Identification of genes and pathways related to atherosclerosis comorbidity and depressive behavior via RNA-seq and bioinformation analysis in ApoE-/- mice

Abstract

Background: Depression is an independent risk factor for atherosclerosis (AS), which can increase the risk of death and disability from AS. However, the mechanism of AS comorbidity with depression is complex.

Methods: ApoE^-/- and C57BL/6J mice were fed with a high-fat diet (model group, N=12 ♂) and a normal diet (control group, N=12 ♂). During the 15-week experimental period, the following tests were performed: coat color score, body weight, and sucrose preference tests (every 2 weeks); open-field test (1^st, 7^th, and 15^th weeks); and light/dark and tail suspension tests (15^th week). Oil Red O and hematoxylin and eosin (HE) stainings were used to assess the area of atherosclerotic status. The levels of triglyceride and total and low-density lipoprotein cholesterol in the serum and secretion of pro-inflammatory cytokines were determined using the enzyme-linked immunosorbent assay. The differentially expressed genes (DEGs) in the hippocampus and prefrontal cortex were screened by RNA-sequencing (RNA-seq) and analyzed using the Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations.

Results: Our findings showed that compared with C57 mice in the control group, ApoE^-/- mice in the model group gradually developed depression-like behavioral changes with elevated blood lipid concentrations, serum inflammatory factor levels, and atherosclerotic plaque formation in the thoracic aorta. Consequently, in the RNA-seq and bioinformatics analysis, the high expression of inflammatory chemokine genes was found in the hippocampus and prefrontal cortex area. The regulation of movement, feeding, and reproduction of the gene expression decreased.

Conclusions: These results indicate that when ApoE^-/- mice were fed a high-fat diet for 15 weeks, depression-like behavioral changes occurred with the formation of atherosclerotic lesions. The RNA-seq, combined with bioinformatics analysis, showed that this AS comorbidity with depressive behavior was associated with the high expression of inflammation-related genes and pathways in the hippocampus and prefrontal cortex.

Keywords: ApoE; Atherosclerosis (AS); RNA; depression.

Figure 1

Observations of general health and depression-like behavioral changes in mice. (A) Measurements of body weight. (B) Coat status test. (C) Sucrose preference test. The ratio of sucrose consumption was used as an index to evaluate the loss of anhedonia. (D) Open-field test, including the center zone time, central zone distance, and total distance. (E) Light/dark test, including the percentage of brightfield dwell time, number of shuttles, darkfield latency, and travel distance. (F) Immobility time of the tail suspension experiment. Data are expressed as mean ± SEM (n=9), compared with the control group, ^#, P<0.05; ^##, P<0.01; ^###, P<0.005; ^####, P<0.001; with the first week in comparison, *, P<0.05; ***, P<0.005.

Figure 2

Assessment of the atherosclerotic status in mice. (A) Mouse plasma lipid levels, including total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C). (B) Plasma inflammatory factors, including IL-1β, IL-6, and TNF-α. (C) Aortic Oil Red O staining. (D) Aortic plaque area statistics. (E) HE stains of aortic coronal section (×100, 100-fold; ×400, 400-fold). Data for (A,B) are expressed as mean ± SEM (n=9), ^###, P<0.005; ^#####, P<0.001. Data for (C,D,E) are expressed as mean ± SEM (n=3), ^#####, P<0.001.

Figure 3

Results of the RNA-seq detection in the hippocampus and the prefrontal cortex in mice before and after AS modeling. (A) DEG expression heat map of the hippocampus; (B) DEG expression heat map of the prefrontal cortex; the X-axis represents different samples, and the Y-axis represents the difference gene; the darker the color, the higher the expression level, and the lighter the color, the lower the expression level. (C) The volcanic product distribution map of the DEG from the hippocampus; (D) the volcanic product distribution map of the DEG from the prefrontal cortex; the X-axis represents the difference in the log 2 conversion, the Y-axis represents the significant difference after the log 10 conversion, the blue represents the downregulated DEG, the red represents the upregulated DEG, and the gray is the non-DEG.

Figure 4

GO annotation the DEG from the hippocampus and the prefrontal cortex in mice. (A) The differential gene GO function classification map of the hippocampus; (B) the differential gene GO function classification map of the prefrontal cortex; the X-axis represents DEG numbers, and the Y-axis represents the GO function classification; (C) differential gene up and down GO function classification map of the hippocampus; (D) differential gene up and down GO function classification map of prefrontal cortex; the X-axis represents the GO function classification, and the Y-axis represents the GO term up-and-down regulation gene number. AS, atherosclerosis; DEG, differentially expressed gene; GO, Gene Ontology.

Figure 5

KEGG classification on the DEG from the hippocampus and the prefrontal cortex in mice. (A) Pathway classification map of differential genes from the hippocampus; (B) pathway classification map of differential genes from the prefrontal cortex; the X-axis represents the proportion of genes, and the Y-axis represents the KEGG functional classification. DEG, differentially expressed gene; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 6

Statistics of pathway enrichment of DEG from the hippocampus and the prefrontal cortex in mice. (A) Enrichment pathway differential gene up-and-down map of the hippocampus; (B) enrichment pathway differential gene up-and-down map of the prefrontal cortex; the Y-axis represents the pathway entry, and the X-axis represents the number of up-and-down genes corresponding to the pathway entry. (C) Statistics of pathway enrichment of DEGs map of the hippocampus; (D) statistics of pathway enrichment of DEG map of the prefrontal cortex; the X-axis represents the enrichment factor, the Y-axis represents the pathway name, and color represents Q value; the smaller the value, the more significant the enrichment result is; the size of the point represents the number of DEG. DEG, differentially expressed gene; KEGG, Kyoto Encyclopedia of Genes and Genomes.