小檗碱联合厚朴酚对THP-1巨噬细胞源性泡沫细胞的干预
|
摘要
研究目的
关于动脉粥样硬化(Atherosclerosis,As)的发病机制一直以来存在争议,近年来得到广泛认可的炎症学说认为:在As早期,因内皮损伤引发炎症反应,导致脂质沉积氧化,单核细胞迁移至内皮以下,在多种细胞因子作用下分化为巨噬细胞,上调清道夫受体的表达,摄取大量ox-LDL,形成泡沫化细胞。既然巨噬细胞泡沫化是As早期的关键事件,那么以巨噬泡沫细胞为靶点对As形成进行早期抗炎干预及调节胆固醇代谢成为防治As的主要方向。
中医对As的致病机理有多种不同看法,有学者提出“湿热化瘀”学说,认为湿热蕴结于血脉是As的主要病理环节,因此治疗上应以清热化湿消瘀法作为主要治则。临床中选用王氏连朴饮加味丹参、赤芍来治疗As病人,取得较好疗效。王氏连朴饮是《霍乱论》温病经典方,方中以黄连、厚朴为君药,黄连能清热化湿,厚朴能燥湿消痰,两味药的合用可以起到清热化湿的功效。
综合上述中、西医两种观点,找出相关联部分,同为As早期的病机,西医认为是炎症反应和巨噬细胞泡沫化,中医认为是湿热蕴结,那么采用清热化湿的方法能不能抑制炎症反应泡沫细胞的形成?在这样的假说下,我们结合临床使用王氏连朴饮治疗As病人,受其启发,研究该方中的主要药效成分,即小檗碱、厚朴酚、和厚朴酚,联合用于THP-1巨噬细胞源性泡沫细胞,研究其对泡沫细胞的炎症反应和胆固醇代谢方面的影响,研究目的在于揭示三种成分的合用能否抑制巨噬细胞的泡沫化过程以及其可能的干预机制,为临床中As防治提供实验数据,也为As的中医病机研究提供参考。
研究方法与内容
(一)THP-1巨噬细胞源性泡沫细胞模型的建立及鉴定
1、以佛波酯诱导THP-1细胞分化为巨噬细胞,并以鸡红细胞吞噬实验验证
2、将THP-1源性巨噬细胞与ox-LDL共孵育建立泡沫细胞模型
3、油红O染色和HPLC-MS法测定胆固醇含量鉴定泡沫细胞模型
(二)小檗碱联合厚朴酚对泡沫细胞的形态及胞内胆固醇的影响
1、MTT法考察药物的给药浓度和给药时间
2、结合油红O染色进行形态学观察
3、HPLC-MS法测定细胞内胆固醇含量
(三)ELISA法检测巨噬细胞TNF-α、IL-1α的表达
以佛波酯诱导THP-1细胞分化为巨噬细胞后给予LPS刺激,并给予药物处理,收集培养上清液,以ELISA试剂盒测定其TNF-α、IL-1α的浓度。
(四)FCM法检测泡沫细胞CD36的表达
以佛波酯和ox-LDL诱导THP-1细胞分化为巨噬泡沫细胞,给予药物处理,收集培养上清液,与FITC标记的鼠抗人CD36抗体孵育,测定细胞CD36表达率。
(五)qRT-PCR法检测泡沫细胞ABCA1 mRNA的表达
用TRIzol法提取各组细胞的总RNA,取2μg的总RNA用Invitrogen公司的SuperScriptⅢ按试剂盒说明书操作合成为cDNA后,置八联管中,每个样品设三个复孔,加入SYBR Green染料,采集各孔荧光,记录Ct值,以β-actin为管家基因,采用相对定量2~(-ΔΔCt)法计算各组细胞中相对于对照组ABCA1mRNA的表达水平。
研究结果
(一)泡沫细胞模型的建立与鉴定
1、泡沫细胞模型的建立:取对数生长期的THP-1单核细胞,用无血清的RPMI 1640基础培养液调整细胞浓度为1×10~6个/mL,接种于6孔培养板,每孔2mL,加佛波酯使终浓度为160nmol/L,诱导24 h,轻轻吸去上清液,换含3%胎牛血清的RPMI 1640培养液,并加入终浓度为80 mg/L的oxLDL,共同孵育48h。
2、泡沫细胞模型的鉴定:
①油红O染色:
将细胞接种于6孔培养板内,诱导建模结束后,吸去上清液,用PBS洗三次,冰冻的4%多聚甲醛固定5min。PBS洗一次,再置于丙二醇中固定5min。轻轻吸去丙二醇,加入0.5%油红O的丙二醇溶液,置60℃烤箱中染色15min,用85%丙二醇冲浸洗5min,再用PBS洗三次,染色后,立即置显微镜下观察并摄像。
②HPLC-MS法测胆固醇含量:
色谱条件:色谱柱:Hypersil C_(18)柱(2.1mm×150mm,3μm);流动相:甲醇-水(90:10);流速:0.5mL/min;柱温:室温;进样量:10μL。质谱条件:离子源:大气压化学电离源(APCI);放电电流5μA;气化温度350℃;离子传输毛细管温度250℃;鞘气(N_2)流速30 arbitrary units;辅助气(N_2)流速5 arbitrary units;正离子方式检测;扫描方式:选择性离子检测(SIM);检测离子:胆固醇,[M+H]~+,m/z369,豆甾醇,[M+H]~+,m/z 395。
(二)小檗碱联合厚朴酚对泡沫细胞的影响
1、由MTT法检测联合用药的IC_(50),确定用于泡沫细胞时的给药浓度为小檗碱5μmol/L、厚朴酚2μmol/L、和厚朴酚2μmol/L,用于巨噬细胞时的给药浓度为小檗碱10μmol/L、厚朴酚4μmol/L、和厚朴酚4μmol/L。给药时间均为24h以内。
2、经ox-LDL诱导后,模型组细胞体积增大,由原来的梭形变为不规则圆形,经油红O染色后,细胞胞内大量红色脂质颗粒;中药组细胞仍呈现梭形,洛伐他汀组细胞呈规则圆形且晶莹透亮,两组细胞经油红O染色后,胞内红色脂质颗粒比模型组少。
3、泡沫细胞分别经洛伐他汀和中药处理24h后,测量两组细胞内胆固醇含量,与模型组相比较,中药组和洛伐他汀组细胞内胆固醇明显减少,说明二者均具有抑制细胞泡沫化的作用;并且中药组与洛伐他汀组相比,无显著性差别。
(三)小檗碱联合厚朴酚、和厚朴酚可抑制THP-1巨噬细胞TNF-α的分泌,并且与阳性对照药地塞米松组无显著性差异。IL-1α因表达量低未检出。
(四)以流式细胞术测定细胞表面CD36的表达,结果表明经PMA和oxLDL刺激分化为巨噬泡沫细胞后CD36表达率为84.6%,小檗碱联合厚朴酚处理后降为13.3%,并且比洛伐他汀组降低更多。
(五)THP-1细胞经ox-LDL诱导成为泡沫细胞后ABCA1 mRNA表达下降,给予洛伐他汀处理后无明显改善,但给予小檗碱、厚朴酚及和厚朴酚联合处理后,ABCA1mRNA比单核细胞上升近一倍,说明联合用药能上调ABCA1表达,促进脂质外流。
结论
小檗碱、厚朴酚及和厚朴酚联合用于THP-1源性泡沫细胞后,细胞内总胆固醇含量减少,巨噬细胞泡沫化受到抑制,其干预机制可能与减少炎症介质、抑制CD36及上调ABCA1表达有关。特别在对ABCA1 mRNA的表达方面,洛伐他汀抑制其表达,不利于脂质外流,而中药的联合使用既能下调CD36减少脂质摄取,又能上调ABCA1促进脂质外流,有利于As的防治,体现了中药多组分联合作用的优势。
Objective
There is always controversy about the pathogenesis of atherosclerosis all round the world.In recent years inflammation theory has been widely recognized.This theory proposes that:atherosclerosis can be considered to represent an inflammatory response of macrophages and lymphocytes to 'invading' pathogenic lipoproteins in the arterial wall.As endothelial injury triggers inflammation and lipid deposition and even oxidation, monocytes migrate under the endothelial cells and differentiate into macrophages under large quantities of proinflammatory cytokines.The latter raise scavenger receptors expression that intake much ox-LDL,and form foam cells.Macrophages,in fact,have essential functions in all phases of atherosclerosis,from development of the fatty streak to processes that ultimately contribute to plaque rupture and myocardial infarction.Since the formation of macrophage-derived foam cells is the key events in the early atherosclerosis, this knowledge and further elucidation might allow the development of treatments targeted at gene expression and cholesterol metabolism within foam cells.The next generation of drugs,possibly targeting inflammatory pathways and mechanisms of cellular cholesterol metabolism and efflux,are awaited with considerable interest.As many complex factors involve in the atherosclerosis formation and development,the single-target treatment shows poor clinical effect.The limited efficacy of current treatment strategies for targeting atherosclerosis and its complications requires new therapeutic options to be explored.The combination therapy of several drugs is often used in clinic.Chinese medicine is characteristics of multi-components and many targets and suitable for treatment atherosclerosis.
Many different opinions about pathogenesis mechanism of atherosclerosis exit in Chinese medicine.Recently,some scholars put forward "damp and hot turning into stagnant blood" theory.They think damp and hot accumulate in blood is the main pathological factor in early atherosclerosis;therefore,the heat-clearing and dampness-resolving therapy should be applied as main principle.Moreover,Wangshi Lianpo Yin plus Salvia miltiorrhiza and paeoniae radix is ammoniated to atherosclerotic patients.
In western medicine view,inflammation and foam cell formation are the key events in early atherosclerosis,while Chinese medicine theory points out damp and heat accumulation is crucial in the early period.Take both views into consideration,we find the linked parts and propose a new hypothesis:foam cell formation may be inhibited by heat-clearing and dampness-resolving treatment.
