Ginkgo Biloba Extract (EGb761) Inhibiting Smooth Muscle Foam Cell Formation And The Underlying Mechanisms

Atherosclerosis is the main cause of ischemic heart disease and stroke,which causes vascular stenosis,tissue and organ blood insufficiency,and threatens health.The intake of excess fat leads to abnormal lipid metabolism,elevated blood lipid level and increasing incidence of atherosclerosis.The characteristic pathological changes of atherosclerosis are the formation of foam cells,which derived from macrophages and vascular smooth muscle cells?SMCs?.Foam cells involve in the occurrence and development of atherosclerosis,so illuminating the mechanism of foam cells formation is of great significance for prevention and treatment of atherosclerosis.Previous studies mainly focused on the foam cell derived from macrophages,while there were few studies on smooth muscle foam cells formation.Ginkgo biloba is a traditional Chinese medicine with a long history.Ginkgo biloba extract?GBE?is widely used as an adjuvant treatment for a variety of clinical cardiovascular diseases due to its anti-oxidative stress and anti-inflammatory function.The protective effect of GBE on atherosclerosis has been reported in clinical and animal experiments,but the mechanism has not been fully elucidated.In particular,the effect and mechanism of GBE on smooth muscle foam cell formation has not been reported.AimsThis study is aimed to investigate the effect of GBE on smooth muscle foam cell formation during atherosclerosis and related molecular mechanisms.Methods and Results1.GBE concentration dependent inhibition of ox-LDL induced smooth muscle foam cell formationSMCs were treated with ox-LDL at different concentrations,and the results of oil red O staining showed that ox-LDL treatment induced the formation of smooth muscle foam cells.However,the co-incubation of EGb761?standardized GBE?significantly inhibited the formation of lipid droplets in a concentration-dependent way.The quantitative measurement of intracellular cholesterol content was consistent with the results of oil red O staining.Treatment with EGb761 inhibited the increase of cholesterol content induced by ox-LDL.Dio-ox-LDL was further used to incubate SMCs,and the fluorescence intensity was detected to reflect the cholesterol uptake ability of SMCs.The results showed that EGb761 reduced the lipoprotein uptake of SMCs in a concentration-dependent way.SMCs were incubated with NBD cholesterol,and the cholesterol content in the medium and the intracellular cholesterol content were detected respectively to reflect the cholesterol efflux of SMCs.The results showed that EGb761 pretreatment could significantly increase the cholesterol efflux ability of SMCs.The effect of EGb761 on cholesterol de novo synthesis in SMCs was then examined.The synthesis of cholesterol is mainly regulated by SREBP2,HMGCR,LDLR,while western blot experiments showed that EGb761 incubation had no effect on the expression of the above genes.Results suggested that the inhibitory effect of EGb761 on the smooth muscle foam cells formation was not related to the cholesterol synthesis,but mainly related to the changes in the cholesterol uptake and efflux of SMCs.2.Effects of GBE on cholesterol uptake in SMCsThe uptake of cholesterol by SMCs depends on the binding of lipoproteins to receptors on the cell membrane.SR-A1,CD36 and LOX-1 are major molecules that mediate the uptake of cholesterol by cells.The expression of SR-A1,CD36 and LOX-1 was up-regulated by ox-LDL treatment in SMCs,while the expression of SR-A1 and LOX-1 was decreased by EGb761 incubation,but the transcription level of CD36 was not affected.Nuclear translocation of transcription factors c-jun and c-Fos and their binding to activator protein 1?AP1?are the main mechanisms to regulate the transcriptional expression of SR-A1.The expression levels of c-jun and c-Fos in the nucleus were detected by Western Blotting,and the expression of c-jun and c-Fos in the nucleus were down-regulated by EGb761 incubation,suggesting that EGb761 may inhibit the expression of SR-A1 by inhibiting the nuclear translocation of c-jun and c-Fos.The expression of LOX-1 was regulated by inflammatory factors.Therefore,the nuclear translocation of NF-?B was detected.The results showed that ox-LDL treatment up-regulated the expression of NF-?B in the cell nucleus,while EGb-761 could down-regulate the expression of NF-?B in the cell nucleus,suggesting that EGb-761 inhibited the expression of LOX-1 by inhibiting the activation of NF-?B.3.Effects of GBE on cholesterol efflux of SMCsAfter cholesterol is absorbed into