【作者】 谢湘竹；

【导师】 赵水平；

【作者基本信息】 中南大学 ， 内科学， 2007， 博士

【摘要】 背景动脉粥样硬化性疾病是严重危害人类健康的主要疾病。肥胖是冠心病的独立危险因素，而脂肪细胞的功能改变是引起肥胖的核心因素，因此，研究脂肪细胞的功能对于肥胖、冠心病的防治具有重要意义。脂肪细胞的功能越来越受到重视，并得到重新认识，目前认为脂肪细胞功能改变与动脉粥样硬化密切相关。脂肪组织是体内最大的能量储存器官，脂肪细胞的主要功能是以甘油三酯的形式储存过多的能量；此外，脂肪细胞还具有重要的内分泌功能，可以分泌一系列脂肪因子，其中很多与动脉粥样硬化的发病机制有关。脂肪组织可通过分泌各种促炎性的脂肪因子，进而引起全身的慢性炎症反应状态。脂联素是由脂肪组织特异性分泌的一种炎症相关性蛋白质，目前认为脂联素是胰岛素抵抗、糖尿病和动脉粥样硬化的保护性因子。炎性细胞因子肿瘤坏死因子-α(TNFα)水平升高与动脉粥样硬化的严重性密切相关，脂肪细胞是体内内源性TNFα的重要来源之一。单核细胞趋化蛋白-1(MCP-1)是单核细胞强有力的趋化因子，也是动脉粥样硬化反应的促发信号，脂肪细胞可在多种刺激物的诱导下产生MCP-1。脂多糖(LPS)是一种公认的炎症刺激物。低密度脂蛋白(LDL)经过氧化修饰后具有致动脉粥样硬化作用，而氧化型低密度脂蛋白(oxLDL)除了参与形成泡沫细胞外，还在局部慢性炎症反应中起一定的作用。与oxLDL相反，高密度脂蛋白(HDL)被认为具有抗动脉粥样硬化及心血管保护作用，但其具体机制尚不十分清楚。研究已经证实，HDL参与调节脂肪细胞内脂质代谢，但HDL对脂肪细胞炎症反应的调节尚未见报导。有研究提示，HDL的主要载脂蛋白之一载脂蛋白A-I(apoA-I)和apoA-I模拟肽是有效的抗炎剂并可能对动脉粥样硬化具有临床治疗潜能，但是apoA-I模拟肽对脂肪细胞是否也具有抗炎作用尚不为人所知。过氧化物酶体增殖物激活受体γ(PPARγ)是主要表达于脂肪组织的一种核受体。核因子κB(NF-κB)调控多种细胞因子的转录，参与了动脉粥样硬化发生发展的多个步骤，在动脉粥样硬化中起重要作用。CCAAT／增强子结合蛋白(C／EBPs)是NF-κB以外的另一类重要的细胞核转录调控因子。目前的研究显示，PPARγ、NF-κB或C／EBPs均不同程度地参与了某些细胞因子的转录调控，其可能也部分地参与调控脂肪细胞脂肪因子的转录与分泌，但具体作用及信号通道仍须进一步的研究。目的观察HDL和apoA-I模拟肽L-4F对炎症状态下3T3-L1脂肪细胞脂联素、TNFα及MCP-1的上清液浓度及其mRNA表达的影响，并探讨其可能的作用机制。方法3T3-L1脂肪细胞促分化成熟后，分成三组：(1)LPS刺激组：脂多糖(LPS)刺激脂肪细胞处于炎症状态，给予不同浓度的HDL(10-100μg／ml)和L-4F(1-50μg／ml)干预，收集细胞和培养上清液，测定细胞上清液脂联素浓度，脂肪细胞脂联素和PPARγmRNA表达水平，及NF-κB活性。(2)oxLDL刺激1小时组：oxLDL刺激脂肪细胞1小时，给予不同浓度的HDL(10-100μg／ml)和阿托伐他汀(0.1-10μM)，及H-89(10μM)+HDL(100μg／ml)干预，收集细胞，测定脂肪细胞TNFα和PPARγmRNA表达水平，IκB蛋白浓度及NF-κB活性。(3)oxLDL刺激不同时间组：oxLDL分别刺激脂肪细胞1、6、12小时，给予不同浓度的HDL(10-100μg／ml)、L-4F(1-50μg／ml)，H-89(10μM)及H-89+HDL(100μg／ml)或H-89(10μM)+L-4F(50μg／ml)干预，收集细胞，测定脂肪细胞MCP-1上清液中的浓度和mRNA表达水平，以及脂肪细胞核因子C／EBPα、β的蛋白表达水平；改良Boyden小室法检测不同干预组上清液对人外周血单核细胞趋化活性的影响。结果1．LPS刺激分化成熟的3T3-L1脂肪细胞使其脂联素分泌和mRNA表达水平分别下降44％和60％(P＜0.05)。2．HDL和L-4F浓度依赖性上调炎症状态下3T3-L1脂肪细胞脂联素的分泌及表达。10、50和100μg／ml HDL分别使脂联素水平升高1倍、1.9倍和3.2倍(P＜0.05)，1、10、50μg／ml L-4F分别使脂联素水平升高1.2倍、2.9倍和3.4倍(P＜0.05)。与LPS刺激组(0.36±0.05)比较，10、50、100μg／ml HDL分别使脂联素mRNA表达升高至0.72±0.13、1.05±0.15、1.51±0.11，而1、10、50μg／m L-4F分别使脂联素mRNA表达升高至0.79±0.12、1.40±0.18、1.58±0.10(P均＜0.05)。3．PPAR7在分化成熟的3T3-L1脂肪细胞有较高水平的表达，与LPS刺激组比较，PPARγ表达在HDL和L-4F干预后明显升高(2.85±0.69 vs 0.38±0.19，2.92±0.96 vs 0.38±0.19；P＜0.05)，而NF-κB活性在HDL和L-4F干预后明显降低(0.48±0.09 vs 1.93±0.59，0.43±0.08 vs 1.93±0.59；P＜0.05)。4．OxLDL(50μg／ml)刺激1小时使3T3-L1脂肪细胞TNFα分泌(2.98±1.10 pg／ml Vs 8.93±3.26 pg／ml，P＜0.001)、mRNA表达(0.218±0.078 vs 0.633±0.157，P＜0.001)及NF-κB活性(1.54±0.44 vs4.08±0.87，P＜0.05)明显增强。5．阿托伐他汀浓度依赖性降低TNFα分泌及mRNA表达，抑制NF-κB活化，并增强PPARγmRNA表达。10μM阿托伐他汀使脂肪细胞oxLDL诱导的TNFαmRNA表达降低56.5％，NF-κB活性减少41.2％，而使PPARγmRNA表达增加至2.83倍。HDL也呈浓度依赖性抑制TNFα分泌及mRNA表达，NF-κB活化和IκB降解。与oxLDL刺激组比较，100μg／ml HDL使TNFαmRNA表达降低64.5％，NF-κB活性减少49％，并明显增加IκB蛋白水平，但对PPARγmRNA表达无影响。HDL的这些抗炎效应能被蛋白激酶A(PKA)抑制剂H-89部分抑制。6．OxLDL(50μg／ml)刺激12小时使脂肪细胞上清液中MCP-1的浓度和表达增加至2.13倍，并使得诱导的单核细胞移动距离明显增力口(69.88±8.19μm vs 136.75±8.03μm，P＜0.001)。7．L-4F和HDL均以浓度依赖的方式减少脂肪细胞MCP-1的表达分泌，降低单核细胞趋化活性。50μg／ml L-4F和100μg／ml HDL分别使MCP-1的表达分泌降低91±6％和79±7％(P＜0.001)。PKA抑制剂H-89(10μM)干预oxLDL刺激的脂肪细胞后MCP-1m RNA的表达也显著减少71±7％(P＜0.001)，但是，在100μg／ml HDL或50μg／ml L-4F作用的基础上，H-89(10μM)的孵育并未使得MCP-1mRNA的表达进一步降低(P＞0.05)。8．