| It is well known that cardiovascular diseases resulted from atherosclerosis(AS) is the principal cause of death in the world.Although the concept of AS had been proposed for nearly 100 years,the onset mechanism of AS remains unclear.It has been clear that the development of AS is an integration process with multiple factors at multilevel.Following expressions of pro-inflammatory factors and activation of immune response,lipid level was increased.At the same time,lipid peroxidation aggravated production of ox-LDL,and macrophage swallow ox-LDL.All these resulted in lipid metabolism disorder and foam cell formation ultimately.The occurrence of macrophage-derived foam cell is the key marker of AS at the early stage.Along with the deepening to the mechanism of AS,we pay more attention to inhibiting foam cell formation as a new policy to cure AS.Panax Notoginseng saponin(PNS) is regarded as the main active constituents, including Rg1,Rb1,R1,etc.Our research indicated that PNS could prevent and cure AS through immunological regulation,anti-inflammation,anti-free radical,etc.Because less study on material foundation and mechanism had been operated,the applications of PNS were restricted.Based on the effectiveness of PNS on AS prevention and curing,we used the modern pharmacological methods and techniques to explore the effects of PNS and monomer combination on the foam cell formation during AS.Methods:1.32 New Japanese male rabbits,divided into 4 groups(including control group, AS model group,high-dose PNS group and low-dose PNS group) randomly,were fed with forages as follows respectively:(1) base forage,(2) high-fat forage(containing cholesterol 0.5%),(3) high-fat forage with low-dose PNS(60mg/kg),(4) high-fat forage with high-dose PNS(120mg/kg).The experiment lasted 12 weeks.Rabbit AS models were confirmed by measuring levels of TC,TG,LDL,MDA and SOD activity in rabbit serum,thoracic aortas staining in SudanⅣsolution and tissue section oil red O staining.2.Macrophages,collected from mouse peritoneal,were divided into five groups after cultured and purified.(1) control group,(2) model group,(3) low-dose PNS group(20μg·ml-1),(4) medium-dose PNS group(40μg·ml-1),(5) high-dose PNS group(80μg·ml-1).Cells were maintained at 37℃in 5%CO2-95%air in an incubator for 72 hours.The cellular lipid accumulation was examined by oil red staining.The cellular contents of total cholesterol(TC) and free cholesterol(FC) were detected by enzymatic colorimetry.The cellular protein was measured by Lowry method.3.According to cholesteryl ester as the index and Rg1,Rb1 and R1 as investigation factors(every factor containing 3 levels:10-4,10-5,10-6M),the effects of Rg1,Rb1 and R1 on the foam cell formation of and their interaction were obtained by orthogonal design experiment.4.Expressions of CD36 mRNA,Adipophilin mRNA,LXRαmRNA,ABCA1 mRNA and ACAT-1 mRNA in macrophage were analyzed by RT-PCR.Expression of CD36 protein in macrophage was determined by western blotting.Results:1.Amount of foam cells accumulated under aortic tunica intima of rabbits in AS model rabbits,and the atherosclerotic plaque square obviously increased.High-dose PNS could reduce AS plague area and decrease foam cell formation under rabbit aortic tunica intimas(p＜0.01)).2.The serum concentrations of TC,TG and LDL-C in AS model group were significantly higher than that in normal control group(p＜0.01).Compared to AS group, high-dose PNS could reduce the serum concentrations of TC,TG and LDL-C markedly (p＜0.01).The level of TC in low-dose PNS group is lower than that in AS group (p＜0.05).3.In AS model group,the serum concentration of MDA was increased,but the activity of SOD was decreased.High-dose PNS could reduce the serum concentration of MDA and enhance the activity of SOD in rabbits(p＜0.01),and low-dose PNS could decreased the level of MDA(p＜0.05).4.Macrophage in vitro culture was almost fusiform and there was no oil red-staining positive cell.Because of uptaking amount of lipid,oil red-staining positive cells were filled with macrophages and the shapes were round or irregular after co-cultured with ox-LDL.In high-dose and medium PNS groups,obvious reduction of oil red-staining positive cells was observed.Compared to model group,there was no significant difference in low-dose PNS group. 