Mechanism Of Cordycepin Inhibiting Foam Cells Formation Via Autophagy

Atherosclerosis is a chronic inflammatory disease owing to lipid metabolism disorders and maladaptive inflammatory response in the large arteries.Macrophages-derived foam cells formation leading to lipid accumulation in the blood vessel wall played a crucial role in the process of development of atherosclerosis.Cell autophagy could resist the stress factors and degrade damage component within cells into small molecules for recycle,maintaining the balance of energy metabolism.Research has shown that macrophage autophagy promote intracellular cholesterol efflux and moderate autophagy could delay the development of Atherosclerosis.Cordycepin,a special bioactive compound obtained from traditional Chinese medicine Ophiocordyceps sinensis,has extensive pharmacological activities such as lowering lipid accumulation,anti-tumor and antiviral.Limited study has shown the effect and mechanism of cordycepin on reducing the risk of atherosclerosis.To systematically explore potential mechanism underlying its inhibition of foam cells formation,we investigated the relationship of the cordycepin,macrophage autophagy and foam cells formation in RAW264.7cells.This article adopted the classic ox-LDL-induced foam cells model to investigate the inhibitory effect of cordycepin on foam cells formation and its molecular mechanism.Oil red O staining and total cholesterol(TC)specific kits were performed to assess cordycepin's influence on cholesterol accumulation;The fluorescence-labeled assay were tested to analyze effect of cordycepin on cholesterol flux;Real-time quantitative PCR detected related genes mRNA expression.Then we tested the relationship of cordycepin and autophagy.Western blot detected p62,LC3 protein expression to determine the level of autophagy,immunofluorescence staining assessed autophagy related protein p62 and LC3;transmission electron microscopy was adopted to investigate the ultrastructure changes of intracellular autophagosome.Finally,after disturbing autophagy and upstream pathways of autophagy for the specificity of the key molecular AMPK and mTOR,we detected p62,LC3,p-AMPK,AMPK,p-mTOR,mTOR related protein levels by Western blot to explore the change of the level of autophagy.We also evaluated the influence of cordycepin on foam cells formation to explore effect and mechanism of cordycepin inhibiting foam cells formation via autophagy mediated by AMPK/mTOR signaling pathway.The results revealed treatment of cordycepin at the dose of 10 ?mol/L significantly inhibited cholesterol accumulation and regulated high-density lipoprotein(HDL)mediated cholesterol flux which proved the effect on inhibiting foam cells formation.RT-PCR results showed that the cordycepin remarkably regulated the cholesterol flux related gene mRNA levels.Above results indicated that cordycepin inhibit foam cells formation.Western blot proved that cordycepin promoted autophagy with the relations of time-effect and dosage-effect through the degradation of p62 and the increase of LC3II/LC3I.Meanwhile,immunofluorescence staining assessed that cordycepin significantly promoted the occurrence of autophagy,TEM revealed that cordycepin obviously increased autophagosome numbers in RAW264.7 cells.The above results indicated that cordycepin significantly promoted the autophagy in RAW264.7 cells.Adopt the corresponding compounds to interfere with the level of autophagy and the expression of mTOR and AMPK,chloroquine which can inhibit cell autophagy significantly reduced the effect of cordycepin promoting autophagy;Selecting leucine to promote mTOR expression,cordycepin can't promote autophagy and increase mTOR phosphorylation levels;After inhibiting the expression of AMPK,the role of cordycepin was suppressed characterized by reducing levels of AMPK phosphorylation,upregulating mTOR phosphorylation,decreasing autophagy levels.The experimental results show that the cordycepin promote autophagy by AMPK/mTOR signaling pathways.Interference the expression of mTOR and AMPK could inhibit cordycepin's ability of promote cell autophagy,while lowering the impact intracellular lipid accumulation,raising cholesterol efflux,inhibiting expression of cholesterol efflux related genes.Above results showed that treatment with cordycepin inhibits foam cells formation in RAW264.7 macrophages by promoting cell autophagy,which is regulated by activity of the AMPK/mTOR signaling pathway.Cordycepin might play a vital role in preventing atherosclerosis.In conclusion,we first proved that cordycepin activate autophagy via AMPK/mTOR pathway,and inhibit foam cells formation which contribute atherosclerosis.The data provided an experimental reference of cordycepin on prevention and treatment on atherosclerosis.The innovation reflected that to inhibiting effect of cordycepin on foam cells formation is closely related to cell autophagy,which provides a new target and ideas in treating atherosclerosis.