Inhibiting NF-κB increases cholesterol efflux from THP-1 derived-foam cells treated with AngⅡ via up-regulating the expression of ATP-binding cassette transporter A1

Objective： To study the role of nuclear factor-kappa B（NF- κB） in cholesterol efflux from THP-1 derived-foam cells treated with Angiotensin Ⅱ（Ang Ⅱ ）. Methods：Cultured THP-1 derived-foam cells were treated with Ang Ⅱ or preincubated with tosyl-phenylalanine chloromethyl-ketone（TPCK） NF- κB inhibitor. The levels of activated NF- κB in the cells were examined by sandwich ELISA, Cellular cholesterol content was studied by electron microscopy scanning and zymochemistry via fluorospectrophotometer and cholesterol effiux was detected by scintillation counting technique. ABCA1 mRNA and protein were quantified by RT-PCR and Western blotting. Results：Addition of TPCK to the cells before Ang Ⅱ stimulation attenuated the response of NF- κB p65 nuclear translocation induced by Ang Ⅱ and showed no peak in foam cells group and caused a reduction in cholesterol content and an increase in cholesterol efflux by 24.1%（P〈 0.05） and 41.1%（P〈 0.05） respectively, when compared with Ang Ⅱ group. In accordance, the ABCA1 mRNA and protein were increased by 30% and 19%（P 〈 0.05） respectively, when compared with Ang Ⅱ group. Conclusion：Ang Ⅱ can downregulate ABCA1 in THP-1 derived-foam cells via NF- K B, which leads to less cholesterol effiux and the increase of cholesterol content with the consequence of the promotion of atherosclerosis.

ATP-binding cassette transporters in progression and clinical outcome of pancreatic cancer: what is the way forward?

Pancreatic ductal adenocarcinoma(PDAC) is one of the most aggressive diseases and is characterized by high chemoresistance, leading to the lack of effective therapeutic approaches and grim prognosis. Despite increasing understanding of the mechanisms of chemoresistance in cancer and the role of ATPbinding cassette(ABC) transporters in this resistance, the therapeutic potential of their pharmacological inhibition has not been successfully exploited yet. In spite of the discovery of potent pharmacological modulators of ABC transporters, the results obtained in clinical trials have been so far disappointing, with high toxicity levels impairing their successful administration to the patients. Critically, although ABC transporters have been mostly studied for their involvement in development of multidrug resistance(MDR), in recent years the contribution of ABC transporters to cancer initiation and progression has emerged as an important area of research, the understanding of which could significantly influence the development of more specific and efficient therapies. In this review, we explore the role of ABC transporters in the development and progression of malignancies, with focus on PDAC. Their established involvement in development of MDR will be also presented. Moreover, an emerging role for ABC transporters as prognostic tools for patients' survival will be discussed, demonstrating the therapeutic potential of ABC transporters in cancer therapy.

ATP-binding cassette subfamily B member 1 (ABCB1) and subfamily C member 10(ABCC1O) are not primary resistance factors for cabazitaxel

Introduction:ATP-binding cassette subfamily B member 1(ABCB1) and subfamily C member 10(ABCCIO) proteins are efflux transporters that couple the energy derived from ATP hydrolysis to the translocation of toxic substances and chemotherapeutic drugs out of cells.Cabazitaxel is a novel taxane that differs from paclitaxel by its lower affinity for ATP-binding cassette(ABC) transporters.Methods:We determined the effects of cabazitaxel,a novel tubulin-binding taxane,and paclitaxel on paclitaxelresistant,ABCB1-overexpressing KB-C2 and LLC-MDR1-WT cells and paclitaxel-resistant,ABCC10-overexpressing HEK293/ABCC10 cells by calculating the degree of drug resistance and measuring ATPase activity of the ABCB1 transporter.Results:Decreased resistance to cabazitaxel compared with paclitaxel was observed in KB-C2,LLC-MDR1-WT,and HEK293/ABCC10 cells.Moreover,cabazitaxel had low efficacy,whereas paclitaxel had high efficacy in stimulating the ATPase activity of ABCB1,indicating a direct interaction of both drugs with the transporter.Conclusion:ABCB1 and ABCC10 are not primary resistance factors for cabazitaxel compared with paclitaxel,suggesting that cabazitaxel may have a low affinity for these efflux transporters.

