Exploring The Molecular Mechanism Of Siwu Decoction In Preventing And Treating Atherosclerosis From The Perspective Of Vascular Smooth Muscl

ObjectivesGlobally,about one third of all deaths in recent years are due to Cardiovascular Diseases(CVDs),and the number of CVD-related deaths is expected to rise to 23 million by 2030.Despite many key advances in the management and treatment of CVDs,CVDs claim more lives than all cancers combined.Atherosclerosis(AS)is the most important cause of CVDSrelated deaths,accounting for nearly 50%of all deaths in developed countries.In the earliest studies,atherosclerosis was generally considered to be an inevitable consequence of aging,which changed with the emergence of the concept of AS a "risk factor" for cardiovascular disease in epidemiological studies beginning in the 1940s.The pathogenesis of AS disease involves a variety of mechanisms.In addition to Endothelial Cells(ECS)damage,another key factor in the development of the disease is the phenotypic transformation of Vascular Smooth Muscle Cells(VSMCs).Under pathophysiological conditions such as injury,VSMCs change from contractile to synthetic,which usually has high proliferative and migratory capacities.Previous studies have shown that Siwu Tang(SWT)can affect the expression of downstream cyclins and regulate the proliferation of ECS through the G-protein coupled estrogen receptor(GPER)mediated pathway,thus generating the protective effect of ECS.In this study,VSMCs and apolipoprotein E(apoe)gene knockout mice were used to conduct related in vivo and experimental studies,and network pharmacological methods combined with GEO database mining were used to explore the mechanism of AS action of SWT.MethodsChapter 1:15 apoe-/-mice and 5 normal C57BL/6J mice of the same strain and week of age were fed high fat diet and maintenance diet respectively for 8 weeks.After 8 weeks,the apoe-/mice were randomly divided into three groups,namely model group,SWT group and positive drug group.After 4 weeks of intragastric gavage,the samples were collected and the body weight of the mice were monitored.The influence of S WT on the morphology and area of AS plaque was analyzed by observing the plaque morphology at the aortic arch of mice in each group,the fat distribution in the plaque and the immunohistochemical staining of α-SMA.The influence of SWT on the level of blood lipid in mice was investigated by detecting the four levels of blood lipid in peripheral blood serum of mice in each group,and the aspartate aminotransferase in peripheral blood serum of mice was tested.AST and alanine aminotransferase(ALT)levels to observe the damage to the liver.Next,Western Blot was used to detect the phenotypic related molecular expression levels of VSMCs in aortic tissues,to explore the molecular mechanism of the prevention and treatment effect of SWT on AS.Chapter 2:Main active compounds of Siwu Tang were screened from TCMSP database,and their corresponding targets were retrieved through TCMSP database and Swisstarget platform.The genes related to AS were screened by GEO database combined with OMIM and DRUGBANK database.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were used to enrich the targets of SWT against AS.Protein-protein Interaction(PPI)networks were constructed.According to the results of network pharmacological analysis,CCK8 assay,Nitric Oxide(NO)assay and Western Blot assay were used to verify the effect of SWT on Oxidized Low Density Lipoprotein.Influence of VSMCs after ox-LDL)induction.Chapter 3:In vitro experiment results showed that 25-1500μg/mL SWT had no significant effect on the cell viability of VSMCs.However,after ox-LDL-induced proliferation,VSMCs treated with high concentration SWT(1000μg/mL),medium concentration SWT(500μg/mL)and low concentrationSWT(100μg/mL)could inhibit the excessive proliferation to a certain extent compared with ox-LDL-induced cells.The cell cycle results showed that SWT could reduce the proportion of cells in G2 phase induced by ox-LDL,and increase the proportion of cells in S phase induced by ox-LDL.In addition,SWT can increase the NO level and eNOS protein expression of VSMCs decreased after ox-LDL induction.ResultsChapter 1:SWT had no significant effect on the distribution of arterial plaques in apoe-/mice,but it could reduce the levels of triglyceride and cholesterol in serum of apoe-/-mice,and had no significant effect on the levels of ALT and AST in serum of mice.SWT can regulate the expression levels of phenotypic related molecules(α-SMA,OPN and SM22α)of VSMCs,and inhibit the phenotypic transition to secretory type of apoe-/-mice VSMCs.Chapter 2:A total of 34 major active compounds of SWT were screened by TCMSP database,corresponding to 231 potential targets,and 40 targets of SWT for the prevention and control of AS were obtained.Biological processes involved in GO enrichment analysis include inflammatory response,regulation of blood pressure,and response to estrogen.KEGG enrichment analysis indicated that SWT mainly acted on TNF-α and NF-κB pathways.PPI network analysis suggested that IL6,TP53,TNF,PTGS2,MMP9 and NOS3 were the core target networks in the network.Chapter 3:In vitro experiment results confirmed that SWT can inhibit ox-LDL-induced excessive proliferation of VSMCs to a certain extent,and can inhibit ox-LDL-induced increase in the proportion of G1 phase cells and decrease in the proportion of S phase cells.In addition,the level of NO produced by VSMCs decreased after ox-LDL induction was significantly increased,which could increase the expression of eNOS protein decreased after ox-LDL induction.ConclusionIn this study,both in vivo and in vitro experiments and combined with network pharmacology were used to obtain the possible core targets and molecular pathways of SWT for the prevention and treatment of AS,and the ox-LDL-induced VSMCs model was used for verification,showing that SWT can inhibit the abnormal proliferation of VSMCs and affect the progress of cell cycle.And enhance the expression level of important molecules including NO and eNOS;In vivo experiments showed that SWT could promote the stability of its plaque.Molecular experiments indicated that the effect of SWT on the prevention and treatment of AS may be played by inhibiting phenotype conversion of vascular smooth muscle,providing reference for further studies on the prevention and treatment of atherosclerosis by SWT.