Esculin Inhibits Lipopolysaccharide-induced Inflammation in RAW264.7 Macrophages by Toll-like receptor 4/nuclear factor κB Signaling Pathway
Objective To investigate the inhibitory effect of esculin on lipopolysaccharide(LPS)induced RAW264.7 macrophages in-flammation by regulating Toll-like receptor 4/nuclear factor κB(TLR4/NF-κB)pathway.Methods RAW264.7 macrophages were cul-tured and treated with LPS to induce inflammatory injury from October 2022 to June 2023.The cells were divided into normal group,LPS-stimulated model group,and esculin low,medium,and high concentration groups(drug concentrations of esculin were 25,100 and 200 µmol/L,respectively).RAW264.7 macrophages was treated with esculin for 12 hours and then induced by LPS for 12 hours.The cell viability was detected by thiazole blue tetrazolium bromide(MTT)method.Apoptosis was detected by Annexin V-fluorescein iso-thiocyanate(FITC)and propyl iodide(PI)apoptosis and necrosis assay combined with cell flow analysis.The expression and release of inflammatory factors were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and enzyme-linked im-munosorbent assay(ELISA),respectively.Protein expression of apoptosis factors,TLR4/NF-κB signaling pathway and nuclear translo-cation of NF-κB were detected by Western blot.Results MTT assay showed that 200 µmol/L esculin treatment significantly increased the proliferation activity of RAW264.7 cells induced by LPS(1.524±0.223).Flow analysis showed that esculin significantly inhibited LPS-induced apoptosis and necrosis[(10.68±3.69)%],and western blot showed that 200 µmol/L esculin significantly promoted B-cell leukemia/lymphoma 2(Bcl-2)[(1.981±0.026)fold]and promoted Bcl-2-associated X(Bax)protein[(1.750±0.016)fold]expression.200 µmol/L esculin significantly decreased the transcription levels of inflammatory factors tumor necrosis factor α(TNF-α)[(1.59±0.14)fold,(267.0±25.5)ng/L],interleukin-1β(IL-1β)[(1.28±0.22)fold,(126.0±19.4)ng/L],and interleukin-6(IL-6)[(1.26±0.13)fold,(113.0±18.4)ng/L]in RAW264.7 cells and the paracrine levels in the culture supernatant.200 µmol/L esculin inhibited LPS-induced TLR4 protein expression,phosphorylation of nuclear factor-κB p65(NF-κB p65),pNF-κB inhibitor(IκB),and NF-κB p65 nuclear transcription in RAW264.7 cells.Conclusion Esculin inhibits LPS-induced apoptosis,necrosis and inflammation in RAW264.7 cells by inhibiting TLR4/NF-κB signaling pathway and NF-κB p65 nuclear translocation.
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