罗格列酮抗动脉粥样硬化的机制研究及2型糖尿病患者单核细胞ABCA1变化的分析
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摘要
一.研究背景
动脉粥样硬化性心血管疾病是全世界发病及死亡的主要原因。流行病学和临床研究表明,血浆高密度脂蛋白胆固醇(HDL-C)和其主要成分载脂蛋白A1(apoA-1)与动脉粥样硬化性心血管病的危险性呈负相关,其主要机制可能是HDL-C和apoA-1促进外周细胞蓄积的胆固醇向肝脏的逆转运,并最终通过胆汁、粪便排出体外,即胆固醇逆转运(RCT)。RCT包括许多环节,其中ATP结合盒转运子A1(ABCA1)介导的巨噬细胞内胆固醇的流出和HDL-C的形成是其始动环节。因此ABCA1与HDL-C水平及动脉粥样硬化(AS)密切相关。上调ABCA1表达显著增加血浆HDL-C水平,同时抑制AS的进展,相反,ABCA1基因的变异或缺失可以使HDL-C水平接近于零,同时促进AS,这些显示ABCA1促进RCT。
近年来研究发现罗格列酮作为PPARγ激动剂,不论是在糖尿病状态还是在非糖尿病状态均可抑制AS的进展,其中一个重要途径是增加细胞ABCA1的表达,由此可以推测,罗格列酮可以通过上调ABCA1表达促进RCT最终发挥抗AS作用,但是这些观点一直没有被证明。因此在本研究中,我们以外周巨噬细胞、脂肪细胞及中心肝细胞对[3H]胆固醇的转出率间接反映RCT水平,以流式细胞术检测细胞表面ABCA1表达,分析巨噬细胞、肝细胞及脂肪细胞ABCA1在兔AS形成过程对RCT的作用并探讨罗格列酮抗AS的新机制。
2型糖尿病患者出现的动脉粥样硬化性心血管合并症被越来越多的学者及临床工作者所重视,以慢性高血糖为特征的脂质代谢紊乱可能是2型糖尿病患者易发AS的一个重要因素。ABCA1与脂质代谢密切相关,其阻止动脉粥样AS进展的作用已经被证实,但目前国内外对2型糖尿病状态下ABCA1的变化却知之甚少,因此对ABCA1的变化分析将为2型糖尿病患者易发AS的机制研究提供一个新的方向。
二.研究目的
我们以兔动脉粥样硬化(AS)模型为研究对象,分析单核/巨噬细胞、脂肪细胞及肝细胞ABCA1在动脉粥样硬化(AS)形成过程中对RCT的作用,同时探讨及证明罗格列酮抗AS的新机制;我们同时在临床上对2型糖尿病患者外周单核细胞ATP结合盒转运子A1的变化进行分析,以发现2型糖尿病状态下ABCA1的变化及意义。
三.方法
基础研究方面:我们用24只新西兰白兔随机分为4组:(1)对照组:单纯喂食高胆固醇饮食;(2)罗格列酮组:在单纯高胆固醇饮食的基础上,予以灌服罗格列酮2mg/d;(3)罗格列酮+辛伐他汀组:在单纯高胆固醇饮食的基础上,予以灌服罗格列酮2mg/d,辛伐他汀10mg/d。(4)格列本脲组:在单纯高胆固醇饮食的基础上,予以灌服格列苯脲2.5mg/d。利用流式细胞术检测外周单核细胞、腹腔巨噬细胞、脂肪细胞和肝细胞ABCA1的表达量,利用液闪计数仪检测腹腔巨噬细胞、脂肪细胞、肝细胞对[3H]胆固醇转出率,利用酶法测定兔的血脂以及主动脉、脂肪、肝脏组织的胆固醇含量,并利用专业图像分析软件分析主动脉粥样硬化斑块的面积,最后进行统计学分析。
临床研究方面:我们分别抽取2型糖尿病和正常对照两组人群(每组40例。2型糖尿病组,男21例,女19例,年龄52.2±10.4岁,就诊于南方医院内分泌科,2型糖尿病诊断明确但未正规服用降脂药物治疗的患者;正常组,男22例,女18例,年龄49.8±12.5岁,就诊于南方医科大学附属南方医院脊柱骨科非高脂血症患者,以上两组均排除高血压、冠心病、严重肝肾功能障碍、肿瘤等疾病,年龄、性别无显著差异。)静脉血2ml,提取外周血单核细胞并利用流式细胞术分析氧化修饰的低密度脂蛋白(Ox-LDL)刺激前后ATP结合核转运子A1的表达变化,同时检测两组人群的血脂水平和2型糖尿病组人群空腹血糖及糖化血红蛋白含量,并对2型糖尿病患者单核细胞ABCA1表达与年龄、血脂、糖代谢的关系进行了研究。
四.结果
1.基础研究
1) 4组动物间血浆HDL-C、apoA1、NHDL-C水平及体重基线无明显差异,6周后所有动物均成功复制动脉粥样硬化模型,且4组动物间体重无明显改变。
2) 6周时,与对照组相比,罗格列酮组外周单核细胞、腹腔巨噬细胞、脂肪细胞、肝细胞ABCA1的表达,血浆HDL-C、apoA1水平及腹腔巨噬细胞、脂肪细胞、肝细胞对[3H]胆固醇的转出率明显升高;NHDL-C水平无明显变化;肝脏、脂肪、主动脉的胆固醇含量以及主动脉AS面积明显减小。
3) 6周时,罗格列酮+辛伐他汀组出现类似罗格列酮组的变化,但不同的是,罗格列酮+辛伐他汀组血浆NHDL-C水平较对照组明显下降,并且与罗格列酮组相比,罗格列酮+辛伐他汀组外周单核细胞、腹腔巨噬细胞、脂肪细胞、肝细胞ABCA1的表达,血浆HDL-C、apoA1水平以及腹腔巨噬细胞、脂肪细胞、肝细胞对[3H]胆固醇的转出率均增加得更加明显,同时肝脏、脂肪、主动脉的胆固醇含量和主动脉AS面积则更加明显的减少。
4) 6周时,与对照组相比,格列苯脲组外周单核细胞、腹腔巨噬细胞、脂肪细胞、肝细胞ABCA1的表达,血浆HDL-C、apoA1水平及腹腔巨噬细胞(p=0.006)、脂肪细胞、肝细胞对[3H]胆固醇的转出率明显降低;NHDL-C水平无明显变化;肝脏、脂肪、主动脉的胆固醇含量以及主动脉AS面积明显增大。
5)综合4组动物分析,主动脉组织、脂肪组织、肝组织的胆固醇含量均分别与腹腔巨噬细胞、脂肪细胞、肝细胞对[3H]胆固醇的转出率呈负相关;腹腔巨噬细胞、脂肪细胞、肝细胞的ABCA1表达量分别与其对[3H]胆固醇的转出率呈正相关;单核细胞ABCA1的表达量分别与腹腔巨噬细胞、脂肪细胞、肝细胞对[3H]胆固醇的转出率呈正相关。
