【作者】 杨柳；

【导师】 赵水平；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 背景大量研究显示,高密度脂蛋白胆固醇(HDL-C)水平和冠心病的发病率呈负相关。提高HDL-C水平可延缓或预防动脉粥样硬化的发生。HDL-C抗动脉粥样硬化主要与HDL-C在胆固醇逆向转运(RCT)中发挥的关键作用有关。随着生活环境和膳食结构的改变,肥胖病发生率在全球范围内呈迅速上升趋势,肥胖是引起HDL-C降低的常见疾病。他汀类和贝特类药物是目前临床上常见的调脂药,研究表明他汀类和贝特类药物能不同程度的升高HDL-C水平和改善RCT。载脂蛋白M(apoM)是新近发现的一种主要与HDL-C相关的载脂蛋白,对HDL-C的代谢起重要作用,它主要通过对前p-HDL生成的调节来影响人体RCT过程。apoM在HDL-C生成和RCT过程中不可替代。肝细胞核因子-1α(HNF-1α),叉头转录因子A2 (FoxA2)和肝X受体a(LXRa)是调节apoM表达的三个关键核受体。其中,前两者上调apoM的表达,后者下调apoM的表达。目前尚不清楚他汀类和贝特类药物升高HDL-C和改善RCT是否与apoM有关；也不知肥胖状态下低HDL-C血症是否与apoM有关。研究证实肥胖是慢性炎症性疾病,且常伴随低脂联素血症,目前尚无肥胖状态下apoM与炎症因子和脂联素的关系研究。目的观察肥胖小鼠载脂蛋白M的改变,并探讨肥胖状态下载脂蛋白M与脂联素的关系,并揭示其机制;观察肥胖患者载脂蛋白M水平并探讨其与炎症因子的关系；探讨辛伐他汀和非诺贝特及两者联合对载脂蛋白M表达的影响及其机制。方法一、肥胖小鼠载脂蛋白M的改变及与脂联素关系的探讨1.动物实验32只3周龄雄性C57BL/6 N小鼠,随机分为4组(n=8／组)：(1)对照组：普通饮食,喂养12周；(2)肥胖组：高脂饲料,喂养12周；(3)肥胖干预组：高脂饲料,喂养12周后脂联素干预7天。(4)普通干预组：普通饮食,喂养12周后脂联素干预7天。分别于实验第0周、第12周及脂联素干预后测定体重,血浆脂联素,血糖,血浆胰岛素。实验结束时,取小鼠内脏脂肪并称取重量,取小鼠肝脏组织标本,采用实时定量逆转录聚合酶链反应(实时RT-PCR)和免疫印迹(Western Blot)检测小鼠肝脏载脂蛋白M、FoxA2的mRNA和蛋白的表达。2.细胞实验高浓度胰岛素诱导HepG2细胞构建胰岛素抵抗(IR)肝细胞模型,采用实时RT-PCR和Western Blot检测胰岛素抵抗状态下肝细胞的apoM基因和蛋白表达变化。脂联素分别干预胰岛素抵抗HepG2细胞、HepG2细胞24h,采用实时RT-PCR和Western Blot检测各组细胞载脂蛋白M、FoxA2的mRNA和蛋白的表达。二、肥胖患者载脂蛋白M水平及其与炎症因子的关系研究入选58例健康者和40例肥胖患者作为研究对象,常规测量血压、体重、身高,计算体重指数,抽取空腹静脉血检测其血糖、血脂、空腹胰岛素、血浆apoM、白细胞介素-6(IL-6)、高敏C反应蛋白(hs-CRP)、肿瘤坏死因子α(TNF-α)、血浆脂联素,并检测肱动脉内皮舒张功能(FMD)。三、辛伐他汀和非诺贝特对载脂蛋白M表达的影响及调控机制1.动物实验32只8周龄健康雄性C57BL/6N小鼠,随机分为四组(n=8／组)：(1)对照组：正常普通饮食喂养;(2)他汀组：辛伐他汀(10mg/kg/天)干预4周；(3)贝特组：非诺贝特(100mg/kg/天)干预4周；(4)联合组：辛伐他汀(10mg/kg/天)和非诺贝特(100mg/kg/天)干预4周；分别在实验前(0周)、实验4周测定空腹血脂水平。4周后取动物肝脏组织标本,采用实时RT-PCR和免疫印迹(Western Blot)测定肝脏apoM的基因和蛋白表达,同时采用实时RT-PCR和Western Blot测定肝脏HNF-1α和LXRa的基因和蛋白表达。2.细胞实验分别以不同浓度的辛伐他汀(0、1、5、10、25μmol/L)和非诺贝特(0、50、100mmol/L)及辛伐他汀(5.0μmol/L)+非诺贝特(50mmol/L)、辛伐他汀(25μmol /L)+HNF-1α抑制剂、辛伐他汀(25μmol/L)+LXRα激动剂、非诺贝特(100mmol /L)+HNF-1α抑制剂、非诺贝特(100mmol/L)+LXRa抑制剂干预HepG2细胞24h。提取各组细胞总RNA和蛋白质,分别采用实时RT-PCR和Western Blot检测apoM的mRNA和蛋白的表达。采用实时RT-PCR和Western Blot检测HNF-1α和LXRα的基因和蛋白表达。结果一、肥胖小鼠载脂蛋白M的改变及与脂联素关系的探讨1.与对照组比较,肥胖组、肥胖干预组小鼠体重显著高于对照组(P<0.05),肥胖组的内脏脂肪重量、空腹血糖、空腹胰岛素、胰岛素抵抗指数(HOMA-IR)显著高于对照组(P均<0.05)；与对照组比较,肥胖组血浆脂联素水平显著下降(P<0.05);肥胖干预组血糖、血浆胰岛素、胰岛素抵抗指数显著低于肥胖组(P均<0.05),肥胖干预组血浆脂联素水平显著高于肥胖组(P<0.05),肥胖干预组内脏脂肪重量与肥胖组比较无统计学差异。2.与对照组比较,肥胖组肝脏载脂蛋白M的mRNA和蛋白表达显著下降(P<0.05)。3.与对照组比较,肥胖组肝脏FoxA2的mRNA和蛋白表达显著下降(P<0.05)。4.脂联素干预后的肥胖干预组小鼠载脂蛋白M的mRNA和蛋白表达显著高于肥胖组(P<0.05),脂联素作用于普通干预组小鼠载脂蛋白M的mRNA和蛋白表达与对照组比较无统计学差异(P>0.05)。5.脂联素干预后的肥胖干预组小鼠FoxA2的mRNA和蛋白表达,血浆脂联素水平显著高于肥胖组(P<0.05)；空腹血糖、空腹胰岛素、HOMA-IR均显著低于肥胖组(P<0.05)。脂联素作用于普通干预组小鼠FoxA2的mRNA和蛋白表达与对照组比较无统计学差异(P>0.05)。6.胰岛素抵抗HepG2细胞较HepG2细胞载脂蛋白M、FoxA2的mRNA和蛋白表达均显著下降(P均<0.05)。7.脂联素干预后,胰岛素抵抗HepG2细胞载脂蛋白M、FoxA2的mRNA和蛋白表达显著增加(P均<0.05)。8.脂联素干预后HepG2细胞载脂蛋白M、FoxA2的mRNA和蛋白表达无显著变化(P均>0.05)。二、肥胖患者载脂蛋白M水平及其与炎症因子的关系研究1.与对照组比较,肥胖患者的血浆apoM、HDL-C、血浆脂联素、FMD水平显著降低(P<0.05),空腹胰岛素、IL-6、TNF-α、hs-CRP水平升高(P<0.05)。2.肥胖患者血浆apoM与HDL-C显著正相关(P<0.05),血浆apoM与BMI、胰岛素、胰岛素抵抗指数(HOMA-IR)、IL-6、TNF-α、CRP水平显著负相关(P均<0.05),血浆apoM与血浆脂联素、LDL-C、TC、TG、血压、血糖、FMD无显著相关(P均>0.05)。