Wangshi Lianpo Yin was a classical formula in Cholera Therory,composed with rhizoma coptidis,cortex magnoliae officinalis,rhizoma acroi tatarinowii,rhizoma pinelliae praeparatum,semen sojae praeparatum,fructus gardeniae praeparatum,rhizoma phragmitis. Rhizoma coptidis with heat- clearing effect and cortex magnoliae officinalis with damp-removing effect both served as principle drugs in this formula.Their combination application could clear the heat and remove the dampness.The primary medicative component in rhizoma coptis was berberine,and the primary medicative components in cortex magnoliae officinalis were honokiol and magnolol.As reported,berberine and honokiol have anti-inflammatory effect,what' more,berberine as a noval cholesterol-lowering drug,could improves the lipid-lowering efficacy combination with simvastatin.
As this study was just a preliminary investigation,as well as considering the characteristics of cell medication,we study the intervention of the main medicative components of the two principle drugs in Wangshi Lianpo Yin,namely berberine,honokiol, and honokiol,on THP-1 macrophages-derived foam cells.The research mainly focused on anti-inflammatory and cholesterol metabolism,aiming to reveal the intervention pathway of combination of the three components on macrophages-derived foam cells.This study offered some laboratory research data for clinical prevention and therapy and new drug development for atherosclerosis,and provided reference materials for Chinese medicinal pathogenesis research of atherosclerosis.
Methods
1、Establishment fand identification of foam cell model from THP-1 cell line
(1) THP-1 monocyte was treated with phorbol-12-myristate-13-acetate(PMA) to induce a macrophage phenotype.Cock red blood cells were used to evaluate macrophage phagocytic activity.
(2) The THP-1 macrophages were incubated with ox-LDL to establish foam-cell model.
(3) Red oil O staining for the identification of foam cell model
2、Influence of BMH on the appearances and cholesterol content of foam cells
(1) MTT assay for the measurement of administration dosage and time
(2) Observation under microscope directly or with red oil O staining
(3) Determination of intracellular cholesterol in foam cells by HPLC-MS
3、Enzyme-linked immunosorbent assay
THP-1 cells were differentiated into macrophages with PMA.The macrophages were stimulated with LPS and treated with drugs at the same time.The supematants were collected to measure the content of proinflammatory cytokines.
4、Flow cytometric analysis
THP-1 cells were differentiated into macrophage-derived foam cells with PMA and ox-LDL and treated with drugs.The supernatants were collected and incubated with antibody of FITC Mouse Anti-Human CD36.The expression of the cell surface CD36 molecules in the macrophages was determined by a flow cytometric analysis.
5、Quantitative real-time reverse transcription-PCR
Total RNA was isolated with TRIzol reagent according to the manufacturer's instructions.The total RNA(2μg) was reverse-transcribed by using SuperscriptⅢ(Invitrogen) and random hexamer primers(Invitrogen).Each RNA sample was amplified in triplicate for the genes of interest andβ-actin as a housekeeping marker on a 7300 Sequence Detection System(Applied Biosystems) by using a SYBR-green kit(Invitrogen). The primers used are listed in Table 1.The threshold was set automatically,and a threshold cycle(Ct) was measured for each well.The data were analyzed according to 2~(-△△Ct).
Results
1、Establishment and identification of foam cell model
(1) THP-1 cells in log phase growth were diluted to 10~6cells/mL with serum-free RPMI 1640,and plated in 6-well sterile plastic culture plates,2 milliliters/ well.The differentiation of monocyte into macrophage was induced in the presence of 160nmol/L PMA for 24 hours.Remove the supematant and add RPMI 1640 supplemented with 3% fetal bovine serum.Cholesterol loading was achieved by incubation of the cells for 48 hours in the presence of 80mg/L ox-LDL.
(2) Oil red O staining
Foam cells were washed three times with phosphate-buffered saline(PBS),and then fix in ice cold 4%paraformaldehyde for 5 minutes.Rinse with PBS.Place in absolute propylene glycol for 5 minutes to avoid carrying water into Oil Red O.Stain in oil red O solution for 15 minutes in 60℃oven.Rinse in 85%propylene glycol solution for 5 minutes.Rinse 3 changes of PBS.
(3) Analysis of cholesterol and cholesteryl esters
Liquid chromatography:The sterol samples of 10μL injection volume were separated under isocratic conditions with a methanol-water(90:10,v/v) mixture at room temperature. The chromatographic separation was performed using an Hypersil C_(18) reversed-phase column(2.1mm×150mm,3μm) with a flow rate of 0.5mL/min.Mass spectrometry:The atmospheric pressure chemical ionization(APCI) source was used and the ionization was performed in the positive mode and selected ion monitoring was carried out by monitoring m/z 369 for cholesterol and m/z 395 for stigmasterol.The optimized operating parameters of the APCI-MS interface were as follows:vaporization temperature350℃;source temperature 250℃;discharge current 5μA;sheath gas flow 30 arbitrary(instrument) unit; aux gas flow 5arbitrary unit.Data were processed using the Xcalibur software package version2.0.6f.
2、Effects of berberine,magnolol and honokiol on foam cells
(1) The administration dose and time were determined by MTT assay.5μmol/L Berberine,2μmol/L magnolol and 2μmol/L honokiol were applied to foam cells and 10μmol/L,4μmol/L,4μmol/L respectively were applied to macrophages,and both the treatment time were 24 hours.
(2) The model group cells after incubation with ox-LDL turned bigger and irregular round compared to the spindle macrophages.After red O oil dying,many red lipid particles were observed inside the cells under microscope.Chinese medicine group cells still presented spindle,and lovastatin group cells turned round,translucent and bright.Red lipid particles in both drug-treated group cells were less than the model group.
(3) The foam cells were treated with lovastatin and herbal treatment respectively for 24h, and both significantly reduced intracellular cholesterol content.That showed that they had the effect of inhibiting foam cell formation.And the Chinese medicine group had no significant difference compared to lovastatin group.
3、The LPS-induced TNF-αproduction was determined in THP-1 macrophages by ELISA. The combination of Chinese medicine reduced the level of TNF-αand there was on significant difference compared to dexamethasone group.
4、CD36 expression was measured by flow cytometry in THP-1 derived foam cells that had been incubated with ox-LDL alone or in combination with drug.Data showed that CD36 expression in Chinese medicine decreased from 84.6%to 13.3%,even less than lovastatin group.
5、ABCA1 mRNA level was down-regulated 3-fold by ox-LDL at a concentration of 80mg/mL and was slightly increased after lovastatin treatment,but still lower 50% compared with control group.In contrast,the combination of berberine,magnolol and honokiol up- regulated the ABCA1 mRNA level 2-fold compared with control group.That implied the combination treatment promoted cholesterol efflux.
Conclusion
The combination of berberine,magnolol and honokiol inhibited the formation of foam cell,mainly in regard to inhibiting inflammation,reducing CD36 expression and up-regulating ABCA1 mRNA.
Lovastatin,a commonly used cholesterol-lowering drug,suppressed CD36 expression, and concomitantly down-regulated ABCA1 mRNA levels.As ABCA1 redirected macrophage cholesterol handling towards reverse cholesterol transport,which contributed to cholesterol efflux,the decrease of ABCA1 by lovastatin was unfavorable for anti-atherosclerotic action.The combination of berberine,magnolol and honokiol,however, showed dual effects on lipid metabolism in addition to anti-inflammatory actions.Its anti-atherosclerotic mechanisms were down-regulated CD36 and ox-LDL uptake and stimulated ABCA1 and cholesterol efflux.That demonstrated the advantage of Chinese herbal drug's synergistic effect on various targets.
