Gal-3 Mediates AS Inflammatory Mechanism By Activating NLRP3 Inflammasome And The Protective Effect Of Quercetin

Objective: Inflammatory response is one of the main pathological mechanisms of the development of atherosclerosis.This study aims to explore the possible pathways of inflammatory response in atherosclerosis as well as the protective effect and possible mechanism of quercetin in the early stage of atherosclerosis in combination with proteomics technology.Methods:1.In vivo experiment sixty male ApoE knockout C57 mice(18.7 ± 0.9 g)were randomly divided into three groups: the control group(Ct),high fat diet group(HFD),HFD + quercetin 100 mg/kg bw group(HFD + Q),High fat diet was composed by 21% fat and 0.15% cholesterol.After 16 weeks,blood samples and aortic samples were collected.Blood samples were used to measure total cholesterol(TC),total triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C)and arterial tissue samples were used for Oil Red O,VVG and H&E staining.2.Proteomics: After 16 weeks,nine mice were randomly selected in each group for aorta.The protein,Gal-3 researched in this study,was determined by the standard of FD > 1.2 or FD < 5/6 and P < 0.05 combined with Biological Process(BP),Molecular Function(MF),Cell Component(CC).And protein-protein interaction network was used to look for the proteinal association between Gal-3 and NLRP3.3.In vitro experiments :(1)RAW264.7 cells were cultured treated with 0、25、50 μg/ml ox-LDL.The expressions of Gal-3,NLRP3,caspase-1 and IL-1β were determined by western blotting.(2)RAW264.7 cells were divided into six groups: control group,model group(ox-LDL 50 μg/ml),quercetin group(50 μM),TD139 group(10 μM),ox-LDL + quercetin group and ox-LDL + TD139.The measurement of TC and TG,Oil Red O staining,immunofluorescence and western blotting were used to determin the expressions of Gal-3,NLRP3,pro-caspase-1,caspase-1,pro-IL-1β,IL-1β.(3)RAW264.7 cells were divided into five groups: control group,model group(ox-LDL 50 μg/ml),ox-LDL + rGal-3 group(rGal-3 5 μg/ml),ox-LDL + rGal-3 + quercetin group(quercetin 50 μM),and ox-LDL + quercetin group.Expression of Gal-3,NLRP3,pro-caspase-1,caspase-1,pro-IL-1β,and IL-1β was determined by western blotting.Results:1.In vivo,compared with Ct group,the result of Oil Red O staining showed that the number and eare of the arterial plaques were larger in HFD model group.The result determined by VVG showed that arterial fibrin ruptured and H&E staining showed that inflammatory response was very serious in model group,too.However,quercetin administration could effectively alleviate these changes induced by HFD.In addition,quercetin administration could significantly increase the level of HDL-C by 31.0%(P < 0.01),while it could decrease the level of LDL-C,TC and TG by 24.9%(P < 0.01),16.1%(P < 0.01)and 29.7%(P < 0.001),respectively.2.According to the criteria of FD > 1.2 or FD < 5/6 and P < 0.05 as well as the purpose for our study,Gal-3,one of the proteins with the most significant changes,was selected.Compared with control group,Gal-3 was increased by 2.18 times in HFD group,and decreased by 0.77 times with the administration of quercetin.The result of immunohistochemistry also showed that quercetin could effectively reduce the expression level of gal-3,and the expression trend of NLRP3 was consistent with that of gal-3.3.In vitro,when RAW264.7 cells were treated with ox-LDL,the expression of Gal-3 could be up-regulated significantly by 248.1%(P < 0.05),meanwhile NLRP3,caspase-1,IL-1β were up-regulated by 349.5%(P < 0.05)、401.7%(P < 0.05)、203.4%(P < 0.05),respectively.Accordingly,the administration of quercetin could reduce the inflammatory response.4.In vitro,it showed that administration of quercetin could significantly reduce the intracellular TC,TG by 47.9%(P < 0.001),22.9%(P < 0.01),respectively.And the inhabitor of Gal-3 could also significantly reduce TC,TG by 43.8%(P < 0.01),23.3%(P < 0.01),respectively.Oil Red O staining showed that both quercetin and the inhibitor of gal-3 could effectively reduce lipid retention in cells,which could suppress the formation of foam cells from macropages.5.The inhibitor of Gal-3 could reduce inflammatory responses and down-regulate the expression of NLRP3,pro-caspase-1,caspase-1,pro-IL-1β and IL-1β by 48.3%(P < 0.01),37.1%(P < 0.05),81.9%(P < 0.001),41.35%(P > 0.05),and 42.9%(P < 0.01),respectively.Recombinant Gal-3(rGal-3)could aggravate the inflammatory response and activate the NLRP3 inflammasome.Quercetin administration could inhibit the activation of NLRP3 inflammasome and reduce the expression of NLRP3,pro-caspase-1,caspase-1,pro-IL-1β,IL-1β by 57.1%(P < 0.001),55.6%(P < 0.001),62.5%(P < 0.01),45%(P < 0.001),57%(P < 0.001),indicating that quercetin was involved in the Gal-3-NLRP3 inflammasome signaling pathway to regulate the cellular inflammatory response process.Conclusions: Quercetin may reduce the inflammatory response by mediating the Gal-3-NLRP3 inflammasome signaling pathway,thus having a certain protective effect on atherosclerosis.