The Study Of Wendan Decoction On In Vivo Effective Components And Anti-atherosclerotic Mechanism Based On The Phlegm Syndrome Theory In TCM Of Coronary Heart Disease

ObjectiveAs the pathological foundation and the key link in many cardiovascular diseases,prevention and treatment of atherosclerosis has become a major issue with common concern in the world.According to the theory of traditional Chinese medicine,phlegm turbidity is the foundation of atherosclerotic lesion formation.Throughout history,experts in Chinese medicine emphasized that treatment of phlegm is one of the important ways in treatment of coronary heart disease.Wendan decoction was first showed in the Tang dynasty and widely used in clinical treatment of phlegm syndrome.This study is aim to evaluate the effect of Wendan decocotion on hyperlipidemia and coronary heart disease based on the papers of clinical randomized and controlled trials.This study is also design to explore the role of cholesterol transport in the anti-atherosclerotic effects of Wendan decoction and its in vivo effective components by establishing cell and mouse models related with phlegm syndrome in TCM of coronary heart disease.This study will not only provide more comprehensive scientific experimental evidence for clinical application of Wendan decoction,but also provide foundation for further development of anti-atherosclerotic Chinese medicine with a clear target and a material base.Methods1.Clinical papers researchThe meta-analysis of randomized and controlled trials was apply for evaluating the effects of Wendan decoction on hyperlipidemia and coronary heart disease basing on the comprehensively electronic database search.2.Cell researchThis research was design to explore the effect of Wendan decoction and its in vivo effective components,including isosakuranetin,glycyrrhetinic acid,naringenin,hesperetin,meranzin,on early stage of atherosclerosis based on the foam cells induced by ox-LDL.First,the effects of different concentrations of treatments on cell viability were examined by MTT.Then the effects of Wendan decocotion and its in vivo effective components on intracellular lipid accumulation and formation of foam cells were observed on oil red o staining.The in vivo effective components with significant efficacy on the formation of foam cells were selected for next experiments.The cholesterol efflux,cholesterol level,free cholesterol level and cholesterol esterification rate were examined using a cholesterol efflux assay,total cholesterol assay and free cholesterol assay,respectively.The levels of macrophages membrane proteins,including CD36,SR-A,ABCA1,SR-BI,were quantified by western blot.3.Animal researchThis research was design to explore the effects of Wendan decoction on atherosclerosis based on Apo E-/-mice with high fat diet.The weight and serum lipid profiles of mice were analyzed.And the atherosclerotic plaque buildup in aortic root and whole aorta were observed on HE staining and oil red o staining,respectively.The protein levels related to cholesterol transport in aorta(ABCA1,caveolin-1)and liver(SR-BI,CD36)were quantified by western blot.Results1.Clinical papers researchThe meta-analysis results of the effects of Wendan decoction on hyperlipidemia showed that the response rates and reduced levels of TG and LDL-C by treatment with Wendan decoction were better than treatment with regular western medicine(P < 0.05);However there were no statistical significance between treatment with Wendan decoction and regular western medicine in the reduced levels of TC as well as the increased levels of HDL-C(P>0.05).The result of subgroup analysis showed that there was no statistical significance between treatment with Wendan decoction and regular western medicine in regulating blood fat in patients with nonalcoholic fatty liver(P>0.05).Besides,the response rates and reduced levels of TG,TC,LDL-C by treatment with Wendan decoction plus regular western medicine were better than treatment with regular western medicine alone(P<0.05).As to adverse reactions,only one paper referred to stomach discomfort induced by Wendan decoction,which means the good safety and tolerability of Wendan decoction.The meta-analysis results of the effects of Wendan decoction on coronary heart disease showed that compared to treatment with regular western medicine alone,treatment with Wendan decoction plus regular western medicine exerted the better effects on the response rates,electrocardiogram,traditional Chinese syndrome,and the levels of CRP(P<0.05).Because of the variant between studies and the less studies included in analysis,it could not draw a defined conclusion on the difference between treatment with Wendan decoction plus regular western medicine and regular western medicine on the levels of LDL-C in patients with coronary heart disease yet.There were 4 papers with safety description demonstrating the good safety and tolerability of Wendan decoction without any adverse reactions.2.Cell researchWendan decoction concentration-dependently reduced the formation of foam cells and the levels of intracellular cholesterol,cholesterol ester,and cholesterol esterification rate(P<0.01 VS.Control group).However there was no statistical significance between Wendan decoction group and control group in