2.临床研究
1)与对照组相比,2型糖尿病组AS的危险因素TG、NHDL-C水平显著升高,抗AS的因素HDL-C、apoA-1的水平显著降低。
2)与对照组相比,2型糖尿病组外周单核细胞ABCA1的表达显著降低,经Ox-LDL刺激24h后ABCA1的改变量(刺激后表达量/初始表达量)明显增大,但最终表达量仍显著小于对照组。
3) 2型糖尿病组ABCA1的表达变化与年龄、空腹血糖(FPG)和糖化血红蛋白(HbA1C)无明显相关性,但2型糖尿病组ABCA1的改变量与血浆HDL-C呈正相关。
五.结论
1.单核/巨噬细胞、脂肪细胞、肝细胞ABCA1表达是RCT的正性调节剂;
2.在非糖尿病的AS状态下,罗格列酮通过上调单核/巨噬细胞、脂肪细胞和肝细胞ABCA1的表达促进RCT,最终抑制AS;
3.单核细胞ABCA1是RCT的一个间接和可信的反映指标;
4.2型糖尿病患者体内已存在紊乱的血脂谱及损伤的RCT,从而具有诱发AS及CAD的基础。
Study Background
Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality worldwide.Numerous epidemiological studies have established high-density lipoprotein cholesterol(HDL-C) and its major protein apolipoprotein A1 (apoA-1) are inversely associated with the risk of atherosclerotic vascular disease. One major mechanism by which HDL and apoA-1 protect against atherosclerosis is probably by promoting reverse cholesterol transport(RCT) from periphery to the liver, where it can be excreted from the body directly or indirectly after conversion into bile acids eventually.It is well known that RCT is a process of anti-atherosclerosis,and involves multiple steps,beginning with the cholesterol efflux of macrophages and the formation of HDL by ATP-binding cassette transporter A1(ABCA1).So,ABCA 1 has a significant relation with HDL and atherosclerosis.Overexpression of ABCA1 dramatically increases plasma HDL-C levels and inhibits atherosclerosis.Conversely, gene mutation or deletion of ABCA1 has little or no plasma HDL-C levels and markedly increases atherosclerosis.These data suggest that ABCA1expression promotes RCT.
Recently,many studies have shown that rosiglitazone,a peroxisome proliferator-activated receptorγagonist,plays a role in anti-atherosclerosis not only in type 2 diabetes mellitus but also in nondiabetics.One important way is that rosiglitazone increases ABCA1 expression and plasma HDL-C levels.So,we suppose that one mechanism of rosiglitazone for anti-atherosclerosis is probably by promoting RCT,but it has never been proven.Here,process of RCT is indirectly reflected by cholesterol efflux rates from hepatic and extrahepatic cells determined through measuring release of radioactivity from[3H]-cholesterol prelabeled cells into medium by liquid scintillation counting and ABCA1 expression were evaluated by flow cytometry,we analyze the effect of ABCA1 in peritoneal macrophages, hepatocytes and adipocytes on RCT and explore the mechanism of rosiglitazone for anti-atherosclerosis.
