Anti-atherosclerosis Effects Of Diol Ginsenoside Derivatives And Its Mechanism

Atherosclerosis（AS）is the main pathological basis and the key link of cardiovascular disease（CVD）.Severe and acute diseases such as stroke and myocardial infarction caused by plaque rupture seriously endanger life and health.Although there have been extensive studies on AS,its pathogenesis has not been fully elucidated.At present,the main clinical first-line anti-atherosclerotic drugs are statins,but the morbidity,mortality and disability rates of AS are still high.Therefore,there is an urgent need to develop new and effective drugs for the prevention and treatment of AS.Lipid metabolism disorder,chronic inflammation and endothelial dysfunction are all important factors in the pathogenesis and progression of AS.In order to remove abnormal lipid accumulation under the vessel endothelium,the immune system is triggered.Immune cells,such as monocytes,dendritic cells and neutrophils,recruit to the abnormal vascular areas and migrate through the endothelial barrier to the subendothelium to cause inflammation.Meanwhile,damaged endothelial cells release pro-inflammatory cytokines to exacerbate the inflammatory response.If the peripheral hyperlipidemia environment persist,the inflammatory response will be further aggravated,leading to the formation of necrotic centers of large amounts of lipids,foam cells,inflammatory cells,and cell debris in the lesion,and the formation of atheromatous plaques.The plaque further expands and ruptures,causing platelet aggregation and thrombosis,resulting in vascular stenosis or even occlusion,and ultimately leading to the occurrence of acute cardiovascular diseases.Ginsenoside Compound K（CK）is the main metabolite of ginseng,panax notoginseng and other valuable Chinese medicines.Studies have found that it has anti-tumor and hypoglycemic effects.Previous studies in our laboratory have found that CK also has anti-atherosclerosis effect,but its activity still has great room for improvement.Our research group has carried out molecular optimization and structural modification of CK in the past,and compounds CKN and CK201 with better anti-atherosclerosis potential have been obtained.On this basis,the anti-atherosclerosis activities of compounds CKN and CK201were evaluated and their mechanisms were investigated in this paper.This study is divided into two parts.The first part investigates the prevention and treatment of AS by CKN and its mechanism.The second part investigates the prevention and treatment of AS by CK201 and its mechanism.Part Ⅰ Anti-atherosclerosis effects of diol ginsenoside derivative CKN and its mechanismMethods:1.Pharmacodynamic evaluation of CKN ameliorating atherosclerosis in ApoE^-/-mice induced by high-fat dietApoE^-/-mice aged 8 weeks were randomly divided into 5 groups:control group,model group,atorvastatin group,CK group and CKN group.High-fat diet was fed for 10 weeks to establish AS model,and Atorvastatin calcium,CK and CKN solutions of 3mg/kg were intraperitoneally injected at the same time.The aorta and brachiocephalic trunk of ApoE^-/-mice were stained with oil red O to detect the area of atherosclerotic plaque.The livers of ApoE^-/-mice were collected,and the liver lipid deposition and liver function were detected by tissue sections and liver function tests.2.Mechanism of anti-atherosclerosis effect of CKN2.1 In vivo study on the anti-atherosclerotic mechanism of CKNThe aorta of ApoE^-/-mice was taken for transverse section,and the lesions in the aortic vascular plaque area were observed by HE staining.The macrophage infiltration and ABCA1expression in the aortic plaque area were observed by immunofluorescence.The expressions of LXRα,ABCA1,IL-1βand TNF-αin abdominal aorta were detected by Western blot.The levels of total cholesterol（TC）,triglyceride（TG）,high-density lipoprotein cholesterol（HDL-C）,low-density lipoprotein cholesterol（LDL-C）and the levels of inflammatory factors IL-1βand TNF-αwere detected.2.2 In vitro study on the anti-atherosclerotic mechanism of CKNRAW264.7 macrophages were induced by ox-LDL to establish foam cell model,and the cells were treated with CKN（3,10,30μmol/L）.Oil red O staining was used to evaluate the effect of CKN on foam cell formation.The expression of IL-1βand TNF-αin cell culture medium was detected by ELISA.To explore whether the anti-AS mechanism of CKN was related to LXRα,LXRαantagonist SR9243（10μmol/L）was used to pretreat the cells,and the expressions of LXRαand ABCA1 in the cells were detected by Western blot and immunofluorescence.In order to explore the interaction mode of CKN and LXRα,the expression of LXRαin cells and nucleus was detected by immunofluorescence,the binding between CKN and LXRαprotein was detected by molecular docking assay,and the stability of LXRαprotein was detected by thermal shift assay.Results:1.CKN can significantly inhibit the formation of atherosclerotic plaque in aorta and brachiocephalic trunkCKN could significantly reduce atherosclerotic plaque area and inhibit atherosclerotic plaque formation in aorta and brachiocephalic trunk.CKN can reduce liver lipid accumulation and has no significant effect on liver