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Disulfide-Bond Proteomics Reveals Functional Reprogramming of NR4A2 after Myocardial Infarction

WEI Dongsheng^1,2, LI Han^1,2, LIU Xiaosheng^1,2, CAO Huimin^1,2, MA Yixin^1,2, LIU Yuting^1,2, QI Jiajie^1,2, ZHANG Zhe^1,2,3*

(¹First Clinical College, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China; ²Key Laboratory of Viscera-State Theory and Applications of Traditional Chinese Medicine, Ministry of Education, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China; ³Key Research Laboratory of Phlegm-Blood Stasis Syndrome in Vascular Diseases, National Administration of Traditional Chinese Medicine, Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China)

Abstract:

MI (myocardial infarction) is characterized by enhanced oxidative stress and inflammatory re sponses in cardiomyocytes, while the underlying molecular mechanisms remain incompletely understood. This study aimed to investigate the dynamic changes of protein disulfide-bond modifications after MI based on disulfide bond proteomics and to explore the functional reprogramming of the transcription factor NR4A2. A rat MI model was established by left anterior descending coronary artery ligation, and myocardial tissues were collected at 3 and 5 days post-surgery. HE (hematoxylin-eosin) staining was performed to evaluate myocardial injury. DSBPs (dif ferentially modified disulfide-bond proteins) were identified by disulfide-bond proteomics followed by GO, KEGG, and PPI analyses. NR4A2 disulfide modification was examined by immunoprecipitation and non-reducing/reduc ing SDS-PAGE. In vitro, an OGD (oxygen-glucose deprivation) model was constructed, and CCK8 assay was used to determine the safe concentration of 4-PBA. ER stress-related proteins and NR4A2 disulfide configuration were analyzed, along with the mRNA and protein expression of inflammatory markers IL-10, NF-κBIA, CXCL10, IL-1β, and IL-6. HE staining showed that myocardial structure was markedly damaged after MI, with scattered inflamma tory infiltration at day 3 and extensive necrosis and inflammatory cell aggregation at day 5. Disulfide-bond pro teomics revealed significant alterations in the myocardial disulfide modification profile after MI. NR4A2 exhibited increased disulfide modification and expression at day 3, forming complexes with RXR and PPAR, while both the modification level and interaction weakened at day 5. SDS-PAGE confirmed the reversibility of NR4A2 disulfide modification. In the OGD model, NR4A2 disulfide bonding was impaired and accompanied by enhanced ER stress. Treatment with 4-PBA effectively alleviated ER stress, restored NR4A2 disulfide configuration, and reduced ROS levels. Inflammatory analysis showed that OGD downregulated IL-10 and NF-κBIA while upregulating CXCL10, IL-1β, and IL-6, which were partially reversed by 4-PBA intervention. This study reveals that NR4A2 undergoes disulfide bond-dependent structural and functional reprogramming during the early stage of MI, in which ER stress plays a key regulatory role. The dynamic changes in NR4A2 disulfide modification may affect myocardial injury through inflammatory transcriptional networks, providing a potential therapeutic target for post-MI treatment.

CSTR: 32200.14.cjcb.2026.02.0008