Atherosclerotic cardiovascular complications in type 2 diabetes arouse the interests of many scholars.It is easy to form atherosclerosis in type 2 diabetes attributable to the lipids metabolism with hyperglycemia.ABCA1 has a close correlation with lipids metabolism,and the effect on anti-atherosclerosis has been proved.But the research between ABCA1 and type 2 diabetes mellitus is little, thereby the change research in ABCA1 will provide a new direction in the mechanism of type 2 diabetes mellitus and atherosclerosis.
Objectives
The aim of foundation research was to analyze the effect of ABCA1 expressions in monocytes/macrophages,hepatocytes and adipocytes on RCT and to research the mechanism of rosiglitazone for anti-atherosclerosis in atherosclerotic rabbits.In clinic, we analyze the change of ABCA1 of peripheral monocytes in patients with type 2 diabetes in order to illuminate the significance of ABCA1 in type 2 diabetes mellitus.
Methods
In foundation research,twenty-four rabbits were randomly divided into four groups:(1) control group(n=6):only high cholesterol diet for 6 weeks;(2) rosiglitazone group(n=6):the same cholesterol diet plus rosiglitazone(2mg/d) for 6 weeks;(3) rosiglitazone&simvastatin group(n=6):the same cholesterol diet plus rosiglitazone(2mg/d) and simvastatin(10mg/d) for 6 weeks;(4) glibenclamide group (n=6):the same cholesterol diet plus glibenclamide(2.5mg/d) for 6 weeks.ABCA1 expressions in peritoneal macrophages,hepatocytes and adipocytes were evaluated by flow cytometry and cholesterol effiux rates from them were determined through measuring release of radioactivity from[3H]-cholesterol prelabeled cells into medium by liquid scintillation spectrometry.Enzymatic and immune method were used to assay plasma lipids levels and cholesterol contents in liver,aorta and adipose tissues, and then,area of atherosclerotic plaque in aorta was calculated by professional image analysis software.
In clinic research,the people blood sample(2ml) is obtained and divided into two groups(40 cases each):the type 2 diabetes group,the patients(21males, 33females,52.2±10.4years) were diagnosed type 2 diabetes mellitus in endocrinology branch of Nanfang Hospital but without the regular treatment by lipid-lowering drugs,and the control group,the patients(22males,18females, 49.8±12.5years) visited in orthopedic spine branch of Nanfang Hospital and excluded hyperlipidemia.Above two groups,hypertension,CAD,Serious liver and kidney dysfunction and tumor were all excluded.There was no difference in age and gender of the two groups.And then,monocytes of the blood was extracted and ABCA1 expression in monocytes before and after stimulation of the oxidized low density lipoprotein was detected with flow cytometer in type 2 diabetes group and the control group.Plasma lipids levels in the different group and concentrations of fasting plasma glucose and glycosylated hemoglobin in type 2 diabetes group were assayed and the correlation of ABCA1expression in type 2 diabetes group with age,blood lipids and glycometabolism was analyzed.
Results
1.Foundation research
1) There were no significant differences in body weight among the four groups at the baseline.After 6 weeks of experiment,all atherosclerotic models of rabbit were created successfully,and there was no difference in body weight among the four groups.
2) Compared with control group,in rosiglitazone group,ABCA1 expressions in peripheral mononuclear cells,peritoneal macrophages,adipocytes and hepatocytes; [~3H]cholesterol efflux rates in peritoneal macrophages,adipocytes and hepatocytes, and serum levels of HDL-Cand apoA-1 were all increased,while contents of cholesterol in aorta,adipose and liver tissues and area of atherosclerosis in aorta were all decreased,and there was no difference in NHDL-C level.
3) The effects of rosiglitazone&simvastatin group were similar with rosiglitazone group,however,in difference,the serum NHDL-C level was decreased in rosiglitazone&simvastatin group as compared with control group.Compared with rosiglitazone group,in rosiglitazone&simvastatin group,ABCA1 expressions in peripheral mononuclear cells,peritoneal macrophages,adipocytes and hepatocytes, [~3H]cholesterol efflux rates in peritoneal macrophages,adipocytes and hepatocytes, and serum levels of HDL-C and apoA-1 were all higher,while contents of cholesterol in aorta,adipose and liver tissues and area of atheroscler osis in aorta were all lower.
4) Compared with control group,in glibenclamide group,ABCA1 expressions in peripheral mononuclear cells,peritoneal macrophages,adipocytes and hepatocytes, [~3H]cholesterol efflux rates in peritoneal macrophages,adipocytes and hepatocytes, and serum levels of HDL-C and apoA-1 were all decreased,while contents of cholesterol in aorta,adipose and liver tissues and area of atherosclerosis in aorta were all increased,and there was no difference in NHDL-C level.
5) In all experimental animals,cholesterol contents in aorta,adipose and liver tissues respectively had a negative correlation with[3H]cholesterol efflux rates from peritoneal macrophages,adipocytes and hepatocytes which were positively associated with ABCA1 expressions in them severally.ABCA1 expression in monocytes respectively had a positive correlation with[3H]cholesterol efflux rates from peritoneal macrophages,hepatocytes and adipocytes.
