The Effect And Mechanism Study Of Idebenone In Anti-atherosclerosis

Research BackgroundIchaemic cerebrovascular diseases in China have the characteristics of high morbidity,morbidity,mortality and disability rate,which is an important factor affecting the life span of Chinese people and brings immeasurable obstacles to Chinese families and social and economic aspects.In 2013,China carried out the first national epidemiological survey of cerebrovascular diseases in seven regions,which was also the largest epidemiological survey with the largest sample size in the world.From the survey results,we can see that the incidence and prevalence of stroke were 1287.3/100,000 and 274.4/100,000 respectively,and the mortality rate of stroke was 126.4/100,000.In the epidemiological survey of stroke,ischemic cerebrovascular disease accounted for 77,8%of all strokes,ranking first.lschemic stroke is a focal or general cerebral ischemia due to insufficient blood supply to the cerebrovascular vessels,resulting in necrosis of the brain cells and resulting in the corresponding clinical manifestations,including transient ischemic attack and cerebral infarction.According to the Chinese CISS classification,perforating artery type and large artery type related to atherosclerosis account for about 70%.Plaque formation in the head and neck arteries is a severe stage of the development of atherosclerosis,and stroke with head and neck arterial stenosis is the main type leading to severe disability and death.Atherosclerotic lesions cause cerebral ischemia symptoms mainly through the following mechanisms:(1)During the progression of atherosclerotic plaques,cholesterol crystals or other atherosclerotic debris may continually fall off,and the debris itself may form an embolus An embolism flows to the distal intracranial blood vessel;(2)After the debris is shed,collagen and other prothrombotic substances in the plaque are exposed,and the continuous shedding after thrombosis leads to repeated embolization of the distal blood vessel;(3)stenosis causes distal brain tissue Low blood flow perfusion;(4)The destruction of the arterial wall structure causes vascular dissection or subintimal hematoma and other causes leading to blood vessel stenosis or occlusion.This shows that ischemic stroke is closely related to atherosclerosis.Atherosclerosis(atherosclerosis,AS)is the common pathological basis of cardiovascular and cerebrovascular diseases and an important cause of death.Pathological studies have shown that the occurrence and development of atherosclerosis include lipid infiltration,platelet activation,thrombosis,intimal injury,inflammatory response,oxidative stress,and activation of vascular smooth muscle cells.Although AS has different pathogenesis mechanisms,such as the theory of lipid penetration,the theory of macrophage receptor loss,the theory of smooth muscle mutation,the theory of mitochondrial damage,the theory of inflammatory response,and the theory of immunology,etc.,no single theory can be fully explained separately The occurrence and development of AS.Mitochondria are important risk factors for cardiovascular and cerebrovascular diseases.Mitochondrial dysfunction can affect AS progression by aggravating the body's oxidative stress,inflammatory response,apoptosis and cholesterol accumulation and other pathological processes,and plays an important role in regulating AS cardiovascular and cerebrovascular diseases..Mitochondria are the main sites for the production of oxygen free radicals(ROS)in cells.Impaired mitochondrial function will further increase the generation of ROS and induce vascular endothelial cell dysfunction,which leads to the formation of atherosclerosis.Mitochondria are considered to be an important link in oxidative stress and endothelial cell dysfunction,and an important factor in promoting the formation and development of atherosclerotic lesions.The relationship between mitochondria and atherosclerosis will be explained by the following four aspects.1.Mitochondrial ROS and atherosclerosisPromoting the progression of atherosclerosis AS risk factors can increase the production of mitochondrial oxygen free radicals(mtROS).Not only can mtROS directly cause mitochondrial damage and dysfunction,but ROS can also be combined with NO to generate nitrous peroxide Ion,the latter can dose-dependently inhibit the synthesis of mitochondrial proteins,resulting in a reduction in cellular ATP levels and mitochondrial reduction function.At the same time,mitochondrial arginase II can control the biosynthesis of NO in the body by regulating the level of arginine in the cell.At the same time,because mitochondrial DNA lacks histones and has weak repair activity,mitochondrial DNA is also an important target molecule for mtROS damage.2.Mitochondrial-derived inflammation and atherosclerosisMitochondrial damage and increased mitochondrial oxidative stress can promote the occurrence of related inflammation.Related studies have shown that NLRP3 inflammatory bodies are closely related to atherosclerosis,which can be activated either directly by mtROS or by damaged mitochondrial DNA.After LDLR--mice were fed a high-cholesterol diet,if they transplanted the bone marrow of NLRP3-deficient mice,ASC-deficient mice,or IL-1?