【作者】 魏艳玲；

【导师】 李晓辉；

【作者基本信息】 第三军医大学 ， 药理学， 2010， 博士

【摘要】 心血管疾病具有高患病率、高致残率、高死亡率的特点,其发病率和死亡率已超过肿瘤性疾病而跃居第一。心肌血管重构是心血管疾病发生发展的病理基础,心肌重构能够导致猝死、心肌梗死、心律失常等心血管意外事件,其中由重构而心衰是心血管疾病患者死亡的主要原因之一。血管重构是引起血管疾病和循环功能紊乱的病理基础,是引起高血压各种严重并发症的关键环节。对于心肌血管重构的病因研究目前主要包括机械刺激、神经体液因素等,仅仅是围绕成年个体展开,尚未完全阐明。近年来包括我室在内的关于炎症在心血管疾病发病中重要作用的发现是对心血管疾病病因寻找中最重要的进展之一,我室经过近7年的孕期炎症免疫刺激与子代心血管疾病之间关系的探索研究,成功地建立了大鼠孕期腹腔注射脂多糖(lipopolysaccharide,LPS)及酵母多糖(zymosan)引起子代高血压和体重增加的动物模型,并在一系列实验的基础上,提出了高血压病因探索的新假说:原发性高血压的形成可能要追溯来自于孕期的炎症免疫刺激所致,这一发现作为特别首报发表于2007年期刊《Acta Pharmacologica Sinica》。进一步研究发现子代动物出现了肾素-血管紧张素及内皮素系统的高表达,这一系列神经体液因素的改变,是心肌、血管重构发生的重要危险因素,因此,我们推测孕期的炎症免疫刺激可能会导致子代大鼠的心肌、血管重构,从而可能引发患者更为严重的心血管事件发生甚至死亡,据此我们在原有实验基础上,将所提出的假说拟进行范围上的延伸,即:原发性高血压、心肌血管重构等心血管疾病的形成可能要追溯来自于孕期的炎症免疫刺激所致。既往对于高血压、心肌血管重构等心血管疾病的预防和控制都是在病人成年以后,疾病已经发生了,才开始进行对症处理,该理论的提出将会扩大以往人们对于原发性高血压、心肌血管重构预防和治疗的时间窗。方法:1. 24只通过检查阴栓获得准确妊娠时间的怀孕SD大鼠,随机分为3组,三组孕鼠分别在妊娠8、10、12天,腹腔注射0.79mg/kgLPS(LPS组);在孕第8、10、12天腹腔注射LPS 0.79mg/kg,第8-14天注射LPS后每天腹腔注射PDTC 100 mg/kg(LPS+PDTC组);在孕第8-14天腹腔注射无菌生理盐水1ml无菌生理盐水(对照组);因前期实验发现孕期8-14天单纯注射PDTC,对于孕鼠和仔鼠均没有任何影响,故此本实验未设PDTC组。子代大鼠随机选取用于该实验(雄性:对照组,n=18;LPS组,n=18;L+P组,n=18;雌性:对照组,n=18;LPS组,n=18;L+P组,n=18)。2.通过以下方法对孕期炎症免疫刺激及同时给予NF-κB抑制剂后导致子代大鼠的心血管功能变化进行评价:①大鼠血压的测定采用无创尾套法;②超声心动图评价大鼠的左心室、右心室结构和心功能;③心肥厚指数及左心室肥厚指数的测定评价大鼠的心室肥厚程度;④主动脉重构的评价采用病理切片HE染色,图像分析测定大鼠主动脉血管中层壁厚(MT)和内径(LD)并计算二者比值(MT/LD)的方法;⑤HE染色,光镜下观察大鼠心肌、血管平滑肌组织形态学改变;⑥透射电镜观察大鼠心肌细胞、血管平滑肌细胞超微结构的改变。3.用放射免疫法检测血浆ET-1水平、硝酸还原酶法测定血清NO含量、用TdT介导的缺口末端标记法(TUNEL)检测左心室心肌细胞凋亡的变化,用免疫组织化学技术观察心肌、血管平滑肌NF-κBp65的激活、血管平滑肌细胞增殖细胞核抗原(PCNA),左心室Bcl-2抗原和Bax抗原表达的变化。结果:1.对照组、LPS组、L+P组每只孕鼠生育仔鼠的数量、雌雄比例及胎鼠出生体重,各组之间均没有明显统计学差异(P>0.05)。2.孕期炎症免疫刺激对于仔鼠血压的影响: LPS组仔鼠从2月龄起,同对照组仔鼠相比较,无论雌雄,血压均出现具有统计学意义的升高(P<0.05),该升高趋势一直持续到8月龄,在6月龄达到高血压标准,而在L+P组,则有效阻止了其血压统计学意义上的升高。3.孕期炎症免疫刺激对于仔鼠心室重构的影响:(1) LPS模型组仔鼠有明显的左心室重构现象:与正常组相比,LPS模型组仔鼠无论雌雄,在4月龄时出现了左室后壁厚度(PWT)的增厚,8月龄时则出现了左室后壁厚度(PWT)、室间隔厚度(IVST)、心肌肥厚指数(HMI)、左心室肥厚指数(LVMI)明显增高(p<0.05);而在L+P组,则有效改善了这一改变。(2) LPS模型组仔鼠有明显的心肌舒张功能障碍:与正常组相比,LPS模型组仔鼠无论雌雄,在8月龄时均出现了E/A指数、Tei指数的升高(p<0.05);而在L+P组,则有效改善了这一改变。(3)病理及电镜:①LPS模型组仔鼠,无论雌雄,心肌组织形态及心肌细胞超微结构受到损伤,光镜下可见心肌细胞明显浊肿,空泡样变化;细胞核肥大或固缩;细胞内容物呈颗粒状,断裂融合。电镜下可见心肌细胞肿大,核肥大、畸形、溶解等呈不规则变化;肌浆网扩张;线粒体增生明显,排列紊乱,不同程度(轻、中、重)肿胀,内有空泡形成。②在L+P组,孕期PDTC腹腔注射,能明显改善上述病理改变及心肌细胞超微结构,心肌组织形态及心肌细胞超微结构基本正常。4.孕期炎症免疫刺激对于仔鼠血管重构的影响:(1) LPS模型组仔鼠血管平滑肌存在明显重构现象:与对照组相比,4月龄和8月龄时,LPS组胸主动脉MT明显增加、LD也明显增大,而MT/LD值也明显增高(p<0.05),表明LPS组大鼠胸主动脉在4月龄时即存在着明显的血管重构现象。而在L+P组仔鼠则明显改善了这一现象。(2)病理及电镜:①LPS模型组仔鼠,无论雌雄,平滑肌细胞肥大增生明显,排列紊乱,细胞核明显增多。电镜下可见管壁中膜平滑肌细胞明显肥大,核形态不规则,核内常染色质丰富,核浆间隙增大,肌膜下吞饮小泡减少,肌丝成分少,细胞外纤维成分增多,平滑肌细胞内线粒体肿胀,密度低,呈现嵴溶解和空泡样变性,粗面内质网增多。②在L+P组,孕期PDTC腹腔注射,能明显改善上述病理改变及血管平滑肌细胞超微结构,血管平滑肌组织形态及平滑肌细胞超微结构基本正常。5.对孕期炎症免疫刺激导致仔鼠心肌血管重构的机制研究;(1) LPS模型组子代大鼠第2、4、6、8月龄血浆ET-1较对照组均显著升高(P<0.05),至8月龄子鼠出现心肌血管重构时,其水平升高加快,LPS组血浆ET-1水平为(151.07±19.03)μmol/L,较对照组(78.56±12.39)μmol/L和L+P组的(84.78±5.71)μmol/L明显升高接近1倍。