Effect Of Euryale Ferox Seed Shell-derived Corilagin On LPS-induced Inflammation Of RAW264.7 Macrophages

Euryale ferox is a plant of Nymphaeaceae family,which is originally grown in Southeast Asia and China.Previous studies found that Euryale ferox shell extract had anti-tumor,antioxidant and antibacterial activities.Corilagin is a kind of ellagic tannin isolated and purified from Euryale ferox seed shell in our laboratory.Studies have found that corilagin has liver-protecting,anti-tumor,antioxidant and anti-inflammatory activities.This paper studies the effect of corilagin on LPS-induced inflammatory response of macrophages from biochemical factors,transcription and protein levels.The RAW264.7 cells were stimulated by LPS with appropriate concentration to establish an in vitro inflammatory model using commonly used cell models for screening anti-inflammatory drugs.The experiment was divided into normal control group,model group(LPS group),experimental group(LPS combined with corilagin treatment group)and dexamethasone positive control group.The dose range of corilagin without significant cytotoxicity was screened by CCK-8 method.The content of NO was determined by Griess method.The contents of TNF-α,IL-6,IL-1β and IL-10 were determined by ELISA to evaluate the effect of corilagin on the secretion of inflammatory factors.Flow cytometry was used to detect the content of reactive oxygen species in cells.Q-RT-PCR was used to detect TNF-α,IL-6,IL-1β,COX-2,i NOS gene expression levels and the gene expression level related to NF-κB,MAPK,PI3K-AKT signaling pathway.Westen-Blot detected the protein expression levels related to NF-κB,MAPK and PI3K-AKT signaling pathways.The main experimental results are as follows:1.Effect of Euryale ferox seed shell-derived corilagin on secretion of inflammatory factors.LPS-induced RAW264.7 cells were treated with 25 μmol/L,50 μmol/L,and 100 μmol/L Euryale ferox seed shell-derived corilagin,and the secretion of NO,TNF-α,IL-6,IL-1β,and IL-10 in the cell supernatants were significantly reduced in a dose-dependent manner.Flow cytometry was used to detect intracellular reactive oxygen species.It was found that the treatment with different concentrations of corilagin reduced the ROS content of RAW264.7 cells induced by LPS.Preliminary results show that corilagin can play an anti-inflammatory role by reducing the contents of NO,TNF-α,IL-6,IL-1β,IL-10 and ROS in RAW264.7 cells induced by LPS.2.Effect of corilagin on LPS-induced Expression of Inflammatory Factor Gene in RAW264.7 Cells.The expression levels of TNF-α,IL-6,IL-1β,COX-2 and i NOS genes were detected by q-RT-PCR.It was found that the expression levels of TNF-α,IL-6,IL-1β,COX-2 and i NOS genes induced by LPS in RAW264.7 cells were significantly reduced after being treated with different concentrations of corilagin,and were dose-dependent.The results showed that corilagin could play an anti-inflammatory role by reducing the expression of TNF-α,IL-6,IL-1β,COX-2 and i NOS genes in RAW264.7 cells induced by LPS.3.The Effect of Euryale ferox Seed Shell-derived corilagin on Expression of NF-κB,MAPK and PI3K-AKT Signaling Pathway Related Genes and Proteins.50 μmol/L Euryale ferox seed shell-derived corilagin interferes with LPS-induced RAW264.7 cells.The q-RT-PCR detection results show that compared with the model group,the Euryale ferox seed shell-derived corilagin intervention group increases the expression of NF-κB,P65,ERK,JNK,P38,PI3 K and AKT genes,significantly reduces the expression of IKB-α,indicating that Euryale ferox seed shell-derived corilagin may alleviate lipopolysaccharide tolerance through NF-κB,MAPK and PI3K-AKT signaling pathway and play an immunomodulatory role.Western-Blot test results showed that compared with the model group,gordon euryale seed shell-derived corilagin intervention group decreased the phosphorylation of IκB-α and increased the phosphorylation of P65 and JNK proteins.The results showed that gordon euryale seed shell-derived corilagin can alleviate lipopolysaccharide tolerance through NF-κB and MAPK signaling pathways and play an immunomodulatory role.