Together, our results demonstrated Cpn as a potential therapeutic agent for the prevention and treatment of atherosclerosis, and the autophagic activity presents a novel mechanism for Cpn-mediated atheroprotection. 0.01, ### Laquinimod (ABR-215062) 0.001, vehicle vs. part, in the atheroprotective role of Cpn. Further investigations using different autophagy inhibitors and specific siRNAs for AMP-activated protein kinase (AMPK) gamma1 subunit indicated that Cpn may stimulate macrophage autophagy through AMPK-mTOR pathway. Together, our results exhibited Cpn as a potential therapeutic agent for the prevention and treatment of atherosclerosis, and the autophagic activity presents a novel mechanism for Cpn-mediated atheroprotection. 0.01, ### 0.001, vehicle vs. normal, * 0.05, ** 0.01, *** 0.001, test group vs. vehicle group. TC, total cholesterol; TG, triglycerides; LDL-c, low-density lipoprotein cholesterol; HDL-c, high-density lipoprotein cholesterol; MCP-1, monocyte chemotactic protein-1; TNF, tumor necrosis factor ; IL-6, interleukin-6. The atherosclerotic mice were accompanied with marked dyslipidemia and hyperlipidemia, as revealed by significant increase in serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c) and glucose as well as increased levels of liver TC and TG (Physique ?(Physique1C1C and ?and1D).1D). Administration of Cpn dose-dependently ameliorated all the parameters for dyslipidemia and hyperlipidemia (Physique ?(Physique1C1C and ?and1D),1D), which was in accordance with previous reports [14C16]. Simultaneously, the increased levels of serum monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor (TNF) and interleukin-6 (IL-6) in HFD-fed apoE-/- mice were also largely suppressed (Physique ?(Figure1E).1E). These data suggest that Cpn is effective to eliminate multiple risk factors and thus prevent the incidence and progress of atherosclerosis Cordycepin inhibits foam cell formation by regulating intracellular cholesterol homeostasis Lipid deposition in macrophage induces foam cell formation and ultimately accelerates the development of atherosclerosis [6]. Although the inhibitive actions of Cpn on dyslipidemia and inflammation had been exhibited [15C17], its effects on foam cell formation have not been reported. PROML1 We decided oxLDL-induced foam cell formation by using oil-red O staining and intracellular TC quantification. Supplemented with Cpn (0.1-10 M) significantly decreased oxLDL-elicited lipids deposit and TC accumulation in RAW264.7 macrophages (Figure ?(Figure2),2), showing clear inhibiting effects on foam cell formation. The inhibiting efficacy of Cpn (10 M) was comparable to that of simvastatin, a popular drug for the prevention and treatment of atherosclerosis. These results suggested that inhibition of macrophage-derived foam cell formation may participate in the preventing effect of Cpn on atherosclerosis development. Open in a separate window Physique 2 Cordycepin (Cpn) Laquinimod (ABR-215062) inhibited oxidized low-density lipoprotein (oxLDL)-elicited foam cell formation in RAW2647 cells. RAW264.7 cells were elicited by oxLDL for 24 h with or without supplementation of Cpn or Laquinimod (ABR-215062) simvastatin. Cells were then stained with oil-red O, and the representative staining pictures (A), the absorptance at 358 nm (B), and intracellular total cholesterol content (C) were acquired. Bar = 50 m. Values are means SEM of at least three experiments. ### 0.001, vehicle vs. blank, * 0.05, ** 0.01, *** 0.001, test group vs. vehicle group. The cholesterol homeostasis is usually a key regulator in the development of foam cells. Inhibiting cholesterol uptake and stimulating cholesterol efflux are two effective approaches to suppress foam cell formation. We assessed the effect of Cpn around the cholesterol homeostasis in macrophages using NBD-cholesterol-based fluorescence assays. As shown Laquinimod (ABR-215062) in Physique ?Physique3A,3A, Cpn remarkably inhibited cholesterol uptake by RAW264.7 macrophages in a dose- and time-dependent manner. Simultaneously, Cpn significantly promoted cholesterol efflux from macrophages with an efficiency higher than that of rosiglitazone, a PPAR agonist that is known to promote cholesterol efflux (Physique ?(Figure3B).3B). Realtime quantitative PCR also shown that Cpn switched the mRNA levels of cholesterol-uptaking genes (SR-A and SR-B) and cholesterol-effluxing genes (PPAR, LXR, ABCA1 and ABCG1) to the intracellular cholesterol-reducing side (Physique ?(Physique3C3C and ?and3D),3D), indicating that Cpn might prevent foam cell formation through regulating cholesterol uptake and efflux. Open in a separate window Physique 3 Cordycepin (Cpn) regulated intracellular cholesterol homeostasis by inhibiting cholesterol uptake (A), promoting cholesterol efflux (B) and modulating mRNA levels of genes that involved in cholesterol uptake (C) and efflux (D) in RAW264. 7 macrophages. Values are means SEM of at least three experiments. ### 0.001, vehicle vs. blank, * 0.05, ** .