Mechanism Of Scutellarin Anti-atherosclerosis Through PPARα/γ-LXRα-ABCA1 Pathway

Atherosclerosis is a common disease that threats to human health.The main pathological manifestations of atherosclerosis are arterial intimal lipid deposition,inflammatory cell infiltration,smooth muscle cell and matrix hyperplasia,intimal thickening,stenosis and thrombosis and so on.It is a complex pathological process of abnormal multi-factor participation and multi-gene regulation.It is generally believed that cholesterol and cholesteryl esters aggregating in macrophages and foaming play an important role in the formation of atherosclerosis.So the factors affecting cholesterol synthesis,uptake,efflux and balance of cellular cholesterol is the key to study the mechanism of atherosclerosis.The reverse transport of cholesterol is an important pathway leading to the efflux of intracellular cholesterol.ABCA1 plays a key regulatory role in cholesterol reverse transport,sterol metabolism,and high-density lipoprotein metabolism.ABCA1 also regulated by a variety of factors such as liver X receptors(LXRα and β),a member of nuclear receptor family,peroxisome proliferator activated receptor(PPARα,β,γ),sugar Corticosteroid receptor(GR)and cyclic adenosine monophosphate(cAMP),cytokines(TNF-α,IL-1β)and some protein kinases.A number of studies show that,enhancing ABCA1 expression can promote intracellular cholesterol efflux to exert anti-atherosclerotic effects.ABCA1 can be used as an effective intervention target for the prevention and treatment of As and cardiovascular disease.At present,the anti-As and cholesterol-lowering drugs in clinic are widely used including fibrates,statins,probucol and CETP inhibitors etc.The above drugs all have some side effects in the prevention and treatment of cardiovascular diseases.In particular,statins can not only specifically reduce endogenous cholesterol synthesis by inhibiting intracellular HMG-CoA reductase,but also enhance cholesterol efflux by enhancing the expression of LXRα and ABCA1,and also promote the synthesis of hepatocyte ApoAI HDL.However,the long-term use of these drugs might lead to myositis and liver dysfunction and other adverse reactions.Therefore,it is of great significance to develop new anti As strategies,such as the effect and mechanism of Chinese traditional Chinese medicine in the prevention and control of As.Previous studies on prescriptions of traditional Chinese medicine were difficult to clarify its mechanism of action.So it has a remarkable clinical effect,but also difficult to be recognized by international academic.Therefore,in recent years,many scholars have turned their attention to single-component,chemically-defined traditional Chinese medicine monomer and made great achievement.A lot of research suggests that curcumin,berberine,baicalin and other traditional Chinese medicine monomer can exert anti-As effect by lowering blood fat,inhibiting inflammation,reducing oxidative stress,inhibiting vascular smooth muscle cell proliferation and improving microcirculation.In this study,we summarized the main traditional Chinese medicine(TCM)monomer of the prescriptions which are effective in clinical treatment,through the observation and investigation of pre-clinical cases.Seven TCM Monomers were finally selected for further study.In the first part,we used qPCR,oil red O staining,computer modeling molecular docking and gene expression microarray to screen effective TCM monomer and its possible target and signal pathway.Then,in the second part,we explored the scutellarin,which was screened by quantitative real-time PCR,Western-blot,Oil Red O staining and fluorescence spectrophotometry,determined the optimal concentration and optimal time of action.Through transected siRNA,the mechanism of action of scutellarin on anti-As was clarified through the PPARα-LXRα-ABCA1 and PPARγ-LXRα-ABCA1 pathway to promote the reverse transport of cholesterol.Finally,in the third part,APOE-/-mice were used to establish animal models.To determine scutellarin will affect lipid accumulation in the liver and aortic and aortic sinus plaque area.The mRNA and protein expression of each gene in PPARα/γ-LXRα-ABCA1 pathway was observed in mouse peritoneal macrophages and liver tissue also aortic tissue to explore