Pharmacological regulation of arachidonic acid metabolism for resolution of inflammation and suppression of cardiac injury in rodent models of myocardial infarction

Abstract

Arachidonic acid metabolites are important proinflammatory and resolving mediators in myocardial infarction. Thus, pharmacological regulation of arachidonic acid metabolism is of importance in the therapy of myocardial injury. The aim of the present thesis was to discover small molecules for the regulation of arachidonic acid metabolism towards the resolution of inflammation and suppression of cardiac injury after myocardial infarction. Firstly, we investigated whether selective degradation of leukotriene B4 (LTB4) could suppress neutrophil-mediated inflammation after myocardial infarction. Current anti-inflammatory therapies are mainly designed to control the production of proinflammatory lipid mediators, and thereby restricted by the lack of specificity and the occurrence of side-effects. The present study focused on the degradation of LTB4 by leukotriene B4-12-hydroxydehydrogenase (LTB4DH). For inducing LTB4DH expression, we have sequentially isolated gallic acid from Radix Paeoniae Rubra and calycosin and formononetin from Radix Astragali. Calycosin or formononetin in combination with gallic acid not only induced LTB4DH expression but also attenuated the survival and chemotaxis of neutrophils in response to LTB4. In mouse model of myocardial infarction induced by isoproterenol, calycosin and gallic acid synergistically suppressed neutrophil infiltration and protected cardiac integrity. Secondly, we investigated whether gallic acid derivative could induce LTB4DH for selective degradation of leukotriene B4 (LTB4). We synthesized gallic acid-l-leucine (GAL) conjugate and investigate the effects of GAL conjugate on LTB4DH induction and macrophage phagocytosis. We found that GAL conjugate stimulated RAW264.7 macrophages to engulf fluorescently labeled latex beads or apoptotic H9c2 cells. We further discovered that GAL conjugate enhanced macrophage phagocytosis through sequential activation of Nrf2/HO-1/LTB4DH signaling pathways. Thirdly, we investigated whether arachidonic acid metabolites formed covalent conjugates with cellular proteins. We employed ω-alkynyl arachidonic acid as probe to treat macrophages and isolate lipid-protein adducts by click chemistry “azide-alkyne cycloadduction” and affinity isolation. We identified glycolytic enzyme pyruvate kinase M2 (PKM2) as a predominant target protein and 15-keto-PGF2α as the specific metabolite. 15-keto-PGF2α promoted macrophage M2 polarization, enhanced macrophage phagocytosis and reduced cardiac infarct size in mice after ischemia reperfusion. Mechanistic studies revealed that 15-keto-PGF2α inhibited dimerization, phosphorylation and nuclear translocation of PKM2, thereby suppressing HIF-1α expression and STAT3 phosphorylation. Finally, we investigated whether ω-alkynyl arachidonic acid regulated macrophage polarization and attenuate myocardial injury. We found that ω-alkynyl arachidonic acid promoted anti-inflammatory macrophage M2 polarization in acute myocardial infarction via regulating the cross-talk between PKM2, HIF-1α and iNOS. In conclusion, the present thesis investigated the important roles of arachidonic acid metabolites the resolution of inflammation and reduction of cardiac damage after myocardial infarction. The key findings from the present thesis include: 1) calycosin and gallic acid synergistically attenuate neutrophil-mediated inflammation and injury in isoproterenol-induced myocardial infarction via inducing LTB4DH; 2) GAL conjugate promoted macrophage phagocytosis via induction of Nrf2/HO-1/LTB4DH signaling pathway; 3)15-keto-PGF2α promoted macrophage M2 polarization to diminish cardiac injury via covalent interaction with PKM2; 4) ω-alkynyl arachidonic acid promoted M2 polarization and phagocytosis of macrophages via regulating the cross-talk between PKM2, HIF-1α and iNOS. These results may promote further investigations on arachidonic acid metabolism for new drugs against myocardial infarction.