肝脏MsrA高表达对小鼠脂质代谢及动脉粥样硬化的影响

发布时间：2018-06-25 06:58

本文选题：MsrA + 甲硫氨酸亚砜　；参考：《武汉大学》2015年博士论文

【摘要】：研究背景脂质代谢紊乱和炎症是动脉粥样硬化(atherosclerosis, As)形成的公认危险因素。肝脏作为机体脂质代谢和炎症控制的重要器官,与As的发生发展密切相关。调节肝脏脂质代谢及炎症是防治As的有效策略。最新研究发现甲硫氨酸残基氧化修饰(甲硫氨酸亚砜)可通过影响某些特定蛋白质的结构与功能,参与多种氧化相关疾病的发生与发展。而MsrA作为机体内一道特殊的蛋白抗氧化防护屏障,可特异性还原S型甲硫氨酸亚砜(Methionine S-sulfoxide, MetSO),在调节蛋白功能及细胞氧还状态平衡的过程中扮演重要的角色。因此,本研究拟将MsrA作为一个调控靶点,干预肝脏氧还状态,观察肝脏hMsrA高表达对As模型鼠炎症、脂质代谢及As斑块的影响并探究其相关机制。方法体外研究：采用脂质体法分别将构建的慢病毒表达载体质粒pWPI-hMsrA-GFP及对照质粒pWPI-GFP瞬时转染传代培养的人肝癌HepG2细胞系,荧光显微镜观察细胞报告基因GFP表达状态,蛋白免疫印迹检测MsrA表达水平。利用透膜荧光探针二氢溴乙啶(氧化转化为溴化乙啶)荧光显微镜下检测细胞红色及绿色荧光,ImageJ软件计算转染细胞的平均荧光强度分析胞内活性氧簇(ROS)的水平;实时定量PCR (Q-PCR)及蛋白免疫印迹检测HepG2细胞炎症和脂质代谢相关基因表达的水平,观察hMsrA高表达对HepG2细胞炎症及脂代谢的影响。体内研究：将慢病毒表达载体pWPI-GFP/pWPI-hMsrA-GFP和包装质粒PCMV/PD2G,通过磷酸钙法共转染293T细胞,包装形成慢病毒颗粒(Lv-GFP/Lv-MsrA-GFP),收集、浓缩获得高滴度慢病毒颗粒浓缩液。先后采用载脂蛋白E基因敲除(apoE-/-)小鼠或清道夫受体BI基因敲除(SR-BI-/-)小鼠作为As模型鼠,各自随机分两组,每组8只,分别经眼球后静脉丛注射pWPI-GFP慢病毒(Lv-GFP对照组)和含hMsrA的重组慢病毒(Lv-MsrA-GFP组)；慢病毒颗粒输注两周后,小鼠采用AIN-76A西方膳食高脂喂养12周,加速As进程。实验小鼠自病毒注射两周后每间隔3-4周采血,酶法监测血浆总胆固醇(TC)、甘油三酯(TG)、游离胆固醇(FC)、高密度脂蛋白胆固醇(HDL-C)等血脂水平；快速蛋白液相色谱(fast protein liquid chromatography, FPLC)分析血浆脂蛋白胆固醇分布；血浆超氧化物歧化酶(SOD)采用试剂盒检测；血浆对氧磷酯酶(PON1)活性利用对氧磷为底物,采用连续动态法监测；异丙醇法抽提粪便脂质并检测粪胆固醇含量。病毒注射14周后小鼠安乐死,从主动脉根部连续冰冻切片及主动脉胸腹段en face剖面进行油红O染色分析斑块面积。采取小鼠肝脏组织行组织化学检测肝脏MsrA表达水平；酶法分析肝脏脂质含量；实时荧光定量PCR检测小鼠肝脏炎症及脂质代谢相关基因的mRNA水平；蛋白免疫印迹检测肝脏脂质代谢相关基因和血浆中载脂蛋白AI(apoAI)、PON1及血清淀粉样蛋白A(SAA)的蛋白水平。结果1. hMsrA高表达可降低HepG2细胞ROS水平,抑制炎症因子肿瘤坏死因子α(TNFα)和白细胞介素-6(IL-6)的产生,也可显著上调胆固醇代谢相关基因肝X受体α(LXRα)、ATP结合盒转运蛋白Al(ABCA1)及SR-BI的基因表达,但对低密度脂蛋白受体(LDLR)的基因表达未产生明显影响。结果表明高水平hMsrA可通过发挥抗氧化作用,降低ROS水平,影响HepG2细胞炎症及脂代谢相关基因表达。2.GFP荧光检测及MsrA的免疫组化均显示,慢病毒注射可使小鼠肝脏高表达hMsrA,实验中小鼠的体重、脾重及血浆谷丙转氨酶(ALT)活性无明显变化,说明肝脏高表达hMsrA并未对小鼠免疫及肝功能产生明显不良影响。与Lv-GFP小鼠相比,Lv-MsrA-GFP apoE-/-小鼠肝脏MsrA高表达3.4倍;而Lv-MsrA-GFP组SR-BI-/-小鼠肝脏MsrA水平是对照组SR-BIA鼠的2.38倍,即肝脏hMsrA高表达实验动物模型构建成功。3.肝脏hMsrA高表达对高脂喂养apoE-/-小鼠的影响：1)与Lv-GFP组相比,Lv-MsrA-GFP组小鼠肝脏炎症因子TNFa/IL-6的mRNA水平明显下调,血浆抗氧化酶SOD、PON1的活性显著提高,血浆PON1、apoAI的蛋白含量也明显增加,而血浆急性应激蛋白因子SAA水平显著下降；2)肝脏hMsrA高表达可明显降低TC、TG,FPLC分析脂蛋白分布可见血脂水平主要是血浆VLDL/LDL水平的降低,HDL-C并未见显著变化；3)肝脏hMsrA高表达可通过上调肝脏胆固醇代谢相关基因SR-BI、apoAI、LXRa、ABCA1/G8及胆固醇7/27α-羟化酶(CYP7A1/27A1)的表达,并协同调节肝脏胆固醇酯水解酶(CEH)和乙酰辅酶A乙酰转移酶(ACAT)的mRNA和/或蛋白水平,显著增强apoE-/-小鼠肝脏对胆固醇的选择性摄取、转化及胆道排泄,提高粪便胆固醇的含量(增加24.6%),减少肝脏脂质沉积；4)肝脏hMsrA高表达显著下调脂肪合成相关酶：乙酰辅酶A羧化酶a (ACCa)/脂肪酸合成酶(FASN)的mRNA水平,抑制TG合成,降低肝脏及血浆TG含量；5)肝脏hMsrA高表达可显著减缓高脂喂养的apoE-/-小鼠动脉粥样硬化斑块的进程：与Lv-GFP组相比,Lv-MsrA-GFP组实验小鼠主动脉根部横截面切片的平均斑块面积明显减少(0.50±0.06 vs 0.66±0.06 mm2; P0.01);胸腹主动脉en face剖面分析结果显示,Lv-MsrA-GFP组小鼠主动脉弓及主动脉斑块面积均明显减小(P0.01,P0.05)。上述结果提示：肝脏hMsrA高表达可显著改善apoE-/-小鼠肝脏及循环中的氧还状态,抑制炎症,并促进肝脏胆固醇摄取、转化及胆道排泄,抑制TG合成,从而改善脂质代谢,降低肝脏脂质沉积,阻抑As斑块进程。4.肝脏hMsrA高表达对高脂喂养SR-BI-/-小鼠的影响：1)与Lv-GFP组相比,Lv-MsrA-GFP组小鼠肝脏炎症因子TNFa/IL-6的mRNA水平明显下调,血浆急性蛋白因子SAA水平明显降低,而血浆抗氧化酶PON1的活性显著提高。