【作者】 荣爽；

【导师】 刘烈刚；

【作者基本信息】 华中科技大学 ， 营养与食品卫生学， 2012， 博士

【摘要】 动脉粥样硬化(atherosclerosis, AS)是心脑血管疾病的重要病理生理基础,给人类带来了沉重的疾病和经济负担。氧化应激在AS的启动和发生发展过程中发挥重要的作用,抗氧化剂的早期干预对于AS的防治可能具有积极的意义。原花青素是植物体内天然的抗氧化物质,具有抗氧化、抗炎、抗突变、抗肿瘤、调节血脂等多种生物学活性。研究证实其抗氧化的效果要远远优于维生素C、维生素E和p-胡萝卜素等传统抗氧化剂。荔枝壳原花青素(Procyanidins extracted from the litchi pericarp, LPPC)是华中农业大学食品科技学院天然作物实验室率先从荔枝果壳中提取出的一种以A型为主的原花青素。LPPC来源天然丰富,变废为宝,体外研究证实其具有良好的抗氧化清除自由基的能力,然而有关其抗AS的功效研究尚是空白。为了探讨LPPC对AS的改善作用及其相关机制,本研究采用高脂饮食(high fat diet,HFD)饲养载脂蛋白E基因敲除(apolipoprotein E knockout, ApoE KO)小鼠24周建立AS模型,观察LPPC对AS的改善效果；并从脂代谢调节、氧化抗氧化系统、以及一氧化氮系统的改变等方面探讨LPPC防治AS的可能机制。第一部分LPPC对动脉粥样硬化的保护作用目的：探讨LPPC对ApoE KO小鼠动脉粥样硬化病变程度的影响。方法：50只ApoE KO小鼠(6周龄,雄性,体重18-22 g)随机分为2组,即为ApoE KO组及LPPC干预组(ApoE KO+LPPC)。对照组为25只相同遗传背景下野生型C57BL/6小鼠。ApoE KO小鼠给予HFD(脂肪21%,蛋白质20%,胆固醇0.15%)喂养,对照组小鼠给予普通饲料喂养。LPPC组每日给予新鲜配制的LPPC溶液(100mg/kg/d)灌胃干预。实验共进行24周,期间每周记录体重及进食量。实验结束时每组随机取12只小鼠灌注后取血管标本,分别用于主动脉血管整体油红O染色、主动脉弓横截面冰冻切片油红O染色以及免疫组化检测,观察主动脉血管内粥样斑块的形成情况及巨噬细胞和平滑肌细胞的表达情况。结果：(1)血管整体油红O染色结果显示对照组WT小鼠血管内壁无红色染色斑块,ApoE KO小鼠血管内壁可见大面积连续红色粥样病变斑块,提示经过24周HFD干预ApoE KO小鼠成功建立了AS模型。LPPC干预组小鼠血管内壁同样可见粥样硬化病变斑块,但斑块呈现不连续散在分布,与模型组小鼠相比,斑块面积明显减少。采用ImagePro软件对粥样斑块部位进行测量分析,以红色斑块占动脉整体面积的百分比来代表AS的病变程度,结果提示LPPC干预后小鼠血管内壁粥样硬化斑块面积明显减少,差异具有统计学意义(ApoE KO:56.9%±8.1%, ApoE KO+LPPC:32.6%±9.2%,P<0.01)。(2)血管横截面冰冻切片油红O染色以及石蜡切片HE染色的结果提示LPPC能够显著减少ApoE KO小鼠横截面粥样硬化斑块的面积,有效改善血管内壁粥样斑块的聚集。(3)免疫组化的结果显示ApoE KO小鼠主动脉内膜和中膜下有大量的巨噬细胞聚集,LPPC干预后巨噬细胞阳性率明显减少。LPPC未能显著改变平滑肌细胞的表达情况。结论：LPPC能够有效减少动脉粥样硬化斑块的形成,改善AS病变斑块中巨噬细胞的聚集,显示出良好的抗AS发生发展的作用。第二部分LPPC改善动脉粥样硬化的相关机制研究第一节LPPC通过调节脂质代谢紊乱发挥抗动脉粥样硬化的作用目的：探讨LPPC对ApoE KO小鼠体内脂质代谢紊乱的调节作用。方法：各组小鼠行眼眶静脉丛取血后分离血浆用于血脂水平的检测；采用Folch法(即三氯甲烷／甲醇液相分离法)测定肝脏组织中总胆固醇(total cholesterol, TC)及甘油三酯(triglyceride, TG)的含量；实时定量PCR检测各组小鼠肝脏内脂代谢关键基因的表达。结果：(1)与对照组相比,ApoE KO小鼠血浆TC和低密度脂蛋白胆固醇(low density lipoprotein-cholesterol, LDL-C)的水平显著升高(P<0.01),表现为明显的高胆固醇血症,LPPC干预后血浆TC和LDL-C的水平显著下降(P<0.01)。与对照组相比,ApoEKO小鼠血浆TG的含量略微上升(差异无统计学意义),LPPC干预后ApoE KO小鼠血浆TG的水平显著下降(P<0.05)。与对照组相比,ApoE KO小鼠血浆中HDL-C的水平显著下降(P<0.05),然而LPPC未能显著升高血浆中HDL-C的水平。(2)与对照组相比,ApoE KO小鼠肝脏中TC的含量显著上升(P<0.05),TG的含量略微上升(差异无统计学意义),LPPC显著降低ApoE KO小鼠肝脏中TC和TG的含量(P<0.05)。(3)通过对肝脏脂代谢相关关键基因的检测发现,LPPC明显增加ApoE KO小鼠肝脏内PPARa mRNA的表达水平(P<0.05),增强肝脏内脂肪酸氧化的能力；LPPC显著降低ApoE KO小鼠肝脏中HMG-CoA还原酶mRNA的表达水平(P<0.05),减少肝脏内TC的合成；LPPC显著增加ApoE KO小鼠肝脏中ABCA1(P<0.01)及LXRα(P<0.05) mRNA的表达,增加胆固醇外流；此外,LPPC还能够显著增加脂代谢关键核因子FXR和SHP mRNA的表达。结论：LPPC干预显著降低ApoE KO小鼠血浆TC、TG及LDL-C的水平,降低肝脏TC及TG的含量,有效调控脂代谢相关关键基因的表达,表现出良好的改善脂质代谢的功能。调节脂代谢紊乱可能是LPPC发挥抗AS作用的机制之一。第二节LPPC通过调节一氧化氮系统平衡发挥抗动脉粥样硬化的作用目的：探讨LPPC对ApoE KO小鼠一氧化氮(nitric oxide, NO)系统的调节作用。方法：进行血浆NO水平、内皮素(endothelin-1, ET-1)水平以及诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)活性的检测;进行主动脉弓横截面石蜡切片iNOS和内皮型一氧化氮合酶(endothelial NOS, eNOS)的免疫组化检测；进行主动脉血管组织iNOS和eNOS的实时定量PCR检测和western blot检测；使用ELISA的方法进行血浆血管细胞粘附因子(vascular cell adhesion molecules-1, VCAM)水平的检测。