C25-140

Dehydrocorydaline Protects Against Sepsis-Induced Myocardial Injury Through Modulating the TRAF6/NF-κB Pathway

We aim to investigate the effect and mechanism of dehydrocorydaline (Deh), an alkaloidal component isolated from Rhizoma corydalis, in the treatment of sepsis-mediated myocardial injury. Lipopolysaccharide (LPS) was taken to construct an in-vitro sepsis-myocardial injury models H9C2 cardiomyocytes. The in-vivo model of sepsis in C57BL/6 mice was induced by intraperitoneal injection of Escherichia coli (E. coli). The in-vitro and in-vivo models were treated with Deh in different concentrations, respectively. Hematoxylin-eosin (HE) staining, Masson staining, and immunohistochemistry (IHC) staining were taken to evaluate the histopathological changes of the heart. ELISA was applied to evaluate the levels of inflammatory factors, including IL-6, IL-1β, TNFα, IFNγ, and oxidized factors SOD, GSH-PX in the plasma or culture medium. Western blot was used to measure the expressions of Bax, Bcl2, Caspase3, iNOS, Nrf2, HO-1, TRAF6, NF-κB in heart tissues and cells. The viability of H9C2 cardiomyocytes was detected by the CCK8 method and BrdU assay. The ROS level in the H9C2 cardiomyocytes were determined using immunofluorescence. As a result, Deh treatment improved the survival of sepsis mice, reduced TUNEL-labeled apoptosis of cardiomyocytes. In vitro, Deh enhanced the viability of LPS-induced H9C2 cardiomyocytes and inhibited cell apoptosis. Additionally, Deh showed significant anti-inflammatory and anti-oxidative stress functions via decreasing IL-1β, IL-6, TNFα, and IFNγ levels, mitigating ROS level, up-regulating Nrf2/HO-1, SOD, and GSH-PX expressions dose-dependently. Mechanistically, Deh inhibited TRAF6 expression and the phosphorylation of NF-κB p65. The intervention with a specific inhibitor of TRAF6 (C25-140) or NF-κB inhibitor (BAY 11-7082) markedly repressed the protective effects mediated by Deh. In conclusion, Deh restrains sepsis-induced cardiomyocyte injury by inhibiting the TRAF6/NF-κB pathway.

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

Constitutive NF-κB signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-κB pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys⁶³-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6-Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NF-κB activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6-Ubc13 interaction and thereby counteract NF-κB signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6-Ubc13 interaction that reduces TRAF6-Ubc13 activity both in vitro and in cells. We found that this compound, C25-140, impedes NF-κB activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical in vivo mouse models. Hence, the first-in-class TRAF6-Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases.

TLR4/TRAF6/NOX2 signaling pathway is involved in ventilation-induced lung injury via endoplasmic reticulum stress in murine model

In ventilation-induced lung injury (VILI), prolonged nonpathogen-mediated inflammation is triggered as a result of alveolar hyperinflation. In our previous study, we suggested that endoplasmic reticulum (ER) stress-mediated inflammation was involved in VILI, but how ER stress is triggered remains unknown. Toll-like receptor 4 (TLR4) activation plays an important role in mechanical ventilation (MV)-induced lung inflammation, however, it is unknown whether ER stress is activated by TLR4 to participate in VILI. In this study, C57BL/6 mice were exposed to MV with high tidal volumes (HTV 20 ml/kg). Mice were pretreated with TAK-242 the TLR4 inhibitor, C25-140, the TRAF6 inhibitor, or GSK2795039, the NOX2 inhibitor. Lung tissue and bronchoalveolar lavage fluid (BALF) were collected to measure lung injury, inflammatory responses and mRNA/protein expression associated with ER stress and the TLR4/TRAF6/NOX2 signaling pathway. Our results indicate that MV with HTV caused the TLR4/TRAF6/NOX2 signaling pathway activation and production of large amounts of ROS, which led to ER stress and NF-κB mediated inflammation in VILI. Furthermore, TLR4/TRAF6/NOX2 signaling pathway inhibition attenuated ER stress response and alleviate lung injury in mice.