2)SR-BI-/-小鼠肝脏hMsrA高表达可明显降低血浆TG、FC,提升血浆HDL-C水平,但对血浆TC影响不明显；3) SR-BI-/-鼠肝脏hMsrA高表达也可通过下调ACCa/FASN的表达,抑制TG合成；4) SR-BI-/-鼠肝脏hMsrA高表达不再上调肝脏胆固醇代谢相关基因apoAI、LXRa-ABCA1/G8及CYP7A1/27A1的表达,不改变小鼠粪便/肝脏胆固醇含量；但肝脏hMsrA高表达可通过上调肝脏ACAT、 LXRa、ABCA1及血浆apoAI蛋白水平,与降低高脂喂养SR-BI-/-鼠肝脏和血浆FC水平有关。以上结果证实：肝脏hMsrA高水平改善SR-BI小鼠氧还状态,抑制炎症,并抑制TG合成,降低肝脏/血浆FC比例,但不改变血浆/肝脏/粪便TC水平,原因可能与小鼠SR-BI缺陷无法调节肝脏胆固醇的选择摄取有关。结论MsrA作为胞内一种特殊抗氧化酶,其肝脏高表达可显著降低小鼠肝脏组织局部和机体循环系统的氧化状态,发挥抗炎和调节脂质代谢的作用,从而降低小鼠肝脏脂质沉积,及阻抑动脉粥样硬化斑块的进程。其调脂作用主要涉及：促进SRBI介导的肝脏胆固醇摄取、胆固醇转化及胆固醇胆道排泄而降肝脏/血浆胆固醇水平；降低TG合成,一定条件下协调胆固醇的酯化及胆道排泄过程。总之,肝脏MsrA可有效抗氧化抗炎、改善脂质代谢,有望成为防治动脉粥样硬化相关心血管疾病的重要靶向蛋白。
[Abstract]:Background lipid metabolism disorder and inflammation are recognized as a risk factor for the formation of atherosclerosis (As). The liver is an important organ for lipid metabolism and inflammation control, which is closely related to the development of As. Regulation of liver lipid metabolism and inflammation is an effective strategy for the prevention and treatment of As. The latest research has found methionine residues Oxidative modification (methionine sulfoxide) can participate in the occurrence and development of a variety of oxidative related diseases by affecting the structure and function of certain specific proteins, and MsrA, as a special antioxidant protective barrier in the body, can specifically reduce S type methionine sulfoxide (Methionine S-sulfoxide, MetSO), and regulate protein function and the function of protein. Cell oxygen also plays an important role in the process of balance. Therefore, this study intends to use MsrA as a regulatory target to intervene in the state of liver oxygenation, to observe the effect of liver hMsrA high expression on inflammation, lipid metabolism and As plaque in As model rats and to explore its related mechanisms. The virus expression vector plasmid pWPI-hMsrA-GFP and the control plasmid pWPI-GFP were transiently transfected to the human hepatoma HepG2 cell line. The expression of the cell reporter gene GFP was observed by the fluorescence microscope and the expression level of MsrA was detected by the protein immunoblotting. The fluorescence microscopy of the transmembrane fluorescence probe two hydrobromide (oxidation conversion to ethidium bromide) was used for the fluorescence microscopy. Cell red and green fluorescence were measured. ImageJ software calculated the average fluorescence intensity of transfected cells to analyze the level of intracellular reactive oxygen species (ROS); real-time quantitative PCR (Q-PCR) and protein immunoblotting were used to detect the expression level of HepG2 cell inflammation and lipid metabolism related genes. The effect of hMsrA high expression on the inflammation and lipid metabolism of HepG2 cells was observed. Study: the Lentivirus Expression Vector pWPI-GFP/pWPI-hMsrA-GFP and the package plasmid PCMV/PD2G were transfected into 293T cells