Berbamine alleviates chronic inflammatory pain via microglial inhibition and oxidative stress reduction in the anterior cingulate cortex

Chronic pain, defined as persistent pain lasting beyond three months, imposes a severe burden on patient quality of life. Berbamine (BBM), a natural bisbenzylisoquinoline alkaloid with anti-inflammatory and antioxidant properties, is a promising candidate for pain management, although its analgesic mechanism remains unclear. This study investigated the efficacy and mechanism of BBM in a complete Freund's adjuvant-induced mouse model of chronic inflammatory pain. Behavioral testing revealed that BBM significantly increased mechanical and thermal pain thresholds, indicating its effective antinociceptive action. Histological and biochemical analyses showed that BBM inhibited microglial activation in the anterior cingulate cortex (ACC), reduced proinflammatory cytokines, activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, increased the expression of the Nrf2-driven antioxidant proteins heme oxygenase-1 (HO-1) and glutathione peroxidase 4 (GPX4), decreased malondialdehyde (MDA) levels, and enhanced superoxide dismutase (SOD) activity, indicating attenuation of oxidative stress. In lipopolysaccharide (LPS)-stimulated BV2 microglial cells, BBM similarly reduced inflammatory cytokine production, reactive oxygen species, and mitochondrial superoxide while preserving mitochondrial membrane potential and restoring redox balance. Consistent with the in vivo findings, BBM activated Nrf2, thereby promoting the mRNA expression of downstream antioxidants, such as NAD(P)H quinone oxidoreductase 1 (NQO1), HO-1, and SOD. At the protein level, BBM simultaneously upregulated HO-1 and GPX4, suppressed the phosphorylation of nuclear factor kappa-B (NF-κB) p65, and decreased Kelch-like ECH-associated protein 1 (KEAP1) abundance to sustain Nrf2 activation. Collectively, our findings reveal that BBM alleviates chronic inflammatory pain via dual anti-inflammatory and antioxidant effects: it suppresses ACC microglial overactivation to block NF-κB-mediated neuroinflammation, while reducing KEAP1 levels to sustain Nrf2 signaling, thereby increasing antioxidant proteins HO-1, GPX4 and NQO1 transcript levels to mitigate central oxidative stress.