关于动脉粥样硬化(Atherosclerosis,As)的发病机制一直以来存在争议,近年来得到广泛认可的炎症学说认为:在As早期,因内皮损伤引发炎症反应,导致脂质沉积氧化,单核细胞迁移至内皮以下,在多种细胞因子作用下分化为巨噬细胞,上调清道夫受体的表达,摄取大量ox-LDL,形成泡沫化细胞。既然巨噬细胞泡沫化是As早期的关键事件,那么以巨噬泡沫细胞为靶点对As形成进行早期抗炎干预及调节胆固醇代谢成为防治As的主要方向。
中医对As的致病机理有多种不同看法,有学者提出“湿热化瘀”学说,认为湿热蕴结于血脉是As的主要病理环节,因此治疗上应以清热化湿消瘀法作为主要治则。临床中选用王氏连朴饮加味丹参、赤芍来治疗As病人,取得较好疗效。王氏连朴饮是《霍乱论》温病经典方,方中以黄连、厚朴为君药,黄连能清热化湿,厚朴能燥湿消痰,两味药的合用可以起到清热化湿的功效。
综合上述中、西医两种观点,找出相关联部分,同为As早期的病机,西医认为是炎症反应和巨噬细胞泡沫化,中医认为是湿热蕴结,那么采用清热化湿的方法能不能抑制炎症反应泡沫细胞的形成?在这样的假说下,我们结合临床使用王氏连朴饮治疗As病人,受其启发,研究该方中的主要药效成分,即小檗碱、厚朴酚、和厚朴酚,联合用于THP-1巨噬细胞源性泡沫细胞,研究其对泡沫细胞的炎症反应和胆固醇代谢方面的影响,研究目的在于揭示三种成分的合用能否抑制巨噬细胞的泡沫化过程以及其可能的干预机制,为临床中As防治提供实验数据,也为As的中医病机研究提供参考。
研究方法与内容
(一)THP-1巨噬细胞源性泡沫细胞模型的建立及鉴定
1、以佛波酯诱导THP-1细胞分化为巨噬细胞,并以鸡红细胞吞噬实验验证
2、将THP-1源性巨噬细胞与ox-LDL共孵育建立泡沫细胞模型
3、油红O染色和HPLC-MS法测定胆固醇含量鉴定泡沫细胞模型
(二)小檗碱联合厚朴酚对泡沫细胞的形态及胞内胆固醇的影响
1、MTT法考察药物的给药浓度和给药时间
2、结合油红O染色进行形态学观察
3、HPLC-MS法测定细胞内胆固醇含量
(三)ELISA法检测巨噬细胞TNF-α、IL-1α的表达
以佛波酯诱导THP-1细胞分化为巨噬细胞后给予LPS刺激,并给予药物处理,收集培养上清液,以ELISA试剂盒测定其TNF-α、IL-1α的浓度。
(四)FCM法检测泡沫细胞CD36的表达
以佛波酯和ox-LDL诱导THP-1细胞分化为巨噬泡沫细胞,给予药物处理,收集培养上清液,与FITC标记的鼠抗人CD36抗体孵育,测定细胞CD36表达率。
(五)qRT-PCR法检测泡沫细胞ABCA1 mRNA的表达
用TRIzol法提取各组细胞的总RNA,取2μg的总RNA用Invitrogen公司的SuperScriptⅢ按试剂盒说明书操作合成为cDNA后,置八联管中,每个样品设三个复孔,加入SYBR Green染料,采集各孔荧光,记录Ct值,以β-actin为管家基因,采用相对定量2~(-ΔΔCt)法计算各组细胞中相对于对照组ABCA1mRNA的表达水平。
研究结果
(一)泡沫细胞模型的建立与鉴定
1、泡沫细胞模型的建立:取对数生长期的THP-1单核细胞,用无血清的RPMI 1640基础培养液调整细胞浓度为1×10~6个/mL,接种于6孔培养板,每孔2mL,加佛波酯使终浓度为160nmol/L,诱导24 h,轻轻吸去上清液,换含3%胎牛血清的RPMI 1640培养液,并加入终浓度为80 mg/L的oxLDL,共同孵育48h。
2、泡沫细胞模型的鉴定:
①油红O染色:
将细胞接种于6孔培养板内,诱导建模结束后,吸去上清液,用PBS洗三次,冰冻的4%多聚甲醛固定5min。PBS洗一次,再置于丙二醇中固定5min。轻轻吸去丙二醇,加入0.5%油红O的丙二醇溶液,置60℃烤箱中染色15min,用85%丙二醇冲浸洗5min,再用PBS洗三次,染色后,立即置显微镜下观察并摄像。
②HPLC-MS法测胆固醇含量:
色谱条件:色谱柱:Hypersil C_(18)柱(2.1mm×150mm,3μm);流动相:甲醇-水(90:10);流速:0.5mL/min;柱温:室温;进样量:10μL。质谱条件:离子源:大气压化学电离源(APCI);放电电流5μA;气化温度350℃;离子传输毛细管温度250℃;鞘气(N_2)流速30 arbitrary units;辅助气(N_2)流速5 arbitrary units;正离子方式检测;扫描方式:选择性离子检测(SIM);检测离子:胆固醇,[M+H]~+,m/z369,豆甾醇,[M+H]~+,m/z 395。
(二)小檗碱联合厚朴酚对泡沫细胞的影响
1、由MTT法检测联合用药的IC_(50),确定用于泡沫细胞时的给药浓度为小檗碱5μmol/L、厚朴酚2μmol/L、和厚朴酚2μmol/L,用于巨噬细胞时的给药浓度为小檗碱10μmol/L、厚朴酚4μmol/L、和厚朴酚4μmol/L。给药时间均为24h以内。
2、经ox-LDL诱导后,模型组细胞体积增大,由原来的梭形变为不规则圆形,经油红O染色后,细胞胞内大量红色脂质颗粒;中药组细胞仍呈现梭形,洛伐他汀组细胞呈规则圆形且晶莹透亮,两组细胞经油红O染色后,胞内红色脂质颗粒比模型组少。
3、泡沫细胞分别经洛伐他汀和中药处理24h后,测量两组细胞内胆固醇含量,与模型组相比较,中药组和洛伐他汀组细胞内胆固醇明显减少,说明二者均具有抑制细胞泡沫化的作用;并且中药组与洛伐他汀组相比,无显著性差别。
(三)小檗碱联合厚朴酚、和厚朴酚可抑制THP-1巨噬细胞TNF-α的分泌,并且与阳性对照药地塞米松组无显著性差异。IL-1α因表达量低未检出。
(四)以流式细胞术测定细胞表面CD36的表达,结果表明经PMA和oxLDL刺激分化为巨噬泡沫细胞后CD36表达率为84.6%,小檗碱联合厚朴酚处理后降为13.3%,并且比洛伐他汀组降低更多。
(五)THP-1细胞经ox-LDL诱导成为泡沫细胞后ABCA1 mRNA表达下降,给予洛伐他汀处理后无明显改善,但给予小檗碱、厚朴酚及和厚朴酚联合处理后,ABCA1mRNA比单核细胞上升近一倍,说明联合用药能上调ABCA1表达,促进脂质外流。
结论
小檗碱、厚朴酚及和厚朴酚联合用于THP-1源性泡沫细胞后,细胞内总胆固醇含量减少,巨噬细胞泡沫化受到抑制,其干预机制可能与减少炎症介质、抑制CD36及上调ABCA1表达有关。特别在对ABCA1 mRNA的表达方面,洛伐他汀抑制其表达,不利于脂质外流,而中药的联合使用既能下调CD36减少脂质摄取,又能上调ABCA1促进脂质外流,有利于As的防治,体现了中药多组分联合作用的优势。
Objective
There is always controversy about the pathogenesis of atherosclerosis all round the world.In recent years inflammation theory has been widely recognized.This theory proposes that:atherosclerosis can be considered to represent an inflammatory response of macrophages and lymphocytes to 'invading' pathogenic lipoproteins in the arterial wall.As endothelial injury triggers inflammation and lipid deposition and even oxidation, monocytes migrate under the endothelial cells and differentiate into macrophages under large quantities of proinflammatory cytokines.The latter raise scavenger receptors expression that intake much ox-LDL,and form foam cells.Macrophages,in fact,have essential functions in all phases of atherosclerosis,from development of the fatty streak to processes that ultimately contribute to plaque rupture and myocardial infarction.Since the formation of macrophage-derived foam cells is the key events in the early atherosclerosis, this knowledge and further elucidation might allow the development of treatments targeted at gene expression and cholesterol metabolism within foam cells.The next generation of drugs,possibly targeting inflammatory pathways and mechanisms of cellular cholesterol metabolism and efflux,are awaited with considerable interest.As many complex factors involve in the atherosclerosis formation and development,the single-target treatment shows poor clinical effect.The limited efficacy of current treatment strategies for targeting atherosclerosis and its complications requires new therapeutic options to be explored.The combination therapy of several drugs is often used in clinic.Chinese medicine is characteristics of multi-components and many targets and suitable for treatment atherosclerosis.
Many different opinions about pathogenesis mechanism of atherosclerosis exit in Chinese medicine.Recently,some scholars put forward "damp and hot turning into stagnant blood" theory.They think damp and hot accumulate in blood is the main pathological factor in early atherosclerosis;therefore,the heat-clearing and dampness-resolving therapy should be applied as main principle.Moreover,Wangshi Lianpo Yin plus Salvia miltiorrhiza and paeoniae radix is ammoniated to atherosclerotic patients.
In western medicine view,inflammation and foam cell formation are the key events in early atherosclerosis,while Chinese medicine theory points out damp and heat accumulation is crucial in the early period.Take both views into consideration,we find the linked parts and propose a new hypothesis:foam cell formation may be inhibited by heat-clearing and dampness-resolving treatment.
Wangshi Lianpo Yin was a classical formula in Cholera Therory,composed with rhizoma coptidis,cortex magnoliae officinalis,rhizoma acroi tatarinowii,rhizoma pinelliae praeparatum,semen sojae praeparatum,fructus gardeniae praeparatum,rhizoma phragmitis. Rhizoma coptidis with heat- clearing effect and cortex magnoliae officinalis with damp-removing effect both served as principle drugs in this formula.Their combination application could clear the heat and remove the dampness.The primary medicative component in rhizoma coptis was berberine,and the primary medicative components in cortex magnoliae officinalis were honokiol and magnolol.As reported,berberine and honokiol have anti-inflammatory effect,what' more,berberine as a noval cholesterol-lowering drug,could improves the lipid-lowering efficacy combination with simvastatin.
As this study was just a preliminary investigation,as well as considering the characteristics of cell medication,we study the intervention of the main medicative components of the two principle drugs in Wangshi Lianpo Yin,namely berberine,honokiol, and honokiol,on THP-1 macrophages-derived foam cells.The research mainly focused on anti-inflammatory and cholesterol metabolism,aiming to reveal the intervention pathway of combination of the three components on macrophages-derived foam cells.This study offered some laboratory research data for clinical prevention and therapy and new drug development for atherosclerosis,and provided reference materials for Chinese medicinal pathogenesis research of atherosclerosis.
Methods
1、Establishment fand identification of foam cell model from THP-1 cell line
(1) THP-1 monocyte was treated with phorbol-12-myristate-13-acetate(PMA) to induce a macrophage phenotype.Cock red blood cells were used to evaluate macrophage phagocytic activity.
(2) The THP-1 macrophages were incubated with ox-LDL to establish foam-cell model.
(3) Red oil O staining for the identification of foam cell model
2、Influence of BMH on the appearances and cholesterol content of foam cells
(1) MTT assay for the measurement of administration dosage and time
(2) Observation under microscope directly or with red oil O staining
(3) Determination of intracellular cholesterol in foam cells by HPLC-MS
3、Enzyme-linked immunosorbent assay
THP-1 cells were differentiated into macrophages with PMA.The macrophages were stimulated with LPS and treated with drugs at the same time.The supematants were collected to measure the content of proinflammatory cytokines.
