Myriocin reduces atherosclerosis in ApoE-/- mice by inhibiting lipid ⁃ induced integrated stress response

Abstract

Objective To investigate the effect of Myriocin on high fat diet induced integrated stress response and further explore the mechanism. Methods A total of 18 ApoE^-/-mice were fed a high⁃fat diet and randomly divided into control group (n=9, phosphate buffer solution) and Myriocin group (n=9, phosphate buffer solution+Myriocin). The drugs were administered orally for 12 weeks. Serum lipids [total cholesterol (TC), triglyceride (TG), low density lipoprotein⁃cholesterol (LDL⁃C), very low density lipoprotein⁃ cholesterol (VLDL⁃C) and high density lipoprotein⁃cholesterol (HDL⁃C)] were measured. Flow⁃cytometric analysis was used to determine the proportion of lymphocyte antigen 6 complex(Ly ⁃ 6c)^high phenotype monocytes. HE staining was performed to compare the size and detailed composition of atherosclerotic plaques and immunofluorescence staining was used to observe the expression of monocyte chemotactic protein⁃1 (MCP⁃1). Real⁃time fluorescence quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression levels of inflammation related molecules [including pro⁃inflammatory factors, interleukin⁃1β and 6 (IL⁃1β and IL⁃6), tumor necrosis factor⁃α (TNF⁃α), intercellular adhesion molecule⁃1 (ICAM⁃1), vascular cell adhesion molecule⁃1 (VCAM⁃1),vascular endothelial growth factor (VEGF) and anti⁃inflammatory factor, IL⁃10].In addition, the mRNA expression levels of integrated stress response related molecules [including glucose regulated protein 78 (GRP78), protein kinase R⁃like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2 α (eIF2 α), activating transcription factor 4 and 6 (ATF4 and ATF6), endoplasmic reticulum stress ⁃ related apoptosis protein C/EBP homologous protein (CHOP) and Caspase12] were tested. The expression levels of integrated stress response related protein [including eIF2α, ATF4, inositol⁃requiring enzyme 1α (IRE1α) and phosphorylated IRE1α, p65 nuclear factor⁃κB (p65 NF⁃κB) and phosphorylated p65 NF⁃κ B, Caspase12 and cleaved Caspase12] were explored by Western blotting. Results 1) Treatment with Myriocin led to lower level of serum LDL⁃C (t=2.830, P=0.012). 2) Myriocin suppressed monocytes differentiating toward a Ly ⁃ 6c^high phenotype (t=2.866, P=0.011).3) HE staining showed less atherosclerotic lesions at 200 and 300 μm distance away from the aortic valve (t=2.281, P=0.045; t=3.506, P=0.003) and less necrotic core areas (Z=⁃2.870, P=0.004) in the Myriocin group. 4) Immunofluorescence staining showed the reduction of MCP⁃1 protein expression in the Myriocin group; real⁃time fluorescence quantitative PCR showed that IL⁃1β mRNA (t=3.968, P=0.005),TNFα mRNA (t=7.696, P=0.000), ICAM mRNA (t=3.294, P=0.013), VCAM mRNA (t=5.449, P=0.001)and VEGF mRNA (t=2.574, P=0.037) were generally decreased in the Myriocin group, while IL⁃10 mRNAwas increased (t=⁃3.132, P=0.017) in the Myriocin group. 5) Myriocin downregulated PERK mRNA (t=4.174, P=0.004）, eIF2α mRNA (Z=⁃2.692, P=0.007), ATF4 mRNA (t=3.342, P=0.012), ATF6 mRNA(t=5.841, P=0.001) and Caspase12 mRNA (t=7.270, P=0.000). 6) Western blotting showed that Myriocin suppressed the protein expression of eIF2α (t=2.175, P=0.047) and ATF4 (t=2.923, P=0.011),and the phosphorylation of p65 NF⁃ κ B (t=2.909, P= 0.011). Conclusions Myriocin could alleviate atherosclerosis progression of ApoE^-/- mice by reducing integrated stress response and inflammatory response in the arterial walls.

DOI:10.3969/j.issn.1672⁃6731.2020.10.003