The Effect And Mechanism Of Urolithin A On Macrophage-mediated Inflammation And Atherosclerosis In Mice

Urolithin A（Uro A）exhibits anti-inflammation activity in inflammatory cell models.Therefore,there are many researches suggesting that Uro A may have an important protective role in inflammatory disease.Atherosclerosis,is increasingly considered as a chronic inflammatory disease.In the development process of atherosclerosis,macrophages induce the expression and secretion of inflammatory cytokines,which,in turn accelerates the development of atherosclerosis.Therefore,the inhibition of macrophages-mediated vascular inflammation would benefit atherosclerosis therapy.In this study,we investigated the inhibition effect of Uro A on macrophages-mediated inflammation and the potential mechanism,and determined the effect of Uro A on atherosclerosis mouse model for further application that targets inflammation inhibition to treat atherosclerosis.Besides,we also investigated whether Uro A affected gut microbiota composition and try to provide a novel therapy that relates microbiota with atherosclerosis,meanwhile we compared the effect of punicalagin and simvastatin on gut microbiota with the effect of Uro A.In cell model,molecular biological experimental technique was used to measure cell inflammation cytokines expression and protein activation level,or estimates whether autophagy flux was promoted by Uro A in lipopolysaccharide（LPS）-induced macrophages.In animal model,atherosclerosis was established on high fat-induced Apo E^-/-mice,and Uro A was orally administrated at different time point for early and advanced atherosclerosis.By measuring blood lipoprotein level,plaque size,plaque cell construction,we discussed the effect of Uro A on early and advanced stage atherosclerosis.Beside,we collected mouse faeces for 16s r DNA amplicon sequencing.The main results of our study are introduced below:（1）Uro A inhibited LPS-induced macrophage inflammation.J774A.1 cell line was used and stimulated with LPS to induce inflammation.And results showed that Uro A down-regulated the m RNA expression of inflammation cytokines including CXCL10,i NOS,COX2,IFN-βin a concentration dependent manner.Uro A also inhibited TNF-αsecretion and the activation of NLRP3 inflammasome.Beside,Uro A down-regulated the phosphorylation level of MAPK-NF-κB pathway and promoted autophagy.These results suggested that Uro A inhibited LPS induced inflammation via multiple pathway including the inhibition of MAPK pathway and NLRP3 inflammasome,and promotion of autophage flux.（2）Uro A attenuated high fat-induced atherosclerosis on Apo E^-/-mice.Atherosclerosis model was established by feeding Apo E^-/-C57BL/6 mice with high-fat diet,and Uro A gavage administration（50 mg/kg/d）was performed at week 1 and week 9 of the high-fat diet for 8 weeks,respectively,to establish early and advanced atherosclerosis models.The results showed that Uro A had better inhibition effect on early atherosclerosis than on advanced atherosclerosis.Uro A significantly decreased plaque size in aorta especially in aortic sinus,inhibited the proliferation of smooth muscle cell and infiltration of macrophages.In advanced atherosclerosis,Uro A decreased plaque size in whole aorta,but showed no inhibition effect on plaque development around aortic sinus.Neither body weight nor blood lipid level was affected by Uro A in both group.（3）Uro A up-regulated the abundance of Actinobacteria and down-regulated the abundance of Deferrbacteroides.Intestinal microbial composition of early atherosclerosis was analyzed by 16S r DNA amplicon sequencing.Results showed that compared with high-fat diet intervention group,Uro A intervention showed inhibition effect on most gut microbiota growth and decreased the microbiota composition diversity.Especially,Uro A intervention increased the abundance of Actinobacteria as well as Firmicutes,and decreased the abundance of Deferrbacteroides,which was up-regulated by high-fat diet,as well as Bacteriodetes.Both puncalagin and simvastatin intervention increased the diversity of gut microbiota that was decreased by high-fat diet or Uro A intervention.Besides,puncalagin and simvastatin down-regulated the abundance of Actinobacteria.