ZBM30 suppresses atherosclerosis through up-regulating ATP-binding cassette A1 and G1

Atherosclerosis is the most common cause of cardiovascular diseases, such as myocardial infarction and stroke. The aim of this study was to investigate the effects of a novel compound ZBM30 on atherosclerosis in ApoE- deficient mice and its associated mechanism. ApoE-deficient mice （6 weeks old）, fed an atherogenic high-fat and high cholesterol diet for 8 weeks, were divided into three groups. Two groups were orally administrated ZBM30 （10, 30 nag ~ kg-1） daily for 12 weeks, while the control group was administered saline. Atherosclerotic lesions with en face aortas were evaluated by Sudan IV staining, and lesion areas in aortic sinuses were evaluated by oil red O staining. Necrotic core areas and fibrous cap areas in the lesion were evaluated by henaatoxylin and eosin （HE） staining and Masson＇ s trichronae staining in the aorta sinuses. The effects of ZBM30 on cholesterol accumulation in naacrophages and cholesterol transporters： ATP binding cassette A1 （ABCA1） and ATP binding cassette G1 （AB- CG1） were evaluated by oil red O assay, 3H-cholesterol efflux assay, Western blot, and real-time PCR on macro- phage cell lines： Raw 264.7 and THP-1. Inanauno-fluoresces was used to determine the ABCA1 expression in naac- rophage in aorta sinuses. Luciferase reporters of wild type and mutant types of ABCA1 promoter were constructed to determine the regulatory domain of ZBM30 on ABCA1 promoter. Results showed that, compared with the control group, en face lesions in ZBM30 group （ 10, 30 mg · kg^-1 ） were reduced 54.96 ± 10.06% and 71.50 ± 15.37% respectively, and aorta sinus lesions were reduced 41.85 ± 11.21% and 82.23 ± 8.25% respectively. Necrotic core areas in the ZBM30 group were markedly reduced and fibrous cap areas were not changed. Oil red O staining and 3 H-cholesterol efflux assays on Raw 264.7 cell line revealed that ZBM30 significantly attenuated the cholesterol accumulation in naacrophages by enhancing apolipoprotein AI and HDL mediated cholesterol efflux. Furthermore, ZBM30 induced the protein and naRNA expression of cholesterol transporters such as ABCA1 and ABCG1. Inanauno- fluoresces experiment revealed that ZBM30 induced the ABCA1 expression in naacrophage in the lesion, which is consistent with the results in vitro. Luciferase reporter assay revealed that ZBM30 exerted its effect on ABCA1 via liver X receptor （LXR） binding domain. In conclusion, ZBM30 suppresses atherosclerosis through up-regulating cholesterol efflux via ABCA1 and ABCG1 transporters in ApoE-deficient mice.

Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

AIM: To evaluate ATP-binding cassette(ABC) transporters in colonic pathophysiology as they had recently been related to colorectal cancer(CRC) development. METHODS: Literature search was conducted on Pub Med using combinations of the following terms: ABC transporters, ATP binding cassette transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohns disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein(P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2(MRP2) and ABCG2/breast cancer resistance protein(BCRP), Abcb1/Mdr1 a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function. RESULTS: Recently, human studies reported thatchanges in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes in relation to colitis was suggested by the animal studies. The finding that colitis was preceded by altered gut bacterial composition suggests that deletion of Abcb1 leads to fundamental changes of hostmicrobiota interaction. Also, high fat diet increases the frequency and severity of colitis in specific pathogenfree Abcb1 KO mice. The Abcb1 KO mice might thus serve as a model in which diet/environmental factors and microbes may be controlled and investigated in relation to intestinal inflammation. Potential molecular mechanisms include defective transport of inflammatory mediators and/or phospholipid translocation from one side to the other of the cell membrane lipid bilayer by ABC transporters affecting inflammatory response and/or function of tight junctions, phagocytosis and vesicle trafficking. Also, diet and microbes give rise to molecules which are potential substrates for the ABC transporters and which may additionally affect ABC transporter function through nuclear receptors and transcriptional regulation. Another critical role of ABCB1 was suggested by the finding that ABCB1 expression identifies a subpopulation of pro-inflammatory Th17 cells which were resistant to treatment with glucocorticoids. The evidence for the involvement of ABCC2 and ABCG2 in colonic pathophysiology was weak. CONCLUSION: ABCB1, diet, and gut microbes mutually interact in colonic inflammation, a well-known risk factor for CRC. Further insight may be translated into preventive and treatment strategies.

High-density lipoprotein and atherosclerosis: Roles of lipid transporters

Various previous studies have found a negative cor-relation between the risk of cardiovascular events and serum high-density lipoprotein(HDL) cholesterol levels. The reverse cholesterol transport, a pathway of choles-terol from peripheral tissue to liver which has several potent antiatherogenic properties. For instance, the particles of HDL mediate to transport cholesterol from cells in arterial tissues, particularly from atherosclerotic plaques, to the liver. Both ATP-binding cassette trans-porters(ABC) A1 and ABCG1 are membrane cholesterol transporters and have been implicated in mediating cholesterol effluxes from cells in the presence of HDL and apolipoprotein A-I, a major protein constituent of HDL. Previous studies demonstrated that ABCA1 and ABCG1 or the interaction between ABCA1 and ABCG1 exerted antiatherosclerotic effects. As a therapeutic approach for increasing HDL cholesterol levels, much focus has been placed on increasing HDL cholesterol levels as well as enhancing HDL biochemical functions. HDL therapies that use injections of reconstituted HDL, apoA-I mimetics, or full-length apoA-I have shown dramatic effectiveness. In particular, a novel apoA-I mi-metic peptide, Fukuoka University ApoA-I Mimetic Pep-tide, effectively removes cholesterol via specific ABCA1 and other transporters, such as ABCG1, and has an an-tiatherosclerotic effect by enhancing the biological func-tions of HDL without changing circulating HDL choles-terol levels. Thus, HDL-targeting therapy has significant atheroprotective potential, as it uses lipid transporter-targeting agents, and may prove to be a therapeutic tool for atherosclerotic cardiovascular diseases.

ABC efflux transporters at blood-central nervous system barriers and their implications for treating spinal cord disorders

The barriers present in the interfaces between the blood and the central nervous system form a major hurdle for the pharmacological treatment of central nervous system injuries and diseases.The family of ATP-binding cassette(ABC)transporters has been widely studied regarding efflux of medications at blood-central nervous system barriers.These efflux transporters include P-glycoprotein(abcb1),‘breast cancer resistance protein'(abcg2)and the various‘multidrug resistance-associated proteins'(abccs).Understanding which efflux transporters are present at the blood-spinal cord,blood-cerebrospinal fluid and cerebrospinal fluid-spinal cord barriers is necessary to determine their involvement in limiting drug transfer from blood to the spinal cord tissue.Recent developments in the blood-brain barrier field have shown that barrier systems are dynamic and the profile of barrier defenses can alter due to conditions such as age,disease and environmental challenge.This means that a true understanding of ABC efflux transporter expression and localization should not be one static value but instead a range that represents the complex patient subpopulations that exist.In the present review,the blood-central nervous system barrier literature is discussed with a focus on the impact of ABC efflux transporters on:(i)protecting the spinal cord from adverse effects of systemically directed drugs,and(ii)limiting centrally directed drugs from accessing their active sites within the spinal cord.

