Anti-Inflammatory Actions of Neuroprotectin D1/Protectin D1 and Its Natural Stereoisomers: Assignments of Dihydroxy-Containing Docosatrienes

摘要

Protectin D1, neuroprotectin D1 when generated by neural cells, is a member of a new family of bioactive products generated from docosahexaenoic acid. The complete stereochemistry of protectin D1 (10,17 S -docosatriene), namely, chirality of the carbon-10 alcohol and geometry of the conjugated triene, required for bioactivity remained to be assigned. To this end, protectin D1europrotectin D1 (PD1) generated by human neutrophils during murine peritonitis and by neural tissues was separated from natural isomers and subjected to liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Comparisons with six 10,17-dihydroxydocosatrienes prepared by total organic and biogenic synthesis showed that PD1 from human cells carrying potent bioactivity is 10 R ,17 S -dihydroxy-docosa-4 Z ,7 Z ,11 E ,13 E ,15 Z ,19 Z -hexaenoic acid. Additional isomers identified included trace amounts of Δ15- trans -PD1 (isomer III), 10 S ,17 S -dihydroxy-docosa-4 Z ,7 Z ,11 E ,13 Z ,15 E ,19 Z -hexaenoic acid (isomer IV), and a double dioxygenation product 10 S ,17 S -dihydroxy-docosa-4 Z ,7 Z ,11 E ,13 Z ,15 E ,19 Z -hexaenoic acid (isomer I), present in exudates. 18O2 labeling showed that 10 S ,17 S -diHDHA (isomer I) carried 18O in the carbon-10 position alcohol, indicating sequential lipoxygenation, whereas PD1 formation proceeded via an epoxide. PD1 at 10 nM attenuated (～50%) human neutrophil transmigration, whereas Δ15- trans -PD1 was essentially inactive. PD1 was a potent regulator of polymorphonuclear leukocyte (PMN) infiltration (～40% at 1 ng/mouse) in peritonitis. The rank order at 1- to 10-ng dose was PD1 ≈ PD1 methyl ester ? Δ15- trans -PD1 10 S ,17 S -diHDHA (isomer I). 10 S ,17 S -dihydroxy-docosa-4 Z ,7 Z ,11 E ,13 E ,15 Z ,19 Z -hexaenoic acid (isomer VI) proved ≥ PD1 in blocking PMN infiltration, but was not a major product of leukocytes. PD1 also reduced PMN infiltration after initiation (2 h) of inflammation and was additive with resolvin E1. These results indicate that PD1 is a potent stereoselective anti-inflammatory molecule.