Anti-atherosclerosis Effect Of Zedoarondiol By Regulating Monocyte Migration And Adhesion

Atherosclerosis（AS）is a chronic inflammatory reaction process,which is one of the main causes of death from cardiovascular diseases.In the early stage of AS plaque formation,monocytes in circulating blood undergo migration under the action of chemokines and adhesion molecules,and are recruited to the site of activation,infection or injury,and then exude to the intima of blood vessels through adhesion to the endothelium.Subsequently,they differentiate into macrophages,which on the one hand promote the secretion of inflammation and cytokines,and on the other hand phagocyte and modify inflammatory lipid particles to form foaming cells,inducing and activating downstream effects,thereby promoting the progression of AS diseases.Because the action sites of monocytes during the occurrence of AS are mainly outside the vascular lumen,the ability of migration and adhesion of monocytes is the key to their function.Zedoarondiol is a natural sesquiterpene compound isolated from zedoary,the representative medicine of blood dispersal.Previous studies have found that Zedoarondiol can inhibit inflammatory response,protect endothelial cells,reduce smooth muscle cell proliferation and other pathological links related to AS.However,whether Zedoarondiol play an anti-AS role by regulating the migration and adhesion process of monocytes has not been reported.Based on this,we propose the hypothesis that Zedoarondiol has an anti-AS effect,and the mechanism is related to its regulation of monocyte migration and adhesion.Based on this hypothesis,this study first explored the anti-AS mechanism of Zedoarondiol based on network pharmacology.The effects of Zedoarondiol on atherosclerotic plaque formation,leukocyte migration and adhesion in ApoE-/-mice were observed using ApoE-/-mice AS model.The HUVECs model injured by ox-LDL and THP-1 monocyte migration model induced by CCL2 were used to observe the effect of Zedoarondiol on monocyte migration and adhesion to endothelial cells.Singlecell RNA sequencing and biological analysis were performed on ApoE-/-mouse aorta cells to explore the key genes or pathways that regulate the migration and adhesion to endothelial cells of monocytes.The molecular mechanism of Zedoarondiol against AS was verified by antagonizing the expression of key genes or pathways in vitro.Part 1 The mechanism of Zedoarondiol in the treatment of atherosclerosis was studied based on network pharmacologyObjective:To explore the potential target and intervention approach of Zedoarondiol,an effective component of zedoary turmeric in the treatment of AS.Methods:According to the 3D structure of Zedoarondiol,the action targets of Zedoarondiol were obtained,and the potential therapeutic targets of Zedoarondiol for AS were obtained through the intersection of Zedoarondiol with disease targets of AS.PPI network of potential therapeutic targets of Zedoarondiol for AS was constructed by String 11.0.Gene function enrichment analysis and visualization were performed by Funrich₃.1.3,and signal pathway enrichment analysis was performed by David V6.8 database.The target and pathway information of Zedoarondiol were imported into Cytoscape_v3.7.2 software to construct the network of "drug components-targetspathway" for Zedoarondiol in the treatment of atherosclerosis.Results:The target of Zedoarondiol was intersected with the target of disease,and 105 potential target of Zedoarondiol in the treatment of AS were obtained.Through the construction of PPI network,the top ten genes with access value were:ALB,SRC,HRAS,EGFR,MAPK8,IGF1,HSP90aa1,MMP9,CASP3,and MAPK14.GO analysis showed 101 related biological processes with statistical significance.As related biological processes mainly involved EGFR-dependent endothelial signal transduction,P38 MAPK signaling pathway,TGF-β signaling pathway,integrin kinase-related pathway,TNF signaling pathway,etc.KEGG analysis showed that there were 78 signaling pathways related to potential targets,among which the pathways related to the pathogenesis of AS mainly included PI3K-Akt signaling pathway,adhesion junction-related pathway,VEGF signaling pathway,MAPK signaling pathway,chemokine signaling pathway,etc.The "drug component-target pathway" network showed that a total of 21 signaling pathways had 197 associations with 23 genes of atherosclerotic genes.Conclusions:Zedoarondiol may regulate biological processes such as P38 MAPK signal transduction,integrin