How to overcome ATP-binding cassette drug efflux transporter-mediated drug resistance?

P-glycoprotein(ABCB1),multidrug resistance protein-1(ABCC1)and breast cancer resistance protein(ABCG2)belong to the ATP-binding cassette(ABC)superfamily of proteins that play an important physiological role in protection of the body from toxic xenobiotics and endogenous metabolites.Beyond this,these transporters determine the toxicity profile of many drugs,and confer multidrug resistance(MDR)in cancer cells associated with a poor treatment outcome of cancer patients.It has long been hypothesized that inhibition of ABC drug efflux transporters will increase drug accumulation and thereby overcome MDR,but until now no approved inhibitor of these transporters is available in the clinic.In this review we present molecular strategies to overcome this type of drug resistance and discuss for each of these strategies their promising value or indicate underlying reasons for their limited success.

ATP结合盒转运蛋白B1与羧酸酯酶1基因多态性对达比加群酯药物谷浓度的影响

目的探讨ATP结合盒转运蛋白B1(ABCB1)及羧酸酯酶1(CES1)基因多态性与服用达比加群酯的患者药物谷浓度的相关性。方法选取2018年3月-2021年11月在福州某医院诊断为非瓣膜性房颤的69例患者作为研究对象。检测患者ABCB1及CES1的单核苷酸多态性位点以及基因型,采用抗Ⅱa因子显色法测定患者服用达比加群酯后药物的谷浓度,分析ABCB1及CES1基因多态性与谷浓度的相关性,进一步研究相关位点对谷浓度达标的影响。结果本研究共发现11个单核苷酸多态性位点,且ABCB1 rs2235013与ABCB1 rs2235033处于连锁不平衡状态(D’=0.96,r2=0.82)。但ABCB1基因多态性对达比加群谷浓度无显著影响(P>0.05)。CES1 rs2302719突变型(TG,GG)患者的达比加群谷浓度高于野生型(TT)(51.98±8.06 vs 42.44±3.88,57.71±9.06 vs 42.44±3.88),并且CES1 rs2302719突变型(TG,GG)是患者谷浓度达标的保护因素,结果具有统计学意义(P<0.05)。结论ABCB1基因多态性对达比加群谷浓度的影响还需进一步探索。CES1 rs2302719次要等位基因(G)可增加达比加群谷浓度,并且不良反应的发生率可能更低。

X连锁肾上腺脑白质营养不良1家系报告并文献复习

目的分析1例X连锁肾上腺脑白质营养不良(X-linked adrenoleukodystrophy,X-ALD)患者家系中发现的ATP结合盒超家族D亚家族1(ABCD1)基因突变位点,结合患者家系临床资料及相关文献,探讨X-ALD的诊断、治疗及预后。方法回顾性分析2021年11月就诊我院神经内科的1例X-ALD患者的临床资料及其家系情况,并检索国内外相关文献进行复习讨论。结果患者主要表现为皮肤发黑、头晕头痛、全身不适、乏力、疲惫、低血压、食欲减退、睡眠欠佳等肾上腺皮质功能减退症状及双下肢轻瘫的神经系统异常。其父母、一兄一姐、其兄之子及其姐之子均未见相关临床表现。患者颅脑及脊髓MRI检查未见异常,基因检测示患者ABCD1基因存在一处半合子突变(c.1628C>T),其母ABCD1基因存在杂合突变,其父ABCD1基因未见异常。结论X-ALD存在多种类型,可出现肾上腺皮质功能减退及中枢神经系统异常症状,本例X-ALD患者为肾上腺脊髓神经病型,ABCD1基因第6号外显子的1处突变(c.1628C>T:p.P543L)为本患者致病原因,MRI在X-ALD的诊断中或无决定性作用。

