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

Objective: To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms. Methods: 3-[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 inflflammation model in vitro, the production of proinflflammatory cytokines interleukin (IL)-6 and tumor necrosis factor a (TNF-a) 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.B) a, and phospho-I.Ba (p-I.Ba, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-.B p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafifish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflflammation 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-inflflammatory effects of ethyl lithospermate and its probable mechanisms in vivo. Results: The 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-a ( P< 0.05 or P< 0.01), decreased I.Ba degradation and phosphorylation ( P< 0.05) as well as the nuclear translocation of NF-.B p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells ( P< 0.01). Ethyl lithospermate also decreased inflflammatory 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-a, I.Ba, STAT3, and NF-kappa B in LPS-stimulated zebrafifish ( P< 0.01). Conclusion: Ethyl lithospermate exerts anti-Inflflammatory effected by inhibiting the NF-.B and STAT3 signal pathways in RAW 264.7 macrophages and zebrafifish.