3-carbamoyl proxyl nitroxide attenuates CCl4-induced liver fibrosis in mice through antioxidant-inflammatory regulation of TLR4/NF-κB signaling pathway

Abstract

Abstract Liver fibrosis is a dynamic pathological consequence of chronic liver injury, in which persistent oxidative stress and inflammation drive progressive extracellular matrix deposition. Cyclic nitroxide radicals exhibit diverse biological activities, but their effects on liver fibrosis remain unclear. This study systematically evaluates the therapeutic potential of 3-carbamoyl proxyl nitroxide (3-CP) against carbon tetrachloride (CCl₄)-induced liver fibrosis. In vitro, 3-CP inhibited hepatic stellate cell (HSC) activation, migration, and proliferation, and reduced α-smooth muscle actin (α-SMA) and collagen I (COL1) expression. In a BALB/c mouse model of CCl4-induced liver fibrosis, 20 and 40 mg/kg 3-CP reduced the fibrosis area from 13.6 ± 1.0% (model group) to 6.9 ± 0.9% and 5.7 ± 1.3%, respectively, accompanied by decreased serum transaminase levels, restored liver architecture, and diminished collagen deposition. Mechanistic studies revealed that 3-CP modulated the TLR4/NF-κB signaling pathway, downregulating phosphorylated NF-κB p65 (p-p65) and reducing hepatic mRNA levels of pro-inflammatory (IL-1β, IL-6, TNF-α) and pro-fibrotic (TGF-β) cytokines by approximately 35–55%. Supportive in silico analysis suggested potential interactions between 3-CP and key pathway proteins (TLR4, MyD88, IKKβ, p65, IκBα). These findings indicate that 3-CP represents a promising therapeutic candidate that concurrently addresses oxidative damage and inflammatory signaling during liver fibrogenesis.