2.Clinical research
1) Compared with control group,type 2 diabetes enhanced the plasma levels of TG and NHDL-C which are pro-atherosclerosis,and reduced the serum apoA-1 and HDL-C levels which are anti-atherosclerosis.
2) Type 2 diabetes group up-regulate the ABCA1 as compared with control group,while the change(stimulated expression / initial expression) of ABCA1 after stimulated by oxidized low density lipoprotein was significantly greater in type 2 diabetes group than in control group.But the ultimate expression of ABCA1 in type 2 diabetes group was still smaller than in the control group.
3) In type 2 diabetes group,the ABCA1 had no correlation with age,fasting plasma glucose(FPG),and HbA1C.But the change of ABCA1 was positive correlation with plasma high density lipoprotein cholesterol level.
Conclusions
1.Expressions of ABCA1 in monocytes/macrophages,hepatocytes and adipocytes are positive regulators of RCT.
2.Rosiglitazone promotes RCT by up-regulating ABCA1 expressions in monocytes/macrophages,hepatocytes and adipocytes to repress atherosclerosis.
3.ABCA1expression in monocytes is a credible index which reflects RCT indirectly.
4.Patients with type 2 diabetes already have the injuried lipids spectrum and RCT,thus forming the foundation of pro-atherosclerosis and coronary heart disease.
动脉粥样硬化性心血管疾病是全世界发病及死亡的主要原因。流行病学和临床研究表明,血浆高密度脂蛋白胆固醇(HDL-C)和其主要成分载脂蛋白A1(apoA-1)与动脉粥样硬化性心血管病的危险性呈负相关,其主要机制可能是HDL-C和apoA-1促进外周细胞蓄积的胆固醇向肝脏的逆转运,并最终通过胆汁、粪便排出体外,即胆固醇逆转运(RCT)。RCT包括许多环节,其中ATP结合盒转运子A1(ABCA1)介导的巨噬细胞内胆固醇的流出和HDL-C的形成是其始动环节。因此ABCA1与HDL-C水平及动脉粥样硬化(AS)密切相关。上调ABCA1表达显著增加血浆HDL-C水平,同时抑制AS的进展,相反,ABCA1基因的变异或缺失可以使HDL-C水平接近于零,同时促进AS,这些显示ABCA1促进RCT。
近年来研究发现罗格列酮作为PPARγ激动剂,不论是在糖尿病状态还是在非糖尿病状态均可抑制AS的进展,其中一个重要途径是增加细胞ABCA1的表达,由此可以推测,罗格列酮可以通过上调ABCA1表达促进RCT最终发挥抗AS作用,但是这些观点一直没有被证明。因此在本研究中,我们以外周巨噬细胞、脂肪细胞及中心肝细胞对[3H]胆固醇的转出率间接反映RCT水平,以流式细胞术检测细胞表面ABCA1表达,分析巨噬细胞、肝细胞及脂肪细胞ABCA1在兔AS形成过程对RCT的作用并探讨罗格列酮抗AS的新机制。
2型糖尿病患者出现的动脉粥样硬化性心血管合并症被越来越多的学者及临床工作者所重视,以慢性高血糖为特征的脂质代谢紊乱可能是2型糖尿病患者易发AS的一个重要因素。ABCA1与脂质代谢密切相关,其阻止动脉粥样AS进展的作用已经被证实,但目前国内外对2型糖尿病状态下ABCA1的变化却知之甚少,因此对ABCA1的变化分析将为2型糖尿病患者易发AS的机制研究提供一个新的方向。
二.研究目的
我们以兔动脉粥样硬化(AS)模型为研究对象,分析单核/巨噬细胞、脂肪细胞及肝细胞ABCA1在动脉粥样硬化(AS)形成过程中对RCT的作用,同时探讨及证明罗格列酮抗AS的新机制;我们同时在临床上对2型糖尿病患者外周单核细胞ATP结合盒转运子A1的变化进行分析,以发现2型糖尿病状态下ABCA1的变化及意义。
三.方法
基础研究方面:我们用24只新西兰白兔随机分为4组:(1)对照组:单纯喂食高胆固醇饮食;(2)罗格列酮组:在单纯高胆固醇饮食的基础上,予以灌服罗格列酮2mg/d;(3)罗格列酮+辛伐他汀组:在单纯高胆固醇饮食的基础上,予以灌服罗格列酮2mg/d,辛伐他汀10mg/d。(4)格列本脲组:在单纯高胆固醇饮食的基础上,予以灌服格列苯脲2.5mg/d。利用流式细胞术检测外周单核细胞、腹腔巨噬细胞、脂肪细胞和肝细胞ABCA1的表达量,利用液闪计数仪检测腹腔巨噬细胞、脂肪细胞、肝细胞对[3H]胆固醇转出率,利用酶法测定兔的血脂以及主动脉、脂肪、肝脏组织的胆固醇含量,并利用专业图像分析软件分析主动脉粥样硬化斑块的面积,最后进行统计学分析。
