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Objective: To analyze the contents of different kinds of fatty acids in carotid atherosclerotic plaques. Methods: A total of 24 patients who underwent carotid endarterectomy at the Second Affiliated Hospital of Naval Medical University from October 2021 to September 2022 due to moderate and severe carotid artery stenosis were retrospectively enrolled, including 20 males and 4 females, with a median age[ M ( Q 1 , Q 3 )] of 68.5 (63.5, 72.3) years. According to the symptoms of cerebral ischemia, the patients were divided into a symptomatic group (12 cases) and an asymptomatic group (12 cases). Regarding the pathological characteristics, the patients were divided into a stable group (14 cases) and a vulnerable group (10 cases) according to carotid plaque pathology scores. The expression differences of different types of fatty acids in carotid plaques were analyzed by targeted fatty acid metabolomics technology based on ultra-performance liquid chromatography-mass spectrometry (UPLC-ESI-MS/MS) analysis. Results: In the 24 samples, the median amount of fatty acids [ M ( Q 1 , Q 3 )] was 1 113 (330, 5 897) ng/g. A total of 13 medium and long-chain fatty acids were detected, including saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids. The content of saturated fatty acids was 584 (290, 9 888) ng/g, accounting for the highest proportion of 51.8%. The content of polyunsaturated fatty acids was 1 444 (393, 4 264) ng/g, accounting for 44.4%. The content of monounsaturated fatty acids was 2 793 (1 558, 3 247) ng/g, accounting for 3.8%. The contents of linoleic acid, α-linolenic acid and oleic acid in carotid plaques in the symptomatic group were 1 760 (581, 3 006), 682 (527, 886) and 2 081 (1 358, 2 907) ng/g, respectively, which were lower than those in the asymptomatic group 3 149 (2 226, 4 683), 1 423 (964, 2 270) and 3 178 (2 352, 3 993) ng/g (all P <0.05). The contents of linoleic acid, α-linolenic acid and oleic acid in carotid plaques in the vulnerable group were 1 537 (588, 2 921), 649 (477, 850) and 2 081 (1 129, 2 831) ng/g, respectively, which were lower than those in the stable group 3 149 (2 047, 4 416), 1 423 (940, 2 184) and 3 091 (2 201, 3 973) ng/g (all P <0.05). There were no significant differences in the contents of 11, 14-eicosadienoic acid, γ-linolenic acid, eicosapentaenoic acid, arachidonic acid, erucic acid, margaric acid, pentadecanoic acid, stearic acid, dodecanoic acid and palmitic acid (all P >0.05). Conclusions: Saturated fatty acids are the main type in carotid plaques. The contents of oleic acid, α-linolenic acid and linoleic acid decrease in vulnerable plaques.

Atherosclerotic plaques belong to the common vascular disease in the aged, which rupture will lead to acute thromboembolic diseases, the leading cause of fatal cardiovascular events. Accumulating evidence indicates that the lncRNAs-miRNAs-mRNA regulatory network plays a critical role in atherosclerosis. Based on RNA sequencing (GSE207252), we constructed expression profiles of lncRNAs, microRNAs, and mRNA in the carotid plaque of atherosclerosis patients and analyzed differentially expressed genes (DEGs). We identified three candidate lncRNAs using two algorithms (LASSO and SVM-RFE): lnc&#95;GLRX3, lnc&#95;FGF7-5, and DISC1FP1). LNCipedia, TargetScan, and miRDB databases were used to predict target miRNAs of lncRNAs and target genes of miRNAs. Gene ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Set Enrichment Analysis (GSEA) analysis of DEGs was carried out using the R package clusterProfiler. A PPI network was constructed using the STRING website and visualized by Cytoscape. According to the "MCC" method of the plug-in cytoHubba in Cytoscape, ERCC4 was the top hub gene of the PPI network. We constructed a lncRNA&#95;FGF7-5/lncRNA&#95;GLRX3-miR-2681-5p-ERCC4 regulatory network for carotid plaque using lncRNA-miRNA and miRNA-mRNA pairs. Next, lncRNA&#95;FGF7-5 and lncRNA&#95;GLRX3 targeted miR-2681-5p directly to upregulate ERCC4 expression. Silencing of lncRNA&#95;FGF7-5 and lncRNA&#95;GLRX3 promoted apoptosis and TP53 expression in HUVECs treated with ox-LDL; however, these effects were reversed by ERCC4-overexpression. Taken together, these findings indicated that lncRNA&#95;FGF7-5 and lncRNA&#95;GLRX3 together reduced atherosclerosis-induced apoptosis of HUVECs via targeting miR-2681-5p to increase ERCC4 expression, thereby preventing the formation of carotid plaque and finally inhibiting atherosclerosis progression.

Objective: To explore a modified method for constructing a mouse model of instable carotid plaque and provide the mouse model for simulating the development of human instable carotid plaque. Methods: Twenty-four low density lipoprotein receptor deficiency (LDLr-/-), C57BL/6, male mice were randomized into two groups according to computer-generated table, tandem constriction of carotid artery+ high cholesterol diet (tandem surgery group, n= 12); sham surgery+ high cholesterol diet(sham surgery group, n= 12). After 12 weeks of consecutive feeding, murine carotid artery were collected and analyzed by carotid ultrasound, pathological examination to assess the formation and stability of atherosclerotic plaque. Results: No statistical difference in body weight and blood lipid level between the two groups was observed (all P> 0.05). After surgery, through ultrasound biomicroscopy, obvious stenosis at the two sites of tandem constriction and atherosclerotic plaque between the two sites were observed in tandem surgery group. By pathological examination, no plaque was formed in carotid artery in sham surgery group. The lipid area in the stable plaque of innominate artery in sham surgery group and the plaque of carotid artery in tandem surgery group were (4.8±0.6) ×10(4),(10.2±1.1)×10(4) μm(2), respectively. The difference was statistically significant between the two groups ( t= 12.023, P< 0.001). In addition, the thickness of fibrous cap in the above groups were (122.4±17.8), (41.3±20.2) μm, respectively. The difference was statistically significant either ( t= 9.224, P< 0.001). The region of necrotic core were (3.3±0.4)×10(4),(8.1±0.9)×10(4) μm(2).The difference was statistically significant as well ( t= 13.456, P< 0.001). The percentage of macrophages in innominate artery of sham surgery group and in carotid artery of tandem surgery group were (20.8±5.2)%, (38.6±6.4)%, respectively.The percentage of vascular smooth muscle cells were (32.5±7.3)%,(21.2±5.1)%, respectively ( t= 6.114,3.585, all P< 0.05). The results indicated that the plaque in tandem surgery group had severer inflammatory response. Conclusion: Through tandem constriction surgery upon carotid artery in LDL-/-mouse, instable carotid atherosclerotic plaque can be induced, which is less time-consuming, replicable and effective.