/?-deficient mice,the AS plaque lesions were reduced compared to the control group,and their plaques were reduced.The level of inflammation in the block was significantly reduced.Recent studies have shown that cholesterol crystals are also an endogenous danger signal,which can directly activate NLRP3 inflammation and contribute to the inflammatory development of AS.3.Mitochondrial pathway apoptosis and atherosclerosisApoptosis is an important factor affecting the development of AS.Early apoptosis of endothelial cells can weaken the barrier effect of the intima,lipid is easily deposited under the intima and initiates the occurrence of AS.A large amount of apoptosis in the late stage can lead to the formation and rupture of vulnerable plaques and affect the stability of plaques,which is the main cause of the clinical acute symptoms in the late stage of AS lesions.Apoptosis in the mitochondrial pathway causes mitochondria to oxidize cytochrome C to produce mtROS.mtROS can also cause the opening of the mitochondrial permeability transition pore,which reduces the permeability of the mitochondrial membrane,thereby releasing cytochrome C and activating caspase-9 in the cytoplasm,which ultimately leads to Apoptosis.4.Mitochondrial disorder leads to obstruction of cholesterol transport and atherosclerosisThe outflow of cholesterol from foam cells on the arterial wall is an important mechanism for the body to resist AS.In this process,ATP binding cassette transporter A1 promotes cholesterol and phospholipid outflow to poA-? and apoE,while ATP binding cassette transporter G1 promotes cholesterol outflow to high-density lipoprotein.Under normal circumstances,sterol-27-hydroxylase on the inner mitochondrial membrane can convert cholesterol to 27-hydroxylated sterol,thereby promoting cholesterol outflow in the cell.The transport of cholesterol from the outer mitochondrial membrane to the inner membrane is an important condition for the activation of sterol-27-hydroxylase.When mitochondrial dysfunction occurs,cholesterol transport on the mitochondrial membrane may be abnormal,thereby affecting sterol-27-hydroxylase activity and cholesterol outflow.Improving mitochondrial function and mitochondrial cholesterol transport will help cholesterol efflux from macrophage-derived foam cells and stabilize AS plaques.Therefore,the use of mitochondrial-targeted antioxidant therapeutic drugs can inhibit the generation of excessive ROS,while improving mitochondrial function and reducing inflammation,thereby achieving the effect of preventing and treating atherosclerosis.Idebenone,6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinon e.It started as an intelligent promoting drug and was developed and marketed in Japan in 1986.Its main mechanism of action is to activate brain mitochondrial respiratory activity,improve cerebral energy metabolism of cerebral ischemia,improve the utilization of glucose in the brain,and produce ATP in the brain.Increase;At the same time improve the metabolism of neurotransmitter serotonin in the brain,has a strong antioxidant and scavenging free radicals.Idebenone is a synthetic analogue of coenzyme Q10 with the same benzoquinone ring-like structure,and its short alkyl side chain replaces the isoprenoid side chain of coenzyme Q10,which is more likely to pass through the biofilm and blood-brain barrier.Previous studies have shown that idebenone has the effect of protecting mitochondria,preventing apoptosis of vascular endothelial cells,clearing mitochondrial ROS and reducing inflammation.Idebenone is an antioxidant and an electron carrier.Its benzoquinone ring has two forms:oxidized and reduced(hydroquinone).The oxidized type accepts two electrons to reduce to generate hydroquinone,which plays the role of antioxidant and electron transfer.The oxidized form accepts an electron to generate an unstable semiquinone,which promotes oxidative stress.A number of studies have found that idebenone can effectively scavenge oxygen free radicals,and idebenone can bind to mitochondrial complex ?,in the electron transport chain,transfer electrons to complex ?,produce ATP,reduce ROS generation.Unlike coenzyme Q10,idebenone can bypass the electron transport chain independent of mitochondrial complex ?,such as complex?