| Atherosclerosis（AS）is the underlying pathological process of severe cardiovascular and ischemic stroke（IS）.Lipid deposition and fibrosis in arteries and inflammation are important triggers of atherosclerosis progression.With the development of social economy and the improvement of material level,hyperlipidemia,hyperglycemia and hypertension caused by high energy intake and high fat diet are the causes of many cardiovascular diseases.The deposition of lipids in blood vessels,such as cholesterol,and the inflammatory reaction caused by cholesterol are important factors inducing atherosclerosis.Macrophages play a role in the development of many diseases by enhancing the immune response.Macrophage inflammation is an important physiological process in the formation and rupture of atherosclerotic plaques.Micro RNA（mi RNA）is a kind of short endogenous non-coding ribonucleic acid（RNA）,which is composed of about 22 nucleotides.Its function is to regulate gene expression after transcription.It can be seen in a variety of acute and chronic inflammatory reactions in human body.mi R-223-3p is an important mi RNA involved in the inflammatory response in the human body.Studies have shown that inhibition of mi R-223-3p expression in macrophages promotes inflammation,while promoting diet-induced adipose tissue inflammation.Therefore,mi R-223-3p may play a role in atherosclerotic disease process by regulating macrophage inflammatory response.Objective:This study deeply explored the function of mi R-223-3p and studied its molecular mechanism.Methods:1.Venous blood and carotid artery tissues were collected from the patients included in the study,and they were divided into experimental group and control group according to whether there was atherosclerosis.The levels of mi R-223-3p in serum,carotid plaques or tissues were detected by q PCR,and the expression level of mi R-223-3p was quantified by 2-ΔCTwith U6 gene as internal control.The morphological differences between the experimental group and the control group were studied by Movat staining.2.Specific Pathogen-Free Organisms（SPF）mice were randomly divided into control group and high-fat diet group,which were fed with normal diet and high-fat diet（High-Fat Diets）,P.After 12 weeks of feeding,the aorta tissues of the two groups were taken to make paraffin sections（Formalin Fixed Paraffin Embedded,FFPE）,and the morphological differences of the aorta cells of the two groups were observed by Movat staining.3.The localization of mi R-223-3p in intimal macrophages（CD68+）and atherosclerotic lesions in unstable plaques was identified by Fluorescent in Situ Hybridization（FISH）.Apo E knockout（Apo E-/-）mice were transformed by tail vein injection with adeno-associated virus serotype 9 vector to overexpress mi R-223-3p.Movat staining method was used to stain the aortic root and muscular layer of mice to observe the difference of cell morphology.The expression ofα-actin,CD68+and tumor necrosis factor-α（TNF-α）in mi R-223-3p over-expressed macrophages was detected by q PCR.4.The potential genes and pathways of mi R-223-3p were speculated by bioinformatics analysis,and GSE34822 data sets related to AS were downloaded from public databases,and the target genes of mi R-223-3p were predicted by online tools,and the regulatory effects of mi R-223,3p on downstream target genes were studied by in vivo and in vitro experiments.In mi R-223-3p mimics and normal control（NC）mice,the levels of inflammatory cytokines and p-ERK1/2 expression were assessed by double immunofluorescence labeling and Western Blot.5.In vitro,mi R-223-3p mimics and NC were transferred into RAW264.7macrophages cultured in medium containing oxidized low density lipoprotein（ox-LDL）.Platelet activating factor C16（C16-PAF）,an ERK1/2 agonist,was added to the control group and the intervention group,and the protein levels of inflammatory cytokines were detected by Western blot.In this study,all experiments were independently repeated for 3 times,and the experimental data were statistically analyzed by Graph Pad Prism 9.02software,and the experimental data were expressed as mean±standard deviation（SD）.Differences between the control and experimental groups were assessed by one-way analysis of variance（ANOVA）or t-test（Student’s t-test）.For P<0.05,with statistical.Results:Compared with the control group,the expression level of mi R-223-3p in carotid artery plaque and venous serum of AS patients decreased by50%;there was a statistical difference in mi R-223-3p between stable plaque and unstable plaque.FISH results indicate that mi R-223-3p colocalizes with CD68+macrophages in unstable plaque AS lesions of AS patients.Compared with the NC group,the severity of atherosclerotic lesions in the aortic tissue of mice injected with mi R-223-3p mimics through the tail vein was significantly reduced,and the atherosclerotic lesion area was reduced from 3452±353μm2to 2292±192μm2;In the mi R-223-3p mimic group,the number of positive cells in the plaque area of macrophages（CD68+）was significantly reduced by nearly 50%,whileα-Actin（SMA）staining was increased by about 40%.ELISA results showed that the relative fluorescence density of TNF-αin the immunopositive plaque tissue of mice in the mi R-223-3p mimic group decreased from 1.05±0.04 to 0.76±0.03 compared with the NC group,indicating that mi R-223-3p inhibited the formation of as plaque by inhibiting the expression level of immune inflammatory factors in macrophages.According To the binding principle of mi RNA and m RNA,the target gene of mi R-223-3p was analyzed by online tools,and the results showed that mitogen-activated protein kinase kinase 1（Mitogen-activated protein kinase kinase 1,MAP2K1,MEK1）may be the target gene of mi R-223-3p.Phosphorylated extracellular signal-regulated kinase（Phosphory lated extracellular signal regulated kinase,p-ERK1/2）protein content was significantly reduced in macrophages transfected with mi R-223-3p mimics,The p-ERK1/2:total-ERK1/2 ratio decreased from 1.1 to 0.4,indicating that the mi R-223-3p mimetic reduced the activation of the MEK1/ERK2 signaling pathway in vitro and in vivo.Agonist of MEK1/ERK1/2 signaling（C16-PAF）reversed mi R-223-3p-mediated inhibition of MEK1/ERK1/2 signaling and inflammation.Conclusions:In conclusion,our results demonstrate that mi R-223-3p can negatively regulate the inflammatory response of macrophages through MEK1/ERK1/2 signaling pathway and play an important role in the progression of atherosclerosis.Therefore,mi R-223-3p becomes a new potential therapeutic target to prevent and alleviate atherosclerosis.To provide a theoretical basis for the pathogenesis of atherosclerosis and a possible therapeutic target. |
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