Study On The Regulation Of NLRP3 Signaling Pathway In Macrophages By Flavonoids And The Mechanisms Of Alleviation Of DSS-induced Colitis In Mice

The Nod-like receptor(NLR)is one of recognition receptor in the innate immune system.Upon activation by pathogens or danger signals,NLRs form a variety of cytoplasmic protein complexes with a molecular weight of 100-700 k Da.The activation of NLRP3 inflammasome is closely related to the occurrence of intestinal inflammatory diseases.Therefore,the NLRP3 signaling pathways have been widely concerned in recent years.In this paper,both in vitro and in vivo experiments were carried out to investigate the effects of flavonoids on NLRP3 signaling pathway in RAW264.7 and THP-1 cells and mouse colitis induced by DSS.The IC50 of Rutin,Quercetin,Kaempferol and Dihydromyricetin on RAW264.7 and THP-1 cells was measured and analyzed.The RAW264.7 macrophages and PMA-induced THP-1 cells were pretreated with the above four flavonoids before LPS and ATP stimulation.IL-1β,TNF-α,IL-6,and IL-10 expressions of proteins and m RNA were detected by ELISA and PCR;NLRP3 NF-κB and MAPKs signaling pathways were detected by Western-blot.Seventy C57BL/6 female mice were randomly divided into 7 groups(n=10): control group,DSS(3.5%,W/V)group,Rutin low-dose group(30 mg/kg),Rutin high-dose group(100mg/kg),low-dose Quercetin group(30mg/kg),high-dose Quercetin group(100mg/kg)and positive mesalazine group(30mg/kg)group.The Rutin and Quercetin groups were intragastric administrated 14 days.The control and model groups were given same volu me of saline.On the 8th day,besides the control group,the other groups were given DSS solution.Acute colitis in mice was induced by continuous drinking DSS for 7 days.In the positive group,Mesalazine was administered twice a day,and the DAI index was detected every day.On the 14 th day,mice were sacrificed and peripheral blood was collected to determine the content of IL-1β,TNF-α,IL-6,and IL-10.The length of the colon and the changes of IL-1β,TNF-α,IL-6 and IL-10 m RNA were detected.Parts of the colon tissues were fixed in formalin solution for H&E stained.Results: Rutin and quercetin can significantly inhibit the expression of NLRP3 and the phosphorylation of MAPKs-NF-κB in RAW264.7 cells.Rutin and quercetin inhibit the expression both IL-1β protein and m RNA in THP-1 and RAW264.7 cells.In vivo experiment showed that rutin and quercetin significantly reduced the m DAI value,increased the length of colon as compared with DSS group.In addition,IL-1β protein and m RNA expression were also decreased in rutin and quercetin groups.Conclusions: Flavonoids can inhibit both the expression of NLRP3 an d caspase-1 in macrophages.Rutin have significant mitigative effects on colitis induced by DSS.