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 PGE(2) 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 mu M) suppressed LPS + IFN gamma-induced release of TNF alpha, IL-6 and IL-1 beta, as well as NO/iNOS and PGE(2)/COX-2 levels in RAW264.7 cells. Furthermore, there was a reduction in phospho-I kappa B and phospho-p65 proteins, accompanied by a reduction in total I kappa B in RAW264.7 cells. Further studies showed that MTC also produced a reduction in NF-kappa B DNA binding and luciferase activity. Treatment of RAW264.7 cells with MTC (5-20 mu M) 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 TNF alpha, IL-6, IL-1 beta, MCP-1 and RANTES, while enhancing glucose uptake and activation of AMPK alpha. 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.