(2) LPS模型组大鼠的心肌和血管平滑肌NF-κBp65被激活,从胞浆中转移到细胞核,阳性细胞比率明显较对照组和L+P组增多(P<0.01),在正常心肌和血管平滑肌组织中,则基本没有NF-κBp65表达。(3) LPS组子代大鼠左心室心肌细胞TUNEL阳性心肌细胞的数量较对照组和L+P组明显增多(P<0.01),左心室心肌细胞的Bcl-2抗原的表达明显下调(P<0.01)、而心肌细胞的Bax抗原的表达则明显上调(P<0.01)。(4)子代大鼠第2、4、6、8月龄血清NO各组之间较对照组无统计学差异(P>0.05),与对照组和L+P组相比,LPS组子代大鼠胸主动脉PCNA表达增加显著增加(P<0.01),说明其血管平滑肌细胞增殖明显。结论:1.孕期暴露于LPS后,子代大鼠6月龄时高血压形成,成功的建立了孕期炎症刺激导致子代高血压的大鼠动物模型。2.孕期暴露于LPS后,子代大鼠4月龄时出现了向心性重构现象,8月龄出现了向心性肥厚并伴有心肌舒张功能障碍,说明孕期LPS刺激导致了子代大鼠心肌重构,其发生于高血压形成之前并在高血压形成后明显加重。3.孕期暴露于LPS后,子代大鼠4月龄时出现了血管重构,8月龄时重构更加明显,表明孕期LPS刺激导致了子代大鼠的血管重构,同心肌重构一样发生于高血压形成之前,并在高血压形成后明显加重。4.孕期的炎症免疫刺激导致了子代大鼠心肌、血管重构的发生,其发病机制可能与血浆ET-1水平的升高和NF-κBp65的激活有关;其中子代大鼠心肌重构的机制,可能还与心肌细胞凋亡增多有关;血管重构的机制,可能还与ET-1/NO失衡,血管平滑肌细胞增殖增多有关。5.在孕期暴露于LPS同时给予NF-κB的抑制剂PDTC的子代大鼠,有效改善了血压升高、心肌血管重构的现象,NF-κB可能是生命早期有效防治子代心血管疾病发生的靶点之一。更多还原

【Abstract】 BACKGROUD: Cardiovascular disease ranks among the leading causes of morbidity and mortality in adult populations in most countries in the world. Hypertension is a major risk factor of cardiovascular diseases and its underlying pathogenetic mechanisms are still not well elucidated. Significant progress in understanding the etiology of cardiovascular disease has come from recent recognition that inflammation plays a key role in its development. Accumulating clinical evidences have implied that inflammation was also related to hypertension to some extent. Sesso and his colleagues reported a positive relationship between increased serum levels of C-reactive protein and the risk for development of incident hypertension in participants of the Women’s Health Study. Bautista et al discovered the serum level of IL-6 elevated in hypertensive patients.Moreover, Events in utero appear to have a significant role in the development of cardiovascular dysfunction in adulthood. Emerging evidence indicates that there are multiple causes of hypertension and that adverse conditions experienced in utero can increase the incidence of hypertension in adulthood. Systemic inflammatory response during pregnancy represents one form of stressful event for the fetus. In a previous study, we showed that prenatal exposure to lipopolysaccharide (LPS), commonly used to mimic prenatal infection, resulted in hypertension in rat offspring. We mentioned the hypothesis that maternal inflammation could induce hypertension in offspring. However, does the stressful event of maternal inflammation only result in hypertension in offspring? Are there anyother cardiovascular events come out accompanied with hyepertension? Therefore, in the present study, we investigated the prolonged hypothesis that, as a common pathophysiological phenomenon, maternal inflammatory stress can not only cause hypertension but also can cause hypermyotrophy in adult offspring.AIM: The study is designed to study the effects of prenatal exposure to LPS on the function and