临床研究方面:我们分别抽取2型糖尿病和正常对照两组人群(每组40例。2型糖尿病组,男21例,女19例,年龄52.2±10.4岁,就诊于南方医院内分泌科,2型糖尿病诊断明确但未正规服用降脂药物治疗的患者;正常组,男22例,女18例,年龄49.8±12.5岁,就诊于南方医科大学附属南方医院脊柱骨科非高脂血症患者,以上两组均排除高血压、冠心病、严重肝肾功能障碍、肿瘤等疾病,年龄、性别无显著差异。)静脉血2ml,提取外周血单核细胞并利用流式细胞术分析氧化修饰的低密度脂蛋白(Ox-LDL)刺激前后ATP结合核转运子A1的表达变化,同时检测两组人群的血脂水平和2型糖尿病组人群空腹血糖及糖化血红蛋白含量,并对2型糖尿病患者单核细胞ABCA1表达与年龄、血脂、糖代谢的关系进行了研究。
四.结果
1.基础研究
1) 4组动物间血浆HDL-C、apoA1、NHDL-C水平及体重基线无明显差异,6周后所有动物均成功复制动脉粥样硬化模型,且4组动物间体重无明显改变。
2) 6周时,与对照组相比,罗格列酮组外周单核细胞、腹腔巨噬细胞、脂肪细胞、肝细胞ABCA1的表达,血浆HDL-C、apoA1水平及腹腔巨噬细胞、脂肪细胞、肝细胞对[3H]胆固醇的转出率明显升高;NHDL-C水平无明显变化;肝脏、脂肪、主动脉的胆固醇含量以及主动脉AS面积明显减小。
3) 6周时,罗格列酮+辛伐他汀组出现类似罗格列酮组的变化,但不同的是,罗格列酮+辛伐他汀组血浆NHDL-C水平较对照组明显下降,并且与罗格列酮组相比,罗格列酮+辛伐他汀组外周单核细胞、腹腔巨噬细胞、脂肪细胞、肝细胞ABCA1的表达,血浆HDL-C、apoA1水平以及腹腔巨噬细胞、脂肪细胞、肝细胞对[3H]胆固醇的转出率均增加得更加明显,同时肝脏、脂肪、主动脉的胆固醇含量和主动脉AS面积则更加明显的减少。
4) 6周时,与对照组相比,格列苯脲组外周单核细胞、腹腔巨噬细胞、脂肪细胞、肝细胞ABCA1的表达,血浆HDL-C、apoA1水平及腹腔巨噬细胞(p=0.006)、脂肪细胞、肝细胞对[3H]胆固醇的转出率明显降低;NHDL-C水平无明显变化;肝脏、脂肪、主动脉的胆固醇含量以及主动脉AS面积明显增大。
5)综合4组动物分析,主动脉组织、脂肪组织、肝组织的胆固醇含量均分别与腹腔巨噬细胞、脂肪细胞、肝细胞对[3H]胆固醇的转出率呈负相关;腹腔巨噬细胞、脂肪细胞、肝细胞的ABCA1表达量分别与其对[3H]胆固醇的转出率呈正相关;单核细胞ABCA1的表达量分别与腹腔巨噬细胞、脂肪细胞、肝细胞对[3H]胆固醇的转出率呈正相关。
2.临床研究
1)与对照组相比,2型糖尿病组AS的危险因素TG、NHDL-C水平显著升高,抗AS的因素HDL-C、apoA-1的水平显著降低。
2)与对照组相比,2型糖尿病组外周单核细胞ABCA1的表达显著降低,经Ox-LDL刺激24h后ABCA1的改变量(刺激后表达量/初始表达量)明显增大,但最终表达量仍显著小于对照组。
3) 2型糖尿病组ABCA1的表达变化与年龄、空腹血糖(FPG)和糖化血红蛋白(HbA1C)无明显相关性,但2型糖尿病组ABCA1的改变量与血浆HDL-C呈正相关。
五.结论
1.单核/巨噬细胞、脂肪细胞、肝细胞ABCA1表达是RCT的正性调节剂;
2.在非糖尿病的AS状态下,罗格列酮通过上调单核/巨噬细胞、脂肪细胞和肝细胞ABCA1的表达促进RCT,最终抑制AS;
3.单核细胞ABCA1是RCT的一个间接和可信的反映指标;
4.2型糖尿病患者体内已存在紊乱的血脂谱及损伤的RCT,从而具有诱发AS及CAD的基础。
Study Background
Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality worldwide.Numerous epidemiological studies have established high-density lipoprotein cholesterol(HDL-C) and its major protein apolipoprotein A1 (apoA-1) are inversely associated with the risk of atherosclerotic vascular disease. One major mechanism by which HDL and apoA-1 protect against atherosclerosis is probably by promoting reverse cholesterol transport(RCT) from periphery to the liver, where it can be excreted from the body directly or indirectly after conversion into bile acids eventually.It is well known that RCT is a process of anti-atherosclerosis,and involves multiple steps,beginning with the cholesterol efflux of macrophages and the formation of HDL by ATP-binding cassette transporter A1(ABCA1).So,ABCA 1 has a significant relation with HDL and atherosclerosis.Overexpression of ABCA1 dramatically increases plasma HDL-C levels and inhibits atherosclerosis.Conversely, gene mutation or deletion of ABCA1 has little or no plasma HDL-C levels and markedly increases atherosclerosis.These data suggest that ABCA1expression promotes RCT.