4、Flow cytometric analysis
THP-1 cells were differentiated into macrophage-derived foam cells with PMA and ox-LDL and treated with drugs.The supernatants were collected and incubated with antibody of FITC Mouse Anti-Human CD36.The expression of the cell surface CD36 molecules in the macrophages was determined by a flow cytometric analysis.
5、Quantitative real-time reverse transcription-PCR
Total RNA was isolated with TRIzol reagent according to the manufacturer's instructions.The total RNA(2μg) was reverse-transcribed by using SuperscriptⅢ(Invitrogen) and random hexamer primers(Invitrogen).Each RNA sample was amplified in triplicate for the genes of interest andβ-actin as a housekeeping marker on a 7300 Sequence Detection System(Applied Biosystems) by using a SYBR-green kit(Invitrogen). The primers used are listed in Table 1.The threshold was set automatically,and a threshold cycle(Ct) was measured for each well.The data were analyzed according to 2~(-△△Ct).
Results
1、Establishment and identification of foam cell model
(1) THP-1 cells in log phase growth were diluted to 10~6cells/mL with serum-free RPMI 1640,and plated in 6-well sterile plastic culture plates,2 milliliters/ well.The differentiation of monocyte into macrophage was induced in the presence of 160nmol/L PMA for 24 hours.Remove the supematant and add RPMI 1640 supplemented with 3% fetal bovine serum.Cholesterol loading was achieved by incubation of the cells for 48 hours in the presence of 80mg/L ox-LDL.
(2) Oil red O staining
Foam cells were washed three times with phosphate-buffered saline(PBS),and then fix in ice cold 4%paraformaldehyde for 5 minutes.Rinse with PBS.Place in absolute propylene glycol for 5 minutes to avoid carrying water into Oil Red O.Stain in oil red O solution for 15 minutes in 60℃oven.Rinse in 85%propylene glycol solution for 5 minutes.Rinse 3 changes of PBS.
(3) Analysis of cholesterol and cholesteryl esters
Liquid chromatography:The sterol samples of 10μL injection volume were separated under isocratic conditions with a methanol-water(90:10,v/v) mixture at room temperature. The chromatographic separation was performed using an Hypersil C_(18) reversed-phase column(2.1mm×150mm,3μm) with a flow rate of 0.5mL/min.Mass spectrometry:The atmospheric pressure chemical ionization(APCI) source was used and the ionization was performed in the positive mode and selected ion monitoring was carried out by monitoring m/z 369 for cholesterol and m/z 395 for stigmasterol.The optimized operating parameters of the APCI-MS interface were as follows:vaporization temperature350℃;source temperature 250℃;discharge current 5μA;sheath gas flow 30 arbitrary(instrument) unit; aux gas flow 5arbitrary unit.Data were processed using the Xcalibur software package version2.0.6f.
2、Effects of berberine,magnolol and honokiol on foam cells
(1) The administration dose and time were determined by MTT assay.5μmol/L Berberine,2μmol/L magnolol and 2μmol/L honokiol were applied to foam cells and 10μmol/L,4μmol/L,4μmol/L respectively were applied to macrophages,and both the treatment time were 24 hours.
(2) The model group cells after incubation with ox-LDL turned bigger and irregular round compared to the spindle macrophages.After red O oil dying,many red lipid particles were observed inside the cells under microscope.Chinese medicine group cells still presented spindle,and lovastatin group cells turned round,translucent and bright.Red lipid particles in both drug-treated group cells were less than the model group.
(3) The foam cells were treated with lovastatin and herbal treatment respectively for 24h, and both significantly reduced intracellular cholesterol content.That showed that they had the effect of inhibiting foam cell formation.And the Chinese medicine group had no significant difference compared to lovastatin group.
3、The LPS-induced TNF-αproduction was determined in THP-1 macrophages by ELISA. The combination of Chinese medicine reduced the level of TNF-αand there was on significant difference compared to dexamethasone group.
4、CD36 expression was measured by flow cytometry in THP-1 derived foam cells that had been incubated with ox-LDL alone or in combination with drug.Data showed that CD36 expression in Chinese medicine decreased from 84.6%to 13.3%,even less than lovastatin group.
5、ABCA1 mRNA level was down-regulated 3-fold by ox-LDL at a concentration of 80mg/mL and was slightly increased after lovastatin treatment,but still lower 50% compared with control group.In contrast,the combination of berberine,magnolol and honokiol up- regulated the ABCA1 mRNA level 2-fold compared with control group.That implied the combination treatment promoted cholesterol efflux.
Conclusion
The combination of berberine,magnolol and honokiol inhibited the formation of foam cell,mainly in regard to inhibiting inflammation,reducing CD36 expression and up-regulating ABCA1 mRNA.
Lovastatin,a commonly used cholesterol-lowering drug,suppressed CD36 expression, and concomitantly down-regulated ABCA1 mRNA levels.As ABCA1 redirected macrophage cholesterol handling towards reverse cholesterol transport,which contributed to cholesterol efflux,the decrease of ABCA1 by lovastatin was unfavorable for anti-atherosclerotic action.The combination of berberine,magnolol and honokiol,however, showed dual effects on lipid metabolism in addition to anti-inflammatory actions.Its anti-atherosclerotic mechanisms were down-regulated CD36 and ox-LDL uptake and stimulated ABCA1 and cholesterol efflux.That demonstrated the advantage of Chinese herbal drug's synergistic effect on various targets.
引文
[1]杨永宗,阮长耿,唐朝枢,等.动脉粥样硬化性心血管病基础与临床[P],北京:科学出版社,2004,第一版
[2]Ross R.Atherosclerosis:an inflammatory disease[J].N Engl J Med,1999,340(2):115-126
[3]Duguid JB.Pathogenesis of atherosclerosis[J].Lancert,1949,2:925-927
[4]Libby P.Inflammation in atherosclerosis[J].Nature,2002,420:868-874
[5]Dargel R.The lipid infiltration theory of atherosclerosis[J].Z Med Lab Diagn,1989,30(5):251-255.
[6]Ross R,Glomset JA.The pathogenesis of atherosclerosis[J].N Engl J Med.1976,295(7):369-477,295(8):420-426
[7]McCully KS.Hmocysteine theory of arteriosclerosis by homocysteinemia[J].Atherosclerosis Rev,1983,11:157
[8]Steinberg D.Lipoprotein and atherosclerosis[J].Arteriosclerosis,1983,3:283-301
[9]Cooke JP,Oka RK.Atherogenesis and the arginine hypothesis[J].Curr Athero Rep,2001,3:252-259
[10]Pober JS,Cotran RC.Cytokines and endothelial cell biology[J].Physiol Rev,1990,70:427-451
[11]Benditt EP,Bendett JM.Evidence for a monoclonal origin of human atherosclerotic plaques[J].Proc Natl Acad Sci USA,1973,70(6):1753-1756
[12]DeFilippi C,WassermanS,RosanioS,et al.Cardiac troponin T and C-reactive protein for predicting prognosis,coronary atherosclerosis,and cardiomyopathy in patients undergoing long-term hemodialysis[J].JAMA,2003,290:353-359
[13]Tedgui A,Mallat Z.Cytokines in atherosclerosis:pathogenic and regulatory pathways[J].Physiol Rev,2006,86(2):515-581
[14]Viljoen A.New approaches in the diagnosis of atherosclerosis and treatment of cardiovascular disease[J].Recent Pat Cardiovasc Drug Discov.2008,3(2):84-91
[15]李玉洁,杨庆,朱晓新.抗炎-中药治疗动脉粥样硬化的有效途径[J].世界科学技术-中医药现代化,2007,9(2):22-28
[16]Sch(o|¨)nbeck U,Libby P.Inflammation,immunity,and HMG-CoA reductase inhibitors:statins as antiinflammatory agents?[J].Circulation,2004,109(21 Suppl 1):Ⅱ18-26
[17]Kleemann R,Zadelaar S,Kooistra T.Cytokines and atherosclerosis:a comprehensive review of studies in mice[J].Cardiovasc Res,2008,79(3):360-376
[18]Mestas J,Ley K.Monocyte-endothelial cell interactions in the development of atherosclerosis[J].Trends Cardiovasc Med,2008,18(6):228-232
[19]Li AC,Glass CK.The macrophage foam cell as a target for therapeutic intervention[J].Nat Med.2002,8(11):1235-1242
[20]Mangge H,Hubmann H,Pilz S,et al.Beyond cholesterol--inflammatory cytokines,the key mediators in atherosclerosis[J].Clin Chem Lab Med,2004,42(5):467-474
[21]Shibata N,Glass CK.Regulation of macrophage function in inflammation and atherosclerosis[J].J Lipid Res.2009,50 Suppl:S 277-281
[22]Schulte S,Sukhova GK,Libby P.Genetically programmed biases in Th1 and Th2 immune responses modulate atherogenesis[J].Am J Pathol.2008,172(6):1500-1508
[23]Gewaltig J,Kummer M,Koella C,et al.Requirements for CD8 T-cell migration into the human arterial wall[J].Hum Pathol,2008,39(12):1756-1762
[24]Kaartinen M,Penttila A,kovanen PT.Accumulation of activated mast cell in the shoulder region of human coronary atheroma,the predilection site of atheromatous rapture[J].Circulation,1994,90(4):1669-1678
[25]梁萍,孙雷,唐建武,等.细胞间粘附分子1、血管细胞粘附分子1和肿瘤坏死因子在人动脉粥样硬化病灶中的表达及意义[J].中国动脉硬化杂志,2004,12(4):427-429
[26]Coll B,Alonso-Villaverde C,Joven J.Monocyte chemoattractant protein-1 and atherosclerosis:is there room for an additional biomarker?[J].Clin Chim Acta,2007,383(1-2):21-29
[27]Apostolakis S,Vogiatzi K,Krambovitis E,et al.IL-1 cytokines in cardiovascular disease:diagnostic,prognostic and therapeutic implications[J].Cardiovasc Hematol Agents Med Chem,2008,6(2):150-158
[28]Rubic T,Lorenz RL.Downregulated CD36 and oxLDL uptake and stimulated ABCA1/G1 and cholesterol efflux as anti-atherosclerotic mechanisms of interleukin-10[J].Cardiovasc Res.2006,69(2):527-535
[29]Brewer HB Jr.The lipid-laden foam cell:an elusive target for therapeutic intervention[J].J Clin Invest.2000,105(6):703-705
[30]徐晓利,马涧泉.医学生物化学[P],北京:人民卫生出版社,第一版
[31]Henriksen T,Mahoney EM,Steinberg D.Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells:recognition by receptors for acetylated low density lipoproteins[J].Proc Natl Acad Sci U S A,1981,78(10):6499-6503
[32]Kita T,Kume N,Minami M,et al.Role of oxidized LDL in atherosclerosis[J].Ann N Y Acad Sci,2001,947:199-205
[33]张小梅,翟桂兰.炎症因子与动脉粥样硬化的研究进展[J].牡丹江医学院学报,200,29(1):91-93
[34]Portugal LR,Fernandes LR,Pietra Pedroso VS,et al.Influence of low-density lipoprotein(LDL)receptor on lipid composition,inflammation and parasitism during Toxoplasma gondii infection[J].Microbes Infect,2008,10(3):276-284
[35]Kunjathoor VV,Febbraio M,Podrez EA,et al.Scavenger receptors class A-Ⅰ/Ⅱ and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages[J].J Biol Chem,2002,277(51):49982-49988
[36]Small DM.Cellular mechanisms for lipid deposition in atherosclerosis[J].New EngI J Med 1977,297:873-877,924-929
[37]康莉,王家富,商战平.与动脉粥样硬化密切相关的清道夫受体[J].泰山医学院学报,2007,28(3):232-235
[38]Endemann G,Stanton L,Madden K,et al.CD36 is a receptor for oxidized low density lipoprotein [J].J Biol Chem,1993,268:11811-11816
[39]Febbraio M,Podrez EA,Smith JD,et al.Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice[J].J Clin Invest,2000,105:1049-1056
[40]Kashiwagi H,Tomiyama Y,Kosugi Y,et al.Identification of the molecular defects in a subject with type I CD36deficiency[J].Blood,1994,83:3545-3552.
