Melatonin inhibits matrix metalloproteinase-9 (MMP-9) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 and BV2 cells and a mouse model of meningitis
Chih-Hao Tien1†, Chang Che-Chao1†, E-Jian Lee1*, Wei-Sheng Juan1, Ying-Hsin Chen1, Yu-Chang Hung1, Tsung-Ying Chen1,2, Hung-Yi Chen1,3, Tian-Shung Wu3,4
1Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan.
2Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan.
3Institute of Pharmacy, China Medical University, Taichung, Taiwan.
4Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.
*Correspondence should be addressed to: E-Jian Lee, M.D., M.Sc., Ph.D., Professor in Neurosurgery, Department of Surgery, National Cheng Kung University Medical College and Hospital, 138 Sheng-Li Road, Tainan 70428, Taiwan. Telephone:
+886-6-235-3535 ext.5186; Fax: +886-6-276-6676. Email: [email protected]
Abstract
We explored anti-inflammatory potential of melatonin against the
lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro. RAW 264.7 and BV2 cells were stimulated by LPS, followed by the treatment of melatonin or vehicle at various time intervals. In a mice model of meningitis induced by LPS, melatonin (5 mg/kg) or vehicle was intravenously injected at 30 min postinsult. The activity of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) were determined by gelatin zymography. Nuclear factor-kappa B (NFκB) translocation and binding activity were
determined by immunocytochemistry and electrophoretic mobility shift assay (EMSA).
Our results showed that either pretreatment or co-treatment with melatonin at 50-500 µM effectively inhibited the LPS-induced MMP-9 activation in the RAW 264.7 and BV2 cells (P<0.05, respectively). This melatonin-induced MMP-9 inhibition remained effectively, when treatment was delayed up to 2 and 6 hrs postinsult for RAW 264.7 and BV2 cells, respectively (P<0.05 for both groups). Additionally, melatonin significantly attenuated the rises of circulatory and cerebral MMP-9 activity (P<0.05, respectively), and improved the loss of body weight (P<0.05) in mice subjected to meningitis. Moreover, melatonin (50 µM) effectively inhibited nuclear factor-kappa B (NFκB) translocation and binding activity in the LPS-treated RAW 264.7 and BV2 cells (P<0.05, respectively). These results demonstrate direct inhibitory actions of melatonin against post-inflammatory NFκB translocation and MMP-9 activation, and highlight its ability to inhibit systemic and cerebral MMP-9 activation following brain inflammation.
Key Words: Lipopolysaccharide; matrix metalloproteinase-9; nuclear factor-kappa B; neuroinflammation; and melatonin.
Introduction
Recent studies indicate that matrix metalloproteinases (MMPs) plays a pivotal role in the pathogenesis in a variety of disease entities, including ischemic stroke, inflammation, gastric ulcer, cancer invasion, wound healing and
endometriosis [1-6]. Following cerebral ischemia-reperfusion, increased MMP-9 activity and expression have been observed at 12-48 hr postinsult [6, 7]. The event not only increases the permeability of the blood-brain barrier (BBB), but also exaggerates postischemic endothelial damage and further cleaves protein
components of the extracellular matrix (ECM) such as collagen, proteoglycan and basal laminin [7-9]. Consequently, activated MMPs increase transmigration of blood and inflammatory cells and allow large, toxic molecules entering into the brain parenchyma so as to exaggerate post-insult brain edema, hemorrhagic
transformation and neuronal damage [10, 11].
One strategy to treat ischemic stroke patients is, therefore, to attenuate MMP activation during the acute phase of stroke and, therefore, to improve
neurologic outcome by reducing secondary brain edema and hemorrhagic insults [12-14]. Redox-sensitive MMP protein expression requires activation of both extracellular-regulated kinase 1/2 (ERK1/2) and nuclear factor-kappa B (NFκB) pathways [7, 15-20]. Melatonin (N-aceyl-5-methoxytryptamine) has a variety of actions that can effectively inhibit acute MMP activation and expression following ischemic stroke [6, 7, 10, 13, 14, 21-25]. This natural neuroprotectant and its metabolites are potent free radical scavengers and antioxidants [26, 27]. The agent also have potential to inhibit the ERK1/2- and nuclear factor-kappa B (NFκB)-driven signaling and may, therefore, exhibit robust protective and anti-inflammatory actions in different organs against a variety of insults [7, 28-32]. We have previously demonstrated that melatonin reduced oxidative damage, protected against both gray, For Peer Review white matter and dendritic pathology, and improved
neurobehavioral and electrophysiological outcomes following experimental stroke [33-35]. We have also documented that melatonin improved the preservation of early and late increases in the BBB permeability and reduced the risk of
hemorrhagic transformation following ischemic stoke in mice [10, 13]. More
recently, we have shown that melatonin effectively modulates plasminogen/plasmin system and endogenous MMP-9 inhibitors and, therefore, inhibits systemic
leukocyte transmigration and local microglial activation by decreasing MMP activation following transient focal cerebral ischemia [7, 14]. It was, however, not known whether the melatonin-mediated MMP-9 inhibition observed in vivo might have simply reflected its potent cytoprotection against ischemic stroke. In particular, the melatonin-mediated infarction volume reduction might well explain the
concomitant decreases in the MMP-9 activity and expression as well as the attenuated brain inflammation observed in a whole animal model.
Additional studies involving the in vitro data for directly evaluating melatonin’s anti-inflammatory ability and its underlying mechanism as well as the in vivo data with sparing of focal brain lesions are, therefore, needed.
In attempt to extend our work, we, herein, investigated direct inhibition ability of melatonin on the NFκB translocation and binding activity and its down-stream MMP-9 activation in the LPS-stimulated RAW 264.7 and BV2 cells, respectively. In addition, we examined the inhibitory potential of melatonin against systemic and cerebral MMP-9 activation following the lipopolysaccharide (LPS)-induced
