Modulation of HMGB1-mediated neuroinflammation and synaptic plasticity by ethyl pyruvate reduces methamphetamine reward and motivation in male rats

• Ethyl pyruvate reduced methamphetamine reinforcement and motivation without affecting natural reward. • It attenuated methamphetamine-induced neuroinflammation, coinciding with reduced HMGB1 translocation and neuronal TLR4 expression. • Ethyl pyruvate decreased neuronal apoptosis and pyroptosis, restored calcium homeostasis, and normalized pathological synaptic plasticity. • Local injections of ethyl pyruvate into the infralimbic cortex reproduced these systemic effects. Methamphetamine (METH) addiction involves neuroinflammatory cascades and maladaptive synaptic plasticity in reward circuits. While high-mobility group box 1 (HMGB1) and its downstream neuroinflammatory pathways contribute to neuropsychiatric disorders, their therapeutic potential in METH addiction remains unexplored. This study investigated whether ethyl pyruvate (EP, an HMGB1 inhibitor) attenuates METH reinforcement and associated neurotoxicity in the rat infralimbic cortex (IL). Male rats self-administering METH received systemic EP (0, 50, or 80 mg/kg, i.p.). Reinforcement, motivation, and reinforcing efficacy were assessed using fixed-ratio 1 (FR1), progressive ratio (PR), and dose–response paradigms. Western blotting quantified HMGB1-TLR4/MyD88/PI3K/Akt/NF-κB signaling, pro-inflammatory cytokines (IL-6, IL-1β, TNF-α), and synaptic proteins in the IL. Neuronal TLR4 and HMGB1 expression, apoptosis (TUNEL, cleaved caspase-3), and pyroptosis (cleaved caspase-1) were assessed by immunofluorescence staining. In vivo calcium dynamics in IL CaMKIIα + neurons were recorded via fiber photometry, and synaptic transmission was assessed via patch-clamp recordings of spontaneous excitatory postsynaptic currents (sEPSCs). Region-specific effects were confirmed by local microinjections of EP. Systemic EP treatment reduced METH intake and motivation and downshifted the dose–response curve without affecting natural reward. EP reversed METH-induced HMGB1 nuclear-to-cytoplasmic translocation and neuronal TLR4 expression, and suppressed downstream neuroinflammatory signaling and cytokine release. Furthermore, EP also reduced METH-associated neuronal apoptosis and pyroptosis in the IL. It also restored calcium homeostasis in IL CaMKIIα + neurons, normalized expression of key synaptic proteins (e.g., NR2A/B, GluA2, PSD95, SNAP25), and reversed METH-induced hyperexcitability (increased sEPSC frequency/amplitude) in IL pyramidal neurons. Critically, local IL microinjection of EP replicated these systemic effects, confirming the IL as a key site of action. The HMGB1 inhibitor EP attenuated METH reinforcement and motivation by targeting the IL to disrupt a neuroinflammatory cascade, prevent apoptosis and pyroptosis, and restore synaptic homeostasis. These findings identify HMGB1 inhibitors as a novel therapeutic target for mitigating the neuropathology and motivational drive underlying METH addiction.