by calcium phosphate method and packaged to form lentivirus particles (Lv-GFP/Lv-MsrA-GFP) and collected and concentrated to obtain high titer lentivirus particle concentrate. It was successively adopted the fat egg white E gene knockout (apoE-/-) mouse or the scavenger receptor BI gene successively. SR-BI-/- mice were randomly divided into two groups, each of which was randomly divided into two groups, 8 in each group. The mice were injected with pWPI-GFP lentivirus (Lv-GFP control group) and hMsrA containing recombinant lentivirus (group Lv-MsrA-GFP), respectively. After two weeks of lentivirus particles infusion, the mice were fed with AIN-76A Western diet high fat for 12 weeks and accelerated As process. Experimental mice were taken from mice. Blood samples were collected every 3-4 weeks after two weeks of injection. Plasma total cholesterol (TC), triglyceride (TG), free cholesterol (FC), high density lipoprotein cholesterol (HDL-C) and other lipid levels were monitored by enzyme method. Plasma lipoprotein cholesterol distribution was analyzed by rapid protein liquid chromatography (fast protein liquid chromatography, FPLC); plasma superoxide dismutase (SOD) (SOD) use the kit test; plasma oxyphosphatase (PON1) activity using oxyphosphoric acid as the substrate, using continuous dynamic monitoring, isopropyl alcohol extraction of fecal lipid and detection of fecal cholesterol content. After 14 weeks of virus injection, mice were euthanized, the continuous frozen section of the aorta and the en face section of the thoracic and abdominal segment of the aorta were stained with oil red O staining. Color analysis of plaque area. The liver MsrA expression level was detected by mouse liver tissue; liver lipid content was analyzed by enzyme method; real-time fluorescence quantitative PCR was used to detect the mRNA level of liver inflammation and lipid metabolism related genes in mice; liver lipid metabolism related genes and plasma apolipoprotein AI (apoAI) were detected by Western blot. The protein level of PON1 and serum amyloid A (SAA). Results the high expression of 1. hMsrA can reduce the ROS level of HepG2 cells, inhibit the production of tumor necrosis factor alpha (TNF a) and interleukin -6 (IL-6), and also significantly up-regulate the cholesterol metabolism related gene liver X receptor alpha (LXR alpha), ATP binding box transporter and substrate. The expression has no obvious effect on the gene expression of low density lipoprotein receptor (LDLR). The results show that high level hMsrA can reduce the level of ROS by exerting antioxidant activity, affecting the expression of.2.GFP fluorescence in HepG2 cell inflammation and lipid metabolism related gene expression and immunohistochemistry of MsrA. Up to hMsrA, the weight of mice, spleen weight and plasma