the mechanism of this pathway in vivo.In this study,we established a method platform for the target screening of anti-As TCM monomer,revealed the mechanism of action of scutellarin for anti-As by promoting the reverse transport of cholesterol through PPARα/γ-LXRα-ABCA1 pathway.This article provided a new idea and method for research on the mechanism of anti-As TCM Monomer.Part 1 Anti-atherosclerosis Chinese medicine monomer and its target screening ObjectivesScreening anti-As TCM monomer from seven TCM monomers,primarily identify its target and possible pathway.MethodsFirst,we establish THP-1 macrophage-derived foam cell model.Foam cells were treated with seven different Chinese herbs.Then real time RT-PCR was performed to evaluate the mRNA expression of inflammatory cytokines(TNF-α、IL-1β and transcription factor NF-κB),Cholesterol effluent protein(ABCA1、ABCG1)、Cholesterol synthesis protein(ABCA1、ABCG1)、Cholesterol absorbed related proteins(SRA1、CD36)and Cholesterol transport-related proteins(CAV-1、NPC1).After preliminary screening of effective TCM monomer,the foam cells were stained by oil red O for analyzing the effect of scutellarin on lipid metabolism.The molecular docking method was used to screen the affinity of seven TCM monomers with the possible pathway regulatory proteins to predict the target.Finally,expression profiling gene chip analysis to predict targets and pathways of drugs purpose of Chinese medicine monomer(scutellarin).ResultsThen qRT-PCR results showed that scutellarin can inhibit the expression of inflammatory factor TNF-α in foam cells in a dose-dependent manner,up-regulate the cholesterol efflux proteins ABCA1 and ABCG1,down-regulate the cholesterol synthesis proteins HMGCR,HMGCS and Cholesterol absorbed related proteins SRA1,CD36.Further oil red O staining showed that 10μM scutellarin significantly reduce intracellular lipid accumulation in foam cells.The high affinity targets of scutellarin screened by molecular docking method were NPC1,PPARα,ABCA1 and HMGCS,suggesting that scutellarin may inhibit cholesterol synthesis through HMGCS and promote intracellular cholesterol efflux through ABCA1.Besides,ABCA1 may modulate by PPARα.The results of gene expression profiles of scutellarin-treated foam cells showed that ABCA1 and PPARα were up-regulated in 310 differentially expressed genes.ConclusionScutellarin play a anti-AS effect by significantly reducing lipid accumulation in foam cells.ABCA1 and PPARα may be the major target of scutellarin.Besides,it may also exert anti-inflammatory effects by down-regulating TNF-α.Part 2 Scutellarin promotes cholesterol efflux from macrophage-derived foam cells via the PPARα/γ-LXRα-ABCA1 pathway ObjectivesExplore the mechanisms of scutellarin on the cholesterol outflow of macrophage-derived foam cells through PPARα/γ-LXRα-ABCA1 pathway.MethodsWe established THP-1 macrophage-derived foam cell model.Foam cells were treated with different concentrations of scutellarin(5μM,10μM,20μM)at different time(0h,12 h,24h,48h).The mRNA and protein levels of ABCA1,LXRα,PPARγ and PPARα in foam cells were detected by qRT-PCR and Western-blot,the rate of cholesterol efflux of foam cells was detected by oil red O staining and fluorescence cholesterol efflux assay.Then LXRα,PPARγ,PPARα or ABCA1 silenced by siRNA respectively.Similarly,mRNA and protein levels of ABCA1,LXRα,PPARγ and PPARα in foam cells were detected by qRT-PCR and Western-blot,oil red O staining and fluorescence cholesterol efflux assay Method to detect foam cell cholesterol efflux rate.ResultsIn concentration and time gradient experiments,mRNA and protein expressions of all the genes were not significantly increased in 5μM group compared with foam cell model group.Oil red O staining and fluorescence cholesterol efflux were also not detected obvious cholesterol efflux.ABCA1 mRNA and protein expression were significantly up-regulated in both concentration and time-dependent manner at both 10μM and 20μM concentrations.LXRα mRNA and protein expression were also significantly up-regulated,but not in concentration and time-dependent manner.The mRNA and protein expressions of PPARγ and PPARα showed significant up-regulation in both drug concentration groups,neither in concentration nor in time-dependent manner.After