Recently,many studies have shown that rosiglitazone,a peroxisome proliferator-activated receptorγagonist,plays a role in anti-atherosclerosis not only in type 2 diabetes mellitus but also in nondiabetics.One important way is that rosiglitazone increases ABCA1 expression and plasma HDL-C levels.So,we suppose that one mechanism of rosiglitazone for anti-atherosclerosis is probably by promoting RCT,but it has never been proven.Here,process of RCT is indirectly reflected by cholesterol efflux rates from hepatic and extrahepatic cells determined through measuring release of radioactivity from[3H]-cholesterol prelabeled cells into medium by liquid scintillation counting and ABCA1 expression were evaluated by flow cytometry,we analyze the effect of ABCA1 in peritoneal macrophages, hepatocytes and adipocytes on RCT and explore the mechanism of rosiglitazone for anti-atherosclerosis.
Atherosclerotic cardiovascular complications in type 2 diabetes arouse the interests of many scholars.It is easy to form atherosclerosis in type 2 diabetes attributable to the lipids metabolism with hyperglycemia.ABCA1 has a close correlation with lipids metabolism,and the effect on anti-atherosclerosis has been proved.But the research between ABCA1 and type 2 diabetes mellitus is little, thereby the change research in ABCA1 will provide a new direction in the mechanism of type 2 diabetes mellitus and atherosclerosis.
Objectives
The aim of foundation research was to analyze the effect of ABCA1 expressions in monocytes/macrophages,hepatocytes and adipocytes on RCT and to research the mechanism of rosiglitazone for anti-atherosclerosis in atherosclerotic rabbits.In clinic, we analyze the change of ABCA1 of peripheral monocytes in patients with type 2 diabetes in order to illuminate the significance of ABCA1 in type 2 diabetes mellitus.
Methods
In foundation research,twenty-four rabbits were randomly divided into four groups:(1) control group(n=6):only high cholesterol diet for 6 weeks;(2) rosiglitazone group(n=6):the same cholesterol diet plus rosiglitazone(2mg/d) for 6 weeks;(3) rosiglitazone&simvastatin group(n=6):the same cholesterol diet plus rosiglitazone(2mg/d) and simvastatin(10mg/d) for 6 weeks;(4) glibenclamide group (n=6):the same cholesterol diet plus glibenclamide(2.5mg/d) for 6 weeks.ABCA1 expressions in peritoneal macrophages,hepatocytes and adipocytes were evaluated by flow cytometry and cholesterol effiux rates from them were determined through measuring release of radioactivity from[3H]-cholesterol prelabeled cells into medium by liquid scintillation spectrometry.Enzymatic and immune method were used to assay plasma lipids levels and cholesterol contents in liver,aorta and adipose tissues, and then,area of atherosclerotic plaque in aorta was calculated by professional image analysis software.
In clinic research,the people blood sample(2ml) is obtained and divided into two groups(40 cases each):the type 2 diabetes group,the patients(21males, 33females,52.2±10.4years) were diagnosed type 2 diabetes mellitus in endocrinology branch of Nanfang Hospital but without the regular treatment by lipid-lowering drugs,and the control group,the patients(22males,18females, 49.8±12.5years) visited in orthopedic spine branch of Nanfang Hospital and excluded hyperlipidemia.Above two groups,hypertension,CAD,Serious liver and kidney dysfunction and tumor were all excluded.There was no difference in age and gender of the two groups.And then,monocytes of the blood was extracted and ABCA1 expression in monocytes before and after stimulation of the oxidized low density lipoprotein was detected with flow cytometer in type 2 diabetes group and the control group.Plasma lipids levels in the different group and concentrations of fasting plasma glucose and glycosylated hemoglobin in type 2 diabetes group were assayed and the correlation of ABCA1expression in type 2 diabetes group with age,blood lipids and glycometabolism was analyzed.
Results
1.Foundation research
1) There were no significant differences in body weight among the four groups at the baseline.After 6 weeks of experiment,all atherosclerotic models of rabbit were created successfully,and there was no difference in body weight among the four groups.
2) Compared with control group,in rosiglitazone group,ABCA1 expressions in peripheral mononuclear cells,peritoneal macrophages,adipocytes and hepatocytes; [~3H]cholesterol efflux rates in peritoneal macrophages,adipocytes and hepatocytes, and serum levels of HDL-Cand apoA-1 were all increased,while contents of cholesterol in aorta,adipose and liver tissues and area of atherosclerosis in aorta were all decreased,and there was no difference in NHDL-C level.