[41]Nicholson A,Pearce SFA,Silverstein R.Oxidized LDL binds to CD36 on human monocyte -derived macrophages and transfected cell lines,Evidence implicating the lipid moiety of the lipoprotein as the binding site[J].Arterioscler Thromb,1995,15:269-275.
[42]BoullierA,Gillotte KL,Horkko S,et al.The binding of oxidized low density lipo protein to mouse CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein[J].J Biol Chem,2000,275:9163-9169
[43]Acton S,Attilio R,Landschultz K,et al.Identification of scavenger receptor SR-B1 as a high density lipoprotein receptor[J].Science,1996,271:518-520
[44]Ohashi R,Mu H,Wang X,et al.Reverse cholesterol transport and cholesterol efflux in atherosclerosis[J].QJM,2005,98(12):845-856
[45]李华波,全智华.ABCA1与PPARs在胆固醇流出中的作用及相关性[J].医学综述,2005,11(11):974-976
[46]Yokoyama S.ABCA1 and biogenesis of HDL[J].J Atheroscler Thromb,2006,13(1):1-15
[47]Oram JF,Lawn RM.ABCA1:the gatekeeper for eliminating excess tissue cholesterol[J].J.Lipid Res,2001,42(8):1173-1179.
[48]Singaraja RR,Bocher V,James ER,et al.Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and ApoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1[J].J Biol Chem,2001,276(36):33969-33979
[49]Van Eck M,Singaraja RR,Ye D,et al.Macrophage ATP-binding cassette transporter Al overexpression inhibits atherosclerotic lesion progression in low-density lipoprotein receptor knockout mice[J].Arterioscler Thromb Vasc Biol,2006,26(4):929-934.
[50]Bald(?)n A,Bojanic DD,Edwards PA.The ABCs of sterol transport[J].J Lipid Res,2009,50Suppl:S80-85
[51]Tavares V,Hirata MH,Hirata RD.Peroxisome proliferator-activated receptor gamma (PPARgamma):molecular study in glucose homeostasis,lipid metabolism and therapeutic approach[J].Arq Bras Endocrinol Metabol.2007,51(4):526-533
[52]Tontonoz P,Nagy L.Regulation of macrophage gene expression by peroxisome-proliferator -activated receptor gamma:implications for cardiovascular disease[J].Cardiovascular disease,1999,10(6):485-490
[53]Chawla A,Boisvert WA,Lee CH,et al.A PAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherosclerosis[J].Mol Cell,2001,7(1):161-171
[54]Argmann CA,Sawyez CG;McNeil CJ,et al.Activation of peroxisome proliferator-activated receptor gamma and retinoid X receptor results in net depletion of cellular cholesteryl esters in macrophages exposed to oxidized lipoproteins[J].Arterioscler Thromb Vasc Biol,2003,23(3):475-482
[55]Sato M,Kawata Y,Erami K,et al.LXR agonist increases the lymph HDL transport in rats by promoting reciprocally intestinal ABCA1 and apo A-I mRNA levels[J].Lipids,2008,43(2):125-131
[56]Chawla A,Boisvert WA,Lee CH,et al.A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis[J].Mol Cell,2001,7(1):161-171
[57]Joseph S B,Castrillo A,Laffitte B A,et al.Reciprocal regulation of inflammation on d lipid metabolism by liver X receptors[J].Nat Med,2003,9(2):213-221
[58]Choudhury RP,Lee JM,Greaves DR.Mechanisms of disease:macrophage-derived foam cells emerging as therapeutic targets in atherosclerosis[J].Nat Clin Pract Cardiovasc Med.2005,2(6):309-315
[59]Webb NR,Moore KJ.Macrophage-Derived Foam Cells in Atherosclerosis:Lessons from Murine Models and Implications for Therapy[J].Curr Drug Targets,2007,8(12):1249-1263
[60]郑杰,田瑞振,刘德山.动脉粥样硬化的中医致病论初探[J].云南中医学院学报,2008,31(2):28-29
[61]许迎春,王化良,丁晶.动脉粥样硬化从毒论治探讨[J].中医杂志,2004,45(6):405-407
[62]张卫娜,管昌益.动脉粥样硬化中医辩证论治研究进展[J].河北北方学院学报:医学版,2008,25(1):72-74
[63]袁中华,杨永宗,杨小毅.平滑肌源性泡沫细胞模型的制备[J].临床与实验病理学杂志,1998,14(1):70-73
[64]李全忠,杨永宗,易光辉,等.U937泡沫细胞模型的建立[J].中国动脉硬化杂志,1999,7(2):152-154
[65]白智峰,成蓓,毛晓波,等.瘦素对THP-1细胞泡沫化过程中ACAT-1表达的影响[J].天津医药,2004,32(10):593-595
[66]王东,丁华,徐红岩,等.洛伐他汀、普罗布考对小鼠巨噬细胞源性泡沫细胞内胆固醇含量的影响[J].中国药理学通报,2004,20(6):680-684
[67]谭健苗,杨永宗,袁中华.巨噬细胞源性与肌源性泡沫细胞的不同特性研究[J].广州医学院学报,2001,29(2):25-30
[68]朱莹,张文高,郑广娟,等.脂欣康胶囊对血管平滑肌细胞源性泡沫细胞CD36表达的影响[J].中国动脉便化杂志,2007,15(7):481-483
[69]Forst T,Wilhelm B,P f(u|¨)tzner A,et al.Investigation of the vascular and pleiotropic effects of atorvastatin and pioglitazone in a population at high cardiovascular risk[J].Diab Vasc Dis Res.2008,5(4):298-30
[70]PAS疗法预防动脉硬化http://cc.39.net/jzzn/0811/19/716415.html
[71]Kong WJ,Wei J,Abidi P,et al.Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins.Nature Medicine,2004,10(12):1344-1351
[72]林培政,杨开清.动脉粥样硬化中医湿热病机再认识[J].新中医.2006,38(3):5-6
[73]林培政,杨开清.清热化湿消瘀法对湿热证模型兔肝脏清道夫受体基因表达的影响[J].中药新药与临床药理,2007,18(1):15-17
[74]Chen FL,Yang ZH,Liu Y,et al.Berberine inhibits the expression of TNF-α,MCP-1,and IL-6 in Ac-LDL-stimulated macrophages through PPARγ pathway[J].Endocrine,2008,33(3):331-337
[75]Kong WJ,Wei J,Zuo ZY.Combination of simvastatin with berberine improves the lipid-lowering efficacy.Metabolism.2008,57(8):1029-1037
[76]Kim BH,Cho JY.Anti-inflammatory effect of honokiol is mediated by PI3K/Akt pathway suppression[J].Acta Phamacol Sin.2008,29(1):113-12
[77]Tsuchiya S,Kobayashi Y,Got Y,et al.Induction of maturation in cultured human monocytic leukemia cells by a phorbol diesterl[J].Cancer Res,1982,42:1530-1536
[78]Fu Y,Luo N,Lopes-Virella MF,et al.The adipocyte lipid binding protein(ALBP/aP2) gene facilitates foam cell formation ha human THP-1 macrophages[J].Atherosclerosis,2002,165(2):259-269
[79]贲长恩,李叔庚,王士平,等.组织化学[M].北京:人民卫生出版社,2001,第1版:169
[80]Fogelman AM,Shechter I,Seager J,et al.Malondialdehyde alteration of low density lipoproteins leads to cholesteryl ester accumulation in human monocyte-macrophages[J].Proc Natl Acad Sci USA,1980,77(4):2214-2218
[81]Palmgr(?)n JJ,T(?)yr(?)s A,Mauriala T,et al.Quantitative determination of cholesterol,sitosterol,andsitostanol in cultured Caco-2 cells by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry[J].J Chromatogr B Analyt Technol Biomed Life Sci,2005,821:144-152
[82]覃丽,秦旭平,王佐,等.普伐他汀对泡沫细胞内胆固醇酯的影响及与小凹蛋白-1的关系[J].生理学报,2006,58(1):47-52
[83]Jak(?)bisiak M,Bruno S,Skierski JS,et al.Cell cycle-specific effects of lovastatin[J].Proc Natl Acad Sci U S A,1991,88(9):3628-3632.
