TRIM21 alleviates sepsis-associated coagulopathy by activating BRD4-mediated autophagy in endothelial cells

To explore the role and mechanism of tripartite-motif protein 21 (TRIM21) in regulating autophagy in sepsis-associated coagulopathy (SAC).

The overexpression vectors of TRIM21 and bromodomain-containing protein 4 (BRD4), si-BRD4, and empty vector were constructed and transfected into human microvascular endothelial cells (HMEC-1). For the in vitro experiments, a sepsis model was established by treating the cells with lipopolysaccharide (LPS) (40 μg/mL) and Nigericin (20 μM) for 0.5 h. The levels of inflammatory and coagulation factors in cells were detected by enzyme-linked immunosorbent assay (ELISA). The intracellular reactive oxygen species (ROS) levels were measured, and cell apoptosis was assessed by flow cytometry. The effect of TRIM21 on BRD4 ubiquitination was verified by co-immunoprecipitation (CO-IP). For the in vivo experiments, a sepsis model was established in mice using the cecal ligation and puncture (CLP) method. At 24 h post-surgery, levels of inflammatory and coagulation factors in lung tissues were detected by ELISA. The pathological changes in lung tissues were observed by hematoxylin and eosin (HE) staining, and apoptosis in lung tissues was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of fibrin in lung tissues was detected by immunohistochemistry (IHC). The expression levels of TRIM21, BRD4, autophagy-related proteins (light chain 3-II (LC3-II), Beclin1, p62), E-cadherin, and Zonulin-1 in cells and lung tissues were examined by Western blot.

LPS and Nigericin were selected at concentrations of 40 μg/mL and 20 μM, respectively. Levels of inflammatory markers (interleukin (IL)-1β, IL-6) and coagulation-related factors (tissue factor (TF), plasminogen activator inhibitor-1 (PAI1), Thrombin) were significantly elevated in LPS and Nigericin stimulated HMEC-1 cells, accompanied by increased ROS levels and apoptotic cells, downregulated LC3-II, Beclin1, E-cadherin, and TRIM21, and upregulated P62, Zonulin-1, and BRD4, which were markedly reversed by the overexpression of TRIM21 or the silence of BRD4. However, when TRIM21 and BRD4 were co-overexpressed, phenotypes and protein expressions were restored to levels observed in the model group. CO-IP results indicated that TRIM21 could promote the ubiquitination of BRD4. In animal experiments, alveolar wall disruption and dissolution, with the presence of inflammatory cells, were observed in the Model group, along with enhanced pulmonary levels of inflammatory factors (IL-1β, IL-6) and coagulation factors (PAI1, Thrombin, TF), upregulated P62 and Zonulin-1, and downregulated E-cadherin, LC3-II, and Beclin1. The number of Fibrin-positive cells was significantly increased, and both apoptosis and LC3-II expression were significantly elevated. These results were markedly reversed by the overexpression of TRIM21.

TRIM21 was found to exert therapeutic effects on SAC by regulating BRD4 to activate autophagy and inhibit coagulation reactions. TRIM21 may serve as a potential therapeutic target for the treatment of SAC.