Foam cells treated with 10μM and 20μM scutellarin,oil red O staining and fluorescence cholesterol efflux rate test results showed that intracellular lipid accumulation was significantly reduced,cholesterol efflux rate was significantly increased;but the effect did not further increased with concentration and time changed.Pathway validation experiments were performed using the optimal concentration of scutellarin at 10μM and the optimal duration of 24 h.When silencing LXRα,PPARγ and PPARα,ABCA1 genes,Real time RT-PCR and Western Blot show that LXRα,PPARγ and PPARα,ABCA1 mRNA and protein expression were significantly down-regulated.Oil red O staining and fluorescence cholesterol efflux assay measure the cholesterol efflux rate,the results showed that the foam cell cholesterol efflux decreased,but intracellular lipid accumulation no evidently reduced.The results showed that silenceing PPARγ and PPARα,both mRNA and protein expression of LXRα and ABCA1 were significantly down-regulated,silenceing LXRα significant down-regulation ABCA1 mRNA and protein expression.The oily red O staining and fluorescence cholesterol efflux assay results are consistent with the above results.ConclusionThe optimal concentration and time of scutellarin for cholesterol ef flux in macrophage-derived foam cells was 10 μM and 24 h.Scutellarin can up-regulate PPARγ,PPARα,LXRα,and ABCA1 mRNA and protein expression in foam cells.silenceing PPARγ,PPARαand LXRα,The expression of ABCA1 i n foam cells induced by Scutellarin was inhibited,the cholesterol efflu x rate also decreased.Scutellarin may promote cholesterol efflux from ma crophage-derived foam cells via the PPARα-LXRα-ABCA1 and PPARγ-LXRα-ABC A1 pathway.Part 3 Effects of scutellarin on atherosclerotic lesions in ApoE-/-mice ObjectivesTo investigate the anti-As effect of scutellarin in atherosclerotic lesion model of ApoE-/-mice.MethodsFor 8 weeks,8 weeks old ApoE-/-mice was fed a high-fat diet and then randomly divided into model group(intraperitoneal injection of the same dose of saline)and Scu drug group(intraperitoneal injection of scutellarin 10mg/kg/day).The changes of body weight were measured after 8 weeks of administration.Oil red O staining was used to observe the frozen section of liver tissue and the aortic sinus,and the aortic resin deposition.The area and ratio of the plaque area of aortic tree and the aortic sinus were calculated by image analysis software Image-pro plus 6.0.The mRNA and protein levels of PPARγ,PPARα,LXRα and ABCA1 in peritoneal macrophages,aorta and liver of mice were detected by qRT-PCR and western-blot.ResultsThere was no significant difference in weight between two groups.Oil red O staining of liver frozen sections showed that the lipid deposition in the liver of Scu group was significantly decreased.Oil red O staining of the aorta and aortic sinus frozen sections showed that plaque area of As model group was significantly larger and irregular than that of Scu drug group.The red lipid droplet area ratio of Scu drug group was reduced dramatically than As Model group.Image analysis software was used to scan the total aortic sinus plaque area and red lipid droplet area.The results showed that the total plaque area and the area of red lipid droplets between Scu drug group and As group was significant.In murine peritoneal macrophages and liver tissues,significant up-regulation of the mRNA and protein levels of PPARα,LXRα and ABCA1 in Scu drug group,while the expression level of PPARγ that was not statistically significant.In mouse aorta tissue,PPARα and ABCA1 mRNA in Scu drug group was significantly up-regulated compared with As model group,while the PPARγ and LXRα mRNA that were not statistically significant.ConclusionScutellarin can reduce the lipid accumulation in the liver of ApoE-/-mice.Scutellarin can reduce the area of atherosclerotic plaque on the intima of the aorta and the lipid deposition in ApoE-/-mice.Scutellarin can also up-regulate the mRNA and protein expression of PPARα,LXRα and ABCA1 in ApoE-/-mice macrophages;It can also up-regulate the mRNA expression of PPARα and ABCA1 in liver tissue and aorta tissue.scutellarin maybe play an anti-atherosclerotic effect by promoting the reverse cholesterol transport via PPARα-LXRα-ABCA1 pathway in ApoE-/-mice.