【作者】 谢江娇；

【导师】 廖玉华；

【作者基本信息】 华中科技大学 ， 心血管内科， 2009， 博士

【摘要】 第一部分PARP抑制剂降低高同型半胱氨酸血症诱导ApoE-／-小鼠动脉粥样硬化斑块面积目的高同型半胱氨酸血症（hyperhomocysteinemia，Hhcy）是动脉粥样硬化的一个重要而独立的危险因子。最近的研究表明多聚ADP核糖合成酶[Poly（ADP-ribose）polymerase，PARP]的活化与Hhcy诱导的内皮功能障碍有关，而后者是Hhcy影响动脉粥样硬化进程的重要机制之一。因此，我们检测PARP抑制剂是否能够降低Hhcy诱导的动脉粥样硬化实验动物模型粥样斑块的面积。方法健康雄性6周龄纯合子ApoE基因缺陷小鼠（ApoE-／-）随机分为普通饮食组或高甲硫氨酸饮食组，每组再分别给予隔天腹腔注射PBS溶解的10mg／kg PARP抑制剂3-aminobenzamide（3-AB）或生理盐水，持续12周。检测血脂及血浆同型半胱氨酸（homocysteine，Hcy）含量，分离心脏及主动脉，测量主动脉窦斑块面积，通过实时定量PCR及Western Blot等方法检测主动脉斑块局部氧化应激相关NADPH氧化酶亚单位p47^phox含量、PARP活性及表达。结果高甲硫氨酸饮食诱导ApoE-／-小鼠产生Hhcy，促进氧化应激相关DNA损伤及PARP活化，显著增加粥样斑块面积。喂以普通饮食的ApoE-／-小鼠能够自发产生高脂血症，PARP抑制剂3-AB虽然对其抑制效果不明显，却能在不影响血浆同型半胱氨酸水平和脂质含量的情况下，有效抑制Hhcy诱导的PARP活化，显著降低ApoE-／-小鼠粥样斑块面积达40％。结论PARP抑制剂3-AB显著降低Hhcy诱导的ApoE-／-小鼠动脉粥样硬化斑块面积，可作为有效抑制粥样斑块进程的治疗方法之一。第二部分PARP抑制剂抑制动脉粥样硬化斑块内NF-κB活化及后续炎症因子表达目的检测多聚ADP核糖合成酶[Poly（ADP-ribose）polymerase，PARP]抑制剂3-aminobenzamide（3-AB）是否能够抑制高同型半胱氨酸血症（hyperhomocysteinemia，Hhcy）诱导的ApoE基因缺陷小鼠（ApoE-／-）动脉粥样硬化斑块内核因子-κB（nuclearfactor-κB，NF-κB）核转移及后续炎症因子的表达。方法健康雄性6周龄纯合子ApoE基因缺陷小鼠（ApoE-／-）随机分为普通饮食组或高甲硫氨酸饮食组，每组再分别给予隔天腹腔注射PBS溶解的10mg／kg PARP抑制剂3-aminobenzamide（3-AB）或生理盐水，持续12周。检测血脂及血浆同型半胱氨酸（homocysteine，Hcy）含量，分离心脏及主动脉，通过Western Blot，免疫组化及实时定量PCR等方法检测主动脉斑块局部胞浆、胞核内及总蛋白中的NF-κB p65、κB抑制蛋白（IκB）及磷酸化IκB（p-IκB）的表达，检测斑块局部炎症因子VCAM-1和MCP-1的表达。结果高甲硫氨酸饮食喂养的ApoE-／-小鼠产生Hhcy，促进主动脉斑块细胞胞核内NF-κB p65及总蛋白中p-IκB的表达增高，即促进NF-κB的核转移，继而提高斑块局部VCAM-1和MCP-1的表达；喂以普通饮食的ApoE-／-小鼠能够自发产生高脂血症，PARP抑制剂3-AB虽然对其抑制效果不明显，却能在不影响血浆同型半胱氨酸水平和脂质含量的情况下，有效抑制伴有Hhcy小鼠NF-κB核内表达及p-IκB的含量，增加胞浆内NF-κB的表达，从而显著降低粥样斑块内炎症因子的表达。结论PARP抑制剂3-AB显著抑制Hhcy诱导的ApoE-／-小鼠动脉粥样硬化斑块内NF-κB核转移及后续炎症因子的表达。第三部分PARP抑制剂促进动脉粥样硬化斑块内细胞凋亡目的检测多聚ADP核糖合成酶[Poly（ADP-ribose）polymerase，PARP]抑制剂3-aminobenzamide（3-AB）对高同型半胱氨酸血症（hyperhomocysteinemia，Hhcy）诱导的ApoE基因缺陷小鼠（ApoE-／-）动脉粥样硬化斑块内细胞凋亡的影响。方法健康雄性6周龄纯合子ApoE基因缺陷小鼠（ApoE-／-）随机分为普通饮食组或高甲硫氨酸饮食组，每组再分别给予隔天腹腔注射PBS溶解的10mg／kg PARP抑制剂3-aminobenzamide（3-AB）或生理盐水，持续12周。检测血脂及血浆同型半胱氨酸（homocysteine，Hcy）含量，分离心脏及主动脉，检测粥样硬化斑块内细胞凋亡情况及相关caspase-3酶蛋白表达情况。结果高甲硫氨酸饮食喂养的ApoE-／-小鼠产生Hhcy，促进主动脉斑块内caspase-3的活化及细胞凋亡；PARP抑制剂3-AB虽然对普通饮食小鼠影响效果不明显，却能在不影响血浆同型半胱氨酸水平和脂质含量的情况下，明显促进Hhcy诱导小鼠斑块内35-KD caspase-3原始蛋白的消耗，即促进caspase-3的活化，进而促进斑块内细胞死亡模式更加倾向于凋亡。结论PARP抑制剂3-AB能够促进Hhcy诱导的ApoE-／-小鼠动脉粥样硬化斑块内细胞凋亡。更多还原

【Abstract】 PartⅠPARP Inhibition Decreases Atherosclerotic Lesion size inApoE-/- Mice with HyperhomocysteinemiaObjective Hyperhomocysteinemia （Hhcy） is an important and independent risk factor foratherosclerosis.Recent studies have shown that Poly （ADP-ribose） polymerase （PARP）activation may be associated with Hhcy-induced endothelial dysfunction,which is animportant mechanism for Hhcy to affect atherosclerotic progress.Thus we investigatedwhether PARP inhibitors may decrease the