Anti-inflammatory and anti-oxidant mechanisms of an MMP-8 inhibitor in lipoteichoic acid-stimulated rat primary astrocytes: involvement of NF-κB, Nrf2, and PPAR-γ signaling pathways.

BACKGROUND: Recent evidence suggests that reactive astrocytes play an important role in neuroinflammation and neurodegenerative diseases. Thus, controlling astrocyte reactivity has been suggested as a promising strategy for treating neurodegenerative diseases. In the present study, we investigated whether a matrix metalloproteinase (MMP)-8 inhibitor, M8I, could control neuroinflammation in lipoteichoic acid (LTA)-stimulated rat primary astrocytes. METHODS: The effects of M8I on the expression of inducible nitric oxide synthase, cytokines, and MMPs were examined in LTA-stimulated rat primary astrocytes by ELISA, RT-PCR, and Western blot analysis. The effects of M8I on reactive oxygen species (ROS) generation and phase II antioxidant enzyme expression were examined by the DCF-DA assay, RT-PCR, and Western blot analysis. The detailed molecular mechanisms underlying the anti-inflammatory and antioxidant effects of M8I were analyzed by the electrophoretic mobility shift assay, the reporter gene assay, Western blot, and RT-PCR analysis. RESULTS: Treatment with LTA, a major cell wall component of Gram-positive bacteria, led to astrocyte activation and induced the expression of inflammatory molecules such as iNOS, COX-2, and pro-inflammatory cytokines. In addition, LTA induced the expression of MMPs such as MMP-1, MMP-3, MMP-8, MMP-9, and MMP-13 in rat primary astrocytes. Based on previous reports showing that MMP-8 plays a role as a proinflammatory mediator in microglia, we investigated whether MMP-8 is also involved in inflammatory reactions of reactive astrocytes. We found that treatment of astrocytes with M8I significantly inhibited LTA-induced expression of iNOS, TNF-α, IL-1ß, IL-6, and TLR-2. In addition, M8I inhibited LTA-induced NF-κB, MAP kinase, and Akt activities, while it increased the anti-inflammatory PPAR-γ activities. Moreover, M8I showed antioxidant effects by suppressing ROS production in LTA- or H2O2-stimulated astrocytes. Interestingly, M8I increased the expression of phase II antioxidant enzymes such as hemeoxygenase-1, NQO1, catalase, and MnSOD by modulating the Nrf2/ARE signaling pathway. CONCLUSIONS: The data collectively suggest the therapeutic potential of an MMP-8 inhibitor in neuroinflammatory disorders that are associated with astrocyte reactivity.

Suppression of neuroinflammation by matrix metalloproteinase-8 inhibitor in aged normal and LRRK2 G2019S Parkinson's disease model mice challenged with lipopolysaccharide.

Microglial priming is caused by aging and neurodegenerative diseases, and is characterized by an exaggerated microglial inflammatory response to secondary and sub-threshold challenges. In the present study, we examined the effects of the matrix metalloproteinase-8 (MMP-8) inhibitor (M8I) on the brain of aged normal and leucine-rich repeat kinase 2 (LRRK2) G2019S Parkinson's disease (PD) model mice systemically stimulated with lipopolysaccharide (LPS). The results indicated that Iba-1 positive microglia and GFAP-positive astrocytes, which were increased by LPS, significantly decreased by M8I in aged normal and PD model mice. M8I also decreased the expression of pro-inflammatory markers in the hippocampus and midbrain of aged normal and PD model mice challenged with LPS, while it also improved the motor coordination of aged normal mice after LPS challenge in rotor rod test and the general crossing locomotor activities of LPS-treated LRRK2G2019S PD mice after LPS challenge in open field test. To assess the effects of M8I in an in vitro priming model, BV2 microglia were pretreated with macrophage colony-stimulating factor (CSF)-1 or interleukin (IL)-34, and subsequently stimulated with LPS or polyinosinic-polycytidylic acid (poly[I:C]). M8I inhibited the LPS- or poly(I:C)-induced production of the tumor necrosis factor-α and nitric oxide, alone or in combination with CSF-1 or IL-34. Collectively, the data suggested that M8I has a therapeutic potential in treating neurodegenerative diseases that are aggravated by systemic inflammation.

Association of MMP-8 with obesity, smoking and insulin resistance.

BACKGROUND: Obesity has been recognized as a state of subclinical inflammation resulting in a loss of insulin receptors and decreased insulin sensitivity. We here studied in vivo the role of circulating matrix metalloproteinase-8 (MMP-8) among young healthy twin adults. Also, in vitro analysis of the cleavage of human insulin receptor (INSR) by MMP-8 was investigated as well its inhibition by doxycycline and other MMP-8 inhibitor, Ilomastat/GM6001, which are broad-spectrum MMP inhibitors. MATERIALS AND METHODS: We analysed serum MMP-8 levels by a time-resolved immunofluorometric assay in obese (n = 34), overweight (n = 76) and normal weight (n = 130) twin individuals. The effect of MMP-8 on INSR and the effects of synthetic MMP-8 inhibitors, doxycycline and Ilomastat/GM6001, were studied by SDS-PAGE. RESULTS: We found that in obese individuals relative to normal weight individuals, the serum MMP-8 levels and MMP-8/TIMP-1 ratio were significantly increased (P = 0·0031 and P = 0·031, respectively). Among normal weight and obese individuals, also smoking significantly increases serum MMP-8 and MMP-8/TIMP-1 ratio. In vitro, we found that INSR was degraded by MMP-8 and this was inhibited by doxycycline and Ilomastat/GM6001. CONCLUSIONS: Obesity associated with elevated circulating MMP-8 found among young adults may contribute to progression of insulin resistance by cleaving INSR. This INSR cleavage by MMP-8 can be inhibited by synthetic MMP-8 inhibitors such as doxycycline. In addition to obesity, also smoking independently explained increased MMP-8 levels. Our results suggest that MMP-8 is an essential mediator in systemic subclinical inflammatory response in obesity, and a potential drug target.

