Pinocembrin Inhibits P2X4 Receptor–Mediated Pyroptosis in Hippocampus to Alleviate the Behaviours of Chronic Pain and Depression Comorbidity in Rats

Neuroinflammation is critical to the comorbidity of chronic pain and depression. Pyroptosis is an inflammatory cell death that is different from apoptosis. Activation of the P2X4 receptor leads to inflammation and is involved in chronic pain and depression. Pinocembrin (5,7-dihydroxyflavanone) is a natural flavonoid compound with anti-inflammatory, antioxidant and neuroprotective effects. In this study, an animal model of chronic pain and depression comorbidity was used to explore the therapeutic effect of pinocembrin in P2X4-mediated pyroptosis. The results showed that nociceptive behaviours and depression-like behaviours were obvious in the model rats induced by chronic constrictive injury (CCI) and chronic unpredictable mild stimulus (CUMS). In the model rats, the mRNA and protein levels of the P2X4 receptor in the hippocampus were increased, and the coexpression of P2X4 and the astrocyte marker glial fibrillary acidic protein (GFAP) in the hippocampus was increased. The protein content of connexion 43 (Cx43), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 was increased. The serum content of IL-1β and the mRNA and protein expression of IL-1β were increased. The protein content of p-P38MAPK was increased. After treatment with pinocembrin in the model rats, these behavioural changes were improved, and the mRNA and protein levels of the above indicators were decreased. The results of molecular docking confirmed that the affinity of pinocembrin and the P2X4 receptor was − 7.8 (kcal/mol). At the same time, pinocembrin inhibited the ATP release and Ca2+ signal release in primary astrocytes and ATP-activated current of HEK293 cells transfected with the pcDNA3.0-EGFP-hP2X4 plasmid. Therefore, pinocembrin relieved nociceptive and depression-like behaviours in rats with chronic pain and depression comorbidity by inhibiting P2X4 receptor–mediated pyroptosis in the hippocampus.Graphical abstractThe mechanism of pinocembrin in treating rats with chronic pain and depression comorbidity. GJ stands for gap junction, and Cx43 is mainly expressed in astrocytes.