Preliminary Study On The Preventive Activity And Mechanism Of Soyasaponins Against Atherosclerotic Plaque In ApoE^-/- Mice

BackgroundCardiovascular diseases(CVDs)are the first killer of human health.Atherosclerosis is an important pathological basis of CVDs.Previous studies have shown that food-derived phytochemicals(such as saponins,polyphenols,etc.)can effectively prevent atherosclerotic diseases.Soyasaponins are members of the family of saponin phytochemicals that are widely distributed in soybeans and their products.It has been attracted much attention because of its wide bioactivities such as anti-inflammation,anti-oxidation,lipid-lowering etc.However,till now no report is available regarding soyasaponins’ antagonistic bioactivity against atherosclerotic plaques and its underlying mechanism.ObjectivesIn this study,we established a model of atherosclerosis by feeding ApoE-/-knockout mice with high fat diet,and investigated the effect of soyasaponin intervention on atherosclerotic plaque f’ormation,and further analyzed lipid metabolism,oxidative stress and inflammation.We aimed to study the antagonistic bioactivity of soyasaponins against atherosclerotic plaque and preliminarily clarify its underlying mechanisms.This study provided theoretical basis for the rational use of soyasaponin in preventing CVDs,and also provided clues for developing soyasaponins-based functional foods or medicines.MethodsSixty 6-week-old male ApoE-/-mice were averagely and randomly divided into the 6 groups.The mice in control group were fed high fat diet containing 0.15%cholesterol and 45%fat(HFD).Four groups of mice were used for soyasaponins intervention and fed with HFD supplemented with 10 or 20μmol/kg BW soyasaponin A1(SS-A1)or soyasaponin A2(SS-A2),respectively.Mice in the simvastatin intervention group were fed with HFD supplemented with 10 μmol/kg BW simvastatin.All mice were given free access to feed and water for 24W.During the intervention period,body weight and feed consumption was monitored once a week.Glucose tolerance test was performed one week before the end of the experiment.Upon the end of the experiment,the arterial tissues(aorta,innominate artery,and aortic root)were separated and collected to prepare pathological sections to evaluate the degree of atherosclerotic plaques.Lipid profiles in feces and serum,and Ox-LDL and inflammatory markers(TNF-a,mcp-1 and hs-crp)levels in serum were detected by enzymatic methods.The expression levels of TLR4,MyD88,p65,p-p65,IkBα and p-IKBα in fresh aortic tissues were determined by western blot.Immunohistochemistry and imunofluorescence were used to semi-quantitatively determine the expression levels of TLR4 and MyD88 in the sections of innominate artery and aortic root.Results(1)There was no significant difference in growth(initial body weight,final body weight,and average body weight gain)and energy intake between mice from different groups(P>0.05).(2)Soyasaponins A1and A2 significantly reduced the proportion of plaques in the aortic roots and innominate arteries of ApoE-/-mice(P<0.05),but had no effect on the proportion of lipid plaques in the aorta(P>0.05).Simvastatin significantly decreased the proportion of plaques in the aortic roots(P<0.05),but produced no effect on the proportion of plaques in the aorta and innominate arteries(P>0.05)(3)Soyasaponins A1 and A2 significantly lowered LDL-C and TC in serum(P＜0.05).Soyasaponin A1 significantly reduced TG and increased HDL-C(P<0.05)Soyasaponin A2 did not change HDL-C level(P>0.05),whereas 20μmol/kg BW of soyasaponin A2 reduced TG(P<0.05).Simvastatin significantly lowered LDL-C and elevated HDL-C in serum(P<0.05),but had no effect on the HDL-C level(P>0.05).Furthermore,20μmol/kg BW soyasaponin A1 increased the excretion of TG in feces and 10μmol/kg BW soyasaponin A2 promoted the excretion of TC in feces(P<0.05)The 20μmol/kg BW soyasaponins A1and A2 both increased the excretion of total bile acids in feces(P<0.05).However,simvastatin had no effect on the excretion of lipids in feces(P>0.05).In addition,20μmol/kg BW of soyasaponin A2 significantly improved the impaired glucose tolerance in mice(P<0.05).(4)Soyasaponin A2 significantly reduced the serum levels of Ox-LDL in mice(p<0.05).(5)Soyasaponins(A1 and A2)and simvastatin all significantly reduced the levels of inflammatory markers(mcp-1 and hs-crp)in serum(P<0.05),inhibited the phosphorylation of NF-kB p65 and IkBa in aortic tissue(P<0.05),and decreased the mean optical density levels of TLR4 and MyD88 proteins in innominate artery(P<0.05).Soyasaponin A1 and 20 μmol/kg BW of soyasaponin A2 significantly reduced the serum level of TNF-a in mice and decreased the protein expression of TLR4 and MyD88 in aorta tissues(P<0.05).In addition,soyasaponins A1 and A2 had a tendency to reduce the fluorescence intensity of TLR4 and MyD88 in aortic root tissues.Conclusion(1)Soyasaponins(A1 and A2)can reduce the size of lipid plaques in innominate artery and aortic roots of ApoE-/-mice,which indicates that they have the bioactivity of preventing early stage formation of atherosclerotic plaques.(2)In ApoE-/-mice,soyasaponins(A1 and A2)can prevent atherosclerosis through promoting fecal excretion of lipids,improving blood lipid profiles,decreasing serum levels of Ox-LDL and inflammatory markers,and inhibiting TLR4/MyD88/NF-kB signaling pathway.