alanine transaminase (ALT) activity did not change significantly, indicating that the liver high expression hMsrA did not have a significant adverse effect on the immune and liver function of mice. Compared with the Lv-GFP mice, the MsrA in the liver of Lv-MsrA-GFP apoE-/- mice was 3.4 times higher than that in the Lv-MsrA-GFP group, and the MsrA level of the liver of Lv-MsrA-GFP group SR-BI-/- mice was In the control group, 2.38 times the SR-BIA rat, that is, the liver hMsrA high expression experimental animal model was constructed successfully. The effect of the high expression of.3. liver hMsrA on the high fat feeding apoE-/- mice: 1) the mRNA level of the liver inflammatory factor TNFa/IL-6 in the Lv-MsrA-GFP group was significantly lower than that in the Lv-GFP group, and the activity of the plasma antioxidant enzyme SOD, PON1 was significantly increased, and the plasma PON1, and the plasma PON1, were The protein content of poAI increased significantly, while the level of acute stress protein factor SAA in plasma decreased significantly; 2) high expression of hMsrA in the liver could significantly reduce TC, TG, FPLC analysis of lipoprotein distribution showed that blood lipid level was mainly the decrease of plasma VLDL/LDL level, and no significant change in HDL-C, and 3) liver hMsrA high expression could be raised by up regulation of liver cholesterol. The expression of metabolic related genes SR-BI, apoAI, LXRa, ABCA1/G8 and cholesterol 7/27 alpha hydroxylase (CYP7A1/27A1), and co regulated mRNA and / or protein levels of liver cholesterol ester hydrolase (CEH) and acetyl coenzyme A acetyltransferase (ACAT), significantly enhanced the selective uptake of cholesterol in the liver of apoE-/- mice, transformation and biliary excretion, and increased feces. The content of cholesterol (increased by 24.6%), reduced liver lipid deposition, 4) liver hMsrA high expression significantly down regulation of lipid synthesis related enzymes: acetyl coenzyme A carboxylase a (ACCa) / fatty acid synthase (FASN) mRNA level, inhibition of TG synthesis, reducing the liver and plasma TG content; 5) liver hMsrA high expression can significantly slow down high fat feeding apoE-/- small apoE-/- The process of atherosclerotic plaque in rats: compared with the Lv-GFP group, the average plaque area of the aortic root section of the Lv-MsrA-GFP group was significantly reduced (0.50 + 0.06 vs 0.66 + 0.06 mm2; P0.01). The results of the thoracic and abdominal aorta en face profile analysis showed that the area of aortic arch and aortic plaque in Lv-MsrA-GFP mice was obvious. Decrease (P0.01, P0.05). These results suggest that the high expression of hMsrA in the liver can significantly improve the state of oxygen in the liver and circulation of apoE-/- mice, inhibit inflammation, promote