Anti-inflammatory Effects of Empagliflozin and Gemigliptin on LPS-Stimulated Macrophage via the IKK/NF- κ B, MKK7/JNK, and JAK2/STAT1 Signalling Pathways

摘要

Background . Sodium-glucose cotransporter 2 (SGLT2) and dipeptidyl peptidase-4 (DPP-4) inhibitors are glucose-lowering drugs whose anti-inflammatory properties have recently become useful in tackling metabolic syndromes in chronic inflammatory diseases, including diabetes and obesity. We investigated whether empagliflozin (SGLT2 inhibitor) and gemigliptin (DPP-4 inhibitor) improve inflammatory responses in macrophages, identified signalling pathways responsible for these effects, and studied whether the effects can be augmented with dual empagliflozin and gemigliptin therapy. Methods . RAW 264.7 macrophages were first stimulated with lipopolysaccharide (LPS), then cotreated with empagliflozin, gemigliptin, or empagliflozin plus gemigliptin. We conducted quantitative RT-PCR (qRT-PCR) to determine the most effective anti-inflammatory doses without cytotoxicity. We performed ELISA and qRT-PCR for inflammatory cytokines and chemokines and flow cytometry for CD80, the M1 macrophage surface marker, to evaluate the anti-inflammatory effects of empagliflozin and gemigliptin. NF- κ B, MAPK, and JAK2/STAT signalling pathways were examined via Western blotting to elucidate the molecular mechanisms of anti-inflammation. Results . LPS-stimulated CD80 + M1 macrophages were suppressed by coincubation with empagliflozin, gemigliptin, and empagliflozin plus gemigliptin, respectively. Empagliflozin and gemigliptin (individually and combined) inhibited prostaglandin E 2 (PGE 2 ) release and COX-2, iNOS gene expression in LPS-stimulated RAW 264.7 macrophages. These three treatments also attenuated the secretion and mRNA expression of proinflammatory cytokines, such as TNF- α , IL-1 β , IL-6, and IFN- γ , and proinflammatory chemokines, such as CCL3, CCL4, CCL5, and CXCL10. All of them blocked NF- κ B, JNK, and STAT1/3 phosphorylation through IKK α / β , MKK4/7, and JAK2 signalling. Conclusions . Our study demonstrated the anti-inflammatory effects of empagliflozin and gemigliptin via IKK/NF- κ B, MKK7/JNK, and JAK2/STAT1 pathway downregulation in macrophages. In all cases, combined empagliflozin and gemigliptin treatment showed greater anti-inflammatory properties.