Inhibition of ellagic acid on lipopolysaccharids-induced inflammatory responses in RAW264.7 macrophage through TLR4-SRC/MAPK/NF-κB pathway
Objective To determine the anti-inflammation mechanism of ellagic acid based on Toll-like receptor 4(TLR4)-tyrosine kinase(SRC)/mitogen-activated protein kinase(MAPK)/nuclear transcription factor-κB(NF-κB)pathway.Methods Different concentrations(0,0.5,5,25,50,75,and 100 μmol·L-1)of ellagic acid were used to treat RAW264.7 macrophage[or lipopolysaccharids(LPS)-induced RAW264.7 cells]for 24 hours.MTT assay was used to detect the viability of RAW264.7 macrophages and screen the optimal concentration.The experiment was divided into a blank control group,a model group(0.5 mg·L-1 LPS),a dexamethasone group(10μmol·L-1),and three ellagic acid(5,25,and 50 μmol·L-1)treatment groups.Griess method was adopted to detect the content of nitric oxide(NO)in the cell culture supernatant,and enzyme-linked immunosorbent assay(ELISA)for detecting the expression levels of prostaglandin E2(PGE2),interleukin-6(IL-6),interleukin-1β(IL-1β),and tumor necrosis factor-α(TNF-α)in the cell culture supernatant.Western blot was applied to detect the expression levels of inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2)protein,and TLR4-SRC/MAPK/NF-κB pathway-related proteins.Results MTT showed that the intervention concentration of ellagic acid was 5～50μmol·L-1.Compared with the blank control group,the expression levels of PGE2,NO,IL-6,IL-1β,and TNF-α in the model group were significantly increased(P＜0.01);the expression levels of inflammatory markers iNOS and COX-2 protein were significantly increased(P＜0.01),indicating that the inflammatory model was successfully established.Compared with the model group,ellagic acid significantly inhibited LPS-induced NO,PGE2,TNF-α,IL-1β,and IL-6(P＜0.05,P＜0.01),reduced the expression levels of iNOS and COX-2 protein(P＜0.05,P＜0.01),in a concentration-dependent manner.Moreover,it also significantly inhibited the expression levels of TLR4 protein and the phosphorylation levels of SRC,P38,JNK,p65,and IκBα protein,showing a concentration-dependent effect.However,it did not show a significant inhibitory effect on the phosphorylation of ERK1/2 protein.Conclusion Ellagic acid may inhibit the inflammatory responses of LPS-induced RAW264.7 macrophages by regulating the TLR4-SRC/MAPK/NF-κB signaling pathway,while the specific regulatory mechanism requires further verification.
ellagic acidanti-inflammatory effectRAW264.7 macrophageSRC protein kinaseMAPK pathwayNF-κB pathway
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