-? and G3PDH shuttle,etc.,to transfer electrons to the mitochondrial complex in the mitochondrial electron respiratory chain ?.It can protect the cell membrane and mitochondria from oxidative stress damage in the condition of mitochondrial complex I disorder.Therefore,idebenone is found in various neurological diseases involving mitochondrial complex ? dysfunction and oxidative stress damage,such as mitochondrial encephalomyopathy,Friedrich ataxia,and Alzheimer's disease.Certain therapeutic effect.In addition,idebenone is an antioxidant that more easily crosses the biofilm and blood-brain barrier,and its metabolites can be broken down into QS-4,QS-6,and QS-10.Among them,QS-10 is an electron acceptor with a strong affinity for NAD(P)H:quinone oxidoreductase 1,which is a key factor in resisting oxidative stress and inhibiting the development of atherosclerosis.At the same time,related studies have shown that idebenone can attenuate the production of proinflammatory NF-?B and MAPK signaling pathways,both of which are important factors in promoting atherosclerosis.The most important thing is that idebenone has good tolerance and safety.These have confirmed that idebenone can be used as a mitochondrial protective agent and antioxidant for the prevention and treatment of early vascular atherosclerosis.Prospects for use.This study combines in vivo and in vitro experiments to explore whether idebenone can be used as a mitochondrial protection-related antioxidant for the prevention and treatment of atherosclerotic cardiovascular and cerebrovascular diseases.At the same time,we applied proteomics technology to analyze and further explore The molecular mechanism of dibenzoquinone against atherosclerosis.We will elaborate in the following 4 parts:Part ? Idebenone inhibits AopE-/-mice atherosclerotic plaque formation and enhances plaque stabilityPurposesTo explore the effect of idebenone on plaque formation and plaque stability in AopE-/-atherosclerosis model mice.Method1.Preparation of Idebenone SolutionIdebenone is provided by Qilu Pharmaceutical Co.,Ltd.,with a purity of?98%,dissolved in corn oil,and administered by gavage.2.Establishment and grouping of experimental animal models60 7-week-old male AopE-/-gene knockout mice were randomly divided into 4 groups after adapting to a high-fat diet for 1 week:(1)high-fat diet group;(2)high-fat diet+idebenone low Dose group(100mg/kg/d);(3)High-fat diet+idebenone medium-dose group(200mg/kg/d);(4)High-fat diet+idebenone high-dose group(400mg/kg/d).After 16 weeks of intervention with idebenone,all mice were euthanized after weighing,blood of each group of mice was collected,8 mice were collected of tissues and placed in 4%paraformaldehyde for pathological staining,7 mice Retain the tissues and put them into liquid nitrogen for quick freezing to be used in related molecular biology experiments.3.Blood index testingBlood index testing detected by enzymatic method.4.Pathological staining of tissue specimensThe aorta was stained with general oil red O,and the frozen sections of aortic root were subjected to hematoxylin-eosin(HE)staining,oil red O staining,saturated picric acid-sirius scarlet staining and corresponding immunohistochemical staining.Aortic plaque load,aortic root plaque cross-sectional area,plaque lipid,collagen,macrophages,smooth muscle cells,and inflammation-related factors were measured.5.Statistical methodsResult1.Basic situation of AopE-/-atherosclerosis model miceAt the end of the experiment,all mice successfully passed the experiment,and there was no significant difference in the weight of the mice between the groups.The hematological test indicators include serum TG,TC,LDL-C,HDL-C and blood glucose levels,and there is no statistical difference between the mice in each group.2.The effect of idebenone on the gross load and cross-sectional area of atherosclerotic plaqueThe plaque morphology was observed by HE staining of frozen sections of aortic roots,and the plaque cross-sectional area was measured.Compared with the high-fat diet group,the plaque cross-sectional area of each idebenone dose intervention group was significantly reduced.3.The effect of idebenone on the composition of atherosclerotic plaqueHistopathological staining results showed that,compared with the high-fat diet group,each idebenone dose intervention group can significantly reduce the percentage of lipid and macrophage content in plaque,and