Central post-stroke pain (CPSP) is a chronic neuropathic condition driven by central sensitization, often resulting in poor clinical outcomes. Neural circuits play a critical role in modulating chronic pain. To elucidate the mechanisms underlying CPSP, we established a mouse model via intracranial injection of type IV collagenase. cFos immunofluorescence and in vivo calcium imaging identified pain-associated activated nuclei. Using viral tracing, optogenetics, chemogenetics, and behavioral assays, we mapped a neural circuit comprising the ventral posterolateral thalamic nucleus (VPL), the hindlimb primary somatosensory cortex (S1HL), and the anterior cingulate cortex (ACC). In CPSP mice, ipsilateral S1HL^CaMKIIα and ACC^CaMKIIα neurons exhibited robust activation. Chemogenetic manipulation further demonstrated that activation of these neurons induced pain behaviors, whereas their inhibition alleviated pain. Notably, specific activation of the S1HL^CaMKIIα-ACC^CaMKIIα circuit produced mechanical allodynia, and optogenetic stimulation of VPL^CaMKIIα projections to S1HL similarly evoked pain responses while enhancing ACC neuronal firing. These findings underscore the critical role of the VPL-S1HL-ACC circuit in pain abnormalities and provide novel insights into the central sensitization underlying CPSP, suggesting promising therapeutic strategies for its management.

Chemotherapy-induced neuropathic pain poses significant clinical challenges and severely impacts patient quality of life. Sodium ion channels are crucial in regulating neuronal excitability and pain. Our research indicates that the microRNA-30b (miR-30b) in rat dorsal root ganglia (DRG) contributes to chemotherapy-induced neuropathic pain by regulating the Nav1.6 protein. Additionally, ten-eleven translocation methylcytosine dioxygenase 1 (TET1) plays a crucial role in pain generation by altering gene expression. We established a chemotherapy-induced neuropathy model using intraperitoneal oxaliplatin (OXA) injections and measured TET1 and Nav1.6 protein in the DRG. Using lentivirus and Tet1^flox/flox mice, we modulated TET1 expression and assessed pain behaviors, DRG neuronal excitability, Nav1.6 currents, miR-30b-5p, and demethylation of the Mir30b promoter region. We employed chromatin immunoprecipitation to pinpoint TET1-binding sites on the Mir30b promoter. The impacts of miR-30b agomir or antagomir on Nav1.6 expression and pain responses were assessed postintrathecal injections. The results showed that OXA reduced TET1, increasing neuronal excitability, Nav1.6 currents, and miR-30b-5p in the DRG. TET1 knockdown exacerbated these effects and induced pain behaviors. Conversely, TET1 overexpression reversed these effects. TET1 also targeted and enhanced demethylation at the Mir30b promoter (-1103 bp to -1079 bp). miR-30b agomir reduces Nav1.6, whereas miR-30b antagomir reverses TET1's effects on Nav1.6 and pain. In OXA-induced neuropathy, decreased TET1 reduces miR-30b, elevating Nav1.6 expression and currents and contributing to pain. We hypothesize that TET1 mediates this process by regulating the demethylation of the Mir30b promoter.

Central post-stroke pain (CPSP) is a debilitating neuropathic condition that significantly impairs quality of life and is challenging to manage. Salvia miltiorrhiza (Danshen), a traditional Chinese herb, has demonstrated efficacy against neuropathic pain, but its active analgesic components and underlying mechanisms remain unclear. This study investigated the therapeutic potential of Danshenol B, an active component of Danshen, in a CPSP mouse model, focusing on its mechanism of action via the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PIK3CG)/NLR family pyrin domain-containing protein 3 (NLRP3) signaling pathway. A CPSP model was established in mice, and RNA sequencing of the ipsilateral ventral posterolateral thalamic nucleus/ventral posteromedial thalamic nucleus (VPL/VPM) was performed to identify differentially expressed genes (DEGs). Network pharmacology analysis linked these DEGs to known Danshen components, and molecular docking of 42 Danshen constituents was conducted to identify strong ligand-target interactions. Danshenol B was then administered (5, 10, 50 mg/kg) to CPSP mice to evaluate its analgesic effects, and thalamic PIK3CG and NLRP3 protein levels were measured to assess pathway involvement. Additionally, PIK3CG and NLRP3 expression were manipulated (via overexpression or knockdown) to determine their roles in CPSP and their regulatory relationship. In the CPSP model, 409 DEGs were identified in the ipsilateral VPL/VPM. Network pharmacology revealed 21 Danshen-derived compounds potentially targeting 11 of these DEGs. Molecular docking highlighted Danshenol B as a top candidate, showing strong binding (-9.127 kcal/mol) to PIK3CG. In CPSP mice, Danshenol B (50 mg/kg) significantly alleviated CPSP and suppressed the PIK3CG/NLRP3 pathway. The overexpression of PIK3CG increased NLRP3 and negated the effects of Danshenol B, whereas its knockdown alleviated CPSP and reduced NLRP3. Notably, simultaneous overexpression of NLRP3 attenuated the analgesic effects induced by PIK3CG knockdown, further confirming that NLRP3 functions downstream of PIK3CG in mediating CPSP. Danshenol B alleviates CPSP in mice by suppressing the PIK3CG/NLRP3 signaling pathway, elucidating its analgesic mechanism and highlighting its potential as a novel therapeutic candidate for CPSP.