The Potential Mechanism Of Artemisinin Attenuating Atherosclerosis In High-fat Diet-fed ApoE-/-Mice By Promoting Macrophage Autophagy Via AMPK/mTOR/ULK1 Pathway

Objective:Atherosclerosis is a disease with high mortality and disability.Therefore,in-depth study of its pathogenesis and treatment measures has become an important worldwide topic.Lipid metabolism disorderis considered to be an important factor in the pathogenesis of AS.The changes of autophagy activity and immune inflammatory response have also been widely concerned in recent years.Deficiency of autophagy promotes the lipid deposition.These phenomena have been confirmed by animal experiments.Autophagy is involved in the progress of many diseases.AMPK/mTOR/ULK1 pathway is an important signal pathway regulating autophagy.AMPK is a key molecule to regulate energy metabolism,and its activity is affected by many factors in vivo.MTOR is a target molecule of rapamycin in mammalian cells.It is a member of phosphatidylinositol 3-kinase-related protein kinase family,and is involved in regulating the growth and autophagy of various cells.AMPK can negatively regulate mTOR phosphorylation and thus up regulate autophagy.Activation of AMPK or inhibition of mTOR may be a new target for as prevention and treatment.In our previous studies,we found that artemisinin,a classical antimalarial drug,can improve as through NF-?B pathway;at the same time,artemisinin's pathway of action on tumor cells in the literature is also related to this,and some studies also observed that the change of autophagy state is involved.So we need to find out whether the drugs that affect macrophage autophagy can be the treatment of AS?How about the specific way?We study whether ART can reduce AS by influencing macrophage autophagy through AMPK/mTOR/ULK1pathway.Methods:In order to exlpore the effect of ART on macrophage autophagy and specific signal pathway in AS,we designed in vivo and in vitro studies to observe the distribution and concentration changes of corresponding proteins.The specific mechanism of ART affecting as was observed by establishing animal model.Male ApoE^-/-mice?8 weeks old?with C57BL6 as the background were randomly divided into three treatment groups,which were fed with HFD for 8 weeks,at the same time,two concentrations of ART were given,and then the materials were taken.The level of LDL was measured,MCP-1,IL-6,TNF-?and IFN-?were detected by enzyme linked immunosorbent assay,the morphological changes of aortic root?cross section?cells were observed by HE staining,and the atherosclerotic lesions in aortic?one,longitudinal section?and aortic root?cross section?were detected by oil red O staining.F4/80 positive cells in aortic root were counted by immunofluorescence.In vitro,we cultured RAW264.7 macrophages and divided them into 5 groups.Four groups were given oxLDL culture,three groups were given ART,two groups were given adenovirus silenced AMPK and control.The levels of MCP-1,IL-6,TNF-?and IFN-?were detected by ELISA.The expression of AMPK,p-ampk?thr172?,mTOR,p-mTOR?s2448?,ULK1,p-ulk1?ser757?,LC3II/I and p62were detected by WB.The corresponding tests were repeated in bone marrow macrophages using AMPK inhibitor compound C and autophagy inhibitor 3mA.Oil red O staining was used to detect lipid aggregation.Autophagy was detected by fluorescence double staining ofRFP-GFP.Results:In the aortic tissue and the supernatant,the contents of MCP-1,IFN-?,IL-6and TNF-?in the ART group were significantly higher than those in AS group,whilethe effect of high concentration ART was more obvious.The contents of theinflammatory factors increased after AMPK was interfered.Oil red O staining of the whole aorta was used to observe the atherosclerotic condition of the whole aorta.In AS group,the atherosclerotic plaques were widely distributed and aggregated in the aortic arch,thoracic aorta and abdominal aorta.Compared with AS group,the area and distribution of plaque formation in ART group were significantly reduced.After the cells were treated with oxLDL,lipid droplets accumulated in the cells,when ART administrated decreased.But Interfering with AMPK,the result is diffrent.Autophagy detection:after the cells were treated with oxLDL,autophagy did not change significantly;autophagy body formation increased significantly in ART treatment group;autophagy was inhibited to some extent after the interference with AMPK.WB detection of ARTsignificantly promoted the accumulation of autophagy related protein LC3II and the degradation of p62.The inhibition of AMPK blocked the effect of ART.WB detection of AMPK,mTOR,ULK1pathway related proteins found that silencing AMPK blocked the effect of ART on these proteins.After the induction of Ox LDL,the fluorescence intensity of autophagy decreased,while ART treatment enhanced the fluorescence intensity of yellow labeled autophagy.This phenomenon will be blocked by AMPK silence.Conclusion:In the process of AS in ApoE^-/-mice,the expression of autophagy related proteinLC3II/I decreased,and the expression of p62 increased,reflecting the decreased autophagy function of macrophages and the increased expression of inflammatory factors.ART can promote the phosphorylation of mTOR and ULK1,enhance the expression of LC3II and reduce p62 by activating AMPK.After silencing AMPK,the effect is also weakened.Similar results can be achieved with autophagy inhibitors or AMPK inhibitors.It indicates that ART can improve macrophage autophagy,inhibit the transformation of macrophages into foam cells and reduce the formation of atherosclerotic plaques in atherosclerotic mice.AMPK is a key target for phosphorylation of mTOR and ULK1.Our study shows that ART can promote macrophage autophagy and alleviate atherosclerosis through AMPK/mTOR/ULK1 pathway.