Benpyrine racemate

Benpyrine directly binds to TNF-α and blocks TNF-α-trigged signaling activation.^[1]

RAW 264.7 cells were grown to 50−70% confluence in a glass chamber, pretreated with DMSO or benpyrine for 12 h, and then stimulated with 10 ng/mL TNF-α for 2 h. Cell treatments were terminated by washing the cells with PBS, followed by fixation with freshly prepared 4% paraformaldehyde in PBS for 20 min. The fixed cells were washed three times with PBS and then permeabilized with 0.25% Triton X-100 in PBS for 10 min. After blocking with 2 mg/mL BSA for 1 h at room temperature, we added the NF-κB/p65 antibody at a 1:1000 dilution at 4 °C overnight, washed three times with PBS, then incubated with a 1:500 dilution of the secondary antibody for 1 h at room temperature in the dark. Finally, 4′,6-diamidino-2-phenyl indole (DAPI) was used to stain the nuclei at 37 °C for 30 min in the dark.

Benpyrine attenuates TNF-α-induced inflammation in endotoxemic murine model, thereby reducing liver and lung injury, and significantly reliefs the symptoms of collagen-induced arthritis and imiquimod-induced psoriasiform inflammation in mice.^[1]