50μg／ml oxLDL刺激对脂肪细胞C／EBPα的表达无明显影响，但增加C／EBPβ蛋白量，该作用呈时间依赖性；H-89、L-4F和HDL干预均降低脂肪细胞C／EBPβ的蛋白表达量。结论1．LPS和oxLDL均能诱导分化成熟的3T3-L1脂肪细胞产生炎症状态，使其表达和分泌脂联素水平下降，使TNFα及MCP-1的表达和分泌增强。2．HDL和L-4F能上调LPS刺激炎症状态下脂肪细胞脂联素的表达和分泌，PPARγ和NF-κB可能参与了HDL和L-4F上调脂联素的效应过程。3．阿托伐他汀通过NF-κB和PPARγ途径抑制oxLDL诱导的3T3-L1脂肪细胞TNFα分泌及mRNA表达。4．HDL能抑制oxLDL诱导的3T3-L1脂肪细胞TNFα分泌和mRNA表达，其抗炎作用强度与阿托伐他汀相似。PKA-IκB-NF-κB信号通路是其中作用途径之一，该效应不需要HDL与oxLDL的直接接触作用。5．OxLDL时间依赖性地诱导脂肪细胞C／EBPβ的蛋白表达。6．L-4F和HDL均以浓度依赖的方式对抗oxLDL对MCP-1的影响，cAMP／PKA-C／EBPβ信号通道可能是L-4F和HDL的作用途径之一。更多还原

【Abstract】 BackgroundAtherosclerosis diseases are the leading cause threatening humanhealth. Obesity is an independent risk factor for coronary heart disease(CHD). Since dysfunction of adipocytes plays a key role for obesity,studying function of adipocytes is of great significance for the preventionand treatment of obesity and CHD. Adipocyte functions have been furtherexplored and recognized recently. Dysfunctional adipocytes have beenthought to be in close relation with atherosclerosis.Adipose tissue is the largest energy reservoir in the body. It storesexcess energy in form of triglyceride. Moreover, adipose tissue showsactive endocrine function that secrets various adipokines, many of whichmight play significant roles in atherogenesis. Adipose tissue may be asignificant contributor to increase systemic chronic inflammation throughsecreting a number of pro-inflammatory adipokines. Adiponectin, anadipose-specific secreted and inflammation-related protein, is a protectivefactor for insulin-resistance, diabetes and atherosclerosis. The elevatedlevel of tumor necrosis factor-α(TNFα), an inflammatory cytokine, isclosely associated with the severity of atherosclerosis. The adipose tissueis a significant source of endogenous TNFαproduction. Monocytechemoattractant protein-1 (MCP-1) is a potent chemotactic factor formonocytes and may serve as a signal that triggers inflammation ofatherosclerosis. Adipocytes may be induced to produce MCP-1 withseveral types of stimulators.Lipopolysaccharide (LPS) is an acknowledged inducer ofinflammation. Low-density lipoproteins (LDLs) are thought to becomeatherogenic after undergoing oxidative modifications. In addition to theirrole in foam cell formation, oxidized low-density lipoproteins (oxLDL) may participate in local chronic inflammatory response. High densitylipoproteins (HDLs), in contrast to LDLs, are antiatherogenic andcardioprotective, but the detailed molecular mechanisms remain poorlyunderstood. The effects that HDL regulates the intracellular lipidsmetabolism in adipocytes have been confirmed. However, the effects ofHDL on regulating adipocytes inflammation have not been investigatedso far. Apolipoprotein A-I (apoA-I), the main protein of HDL and apoA-Imimetic peptide are potent anti-inflammatory agents that may havetherapeutic potential. But whether apoA-I mimetic peptide showsanti-inflammatory effects in adipocytes is not understood.Peroxisome proliferator-activated receptorγ(PPARγ) is a nuclearreceptor which mainly be expressed in adipose tissue. Nuclear factor-κB(NF-κB) is a crucial transcription