kinase-related signal transduction,EGFR-dependent endothelial signal transduction,TGF-β signal transduction,TNF signal transduction,etc.The occurrence and development of AS could be interfered by MAPK signaling pathway,PI3K-Akt signaling pathway,adhesion signaling pathway and chemokine signaling pathway.Part 2 Effects of Zedoarondiol on atherosclerotic plaques in ApoE-/-miceObjective:To observe the effect of Zedoarondiol on reducing atherosclerosis.Methods:AS model was established by high fat feeding ApoE-/-mice.The experiment was divided into 4 groups:model group（n=12）,Zedoarondiol（n=12）administration group（n=12）,atorvastatin calcium tablet（n=20）positive control group（n=12）and C57BL/6 normal control group（n=12）.After 8 weeks of high-fat feeding,continuous gavage was given for 10 weeks.The changes of blood lipid levels in mice were detected by biochemical method.The expression levels of chemokine MCP-1 and inflammatory cytokines TNF-α,IL-6,IL-1β and IL-17A were detected by flow cytometry.The formation of AS plaques in full-length aorta and aortic root was assessed by oil red staining.Microcirculation method was used to detect the adhesion level of leukocytes and endothelial cells.The expression levels of adhesion molecules ICAM-1 and VCAM-1 were detected by ELISA.Results:After 10 weeks of administration,compared with model group,serum TC and TG contents in Zedoarondiol group were decreased（P<0.05）;Compared with model group,serum LDL-C content of Zedoarondiol group was decreased and serum HDL-C content was increased,but there was no significant difference compared with model group（P>0.05）.Zedoarondiol significantly down-regulated the levels of chemokine MCP-1（P<0.001）and inflammatory cytokines TNF-α and IL-1β（P<0.05）.Compared with model group,serum IL-6 and IL-17A were decreased in Zedoarondiol group,but there was no statistical difference（P>0.05）.After 10 weeks of administration,oil red staining of frozen sections of aorta and aortic root showed that compared with model group,the plaque area of aorta and aortic root in Zedoarondiol group was significantly reduced（P<0.05）.The results of microcirculation showed that Zedoarondiol could increase the movement rate of white blood cells（WBC）in the mesenteric venules of ApoE-/-mice（P<0.05）,and reduce the number of WBCendothelial cell adhesion（P<0.05）.Compared with model group,the expression levels of serum adhesion molecules ICAM-1 and VCAM-1 in Zedoarondiol group were significantly decreased（P<0.001）.Meanwhile,Elisa showed that Zedoarondiol could also reduce the expression of ICAM-1 and VCAM-1 in vascular endothelium（P<0.05）.Conclusions:Zedoarondiol inhibited the ApoE-/-mice induced by high fat diet,reduced serum TC,TG and inflammation factors such as MCP-1,IL-1β,IL-10,TNF-α,reduced the plaque formation of aorta and aortic root in ApoE-/-mice,improved white blood cells movement rate and white blood cells adhesion to endothelial cells in microcirculation,reduced serum adhesion molecules VCAM-1 and ICAM-1.Part 3 Effects of Zedoarondiol on monocyte migration and adhesion to endothelial cellsObjective:To observe the effects of Zedoarondiol on THP-1 monocyte migration and adhesion to endothelial cells induced by chemokine CCL2.Methods:Monocyte migration models induced by chemokine MCP-1（CCL2）were established.The migration of monocytes was detected by Transwell assay.OxLDL（50μg/mL）was added to HUVECs for 24 h to establish endothelial cell injury model.The effect of Zedoarondiol on the monocytes adhesion to endothelial cells was detected by immunofluorescence assay.The effects of Zedoarondiol on the expression of endothelial cell adhesion molecules VCAM-1 and ICAM-1 were detected by Western Blot.Results:Compared with model group,Zedoarondiol 20μg/mg significantly reduced the migration of monocytes under the action of chemokine CCL2,and the migration rate decreased by 62%（P<0.001）and 47%（P<0.001）,respectively.Compared with ox-LDL group,Zedoarondiol（20,40μg/mg）decreased the adhesion reaction of THP-1 cells to HUVECs（P<0.05）.Compared with ox-LDL group,Zedoarondiol could down-regulate the protein expression of VCAM-1 and ICAM-1（P<0.05,P<0.001）.The expression of E-selectin was down-regulated by Zedoarondiol 40μg/mL（P<0.05）.Conclusions:Zedoarondiol inhibited the migration and adhesion of monocyte,and regulated the expression levels of endothelial cell adhesion molecules VCAM-1,ICAM-1 and E-selectin.Part 4 The molecular mechanism of Zedoarondiol against atherosclerosis based on single cell RNA sequencingObjective:To explore