对照组apoM与HDL-C显著正相关(P<0.05),血浆apoM与BMI显著负相关(P<0.05),血浆apoM与胰岛素、胰岛素抵抗指数(HOMA-IR)、IL-6、TNF-α、CRP血浆脂联素、LDL-C、TC、TG、血压、血糖、FMD均无显著相关(P均>0.05)。三、辛伐他汀和非诺贝特对载脂蛋白M表达的影响及调控机制1.与对照组比较,药物治疗组HDL-C均显著升高,联合组升高程度高于他汀组和贝特组(P<0.05)；药物治疗组apoM的基因和蛋白均显著升高,联合组升高程度高于他汀组和贝特组(P<0.05)：细胞实验显示,辛伐他汀和非诺贝特均呈剂量依赖性上调apoM的表达,联合组升高程度高于他汀组和贝特组(P<0.05)。辛伐他汀+HNF-1α抑制剂组、辛伐他汀+LXRα激动剂组apoM的基因和蛋白水平均显著低于同浓度辛伐他汀组(P<0.05)；非诺贝特+HNF-1α抑制剂组apoM的基因和蛋白水平均显著低于同浓度非诺贝特组(P<0.05),非诺贝特+LXRα抑制剂组apoM的基因和蛋白水平均显著高于同浓度非诺贝特组(P<0.05)。2.与对照组比较,药物治疗组HNF-1α的基因和蛋白均显著升高,联合组升高程度高于他汀组和贝特组(P<0.05)；细胞实验显示,辛伐他汀和非诺贝特均呈剂量依赖性上调HNF-1α的基因和蛋白表达,联合组升高程度高于他汀组和贝特组(P<0.05)。3.他汀组LXRα的基因和蛋白表达显著降低(P<0.05),贝特组LXRα的基因和蛋白表达显著升高(P<0.05),联合组LXRα的基因和蛋白表达与对照组比较无显著差异(P<0.05)；细胞实验显示,辛伐他汀呈剂量依赖性下调LXRα的基因和蛋白表达(P<0.05),非诺贝特呈剂量依赖性上调LXRα的基因和蛋白表达(P<0.05),联合组LXRα的基因和蛋白表达与对照组无显著差异(P<0.05)。结论1.肥胖小鼠载脂蛋白M、FOXA2基因和蛋白表达明显下降,载脂蛋白M下降可能与FOXA2表达降低有关。2.肥胖小鼠血浆脂联素水平显著下降,内脏脂肪重量、空腹血糖、空腹胰岛素、HOMA-IR显著增加。3.脂联素可上调肥胖小鼠载脂蛋白M表达,其机制可能与脂联素改善胰岛素抵抗,进而上调FOXA2表达有关。4.脂联素可上调胰岛素抵抗HepG2细胞载脂蛋白M表达,却对HepG2细胞载脂蛋白M表达无影响,这提示脂联素对载脂蛋白M的调控可能是间接通过影响胰岛素抵抗来实现的。5.肥胖患者血浆apoM显著降低,apoM水平降低可能是肥胖患者HDL-C下降的潜在机制之一。6.肥胖患者apoM水平与hs-CRP、TNF-α、IL-6水平密切相关,apoM可能受到这些炎症因子的调控,其机制可能与炎症因子促进胰岛素抵抗有关。7.肥胖患者apoM水平与FMD无显著相关,提示apoM还不能作为早期动脉粥样硬化预测因子。8.辛伐他汀和非诺贝特升高HDL-C可能与上调apoM表达有关,两者联合疗效更显著。9.辛伐他汀和非诺贝特上调apoM表达的机制与两者调控HNF-1α和LXRa有关,两药具有互补性：前者上调HNF-1α抑制LXRa,后者上调HNF-1α和LXRa,这可解释联合用药比单药更能显著升高HDL-C和apoM。更多还原

【Abstract】 BackgroundHigh-density lipoprotein cholesterol (HDL-C) level is negatively correlate with the incidence of coronary heart disease.Increased HDL-C levels can delay or prevent the occurrence of atherosclerosis.HDL-C against atherosclerosis is primarily related to its role in reverse cholesterol transport (RCT). In recent years, the worldwide epidemic of obesity has become a public health problem of global concern.Obesity is accompnied with lower HDL-C.Statins and fibrates are widely used in clinical lipid-lowering drugs which can elevate HDL-C levels and improve the RCT.Apolipoprotein M (apoM) is a newly discovered apolipoprotein mainly associated with HDL-C,which play an important role in the metabolism of HDL-C.It affects the RCT process mainly through the regulation of pre-β-HDL. Hepatocyte nuclear factor-1α(HNF-1α), forkhead box A2(FOXA2) and liver X receptor-α(LXRα) are three key nuclear receptors regulating apoM expression.The first two increase the expression of apoM, the latter reduce apoM expression.It is unclear whether the effect of statins and fibrates on HDL-C and RCT related to apoM or not;It is not clear whether apoM changes in the state of obesity.Obesity is a chronic inflammatory disease, In addition, obesity often accompanied with hypoadiponectinemia.However, there is no reports about the relationship between inflammatory factors, adipone-ctin and apolipoprotein M.ObjectiveTo observe the changes of apolipoprotein M in obese mice and to explore the relationship with adiponectin and its mechanism;To observe the level of apoM in obesity and explore the relationship between apoM and inflammatory factor, vascular endohelial function.To examine the effects