5.The contents of TC,FC,CE(cholesteryl ester) and CE/TC ratio in model group were greatly higher than that in control group(p＜0.01).Compared with model group,the contents of TC,FC,CE and CE/TC ratio were significantly reduced in PNS high and medium dose groups(p＜0.01).Meanwhile,there were obvious differences among PNS groups.6.Orthogonal experiment indicated that Rg1,Rb1and R1 were key responsive factors,and there were interaction between Rg1×Rb1 and R1×Rb1(p＜0.01).The optimized monomer combination was Rg1:R1:Rb1=10-5:10-5:10-6M。7.Compared with model group,PNS and monomer combination could reduce the ratio of CE/CT significantly.There were no obvious differences between PNS group and monomer combination group.8.CD36 mRNA expression in macrophages was increased remarkably after treated with ox-LDL(p＜0.01).Compared with model group,PNS(p＜0.01),single substrates compatibility(p＜0.01),Rg1(p＜0.05) and R1(p＜0.05) could decrease CD36 mRNA expression in macrophages induced by ox-LDL.There were no obvious differences among PNS group,monomer combination group,Rg1 group and R1 group.9.Treated with ox-LDL,Adipophilin mRNA expression increased remarkably in macrophages(p＜0.01).Compared with model group,PNS,monomer combination,Rg1, Rb1 and R1 didn't affect Adipophilin mRNA expression induced by ox-LDL.10.Treated with ox-LDL,LXRαmRNA expression was increased in macrophages (p＜0.05).Compared with model group,PNS,monomer combination,Rg1 and R1 could increased LXRαmRNA expression induced by ox-LDL(p＜0.01).LXRαmRNA expressions in PNS group and monomer combination group had no significant differences. To some extent,LXRαmRNA expressions in Rg1 group(p＜0.05) and R1 group(p＜0.05) were lower than that in PNS group.11.Treated with ox-LDL,ABCA1 mRNA expression was increased in macrophage (p＜0.05).Compared with model group,PNS(p＜0.01),monomer combination group (p＜0.01) and R1(p＜0.05) could enhanced ABCA1 mRNA expression induced by ox-LDL in its degree.Compared with PNS group,there were no difference in monomer combination group and R1group.12.Treated with ox-LDL,macrophages ACAT-1 mRNA expression increased remarkably(p＜0.01).Compared with model group,PNS,monomer combination group and Rb1 could decreased ACAT-1 mRNA expression induced by ox-LDL(p＜0.01),and there was no significant difference among them.13.CD36 protein expression in model group macrophages was much higher than that in control group(p＜0.01).PNS(p＜0.01),monomer combination group(p＜0.01), Rg1(p＜0.05) and R1(p＜0.05) remarkably reduced CD36 protein expression induced by ox-LDL,and there was no significant difference among them.Conclusions1.PNS could cure AS and prevent formation of foam cell through the pathway of relieving oxidative stress,modulating blood lipid and inhibiting phagocytose function of macrophage.2.PNS and monomer combination could inhibit macrophage-derived foam cell formation induced by ox-LDL.3.PNS and monomer combination could down-regulate the expressions of CD36 mRNA,ACAT-1 mRNA and CD36 protein,and up-regulate the expressions of ABCA1 mRNA and LXRαmRNA.Rg1 could down-regulate CD36 mRNA and CD36 protein expressions and up-regulate LXRαmRNA expression.Rb1 could down-regulate ACAT-1 mRNA expression.R1 could down-regulate CD36 mRNA and protein expressions and up-regulate ABCA1 mRNA and LXRαmRNA expressions.So, cholesterol ingestion into macrophage was decreased and the excretion was increased. Above all,PNS,monomer combination and monomer had effects on the foam cell formation at multi -targets.4.The optimized monomer combination ratio is Rg1:R1:Rb1=10-5:10-5:10-6 in this experiment.The effect of monomer combination on foam cell formation is the same as that of PNS.So,we can preliminary predict that Rg1,Rb1 and R1 may be the indispensable material foundation for PNS preventing and curing AS.
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