3) The effects of rosiglitazone&simvastatin group were similar with rosiglitazone group,however,in difference,the serum NHDL-C level was decreased in rosiglitazone&simvastatin group as compared with control group.Compared with rosiglitazone group,in rosiglitazone&simvastatin group,ABCA1 expressions in peripheral mononuclear cells,peritoneal macrophages,adipocytes and hepatocytes, [~3H]cholesterol efflux rates in peritoneal macrophages,adipocytes and hepatocytes, and serum levels of HDL-C and apoA-1 were all higher,while contents of cholesterol in aorta,adipose and liver tissues and area of atheroscler osis in aorta were all lower.
4) Compared with control group,in glibenclamide group,ABCA1 expressions in peripheral mononuclear cells,peritoneal macrophages,adipocytes and hepatocytes, [~3H]cholesterol efflux rates in peritoneal macrophages,adipocytes and hepatocytes, and serum levels of HDL-C and apoA-1 were all decreased,while contents of cholesterol in aorta,adipose and liver tissues and area of atherosclerosis in aorta were all increased,and there was no difference in NHDL-C level.
5) In all experimental animals,cholesterol contents in aorta,adipose and liver tissues respectively had a negative correlation with[3H]cholesterol efflux rates from peritoneal macrophages,adipocytes and hepatocytes which were positively associated with ABCA1 expressions in them severally.ABCA1 expression in monocytes respectively had a positive correlation with[3H]cholesterol efflux rates from peritoneal macrophages,hepatocytes and adipocytes.
2.Clinical research
1) Compared with control group,type 2 diabetes enhanced the plasma levels of TG and NHDL-C which are pro-atherosclerosis,and reduced the serum apoA-1 and HDL-C levels which are anti-atherosclerosis.
2) Type 2 diabetes group up-regulate the ABCA1 as compared with control group,while the change(stimulated expression / initial expression) of ABCA1 after stimulated by oxidized low density lipoprotein was significantly greater in type 2 diabetes group than in control group.But the ultimate expression of ABCA1 in type 2 diabetes group was still smaller than in the control group.
3) In type 2 diabetes group,the ABCA1 had no correlation with age,fasting plasma glucose(FPG),and HbA1C.But the change of ABCA1 was positive correlation with plasma high density lipoprotein cholesterol level.
Conclusions
1.Expressions of ABCA1 in monocytes/macrophages,hepatocytes and adipocytes are positive regulators of RCT.
2.Rosiglitazone promotes RCT by up-regulating ABCA1 expressions in monocytes/macrophages,hepatocytes and adipocytes to repress atherosclerosis.
3.ABCA1expression in monocytes is a credible index which reflects RCT indirectly.
4.Patients with type 2 diabetes already have the injuried lipids spectrum and RCT,thus forming the foundation of pro-atherosclerosis and coronary heart disease.
引文
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4. Groen AK, Oude Elferink RP, Verkade HJ, et al. The ins and outs of reverse cholesterol transport. Ann Med, 2004, 36(2): 135-145
5. Brooks-Wilson A, Marcil M, Clee S.M., et al., Mutations inABCl in Tangier disease and familial high-density lipoproteindeficiency. Nat. Genet, 1999, 22 (4): 336-345
6. Tall AR. Role of ABCA1 in cellular cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol, 2003, 23(5): 710-711
7. Yokoyama S, Assembly of high-density lipoprotein. Arterioscler Thromb Vasc Biol,2006,26(1): 20-27
8. Oram JF. HDL apolipoproteins and ABCA1: partners in the removal of excess cellular cholesterol. Arterioscler. Arterioscler Thromb Vasc Biol, 2003, 23(5) : 720-727
9. Schmitz G, Kaminski WE. ATP-binding cassette (ABC) transporters in atherosclerosis. Curr Atheroscler Rep, 2002, 4 (3): 243-251
10. Related Articles, LinksSingaraja RR, Fievet C, et al. Increased ABCA1 activity protects against atherosclerosis. J Clin Invest, 2002,110(1): 35-42
11. Seglen PO. Prepapercentagen of isolated rat liver cells. Methods Cell Biol, 1976; 13: 29-83
12. Ceriello A. Thiazolidinediones as anti-inflammatory and anti-atherogenic agents. Diabetes Metab Res Rev, 2008, 24(1): 14-26
13. Sidhu JS, Kaposzta Z, Markus HS, et al. Effect of rosiglitazone on common carotid intima-media thickness progression in coronary artery disease patients without diabetes mellitus. Arterioscler Thromb Vasc Biol, 2004, 24(5): 930-934
14. Calkin AC, Forbes JM, Smith CM, et al. Rosiglitazone attenuates atherosclerosis in a model of insulin insufficiency independent of its metabolic effects. Arterioscler Thromb Vasc Biol, 2005, 25(9): 1903-1909