construction of cardiac and vascular in offspring in rats. In addition, the effects of an inhibitor of the nuclear transcription factor NF-κB (pyrrolidine dithiocarbamate, PDTC) on this process were assessed.METHODS:1. Twenty-four time-mated Sprague-Dawley (SD) rats’dams were randomly divided into three groups: a control group, an LPS group, and an LPS+PDTC group. The rats in these groups were intraperitoneally administered vehicle, 0.79mg /kg LPS, or LPS plus 100mg/kg PDTC, respectively. LPS was given on the 8th, 10th and 12th days, whereas PDTC was given from the 8th to the 14th day during gestation. The pups were randomly chosen to be included in this study (males: controls, n=18; LPS-treated, n = 18; LPS+PDTC-treated, n=18; females: controls, n =18; LPS-treated, n = 18; LPS+PDTC-treated, n=18).2. The pups’systolic blood pressure (SBP) was measured once every tow months from the 2th month to 8th month with tail-cuff method. At the end of 4th and 8th month of the experiment, the heart mass index (HMI) and the LV mass index (LVMI) was evaluated. In addition, LV and RV structure and function, vascular structure and their microstructure and ultrastructure were examined using echocardiography, routine light microscopy and transmission electron microscopy, respectively.3. The blood plasma level of endothelin-1 (ET-1) was measured by RIA, and the serum level of nitric oxide (NO) was measured by nitrate reductase method.4. The expression of NF-κBp65, Bcl-2 and Bax antigen in left ventricles and the NF-κBp65, PCNA on VSMCs were done through immunohistochemical technique. The TdT mediated nick dUTP end labeling (TUNEL) technique was used to observe the apoptosis of cardiomyocytes in left ventricles.RESULTS1. There were no significant differences in the number of progeny per dam (P>0.05). Meanwhile, no significant differences were discovered about the ratio of male births to female births in each litter (P>0.05). The body weights of newborn pups did not differ much between the LPS, LPS+PDTC and control groups (P>0.05).2. All offspring to LPS exposed dams showed increased systolic blood pressure (P<0.05). They reached the standard of hypertension at the end of 6th month.3. As compared with the control group, prenatal exposure to LPS increased LV posterior wall thickness(PWT) on 4th month in offspring, and the thickness of the interventricular septum(IVST), LVMI, E/A index and Tei index(P<0.05) on 8th month in offspring were singnificantly increased in LPS group.There were no change in LPS+PDTC group(P>0.05).4. In prenatal LPS-treated offspring, the microstructure of left ventricles was as follows: the cardiomyocytes hypertrophy, interstitium fibrosis and myofibrilla lined up in disorder. However, prenatal treated with PDTC improved these morphological changes to some