【作者】 王术玲；

【导师】 王培训；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2009， 博士

【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1×10⁶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 C₁₈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（N₂）流速30 arbitrary units;辅助气（N₂）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IC₅₀,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。更多还原

【Abstract】 ObjectiveThere 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.Methods1、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 model2、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-MS3、Enzyme-linked immunosorbent assayTHP-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 analysisTHP-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-PCRTotal 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.Results1、Establishment and identification of foam cell model（1） THP-1 cells in log phase growth were diluted to 10⁶cells/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 stainingFoam 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 estersLiquid 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 C₁₈ 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.ConclusionThe 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.更多还原