Fig. 5: Repeated mild stress reversed stress-induced gene expression profiles in the vSub in young mice.

a–e PCA (a) of DEGs in young mice, illustrating the variance between CON versus CRST (PC1; 47.95%) and CRST vs. RST7d + RS5 (PC2; 19.83%). Gene expression patterns in RRHO map (b). RRHO map visualizing differential gene expression in CON versus CRST (x axis) and CRST versus CRST + RS5 (y axis) (c). The number (%) of identified genes within each quadrant (d). Flow charts (e) of selecting the top-50% ranked genes within each quadrant. f–k K-means clustering identified eight functional groups within the CRST-upregulated and RS5-downregulated genes (quadrant A) in young mice (f). Top BP terms for cluster 1 of these RS5-downregulated genes (g). GSVA-selected gene sets identified among these genes across the CON versus CRST and CRST versus CRST + RS5 groups, including stress response (h) and signal transduction (i), with their interaction network (j) and heatmaps (k) showing gene expression changes. l–q K-means clustering revealed two functional groups within the CRST-downregulated and RS5-upregulated genes (quadrant D) in young mice (l). Top BP terms for cluster 1 of these RS5-upregulated genes (m). GSVA-selected gene sets identified within these genes across the CON versus CRST and CRST versus CRST + RS5 groups, including multicellular development (n) and cell signaling (o), with their interaction network (p) and heatmaps (q) showing gene expression alterations. Violin plots are shown with overlaid boxplots displaying the mean and quartiles. The boxplot whiskers indicate 1.5 times the interquartile range from the first and third quartiles. Differences in GSVA scores among groups were assessed using one-way ANOVA followed by Tukey’s HSD post-hoc test. *P < 0.05.