EFFECT OF PROBUCOL ON ATHEROSCLEROSIS IN ApoE-/- MICE AND ITS MECHANISM

Abstract

Objective To investigate the effect of probucol on the progression of atherosclerosis in ApoE-/- mice by inhibiting macrophage M1 polarization and its mechanism. Methods A total of 20 ApoE-/- mice were randomly divided into control group and probucol group. Both groups were given high-fat diet, and the mice in the probucol group were given probucol by gavage once every other day, while those in the control group were given an equal volume of normal saline by gavage. After 12 weeks of intervention, blood samples were collected from the apex of the heart after anesthesia, and the mice were sacrificed by cervical dislocation to collect the aorta vessels. A biochemical analyzer was used to measure blood lipids and liver and renal functions, and ELISA was used to measure the serum levels of inflammatory factors (IL-1β, IL-6, and TNF-α). Oil red O staining was performed for the aorta to measure the plaque area of atherosclerosis; the aortic root was collected to prepare frozen sections, and then oil red O staining was used to measure plaque area and lipid deposition; Masson staining was used to measure plaque stability. Immunof-luorescence assay was used to observe macrophages, smooth muscle cells, collagen fibers, and macrophage M1 polarization in plaques, and quantitative real-time PCR was used to measure the mRNA expression levels of inflammatory factors in aortic plaques. Primary peritoneal macrophages were collected from C57BL/6J mice and divided into control group and probucol group. The macrophages in the control group were treated with lipopolysaccharide (LPS) combined with interferon-γ (IFN-γ), and those in the probucol group were treated with probucol in addition to the treatment in the control group. ELISA and RT-qPCR were used to measure the mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and macrophage M1 polarization mar-kers (iNOS and CD86). Results Compared with the control group, the probucol group had significant improvements in the se-rum levels of triglyceride, total cholesterol, and low-density lipoprotein cholesterol (t=2.445-4.024,P<0.05) and a significant reduction in aortic plaque area (t=3.599,2.954,P<0.05), as well as a significant reduction in lipid deposition, significant increases in smooth muscle and collagen fiber, and significant reductions in macrophages and their M1 polarization markers in aortic root plaques. Compared with the control group, the probucol group had significant reductions in the levels of the inflammatory factors IL-1β, IL-6, and TNF-α in serum and aortic root plaques (t=2.388-4.891,P<0.05). Compared with the control group, the probucol group of primary peritoneal macrophages had significant reductions in the levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α (t=4.238-9.580,P<0.05) and the mRNA expression levels of iNOS and CD86 (t=3.251,20.860,P<0.05). Conclusion Probucol can delay the progression of atherosclerosis and promote plaque stability in ApoE-/- mice, possibly by inhibiting macrophage M1 polarization and alleviating plaque inflammatory response.

Keywords

• atherosclerosis|probucol|macrophages|inflammation|disease models, animal