Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-B and STAT3 Pathways in RAW 264.7 Cells and Zebrafish

ObjectiveTo explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms.Methods3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 mu mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-alpha) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-kappa B) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-kappa B (I kappa B) alpha, and phospho-I kappa B alpha (p-I kappa B alpha, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-kappa B p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo.ResultsThe non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 mu mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-alpha(P<0.05 or P<0.01), decreased I kappa B alpha degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-kappa B p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-alpha, I kappa B alpha, STAT3, and NF-kappa B in LPS-stimulated zebrafish (P<0.01).ConclusionEthyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-kappa B and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.