significantly increase the smooth muscle cell and collagen content in plaque.The content percentage significantly reduces the content percentage of inflammation-related factor TNF-?.Chapter summaryIn the experiment of AopE-/-atherosclerosis model mice,idebenone can inhibit the formation of atherosclerotic plaques and improve the plaque tissue composition to make it more stable.Part ? Proteomics Study and Bioinformatics Analysis of Idebenone in anti-atherosclerosisPurposeIn order to further explore the molecular mechanism of the development of idebenone against atherosclerosis,we performed proteomics analysis on the high-fat diet group and the high-dose group of idebenone.Material1.We randomly selected three AopE-/-mouse aortic tissues from the high-dose idebenone group(IDE-H)and high-fat diet group(HFD)for proteomic analysis and bioinformatics analysis.2.Protein extractionThe aortic tissue protein of mice in the high-dose idebenone group and the high-fat diet group was extracted and its concentration was detected.3.Trypsin cleavageAdd lysis treatment to the protein solution.4.TMT markThe peptide TMT digested by pancreatin was labeled and then freeze-dried in vacuo.5.Liquid chromatography-mass spectrometry analysisAfter separation by ultra-high performance liquid phase system,the peptides were injected into the NSI ion source for ionization and mass spectrometry analysis.Results1.Overview of proteomics analysisTaking 1.3 times as the differential expression threshold,351 proteins in the IDE-H/HFD comparison group were up-regulated and 379 proteins were down-regulated.2.Sample repeatability testPrincipal component analysis(PCA),relative standard deviation(RSD)and Pearson's Correlation Coefficient(Pearson's Correlation Coefficient)three statistical analysis methods were used to evaluate the quantitative repeatability of protein.All the samples sent for inspection have good repeatability.3.Functional classification of differentially expressed proteins3.1 The difference proteome between IDE-H group and HFD group should be located in the mitochondria of cells(22.88%),extracellular matrix(22.6%)and cytoplasm(20.41%),which also indirectly reflects that idebenone can mainly change mitochondrial protein Thus affecting the occurrence and development of atherosclerosis.3.2 Through comparative analysis of databases,COG/KOG functional classification statistics of differentially expressed proteins were found,and the differential proteins were mainly involved in(1)energy production and conversion;(2)amino acid transport and metabolism;(3)lipid transport and metabolism and(4)Signal transduction mechanism pathway.4.Functional enrichment analysis of differentially expressed proteins4.1 GO enrichmentWe performed differential protein enrichment analysis on the three major categories(Biological Process,Cellular Component,Molecular Function)in the GO classification.We can find that in the Biological Process,proteins with larger fold multiples are mainly enriched in(1)the process of acyl-CoA biosynthesis;(2)the metabolic process of acetyl-CoA;(3)aerobic respiration;(4)respiratory electrons Transmission chain;(5)Acute inflammatory response.In Cellular Component,the proteins with larger fold multiples are mainly enriched in(1)oxidoreductase complex;(2)mitochondrial respiratory chain;(3)mitochondrial respiratory chain complex I;(4)NADH dehydrogenase complex;(5)Mitochondrial membrane protein complex.In Molecular Function,proteins with larger fold multiples are mainly enriched in(1)pyruvate dehydrogenase(NAD+)activity;(2)immunoglobulin receptor binding;(3)electron carrier activity;(4)NADH dehydrogenation Enzyme activity;(5)NADH dehydrogenase(ubiquinone)activity.4.2 KEGG channel enrichmentThe KEGG pathway obtained by enrichment analysis mainly involves(1)fatty acid metabolism;(2)oxidative phosphorylation;(3)PPAR signaling pathway.4.3 Enrichment of protein domainsThe domains of higher difference fold proteins in IDE-H group and HFD group are mainly concentrated in(1)MCM domain;(2)MCM OB domain;(3)MCM N-terminal domain.Chapter summaryTaking 1.3 times as the differential expression threshold,a total of 351 proteins were up-regulated and 379 proteins were down-regulated in the IDE-H/HFD comparison group.Through proteomics analysis of the aortic tissues of mice in the HFD group and IDE-H group,it was found that the differential proteins were mainly located in mitochondria;through GO analysis and KEGG analysis,the differential proteins were mostly involved in oxidative respiration,mitochondrial electron transport chain and inflammation Reaction,which laid a theoretical foundation for us to seek the target of