liver cholesterol uptake, transform and bile duct excretion, inhibit TG synthesis, thus improve lipid metabolism, reduce liver lipid deposition, and inhibit the high expression of hMsrA in the As plaque process of.4.. The effect on high fat feeding SR-BI-/- mice: 1) compared with the Lv-GFP group, the mRNA level of liver inflammatory factor TNFa/IL-6 in the Lv-MsrA-GFP group was obviously down, the plasma acute protein factor SAA level was significantly reduced, and the activity of the plasma antioxidant enzyme PON1 was significantly increased.2) and the high expression of hMsrA in the liver of the SR-BI-/- mice could obviously reduce the TG, FC, and ascension of the plasma. The plasma HDL-C level was not significantly affected by the plasma TC; 3) the high expression of hMsrA in the liver of SR-BI-/- rats could also reduce the expression of ACCa/FASN and inhibit the synthesis of TG; 4) the high expression of hMsrA in the liver of SR-BI-/- mice no longer up-regulated the expression of cholesterol metabolism related genes of liver, LXRa-ABCA1/G8 and CYP7A1 /27A1, and did not change the cholesterol and liver cholesterol of mice. The high expression of hMsrA in the liver can be related to the increase of liver ACAT, LXRa, ABCA1 and plasma apoAI protein levels, which are related to the reduction of FC levels in the liver and plasma of SR-BI-/- rats fed by high fat diet. The above results confirm that the high level of liver hMsrA improves the oxygen state of the SR-BI mice, inhibits the inflammation, inhibits the synthesis of TG, reduces the FC proportion of the liver / plasma, but does not change the liver / plasma FC ratio. The level of plasma / liver / fecal TC may be related to the failure to regulate the selective uptake of liver cholesterol by SR-BI deficiency in mice. Conclusion MsrA is a special antioxidant enzyme in the cell. The high expression of the liver can significantly reduce the oxidation state of the local and circulatory system of the liver tissue in mice, and play the role of anti-inflammatory and regulating lipid metabolism. The lipid deposition of the liver and the inhibition of atherosclerotic plaques are reduced. The lipid regulating effect is mainly involved in promoting SRBI mediated cholesterol uptake, cholesterol conversion and cholesterol excretion, reducing liver / plasma cholesterol levels, reducing TG synthesis, and coordinating cholesterol esterification and biliary excretion under certain conditions. In conclusion, liver MsrA is an effective target protein for the prevention and treatment of atherosclerotic cardiovascular diseases, which is effective in anti-oxidation, anti-inflammatory and improving lipid metabolism.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R543.5

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