Study On The Action Mechanism Of Narirutin Inhibiting NLRP3 Inflammasome Activation

Background: Citrus peel(Chenpi)has been long time used in both food and traditional medicine to treat common cold,dyspepsia,cough,and phlegm in Asia.Narirutin,flavanone-7-O-glycoside,is the major flavonoid found in citrus peel.Narirutin is known to have various bioactivities,including anti-oxidative,anti-allergic,and anti-inflammatory activities.Although there are several reports on the anti-inflammatory effects of nariruitn,the action mechanism of narirutin targeting NLRP3 inflammasome has not been reported.NLRP3 inflammasome is a key factor mediating the inflammatory response of the innate immune system.Growing studies have reported that the NLRP3 inflammasome is involved in the occurrence and development of various inflammatory diseases.Until now,more than 70 natural products that inhibit the activity of the NLRP3 inflammasome have been identified,but the research on small molecule inhibitors of the NLRP3 inflammasome is still in its early stages.Aim of the study: NLRP3 inflammasome plays an important role in the occurrence and development of inflammation-related diseases.Studying the action mechanism of NLRP3 inflammasome not only helps to deepen the understanding of the occurrence and development of inflammatory diseases,but also provides new insights for the development of new drugs for inflammatory diseases.The purpose of this study is to reveal the action mechanism of narirutin inhibiting the occurrence and development of NLRP3-mediated inflammation.Methods: 1.In vitro experiments,a number of experimental methods were used to reveal the mechanism of narirutin inhibiting NLRP3 inflammasome activation.PMA-differentiated THP-1 macrophages and BMDMs were treated with narirutin(100-300 μM)and then stimulated by LPS and ATP.(1)The effect of narirutin on cell viability was evaluated by MTT assay and CCK-8 assay,and the effect of narirutin on IL-1β,IL-6 and TNF-α secretion was detected by ELISA assay.(2)The effects of narirutin on IL-1β maturation and caspase-1 cleavage activation was detected by western blotting assay.(3)Pyroptosis was evaluated by LDH release assay and PI/Hoechst double staining assay.Additionally,the effect of narirutin on GSDMD cleavage which is a critical event in NLRP3-mediated pyroptosis was analyzed by western blotting assay.(4)ASC oligomerization was investigated through ASC speck immunofluorescence staining and through western blotting assay of ASC proteins treated with a chemical cross-linking reagent.(5)RT-PCR,western blotting,and immunofluorescence assays were exploited to investigate the effects of narirutin on the expression of NLRP3,IL-1β,caspase-1 and ASC at the NLRP3 inflammasome priming step,and on the regulatory signalling pathways(NF-κB,MAPK and PI3K/AKT).(6)The effect of narirutin on NLRP3-ASC interaction was examined by co-immunoprecipitation and immunofluorescence assays.2.In vivo experiments,alum-induced peritonitis and DSS-induced colitis mice were used as the inflammation animal models.(1)Narirutin was pre-administered at a dose of 100 and 300 mg/kg by oral gavage for 6 days to C57BL/6 mice before inducing peritonitis.Thereafter,peritonitis was induced by intraperitoneal injection of 40 mg/kg alum crystals.The effect of narirutin on the recruitment of inflammatory immune cells into peritoneal cavities was detected by flow cytometry analysis.ELISA assay was used to evaluate the effect of narirutin on the secretion of IL-1β,IL-6 and TNF-α into serum and peritoneal fluids.The effects of narirutin on the expression of NLRP3 and IL-1β,and on NF-κB and MAPK signaling pathways were investigated by western blotting assay of peritoneal exudate cells.(2)To induce colitis in C57BL/6 mouse,narirutin at a dose of 100 and 300 mg/kg was pre-administered for 7 days by oral gavage.Then,following 7 days,the mice were fed with the water containing 4% DSS while the treatment of narirutin was continued.The effect of narirutin on inflammatory symptoms was evaluated by measuring body weight,disease activity index,and colon length,and by H&E staining of mouse colon tissue.The levels of IL-1β,IL-6 and TNF-α in serum,and IL-1β expression in colon tissue were detected by ELISA and immunohistochemical analysis,respectively.Western blotting assay of colon tissue proteins was performed to investigate the effect of narirutin on the expression of NLRP3 and IL-1β,and on NF-κB and MAPK signaling pathways.Results:(1)Narirutin had no toxicity even up to the concentration of 500 μM in cell culture and the dose of 300 mg/kg oral administration in mice.(2)Narirutin at the final concentration of 100-300 μM inhibited pro-inflammatory cytokines secretion in LPS/ATP stimulated macrophages,including IL-1β,IL-6,and TNF-α.(3)Narirutin suppressed NLRP3-related pro-inflammatory events in LPS/ATP stimulated macrophages,including mature IL-1β and active caspase-1 production,GSDMD-NT generation and pyroptosis,and ASC oligomerization.(4)Narirutin decreased the expression of NLRP3 and IL-1β in LPS-priming step through inhibiting NF-κB,MAPK and PI3K/AKT signaling pathways.(5)Narirutin inhibited NLRP3-ASC interaction to suppress NLRP3 inflammasome assembly.(6)Oral administration of narirutin at a dose of 300 mg/kg suppressed the pro-inflammatory responses including the recruitment of inflammatory immune cells and the secretion of IL-1β,IL-6 and TNF-α into peritoneal cavities in alum-induced mice peritonitis.(7)Oral administration of narirutin at a dose of 300 mg/kg alleviated inflammation symptoms in DSS-induced mice,as indicated by the changes of body weight,disease activity index,and colon length and histopathological analysis.(8)Narirutin decreased the activation of NF-κB and MAPK signaling pathways and the expression of NLRP3 and IL-1β during the inflammation induced by alum or DSS in mice.Conclusion: The present study concludes that narirutin exerts anti-inflammatory activity through NLRP3 inflammasome inhibition in vitro and in vivo.In detail,narirutin suppresses the priming process of NLRP3 inflammasome via inhibiting NF-κB,MAPK and PI3K/AKT signaling pathways,as well as inflammasome assembly via interfering NLRP3-ASC interaction.These findings would contribute to understand the anti-inflammation effects of narirutin-rich foods or traditional medicines,and provide a potential pharmacological therapeutic strategy for the prevention or treatment of NLRP3-associated diseases.