靶向肝X受体对PD-1单抗治疗原发性肝癌的增敏作用

目的:探索原发性肝癌中肝脏X受体(LXR)与程序性死亡配体1(PD-L1)表达的相关性;明确LXR激动剂(T09)对于PD-1单抗治疗的影响。方法:采用生物信息分析方法分析肝癌中LXR及其下游关键基因ATP结合盒转运体A1(ABCA1)表达和PD-L1表达的相关性。采用免疫组化染色在肝癌组织样本中验证LXR和PD-L1蛋白水平表达的相关性。采用慢病毒介导的RNA干扰技术分析肝癌细胞中LXR沉默对PD-L1蛋白水平表达的影响。建立免疫健全小鼠C57BL/6小鼠Hepa1-6肝癌原位瘤移植模型,分别给予T09和(或)PD-1单抗治疗,测量干预3周后的各组皮下瘤体积。结果:生物信息结果显示,LXR(r=0.20,P=0.021)及其下游关键基因ABCA1(r=0.12,P<0.001)表达和PD-L1表达呈正相关。免疫组化结果显示,高表达LXR的患者往往合并PD-L1高表达,两者表达具有相关性,差异有统计学意义(P<0.05)。通过慢病毒介导的RNA干扰技术敲低了肝癌细胞系中LXR的表达后,PD-L1的表达水平亦显著下调。动物实验显示与对照组、T09组、PD-1单抗组相比,T09+PD-1单抗组小鼠皮下瘤体积显著减小(P<0.001)。结论:肝癌中LXR表达与PD-L1表达具有显著相关性,LXR激动剂可以显著增加PD-1单抗治疗肝癌的敏感性。

Protective Effect of Irbesartan on ATP Binding Cassette Transporter A1 in THP-1 Derived Macrophages

Objectives To explore the protective effect of irbesartan （lrb） under the interference with angiotensin II （Ang II） on ABCA1. Methods Electron microscopy was used to detect the morphous of foam cells. The expression of ABCA1 mRNA and its protein were determined by RT-PCR and Western blotting, respectively. The variance of cellular cholesterol content was measured by zymochemistry via-fluorospeetrophotometer. Results A positive facilitative effect of Ang II on the formation of foam cells was observed. Total cholesterol was increased significantly by Ang II, the expression of ABCA1 was down-regulated obviously by Ang II; Irb could protect ABCA1 against the lesion of Ang II; Total cellular cholesterol content was reduced significantly in Irb ＋ Ang II group; However, considerable alteration about the cholesterol content and the expression of ABCA1 were not observed in Irb group without incubation with Ang II. Conclusions Irb could protect ABCA1 against the lesion of Ang II, which may contribute to its anti-atherosclerotic properties. （ S Chin J Cardiol 2009; 10（4） ： 227 -233）

Efficacy of Shoushen granule on adenosine triphosphate binding cassette transporter A1, proprotein convertase subtilisin/kexin type 9 and toll-like receptor 4/nuclear factor kappa-B signaling pathway in ApoE-knockout mice

