Circ＿0043837 Participates In Atherosclerosis By Regulating Oxidative Stress Of Macrophages

Objective:Atherosclerosis is a chronic inflammatory disease.The most common cause of cardiovascular and cerebrovascular diseases is the increase of plaque volume and the rupture of unstable plaque caused by the continuous progress of atherosclerosis.Macrophages play an important role in atherosclerosis.Circular RNAs(circ RNAs)are a new class of non-coding RNA,which can participate in the process of atherosclerosis through a variety of mechanisms.Previous studies have shown that oxidative stress of macrophages plays a key role in atherosclerosis.However,we previously found that the expression of circ＿0043837 was reduced in the plasma of patients with large atherosclerosis(LAA)type cerebral infarction.Therefore,our research aims to explore its function in macrophages and whether it can participate in the process of atherosclerosis by influencing the oxidative stress of macrophages.Methods:1、Human acute mononuclear leukemia(THP-1)cells were cultured in vitro and induced into macrophages by phorbol ester(PMA).The morphological characteristics of THP-1 cells and macrophages were observed using an inverted microscope.2 、 Use oxidized low density lipoprotein(ox LDLs)to treat macrophages to establish foam cell model,and use oil red O staining to verify the establishment of foam cell model.3、Utilizing Cirular RNA interactome(Circ Interatome)database to analyze circ＿0043837 for analysis,search for mi RNAs that bind to target genes,and use the Target Scan database to predict the target genes of mi RNAs,conduct GO and KEGG enrichment analysis on the target genes.4、The production of reactive oxygen species(ROS)in macrophages and foam cells were detected by fluorescence staining and microplate reader,real time fluorescence quantitative PCR(q RT PCR)was used to verify the expression of m RNA between interleukin-6(IL-6)and tumor necrosis factor α(TNF-α)in macrophages and foam cells.5、Through Lentivirus transfection verification,transfer lentivirus plasmid of carrying the circle＿0043837 gene into THP-1 cells,q RT-PCR validation of the circ＿0043837 differential expression after transfection.6 、 Construct control group(macrophage group),ox-LDL group,ox-N.C.group(blank plasmid group),and ox-circ＿0043837up group(overexpressing circ＿0043837 group),quantitative detection of ROS production between groups using fluorescence staining and enzyme-linked immunosorbent assay,verify the m RNA expression level of IL-6 and TNF-α through q RT-PCR.Results:1、THP-1 cells cultured in vitro were induced by PMA to form macrophages.Through inverted microscopy,THP-1 cells were observed to be circular,semi transparent suspended cells.After 24 hours of induction,most of the cells changed from suspended growth to adherent growth.After 48 hours of induction,almost all cells adhered to the wall and grew,mostly in an elliptical or irregular shape,with short extended pseudopodia.2、After the macrophages were treated with ox-LDLs to form foam cells,oil red O staining showed that it was consistent with the characteristics of foam cells,and the foam cell model was successfully constructed.3、GO function enrichment analysis showed that the functions of target genes related to oxidative stress included regulating the activities of guanosine triphosphate(GTP)enzyme and nucleoside triphosphatase.KEGG enrichment analysis showed that target genes involved in oxidative stress related pathways include energy metabolism and lipid metabolism.4、The results of fluorescence staining showed that the red fluorescence in the macrophage group was weak,while the red fluorescence in the foam cell group was strong.The quantitative detection results of the microplate reader showed that the ROS production in the macrophage group was lower than that in the foam cell group(P<0.05).QRT-PCR was used to verify the m RNA expression level of IL-6 and TNF-α in macrophages and foam cells were decreased.5、Lentivirus transfection verification results show that,inverted fluorescence microscope observation of visible green fluorescence,indicates carrying an upregulated circ＿0043837 gene of lentivirus plasmid was successfully transferred into and expressed green fluorescent protein.The q RT-PCR results showed that compared to the control group,circ＿0043837up group expression increased(P<0.05).6、Compared with ox-N.C.group,after up-regulating circ＿0043837 gene,the production of ROS in ox-circ＿0043837up group was reduced(P<0.05).Compared with ox-N.C.group,after up-regulating circ＿0043837 gene,the m RNA expression of IL-6、TNF-α in ox-circ＿0043837up group were decreased(P<0.05).Conclusion:1.In the foam cell model,the production of ROS was increased,Increased response to oxidative stress compared to before.2.After up-regulating circ＿0043837 gene,the m RNA expression of IL-6、TNF-αin ox-circ＿0043837up group were decreased,circ＿0043837 may be involved in regulating the inflammatory response of macrophages.3.After up-regulating circ＿0043837 gene,the production of ROS in ox-circ＿0043837up group was reduced,circ＿0043837 may be involved in regulating oxidative stress in macrophages.