[84]N(u|¨)bel T,Dippold W,Kleinert H.Lovastatin inhibits Rho-regulated expression of E-selectin by TNFalpha and attenuates tumor cell adhesion.FASEB J,2004,18(1):140-142
[85]Zhong WB,Wang CY,Chang TC,et al.Lovastatin Induces Apoptosis of Anaplastic Thyroid Cancer Cells via Inhibition of Protein Geranylgeranylation and de Novo Protein Synthesis[J].Endocrinology,2003,44(9):3852-3859
[86]司徒镇强,吴军正.细胞培养[P],世界图书出版公司,1996年,第1版:134-135
[87]周程,胡思安,龚昭,等.胆囊癌对化疗药物敏感性的研究[J].中华现代外科学杂志,2005,2(20):1830-1832
[88]赵书刚,陈昕,雷开键.连朴饮对湿热夹瘀型动脉粥样硬化患者血脂及内皮功能影响的研究[J].陕西中医,2008,29(6):660-662
[89]Jantova S,Cipak L,Letasiova S.Berberine induces apoptosis through a mitochondrial/caspase pathway in human promonocytic U937 cells[J].Toxicol In Vitro,2007,21(1):25-31
[90]Ikai T,Akao Y,Nakagawa Y,et al.Magnolol-induced apoptosis is mediated via the intrinsic pathway with release of AIF from mitochondria in U937 cells[J].Biol Pharm Bull,2006,29(12):2498-2501
[91]薛芳,李世辉,潘崚.和厚朴酚对U937细胞的体外作用[J].中成约,2007,29(11):1582-1585
[92]Li H,Li X,Duan L,et al.Inhibition of lovastatin on proliferation and expression of proinflammatory cytokines in cultured human glomerular mesangial cells[J].Chin Med J(Engl),2003,116(9):1366-1369
[93]Moyer CF,Sajuthi D,Tulli H,et al.Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis[J].Am J Pathol,1991,138(4):951-960
[94]Mei CL,Chen ZJ,Liao YH,et al.Interleukin-10 inhibits the down-regulation of ATP binding cassette transporter Al by tumour necrosis factor-alpha in THP-1 macrophage-derived foam cells[J].Cell Biol Int,2007,31(12):1456-1461
[95]Xiao N,Yin M,Zhang L,et al.Tumor necrosis factor-alpha deficiency retards early fatty-streak lesion by influencing the expression of inflammatory factors in apoE-null mice[J].Mol Genet Metab,2009,96(4):239-244
[96]曾颖,,谭玉林,易光辉,等.阿托伐他汀对THP-1巨噬细胞脂质蓄积及CD36表达的影响[J].中国动脉硬化杂志,2007,15(10):729-732
[97]Umetani N,Kanayama Y,Okamura M,et al.Lovastatin inhibits gene expression of type Ⅰ scavenger receptor in THP-1 human macrophages[J].Biochim Biophys Acta,1996,1303(3):199-206
[98]Fuhrman B,Koren L,Volkova N,et al.Atorvastatin therapy in hypercholesterolemic patients suppresses cellular uptake of oxidized-LDL by differentiating monocytes[J].Atherosclerosis,2002,164(1):179-185
[99]Febbraio M,Podrez EA,Smith JD et al.Targeted disruption of the class B scavenger receptor CD36protects against atherosclerosis lesion development in mice[J].J.Clin Invest,2000,105(8):1049-1056
[100]Febbraio M,Guy E,Silverstein RL.Stem cell transplantation reveals that absence of macrophage CD36 is protective against atherosclerosis[J].Arterioscler Thromb Vasc Biol,2004,24(12):2333-2338
[101]Kashiwagi H,Tomiyama Y,Kosugi Y.Identification of the molecular defects in a subject with type Ⅰ CD36deficiency[J].Blood,1994,83(12):3545-552
[102]Nakata A,Nakagawa Y,Nishida M,et al.CD36,a novel receptor for oxidized low-density lipoproteins,is highly expressed on lipid-laden macrophages in human atherosclerofic aorta[J].Arterioscler Thromb Vasc Biol,1999,19(5):1333-1339
[103]Lim HJ,Lee S,Lee KS,et al.PPARgamma activation induces CD36 expression and stimulates foam cell like changes in rVSMCs[J].Prostaglandins Other Lipid Mediat,2006,80(3-4):165-174
[104]Tontonoz P,Nagy L,Alvarez JG,et al.PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL[J].Cell,1998,93(2):241-252
[105]孙颖,常志文.B类清道夫受体CD36与动脉粥样硬化研究进展[J].心脏杂志2008,20(2):232-234
[106]莫中成,易光辉,陈欣,等.氧化低密度脂蛋白对THP-1巨噬细胞CD36表达的影响[J].医学研究生学报,2006,19(1):6-10
[107]张红明,何作云.干扰素γ对巨噬细胞CD36表达的影响[J].中国血液流变学杂志,2007,17(1):11-13
[108]陈三姝,徐敏,王芳等.小檗碱对2型糖尿病大鼠巨噬细胞ox-LDL、CD36利PPARγ的影响[J].中国糖尿病杂志,2008,16(7):434-437
[109]Qiu G,Hill JS.Atorvastatin inhibits ABCA1 expression and cholesterol efflux in THP-1 macrophages by an LXR-dependent pathway[J].J Cardiovasc Pharmacol,2008,51(4):388-395
[110]Llaverias G.Lacasa D,Vinals M,et al.Reduction of intracellular cholesterol accumulation in THP-1macrophages by a combination of rosiglitazone and atorvastatin[J].Biochem Pharmacol,2004,68(1):155-163
[111]Yan D,M(?)yr(?)np(?) MI,Wong J,et al.OSBP-related protein 8(ORP8) suppresses ABCA1 expression and cholesterol efflux from macrophages[J].J Biol Chem,2008,283(1):332-340
[112]Zhu Y,Liao H,Xie X,Yuan Y,et al.Oxidized LDL downregulates ATP-binding cassette transporter-1 in human vasctlar endothelial cells via inhibiting liver X receptor(LXR)[J].Cardiovasc Res,2005,68(3):425-432
[113]Tang CK,Yi GH,Yang JH,et al.Oxidized LDL upregulated ATP binding cassette transporter-1 in THP-1macrophages[J].Acta Pharmacol Sin,2004,25(5):581-586
[114]Sone H,Shimano H,Shu M,et al.Statins downregulate ATP-binding-cassette transporter A1 gene expression in macrophages[J].Biochem Biophys Res Commun,2004,316(3):790-794
[115]Srivastava N.ATP binding cassette transporter A1--key roles in celluar lipid transport and atherosclerosis[J].Mol Cell Biochem,2002,237:155-164
[116]Pfaffl MW.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Res,2001,29(9):2002-2007
[117]Clarke MC,Bennett MR,Cause or Consequence:What Does Macrophage Apoptosis Do in Atherosclerosis?[J].Arterioscler Thromb Vasc Biol,2009,29(2):153-155
[118]Seimon T,Tabas I.Mechanisms and Consequences of Macrophage Apoptosis in Atherosclerosis[J].J Lipid Res,2009,50 Suppl:S382-387
[2]Ross R.Atherosclerosis:an inflammatory disease[J].N Engl J Med,1999,340(2):115-126
[3]Duguid JB.Pathogenesis of atherosclerosis[J].Lancert,1949,2:925-927
[4]Libby P.Inflammation in atherosclerosis[J].Nature,2002,420:868-874
[5]Dargel R.The lipid infiltration theory of atherosclerosis[J].Z Med Lab Diagn,1989,30(5):251-255.
