Zanthoxylum zanthoxyloides inhibits lipopolysaccharide- and synthetic hemozoin-induced neuroinflammation in BV-2 microglia: roles of NF-κB transcription factor and NLRP3 inflammasome activation.

OBJECTIVES: The effects of a root extract of Zanthoxylum zanthoxyloides on neuroinflammation in BV-2 microglia stimulated with LPS and hemozoin were investigated. METHODS: ELISA, enzyme immunoassay and Griess assay were used to evaluate levels of cytokines, PGE2 and NO in culture supernatants, respectively. Microglia-mediated neurotoxicity was evaluated using a BV-2 microglia-HT-22 neuron transwell co-culture. KEY FINDINGS: Treatment with Z. zanthoxyloides caused reduced elevated levels of TNFα, IL-6, IL-1ß, NO and PGE2, while increasing the levels of IL-10. In addition, there were reduced levels of iNOS and COX-2 proteins. This was accompanied by a prevention of microglia-mediated damage to HT-22 mouse hippocampal neurons. Z. zanthoxyloides reduced elevated levels of phospho-IκB and phospho-p65, while preventing degradation of IκB protein and DNA binding of p65. Further mechanistic studies revealed that Z. zanthoxyloides reduced the levels of pro-IL-1ß and IL-1ß in hemozoin-activated BV-2 microglia. This was accompanied by a reduction in caspase-1 activity and NLRP3 protein expression. Bioassay-guided fractionation resulted in the isolation of skimmianine as an anti-inflammatory compound in Z. zanthoxyloides. CONCLUSION: This is the first report showing the inhibition of neuroinflammation in LPS- and hemozoin-activated BV-2 microglia by the root extract of Z. zanthoxyloides by targeting the activation of both NF-κB and NLRP3 inflammasome.

Methyl 3,4,5-trimethoxycinnamate suppresses inflammation in RAW264.7 macrophages and blocks macrophage-adipocyte interaction.

Methyl 3,4,5-trimethoxycinnamate (MTC) is a bioactive natural phenylpropanoid. We evaluated anti-inflammatory effects of synthetic MTC in RAW264.7 macrophages and RAW264.7-3T3-L1 adipocytes co-culture. Levels of cytokines and chemokines, as well as NO and PGE2 in cell supernatants were analysed using ELISAs, Griess assay and enzyme immunoassays, respectively. In-cell cytoblot was used to assess levels of proteins; while DNA binding and reporter gene assays were used to measure transcription factor DNA binding and transcriptional activities, respectively. Glucose uptake in adipocytes was evaluated with 2-deoxy-2-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl) amino]-D-glucose uptake. MTC (5-20 µM) suppressed LPS + IFNγ-induced release of TNFα, IL-6 and IL-1ß, as well as NO/iNOS and PGE2/COX-2 levels in RAW264.7 cells. Furthermore, there was a reduction in phospho-IκB and phospho-p65 proteins, accompanied by a reduction in total IκB in RAW264.7 cells. Further studies showed that MTC also produced a reduction in NF-κB DNA binding and luciferase activity. Treatment of RAW264.7 cells with MTC (5-20 µM) resulted in enhanced DNA binding of Nrf2 and an increase in ARE-luciferase activity. In a macrophage-adipocyte co-culture, the compound reduced the release of TNFα, IL-6, IL-1ß, MCP-1 and RANTES, while enhancing glucose uptake and activation of AMPKα. Our results suggest that MTC produced anti-inflammatory and antioxidant activities in macrophages. MTC also prevented inflammation in macrophage-adipocyte co-culture. The effect of MTC on glucose uptake in adipocytes is proposed to be linked to activation of AMPK.