factor, which controls the transcriptionof many genes with an established role in atherosclerosis.CCAAT/enhancer binding proteins (C/EBPs) is another important kind ofnuclear transcription factor. It was shown that PPARγ、NF-κB andC/EBPs all participate in the transcription control of some cytokines. Andthey may have actions on the regulation of transcription and secretion ofadipokines at least in part, which need to be further studied.ObjectiveThe aim of this study was to evaluate the effect of HDL and L-4F,an apoA-I mimetic peptide, on the secretion and expression ofadiponectin, TNFαand MCP-1 in fully differentiated 3T3-L1adipocytes with inflammatory status, and to elucidate the possiblemechanisms,MethodsFully differentiated 3T3-L1 adipocytes were divided into threegroups: (1)LPS stimulated group: Fully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentrations of HDL(10-100μg/ml) or L-4F (1-50μg/ml) with 100μg/L LPS (n=3). Evaluatethe levels of adiponectin in supernatant, the mRNA expression ofadiponectin and PPARγ, and the activity of NF-κB. (2) OxLDLstimulated group (for 1h): Fully differentiated 3T3-L1 adipocytes wereincubated in the medium containing various concentrations of HDL (10-100μg/ml) or atorvastatin (0.1-10μM) with oxLDL (50μg/ml)stimulated for 1 hour, with/without H-89 (10μM) preincubated. TNFαand PPARy mRNA expression, NF-κB activity and IκB protein level wereevaluated. (3) OxLDL stimulated group (for 1h、6h、12h): Fullydifferentiated 3T3-L1 adipocytes were incubated in the mediumcontaining various concentrations of HDL (10-100μg/ml) or L-4F (10-100μg/ml) with oxLDL (50μg/ml) stimulated for 1h, 6h or 12h,with/without H-89 (10μM) preincubated. The concentrations of MCP-1in supernatant, the mRNA expression of MCP-1, the levels of C/EBPαand C/EBPβwere evaluated. The monocyte chemotaxis assay wasperformed by micropore filter method using a modified Boyden chamber.Results1. The secretion and mRNA expression of adiponectin decreased by44％and 60％after LPS stimulation in full differentiated 3T3-L1adipocytes (P＜0.05).2. HDL and L-4F dose-dependently increased adiponectin secretionand mRNA expression. Compared with LPS stimulation, treatment withHDL at 10, 50, 100μg/ml significantly enhanced adiponectin secretionabout 1, 1.9, 3.2-fold respectively (P＜0.05), and treatment with apoA-Imimetic peptide at 1, 10, 50μg/ml significantly enhanced adiponectinsecretion about 1.2, 2.9, 3.4-fold respectively (P＜0.05). Compared withLPS stimulated group (0.36±0.05), adiponectin mRNA expression increased to 0.72±0.13, 1.05±0.15 and 1.51±0.11 respectively by 10, 50and 100μg/ml HDL and to 0.79±0.12, 1.40±0.18 and 1.58±0.10respectively by 1, 10 and 50μg/ml L-4F (all P＜0.05).3. PPARγpresents high level expression in fully differentiated3T3-L1 adipocytes. Compared with LPS stimulated group, HDL andL-4F significant enhanced PPARγmRNA expression (2.85±0.69 vs0.38±0.19, 2.92±0.96 vs 0.38±0.19; P＜0.05) and the activity of NF-κBdecreased significantly after using HDL and L-4F (0.48±0.09 vs1.93±0.59, 0.43±0.08 vs 1.93±0.59; P＜0.05).4. OxLDL