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A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways.

ABSTRACT: To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro.Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and COX-2, proinflammatory cytokines, NF-?B and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA. The morphological changes of microglia and nuclear translocation of NF-?B were visualized using phase contrast and fluorescence microscopy, respectively.Pretreatment with MCAP (0.1, 1, 10 ?mol/L) dose-dependently inhibited LPS-induced expression of iNOS and COX-2 in BV2 microglia cells. Similar results were obtained in primary microglia pretreated with MCAP (0.1, 0.5 ?mol/L). MCAP dose-dependently abated LPS-induced release of TNF-?, IL-6 and IL-1?, and mitigated LPS-induced activation of NF-?B by reducing the phosphorylation of I?B? in BV2 microglia cells. Moreover, MCAP attenuated LPS-induced phosphorylation of p38 MAPK, whereas SB203580, a p38 MAPK inhibitor, significantly potentiated MCAP-caused inhibition on the expression of MEF-2 (a transcription factor downstream of p38 MAPK).MCAP exerts anti-inflammatory effects in murine microglia in vitro by inhibiting the p38 MAPK and NF-?B signaling pathways and proinflammatory responses. MCAP may be developed as a novel agent for treating diseases involving activated microglial cells.

SUBMITTER: Kim BW

PROVIDER: S-EPMC4775849 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways.

Kim Byung-Wook BW More Sandeep Vasant SV Yun Yo-Sep YS Ko Hyun-Myung HM Kwak Jae-Hwan JH Lee Heesoon H Suk Kyoungho K Kim In-Su IS Choi Dong-Kug DK

Acta pharmacologica Sinica 20160201 3

<h4>Aim</h4>To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro.<h4>Methods</h4>Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and COX-2, proinflammatory cytokines, NF-κB and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA. The morphological changes of ...[more]

PMID: 26838070

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Project description:BackgroundMicroglial cells play an important role in the immune system in the brain. Activated microglial cells are not only injurious but also neuroprotective. We confirmed marked lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression in microglial cells in pathological lesions in the neonatal hypoxic-ischemic encephalopathy (nHIE) model brain. LOX-1 is known to be an activator of cytokines and chemokines through intracellular pathways. Here, we investigated a novel role of LOX-1 and the molecular mechanism of LOX-1 gene transcription microglial cells under hypoxic and ischemic conditions.MethodsWe isolated primary rat microglial cells from 3-day-old rat brains and confirmed that the isolated cells showed more than 98% Iba-1 positivity with immunocytochemistry. We treated primary rat microglial cells with oxygen glucose deprivation (OGD) as an in vitro model of nHIE. Then, we evaluated the expression levels of LOX-1, cytokines and chemokines in cells treated with or without siRNA and inhibitors compared with those of cells that did not receive OGD-treatment. To confirm transcription factor binding to the OLR-1 gene promoter under the OGD conditions, we performed a luciferase reporter assay and chromatin immunoprecipitation assay. In addition, we analyzed reactive oxygen species and cell viability.ResultsWe found that defects in oxygen and nutrition induced LOX-1 expression and led to the production of inflammatory mediators, such as the cytokines IL-1β, IL-6 and TNF-α; the chemokines CCL2, CCL5 and CCL3; and reactive oxygen/nitrogen species. Then, the LOX-1 signal transduction pathway was blocked by inhibitors, LOX-1 siRNA, the p38-MAPK inhibitor SB203580 and the NF-κB inhibitor BAY11-7082 suppressed the production of inflammatory mediators. We found that NF-κB and HIF-1α bind to the promoter region of the OLR-1 gene. Based on the results of the luciferase reporter assay, NF-κB has strong transcriptional activity. Moreover, we demonstrated that LOX-1 in microglial cells was autonomously overexpressed by positive feedback of the intracellular LOX-1 pathway.ConclusionThe hypoxic/ischemic conditions of microglial cells induced LOX-1 expression and activated the immune system. LOX-1 and its related molecules or chemicals may be major therapeutic candidates. Video abstract.

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Dataset Information

A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways.

Publications

A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways.

Similar Datasets

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