【作者】 唐任宽；

【导师】 黄爱龙； 阮雄中；

【作者基本信息】 重庆医科大学 ， 内科学， 2008， 博士

【摘要】 目的低度慢性系统性炎症反应是代谢综合征（MS）的核心点和几项组成成份（如血糖、血脂及尿酸等）的连接点，炎症因子参与脂质代谢障碍的全过程。固醇调节元件结合蛋白（SREBPs）是核转录因子，SREBP裂解激活蛋白（SCAP）是内质网上的膜蛋白，SCAP对SREBPs的活性起着重要的调节作用；细胞是通过一个依赖于胞内胆固醇水平的反馈调节系统来控制胆固醇内稳态平衡，即主要通过SCAP-SREBPs-LDLR之间的相互作用实现对细胞内脂质稳态平衡的调节。体外实验证实炎症因子可通过干扰胆固醇介导的低密度脂蛋白受体反馈调节来促进外周细胞，如血管平滑肌细胞（VSMCs）的胆固醇摄入，从而导致胆固醇在外周细胞的异常聚集而形成泡沫细胞。乙酰CoA羧化酶（ACC）与脂肪酸合成酶（FAS）是甘油三酯（TG）合成的关键酶，肝脏TG的代谢是通过SCAP-SREBPs-ACC/FAS途径实现的，肝脏脂质代谢障碍主要是TG在肝细胞内异常聚集而形成以大泡性为主的肝细胞脂肪变性，可伴有炎症细胞浸润和/或纤维组织增生，炎症因子是否干扰ACC与FAS的表达而导致TG代谢障碍的机制目前仍不清楚。本实验在过去体外细胞实验的基础上，建立慢性炎症动物模型，探讨炎症状态下血脂代谢紊乱情况；炎症因子是否通过干扰SCAP-SREBPs介导的LDLR反馈调节促进脂质摄取，从而导致主动脉与肝脏脂质聚集。材料和方法利用8周龄C57BL/6J小鼠皮下注射酪蛋白（Casein）制作小鼠慢性炎症模型，分别给予普通饮食、高脂饮食饲养20周左右，提取血液、肝脏及主动脉进行下列实验；用酶学方法检测各组小鼠IL-6及血脂（包括TC、LDL-C、HDL-C及TG）水平；HE染色、ORO染色及Masson三色染色观察肝脏与主动脉形态学改变；根据NAFLD、NASH分级与分期标准对各组小鼠肝脏进行分级和分期；利用Real-time PCR技术定量检测肝脏LDLR、SCAP、SREBP2、SREBP1、ACC及FAS mRNA水平以及主动脉LDLR、SCAP、SREBP2mRNA水平； Western blotting检测肝脏LDLR、SCAP、SREBP1蛋白及主动脉LDLR、SCAP、SREBP2蛋白表达水平。结果1. C57BL/6J小鼠皮下注射Casein后，普通饮食和高脂饮食小鼠血清IL-6均显著升高。2.在普通饮食组中除单纯普通饮食小鼠血清TC轻微下降外，LDL、HDL、及TG均明显下降；高脂饮食各项血脂指标（TC、LDL、HDL及TG）均明显下降。这些数据说明慢性炎症状态可导致血脂紊乱。3.与普通饮食组相比，单纯高脂饮食显著抑制主动脉和肝脏LDLR、SCAP和SREBP2mRNA与蛋白的表达，但在慢性炎症状态下，LDLR、SCAP和SREBP2mRNA与蛋白的显著提高，这提示慢性炎症状态可干扰细胞胆固醇代谢。4.单纯高脂饮食组小鼠SREBP1、ACC及FAS基因表达升高，在慢性炎症状态下，SREBP1、ACC及FAS表达进一步提高，肝细胞摄取大量的TG，从而导致肝细胞脂肪变性、气球样变、肝细胞坏死及纤维组织增生，即形成NAFLD或者NASH。5．单纯高脂饮食组小鼠主动脉根部内膜明显增厚，大量泡沫细胞形成及动脉粥样硬化斑块形成；在慢性炎症状态下，主动脉根部动脉粥样硬化斑块明显增厚，斑块面积更大，这说明炎症可加重主动脉损害。1结论1．IL-6是炎症反应的敏感标记物，IL-6的高低与炎症反应程度密切相关,血清IL-6显著升高提示慢性炎症模型成功建立。2．体内慢性炎症状态通过干扰SCAP-SREBP2-LDLR反馈调节系统，促进脂质摄取，从而导致主动脉与肝脏脂质聚集。这些数据表明慢性炎症状态可明显降低血脂水平；慢性炎症状态下没有一个安全的血浆胆固醇浓度，血浆胆固醇水平并不与细胞内胆固醇水平呈正相关。3．炎症状态促进SREBP1、ACC及FAS的表达而使肝细胞内大量TG聚集，导致肝细胞脂肪变性，坏死及纤维化，故炎症状态可加重肝脏损害。更多还原