15. Samaha FF, Szapary PO, Iqbal N, et al. Effects of rosiglitazone on lipids, adipokines, and inflammatory markers in nondiabetic patients with low high-density lipoprotein cholesterol and metabolic syndrome. Arterioscler Thromb Vasc Biol, 2006, 26(3): 624-630
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23. Nedvidkova J, Smitka K, Kopsky V, et al. Adiponectin, an adipocyte-derived protein. Physiol Res, 2005, 54(2): 133-140
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2. Rader DJ. Regulation of reverse cholesterol transport and clinical implications. Am J Cardiol, 2003, 92(4): 42-49
3. Brewer HB. High-density lipoproteins: a new potential therapeutic target for the prevention of cardiovascular disease. Arterioscler Thromb Vasc Biol, 2004, 24(3): 387-391
4. Groen AK, Oude Elferink RP, Verkade HJ, et al. The ins and outs of reverse cholesterol transport. Ann Med, 2004, 36(2): 135-145
5. Brooks-Wilson A, Marcil M, Clee S.M., et al., Mutations inABCl in Tangier disease and familial high-density lipoproteindeficiency. Nat. Genet, 1999, 22 (4): 336-345
6. Tall AR. Role of ABCA1 in cellular cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol, 2003, 23(5): 710-711
7. Yokoyama S, Assembly of high-density lipoprotein. Arterioscler Thromb Vasc Biol,2006,26(1): 20-27
8. Oram JF. HDL apolipoproteins and ABCA1: partners in the removal of excess cellular cholesterol. Arterioscler. Arterioscler Thromb Vasc Biol, 2003, 23(5) : 720-727
9. Schmitz G, Kaminski WE. ATP-binding cassette (ABC) transporters in atherosclerosis. Curr Atheroscler Rep, 2002, 4 (3): 243-251
10. Related Articles, LinksSingaraja RR, Fievet C, et al. Increased ABCA1 activity protects against atherosclerosis. J Clin Invest, 2002,110(1): 35-42
11. Seglen PO. Prepapercentagen of isolated rat liver cells. Methods Cell Biol, 1976; 13: 29-83
12. Ceriello A. Thiazolidinediones as anti-inflammatory and anti-atherogenic agents. Diabetes Metab Res Rev, 2008, 24(1): 14-26
13. Sidhu JS, Kaposzta Z, Markus HS, et al. Effect of rosiglitazone on common carotid intima-media thickness progression in coronary artery disease patients without diabetes mellitus. Arterioscler Thromb Vasc Biol, 2004, 24(5): 930-934
14. Calkin AC, Forbes JM, Smith CM, et al. Rosiglitazone attenuates atherosclerosis in a model of insulin insufficiency independent of its metabolic effects. Arterioscler Thromb Vasc Biol, 2005, 25(9): 1903-1909
15. Samaha FF, Szapary PO, Iqbal N, et al. Effects of rosiglitazone on lipids, adipokines, and inflammatory markers in nondiabetic patients with low high-density lipoprotein cholesterol and metabolic syndrome. Arterioscler Thromb Vasc Biol, 2006, 26(3): 624-630
16. Zhao SP, Yang J, Li J, et al. Effect of niacin on LXRalpha and PPARgamma expression and HDL-induced cholesterol efflux in adipocytes of hypercholesterolemic rabbits. Int J Cardiol, 2008, 124(2): 172-178
17. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem, 1957, 226(1) : 497-509
18. Haug A, Hostmark AT, Lipoprotein lipases, lipoproteins and tissue lipids in rats fed fish oil or coconut oil. J Nutr, 1987,117(6): 1011-1017
19. Singaraja RR, Van Eck M, Bissada N, et al. Both hepatic and extrahepatic ABCA1 have discrete and essential functions in the maintenance of plasma high-density lipoprotein cholesterol levels in vivo. Circulation, 2006, 114(12) : 1301-1309
20. Singaraja RR, Stahmer B, Brundert M, et al. Hepatic ATP-binding cassette transporter Al is a key molecule in high-density lipoprotein cholesteryl ester metabolism in mice. Arterioscler Thromb Vasc Biol. 2006, 26(8): 1821-1827
21. Brewer HB Jr, Santamarina-Fojo S. Clinical significance of high-density lipoproteins and the development of atherosclerosis: focus on the role of the adenosine triphosphate-binding cassette protein A1 transporter. Am J Cardiol, 2003,92:10-16
22. Wu ZH, Zhao SP. Adipocyte: a potential target for the treatment of atherosclerosis. Med Hypotheses, 2006, 67(1):82-86
23. Nedvidkova J, Smitka K, Kopsky V, et al. Adiponectin, an adipocyte-derived protein. Physiol Res, 2005, 54(2): 133-140
24. Diez JJ, Iglesias P. The role of the novel adipocyte-derived hormone adiponectin in human disease. Eur J Endocrinol, 2003, 148(3):293-300
25. Tiikkainen M, Hakkinen AM, Korsheninnikova E, et al. Effects of rosiglitazone and metformin on liver fat content, hepatic insulin resistance, insulin clearance, and gene expression in adipose tissue in patients with type 2 diabetes. Diabetes, 2004, 53(8):2169-2176
26. Salinas CA, Cruz-Bautista I, Mehta R, et al. The ATP-binding cassette transporter subfamily A member 1 (ABC-A1) and type 2 diabetes: an association beyond HDL cholesterol. Curr Diabetes Rev, 2007 , 3(4): 264-267
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