idebenone against atherosclerosis.Idebenone's resistance to atherosclerosis may involve protein-related pathways such as SIRT3,SOD2,and NLRP3,which require further verification in vivo and in vitro.Part ? Idebenone inhibits atherosclerotic plaque formation by activating SIRT3-SOD2-NLRP3 pathway in AopE-/-micePurposeIn this part of the project,by constructing a mouse model of atherosclerosis,it is verified whether idebenone can change SIRT3-SOD2-NLRP3 pathway-related proteins,thereby inhibiting the occurrence and development of atherosclerosis.Method1.Establishment and grouping of experimental animal modelsThe same as the first part of mouse grouping and model building method.2.Molecular biological testing of tissue specimens.The total protein of aortic tissue was extracted from each group of mice,and the changes of SIRT3,FOXO3A,SOD2,NLRP3,CASPASE1 and IL-1?indexes at each protein level were detected by Western Blot method.3.Immunohistochemical staining of frozen sections.Immunohistochemical staining of SIRT3,SOD2,NLRP3 indexes was performed on frozen sections of aortic root tissue.4.ELISA analysisWe used ELISA experiments to analyze the changes in the level of IL-1? index among groups.Result1.Preliminary verification of changes in SIRT3-SOD2-NLRP3 pathway-related proteins in proteomics results.The remaining aorta of the 6 mice sent to IDE-H group and HFD group were examined by Western blot.The results of Western blot analysis of SIRT3,SOD2 and NLRP3 proteins were found to be consistent with proteomics data analysis.2 Idebenone affects SIRT3-SOD2-NLRP3 protein expression in atherosclerotic plaques in mice:2.1.Western Blot statistical results showed that compared with the high-fat diet group,the expression levels of SIRT3,FOXO3A,and SOD2 protein in plaques of various concentrations of idebenone group were significantly increased.The results of immunohistochemical staining showed that Compared with the lipid diet group,the percentage of SIRT3 in plaques of each concentration of idebenone group increased significantly.In addition,the percentage of SOD2 in plaques of each concentration of idebenone group also increased significantly.2.2 The protein content of NLRP3 and CASPASE1(P20)in each concentration of idebenone group was significantly reduced;immunohistochemical staining statistical results showed that compared with the high-fat diet group,each The percentage of NLRP3 content in plaques in the concentration idebenone group was significantly reduced.2.3 Western Blot and ELISA analysis results showed that:compared with the high-fat diet group,the expression level of IL-1? in each concentration of idebenone group was significantly reduced.Chapter summaryIn ApoE-/-atherosclerosis model mice,in the pre-intervention group of idebenone at various concentrations,it was found that idebenone can activate the expression of SIRT3,increase the levels of FOXO3A,SOD2 protein,and further inhibit the activation of NLRP3,Reduce the expression of downstream inflammatory factors such as CASPASE1,IL-1?,and further inhibit the occurrence and development of atherosclerosis.Part ? HUVECs model validates the mechanism of idebenone in anti-atherosclerosisPurposeIn this part of the study,in vitro experiments were conducted to construct a human umbilical vein blood endothelial cell injury model to further verify the molecular mechanism of idebenone against atherosclerotic plaque formation.Method1.Culture and identification of human umbilical vein endothelial cells.2.The establishment of human venous blood endothelial cell injury model and related experimental detection(1)? normal control group;? 10 pM cholesterol group;? 10 ?M cholesterol+0.2 ?M idebenone group.(2)CCK-8 was used to detect the proliferation of vascular endothelial cells in each group.(3)Using a microplate reader to detect the MDA,SOD2,IL-1? and JC-1 mitochondrial membrane potential activity levels in each group of cells.(4)MitoSOX dye was used to detect the change of mitochondrial ROS level.(5)Western blot was used to detect the expression levels of Bcl-2,Bax,caspase-3 and cleared caspase-3 protein in each group of cells,and to detect the level of apoptosis in each group.3.Use si-SIRT3 to explore the molecular mechanism of idebenone against atherosclerosis.(1)Divide HUVECs into 4 groups:?Si-control group;?Si-control+10?M cholesterol group;?Si-control+10?M cholesterol+0.2?M idebenone group;?Si-SIRT3+10?M cholesterol+0.2?M ideben Quinone group.