| The present study,using techniques and methods of MTT,Oil Red O staining,cholesterol content determination,immunofluorescence,Western Blotting,ELISA,H&E staining,blood lipid level and biochemical analysis,wheat germ agglutinin(WGA)staining,Masson staining,immunohistochemistry,etc.,and choosing Raw264.7 macrophage cells as experimental subjects,voltage-gated potassium channel Kv1.3 specific inhibitor PAP-1 as drug,discovers and demonstrates that PAP-1 can reduce Recombinant Macrophage scavenger receptor 1(MSR1)and Thrombospondin receptor(CD36)levels and up-regulate ATP-binding cassette transporter A1(ABCA1),ATP-binding cassette,sub-family G member 1(ABCG1)protein expressions based on the reverse cholesterol transport pathway to improve the Raw264.7 macrophage-derived foam cells of the atherosclerotic vitro model.Then through establishment of the AS(Atherosclerosis)model in rats,we find that PAP-1 can improve the carotid artery pathology,decrease blood lipid levels related to cholesterol.We also find the effect of PAP-1 in AS rats related to the down-regulating of CD36,MSR1 and up-regulating of ABCA1,ABCG1 protein expressions.At the same time,we find that PAP-1 can inhibit and reduce the heart damage caused by AS,including cardiomyocyte hypertrophy and increased heart wall thickness,myocardial fibrosis and inflammation.The results are as follows:1.The effect of PAP-1 to macrophage-derived foam cells based on reverse cholesterol transport pathwayRaw264.7 macrophage cells are pretreated with 100 nM PAP-1 for 2 h and exposed to 60μg/mL ox-LDL for 24 h.We use MTT method to detect cell viability,Oil Red O staining method to observe intracellular lipid accumulation,enzymatic method to detect TC and FC leves in cells,Western Blotting combined with immunofluorescence technology to detect MSR1,CD36,ABCAl,ABCG1 pathway protein expressions.The results show that ox-LDL induce the decreasing of cell viability in Raw264.7,stimulate lipid intake,add TC/FC intake,increase CD36,MSR1 and down-regulate ABCA1 and ABCG1 protein expressions in a concentration-dependent manner;PAP-1 inhibits the decline of foam cell viability,reduces the intracellular lipid accumulation and the cholesterol content.In addition,Western Blotting combined with immunofluorescence technology show that PAP-1 can suppress the formation of foam cells by up-regulating or down-regulating the expression of ABCA1,ABCG1,MSR1,CD36 to achieve the balance of lipid transport in Raw264.7 cell.Tip:PAP-1 can promote the efflux of cholesterol from foam cells through the cholesterol reverse transport pathway,reverse the formation of foam cells,and improve the vitro foam cell model of AS.2.The effect of PAP-1 in AS rats based on reverse cholesterol transport pathwayUse Carotid artery balloon injury combined with high-fat feeding to establish AS rats.The 20 rats are adaptively fed for one week,randomly divide the rats into a blank control group(4 rats)and a model group(16 rats).During the period,the blank control group are ordinary fed,and treat the model rats with balloon damage combined with high-fat feed to establish AS rat model.Then Model rats are randomly divided into 3 groups:Model group,PAP-1 group and AV(Atorvastatin)group as positive controls.Inject Antibiotics intraperitoneally for 3 consecutive days after operation,once a day.Afterwards,start to give the drug.PAP-1 group:intraperitoneal injection of 3 mg/(kg·d)(dissolved in 1%DMSO,99%corn oil);AV group:intragastric administration of 10 mg/(kg·d),dissolved in corn oil.At the same time,the same volume of corn oil is taken orally and intraperitoneally to the rats in the model group and the blank control group.Observe the general state of the experimental rats(weight,spirit,hair color,etc.).At the end of the 12th week,Vevo 3100 high-resolution animal ultrasound imaging system is used to observe ventricular ultrasound images and calculate cardiac function.Then collect blood samples of each group of rats and the vascular tissues of the carotid artery.Use H&E staining to analyze the pathological changes of the injured carotid artery in rats,Western Blotting to detect the expressions of CD36,MSR1,ABCG1 and ABCA1 protein levels,enzymatic detection of serum TC,FC,TG,LDL-C contents to evaluate the changes of blood lipid in rats,ELISA method to detect serum inflammatory factor IL-1β,IL-6,TNF-α levels to investigate the accumulation of inflammation.We find that PAP-1 reduces the rapid increase of body weight between AS rats,declines the thickness and lipid deposition of the carotid artery between AS rats,also lowers the IL-1β,IL-6,TNF-α inflammation levels and TC,FC,TG,LDL-C levels of AS rats.Through Western Blotting experiment,it indicates that the protection of PAP-1 on AS connect with the down-regulating of CD36/MSR1 expression and up-regulating of ABCA1/ABCG1 protein expression.Tip:PAP-1 attenuates the pathology of the carotid artery in AS rats by promoting the reverse cholesterol transport pathway.3.The cardioprotection of PAP-1 in AS ratsFurther,we use wheat germ agglutinin(WGA)staining to measure the cross-sectional area of cardiomyocytes.Masson staining and Immunohistochemical staining are used to detect the degree of fibrosis and the response of inflammation in myocardial tissue sections.We find that PAP-1 efficiently decreases cardiac enlargement and reduces collagen fibrosis including type Ⅰand type Ⅲ collagen levels then significantly inhibits myocardial inflammatory responses which manifested by the reduction of IL-1β,IL-6 and TNF-α inflammatory cytokines in myocardial tissue sections.Tip:PAP-1 may have a role in cardioprotection in AS rats by suppressing the enlargement of cardiomyocyte hypertrophy and heart wall thickness,reducing inflammation and fibrosis in heart.Reverse cholesterol transport refers to the process by which high-density lipoprotein(HDL)transports the cholesterol in foam cells and atherosclerotic plaques to the liver for catabolism and elimination from the body.It is one of the important mechanisms for the normal body to maintain the balance of lipid metabolism.Cholesterol metabolism disorder is one of the main causes of AS,and clinical data shows that the most commonly used lipid-lowering drugs,statins,have common side effects that cause muscle soreness,weakness,muscle inflammation,and liver toxicity.Therefore,how to develop therapeutic drugs for AS through the reverse cholesterol transport pathway has potential.In basic research,it finds that the voltage-gated potassium channel Kv1.3 specific blocking antibody hKv1.3-E314 can reverse the foam cell model of AS based on the cholesterol reverse transport pathway;and in vivo studies it finds that Kv1.3 blockade can reduce the signs of obesity,this provides us with ideas for applying Kv1.3 blocker to prevent and treat AS.PAP-1 is a new type of small molecule with high affinity for Kv1.3 with an EC50 of only 2 nM,and its selectivity for Kv1.3 is 33-125 times that of other members in Kvl family.At present,the literature has pointed out that PAP-1 has the safety in vivo and in vitro application and can inhibit the lipid deposition in carotid artery of AS rats to a certain extent,but the exact molecular mechanism is unknown.Therefore,this study explores the effect of PAP-1 on AS cells and animal models through the reverse cholesterol transport pathway,provides evidence for the mechanism of PAP-1 against AS,and provides ideas for the application and development of PAP-1 or other K1.3 inhibitors in the treatment of AS. |