stimulation for 1 hour induced a significant enhancementof TNFαsecretion (2.98±1.10 pg/ml vs 8.93±3.26 pg/ml, P＜0.001),TNFαmRNA expression (0.218±0.078 vs 0.633±0.157, P＜0.001) andNF-κB activity (1.54±0.44 vs 4.08±0.87, P＜0.05) in 3T3-L1 adipocytes.5. Atorvastatin reduced TNFαsecretion and mRNA expression,inhibited NF-κB activation, and enhanced PPARγmRNA expression in adose-dependent manner. Treatment with atorvastatin at 10μMsignificantly suppressed TNFαmRNA expression by 56.5％, inhibitedNF-κB activation by 41.2％, and increased PPARy mRNA expression toabout 2.83-fold, as compared with oxLDL stimulated group. HDLdose-independently inhibited TNFαsecretion and mRNA expression,NF-κB activation and IκB degradation. Treatment with HDL at 100μg/mlsuppressed TNFαmRNA expression by 64.5％, inhibited NF-κBactivation by 49％, and stabilized IκB significantly, as compared withoxLDL stimulated group, but had no effect on PPARγmRNA expression.The anti-inflammatory effect of HDL could be partially abolished byPKA inhibitor H-89.6. OxLDL stimulation for 12 hours induced a significant increase ofMCP-1 secretion and expression to 2.13-fold in 3T3-L1 adipocytes. And the mean of migration distances of human peripheral blood monocyteinduced by oxLDL was significantly longer than that of the randommigration group (69.88±8.19μm vs 136.75±8.03μm, P＜0.001).7. L-4F and HDL decreased MCP-1 secretion and expression in adose-dependent manner as well as reduced the chemotactic activity ofmonocyte. 50μg/ml L-4F and 100μg/ml HDL decreased MCP-1secretion and expression by 91±6％and 79±7％respectively (P＜0.001).PKA inhibitor H-89 (10μM) significantly reduced oxLDL-inducedMCP-1 expression by 71±7％(P＜0.001), but no further decreasingoccurred when H-89 (10μM) adding with the existing of L-4F(50μg/ml)or HDL(100μg/ml) (P＞0.05).8. OxLDL (50μg/ml) stimulation had no marked influence onC/EBPαexpression, but increased C/EBPβlevel in a time-dependentmanner. H-89, HDL and L-4F all attenuated oxLDL-induced C/EBPβlevel in 3T3-L1 adipocytes.Conclusions1. Full differentiated 3T3-L1 adipocytes would be induced to be ininflammatory status after LPS or oxLDL stimulation, which showed thatadiponectin secretion and expression decreased and TNF a and MCP-1secretion and expression increased.2. HDL and L-4F could upregulate the secretion and mRNAexpression of adiponectin in 3T3-L1 adipocytes in inflammatory status.PPARγand NF-κB pathway may participate in the process.3. Atorvastatin inhibited TNFαsecretion and mRNA expression inoxLDL-stimulated 3T3-L1 adipocytes by NF-κB and PPAR7 signalingpathways.4. HDL could suppress TNFαsecretion and mRNA expression inoxLDL-stimulated 3T3-L1 adipocytes by PKA-IκBα-NF-κB signaling pathway, without any contact between HDL and oxLDL. The intensity ofanti-inflammatory effect of HDL was similar to that of atorvastatin.5. OxLDL induced C/EBPβexpression in a time-dependentmanner.6. L-4F and HDL dose-dependently counterbalanced the influence ofoxLDL on MCP-1. The cAMP/PICA-C/EBPβsignaling pathway mayparticipate in the process.更多还原