Molecular and Immunohistochemical Analyses of PLIN4 in the Spinal Cord and Brain During the Development of Electroacupuncture-Induced Analgesic Tolerance in Mice.

BACKGROUND: Electroacupuncture (EA) is an important non-pharmacological approach for pain management in veterinary and human medicine. However, electroacupuncture-induced analgesic tolerance (EAT) limits its long-term efficacy, and the underlying mechanisms remain unclear. Perilipin 4 (Plin4) is a lipid droplet-associated protein involved in lipid storage, lipid droplet dynamics, and cellular lipid homeostasis. Emerging evidence suggests that it also plays a role in neuronal lipid metabolic changes and pain modulation. OBJECTIVES: To investigate the role of Plin4 in the development of EAT and explore its potential as a molecular target for attenuating this limitation. METHODS: EAT was induced in male mice by daily 100 Hz EA stimulation for 7 consecutive days. Plin4 expression levels in the amygdala, periaqueductal grey, and spinal cord were examined, and immunofluorescence was used to determine cellular localization. The functional role of Plin4 was assessed by intrathecal Plin4 knockdown in mice and by Plin4 overexpression or knockdown in N2a cells. A total of 99 male mice were used in the primary experiments, with an additional 24 mice used for siRNA knockdown validation in the Supporting Information. RESULTS: Repeated EA induced analgesic tolerance and markedly upregulated Plin4 in the spinal cord, amygdala, and periaqueductal grey. In the spinal cord, increased Plin4 was mainly localized to spinal cord dorsal horn neurons. Intrathecal Plin4 knockdown delayed EAT onset and increased the pain threshold. In N2a cells, Plin4 expression was positively associated with lipid droplet formation. CONCLUSIONS: Spinal neuronal Plin4 contributes to the development of EA-induced analgesic tolerance, possibly by altering neuronal lipid homeostasis. Plin4 may represent a potential molecular target for attenuating tolerance, prolonging EA-induced analgesia, and optimizing EA-based pain management strategies in veterinary medicine.