A robust classification-dependent multi-molecular modelling study on some biphenyl sulphonamide based MMP-8 inhibitors.

Matrix metalloproteinases (MMPs) are a group of zinc and calcium-dependent endopeptidases, which contribute to different physiological and biological activities via extracellular matrix (ECM) degradation. Matrix metalloproteinase-8 (MMP-8) belongs to type-II collagenases of the MMP family that has contribution in several physiological disorders such as cardiovascular diseases, joint, renal, digestive and respiratory disorders as well as in cancer. In clinical study, MMP-8 is found to be associated with periodontal disease condition. Therefore, MMP-8 specific inhibitors should be developed to target these disorders. The biphenyl sulphonamide (BPS) moiety is one of the crucial structural characteristics found in several MMP-8 inhibitors. Here, different classification-based molecular modelling methods were used to explore the structural features that lead to the activity variation of a series of MMP-8 inhibitors possessing a BPS moiety. Our current classification-based structural analysis of these BPS-derived MMP-8 inhibitors was able to identify the importance of several structural features such as the tetrahydroisoquinoline and N-Boc pyridyl groups, which have positive influences on MMP-8 inhibition. This study was also reflected the importance of the zinc-binding groups (ZBGs) like the hydroxamate and phosphonate for potent and sub-nanomolar range MMP-8 inhibition, which may benefit the development of highly potent MMP-8 inhibitors.

Regulation of neuroinflammation by matrix metalloproteinase-8 inhibitor derivatives in activated microglia and astrocytes.

Matrix metalloproteinases (MMPs) play a pivotal role in neuroinflammation that is associated with neurodegenerative diseases. Our group recently reported that MMP-8 mediates inflammatory reactions by modulating the processing of TNF-α. To improve the efficacy of the currently available MMP-8 inhibitor (M8I), we have synthesized structurally modified M8I derivatives (comp 2, 3, 4, 5) and compared their efficacy with original compound (comp 1). Among M8I derivatives, comp 2, 3, and 5 inhibited the production of NO, ROS, and IL-6 more efficiently than the original compound in lipopolysaccharide (LPS)-stimulated microglia. When we compared the anti-inflammatory mechanisms of the most effective derivative, comp 3, with comp 1, comp 3 suppressed the mRNA expression of iNOS and cytokines more efficiently than comp 1. Although comp 1 inhibits only TNF-α processing, comp 3 additionally inhibits the expression of TNF-α. Both compounds inhibited LPS-induced activity of MAP kinases, NF-κB, and AP-1, while they increased heme oxygenase-1 expression by upregulating AMPK-Nrf2 signaling. Overall, the effect of comp 3 on anti-inflammatory signaling was much stronger than comp 1. We verified the anti-inflammatory effects of comp 1 and 3 in the LPS-injected mouse brain and primary cultured astrocytes. Comp 1 and 3 suppressed microglial activation, astrogliosis, and proinflammatory gene expression in the brain. Moreover, the compounds inhibited proinflammatory gene expression in the cultured astrocytes. Collectively, our data suggest that the MMP-8 inhibitor may be a promising therapeutic agent for neuroinflammatory disorders.

Matrix Metalloproteinase-8 is a Novel Pathogenetic Factor in Focal Cerebral Ischemia.

The neutrophil collagenase matrix metalloproteinase-8 (MMP8) is a recently identified member of MMPs that have important roles in various inflammation-related disorders. Previously, we identified MMP8 as a new neuroinflammatory mediator in activated microglia by regulating TNF-α productivity. Here, we present evidence that MMP8 is a critical factor for brain damage in transient focal cerebral ischemia by modulating neuroinflammation likely microglial activation and TNF-α production. Biochemical analyses showed upregulation of MMP8 expression at mRNA and protein levels in transient middle cerebral artery occlusion/reperfusion (M/R)-challenged brains. Furthermore, double immunolabeling showed that MMP8 expression was upregulated in the activated microglia of M/R-challenged brains. Assessment of infarct volume, neurological score, and survival/death of neural cells revealed that administration of an MMP8 inhibitor (M8I) immediately after reperfusion reduced brain damage. Histological analyses showed that microglial activation and TNF-α expression in ischemic conditions was abrogated by exposure to M8I, as demonstrated in our previous study using cultured microglia. These outcomes from a pharmacological approach were reaffirmed by a genetic approach using a lentiviral system. Intracerebroventricular microinjection of MMP8-specific shRNA lentivirus reduced the extent of ischemia-induced brain damage, as assessed by infarct volume, neurological score, microglial activation, and TNF-α expression. These results suggest a novel pathogenetic role of MMP8 and implicate modulation of its activity as a tractable strategy for therapies against cerebral ischemia.