[6]Ross R,Glomset JA.The pathogenesis of atherosclerosis[J].N Engl J Med.1976,295(7):369-477,295(8):420-426
[7]McCully KS.Hmocysteine theory of arteriosclerosis by homocysteinemia[J].Atherosclerosis Rev,1983,11:157
[8]Steinberg D.Lipoprotein and atherosclerosis[J].Arteriosclerosis,1983,3:283-301
[9]Cooke JP,Oka RK.Atherogenesis and the arginine hypothesis[J].Curr Athero Rep,2001,3:252-259
[10]Pober JS,Cotran RC.Cytokines and endothelial cell biology[J].Physiol Rev,1990,70:427-451
[11]Benditt EP,Bendett JM.Evidence for a monoclonal origin of human atherosclerotic plaques[J].Proc Natl Acad Sci USA,1973,70(6):1753-1756
[12]DeFilippi C,WassermanS,RosanioS,et al.Cardiac troponin T and C-reactive protein for predicting prognosis,coronary atherosclerosis,and cardiomyopathy in patients undergoing long-term hemodialysis[J].JAMA,2003,290:353-359
[13]Tedgui A,Mallat Z.Cytokines in atherosclerosis:pathogenic and regulatory pathways[J].Physiol Rev,2006,86(2):515-581
[14]Viljoen A.New approaches in the diagnosis of atherosclerosis and treatment of cardiovascular disease[J].Recent Pat Cardiovasc Drug Discov.2008,3(2):84-91
[15]李玉洁,杨庆,朱晓新.抗炎-中药治疗动脉粥样硬化的有效途径[J].世界科学技术-中医药现代化,2007,9(2):22-28
[16]Sch(o|¨)nbeck U,Libby P.Inflammation,immunity,and HMG-CoA reductase inhibitors:statins as antiinflammatory agents?[J].Circulation,2004,109(21 Suppl 1):Ⅱ18-26
[17]Kleemann R,Zadelaar S,Kooistra T.Cytokines and atherosclerosis:a comprehensive review of studies in mice[J].Cardiovasc Res,2008,79(3):360-376
[18]Mestas J,Ley K.Monocyte-endothelial cell interactions in the development of atherosclerosis[J].Trends Cardiovasc Med,2008,18(6):228-232
[19]Li AC,Glass CK.The macrophage foam cell as a target for therapeutic intervention[J].Nat Med.2002,8(11):1235-1242
[20]Mangge H,Hubmann H,Pilz S,et al.Beyond cholesterol--inflammatory cytokines,the key mediators in atherosclerosis[J].Clin Chem Lab Med,2004,42(5):467-474
[21]Shibata N,Glass CK.Regulation of macrophage function in inflammation and atherosclerosis[J].J Lipid Res.2009,50 Suppl:S 277-281
[22]Schulte S,Sukhova GK,Libby P.Genetically programmed biases in Th1 and Th2 immune responses modulate atherogenesis[J].Am J Pathol.2008,172(6):1500-1508
[23]Gewaltig J,Kummer M,Koella C,et al.Requirements for CD8 T-cell migration into the human arterial wall[J].Hum Pathol,2008,39(12):1756-1762
[24]Kaartinen M,Penttila A,kovanen PT.Accumulation of activated mast cell in the shoulder region of human coronary atheroma,the predilection site of atheromatous rapture[J].Circulation,1994,90(4):1669-1678
[25]梁萍,孙雷,唐建武,等.细胞间粘附分子1、血管细胞粘附分子1和肿瘤坏死因子在人动脉粥样硬化病灶中的表达及意义[J].中国动脉硬化杂志,2004,12(4):427-429
[26]Coll B,Alonso-Villaverde C,Joven J.Monocyte chemoattractant protein-1 and atherosclerosis:is there room for an additional biomarker?[J].Clin Chim Acta,2007,383(1-2):21-29
[27]Apostolakis S,Vogiatzi K,Krambovitis E,et al.IL-1 cytokines in cardiovascular disease:diagnostic,prognostic and therapeutic implications[J].Cardiovasc Hematol Agents Med Chem,2008,6(2):150-158
[28]Rubic T,Lorenz RL.Downregulated CD36 and oxLDL uptake and stimulated ABCA1/G1 and cholesterol efflux as anti-atherosclerotic mechanisms of interleukin-10[J].Cardiovasc Res.2006,69(2):527-535
[29]Brewer HB Jr.The lipid-laden foam cell:an elusive target for therapeutic intervention[J].J Clin Invest.2000,105(6):703-705
[30]徐晓利,马涧泉.医学生物化学[P],北京:人民卫生出版社,第一版
[31]Henriksen T,Mahoney EM,Steinberg D.Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells:recognition by receptors for acetylated low density lipoproteins[J].Proc Natl Acad Sci U S A,1981,78(10):6499-6503
[32]Kita T,Kume N,Minami M,et al.Role of oxidized LDL in atherosclerosis[J].Ann N Y Acad Sci,2001,947:199-205
[33]张小梅,翟桂兰.炎症因子与动脉粥样硬化的研究进展[J].牡丹江医学院学报,200,29(1):91-93
[34]Portugal LR,Fernandes LR,Pietra Pedroso VS,et al.Influence of low-density lipoprotein(LDL)receptor on lipid composition,inflammation and parasitism during Toxoplasma gondii infection[J].Microbes Infect,2008,10(3):276-284
[35]Kunjathoor VV,Febbraio M,Podrez EA,et al.Scavenger receptors class A-Ⅰ/Ⅱ and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages[J].J Biol Chem,2002,277(51):49982-49988
[36]Small DM.Cellular mechanisms for lipid deposition in atherosclerosis[J].New EngI J Med 1977,297:873-877,924-929
[37]康莉,王家富,商战平.与动脉粥样硬化密切相关的清道夫受体[J].泰山医学院学报,2007,28(3):232-235
[38]Endemann G,Stanton L,Madden K,et al.CD36 is a receptor for oxidized low density lipoprotein [J].J Biol Chem,1993,268:11811-11816
[39]Febbraio M,Podrez EA,Smith JD,et al.Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice[J].J Clin Invest,2000,105:1049-1056
[40]Kashiwagi H,Tomiyama Y,Kosugi Y,et al.Identification of the molecular defects in a subject with type I CD36deficiency[J].Blood,1994,83:3545-3552.
[41]Nicholson A,Pearce SFA,Silverstein R.Oxidized LDL binds to CD36 on human monocyte -derived macrophages and transfected cell lines,Evidence implicating the lipid moiety of the lipoprotein as the binding site[J].Arterioscler Thromb,1995,15:269-275.
[42]BoullierA,Gillotte KL,Horkko S,et al.The binding of oxidized low density lipo protein to mouse CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein[J].J Biol Chem,2000,275:9163-9169
[43]Acton S,Attilio R,Landschultz K,et al.Identification of scavenger receptor SR-B1 as a high density lipoprotein receptor[J].Science,1996,271:518-520
[44]Ohashi R,Mu H,Wang X,et al.Reverse cholesterol transport and cholesterol efflux in atherosclerosis[J].QJM,2005,98(12):845-856
[45]李华波,全智华.ABCA1与PPARs在胆固醇流出中的作用及相关性[J].医学综述,2005,11(11):974-976
[46]Yokoyama S.ABCA1 and biogenesis of HDL[J].J Atheroscler Thromb,2006,13(1):1-15
[47]Oram JF,Lawn RM.ABCA1:the gatekeeper for eliminating excess tissue cholesterol[J].J.Lipid Res,2001,42(8):1173-1179.
[48]Singaraja RR,Bocher V,James ER,et al.Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and ApoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1[J].J Biol Chem,2001,276(36):33969-33979
[49]Van Eck M,Singaraja RR,Ye D,et al.Macrophage ATP-binding cassette transporter Al overexpression inhibits atherosclerotic lesion progression in low-density lipoprotein receptor knockout mice[J].Arterioscler Thromb Vasc Biol,2006,26(4):929-934.
[50]Bald(?)n A,Bojanic DD,Edwards PA.The ABCs of sterol transport[J].J Lipid Res,2009,50Suppl:S80-85
[51]Tavares V,Hirata MH,Hirata RD.Peroxisome proliferator-activated receptor gamma (PPARgamma):molecular study in glucose homeostasis,lipid metabolism and therapeutic approach[J].Arq Bras Endocrinol Metabol.2007,51(4):526-533
[52]Tontonoz P,Nagy L.Regulation of macrophage gene expression by peroxisome-proliferator -activated receptor gamma:implications for cardiovascular disease[J].Cardiovascular disease,1999,10(6):485-490
[53]Chawla A,Boisvert WA,Lee CH,et al.A PAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherosclerosis[J].Mol Cell,2001,7(1):161-171
[54]Argmann CA,Sawyez CG;McNeil CJ,et al.Activation of peroxisome proliferator-activated receptor gamma and retinoid X receptor results in net depletion of cellular cholesteryl esters in macrophages exposed to oxidized lipoproteins[J].Arterioscler Thromb Vasc Biol,2003,23(3):475-482
[55]Sato M,Kawata Y,Erami K,et al.LXR agonist increases the lymph HDL transport in rats by promoting reciprocally intestinal ABCA1 and apo A-I mRNA levels[J].Lipids,2008,43(2):125-131
[56]Chawla A,Boisvert WA,Lee CH,et al.A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis[J].Mol Cell,2001,7(1):161-171
[57]Joseph S B,Castrillo A,Laffitte B A,et al.Reciprocal regulation of inflammation on d lipid metabolism by liver X receptors[J].Nat Med,2003,9(2):213-221
[58]Choudhury RP,Lee JM,Greaves DR.Mechanisms of disease:macrophage-derived foam cells emerging as therapeutic targets in atherosclerosis[J].Nat Clin Pract Cardiovasc Med.2005,2(6):309-315
[59]Webb NR,Moore KJ.Macrophage-Derived Foam Cells in Atherosclerosis:Lessons from Murine Models and Implications for Therapy[J].Curr Drug Targets,2007,8(12):1249-1263
[60]郑杰,田瑞振,刘德山.动脉粥样硬化的中医致病论初探[J].云南中医学院学报,2008,31(2):28-29
[61]许迎春,王化良,丁晶.动脉粥样硬化从毒论治探讨[J].中医杂志,2004,45(6):405-407
[62]张卫娜,管昌益.动脉粥样硬化中医辩证论治研究进展[J].河北北方学院学报:医学版,2008,25(1):72-74
[63]袁中华,杨永宗,杨小毅.平滑肌源性泡沫细胞模型的制备[J].临床与实验病理学杂志,1998,14(1):70-73
[64]李全忠,杨永宗,易光辉,等.U937泡沫细胞模型的建立[J].中国动脉硬化杂志,1999,7(2):152-154
[65]白智峰,成蓓,毛晓波,等.瘦素对THP-1细胞泡沫化过程中ACAT-1表达的影响[J].天津医药,2004,32(10):593-595
[66]王东,丁华,徐红岩,等.洛伐他汀、普罗布考对小鼠巨噬细胞源性泡沫细胞内胆固醇含量的影响[J].中国药理学通报,2004,20(6):680-684
[67]谭健苗,杨永宗,袁中华.巨噬细胞源性与肌源性泡沫细胞的不同特性研究[J].广州医学院学报,2001,29(2):25-30
[68]朱莹,张文高,郑广娟,等.脂欣康胶囊对血管平滑肌细胞源性泡沫细胞CD36表达的影响[J].中国动脉便化杂志,2007,15(7):481-483
[69]Forst T,Wilhelm B,P f(u|¨)tzner A,et al.Investigation of the vascular and pleiotropic effects of atorvastatin and pioglitazone in a population at high cardiovascular risk[J].Diab Vasc Dis Res.2008,5(4):298-30
[70]PAS疗法预防动脉硬化http://cc.39.net/jzzn/0811/19/716415.html
[71]Kong WJ,Wei J,Abidi P,et al.Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins.Nature Medicine,2004,10(12):1344-1351
[72]林培政,杨开清.动脉粥样硬化中医湿热病机再认识[J].新中医.2006,38(3):5-6
[73]林培政,杨开清.清热化湿消瘀法对湿热证模型兔肝脏清道夫受体基因表达的影响[J].中药新药与临床药理,2007,18(1):15-17
[74]Chen FL,Yang ZH,Liu Y,et al.Berberine inhibits the expression of TNF-α,MCP-1,and IL-6 in Ac-LDL-stimulated macrophages through PPARγ pathway[J].Endocrine,2008,33(3):331-337
[75]Kong WJ,Wei J,Zuo ZY.Combination of simvastatin with berberine improves the lipid-lowering efficacy.Metabolism.2008,57(8):1029-1037
[76]Kim BH,Cho JY.Anti-inflammatory effect of honokiol is mediated by PI3K/Akt pathway suppression[J].Acta Phamacol Sin.2008,29(1):113-12
[77]Tsuchiya S,Kobayashi Y,Got Y,et al.Induction of maturation in cultured human monocytic leukemia cells by a phorbol diesterl[J].Cancer Res,1982,42:1530-1536
[78]Fu Y,Luo N,Lopes-Virella MF,et al.The adipocyte lipid binding protein(ALBP/aP2) gene facilitates foam cell formation ha human THP-1 macrophages[J].Atherosclerosis,2002,165(2):259-269
[79]贲长恩,李叔庚,王士平,等.组织化学[M].北京:人民卫生出版社,2001,第1版:169
[80]Fogelman AM,Shechter I,Seager J,et al.Malondialdehyde alteration of low density lipoproteins leads to cholesteryl ester accumulation in human monocyte-macrophages[J].Proc Natl Acad Sci USA,1980,77(4):2214-2218
[81]Palmgr(?)n JJ,T(?)yr(?)s A,Mauriala T,et al.Quantitative determination of cholesterol,sitosterol,andsitostanol in cultured Caco-2 cells by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry[J].J Chromatogr B Analyt Technol Biomed Life Sci,2005,821:144-152
[82]覃丽,秦旭平,王佐,等.普伐他汀对泡沫细胞内胆固醇酯的影响及与小凹蛋白-1的关系[J].生理学报,2006,58(1):47-52
[83]Jak(?)bisiak M,Bruno S,Skierski JS,et al.Cell cycle-specific effects of lovastatin[J].Proc Natl Acad Sci U S A,1991,88(9):3628-3632.
