Study On The Mechanism Of Lupeol Inhibiting The Activation Of NLRP3 Inflammasome

BackgroundUlcerative colitis(UC)is an autoimmune intestinal disease characterized by severe diffuse lesions of the colorectal mucosa and submucosa,characterized by severe diarrhea,abdominal pain,vomiting,and blood in the stool.The incidence of UC has been on the rise in the world and has become a global public health challenge.In recent years,the incidence of UC in China has been increasing year by year,which has gradually approached the level of Europe and the United States,seriously threatening the global human health and daily quality of life.Therefore,it is urgent to explore the pathogenesis and mechanism of UC and to seek effective treatment strategies.A large number of studies have shown that the expression of NLRP3 inflammasome is significantly increased in UC mouse models,and in NLRP3-/-mice,the symptoms of UC induced by DSS are significantly reduced,and the levels of pro-inflammatory cytokines are significantly down-regulated.Therefore,blocking the activation of NLRP3 inflammasome and down-regulating the release of inflammatory cytokines may improve the development of UCTwo signals are required to activate NLRP3 inflammasome.The first signal,the initiation signal,activates the NF-κB pathway and promotes the transcription of NLRP3.The second signal is activated by various stimulating molecules to activate NLRP3 inflammasome,during which NLRP3 is oligonuclized,resulting in the subsequent assembly of NLRP3,ASC,and Caspase-1 into a complex that induces the release of pro-inflammatory cytokines IL-1β and IL-18.The expression of IL-1β is largely dependent on the activation of NLRP3 inflammasome,which can be stimulated by various types of danger signals Inflammation in the body’s normal physiological state,the small body mainly exist in the epithelial cells,macrophages,dendritic cells and other inflammatory cells and immune cells,helps to identify all sorts of Pathogen associated molecular patterns(Pathogen associated molecular pattern,PAMPs)and Danger associated molecular patterns(Dangerous molecular pattern,DAMPs),in order to induce the subsequent adaptive immune response,mediates the body’s inflammatory reaction,and thus play a role of resistance to infection.However,when the inflammasome is overactivated,it is involved in a variety of autoimmune diseases,such as alzheimer’s disease,atherosclerosis,ulcerative colitis and other inflammatory diseases Lupeol is a triterpene found in a wide variety of fruits.Existing data suggest that Lupeol has anti-inflammatory effects and can down-regulate the expression of IL-1β inflammatory factor,but the specific mechanism of Lupeol’s anti-inflammatory effects remains to be further explored.In this study,we investigated the specific regulatory mechanism of Lupeol inhibition of NLRP3 inflammasome activation through in vivo and in vitro experiments,and observed whether Lupeol could improve the acute UC mouse model.ObjectsTo investigate in vitro and in vivo whether Lupeol can improve UC by inhibiting the activation of NLRP3 inflammasome,and to explore its mechanism.Methods1.In vitro:First,Bone marrow-derived macrophage(BMDM)and Peritoneal derived macrophage(PM)were activated with classical stimuli of NLRP3 inflammasome,and Lupeol(25,50,100)was added at the same time.The activation of Bone marrow-derived macrophage(BMDM)and Peritoneal derived macrophage(PM)were treated with different concentrations of Lupeol(25,50,100).Quantitative real-time PCR(qRT-PCR)and Western blot(WB)were used to detect the changes in the levels of genes and proteins in the components of NLRP3 inflammasome in the cells.Meanwhile,the expression of IL-1β、TNF-α in supernatant was detected by ELISA.Then,BMDM was stimulated with different inflammasome stimulants(Nig,MSU,Alum),and the activation of inflammasome and the content of IL-1β in the supergene were tested with WB and ELISA,respectively.At the same time,crosslinking agent was used to detect oligomerization of ASC.Then we used LPS combined with Lupeol of 100μM facilitation to stimulate PM at different times(2h,3h,4h).qRT-PCR and WB were used to detect the changes in inflammatory factor genes and protein levels(detecting the first signal).WB was used to detect the effect of Lupeol on AIM2 