Obacunone Inhibits Proinflammatory Mediators By Targeting MIF Inhibition In LPS-activated Macrophages

Inflammation is an important component of innate immunity and the host response to pathogens. It must be precisely controlled at multiple levels, as excessive inflammation does not benefit organisms and may cause tissue impairment. Macrophages play important roles in inflammation by directly affecting microbial cell killing and releasing chemokines and cytokines to amplify acute inflammation. Obacunone (OBA) is a triterpenoids limonoids compound mainly in the Rutaceae citrus, Dictamnus dasycarpus Turcz and Phellodendron chinense Schneid. Although the latter two herbs have been clinically used for the treatment of inflammation, the anti-inflammatory activity of OBA has been little studied.Herein, the anti-inflammatory effect of OBA on lipopolysaccharide (LPS)-stimulated RAW264.7 cells is demonstrated along with its underlying mechanisms. In LPS-activated RAW264.7 macrophages, the supernatant NO was determined by Griess method. Proteins (IL-6, IL-1β and MCP-1) in the supernatant were determined by ELISA, while other proteins (iNOS, p-JNK/JNK, ERK1/2/ERK1/2, p-p38/p38, MKP-1) in the cytosol were assayed by western blot. The mRNA levels of pro-inflammatory cytokines were measured by RT-PCR. Effects of OBA on the NF-κB and AP-1 activation were determined by luciferase assay. The results from Target-fishing and Docking indicates that OBA can directly bind with MIF (315t). Thus, we next evaluated the effects of OBA on MIF. Recombinant MIF activity assay was performed using L-dopachrome methyl ester as a substrate. The number of macrophages induced by MIF was counted in the chemotaxis assay. MIF kinase activity assay was performed using L-dopachrome as a substrate. MIF (-) RAW264.7 cells were obtained by using MIF shRNA plasmid.Our results show that OBA (25,50,100μM) potently represses the pro-inflammatory mediators, such as iNOS, MCP-1, IL-1β and IL-6 at the transcriptional and translational levels without cytotoxicity, indicating the excellent anti-inflammatory efficacy of OBA in vitro. Further study shows that OBA significantly suppresses the activation of activator protein-1 (AP-1) through inhibiting p38 kinase (p38) phosphorylation levels and increasing the mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) expression post-transcriptionally. Simultaneously, OBA can inhibit recombinant mouse MIF-induced chemotactic response of RAW264.7 cells, and suppress the activity of recombinant mouse MIF in a cell-free system. OBA was docked into the active pocket of 315t via locating between chain A and chain C. Hydrogen bonds are formed with Tyr36, Lys32 and Ile64 in chain A. We also confirmed that MIF is a negative regulator of MKP-1 expression. To verify whether the pro-and anti-inflammatory actions of OBA attribute to the MIF inhibition and exclude the off-target effects by MIF inhibitors, we conditionally knocked down the MIF gene in RAW264.7 cells using MIF shRNA plasmid and obtained stable MIF (-) cell, whose responses to LPS were similar to OBA-treated macrophages, suggesting that OBA exerted anti-and pro-inflammatory activities by directly targeting MIF inhibition.In summary, our data indicates that OBA exert anti-inflammatory effects by suppressing production of IL-1β, IL-6 and MCP-1 at both the transcriptional and translational levels via MKP-1/p38 mediated AP-1 pathway. Further mechanism study showed that the molecular target of the anti-inflammatory activities of OBA is MIF. Our study provides a molecular mechanism for the anti-inflammatory of OBA by targeting MIF inhibition in macrophages for the first time.