OBJECTIVE: To evaluate the efficacy of Shoushen granule, prepared with four Chinese medicinals, on the targeted regulation of adenosine triphosphate binding cassette transporter A1(ABCA1) through proprotein convertase subtilisin/kexin type 9(PCSK9) and toll-like receptor 4(TLR4)/nuclear factor kappa-B(NF-κB) signaling pathway to affect atherosclerosis(AS) in ApoE-knockout(ApoE-/-) mice.METHODS: ApoE-/-mice fed with a high-fat diet were used for AS modeling and divided into Model,Shoushen, and Atorvastatin groups. C57 BL/6 J mice at the same age and background strain were included in the Control group. Western blot and immunohistochemistry were used to measure ABCA1, PCSK9, TLR4, and NF-κB protein expression in mouse aortas. Enzyme-linked immuno sorbent assay was used to measure mouse serum tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), monocyte chemoattractant protein 1(MCP-1), and intercellular cell adhesion molecule-1(ICAM-1) expression. Serum lipid profiles and histopathology were also assessed. Shoushen granule were composed of Heshouwu(Radix Polygoni Multiflori) 15 g, Gouqizi(Fructus Lycii) 15 g, Sheng shanzha(Raw Fructus Crataegus Pinnatifidae) 10 g, and Sanqi(Radix Notoginseng) 3 g.RESULTS: ApoE-/-mice fed with a high-fat diet had notable AS lesions, with reduced ABCA1 and IL-10 levels, elevated PCSK9, TLR4, NF-κB, TNF-α, MCP-1,and ICAM-1 expression, and increased total cholesterol(TC) and low density lipoprotein cholesterol(LDL-C) contents. With drug interventions, the areas of AS plaques were significantly reduced,the ABCA1 and IL-10 levels were increase, while the PCSK9, TLR4, NF-κB, TC, and LDL-C contents,and the TNF-α, MCP-1, and ICAM-1 expression were reduced.CONCLUSION: Shoushen granule effectively interfered with AS development by antagonizing the expression of key factors of the PCSK9 and TLR4/NF-κB signaling pathway to upregulate ABCA1 expression.

ABCA1基因多态性与冠心病相关性研究

为了探讨ATPBindingCassetteTransporter1(ABCA1)基因R219K多态在中国汉族人群中的分布及其与冠心病(coronaryheartdisease,CHD)的关系,采用PCRRFLP方法,对396例CHD患者和417名正常人ABCA1基因R219K多态位点进行分析。结果表明,对照组R219K多态K等位基因及KK基因型的频率(0.465、0.228)较CHD组(0.381、0.162)显著增高(P<0.05);根据发病年龄分组,早发CHD组K等位基因及KK基因型频率(0.34、0.111)明显低于晚发CHD组(0.419、0.205)和对照组(P<0.05),而在对照组和晚发CHD组间无此频率差异显著性(P>0.05);KK基因型患者血浆甘油三酯(TG)水平较RR基因型显著降低(P<0.05);不同基因型患者间血浆高密度脂蛋白胆固醇(HDLC)水平差异无显著性(P>0.05)。提示ABCA1基因R219K多态与CHD存在相关性;KK基因型可能具有对抗动脉粥样硬化的作用,但这种作用不伴有血浆HDLC水平的改变。

Rolipram对THP-1巨噬细胞源性泡沫细胞胆固醇流出和ABCA1表达的影响

目的 以THP 1巨噬细胞源性泡沫细胞为研究对象 ,探讨磷酸二酯酶抑制剂rolipram对THP 1巨噬细胞源性泡沫细胞ABCA1表达及细胞内胆固醇流出的影响。方法 用液体闪烁计数器检测细胞内胆固醇流出 ,运用逆转录 -多聚酶链反应和Westernblot印迹分别检测ABCA1mRNA与ABCA1蛋白的表达 ,采用低pH值EIA法测定细胞内cAMP水平。结果 实验显示PDE4抑制剂rolipram能引起THP 1巨噬细胞源性泡沫细胞cAMP水平、ABCA1表达和apoA 1介导的胆固醇流出成平衡增加 ,而细胞内总胆固醇、游离胆固醇与胆固醇酯明显减少。结论 PDE4抑制剂rolipram增加THP 1巨噬细胞源性泡沫细胞ABCA1表达及细胞内胆固醇流出 。

