Abstract
Objective: To investigate the process by which quercetin suppresses atherosclerosis by upregulating MST1-mediated autophagy in RAW264.7 macrophages. Methods: An in vitro foam cell model was established by culturing RAW264.7 macrophages with oxidized low-density lipoprotein (ox-LDL). The cells were treated with quercetin alone or in combination with the autophagy inhibitor, 3-methyladenine, and autophagy agonist, rapamycin. Cell viability was detected with a CCK-8 kit. Lipid accumulation was detected by oil red O staining, senescence was detected by SA-β-gal (senescence-associated β-galactosidase) staining, reactive oxygen species were detected by ROS assay kit. Autophagosomes and mitochondria were detected by transmission electron microscope (TEM), and expression of MST1, LC3-II/I, Beclin1, Bcl-2, P21, and P16 were detected by immunofluorescence and Western blot. Results: Ox-LDL induced RAW264.7 macrophage-derived foam cell formation, reduced survival, aggravated cell lipid accumulation, and induced a senescence phenotype. This was accompanied by decreased formation of autophagosome; increased expression of P53, P21, and P16; and decreased expression of LC3-II/I and Beclin1. After intervention with quercetin, the cell survival rate was increased, and lipid accumulation and senescence phenotype were reduced. Furthermore, the expression of LC3-II/I and Beclin1 were increased, which was consistent with the ability of quercetin to promote autophagy. Ox-LDL also increased the expression of MST1, and this increase was blocked by quercetin, which provided a potential mechanism by which quercetin may protect foam cells against age-related detrimental effects. Conclusion: Quercetin can inhibit the formation of foam cells induced by ox-LDL and delay senescence. The mechanism may be related to the regulation of MST1-mediated autophagy of RAW264.7 cells.
Highlights
Cardiovascular and cerebrovascular diseases are the leading causes of morbidity and mortality worldwide, and atherosclerosis (AS) is considered to be a driver [1]
We explored the role of QUE in antagonizing the expression of key factors in the Mammalian Ste20-like kinase 1 (MST1) signaling pathway in order to provide a mechanism for the inhibition of autophagy in oxidized low-density lipoprotein (ox-LDL)-induced RAW264.7 foam cells
The expression of P21 and P16 increased, accompanied by an increase in SA-β-gal positive cells. As these markers are associated with cell senescence [5,26], our results suggested that RAW264.7 macrophage-derived foam cells were successfully derived and accompanied by cellular senescence
Summary
Cardiovascular and cerebrovascular diseases are the leading causes of morbidity and mortality worldwide, and atherosclerosis (AS) is considered to be a driver [1]. Macrophage foam cells are an important component of AS lesions and play key roles in the development of AS [2]. Macrophage phagocytosis and metabolism of oxidized low-density lipoprotein (ox-LDL) are increased, and lipids are transported from the cells to the vessel walls. When ox-LDL intake exceeds the capacity of macrophage metabolism, macrophages are transformed into foam cells, which promote the development of AS. Pulse wave velocity acts as a marker of subclinical atherosclerosis, it increases with the gradual increment of ox-LDL [3]. All the above evidence confirms that the occurrence and development of AS are related to aging and senescence. The aging free radical theory emphasizes the role of reactive oxygen species (ROS)-induced cell damage in aging promotion. Mitochondria are the main source of ROS and the primary target of ROS damage, and their status is closely related to the aging phenotype [10]
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