and mechanism of simvastatin, fenofibrate and combination of the two drugs on the expression of apolipoprotein M.Methods1.changes of apolipoprotein M in obese mice and the relationship with adiponectin1) Animal experimentsThree-week-old male C57BL/6N mice (n=32) were randomly divided into 4 groups :control group:normal diet; obese group:high-fat feed, fed for 12 weeks; obesity-intervention group:high-fat feed, fed for 12 weeks and then received an intraperitoneal injection of adiponectin for 7 days.General intervention group:normal diet,received an intraperitoneal injection of adiponectin for 7 days. Body weight, adiponectin, glucose, insulin levels were measured in the 0 week, the 12 week and after the intervention of adiponectin, respectively. Apolipoprotein M, forkhead box A2(FoxA2)mRNA and protein expression were measured by real-time PCR(real-time reverse transcript PCR)and Western blot at end of the experiment.2) cell experimentsHigh concentrations of insulin-induced insulin resistance in HepG2 cells to buid cell model, insulin-resistance HepG2 cells and HepG2 cells were interfered with adiponectin for 24hrs, Apolipoprotein M, FoxA2-mRNA and protein expression were measured in all cell groups.2.Apolipoprotein M levels and its relationship with inflammatory factors in obesityObese patients(n=40)and healthy volunteers(n=58) were included. blood pressure, weight, height were measured.After plasma samples obtained,plasma apoM level was measured by an ELISA method. Besides, fasting plasma lipids, glucose, insulin, Interleukin-6 (IL-6), creactive protein (CRP), tumor necrosis factor-a (TNF-a), adiponectin were measured. Body mass index and insulin resistance index were calculated.Furthermore, brachial artery endothelial function was detected.3.Effects and mechanism of simvastatin and fenofibrate on the expression of apolipoprotein M1) Animal experimentsHealthy male C57BL/6N mice (n=32) were randomly divided into four groups (n= 8 each group):(1)control group, with no special treat- ment; (2)statin group:with simvastatin (10mg/kg/day) for 4 weeks; (3)brate group:with fenofibrate (1 OOmg/kg/day) for 4 weeks; (4) combin-ation group:with simvastatin (10mg/kg/day) and fenofibrate (100mg/kg /day) for 4 weeks;fasting plasma lipid were measured 0 and 4 week. Liver samples were obtained at 4 week.The expression of hepatic apoM, HNF-la and LXRa gene and protein were measured using real time RT-PCR and Western Blot analysis respectively.2) cell experimentsThe HepG2 cells were incubated with different concentration of simvastatin (0、1、5、10、25μmol/L),fenofibrate (0、50、100mmol /L),simvastatin (5.0μmol/L)+fenofibrate(50mmol/L), simvastatin (5.0μmol/L)+HNF-1αinhibitor simvastatin (5.0μmol/L)+LXRa antagonist, fenofibrate(50mmol/L)+HNF-1αinhibitor, fenofibrate (50mmol/L)+LXRa inhibitor for 24h, respectively. Total RNA and protein of HepG2 cells were extracted. The levels of apoM,HNF-la and LXRa gene and protein were measured by real time RT-PCR and Western