the levels of intracellular free cholesterol(P>0.05).Wendan decoction also concentration-dependently increased the efflux rate of intracellular cholesterol(P < 0.01 VS.Control group).Besides,Wendan decoction concentration-dependently reduced the levels of CD36 protein on macrophages membrane(P < 0.01 VS.Control group).Wendan decocotion at concentration of 6 g/L reduced the level of SR-A protein on macrophages membrane(P<0.05 VS.Control group);However,there were no statistical significance between Wendan decoction group at concentrations of 1.5 g/L,3mg/m L and control group in the level of SR-A protein(P>0.05).The treatment of Wedan decoction at concentrations of 3 g/L and 6 g/L significantly increased the levels of ABCA1 protein and SR-BI protein(P<0.05 or P<0.01 VS.Control group);However,there was no statistical significance between Wendan decoction at concentration of 1.5 g/L and control group in the levels of SR-BI protein(P>0.05).Naringenin and meranzin at concentrations of 10?mol/L,50?mol/L,200?mol/L and hesperetin at concentrations of 10?mol/L,50?mol/L,100?mol/L significantly inhibited the formation of foam cells and reduced the levels of TC and CE and CER in THP-1-derived macrophage foam cells(P<0.05 or P<0.01 VS.Control group).However there was no significance in the levels FC between control group and the group of naringenin,hesperetin,and meranzin at different concentrations(P>0.05).Naringenin,hesperetin,and meranzin at different concentrations also significantly increased choleterol efflux in THP-1-derived macrophage foam cells(P<0.01 VS.Control group).Naringenin at concentration of 50?mol/L reduce the levels of SR-A protein on macrophage membrane(P<0.05 VS.Control group);However there were no significance in the levels of SR-A protein between control group and naringenin group at contrations of 10 ?mol/L and 200 ? mol/L(P >0.05).And there was no significance in the levels of SR-A protein between hesperetin,meranzin and control group(P>0.05).Naringenin at different concentrations increased the levels of CD36 protein(P < 0.05 VS.Control group);There were no signifficance in the levels of CD36 protein between treatment with hesperetin,meranzin and control group(P>0.05).Naringenin(10?mol/L,50?mol/L),hesperetin(10?mol/L,50?mol/L,100?mol/L),and meranzin(10?mol/L,50?mol/L,200?mol/L)significantly increased the levels of ABCA1 protein(P<0.05 or P<0.01 VS.Control group).However,there was no significance in the levels of ABCA1 protein between control group and naringenin at concentration of 200?mol/L(P>0.05).Hespretin(50?mol/L,100?mol/L)and meranzin(10?mol/L,50?mol/L)significantly increased the levels of SR-BI protein(P<0.05 or P<0.01 VS.Control group).But there were no significance in the levels of SR-BI protein between control group and naringenin(10?mol/L,50?mol/L,200?mol/L),hesperetin(10?mol/L),or meranzin(200?mol/L)(P>0.05).3.Animal researchWendan decoction at middle dose significantly inhibited the increase of weigh in Apo E-/-mice(P<0.05 VS.Model group).Wendan decoction at different dose significantly reduced the levels of serum TG,TC,and LDL-C in Apo E-/-mice(P<0.05 or P<0.01 VS.Model group).Wendan decoction at different dose reduced the atherosclerotic plaque buildup in whole aorta of Apo E-/-mice(P<0.05 or P<0.01 VS.Model group).Wendan decoction at middle dose and high dose reduced the atherosclerotic plaque buildup in aortic root(P<0.05 VS.Model group).Bedsides,Wendan decoction at different dose increased the levels of ABCA1 protein and reduced the levels of caveolin-1 protein of aorta in Apo E-/-mice(P<0.01 VS.Model group).Wendan decoction at middle dose and high dose increased the levels of SR-BI protein of liver in Apo E-/-mice(P<0.01 VS.Model group).However there was no statistical significance between treatment with Wendan decoction at different dose and model group in the levels of CD36 protein of liver in Apo E-/-mice(P>0.05 VS.Model group).ConclusionWendan decoction has significant effect on hyperlipidemia and coronary heart disease with good safety and tolerability.Atherosclerosis is the pathological foundation of coronary heart disease.The early stages of atherosclerosis is characterized by foam cell formation.Wendan decoction reduces the accumulation of intracellular cholesterol and increases the cholesterol efflux by reducing the protein levels of CD36 and SR-A on macrophage membrane and increasing the protein levels of ABCA1 and SR-BI on macrophage membrane,which reduces the formation of foa cells.Wendan decoction reduces weight,serum lipid levels,and inhibits the formation of atherosclerotic plaques by reducing the levels of caveolin-1 protein of aorta and increasing the levels of ABCA1 protein of aorta and the levels of SR-BI protein of liver in Apo E-/-mice with high fat diet.Besides,naringenin,hesperetin,and meranzin are in keep with the effects of Wendan decoction on the formation of foam cells.This three components could be considered as the anti-athrosclerotic material base of Wendan decoction through promoting cholesterol reverse transport.This results could provide more comprehensive scientific experimental evidence for clinical application of Wendan decoction and the foundation for further development of anti-atherosclerotic Chinese medicine with a clear target and a material base.