inflammasome.Flow cytometry was used to detect changes in Reactive oxygen species(ROS)and WB was used to detect changes in mitochondrial related proteins Immunoprecipitation was used to investigate the influence of Lupeol on the interaction between NEK7 and NLRP3,ASC and NLRP3.NLRP3 ubiquitination and its degradation pathways were then explored2.In vivo:establish Dextran Sodium Sulfate(Dextran Sulfate Sodium,DSS)induced acute mice UC model of C57/the BL6 mice were randomly divided into six groups,respectively,the Normal group(Normal)(DSS),solvent control group,model group(Oil),and Lupeol treatment group(25 mg/kg、50 mg/kg and 100 mg/kg)three concentrations,Normal group drank ddH2O,the rest of the group are drinking 3%DSS,a total of eight days,modeling on the first day for a day.The weight of the mice was recorded at a fixed time every day,and the feces of the mice were monitored for Disease activity index(DAI).The mice were sacrificed on the eighth day.Flow cytometry(FCM)was used to determine the polarization of macrophages and T cells in peripheral blood,spleen and mesenteric lymph nodes.qRT-PCR and WB were used to detect the changes of inflammatory factor genes and proteins in colon tissues.Hematoxylin and eosin(HE)were used to evaluate the pathological changes of the colon.Immunohistochemistry(IHC)was used to detect the changes of macrophages in colon tissues.The expression of inflammatory cytokines in macrophages was detected by Immunofluorescence(IF)Results1.In vitro:we investigated Lupeol inhibition of NLRP3 inflammasome activation and its primary mechanism.After treatment of BMDM and PM with inflammasome stimulants(LPS and ATP),NLRP3 inflammasome was activated,and Lupeol treatment of macrophages significantly inhibited the activation of NLRP3 inflammasome,down-regulated the expression of IL-1β,and showed a concentration dependence,while TNF-α showed no significant change.After BMDM was stimulated by other stimuli activated by NLRP3(Nig,MSU,Alum),both WB and ELISA results showed that Lupeol inhibited the activation of NLRP3 inflammasome and down-regulated IL-1β expression.At the same time,when the PM was stimulated by AIM2 inflammasome stimulus,WB results showed that Lupeol had no obvious effect on AIM2 inflammasome.Lupeol treatment of PM inhibited the formation of ASC oligomerization.Flow cytometry showed that Lupeol reduced the production of mitochondrial ROS.WB results showed that Lupeol up-regulated the expression of mitochondrial related proteins UCP2 and NCF1.At the same time,the mitochondrial separation experiment showed that the expression of cytochrome C in the cytoplasm was down-regulated after Lupeol treatment of PM.The co-immunoprecipitation results showed that Lupeol attenuated the interaction between NLRP3,NEK7 and ASC.The level of NLRP3 ubiquitination was increased after Lupeol treatment of macrophages,and the degradation of NLRP3 was promoted through the autophagy lysosomal pathway.2.In the DSS-induced acute UC mouse model:we observed a significant increase in DAI score in the Lupeol treatment group,as well as a significant increase in body weight and colon length compared with the DSS group,as well as a decrease in inflammatory cell infiltration in the colon tissue and a significant reduction in pathological damage.At the same time,after Lupeol treatment,the expression of M1-type macrophages in the spleen and lymph nodes of the mice decreased,while the M2-type macrophages increased.IHC and IF results showed increased infiltration of colon macrophages and increased expression of inflammasome in DSS group mice.Lupeol treatment significantly reduced the expression of macrophages and inflammasome in colon.qRT-PCR and WB tests showed that the gene and protein expression levels of inflammatory cytokines in the colon of Lupeol treated mice were down-regulated.ConclusionsLupeol by raising the mitochondria related proteins expression to regulate the production of ROS,and at the same time by inhibiting the ASC polymerization and the interaction between ASC,NEK7 and NLRP3,inhibiting NLRP3 inflammasome activation and inflammatory factor secretion,finally through the autophagy-lysosome pathway to promote the degradation of NLRP3 inflammasome thus improving the occurrence/development of inflammatory diseases such as ulcerative colitis.