NF-κB途径介导Chemerin抑制THP-1源性巨噬泡沫细胞ABCAl表达和降低胆固醇外流

目的观察脂肪因子Chemerin是否调节胆固醇流出和巨噬泡沫细胞脂滴蓄积,以及对巨噬泡沫细胞ATP结合盒转运子A1(ATP-binding cassette transporter A1,ABCA1)和酰基辅酶A:胆固醇酰基转移酶-1(Acyl-CoA:choles-terol acyltransferase-1,ACAT1)表达和NF-κB活化的影响。方法佛波酯(phorbol myristate acetate,PMA)诱导THP-1单核细胞分化为巨噬细胞,随后加入胆固醇和ox-LDL培养促进巨噬细胞转化为巨噬泡沫细胞,免疫荧光染色检测巨噬细胞表面抗原CD68的表达,油红O染色后观察泡沫细胞形态,脂肪因子Chemerin干预巨噬泡沫细胞,应用液体闪烁计数仪检测胆固醇流出变化,油红O染色观察细胞内脂滴变化,实时PCR和Western blot检测ABCA1及ACAT1mR-NA和蛋白表达水平。Sandwich ELISA法检测巨噬泡沫细胞NF-κB活化情况。实时PCR检测NF-κB抑制剂四氢化吡咯二硫代氨基甲酸酯(PDTC)预处理后,Chemerin再干预时ABCA1表达的变化。结果 Chemerin抑制巨噬细胞内胆固醇流出,促进脂滴蓄积,下调ABCA1mRNA和蛋白的表达,增加NF-κB活化,差异有统计学意义(均P<0.05),且ABCA1mRNA表达与Chemerin浓度呈负相关,r=-0.936(P<0.05),NF-κB活化程度与ABCA1mRNA表达呈负相关,r=-0.917(P<0.05),但Chemerin对ACAT1mRNA和蛋白的表达无明显影响,PDTC预处理能够逆转ABCA1mRNA表达抑制。结论 Chemerin下调ABCA1mRNA和蛋白的表达,抑制THP-1巨噬泡沫细胞内胆固醇外流,增加细胞内脂滴蓄积,NF-κB活化可能是Chemerin抑制ABCA1mRNA表达的信号通路之一。

ABCA1、ACAT1 DNA甲基化在Hcy致THP-1单核源性泡沫细胞胆固醇流出中作用研究

目的探讨ABCA1、ACAT1 DNA甲基化在同型半胱氨酸(Hcy)致THP-1单核源性泡沫细胞胆固醇流出中的作用。方法复制泡沫细胞模型,用50、100、200、500μmol·L-1Hcy和100μmol·L-1Hcy+叶酸+维生素B12(100+F+V)干预,并设对照组(0μmol·L-1Hcy)。油红O染色观察泡沫细胞形成,巢式降落式甲基化特异性PCR(nMS-PCR)检测ABCA1、ACAT1 DNA甲基化水平,并分析两者比值变化;ELISA-like法检测DNA甲基转移酶(DNMTs)活性;化学比色法分析细胞内胆固醇含量。结果成功复制了泡沫细胞模型;给予不同浓度Hcy刺激后,ABCA1 DNA甲基化改变呈上升趋势,ACAT1 DNA甲基化改变呈下降趋势,给予100+F+V干预后可逆转上述变化,其中ABCA1 DNA甲基化改变更明显;同时,不同浓度Hcy可使泡沫细胞内DNMTs活性和胆固醇含量增加。结论 ABCA1和ACAT1 DNA甲基化的改变参与了Hcy致THP-1单核源性泡沫细胞胆固醇流出,DNMTs可能起关键调控作用。

