title: Gallic acid-l-leucine (GAL) conjugate enhances macrophage phagocytosis via inducing leukotriene B4 12-hydroxydehydrogenase (LTB4DH) expression.
abstract: Timely clearance of apoptotic cells is an important step in the resolution of ongoing inflammation and the restoration of tissue integrity and function after acute myocardial infarction. Natural products gallic acid and l-leucine are well-documented for anti-inflammatory and anabolic effects. We synthesized gallic acid-l-leucine (GAL) conjugate via direct coupling gallic acid and l-leucine. The aim of the present study was to investigate the effect of GAL conjugate on the phagocytotic activity of macrophages. By using murine macrophage cell line RAW264.7 as an in vitro model, we evaluated the effect of GAL conjugate on the phagocytic uptake of fluorescently labeled latex beads and apoptotic cardiomyocyte H9c2 cells. We found that GAL conjugate enhanced the phagocytic activity of macrophage RAW264.7 cells in a concentration-dependent manner. Further mechanistic studies revealed that the effect of GAL conjugate on macrophage phagocytosis was positively correlated with the up-regulation of leukotriene B4 12-hydroxydehydrogenase (LTB4DH) expression at both mRNA and protein levels. By ESI-MS based lipidomics profiling, GAL conjugate increased the enzymatic activities of LTB4DH, leading to the formation of lipid metabolites including 12-oxo-LTB4, 13,14-dh-oxo-PGE2 and 13,14-dh-oxo-PGF2α. Interestingly, GAL conjugate failed to increase macrophage phagocytosis upon silencing of LTB4DH by specific siRNA. Moreover, it appeared that GAL conjugate induced LTB4DH expression via activating the Nrf2/HO-1 pathway. After Nrf-2 was silenced by specific siRNA, GAL conjugate no longer induced LTB4DH expression in the Nrf2-siRNA transfected cells. Taken together, our results suggest that GAL enhances macrophage phagocytosis via sequentially activating Nrf2 and up-regulating LTB4DH expression. Thus, GAL conjugate may serve as a lead compound for the development of new anti-inflammatory drugs.
authors.affiliation: School of Chinese Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
authors.forename: Hung-Fat
authors.initials: HF
authors.lastname: Tse
authors.affiliation: Department of Chemistry, Faculty of Science, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong.
authors.forename: Xuechen
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hung Hom, Hong Kong.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Han
authors.affiliation: School of Chinese Medicine, The University of Hong Kong, Hong Kong. Electronic address: jrong@hkucc.hku.hk.
authors.forename: Jianhui
authors.initials: J
authors.lastname: Rong
mesh_terms: D000429:Alcohol Oxidoreductases / Q000096:biosynthesis*; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D015153:Blotting, Western; D002853:Chromatography, Liquid; D005707:Gallic Acid / Q000494:pharmacology*; D007930:Leucine / Q000494:pharmacology*; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D010587:Phagocytosis / Q000187:drug effects* / Q000276:immunology; D000067996:RAW 264.7 Cells; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry; D015854:Up-Regulation
chemical_list: D000893:Anti-Inflammatory Agents; D005707:Gallic Acid; D000429:Alcohol Oxidoreductases; C082646:leukotriene B4 12-hydroxydehydrogenase; D007930:Leucine
keywords: Cardiomyocytes; Leukotriene B4-12-hydroxydehydrogenase; Macrophages; Nrf-2; Phagocytosis