【Abstract】 Objective The low chronic systematic inflammatory response is thecore and junction point of several composition ingredients（such as bloodglucose, blood lipid and uric acid） about metabolism syndrome (MS).Inflammatory mediators participate in whole process of lipid metabolicdisorder. The Sterol Regulatory Element Binding Proteins (SREBPs) arethe nuclear factors. SREBP Cleavage-activating Protein (SCAP) ismembrane protein on the endoplasmic reticulum. SCAP plays animportant role in regulating SREBPs activity. Intracelluar cholesterolhomeostasis is regulated by a negative feedback system which isdependent on intracellular cholesterol concentration. We havedemonstrated in vitro that inflammatory mediators increased LDL receptor(LDLR)-mediated cholesterol uptake and promoted foam cell formationby disrupting cholesterol-mediated LDLR feedback regulation inperipheral cells, such as vascular smooth muscle cells (VSMCs). Acetylcoenzyme A carboxylase (ACC) and fatty acid synzyme (FAS) are keyenzymes which control triglyceride (TG) synthesis. Both ACC and FAS are regulated by SCAP-SREBP1. The lipid metabolic disorder in liver ismainly characterized by large droplets of fat accumulation, accompaniedby inflammatory cell infiltration and/or fibroplasias. But the mechanismsby which inflammatory mediators disturb ACC and FAS and result in TGmetabolic disorder in liver are still unclear.By using an inflamed mouse model, this project was designed toinvestigate whether inflammatory mediators (i) caused dyslipidaemia;(ii)promoted lipid uptake via LDLR by disrupting SCAP-SREBPs mediatednegative feedback regulation of LDLR, thereby resulting lipidaccumulation in aorta and liver.Materials and methods Using8-week age C57BL/6J mice fed withnormal or atherogenic diet for20weeks respectively, we induced achronic inflammation in mice by injecting10%of casein subcutaneously.The experiments were terminated after20weeks. Plasma samples,collected at the time of sacrifice, were evaluated for lipid profiles (totalserum cholesterol, triglycerides, LDL, HDL, and VLDL). Activation of theinflammatory response was assessed by measurement of serumInterleukin-6(IL-6). Maximal plaque cross-section, plaque area per aorticcircumference, aortic wall thickness, and all other vascular beds includingrenal artery were determined with oil red O staining (ORO). Massontrichrome staining was used to observe the morphological change of liverand aorta. Histological Criteria for NAFLD/NASH was used to assess the grade and stage of fatty liver disease. Total RNA was isolated from theliver and the aorta, and mRNA of LDLR, SCAP, SREBP1,2, ACC andFAS from the liver and the aorta were examined by real-time quantitativePCR. The protein expression of LDLR, SCAP and SREBP1,2in the liveror the aorta was examined by western blotting.Results Serum IL-6was significantly elevated by the caseinsubcutaneous injection in the mice fed by both normal and atherogenic diet.In the mice fed with the normal diet, casein injection slightly decreasedTC level and obviously decreased LDL, HDL and TG levels. In the micefed with the atherogenic diet, all lipids in serum (such as TC, LDL, HDLand TG) are obviously decreased by the casein injection. These datasuggest that chronic inflammatory stress induces a metabolic disorder oflipids.Comparing to the normal diet, the atherogenic diet significantly inhibitedmRNA and protein expression of LDLR, SCAP and SREBP2in the liverand the aorta, however chronic inflammation induced by the caseininjection overrode the suppression of LDLR, SCAP and SREBPs inducedby high cholesterol diet. These data show IM can interfere withintracellular cholesterol metabolism.In the mice fed with atherogenic diet, SREBP1, ACC and FAS mRNAlevels in liver were obviously increased, and these mRNA levels furtherenhanced under the chronic inflammatory condition. Hepatocytes uptaked much TG, resulted in hepatic steatosis, ballooning, cellular necrosis,inflammatory cell infiltration and fibroplasia, namely NAFLD or NASH.Morphological figure showed mice aortic root tunica intima thickened,many foam cell and atherosclerotic plaque formation in the mice fed withatherogenic diet. The atherosclerotic plaque of aortic root were furtherthickened and widened in the mice under chronic inflammatory condition.The data suggest that IM aggravates aorta impairment.Conclusion Casein injection successfully induced a chronicinflammatory stress in the mice by showing that serum IL-6, aninflammatory marker, was significantly increased. The chronicinflammatory stress induced by casein injection promoted lipidaccumulation in aorta and liver by increasing LDLR mediated cholesteroluptake by disrupting SCAP-SREBP2-LDLR mediated feedback regulation.Meanwhile, inflammatory mediators also reduced blood lipid levels.These results suggest that there is not a safe blood plasma cholesterolconcentration under chronic inflammatory condition since the plasmacholesterol level is not associated with intracellular cholesterol extentunder inflammatory stress.The inflammatory mediators caused TG accumulation in liver byincreasing SREBP1, ACC and the FAS gene expression, thereby resultingin liver fat degeneration，accompanied by inflammatory cell infiltrationand/or fibroplasias. These data suggest that inflammatory mediators aggravate the liver injury.更多还原