blot.Results1.Compared with the control group, body weight of obese group and obese intervention group were significantly higher.The weight of visceral fat, fasting glucose, fasting insulin, HOMA-IR in obesity group were significantly higher than control group.Compared with control group, plasma adiponectin levels significantly decreased in obese group.Blood glucose, insulin, insulin resistance index in obesity intervention group was significantly lower than the obese group. Plasma adiponectin levels in obesity intervention group were significantly higher than the obese group.2.compared with control group,apolipoprotein M mRNA and protein expression were significantly decreased in obese mice (all P<0.05);3.compared with control group,FOXA2mRNA and protein expression were significantly decreased in obese mice(all P＜0.05);4.apolipoprotein MmRNA and protein expression were signifi-cantly increased after the intervention of adiponectin in mice(all P＜0.05);5.FOXA2 mRNA and protein expression were significantly increased after the intervention of adiponectin in mice(all P<0.05),Plasma adiponectin levels was significantly increased. Fasting glucose, fasting insulin, HOMA-IR were significantly decreased.(all P＜0.05);6.apolipoprotein M, FOXA2mRNA and protein expression were significantly decreased in insulin-resistance HepG2 cells(all P＜0.05);7.apolipoprotein M, FOXA2mRNA and protein expression increased significantly after the intervention of adiponectin in insulin-resistance HepG2 cells(all P<0.05);apolipoprotein M, FOXA2mRNA and protein expression had no significant change after the intervention of adiponectin in HepG2 cells(all P>0.05)8.Compared with controls, obesity had lower plasma apoM, lower HDL-C, lower plasma adiponectin level(P<0.05). Conversely, Fasting insulin, IL-6, TNF-α, CRP markedly increased in obesity than controls(P<0.05).9.In obesity, apoM was positively related to HDL-C level and negat-ively related to BMI, insulin, HOMA-IR, IL-6, TNF-α, CRP. Plasma apoM had no significant correlation with plasma adiponectin, LDL-C, TC, TG, blood pressure, blood sugar and FMD. In control group,apoM was positively related to HDL-C level and negatively related to BMI, Plasma apoM had no significant correlation with insulin, HOMA-IR, IL-6, TNF-a, CRP,plasma adiponectin, LDL-C, TC, TG, blood pressure, blood sugar, FMD.10.Compared with the control group, plasma HDL-C was significantly elevated in three drug treatment group.Combination group was more effective than statin group and brate group (P< 0.05). The expression of apoM was significantly elevated in three drug treament group. Combination group was more effective than statin group and brate group(P<0.05). both of simvastatin and fenofibrate can dose-dependently increase the expression of apolipoprotein M in HepG2 cells. Combination group obtained more effects than either agent (P< 0.05). The expression of apolipoprotein M in simvastatin+HNF-1αinhibitor, simvastatin+ LXRa antagonist group were significantly lower