Molecular Mechanism Of Artemisinin Inhibiting The Vascular Smooth Muscle Cell Phenotype Switching And Attenuating The Development Of Atherosclerotic Lesions

Objective:Atherosclerosis?AS?is a major pathological process underlying coronary artery disease,cerebral ischemic stroke,and peripheral vascular disease that has been the leading cause of death globally.Despite the advances in drug therapy to reduce hypertension and lower lipids,current clinical interventions remain insufficient for the treatment of atherosclerotic diseases.Due to its complex pathological process,AS continues to impose immense health and economic burdens on society.Many cell types are involved in AS and become dysfunctional during its development and progression.Recently,studies applying the emerging techniques of lineage-tracing and single cell sequencing for unbiased cell identification and tracking have demonstrated that 40–70% of cells in plaques originate from vascular smooth muscle cells?VSMCs?.Furthermore,these cells undergo dedifferentiation and are converted to multiple alternative phenotypes during AS progression,which plays an important role in its development.Therefore,it is necessary to find a drug that can target and modulate the processes involved in AS.Recently,the non-antimalarial effect of artemisinin?ART?has attracted increasing attention from researchers.In the antimalarial clinical application of ART,it has been found to have a mitigating effect on autoimmune diseases.These discoveries have aroused the interest of researchers in ART's therapeutic effect in inflammatory diseases.However,little work has been performed on the therapeutic effect of ART on AS.With the rapid development of high-throughput sequencing technology in recent years,researchers can obtain gene expression profiles for different tissue cells.This can provide comprehensive gene information to investigate the effects and potential targets of drugs for the therapy of diseases.Circular RNA?circ RNA?is a novel class of endogenous noncoding RNAs that do not have the 5' end cap and the 3' poly?A?tail.Circ RNA can form a closed covalent loop structure via precursor m RNA back-splicing.It has been found that circ RNA has high species conservation and tissue specificity and it is not easily cleaved with a nuclease.The intrinsic characteristics of circ RNA suggest that disease-associated circ RNAs are promising diagnostic biomarkers.Circ RNA plays a variety of important biological roles in the process of cell growth,development,and differentiation.However,the functions of most circ RNAs in cardiovascular diseases remain largely unexplored.This study aimed to investigate the pharmacological effect of ART on AS and its underlying mechanism of action.RNA-Seq of the mouse aortas was performed to get an overall in-depth picture of the changes that have taken place at the molecular level.Combined with bioinformatics analysis,the pharmacological mechanism and targets of ART were explored for the anti-AS treatment.By analyzing the expression profile of circ RNA in AS vessels and its function in vascular smooth muscle,the molecular mechanism of ART in anti-AS treatment was investigated.The purpose of this study was to provide a theoretical basis for expanding new clinical indications of ART and a new treatment approach for AS.Methods:In this study,8-week-old male Apo E^-/-mice were selected to create an animal model of AS.Two different doses of ART at 50 and 100 mg/kg/d were administered orally for intervention.AS lesion sizes were determined using Oil Red O and HE staining.Transcriptome sequencing technology?RNA-Seq?was used to detect the expression profile of mouse aorta after treatment with ART.The bioinformatics analysis methods were used to evaluate differentially expressed genes and to perform functional annotation enrichment analysis.The expressions of contractile phenotype markers in the mouse aorta in different treatment groups were detected by q RT-PCR and western blot.MOVAS cells were selected to establish a VSMC phenotype switching model in vitro.Murine platelet-derived growth factor-BB?PDGF-BB?was used to stimulate VSMC proliferation,migration,and phenotypic switching from a contractile phenotype to a pathological synthetic phenotype.The intervention group was pretreated with ART for a further 2 h before exposure to PDGF-BB.Cell proliferation and migration capacity was investigated using wound healing,Transwell,and CCK8 assays.The expressions of contractile phenotype markers in different treatment groups were detected by western blots.The bioinformatics analysis was used to detect the differentially expressed circ RNAs between AS and ART groups in aortic tissue.The q RT-PCR was used to verify the differentially expressed circ RNAs in the mouse aorta among different groups.The subcellular localization of differentially expressed circ RNA in VSMCs was determined using the fluorescent in situ hybridization?FISH?technique and nuclear plasma separation assay.MOVAS cells were transfected by lentivirus to construct the mmu_circ₀000231 overexpression and knockdown cell lines.Then,overexpression and knockdown of mmu_circ₀000231 were performed to observe the effect of mmu_circ₀000231 on the proliferation capacity of smooth muscle cells.The bioinformatics analysis was used to predict the targeted mi RNA.The dual luciferase reporter assay was used to demonstrate the binding of mmu_circ₀000231 and mi R-125b-5p.The mi R-125b-5p inhibitor was transfected to knockdown the mmu_circ₀000231 cell line,which was then used to detect the proliferation capacity via the CCK-8 assay.ART was added to the mmu_circ₀000231 overexpression cell line.The proliferation capacity of VSMCs in each treatment group was assessed using the CCK8 assay.Results:The mice treated with ART for 8 weeks had a significantly smaller plaque area?0.19 ± 0.02 mm2,AS group;0.11 ± 0.01 mm2,ART-L group;0.10 ± 0.01 mm2,ART-H group?.RNA-Seq of aortic tissue revealed a distinct change in gene expression patterns after the mice were treated with ART.The bioinformatics analysis?GO,KEGG,and GSEA?demonstrated that the most prominently enriched pathway was a set of genes involved in the VSMC contractile function.Validation in larger samples found that m RNA and protein expression levels of VSMC contractile phenotype markers ?SMA,SM22?,calponin1,and SMMHC in ART-treated mice were significantly increased?P<0.05?.Using an in vitro cell model,it was demonstrated that ART could effectively reverse PDGF-activated MOVAS migration and proliferation and elevate the level of proteins involved in the contractile phenotype.RNA-Seq of aortic tissue showed that mmu_circ₀000231 was the most significantly downregulated circ RNA in the ART-treated group.The FISH technique and nuclear plasma separation assay showed that mmu_circ₀000231 was mainly distributed in the cytoplasm.The proliferation ability was reduced in the constructed VSMCs?P<0.05?,which have been knocked down with mmu_circ₀000231.Dual luciferase reporter assay results showed that mmu_circ₀000231 could target mi R-125b-5p and confirmed their binding site sequence.After simultaneously knocking down the expression of mmu_circ₀000231 and mi R-125b-5p in VSMCs,the effect of mmu_circ₀000231 on the proliferation capability of VSMCs was weakened?P<0.05?.The ART treatment group reduced the expression of mmu_circ₀000231 in VSMCs.After the overexpression of mmu_circ₀000231,the anti-proliferative effect of ART on VSMCs was reduced?P<0.05?.Conclusion : 1.ART attenuated the development of AS lesions in Apo E^-/-mice.Transcriptome sequencing analysis indicated that the VSMC contraction functional set was the most significantly changed pathway after ART intervention;2.ART suppressed PDGF-induced VSMC migration,proliferation,and de-differentiation.ART also inhibited VSMC phenotype switching;3.Mmu_circ₀000231 promoted the proliferation ability of VSMCs by binding mi R-125b-5p.ART inhibited the proliferation ability of VSMCs by negatively regulating mmu_circ₀000231.