atherosclerotic plaque size in anHhcy-induced experimental animal model with atherosclerosis.Methods Six-week-old homozygous apolipoprotein E-deficient （ApoE-/-） male mice fedwith a normal diet or high methionine-diet were randomly received intraperitonealinjections of 10mg/kg 3-aminobenzamide （3-AB,a kind of PARP inhibitor） dissolvedin PBS,or physiological saline every other day for 12 weeks.Plasma homocysteine（Hcy） levels and lipids contents were measured.Atherosclerotic lesion sizes,thephosphorylation of p47^phox subunit of NADPH oxidase and the expression of PARPprotein and PARP activity were detected. Results Our data demonstrated that the ApoE-/- mice fed with high methionine-dietgenerated Hhcy,which subsequently promoted the oxidative stress-associated DNAdamage and PARP activation,increased atherosclerotic lesion size significantly.Although PARP inhibition by 3-AB did not markedly inhibit the plaque developmentin ApoE-/- mice with spontaneous hyperlipidemia by feeding with a normal diet,itsignificantly reduced atherosclerotic lesion size by 40% in Hhcy-inducedatherosclerosis without affecting plasma homocysteine levels and lipid contents,effectively suppressed PARP activation.Conclusions Our results suggest that PARP inhibition attenuates the atherosclerotic plaquesize in the hyperhomocysteinemic conditions,indicates 3-AB may prove beneficial forthe treatment of atherosclerosis. PartⅡPARP Inhibition Suppress Nuclear Translocation ofNF-κB and Subsequent Production of Inflammatory Factors withinAtherosclerotic PlaquesObjective To investigate whether PARP [Poly （ADP-ribose） polymerase] inhibitor3-aminobenzamide （3-AB） may suppress the nuclear translocation of nuclear factor（NF）-κB and subsequent production of inflammatory factors in the plaque lesions in anHhcy （hyperhomocysteinemia）-induced experimental animal model withatherosclerosis.Methods Six-week-old homozygous apolipoprotein E-deficient （ApoE-/-） male mice fedwith a normal diet or high methionine-diet were randomly received intraperitonealinjections of 10mg/kg 3-aminobenzamide （3-AB,a kind of PARP inhibitor） dissolvedin PBS,or physiological saline every other day for 12 weeks.Plasma homocysteine（Hcy） levels and lipids contents were measured.Then hearts and aortas were removedrapidly.The thoracic and abdominal aorta was quick-frozen in nitrogen for laterextraction of protein and RNA.The expression of NF-κB p65、inhibitors ofκB （IκB）,and phospho-inhibitors ofκB （p-IκB） in total proteins,cytoplasmic extracts andnuclear extracts was measured.The expression of VCAM-1 and MCP-1 withinplaques was also detected by Immunohistochemistry analysis,Westem Blot analysisand real-time reverse-transcription