Trimethylamine N-oxide Promotes Atherosclerosis by Regulating Low-Density Lipoprotein-Induced Autophagy in Vascular Smooth Muscle Cells Through PI3K/AKT/mTOR Pathway.

Abstract

The research aimed to study the mechanism of how trimethylamine N-oxide (TMAO) regulates autophagy to promote atherosclerosis (AS). The AS in vitro model was constructed with vascular smooth muscle cells (VSMCs) treated with ox-LDL. The Cell Counting Kit-8 (CCK-8) trial was chosen to examine VSMCs' absorbance (OD) value. A transmission electron microscope (TEM) was selected for monitoring autophagosomes. Western blotting (WB) was adopted for examining the expression of Beclin-1, p62, LC3, α-SMA, SM22-α, OPN, PI3K, AKT, mTOR, p-PI3K, p-AKT, and p-mTOR proteins. Real-time fluorescent quantitative PCR (RT-qPCR) was accepted for testing the expression of α-SMA, SM22-α, OPN, PI3K, AKT, mTOR, Beclin-1, p62, and LC3 genes. The transwell assay was employed to examine the ability of migration in VSMCs. Oil red O staining assay was accepted to stain lipid droplets in VSMCs. TMAO noticeably promoted autophagy inhibition and the phenotypic transformation of AS. Protein expressions of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, and p62 of the TMAO+ox-LDL group were higher than the ox-LDL group, while Beclin-1 and LC3 were lower than the ox-LDL group. Gene expressions of PI3K, AKT, mTOR, and p62 of the TMAO+ox-LDL group were higher than the ox-LDL group, while Beclin-1 and LC3 were lower than the ox-LDL group. The intervention of LY294002 reversed the regulation of the corresponding proteins and genes. The study proved that TMAO could promote autophagy inhibition of AS via activating the PI3K/AKT/mTOR pathway. It supplied a reliable basis for improving clinical diagnostic methods and developing targeted AS drugs.