结果：(1)与对照组相比,ApoE KO小鼠血浆NO的含量显著增加,LPPC能够显著减少ApoE KO小鼠血浆NO的含量(P<0.01)。与此相反的是ApoE KO小鼠血浆ET-1的水平显著下降(P<0.05),LPPC能够逆转这种下降,明显升高血浆ET-1的水平。(2)免疫组化结果显示ApoE KO小鼠主动脉内膜和中膜下均有大量的iNOS蛋白表达,而LPPC组小鼠主动脉仅可见少量iNOS蛋白表达,说明LPPC能够有效减少主动脉中iNOS的表达。PCR的结果显示,ApoE KO小鼠主动脉组织iNOS mRNA的表达与对照组相比显著增加(P<0.01),LPPC能够显著降低主动脉组织中iNOS mRNA的表达水平(P<0.05)。western blot的结果,ApoE KO小鼠主动脉组织中iNOS蛋白的表达水平与对照组相比显著增加(P<0.05),LPPC能够显著降低主动脉组织中iNOS蛋白的表达水平(P<0.05)。与此一致的是ApoE KO小鼠血浆iNOS的活性与对照组相比显著增加(P<0.01), LPPC能够显著降低血浆iNOS的活性(P<0.05)。(3)与iNOS的表达相反,免疫组化结果显示ApoE KO小鼠主动脉内膜仅有少量的eNOS蛋白表达,LPPC显著增加eNOS蛋白的表达。PCR和western blot的结果也显示ApoE KO小鼠主动脉组织eNOS mRNA和蛋白的表达与对照组相比显著减少,LPPC显著增加主动脉组织eNOS mRNA和蛋白的表达水平(P<0.05)。(4)与对照组相比,ApoE KO小鼠血浆VCAM的水平明显增加(P<0.01),LPPC显著降低血浆VCAM的水平(P<0.01)。结论：ApoE KO小鼠血浆NO含量的大幅度增加可能是由iNOS的异常高表达所导致的,LPPC能够有效改善iNOS异常表达引起的NO大量释放。这种调节NO系统平衡的作用可能是LPPC发挥抗AS作用的机制之一。第三节LPPC通过调节氧化抗氧化系统平衡发挥抗动脉粥样硬化的作用目的：探讨LPPC对ApoE KO小鼠体内氧化抗氧化系统的调节作用。方法：按照试剂盒说明书进行血浆氧化抗氧化系统相关重要指标的检测,如抗氧化剂谷胱甘肽(glutathione, GSH)的水平,抗氧化酶超氧化物歧化酶(superoxide dismutase, SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase, GPx)、过氧化氢酶(catalase, CAT)的活性,脂质过氧化产物丙二醛(malondialdehyde, MDA)的水平等。实时定量PCR检测各组小鼠主动脉组织中NADPH酶亚基(p22phox、p47phox、P67phox、NOX-1、NOX-2/gp91phox和NOX-4)mRNA的表达水平。结果：(1)与ApoE KO组相比,LPPC能够显著增加ApoE KO小鼠血浆GSH的含量(P<0.01)。(2)与对照组相比,ApoE KO小鼠血浆MDA的含量显著增加(P<0.01), LPPC干预后血浆MDA的含量显著减少(P<0.05)。(3)与对照组相比,ApoEKO小鼠血浆SOD的活性显著降低(P<0.05),LPPC干预未能显著增加血浆SOD的活性。与对照组相比,ApoE KO小鼠血浆GPx的活性显著降低(P<0.05),LPPC干预后血浆GPx活性略有上升,但差异无统计学意义。与ApoE KO组相比,LPPC能够显著增加血浆CAT的活性(P<0.05),并且能够显著升高CAT/SOD的比值(P<0.01)。(4)通过对主动脉组织NADPH酶亚基mRNA表达水平的检测发现,与对照组相比,ApoE KO小鼠主动脉组织p22 phox、p47phox、p67phox、NOX-2/gp91phox和NOX-4等亚基m RNA的表达水平显著增加(P<0.05),LPPC能够显著减少主动脉组织中p47phox、p67phox、NOX-2/gp91phox和NOX-4iNOS mRNA的表达水平(P<0.05)。结论：LPPC能够显著提高ApoE KO小鼠体内抗氧化的能力,且能够通过降低NADPH酶的活性抑制主动脉血管组织ROS的产生,这种抑制ROS产生及提高机体抗氧化能力的作用可能是LPPC发挥抗AS作用的机制之一。更多还原

【Abstract】 Atherosclerosis is a common disorder characterized by the accumulation of macrophages and products of lipid and protein oxidation in blood vessels. Oxidative stress, chronic inflammation and endothelial dysfunction play key roles in the development and progression of atherosclerosis.Procyanidins, a group of flavonoids that are commonly found in red wine, grapes, cocoa, and apples, have a broad range of biological activities such as antioxidant, antigenotoxic, antiatherogenic and antihyperglycemic function.Procyanidins extracted from the litchi pericarp (LPPC) used in the present study is thought to be a new source of procyanidins and have been proven to possess strong antioxidant activities in vitro.In the present study, we sought to determined the effects of oral administration of LPPC on the developemt of atherosclerosis in apolipoprotein E knockout (ApoE KO) mice fed a high fat diet for 24 weeks and then explored the underlying mechanisms. Part One Effects of LPPC on Atherosclerotic Lesion Formation in ApoE KO MiceObjective:To determine the ameliorating effects of LPPC on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice.Methods:A total of 50 eight-week-old 18-22 g male ApoE KO mice were randomly divided into two groups with 25 mice in each group:the ApoE KO group and ApoE KO+ LPPC group.25 wild-type (WT, C57BL/6J) mice were considered to be the control group.All ApoE KO mice were fed on a high fat diet (HFD, based on Harlan Teklad diet TD 88137 comprised of 21% fat by weight [42% of calories] and 0.15% cholesterol by weight) for 24 weeks. WT mice were fed a chow diet. Mice in LPPC group were orally administrated with LPPC (100 mg/kg body mass daily) dissolved in distilled water freshly prepared every day throughout the whole experiment. Food consumption and body weight changes were recorded weekly. At the end of the experiment, twelve mice in each group were perfused with PBS via the left ventricle after pentobarbital sacrifice. The aortas (from the bifurcation off the aortic arch to the branching point of the right subclavian and common carotid artery) of four mice in each group were opened longitudinally to expose the endothelial surface for enface Oil Red O staining. The aortic arch of the other four mice in each group were removed and snap-frozen in Tissue-Tek OCT compund for 10μm thickness serial sections, and then stained with Oil red O for atherosclerotic lesion quantification. Aortic arch of another four mice in each group were stored in 10% buffered formalin overnight and embedded in paraffin for the immunohistochemical detection.Results:The ApoE KO mice resulted in a significant increase in mean lesion size compared with the control WT mice. Histomorphometric analysis revealed that LPPC significantly decreased atherosclerotic lesion size in the ApoE KO+LPPC group than in the ApoE KO group (ApoE KO:56.9%±8.1%, ApoE KO+LPPC:32.6%±9.2%, P< 0.01). The atherosclerotic lesion formation in the transverse section of aortic arch stained with Oil red O showed that LPPC intervention also significantly decreased atherosclerotic lesion formation.Conclusion:The present study demonstrated that the supplementation of LPPC to ApoE KO mice ameliorated atherosclerotic plaque development. Part Two Mechanism studySection 1 Effects of LPPC on Lipid disorder in ApoE KO MiceObjective:To investigate whether LPPC could provide protection against atherosclerosis through regulating lipid metabolism in ApoE KO MiceMethod:At the end of experiment, blood samples and liver tissues were collected. Plasma total cholesterol (TC), triglyceride (TG) levels, low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), hepatic TC and TG contents were determined. The mRNA expressions of key genes involved in lipid metabolism were quantified by real-time RT-PCRResults:Compared with WT mice in the control group, ApoE KO mice showed a marked increase in TC (P< 0.01) and LDL-C levels (P< 0.05), while after LPPC administration, the TC and LDL-C contents in plasma were significantly decreased (P< 0.05). Although plasma TG level was diminished a little, the difference had no statistical meaning (P>0.05). LPPC also decrease the plasma TG level significantly; For HDL-C levels, there was no significant difference between ApoE KO and ApoE KO+LPPC group. LPPC also decreased hepatic TC and TG contents significantly. Moreover, LPPC could significantly increase mRNA expressions of farnesoid X receptor (FXR) and small heterodimer partner (SHP) which emerge as key regulators of lipid homeostasis at the transcriptional level, decrease mRNA expressions of 3-hydroxy-3-Methylglutaryl (HMG)-CoA reductase which mediates cholestrol biosynthesis, and increase mRNA expressions of ATP-binding cassette transporter-1 (ABCA1) which modulates cholesterol efflux in the liver of ApoE KO mice.Conclusion:LPPC can effectively regulate key gene expressions involved in lipid metabolism system and thus alleviate the lipid disorder especially hypercholesteromia in ApoE KO mice. These findings indicated that LPPC ameliorated atherosclerosis via regulating gene expression involved in hepatic lipid homeostasis in ApoE KO mice fed a HFD. Section 2 Effect of LPPC on NO, iNOS and eNOS in ApoE KO MiceObjective:To investigate whether LPPC could provide protection against atherosclerosis through regulating the nitric oxide system in ApoE KO MiceMethods:Plasma NO levels and iNOS activity were determined using appropriate kits according to the manufacturer’s instructions. Aortic mRNA expression of iNOS and eNOS were quantified by real-time RT-PCR. Aortic protein expression of iNOS and eNOS were quantified by western blot test. The concentrations of ET-1 and vascular cell adhesion molecule-1 (VCAM) in plasma were measured using ELISA assays.Results:The plasma NO contents in ApoE KO mice were markedly higher than that in control WT mice. LPPC intervention significantly lowered the NO levels (P< 0.01 versus ApoE KO group). Interestingly, plasma concentrations of ET-1 were significantly lower in the HFD ApoE mice compared to the chow WT mice while LPPC intervention reversed this decrease significantly. LPPC also significantly reduce plasma iNOS activities when compared with ApoE KO group (P< 0.01). PCR and Western blotting showed that control WT mice had minimal iNOS mRNA expression and iNOS protein contents in