EZH2-mediated suppression of TIMP1 in spinal GABAergic interneurons drives microglial activation via MMP-9-TLR2/4-NLRP3 signaling in neuropathic pain

Effective management of neuropathic pain remains a significant challenge due to the limited understanding of its underlying mechanisms. We found that the FDA-approved enhancer of zeste homolog 2 (EZH2) inhibitor, EPZ6438, can prevent the development of neuropathic pain caused by chronic constriction injury (CCI). Therefore, we utilized EPZ6438 as a probe to investigate the molecular events involved in the early stage of neuropathic pain. RNA-seq analysis reveals that EPZ6438 significantly upregulates Timp1 transcription in the spinal cord of mice. As a specific endogenous inhibitor of MMP-9, tissue inhibitor of metalloproteinase 1 (TIMP1) levels significantly decrease in the cerebrospinal fluid of both neuropathic pain patients and the CCI rat models. Importantly, intrathecal administration of mouse recombinant TIMP1 protein (rmTIMP1) reverses CCI-induced mechanical and thermal hyperalgesia. Mechanistically, substance P released from primary sensory neurons suppresses TIMP1 in spinal GABAergic interneurons by elevating EZH2 expression, which enhances H3K27me3 enrichment at the Timp1 promoter. Blocking spinal NK1R effectively prevents the downregulation of TIMP1 and alleviates CCI-induced hyperalgesia. The imbalance between TIMP1 and MMP-9 leads to NLRP3 activation in spinal microglia and increases IL-1β maturation via TLR2/4 pathway. TIMP1 injection eliminates MMP-9-induced NLRP3 activation and blocks hyperalgesia, suggesting that TIMP1 is a critical gatekeeper in preventing neuroinflammation during neuropathic pain development.