Study On Artesunate Regulating Atherosclerotic Plaque Inflammasome And Its Mechanism

Among the many pathogenic factors of coronary artery disease,atherosclerosis is currently recognized as the primary factor,so scholars have carried out long-term scientific research on its mechanism.In the exploration of its pathogenesis for more than a hundred years,many related theories have been gradually proposed.However,due to the diversity of such diseases,there is no perfect and effective treatment at present,which still needs to be explored in depth.Previous studies have proved that artesunate has an inhibitory effect on inflammatory response and immunity,and the activation of NLRP3 inflammasome is also involved in the development of atherosclerosis.Thioredoxin interacting protein(TXNIP)can affect the inactivation of NLRP3 inflammasome and alleviate inflammatory response.Bioinformatics analysis confirmed that mir-16-5p was down regulated in diabetes nephropathy,and mir-16-5p could also regulate atherosclerosis genes.In this thesis,the mechanism of artesunate in modulating atherosclerotic plaque inflammasome was studied through clinical,in vivo and in vitro experiments.In order to clarify the role of miR-16-5p in atherosclerosis,peripheral blood samples were collected from 115 patients with coronary heart disease and 33 healthy people to detect the levels of inflammatory factors and the expression level of miR-16-5p and TXNIP in monocytes,and to conduct correlation analysis.The interaction between miR-16-5p and TXNIP was analyzed by online prediction software,and verified by double luciferase activity detection and q RTPCR experiment.The results showed that the levels of serum IL-1β and IL-18 disease were significantly increased,the expression of miR-16-5p in peripheral blood monocytes was significantly decreased,and the expression of TXNIP m RNA was significantly increased in patients with coronary heart.The level of peripheral blood IL-1β and IL-18 in patients with coronary heart disease was positively correlated with the severity of coronary heart disease.The level of miR-16-5p expression in peripheral blood monocytes was negatively correlated with the level of TXNIP expression,and both the level of miR-16-5p expression and the level of TXNIP expression were correlated with the severity of coronary heart disease.Moreover,there is a binding site between miR-16-5p and 3′UTR of TXNIP.TXNIP is a downstream negative regulatory molecule of miR-16-5p,and miR-16-5p could regulate the expression level of TXNIP m RNA.In order to further clarify the downstream mechanism of TXNIP,the open data of atherosclerosis collected from GEO database were used to analyze the downstream mechanism of TXNIP by bioinformatics.WGCNA was used to screen gene modules closely related to atherosclerosis,and query the gene modules most related to TXNIP expression.KEGG pathway enrichment analysis was used to screen specific pathways related to TXNIP related gene modules.Whether TXNIP gene expression was closely related to NLRP3 inflammatory body pathway was analyzed by atherosclerosis related immune abundance query and TXNIP related immune abundance query.The results showed that magenta,purple and red modules were most closely related to the expression of TXNIP.TXNIP related module genes were enriched in B cell receptor signal pathways.The expression of TXNIP was mainly related to immune,inflammatory and other processes.MCPcounter and CIBERSORT were used to analyze the immune infiltration.It was found that the expression of TXNIP was mainly related to monocytes and M2 macrophages.ss GSEA analysis showed that TXNIP gene expression was positively correlated with NLRP3 inflammatory body pathway.In order to further explore the molecular pathway mechanism of artesunate in anti inflammation and anti atherosclerosis,cell experiments were conducted with THP-1 differentiated macrophages.The macrophages were given NLRP3 inflammatory body activator and artesunate,and the levels of inflammatory factors,ASC polymers,TXNIP and the interaction between TXNIP and NLRP3 were detected.The results showed that the expression levels of inflammatory factors downstream of NLRP3 in the supernatant of macrophage culture and in cells were significantly increased.However,artesunate decreased the expression of inflammatory factors downstream of NLRP3 and the production of ASC polymers increased by NLRP3 activator.In addition,artesunate reduced TXNIP expression and the interaction between TXNIP and NLRP3.These results indicated that artesunate inhibited the activation of NLRP3 inflammatory bodies through TXNIP,thereby inhibiting release of macrophage IL-1β and IL-18.In order to further clarify the role of miR-16-5p in the regulation of inflammatory response via TXNIP by artesunate,lentivirus vectors including mimic NC,miR-16-5p mimic,inhibitor NC,miR-16-5pacifier were constructed and transfected into macrophages.Rat atherosclerosis model was established,and artesunate was administered to rats by gavage.The area of atherosclerotic plaque in the aorta of rats was statistically analyzed,and the levels of inflammatory factors and blood lipids were detected in each group of rats,so as to verify the regulatory role of artesunate and miR-16-5p in vivo.The results showed that artesunate inhibited the NLRP3 inflammatory body pathway through miR-16-5p targeting TXNIP,thereby inhibiting the production of inflammatory factors in THP1 induced differentiated macrophages.The levels of inflammatory factor IL-1β and IL-18 in blood of atherosclerosis model rats were higher than those in control group,while artesunate decreased the inflammatory response in a dose dependent manner.In addition,artesunate significantly reduced the area of atherosclerotic plaque and blood lipids.However,interfering with miR-16-5p in rats abolished the improvenment of atherosclerosis by artesunate.The above results indicate that artesunate can reduce the area of atherosclerotic plaque in aorta of atherosclerotic rat model in a dose-dependent manner by up regulating miR-16-5p.Artesunate can reduce blood lipid,lipid accumulation and atherosclerotic plaque formation in a dose-dependent manner by up regulating miR-16-5p.In conclusion,through bioinformatics,clinical,in vivo and in vitro studies,this thesis demonstrated that artesunate inhibited NLRP3 inflammatory bodies through miR-16-5p targeting TXNIP,thereby inhibiting the production of inflammatory factors,reducing blood lipids and delaying the process of atherosclerosis.This study provides a theoretical basis for the treatment of atherosclerosis.