[84]N(u|¨)bel T,Dippold W,Kleinert H.Lovastatin inhibits Rho-regulated expression of E-selectin by TNFalpha and attenuates tumor cell adhesion.FASEB J,2004,18(1):140-142
[85]Zhong WB,Wang CY,Chang TC,et al.Lovastatin Induces Apoptosis of Anaplastic Thyroid Cancer Cells via Inhibition of Protein Geranylgeranylation and de Novo Protein Synthesis[J].Endocrinology,2003,44(9):3852-3859
[86]司徒镇强,吴军正.细胞培养[P],世界图书出版公司,1996年,第1版:134-135
[87]周程,胡思安,龚昭,等.胆囊癌对化疗药物敏感性的研究[J].中华现代外科学杂志,2005,2(20):1830-1832
[88]赵书刚,陈昕,雷开键.连朴饮对湿热夹瘀型动脉粥样硬化患者血脂及内皮功能影响的研究[J].陕西中医,2008,29(6):660-662
[89]Jantova S,Cipak L,Letasiova S.Berberine induces apoptosis through a mitochondrial/caspase pathway in human promonocytic U937 cells[J].Toxicol In Vitro,2007,21(1):25-31
[90]Ikai T,Akao Y,Nakagawa Y,et al.Magnolol-induced apoptosis is mediated via the intrinsic pathway with release of AIF from mitochondria in U937 cells[J].Biol Pharm Bull,2006,29(12):2498-2501
[91]薛芳,李世辉,潘崚.和厚朴酚对U937细胞的体外作用[J].中成约,2007,29(11):1582-1585
[92]Li H,Li X,Duan L,et al.Inhibition of lovastatin on proliferation and expression of proinflammatory cytokines in cultured human glomerular mesangial cells[J].Chin Med J(Engl),2003,116(9):1366-1369
[93]Moyer CF,Sajuthi D,Tulli H,et al.Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis[J].Am J Pathol,1991,138(4):951-960
[94]Mei CL,Chen ZJ,Liao YH,et al.Interleukin-10 inhibits the down-regulation of ATP binding cassette transporter Al by tumour necrosis factor-alpha in THP-1 macrophage-derived foam cells[J].Cell Biol Int,2007,31(12):1456-1461
[95]Xiao N,Yin M,Zhang L,et al.Tumor necrosis factor-alpha deficiency retards early fatty-streak lesion by influencing the expression of inflammatory factors in apoE-null mice[J].Mol Genet Metab,2009,96(4):239-244
[96]曾颖,,谭玉林,易光辉,等.阿托伐他汀对THP-1巨噬细胞脂质蓄积及CD36表达的影响[J].中国动脉硬化杂志,2007,15(10):729-732
[97]Umetani N,Kanayama Y,Okamura M,et al.Lovastatin inhibits gene expression of type Ⅰ scavenger receptor in THP-1 human macrophages[J].Biochim Biophys Acta,1996,1303(3):199-206
[98]Fuhrman B,Koren L,Volkova N,et al.Atorvastatin therapy in hypercholesterolemic patients suppresses cellular uptake of oxidized-LDL by differentiating monocytes[J].Atherosclerosis,2002,164(1):179-185
[99]Febbraio M,Podrez EA,Smith JD et al.Targeted disruption of the class B scavenger receptor CD36protects against atherosclerosis lesion development in mice[J].J.Clin Invest,2000,105(8):1049-1056
[100]Febbraio M,Guy E,Silverstein RL.Stem cell transplantation reveals that absence of macrophage CD36 is protective against atherosclerosis[J].Arterioscler Thromb Vasc Biol,2004,24(12):2333-2338
[101]Kashiwagi H,Tomiyama Y,Kosugi Y.Identification of the molecular defects in a subject with type Ⅰ CD36deficiency[J].Blood,1994,83(12):3545-552
[102]Nakata A,Nakagawa Y,Nishida M,et al.CD36,a novel receptor for oxidized low-density lipoproteins,is highly expressed on lipid-laden macrophages in human atherosclerofic aorta[J].Arterioscler Thromb Vasc Biol,1999,19(5):1333-1339
[103]Lim HJ,Lee S,Lee KS,et al.PPARgamma activation induces CD36 expression and stimulates foam cell like changes in rVSMCs[J].Prostaglandins Other Lipid Mediat,2006,80(3-4):165-174
[104]Tontonoz P,Nagy L,Alvarez JG,et al.PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL[J].Cell,1998,93(2):241-252
[105]孙颖,常志文.B类清道夫受体CD36与动脉粥样硬化研究进展[J].心脏杂志2008,20(2):232-234
[106]莫中成,易光辉,陈欣,等.氧化低密度脂蛋白对THP-1巨噬细胞CD36表达的影响[J].医学研究生学报,2006,19(1):6-10
[107]张红明,何作云.干扰素γ对巨噬细胞CD36表达的影响[J].中国血液流变学杂志,2007,17(1):11-13
[108]陈三姝,徐敏,王芳等.小檗碱对2型糖尿病大鼠巨噬细胞ox-LDL、CD36利PPARγ的影响[J].中国糖尿病杂志,2008,16(7):434-437
[109]Qiu G,Hill JS.Atorvastatin inhibits ABCA1 expression and cholesterol efflux in THP-1 macrophages by an LXR-dependent pathway[J].J Cardiovasc Pharmacol,2008,51(4):388-395
[110]Llaverias G.Lacasa D,Vinals M,et al.Reduction of intracellular cholesterol accumulation in THP-1macrophages by a combination of rosiglitazone and atorvastatin[J].Biochem Pharmacol,2004,68(1):155-163
[111]Yan D,M(?)yr(?)np(?) MI,Wong J,et al.OSBP-related protein 8(ORP8) suppresses ABCA1 expression and cholesterol efflux from macrophages[J].J Biol Chem,2008,283(1):332-340
[112]Zhu Y,Liao H,Xie X,Yuan Y,et al.Oxidized LDL downregulates ATP-binding cassette transporter-1 in human vasctlar endothelial cells via inhibiting liver X receptor(LXR)[J].Cardiovasc Res,2005,68(3):425-432
[113]Tang CK,Yi GH,Yang JH,et al.Oxidized LDL upregulated ATP binding cassette transporter-1 in THP-1macrophages[J].Acta Pharmacol Sin,2004,25(5):581-586
[114]Sone H,Shimano H,Shu M,et al.Statins downregulate ATP-binding-cassette transporter A1 gene expression in macrophages[J].Biochem Biophys Res Commun,2004,316(3):790-794
[115]Srivastava N.ATP binding cassette transporter A1--key roles in celluar lipid transport and atherosclerosis[J].Mol Cell Biochem,2002,237:155-164
[116]Pfaffl MW.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Res,2001,29(9):2002-2007
[117]Clarke MC,Bennett MR,Cause or Consequence:What Does Macrophage Apoptosis Do in Atherosclerosis?[J].Arterioscler Thromb Vasc Biol,2009,29(2):153-155
[118]Seimon T,Tabas I.Mechanisms and Consequences of Macrophage Apoptosis in Atherosclerosis[J].J Lipid Res,2009,50 Suppl:S382-387