PCR.Results Our data demonstrated that the ApoE-/- mice fed with high methionine-dietgenerated Hhcy,which significantly increased the expression of NF-κB p65 in nuclearextracts and p-IκB in total proteins,promoted the nuclear translocation of NF-κB,thereby increased the subsequent production of inflammatory factors within plaquessuch as VCAM-1 and MCP-1.Although PARP inhibition by 3-AB did not markedlyinhibit the nuclear translocation of NF-κB in ApoE-/- mice with spontaneous hyperlipidemia by feeding with a normal diet,it significantly reduced the activity ofNF-κB in Hhcy-induced atherosclerosis without affecting plasma homocysteine levelsand lipid contents,effectively suppressed subsequent production of VCAM-1 andMCP-1.Conclusions Our results suggest that PARP inhibition suppress the nuclear translocation ofNF-κB and subsequent production of inflammatory factors in the atheroscleroticplaques in the hyperhomocysteinemic conditions. PartⅢPARP Inhibition Promotes Apoptosis withinAtherosclerotic PlaquesObjective To investigate whether PARP [Poly （ADP-ribose） polymerase] inhibitor3-aminobenzamide （3-AB） effects on the apoptosis within the atherosclerotic plaquesin an Hhcy （hyperhomocysteinemia）-induced experimental animal model withatherosclerosis.Methods Six-week-old homozygous apolipoprotein E-deficient （ApoE-/-） male mice fedwith a normal diet or high methionine-diet were randomly received intraperitonealinjections of 10mg/kg 3-aminobenzamide （3-AB,a kind of PARP inhibitor） dissolvedin PBS,or physiological saline every other day for 12 weeks.Plasma homocysteine（Hcy） levels and lipids contents were measured.Then hearts and aortas were removedrapidly.The thoracic and abdominal aorta was quick-frozen in nitrogen for laterextraction of protein and RNA.Apoptotic cells were detected by Apoptosis DetectionKit;the expression of caspase-3 within plaques was measured by Western Blotanalysis.Results Our data demonstrated that the ApoE-/- mice fed with high methionine-dietgenerated Hhcy.The apoptotic cells were highly increased in Hhcy condition whichwas detected by Apoptosis Detection Kit.Although PARP inhibition by 3-AB did notmarkedly have effect on apoptosis in ApoE-/- mice with spontaneous hyperlipidemiaby feeding with a normal diet,it significantly incrased the depletion of na（?）ve caspase-3, aggravated the caspase-3 activation,made the mode of cell death be prone to apoptosisin Hhcy-induced atherosclerosis without affecting plasma homocysteine levels andlipid contents.Conclusions Our results suggest that PARP inhibition aggravated the caspase-3 activation,promoted the apoptosis in the atherosclerotic plaques in the hyperhomocysteinemicconditions.更多还原