Ginsenoside Rb1 Enhances Atherosclerotic Plaque Stability By Induction Of Macrophage Autophagy

BackgroudAtherosclerotic plaque rupture and thrombosis are the main cause of acute coronary syndrome.Early intervention of vulnerable plaques of possible rupture is important to prevent acute cardiovascular and cerebrovascular events.Atherosclerosis(AS)is a lipid-driven chronic inflammatory disease,characterized by lipid accumulation and inflammatory cells infiltration,among which macrophage cells play a crucial role in this process.Low-density lipoprotein cholesterol(LDL-C)in the blood is phagocytosed by macrophages and stored in the form of cholesterol esters in lipid droplets,and macrophages filled with a large number of lipids transform into foam cells.It is of great significance to reduce the accumulation of lipid in foam cells for plaque stability.Ginsenoside Rb1(Rb1)is the most abundant active component isolated from panax ginseng.Previous studies have shown many health benefits of Rb1,including inhibition of inflammation,anti-obesity,enhancing insulin sensitivity,and lipid-lowering properties.However,the role of Rb1 in atherosclerosis has not been reported.ObjectiveWe aimed to test the hypothesis that ginsenoside Rb1 mitigates lipid accumulation of foam cells and promotes atherosclerosis plaque stabilization by induction of macrophage autophagy.MethodsIn vitro,primary peritoneal macrophages from C57BL/6 mice were incubated with ox-LDL(100 μg/ml)for 12 h,and further incubated in the absence or presence of different doses of Rbl for another 24 h.Oil red O staining was performed to assess lipid accumulation in macrophages.The expression of LC3II and SQSTM1 was analyzed by western blots;and transmission electron microscopy was used to detect autophagosome.Macrophage autophagic flux was blocked by using autophagy-related gene5(Atg5)small interfering RNA(siRNA)to further explore the role of autophagy in Rbl-related lipid metabolism.In addition,an AMPK inhibitor,compound C was additioned to study the role of AMPK in Rbl-induced autophagy.In vivo,the advanced atherosclerotic plaque model was established in ApoE-/-mice by a high-fat diet.Subsequently,all mice were randomly divided into 2 groups(n=15 per group)for treatment:Rbl,intraperitoneal injected with Rbl(50 mg/kg/d)for 7 weeks.;control,an equal volume of saline.After 7-week treatment,mice were sacrificed and the aorta and heart were harvested.Ultrastructural changes of macrophages,lipids accumulation,collagen and smooth muscle cells were measureed by pathology.The expression of LC3 B and SQSTM1 and phosphorylation of AMPK were detected by western blot.Results1.Rbl reduced lipid accumulation in peritoneal macrophages with the minimum effect dose at 20 μM.2.Rbl treatment significantly increased the ratio of LC3II/LC31 and SQSTM1 degradation,along with increased autophagosomes in or around LDs.3.In macrophages transfected with control siRNA,Rbl(20μM)increased the Atg5 expression and LC3Ⅱ/LC3Ⅰ ratio significantly and reduced lipid droplets accumulation.In contrast,in macrophages transfected with Atg5 siRNA,Rb1 failed to increase the ratio of LC3-Ⅱ/LC3-Ⅰ or attenuate lipid accumulation.4.Rbl notably increased AMPK phosphorylation both in vitro and in vivo;AMPK inhibitor compound C abolished Rbl-induced autophagy in macrophages in vitro.5.Rbl treatment promoted the expression of LC3-Ⅱ protein and SQSTM1 degradation in atherosclerotic lesions.6.Rbl reduced the content of macrophages and lipids and increased that of smooth muscle cells and collagen in the atherosclerotic plaque,thus contributing to decreased plaque vulnerability.ConclusionGinsenoside Rbl reduces lipid accumulation of foam cells and enhances atherosclerotic plaque stabilization by induction of macrophage autophagy.AMPK plays a key role in Rbl-induced autophagy.