title: Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation.
abstract: Tolerance seriously impedes the application of morphine in clinical medicine. Thus, it is necessary to investigate the exact mechanisms and efficient treatment. Microglial activation and neuroinflammation in the spinal cord are thought to play pivotal roles on the genesis and maintaining of morphine tolerance. Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception. Metformin, a biguanide class of antidiabetic drugs and activator of AMPK, has a potential anti-inflammatory effect. The present study evaluated the effects and potential mechanisms of metformin in inhibiting microglial activation and alleviating the antinociceptive tolerance of morphine.
  
  
  
  The microglial cell line BV-2 cells and mouse brain-derived endothelial cell line bEnd3 cells were used. Cytokine expression was measured using quantitative polymerase chain reaction. Cell signaling was assayed by western blot and immunohistochemistry. The antinociception and morphine tolerance were assessed in CD-1 mice using tail-flick tests.
  
  
  
  We found that morphine-activated BV-2 cells, including the upregulation of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, pro-inflammatory cytokines, and Toll-like receptor-4 (TLR-4) mRNA expression, which was inhibited by metformin. Metformin suppressed morphine-induced BV-2 cells activation through increasing AMPK phosphorylation, which was reversed by the AMPK inhibitor compound C. Additionally, in BV-2 cells, morphine did not affect the cell viability and the mRNA expression of anti-inflammatory cytokines. In bEnd3 cells, morphine did not affect the mRNA expression of interleukin-1β (IL-1β), but increased IL-6 and tumor necrosis factor-α (TNF-α) mRNA expression; the effect was inhibited by metformin. Morphine also did not affect the mRNA expression of TLR-4 and chemokine ligand 2 (CCL2). Furthermore, systemic administration of metformin significantly blocked morphine-induced microglial activation in the spinal cord and then attenuated the development of chronic morphine tolerance in mice.
  
  
  
  Metformin significantly attenuated morphine antinociceptive tolerance by suppressing morphine-induced microglial activation through increasing AMPK phosphorylation.
authors.affiliation: Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China.
authors.forename: Yinbing
authors.initials: Y
authors.lastname: Pan
authors.affiliation: Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China.
authors.forename: Xiaodi
authors.initials: X
authors.lastname: Sun
authors.affiliation: Department of Pharmacology, Jiangsu Key Laboratory of Neurodegeneration, Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China.
authors.forename: Lai
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Department of Pharmacology, Jiangsu Key Laboratory of Neurodegeneration, Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China.
authors.forename: Liang
authors.initials: L
authors.lastname: Hu
authors.affiliation: Department of Pharmacology, Jiangsu Key Laboratory of Neurodegeneration, Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China.
authors.forename: Hong
authors.initials: H
authors.lastname: Kong
authors.affiliation: Jiangsu Province Key Laboratory of Anesthesiology, School of Anesthesiology, Xuzhou Medical College, Xuzhou, Jiangsu, 221004, People's Republic of China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Han
authors.affiliation: Department of Pharmacology, Jiangsu Key Laboratory of Neurodegeneration, Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Qian
authors.affiliation: Department of Oncology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, 221000, People's Republic of China.
authors.forename: Chao
authors.initials: C
authors.lastname: Song
authors.affiliation: Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China.
authors.forename: Yanning
authors.initials: Y
authors.lastname: Qian
authors.affiliation: Department of Pharmacology, Jiangsu Key Laboratory of Neurodegeneration, Nanjing Medical University, Nanjing, Jiangsu, 210029, People's Republic of China. painresearch@njmu.edu.cn.
authors.forename: Wentao
authors.initials: W
authors.lastname: Liu
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000701:Analgesics, Opioid / Q000494:pharmacology*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002461:Cell Line, Transformed; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004361:Drug Tolerance; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007249:Inflammation / Q000188:drug therapy* / Q000473:pathology; D008262:Macrophage Activation / Q000187:drug effects; D008687:Metformin / Q000494:pharmacology*; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D017628:Microglia / Q000187:drug effects*; D009020:Morphine / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013116:Spinal Cord / Q000166:cytology
chemical_list: C491049:Aif1 protein, mouse; D000701:Analgesics, Opioid; D002135:Calcium-Binding Proteins; D016207:Cytokines; D007004:Hypoglycemic Agents; D008840:Microfilament Proteins; C409466:NR1 NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D009020:Morphine; D008687:Metformin; D011493:Protein Kinase C; D055372:AMP-Activated Protein Kinases
keywords: AMPK; Cytokines; Glia activation; MAPK; Morphine tolerance