cells,it goes through a process of hydrolysis-lipidation-rehydrolysis,and then is transported to the extracellular environment via membrane transporter ATP binding cassette transporter A1?ABCA1?,ATP binding cassette transporter G1?ABGG1?and scavenger receptor B1?SR-B1?.Incubation of EGb761 increased cholesterol efflux from SMCs,and we further tested the effect of EGb761 on the expression of ABCA1,ABCG1 and SR-B1.Results showed that protein expression of ABCA1 was significantly up-regulated by EGb761 incubation,but the protein expression of ABCG1 and SR-B1 were not affected.Furthermore,qPCR experiments showed that EGb761 incubation increased the transcription level of ABCA1,while EGb761 had no significant effect on the transcription of ABCG1 and SR-B1.4.ERK1/2 involved in regulating the expression of ABCA1 of GBE in SMCsIt is believed that the expression of ABCA1 is negatively regulated by the ERK1/2 signaling pathway.Therefore,we examined the effect of EGb761 incubation on the ERK1/2 signaling pathway in SMCs.Results showed that ox-LDL treatment activated the ERK1/2 signaling pathway and down-regulated the expression of ABCA1,while the EGb761 incubation inhibited the activation of ERK1/2 and restored the expression of ABCA1 in SMCs.Subsequently,we used the U0126?blocking agent of ERK1/2?to incubate the SMCs,and found that the phosphorylation level of erk1/2 decreased and the expression of ABCA1 increased meantime,suggesting that blocking the activation of erk1/2 can simulate the regulation effect of EGb761 on ABCA1.Finally,SMCs were co-incubated with U0126 and EGb761,and it was found that the two had no superimposed effect on the up-regulation of ABCA1,suggesting that EGb761 plays a regulatory role in the up-regulation of ABCA1 by inhibiting the activation of ERK1/2.5.PAQR3 mediated GBE induced the upregulation of ABCA1 in SMCsThe mechanism by which EGb761 inhibits ERK1/2 activation remains unclear,and PAQR3 is an important molecule that regulates the phosphorylation level of ERK1/2.Firstly,we found that the expression of PAQR3 was upregulated in SMCs by treating with EGb761,while the expression of PAQR3 was decreased in SMCs treated with ox-LDL.The results of qPCR also showed that the mRNA level of PAQR3 was regulated by EGb761 and ox-LDL.Subsequently,we treated SMCs with EGb761 while using siRNA to knock down the expression level of PAQR3.The results showed that knockdown of PAQR3 expression eliminated the inhibitory effect of EGb761 on ERK1/2 and the up-regulation effect of ABCA1.Finally,we constructed PAQR3 plasmids and transfected them into SMCs.The results showed that overexpression of PAQR3 inhibited the activity of ERK1/2 and further upregulated the expression of ABCA1.The above results showed that EGb761 could inhibit the activation of ERK1/2 by up-regulating the expression of PAQR3,thus up-regulating the expression of ABCA1.6.PAQR3 mediates GBE up-regulation of cholesterol efflux and inhibition ofsmooth muscle foam cells formationFurthermore,the role of PAQR3 in EGb761 enhancing cholesterol efflux in SMCs was detected.Results showed that knockdown of PAQR3 inhibited the effect of EGb761 on the cholesterol efflux in SMCs,while the overexpression of PAQR3could promote the cholesterol efflux of SMCs.However,the overexpression of PAQR3 and EGb761 showed no synergistic effect in increasing the cholesterol efflux of SMCs.7.GBE inhibited the degree of atherosclerosis in ApoE^-/-mice on high-fat dietAtherosclerosis model was established in ApoE^-/-mice on high-fat diet,and EGb761 was administered by gavage to observe the therapeutic effect on atherosclerosis and the expression of related molecules.Oil red O staining showed that EGb761 treatment significantly reduced the size of atherosclerotic plaques,while Western Blotting showed that EGb761 partially inhibited the up-regulation of SR-A1,CD36 and LOX-1 expression in model group.Meanwhile,EGb761 inhibited the downregulation of PAQR3 expression,inhibited the phosphorylation level of ERK1/2,and restored the expression of ABCA1 in atherosclerosis mice.ConclusionIn this study,we found that GBE inhibited smooth muscle foam cell formation induced by ox-LDL.This effect was related to the fact that GBE inhibits the cholesterol uptake of SMCs and improves the cholesterol efflux of SMCs.GBE inhibits the expression of SR-A1 and LOX-1 by inhibiting the transcriptional activity of c-jun and c-Fos and NF-?b.GBE inhibits the activity of ERK1/2 by upregulation of PAQR3,and up-regulated of ABCA1.This study revealed the anti-atherosclerosis mechanism of GBE,providing experimental evidence for its clinical application.