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-dimethylt...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(12.5–100μmol/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 proinflammatory cytokines interleukin(IL)-6 and tumor necrosis factorα(TNF-α)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-κB)p65,phospho-STAT3(p-STAT3,Tyr705),inhibitor of NF-κB(IκB)α,and phospho-IκBα(p-IκBα,Ser32),and confocal imaging was used to identify the nuclear translocation of NF-κB 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(qR T-PCR)were used to further study the anti-inflammatory 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 100μmol/L.Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01),decreased IκBαdegradation and phosphorylation(P<0.05)as well as the nuclear translocation of NF-κB 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 mR NA expression levels of of IL-6,TNF-α,IκBα,STAT3,and NF-κB in LPS-stimulated zebrafish(P<0.01).Conclusion:Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.展开更多
基金Supported by Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (No.GDHVPS2018)Young Elite Scientists Sponsorship Program by the China Association of Chinese Medicine (No.2019-QNRC2-C14)。
文摘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(12.5–100μmol/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 proinflammatory cytokines interleukin(IL)-6 and tumor necrosis factorα(TNF-α)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-κB)p65,phospho-STAT3(p-STAT3,Tyr705),inhibitor of NF-κB(IκB)α,and phospho-IκBα(p-IκBα,Ser32),and confocal imaging was used to identify the nuclear translocation of NF-κB 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(qR T-PCR)were used to further study the anti-inflammatory 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 100μmol/L.Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01),decreased IκBαdegradation and phosphorylation(P<0.05)as well as the nuclear translocation of NF-κB 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 mR NA expression levels of of IL-6,TNF-α,IκBα,STAT3,and NF-κB in LPS-stimulated zebrafish(P<0.01).Conclusion:Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.