同型半胱氨酸对THP-1单核细胞源性泡沫细胞形成中ABCA1、ACAT1表达的影响

目的探讨同型半胱氨酸(Hcy)对THP-1单核细胞源性泡沫细胞形成中三磷酸腺苷-结合转运子A1(ABCA1)和酰基辅酶A:胆固醇酰基转移酶(ACAT1)表达的影响。方法将THP-1单核细胞与佛波酯(PMA)、氧化低密度脂蛋白(ox-LDL)共同培养,复制泡沫细胞模型,并分别用50、100、200、500μmol/L Hcy和100μmol/L Hcy+叶酸+维生素B12(Vit B12)干预72 h,并设对照组(不加入Hcy)。采用油红O染色检测泡沫细胞的形成。采用酶终点法测定细胞内总胆固醇(TC)、游离胆固醇(FC)和胆固醇酯(CE)含量的变化,观察Hcy对泡沫细胞CE流出的影响。采用荧光定量逆转录-聚合酶链反应(RT-PCR)测定ABCA1、ACAT1 mRNA表达;免疫印迹法检测ABCA1、ACAT1蛋白表达。结果油红O染色显示Hcy加剧了泡沫细胞的形成,但不呈量效关系。实验组(加入50、100、200、500μmol/L Hcy和100μmol/L Hcy+叶酸+Vit B12)泡沫细胞阳性百分率均高于对照组(P<0.05、P<0.01),以100μmol/L Hcy组效应最为明显(P<0.01)。在Hcy的干预下,泡沫细胞胞内TC、FC、CE流出减少,与对照组比较差异有统计学意义(P<0.05),以100μmol/L Hcy组效应最为明显(P<0.01)。100μmol/L Hcy+叶酸+VitB12组与100μmol/L Hcy组比较,前者泡沫细胞形成减少,胆固醇流出增多。RT-PCR结果显示ABCA1 mRNA表达下调,ACAT1 mRNA表达上调,均以100μmol/L Hcy组效应最为明显(P<0.01);免疫印迹法检测ABCA1、ACAT1蛋白的表达与其mRNA表达一致。结论 Hcy下调了ABCA1的表达,上调了ACAT1的表达,促使了泡沫细胞形成。

他汀类药物对神经细胞和神经胶质细胞ABCA1基因表达的影响

目的观察他汀类药物对神经细胞和神经胶质细胞ATP结合盒式转运体1(ATP-binding cassette transporter1,ABCA1)基因表达的影响。方法培养人类神经细胞和神经胶质细胞,分别加入不同浓度的辛伐他汀(si mvastatin)和普伐他汀(pravastatin),孵育不同时间后分别提取细胞总RNA和胞质蛋白,RT-PCR观察ABCA1基因的表达,Western blot检测ABCA1蛋白。结果辛伐他汀和普伐他汀组显著降低了ABCA1基因和蛋白的表达,其中辛伐他汀的作用比普伐他汀明显(辛伐他汀对神经元和神经胶质细胞ABCA1基因表达分别降低95%和75%P<0.05),神经元实验组比神经胶质细胞组更为敏感。结论他汀类药物可以显著降低神经元和神经胶质细胞ABCA1基因和蛋白的表达,ABCA1的降低有可能影响细胞内胆固醇的代谢,对全面评价他汀类药物对高血脂相关基因的影响及用药安全性提供了一定的实验依据。

Amiloride通过抑制缺氧诱导的NHE1表达而延缓calpain介导的ABCA1降解

目的:研究缺氧对钠氢交换体1(NHE1)表达、细胞内钙离子浓度([Ca2+]i)和钙蛋白酶(calpain)活性的影响,探讨NHE1抑制剂阿米洛利(amiloride)对ABCA1降解的影响以及与calpain相关的机制。方法:RAW264.7细胞缺氧0、12、24和48 h。MTT法检测细胞活力,real-time PCR及Western blot检测NHE1的表达。流式细胞术检测[Ca2+]i,荧光素法检测细胞内calpain活性。进而,经缺氧24 h处理的细胞,cycloheximide干预条件下,NHE1抑制剂amiloride处理6 h及12 h,检测ABCA1蛋白含量。最后,给予calpain抑制剂ALLN及细胞内钙螯合剂BAPTA共孵育12 h,检测ABCA1含量及calpain活性。结果:缺氧呈时间依赖方式抑制细胞增殖。缺氧促进NHE1表达上调,增加[Ca2+]i及calpain活性。缺氧加速ABCA1蛋白降解,而amiloride减缓ABCA1蛋白降解。ALLN及BAPTA升高ABCA1蛋白含量,降低calpain活性。结论:Amiloride延缓calpain介导的ABCA1降解,提示缺氧诱导NHE1表达可能至少部分参与ABCA1蛋白降解。