| Purpose: The pathogenesis of neuropathic pain and the reasons for its persistence are unclear.Studies have shown that estrogen plays a role in sex differences in pain sensitivity,but few studies have focused on estrogen receptors.Estrogen receptors are widely distributed in the nervous system and may be important molecules that contribute to pain transduction.We used a rat sciatic nerve injury model(SNI)to simulate chronic pain and investigated the effects of estrogen receptors on nerve pain and neuroinflammatory reactions in the spine dorsal horn and dorsal root ganglion(SDH and DRG).Methods: The expression and distribution of three estrogen receptors in DRG and SDH were detected by immunofluorescence technique.SNI models in normal male rats prepared,intrathecal(i.t.)injections of estrogen receptor-specific agonists and antagonists,G protein-coupled oestrogen receptor(GPER)agonist G1(5 m M)and blocker G15(1m M),and estrogen receptor-α(ERα)agonist PPT(1 m M),were administered to normal male rats.agonist PPT(1 m M)and blocker MPP(1 m M),estrogen receptor-β(ERβ)agonist DPN(5 m M)and blocker PHTPP(1 m M),to identify receptors primarily involved in neuropathic pain based on altered behavior in rats.In addition,we used transcriptomic enrichment analysis to identify relevant metabolic pathways with significantly altered gene enrichment,combined metabolomics to identify key metabolic targets,and elucidated potential mechanisms mediating persistent neuronal sensitization and neuroinflammatory responses in neuropathic pain.Primary culture extracts of microglia and DRG neuronal cells.Patch clamp technique was used to detect excitability of DRG neurons.We used si RNA transfection to silence GPER receptor protein expression in cells,behavioral assays to detect changes in pain threshold.Immunofluorescence,Western blotting and RT-PCR techniques were used to assess the expression of major estrogen receptors and their related signaling molecules and estrogen receptor distribution and function of estrogen receptors in DRG tissue cells.Results:(1)Three estrogen receptors were expressed in small and medium-sized neurons and SDH pain-related regions of DRG.GPER and ERα were significantly increased in the SNI model,but not in SDH.intrathecal injection of GPER-specific agonist G1 induced cold pain and mechanical nociceptive sensitization in normal rats.G15 attenuated cold and mechanical nociceptive sensitization in SNI rats.(2)GPER mainly expressed on neuronal cells and microglia in DRG,and GPER was involved in neuropathic pain by regulating GABAAα2.(3)Transcriptomics revealed that GPER regulated neuropathic pain through metabolic pathways while regulating neuroinflammation.Metabolomic assays showed that GPER induced β-alanine upregulation.(4)Intrathecal injection of β-alanine in normal rats caused GPER increasing and SNI-like symptoms.β-alanine and GPER could bind to interact positively,triggering neuronal excitability and neuroinflammation similar to that of SNI.Inhibition of GPER expression in DRG downregulated i NOS,IL-1β and IL-6 expression,restored GABAAα2 expression,and attenuated SNI-induced nociceptive hypersensitivity and neuroinflammation.Conclusion: GPER expression was increased in neurons and microglia of DRG and regulates GABAAα2 triggering neuropathic pain.GPER regulated β-alanine metabolism and causes β-alanine accumulation.β-alanine binded to GPER generating positive interactions,promoting the secretion of inflammatory factors i NOS,IL-1β and IL-6,producing neuroinflammation and promoting chronic development of pain.Blocking GPER and eliminating β-alanine from DRG neurons and microglia prevented the development of neuropathic pain. |
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