieva L V, Dudanova O P, et al. Role of MMP-2 and MMP-9 in the relationship between inflammation, fibrosis, and apoptosis during progression of non-alcoholic fatty liver disease and diagnostic significance of plasma levels of their active forms[J]. Biochem Mosc, 2024, 89( 11): 1998- 2022. DOI: 10.1134/S0006297924110130.
[29]	Livzan M A, Akhmedov V A, Krolevets T S, et al. The informative value of non-invasive liver fibrosis markers in patients with nonalcoholic fatty liver disease[J]. Ter Arkh, 2016, 88( 12): 62- 68. DOI: 10.17116/terarkh2016881262-68.
[30]	Goyale A, Jain A, Smith C, et al. Assessment of non-alcoholic fatty liver disease(NAFLD) severity with novel serum-based markers: A pilot study[J]. PLoS One, 2021, 16( 11): e0260313. DOI: 10.1371/journal.pone.0260313.

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Screening and validation of diagnostic biomarkers in metabolic dysfunction-associated fatty liver disease based on data-independent acquisition quantitative proteomics

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Screening and validation of diagnostic biomarkers in metabolic dysfunction-associated fatty liver disease based on data-independent acquisition quantitative proteomics

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