PMID- 38061116 OWN - NLM STAT- MEDLINE DCOM- 20240104 LR - 20240207 IS - 1878-1705 (Electronic) IS - 1567-5769 (Linking) VI - 126 DP - 2024 Jan 5 TI - miR-99b-3p/Mmp13 axis regulates NLRP3 inflammasome-dependent microglial pyroptosis and alleviates neuropathic pain via the promotion of autophagy. PG - 111331 LID - S1567-5769(23)01658-2 [pii] LID - 10.1016/j.intimp.2023.111331 [doi] AB - BACKGROUND: Neuropathic pain significantly impairs quality of life, and effective interventions are limited. NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-mediated microglial pyroptosis and the subsequent proinflammatory cytokine production are critical in exacerbating pain. Considering microglial pyroptosis as a potential target for developing specific analgesic interventions for neuropathic pain, our study investigated the pathogenesis and therapeutic targets in this condition. METHODS: In vitro experiments involved the co-culture of the immortalized BV-2 microglia cell line with lipopolysaccharide (LPS) to induce microglial pyroptosis. Differentially expressed microRNAs (miRNAs) were identified using high-throughput sequencing analysis. The downstream target genes of these miRNAs were determined through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and the downstream target genes, combined with miRNAs, were predicted and verified through dual luciferase reporter gene assays. In vivo experiments were conducted to construct a chronic constriction injury (CCI) neuropathic pain model in rats and evaluate the analgesic effects of intrathecal injection of an adeno-associated virus vector (AAV) carrying miR-99b-3p. Gene expression was modulated through mimic or siRNA transfection. Western blot analysis assessed the expression of microglial pyroptosis and autophagy-related proteins, whereas RT-qPCR measured changes in proinflammatory cytokines expression. RESULTS: LPS-stimulated up-regulation of proinflammatory cytokines in microglia, accompanied by NLRP3-dependent pyroptosis, including increased NLRP3, GSDMD-N, Caspase1-p20, and mature-IL-1beta expression. High-throughput sequencing analysis revealed 16 upregulated and 10 downregulated miRNAs in LPS-stimulated microglia, with miR-99b-3p being the most downregulated. KEGG analysis revealed that the target genes of these miRNAs are primarily enriched in calcium, FoxO, and mitogen-activated protein kinase (MAPK) signal pathways. Furthermore, overexpression of miR-99b-3p through mimic transfection significantly inhibited the inflammatory response and NLRP3-mediated pyroptosis by promoting autophagy levels in activated microglia. In addition, we predicted that the 3' untranslated region (UTR) of matrix metalloproteinase-13 (Mmp13) could bind to miR-99b-3p, and knockdown of Mmp13 expression through siRNA transfection similarly ameliorated enhanced proinflammatory cytokines expression and microglial pyroptosis by enhancing autophagy. In vivo, Mmp13 was co-localized with spinal dorsal horn microglia and was suppressed by intrathecal injection of the AAV-miR-99b-3p vector. Moreover, overpressed miR-99b-3p alleviated CCI-induced mechanical allodynia and neuroinflammation while suppressing pyroptosis by enhancing autophagy in the spinal cord of CCI rats. CONCLUSION: miR-99b-3p exerts analgesic effects on neuropathic pain by targeting Mmp13. These antinociceptive effects are, at least in part, attributed to the promotion of autophagy, thereby inhibiting neuroinflammation and NLRP3-mediated pyroptosis in activated microglia. CI - Copyright (c) 2023 Elsevier B.V. All rights reserved. FAU - Gao, Xu AU - Gao X AD - Department of Spine Surgery, Affiliated Hospital of Jining Medical University, 129 Hehua Road, Jining, Shandong Province 272000, China. FAU - Gao, Long-Fei AU - Gao LF AD - Department of Spine Surgery, Affiliated Hospital of Jining Medical University, 129 Hehua Road, Jining, Shandong Province 272000, China. FAU - Zhang, Zhen-Yu AU - Zhang ZY AD - Department of Spine Surgery, Affiliated Hospital of Jining Medical University, 129 Hehua Road, Jining, Shandong Province 272000, China. FAU - Jia, Shu AU - Jia S AD - Clinical Research Team of Spine & Spinal Cord Diseases, Medical Research Center, Affiliated Hospital of Jining Medical University, 89 Guhuai Road, Jining, Shandong Province 272000, China. FAU - Meng, Chun-Yang AU - Meng CY AD - Department of Spine Surgery, Affiliated Hospital of Jining Medical University, 129 Hehua Road, Jining, Shandong Province 272000, China. Electronic address: mengchunyang1600@mail.jnmc.edu.cn. LA - eng PT - Journal Article DEP - 20231206 PL - Netherlands TA - Int Immunopharmacol JT - International immunopharmacology JID - 100965259 RN - 0 (Analgesics) RN - 0 (Cytokines) RN - 0 (Inflammasomes) RN - 0 (Lipopolysaccharides) RN - EC 3.4.24.- (Matrix Metalloproteinase 13) RN - 0 (MicroRNAs) RN - 0 (NLR Family, Pyrin Domain-Containing 3 Protein) RN - 0 (RNA, Small Interfering) RN - EC 3.4.24.- (Mmp13 protein, rat) SB - IM MH - Animals MH - Rats MH - Analgesics MH - Autophagy MH - Cytokines/metabolism MH - Inflammasomes/metabolism MH - Lipopolysaccharides/metabolism MH - Matrix Metalloproteinase 13/metabolism MH - Microglia/metabolism MH - *MicroRNAs/genetics/metabolism MH - *Neuralgia/metabolism MH - Neuroinflammatory Diseases MH - NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism MH - Pyroptosis MH - Quality of Life MH - RNA, Small Interfering/metabolism OTO - NOTNLM OT - Autophagy OT - Microglia OT - Neuropathic pain OT - Pyroptosis OT - miR-99b-3p COIS- Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. EDAT- 2023/12/08 00:42 MHDA- 2023/12/28 06:42 CRDT- 2023/12/07 18:01 PHST- 2023/10/12 00:00 [received] PHST- 2023/11/22 00:00 [revised] PHST- 2023/11/28 00:00 [accepted] PHST- 2023/12/28 06:42 [medline] PHST- 2023/12/08 00:42 [pubmed] PHST- 2023/12/07 18:01 [entrez] AID - S1567-5769(23)01658-2 [pii] AID - 10.1016/j.intimp.2023.111331 [doi] PST - ppublish SO - Int Immunopharmacol. 2024 Jan 5;126:111331. doi: 10.1016/j.intimp.2023.111331. Epub 2023 Dec 6.