Oxymatrine Promotes The Expression Of ABCA1 And ABCG1 Against Atherosclerosis Through YAP/RAR Pathway

[Background and Objective] : The increasing incidence of cardiovascular disease(CVD)is a serious threat to human life and health.Epidemiological studies have confirmed that atherosclerotic(As)cardiovascular disease has become one of the leading causes of death worldwide.As is a pathophysiological process characterized by abnormal lipid metabolism and enhanced inflammatory response.Among them,the formation of foam cells caused by abnormal lipid metabolism in macrophages is the most important pathological basis,so inhibiting the formation of foam cells is an important means to prevent and treat As.ATP-binding cassette transporters(ABCs),as a kind of membrane binding protein with ATP as energy source,can effectively regulate intracellular cholesterol metabolism balance.Of which,ATP-binding cassette transporter A1(ABCA1)and ATP-binding cassette transporter G1(ABCG1)can promote the efflux of intracellular free cholesterol to extracellular Apolipoprotein A-I(Apo A-I)or nascent high-density lipoprotein(n HDL),reduce intracellular lipid accumulation,inhibit the formation of foam cells and the occurrence and development of as-induced cardiovascular disease.A large number of studies have confirmed that up-regulating the expression of ABCA1 and ABCG1 in macrophages can improve dyslipidemia in Apo E-/-mice and effectively inhibit the formation of atherosclerotic plaque in Apo E-/-mice.These results suggest that ABCA1 and ABCG1 are the key proteins to regulate the balance of cholesterol metabolism in Apo E-/-mice.Therefore,it is of great significance to further elucidate the regulatory mechanism of ABCA1 and ABCG1 expression in the prevention and treatment of the occurrence and development of As.Alkaloids extracted from Chinese herbal medicine can promote intracellular lipid efflux,inhibit inflammation and oxidative stress,so alkaloids are widely considered as potential drugs for the treatment of CVD.Oxymatrine(OMT)is an alkaloid extracted from Sophora flavescens,which has been used as a drug in the clinical treatment of chronic hepatitis B.Related studies have confirmed that OMT has a variety of pharmacological activities,such as anti-tumor,anti-inflammation,anti-oxidation,anti-allergy,anti-parasite and anti-virus and so on.At the same time,OMT also has obvious cardiovascular protective effects,such as antagonizing acute myocardial ischemia injury,improving myocardial cell function and hemodynamics in rats with chronic heart failure,reversing ventricular remodeling and so on.These studies suggest that OMT has a potential role in the prevention of as-induced cardiovascular disease,and the regulation of intracellular cholesterol metabolism is the key to the prevention and treatment of atherosclerotic cardiovascular disease.Therefore,the purpose of this study was to investigate whether OMT can regulate the balance of intracellular cholesterol metabolism,and whether this regulation depends on ABCA1 and ABCG1.In addition,proteomic analysis of epididymal adipose tissue of Apo E-/-mice showed that high fat diet led to changes in the expression of Yes-associated protein(YAP).In addition,some researches have pointed that reducing intracellular YAP expression is a potential way to protect cardiovascular function.These findings further inspire us to explore whether the cardiovascular protective effect of OMT is related to the inhibition of YAP expression.[Methods]:CCK8 assay was used to detect the effect of OMT on the cell activity of THP-1-derived macrophages and to screen the appropriate concentration of drug treatment.THP-1 derived macrophages were cultured with OMT at the optimum concentration,and the intracellular lipid content was detected by high performance liquid chromatography(HPLC).The efflux of [3H] labeled cholesterol was detected by liquid scintillation counter,and the m RNA and protein expression of ABCA1,ABCG1 and related proteins were detected by q RT-PCR and western blot.After it was clear that OMT could affect the expression of ABCA1 and ABCG1 in cells,q RT-PCR and western blot were used to detect the expression of ABCA1 and ABCG1-related regulatory proteins after OMT treatment.After the expression of RARαand RARγ was regulated by OMT,THP-1 derived macrophages were treated with RARα inhibitor Ro 41≤5253 or RARγ inhibitor MM11253,and the effects of RARα and RARγ on the regulation of cholesterol efflux by OMT were detected.Subsequently,the virus was transfected into RARα si RNA,RARγ si RNA and YAP overexpression vectors,respectively.after the transfection was confirmed to be effective,the effect of OMT on the expression of related proteins was detected to verify the role of RAR α,RARγ and YAP in the regulation of ABCA1 and ABCG1 expression by OMT.Finally,Apo E-/-mice fed with high fat diet were injected intraperitoneally with OMT of 60mg/kg for different time.the formation of aortic plaques in heart valve of Apo E-/-mice was evaluated by HE,Masson and oil red O staining.The plasma of Apo E-/-mice was collected.The effects of OMT treatment on blood lipid components in mice were detected,and the aorta of Apo E-/-mice was collected,and the expression of ABCA1 and ABCG1 protein in aortic plaques was detected by q PCR and western blot.[Results]:The optimum concentration of THP-1-derived macrophages determined by CCK8 method was 80 μ M.HPLC showed that OMT could effectively reduce the contents of total cholesterol,free cholesterol and cholesterol ester in THP-1-derived macrophages.Subsequently,liquid scintillation counter showed that OMT promoted cholesterol efflux mediated by ABCA1 and ABCG1.QRT-PCR and Western blot confirmed that OMT could effectively up-regulate the expression of ABCA1,ABCG1,RARα and RARγ.The inhibitors of RARα and RARγand si RNA treatment confirmed that OMT regulated the expression of ABCA1 and ABCG1 dependent on RARα.Bioinformatics detection showed that YAP,RARα and ABCA1 interact with each other.Further Western blot analysis confirmed that OMT inhibited the expression of YAP protein and promoted the increase of ABCA1 and ABCG1 expression mediated by RARα,while overexpression of YAP inhibited the expression of RARα,ABCA1 and ABCG1.It is confirmed that OMT promotes the expression of ABCA1 and ABCG1 depends on YAP/RARαpathway.By measuring the staining area of aortic valve sections in Apo E-/-mice,it was found that OMT significantly inhibited the formation of atherosclerotic plaques in mice,and blood lipid analysis showed that OMT increased the plasma HDL-C level in Apo E-/-mice.[Conclusion]:OMT promotes the expression of ABCA1 and ABCG1 and cholesterol efflux from macrophages through YAP/RAR pathway,thus inhibiting the occurrence and development of atherosclerosis.