extent.5. The MT, LD and the media/lumen ratios (MT/LD) were increased significantly in offspring with LPS-exposed dam by comparison of control (P<0.05) on 4th month. However, there were no change in LPS+PDTC group (P>0.05).6. In prenatal LPS-treated rats, the ultrastructure of left ventricular cadiocytes was observed as the mitochondria swell and medullary sheath-like degeneration, the sarcoplasmic reticulum enlarged and the myofilaments dissolved and the Z lines broken. However, prenatal treated with PDTC improved these morphological changes.7. At the end of the 2th, 4th, 6th, 8th month, the blood plasma level of ET-1 of LPS-exposed dam was higher than the control (P<0.05). Until the 8th month, the level of ET-1 was (151.07±19.03)μmol/L, which was nearly doubled comparing with the control (78.56±12.39)μmol/L and LPS+PDTC group (84.78±5.71)μmol/L (P<0.01).8. The NF-κBp65 was activited in left ventricular cadiocytes and VSMCs in LPS group(P<0.01). However, there was almost no expression in control and LPS+PDTC group.9. As compared with the control group, prenatal exposure to LPS induced the apoptosis of cardiomyocytes and the over-expression of the Bax antigen and the low-expression of Bcl-2 antigen in the left ventricle tissues, respectively (P<0.01). However, prenatal treated with PDTC markedly attenuated the above changes.10. Compared with the control, the VSMCs proliferation activity was obviously increased in LPS group (P<0.01). While there were no change in the LPS+PDTC group.11. There were no significant differences in the serum level of NO in offspring among LPS , LPS+PDTC group and control group at the end of the 2th, 4th, 6th, 8th month (P>0.05).CONCLUSION1. Prenatal exposure to LPS results in left ventricle and vasuclar remodeling on the 4th month in offspring in rats.2. The blood plasma level of ET-1 and the expression of NF-κBp65 of LPS-exposed group is higher than the control. These changes might be the potential underlying mechanism of development of hypermyotrophy and vascular remodeling.3. Prenatal LPS exposure induced left ventricle remodeling. The mechanism is associated with the apoptosis in left ventricular cardiomyocytes to some extent. The mechanism may be relative to the overexpression of Bax antigen, low expression of Bcl-2 antigen.4. Prenatal LPS exposure induced vascular remodeling, which is relative to the impressing effect on the proliferation of VSMCs and the disequilibrium between the serum level of ET-1 and NO.5. There were no hypertension, hypermyotrophy and vascular remodeling in LPS+PDTC group. It showed that prenatal treated with inhibitor of the nuclear transcription factor NF-κB (pyrrolidine dithiocarbamate, PDTC) could reversed the effection of maternal inflammation on cardiovascular diseases in offapring in rats.更多还原