Anti-inflammatory effects of Torin2 on lipopolysaccharide-treated RAW264.7 murine macrophages and potential mechanisms

Abstract

ContextTorin2 has various pharmacological properties. However, its anti-inflammatory activity has not been reported. ObjectiveThis study focused on the potential anti-inflammatory properties of Torin2 in lipopolysaccharide (LPS)-evoked RAW264.7 murine macrophages. The study aimed to shed light on the molecular mechanisms that ameliorate these effects. MethodsTorin2 was applied to 100 ng/mL lipopolysaccharide-induced RAW 264.7 macrophages in vitro. Nitric oxide (NO) levels were detected using the Griess reagent kit. Prostaglandin E2 (PGE2), pro-inflammatory cytokines interleukin (IL)-1β, interleukin (IL)-6, and tumor necrosis factor in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Gene expression of pro-inflammatory cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were tested using real-time quantitative polymerase chain reaction (qPCR). Cyclooxygenase-2 and inducible nitric oxide synthase proteins, phosphorylation of mitogen-activated protein kinase (MAPK) subgroups, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, I-kappa-B-alpha (IκBα), and nuclear factor-kappa-B (NF-κB), and activation in extracts were detected via western blotting. Nuclear factor-kappa-B/p65 nuclear translocation was tested using an immunofluorescence assay. ResultsThe results demonstrated that pre-treatment with Torin2 profoundly attenuated the lipopolysaccharide-stimulated levels of nitric oxide and prostaglandin E2, pro-inflammatory cytokines, messenger ribonucleic acid (mRNA), and protein expression of cyclooxygenase-2 and inducible nitric oxide synthase. Collectively, Torin2 pre-treatment notably weakened lipopolysaccharide-induced damage by reducing the phosphorylation of nuclear factor-kappa-B, p38, c-Jun N-terminal kinase, extracellular signal-regulated kinase proteins, and nuclear factor-kappa-B/p65 nuclear translocation. ConclusionNumerous pieces of evidence indicated that Torin2 reversed inflammatory activation by regulating nuclear factor-kappa-B and mitogen-activated protein kinase signaling pathways and provided a tentative potential candidate for preventing and treating inflammatory diseases.