the molecular target of Zedoarondiol in inhibiting AS by single cell RNA sequencing and to clarify its molecular mechanism.Methods:The AS Model was established by ApoE-/-mice fed with high fat.The experiment was divided into three groups:C57BL/6 normal control group,Model group and Zedoarondiol（20mg/kg/d）group.After 8 weeks of high-fat feeding and 10 weeks of continuous intragastric administration,blood was extracted from the inner canthus of mice,the full-length aorta was dissected,and peripheral blood mononuclear cells（PBMC）and aortic single-cell suspension were prepared.According to the instructions of 10X,the number and activity of cells in cell suspension were detected and library construction was carried out,and then computer sequencing and bioassay were performed.Results:We set a threshold of 15%mitochondrial expression ratio,and obtained 24,004 cells after filtration.The first 20 PCA dimensions were selected in the cell clustering,and 9 cell groups were obtained by executing the FindCluster function.Marker genes of all cell groups were calculated using the FindAllMarkers function.Using the obtained high-quality marker genes,the obtained Cell groups were annotated as VSMC,T Cell,MACRO,B Cell,Fibro,Myeloid,Mono,Neut,EC by referring to Mouse Cell Atlas and Cell marker.Intergroup comparison of EC cells showed that the expression of VCAM1,FBLN5,COL5A2,COL18A1,RPS5 and other genes were significantly different from that of AS-related genes,which may be the key genes to regulate the reduction of AS by Zedoarondiol.Intergroup comparison of MONO cells revealed multiple signaling pathways related to AS,including chemokinechemokine receptor pathway,TNF signaling pathway,chemokine-mediated signaling pathway,NF-κB signaling pathway,etc.We took EC cells with subset and subset with higher resolution,and obtained CD36_EC and PECAM1_EC subsets.Mono cells were divided into Cluster 0,Cluster 1 and Cluster 2 subgroups.Functional analysis of EC cell subsets showed that the biological functions were mainly concentrated in cell-cell adhesion,cell-cell matrix adhesion,monocyte migration,endothelial cell migration equal adhesion and migration.Functional analysis of MONO cell subsets showed that they were mainly enriched in chemotaxis,including chemokine-mediated signaling pathways,ERK1 and ERK cascades,and chemotaxis of various cells.Cell communication was enriched in 75 EC and Mono cell communication pathways,among which 4 pathways were related to monocyte migration and adhesion,including CXCL,JAM,ICAM and VCAM signaling pathways.By further cell communication analysis of genes enriched in the pathway,the CXCL12/CXCR4 pathway may be the specific pathway through which Zedoarondiol regulates the migration of monocytes and adhesion to endothelial cells,thus playing an anti-AS role.Conclusions:CXCL12/CXCR4 pathway may be the specific pathway through which Zedoarondiol regulates the migration of monocytes and adhesion to endothelial cells.Part 5 The mechanism of Zedoarondiol against atherosclerosis by regulating CXCL12/CXCR4 pathwayObjective:To verify the mechanism of Zedoarondiol inhibits the chemotactic migration of monocytes and adhesion to endothelial cells by regulating CXCL12/CXCR4 pathway.Methods:Serum CXCL12 and CXCR4 levels of ApoE-/-mice were detected by ELISA.Transwell assay was used to detect the effects of Zedoarondiol on CXCL12induced THP-1 cell migration and adhesion to HUVECs.Western Blot and RT-qPCR were used to detect CXCR4 expression in THP-1 cells.Western Blot was used to detect the expression of key downstream proteins PI3K,AKT and NF-κB of CXCL12/CXCR4 pathway.Results:To verify whether the reduction of monocyte migration and adhesion by Zedoarondiol is related to the CXCL12/CXCR4 pathway,we first detected the expression level of CXCR4,and the results showed that Zedoarondiol decreased serum CXCR4 expression in ApoE-/-mice and CXCR4 protein expression in THP-1 cells induced by CXCL12.Transwell showed that Zedoarondiol inhibited CXCL12-induced THP-1 cell migration and adhesion to HUVECs,while CXCR4 inhibitor AMD3100 antagonized this effect.Detection of key downstream proteins of CXCL12/CXCR4 showed that Zedoarondiol reduced the expression of PI3K,AKT and NF-κB proteins by regulating the CXCL12/CXCR4 pathway.Conclusions:Zedoarondiol regulated the CXCL12/CXCR4 pathway,inhibited the protein expressions of downstream PI3K,AKT and NF-κB,thereby regulating the migration of monocytes and adhesion to endothelial cells and ultimately played an antiAS role.