Knockdown of USP7 alleviates atherosclerosis in ApoE-deficient mice by regulating EZH2 expression

Abstract

Atherosclerosis (AS) is a chronic vascular disease associated with lipid accumulation. Understanding the molecular mechanisms of AS is essential. Ubiquitin-specific protease 7 (USP7) is a deubiquitination enzyme involved in various cellular processes, including lipid metabolism. In this study, we aimed to elucidate the role of USP7 in AS progression and its underlying mechanism using ApoE-deficient mice. We found that USP7 ablation improved the morphological characteristics of AS in these mice. USP7 knockdown reduced inflammation, evidenced by decreases in inflammatory markers IL-6, TNF-α, and IL-1β by 35, 40, and 38%, respectively (p < 0.01). Additionally, USP7 depletion reduced oxidative stress, indicated by a 30% reduction in malondialdehyde levels and increases in superoxide dismutase and glutathione peroxidase levels by 25 and 28%, respectively (p < 0.01). Moreover, USP7 knockdown blocked lipid accumulation in aortic tissue cells. Mechanistically, USP7 knockdown inhibited enhancer of Zeste Homolog 2 (EZH2) expression, thereby suppressing AS progression. In conclusion, USP7 depletion alleviated AS progression in ApoE-deficient mice by targeting EZH2 expression. USP7 may serve as a therapeutic target for AS.

Keywords: EZH2; atherosclerosis; inflammation; oxidative stress; ubiquitin-specific protease 7.

Figure 1

Ablation of USP7 improved the AS morphological characteristics in AS mice. (a) Immunoblot assays showed the expression of USP7 in aortic tissues of normal mice and AS (ApoE-deficient) mice, as well as AS mice infected with sh-NC or sh-USP7 adenovirus plasmids for 24 h. (b) H&E staining showed the aortic morphology in normal mice and AS (ApoE-deficient) mice, as well as AS mice infected with sh-NC or sh-USP7 adenovirus plasmids for 24 h. Scale bar, 100 μm. ***p < 0.001, AS vs normal, ###p < 0.001, AS + sh-USP7 vs AS + sh-NC. NC, negative control; AS, atherosclerosis.

Figure 2

ELISA showed the secretion of IL-6, TNF-α, and IL-1β in the serum (up) and aortic tissues (down) of normal mice and AS (ApoE-deficient) mice, as well as AS mice infected with sh-NC or sh-USP7 adenovirus plasmids for 24 h.

Figure 3

Depletion of USP7 restrained oxidative stress in AS mice. (a) DHE staining showed the oxidative stress (ROS levels) in the aortic tissues of normal mice and AS (ApoE-deficient) mice, as well as AS mice infected with sh-NC or sh-USP7 adenovirus plasmids for 24 h. Scale bar, 200 μm. (b) Levels of MDA, SOD, and GSH-Px in the aortic tissues of normal mice and AS (ApoE-deficient) mice, as well as AS mice infected with sh-NC or sh-USP7 adenovirus plasmids for 24 h, were measured using corresponding kits. ***p < 0.001, AS vs normal, ##p < 0.01, ###p < 0.001, AS + sh-USP7 vs AS + sh-NC. NC, negative control; AS, atherosclerosis.

Figure 4

USP7 knockdown blocked the lipid accumulation of aortic tissues in AS mice. (a) TC and TG levels in the aortic tissues of normal mice and AS (ApoE-deficient) mice, as well as AS mice infected with sh-NC or sh-USP7 adenovirus plasmids for 24 h, were detected using corresponding kits. (b) Oil Red O staining showed the degree of lipid accumulation in the aortic tissues of normal mice and AS (ApoE-deficient) mice, as well as AS mice infected with sh-NC or sh-USP7 adenovirus plasmids for 24 h. Scale bar, 40 μm. ***p < 0.001, AS vs normal, ###p < 0.001, AS + sh-USP7 vs AS + sh-NC. NC, negative control; AS, atherosclerosis.

Figure 5

USP7 knockdown inhibited EZH2 expression in AS mice. (a) Immunoblot assays showed the expression of EZH2 in the aortic tissues of normal mice and AS (ApoE-deficient) mice, as well as AS mice infected with sh-NC or sh-USP7 adenovirus plasmids for 24 h. (b) Immunoblot assays showed the expression of EZH2 in the aortic tissues of AS mice infected with sh-NC or sh-USP7 and EZH2 adenovirus plasmids. (c) ELISA showed the secretion of IL-6, TNF-α, and IL-1β in the aortic tissues of AS mice infected with sh-NC or sh-USP7 and EZH2 adenovirus plasmids for 24 h. (d) Levels of MDA, SOD, and GSH-Px in the aortic tissues of AS mice infected with sh-NC or sh-USP7 and EZH2 adenovirus plasmids for 24 h were measured using corresponding kits. (e) TC and TG levels in the aortic tissues of AS mice infected with sh-NC or sh-USP7 and EZH2 adenovirus plasmids were detected using corresponding kits. **p < 0.01, ***p < 0.001, AS vs normal, ##p < 0.01, ###p < 0.001, AS + sh-USP7 vs AS + sh-NC. NC, negative control; AS, atherosclerosis.