function.2.CKN can up-regulate the expression of ABCA1 in aortic vessels and inhibit inflammatory responseImmunofluorescence of aortic sections showed that CKN significantly reduced macrophage infiltration in the plaque area and promoted ABCA1 expression.Western blot showed that CKN could up-regulate the expression of ABCA1 protein,but had no significant change in the expression of LXRαprotein,and inhibit the expression of inflammatory factors IL-1βand TNF-α.It can reduce plasma total cholesterol content.3.CKN can up-regulate ABCA1 expression and inhibit inflammatory response by regulating LXRαin RAW264.7 macrophagesCKN could significantly inhibit the formation of foam cells and effectively inhibit the expression of inflammatory factors IL-1βand TNF-αin RAW264.7 macrophages.Immunofluorescence and Western blot showed that CKN could up-regulate ABCA1expression by regulating LXRα.4.CKN may bind to LXRαand promote LXRαnuclear translocationCKN promoted LXRαprotein transfer from cytoplasm to nucleus.Molecular docking suggested that CKN could bind to the site of LXRαprotein.Thermal shift experiments suggested that CKN could improve the stability of LXRαprotein under high temperature.Part 2:Anti-atherosclerosis effects of diol ginsenoside derivative CK201 and its mechanismMethods:1.Pharmacodynamic evaluation of CK201 in improving atherosclerosis in ApoE^-/-mice induced by high-fat dietSix weeks old ApoE^-/-mice were randomly divided into 5 groups:control group,model group,CK group,atorvastatin group and CK201 group.ApoE^-/-mice were induced by high-fat diet to establish AS model,and CK,atorvastatin calcium and CK201 solution（3mg/kg）were administered by gavage for 12 weeks,6 times a week.After 12 weeks,the aorta was harvested for oil red O staining to analyze aortic plaque formation.The liver of mice was stained with HE and oil red O to analyze the pathological morphology and lipid accumulation of mice liver.Liver function indexes（AST,ALT）and renal function indexes（urea nitrogen,creatinine and uric acid）were detected to analyze the effects of CK201 on liver and renal function in ApoE^-/-mice fed with high-fat diet.The blood routine indexes of white blood cells,neutrophils,red blood cells,hemoglobin and platelets were detected to analyze the effect of CK201 on the blood system of ApoE^-/-mice induced by high-fat diet.2.Mechanism of anti-atherosclerosis effect of CK2012.1 In vivo study on the anti-atherosclerosis mechanism of CK201Plasma TC and TG levels were measured.The expression of IL-6 and TNF-αin the aorta of ApoE^-/-mice was detected by Western blot,and the effect of CK201 on the vascular inflammation of ApoE^-/-mice was analyzed.2.2 In vitro study on the anti-atherosclerosis mechanism of CK201The endothelial cell injury model was induced by palmitic acid（100μmol/L）,and the cells were treated with CK201（3,10,30μmol/L）.Adhesion assay was used to investigate the effect of CK201 on the recruitment of monocytes by HUVECs cells.Scratch healing assay was used to examine the migration ability of HUVECs cells after CK201 treatment.The effect of CK201 on the expression of inflammatory factors IL-6 and TNF-αwas detected by Western blot.Results:1.CK201 significantly inhibited aortic plaque formation and had no significant effect on the liver and blood systemCK201 could significantly reduce the plaque area and inhibit the formation of aortic plaque.It can improve the lipid accumulation in the liver of ApoE^-/-mice caused by high fat,and has no significant effect on the liver function and blood cells of ApoE^-/-mice.2.CK201 reduced plasma TC level and inhibited vascular inflammationCK201 could significantly reduce plasma TC content in ApoE^-/-mice.CK201 can significantly inhibit the expression of IL-6 and TNF-αin the aorta of ApoE^-/-mice,indicating that CK201 can significantly inhibit the aortic vascular inflammatory response.3.CK201 can inhibit the inflammatory response of endothelial cellsCK201 significantly inhibited the adhesion of HUVECs cells to THP-1 cells.It can promote the migration of HUVECs cells,suggesting that it has the potential to promote the healing of injured vascular endothelium.CK201 inhibited the expression of inflammatory factors IL-6 and TNF-αin HUVECs cells,indicating that CK201 could inhibit inflammatory response.Conclusions:1.Ginsenoside derivative CKN could significantly inhibit the formation of AS plaque in ApoE^-/-mice.The inhibition rate of CKN in aorta plaque was 60.9%,and the inhibition rate of brachiocephalic trunk plaque was 51.8%.The inhibition rate of aortic plaque and brachiocephalic trunk plaque by clinical anti-atherosclerosis drug atorvastatin was 41.8%and27.3%,respectively.Its mechanism is related to reducing blood lipid levels,promoting LXRαnuclear translocation,up-regulating ABCA1 expression,inhibiting foam cell formation and vascular inflammatory response.2.Ginsenoside derivative CK201 could significantly reduce the plaque formation of AS in ApoE^-/-mice,and the plaque inhibition rate was 62.4%,while the plaque inhibition rate of atorvastatin was 26.6%.Its mechanism of action is related to lowering blood lipids,protecting endothelial cells and reducing vascular inflammation.