(2)TUNEL staining;(3)Western blot was used to detect the expression levels of SIRT3,FOXO3A,SOD2,NLRP3,CASPASE1(P20)and IL-1? protein in each group of cells.Result1.Idebenone significantly inhibits cholesterol-induced lipid peroxidation and enhances SOD activity.Compared with the normal group,the endothelial cell proliferation level in the 10 ?M cholesterol group was significantly reduced,the SOD2 activity was significantly reduced,and the level of lipid peroxidation was increased;compared with the 10 ?M cholesterol group,the 0.2 ?M idebenone pretreatment group MDA was added significantly increased,suggesting that idebenone can enhance the ability of cells to scavenge oxygen free radicals and protect cholesterol-induced vascular endothelial cells by inhibiting cell lipid peroxidation.2.Idebenone reduces endothelial cell apoptosis by protecting mitochondrial function and inhibiting oxidative stress levels.2.1 Compared with the endothelial cells in the 10 ?M cholesterol group,the red fluorescence intensity of the pretreatment group with 0.2 ?M idebenone was significantly increased,and the red-green fluorescence ratio was significantly increased.2.2 Compared with the normal cell group,the MitoSOX stained 10?M cholesterol group significantly increased the red fluorescence,reflecting the increased ROS levels in the mitochondria.The red fluorescence intensity of the 0.2?M idebenone pretreatment group was added.Significantly weakened 2.3 The Bcl-2 protein expression level was significantly increased,suggesting that cholesterol caused an increase in endothelial cell apoptosis.Compared with the 10 ?M cholesterol group,the addition of 0.2 ?M idebenone pretreatment group increased the expression levels of Bax(P<0.05).3.Idebenone can activate SIRT3-SOD2-mtROS-NLRP3 pathway and inhibit atherosclerosis3.1 Compared with the Si-control+10 ?M cholesterol group,the Si-control+10?M cholesterol+0.2 ?M idebenone group It shows that idebenone can reduce the ratio of cholesterol-induced endothelial cell apoptosis,which can be suppressed by the small interfering RNA Si-SIRT3.This conclusion can be obtained from the group of Si-SIRT3+10?M cholesterol+0.2?M idebenone The percentage of apoptotic cells is calculated statistically.3.2 Compared with the Si-control+10 ?M cholesterol group,the expression levels of SIRT3,FOXO3A,and SOD2 protein increased in the 0.2 ?M idebenone group,while the expression levels of NLRP3,CASPASE1(P20)and IL-1? protein decreased.Similarly,this phenomenon can be suppressed by the small interfering RNA Si-SIRT3.In the Si-SIRT3+10?M cholesterol+0.2pM idebenone group,the related proteins did not change significantly compared with the Si-control+10?M cholesterol group.Chapter summaryIn the cholesterol-induced endothelial cell injury model of human umbilical vein blood,idebenone can protect mitochondria,improve mitochondrial function,enhance the ability of scavenging oxygen free radicals,reduce the level of mitochondrial oxidative stress,and thus reduce the level of endothelial cell apoptosis.At the same time,idebenone can activate the SIRT3-SOD2-mtROS pathway and reduce the production of inflammatory factors such as NLRP3,thereby protecting injured endothelial cells.ConclusionIn this study,we carried out the corresponding experimental design and work around the effect and molecular mechanism of idebenone on atherosclerosis,and achieved the following results:1.Through in vivo experiments,we confirmed that idebenone drug intervention can inhibit the formation of atherosclerotic plaques in AopE-/-mice.2.We detected the composition of atherosclerotic plaque in AopE-/-mice and found that idebenone can reduce the lipid and inflammatory components of the plaque,increase the content of collagen and smooth muscle cells,and make the plaque more stable.3.We performed proteomics analysis on the aorta of the high-dose idebenone group and the high-fat diet control group of AopE-/-mice.The analysis results can be found:the differential proteins are mostly involved in oxidative respiration,mitochondrial electron transport chain and inflammation Response,which laid a theoretical foundation for us to seek the therapeutic target of idebenone for the treatment of atherosclerosis.4.Through in vivo and in vitro experiments,we confirmed that idebenone can activate the SIRT3-SOD2 pathway,thereby reducing mitochondrial ROS production,reducing the activation of NLRP3 inflammatory bodies,thereby inhibiting the downstream inflammatory response pathway,and ultimately inhibiting AopE-/-The generation of atherosclerotic plaques in mice.In summary,idebenone can be used as a mitochondrial protective agent and antioxidant for the prevention and treatment of atherosclerosis,and because of its fewer side effects and higher tolerance,it is expected to become a new prevention and treatment of atherosclerosis Effective solution.