C23

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia, inflammatory cell infiltration, and pathological angiogenesis, ultimately leading to progressive joint damage. Cold-inducible RNA-binding protein (CIRP) has been implicated in inflammatory responses; however, its role in RA-associated angiogenesis remains unclear. In this study, we demonstrate that CIRP gene knockout or pharmacological inhibition using C23 significantly attenuates arthritis severity and reduces synovial angiogenesis in adjuvant-induced arthritis models. In vitro experiments revealed that CIRP promotes human umbilical vein endothelial cell (HUVEC) tube formation, migration, and invasion by upregulating the expression of C-X-C motif chemokine receptor 4 (CXCR4) and Delta-like ligand 4 (DLL4). Notably, blocking extracellular CIRP with C23 inhibited CIRP-induced angiogenesis in HUVECs. Further investigations showed that Toll-like receptor 4 (TLR4) knockdown suppressed CIRP-induced angiogenesis in HUVECs, indicating that CIRP promotes angiogenesis through a TLR4-dependent mechanism. Subsequent mechanistic studies revealed that CIRP upregulates histone deacetylase 1 (HDAC1) expression via TLR4 in HUVECs. We therefore investigated the role of HDAC1 in CIRP-induced angiogenesis, and found that HDAC1 knockdown significantly inhibited this process. Moreover, CIRP promotes pyruvate kinase M2 (PKM2) nuclear translocation and suppresses PKM2 lactylation through the TLR4-mediated HDAC1 pathway. Furthermore, The PKM2 lysine 62 to arginine (K62R) mutant enhances PKM2 nuclear translocation and promotes angiogenesis. Taken together, these results suggest that HDAC1-mediated PKM2 delactylation modification regulates CIRP-induced angiogenesis in RA.

Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) in patients with cardiogenic shock (CS) inevitably induces systemic ischemia-reperfusion injury, which is accompanied by elevated levels of extracellular cold-inducible RNA-binding protein (CIRP) and an aggravated inflammatory response. However, the specific role of CIRP during VA-ECMO support remains unclear.

CS patients undergoing VA-ECMO were enrolled, and serum CIRP expression levels were measured. A myocardial infarction-induced CS rat model with VA-ECMO was established. Myocardial tissue and serum were analyzed by ELISA, immunohistochemistry, and Western blotting. Cardiac function was assessed via echocardiography. CIRP-knockout rats and the CIRP antagonist C23 were used to investigate CIRP's role, and H9c2 cells were utilized to investigate the effects of recombinant CIRP in vitro.

In VA-ECMO patients, serum CIRP levels were significantly higher in non- survivors compared to survivors. Similarly, in the VA-ECMO-assisted CS rat model,rats with high CIRP expression exhibited greater myocardial injury. Administration of exogenous CIRP significantly exacerbated myocardial injury during VA-ECMO. In contrast, genetic deletion of CIRP attenuated both myocardial injury and inflammation during VA-ECMO. Furthermore, CIRP was associated with activation of the NLRP3 inflammasome and aggravated cardiomyocyte injury. In vivo, treatment with the C23 significantly alleviated myocardial injury and inflammation under VA-ECMO.

In patients with CS supported by VA-ECMO, elevated serum CIRP levels were significantly associated with poor survival outcomes. CIRP aggravated myocardial injury and inflammation, accompanied by NLRP3 activation.Pharmacological inhibition of CIRP mitigated myocardial damage during VA-ECMO, suggesting its potential as a therapeutic target for attenuating cardiac injury in CS patients.