aorta, while ApoE KO mice showed significantly higher iNOS mRNA expression and iNOS protein contents than WT mice. However, LPPC supplementation exhibited significantly lower iNOS mRNA expression and protein contents in aortas than ApoE KO mice. Aortic sections from ApoE KO mice showed greater staining for iNOS than those of WT mice, whereas in ApoE KO mice treated with LPPC, iNOS protein was significantly decreased. Conversely, the protein expression of eNOS was lower in aortic sections of ApoE KO than in control WT mice, however in ApoE KO mice treated with LPPC the expression was increased. Moreover, the mRNA and protein expression of eNOS in aortas of ApoE KO mice were increased by LPPC which further explain the endothelial protective actions of LPPC.Conclusion:These results elucidated that that the excess NO production found in ApoE KO mice may be due to the elevated iNOS activity as well as gene and protein expression, and the antiatherogenetic effect of LPPC may be at least partly through the inhibition of iNOS. Section 3 Effect of LPPC on Oxidative Stress and Antioxidant Systems in ApoE KO MiceObjective:To determine the effect of LPPC on oxidative stress and antioxidant systems in ApoE KO mice.Methods:The GSH levels, MDA contents and the activities of SOD、GPx and CAT in plasma were measured according to the appropriate methods. The mRNA expression of NADPH oxidases ubtypes (p22phox, p47phox, p67phox, NOX-1, NOX-2/gp91phox and NOX-4) in aortas of four mice in each group were detected by RT PCR.Results:LPPC intervention significantly increases the plasma GSH level when compared with ApoE KO group (P<0.01). MDA contents in plasma were significantly elevated in ApoE KO mice than control WT mice. LPPC intervention brought down the MDA levels significantly. There was also quite a deal of difference in the alterations of activities of SOD between control and ApoE KO mice group. Comparing to control, ApoE KO mice had significantly lower SOD enzyme activities in plasma, however, LPPC intervention resulted in no significant change in the activities of SOD. LPPC intervention significantly increases the plasma CAT activities when compared with ApoE KO group (P < 0.05), and also increases the CAT/SOD ratio significantly (P<0.01).The mRNA expression (expressed as percent of control) for NAD(P)H oxidase (p47phox, p67phox, NOX-2/gp91phox and NOX-4) from aortas were higher in ApoE KO mice than those in control mice, however LPPC significantly decreased the expression of NAD(P)H oxidase in ApoE KO miceConclusion:In this study, LPPC showed profoundly antioxidative effects in ApoE KO mice, and the antiatherogenetic effect of LPPC may be partly due to the inhibition of NADPH oxidase-derived oxidative stress.更多还原