than the same concent-ration simvastatin group (P< 0.05). The expression of apolipoprotein M in fenofibrate+ HNF-la inhibitor was significantly lower than the same concentration fenofibrate group (P< 0.05).The expression of apolipo-protein M in fenofibrate+LXRa inhibitor was significantly higher than the same concentration fenofibrate group (P< 0.05).11.Compared with the control group,the expression of HNF-1αwas significantly elevated in three drug treatment group.Combination group was more effective than statin group and brate group (P< 0.05). both of simvastatin and fenofibrate can dose-dependently increase the expression of HNF-la in HepG2 cells.Combination group obtained more effects than either agent (P<0.05).12.Compared with the control group,the expression of LXRa gene and protein was significantly decreased in statin group, the expression of LXRa was significantly elevated in brate group.However, no significant difference in LXRa expression was seen between combination and control (P<0.05).simvastatin can dose-dependently decrease the expression of LXRa in HepG2 cells. Fenofibrate can dose-dependently inc-rease the expression of LXRa in HepG2 cells (P＜0.05).No significant difference in LXRa expression was seen between combination and control in HepG2 cells.Conclusions 1.Apolipoprotein M, FOXA2 gene and protein expression signi-ficantly decreased in obese mice. Decrease apolipoprotein M expression may be related to decreased FOXA2.2.Plasma adiponectin level was significantly decreased in obese mice,visceral fat weight, fasting glucose, fasting insulin, HOMA-IR were significantly increased in obese mice.3.Adiponectin in obese mice can upregulate the expression of apolipoprotein M and its mechanism may be related to its effect on insulin resistance.4.Adiponectin increases apolipoprotein M expression in insulin resistance HepG2 cells, but has no effect on apolipoprotein M expression in HepG2 cells.This suggests that adiponectin regulates apolipoprotein M by indirectly affecting insulin resistance.5.Obese patients had lower apoM plasma. Lower apoM levels may be one of the underlying mechanisms account for lower HDL-C in obese patients.6.ApoM levels was closely related to CRP, TNF-a and IL-6 levels in obese patients. apoM may be regulated by these inflammatory factors, its mechanism may be related to inflammatory factors in the promotion of insulin resistance.7.ApoM levels of obese patients had no significant correlation with FMD, suggesting that apoM can not serve as an early predictor of atherosclerosis.8.Upregulation of apoM expression by simvastatin and fenofibrate may contribute to their effect on HDL-C.Combination is more effective.9.There is a mechanic compensation in regulation of apoM expression, i.e. Simvastatin increases HNF-1αand inhibits LXRa, but fenofibrate simultaneously induces the expression of HNF-1αand LXRa.It indicates the combination of statin and fibrate will obtain more HDL and apoM elevation effects than either agent.更多还原