白藜芦醇对小鼠败血症心脏损害保护作用的机制研究

发布时间：2018-06-12 01:10

本文选题：脂多糖 + 心力衰竭　；参考：《吉林大学》2017年博士论文

【摘要】：研究背景:败血症心肌病(SIC)严重影响败血症预后。目前对SIC缺乏有效的治疗手段,寻找SIC的防治方法至关重要。研究证实,心肌肌浆网钙三磷酸腺苷酶(SERCA2a)功能下调是SIC发生的重要原因,但其下调的机制没有阐明。受磷蛋白(PLB)是调控SERCA2a功能的重要蛋白,在机体内有两种存在形式,单聚体monomer和五聚体pentamer。单聚体能直接与SERCA2a结合,抑制SERCA2a的Ca2+摄取功能,而五聚体被认为是PLB在体内的储存形式,不具有生物功能和活性[6]。上述两种PLB存在形式在体内保持动态平衡。然而,目前还没有研究指出败血症是否能影响心肌PLB的存在形式进而下调SERCA2a功能。白藜芦醇(RSV)对多种心血管疾病中有治疗作用。我们的预实验发现RSV能预防SIC的发生。在这基础上,我们拟研究RSV是否对SIC有治疗作用,并探讨其机制是否与纠正SERCA2a-PLB异常有关。研究目的:1.RSV是否能治疗SIC。2.RSV治疗SIC的机制是否与纠正SERCA2a-PLB有关。3.RSV调控SERCA2a-PLB的机制。研究方法:采用腹腔注射(i.p.)大肠杆菌脂多糖(LPS,sigma corporation,055:B5 serotype,6 mg/kg)的方法建立小鼠SIC模型。雄性C57BL/6J小鼠(8周龄)分为4组,依次是对照组,RSV组,LPS组和LPS+RSV组。建模成功后,用动物心脏超声机测量小鼠心功能指标如:左室射血分数(EF%)、短轴缩短率(FS%)和左室舒张末期内径(LVEDD)等。并分离心肌细胞,测量不同条件下心肌细胞收缩力、钙瞬变曲线和活性氧水平。应用H-E染色观察心肌组织结构是否改变,使用免疫组织化学法检测心肌切片CD45+炎症细胞浸润数量。应用酶-抗体夹心法(ELISA)测量血浆Tn I和TNF-α、心肌ATP、MDA含量和心肌细胞/组织SERCA2a ATP酶活性。应用Western blot和或Real time PC R法检测心肌4-HNE、SERCA2a、NCX、Ry R2、IP3R、TNF-α、PLB、cleaved Caspase-3、Pgc-1α、Nrf-2及下游蛋白(CAT和NQO-1)表达情况。研究结果:1.LPS使EF%、FS%下降50%以上,伴有LVEDD显著增大,而在LPS注射后2小时用RSV治疗能显著纠正上述异常。离体细胞实验,LPS使细胞收缩幅度和钙瞬变峰值显著下降,收缩后恢复时间和钙瞬变衰减时间显著延长,而RSV能明显纠正心肌细胞机械功能和钙释放异常。2.血浆Tn I和心肌cleaved Caspase 3表达在LPS处理的小鼠中没有升高。血浆TNF-α在LPS处理的小鼠中显著升高,伴有心肌组织CD45+炎性细胞浸润明显增加,而心肌组织TNF-α表达水平在各组小鼠之间没有显著差异。RSV不降低血浆TNF-α水平,不减少心肌组织CD45+炎性细胞数量,心肌NCX、Ry R2和IP3R表达水平在各组之间无差异。心肌SERCA2a在LPS处理的小鼠中表达下降,活性降低,而RSV治疗可以上调SERCA2a表达,改善活性。3.LPS使心肌monomer-PLB增多,pentamer-PLB减少,这与LPS引起的氧化还原紊乱有关。LPS上调心肌4-HNE和MDA的含量,下调Nrf-2及下游蛋白(CAT和NQO-1)的表达,RSV治疗能明显减轻上述异常。离体细胞实验进一步发现:RSV能起到和一种ROS清除剂,N-乙酰半胱氨酸相似的作用,减少心肌细胞ROS水平,恢复受到抑制的SERCA2a功能,促进PLB从monomer转变为pentamer。结论:1.RSV能治疗SIC。2.RSV治疗SIC的机制与促进PLB从单聚体向五聚体转变,进而上调心肌SERCA2a功能有关。3.RSV调控PLB构象改变的机制与激活Nrf-2,调控细胞氧化还原有关。创新点:1.RSV通过激活Nrf-2,促进PLB从单聚体向五聚体转变,恢复受抑制的SERC A2a功能,对SIC起到治疗作用。2.本研究有望为白藜芦醇(或Nrf-2的直接激动剂)应用于临床防治SIC提供理论依据。综上所述,本研究首次阐明了RSV治疗SIC的一种新的机制:RSV通过激活Nrf-2及下游基因,调节细胞内氧化还原状态,促进心肌PLB从monomer向pentamer转变,恢复受抑制的SERCA2a功能。
[Abstract]:Background: septicaemia cardiomyopathy (SIC) seriously affects the prognosis of septicemia. Currently, the lack of effective treatment for SIC and the search for the prevention and treatment of SIC are essential. The study confirms that the downregulation of SERCA2a function is an important cause of SIC, but the mechanism of its downregulation is not clarified. The regulation of phosphor protein (PLB) is the regulation. The important protein of SERCA2a function has two forms of existence in the body. Mono monomer and five polymer pentamer. monopolymer can directly combine with SERCA2a to inhibit the Ca2+ uptake of SERCA2a, and the five polymer is considered to be the form of PLB in the body, and does not have biological function and active [6]. in the two PLB existence forms in the body. However, there has been no study on whether septicemia can affect the presence of PLB in the myocardium and further down the SERCA2a function. Resveratrol (RSV) has a therapeutic effect on a variety of cardiovascular diseases. Our pre experiment found that RSV can prevent the occurrence of SIC. On this basis, we will study whether RSV has a therapeutic effect on SIC and is discussed. Whether the mechanism is related to the correction of SERCA2a-PLB abnormalities. The purpose of this study is whether 1.RSV can treat the mechanism of SIC.2.RSV for the treatment of SIC and the mechanism of SERCA2a-PLB related.3.RSV regulation of SERCA2a-PLB. Methods: the method of establishing mice by intraperitoneal injection of (i.p.) coliform lipopolysaccharide (LPS, sigma Corporation, 055:B5, 6) SIC model. Male C57BL/6J mice (8 weeks of age) were divided into 4 groups, in turn, the control group, the RSV group, the LPS group and the LPS+RSV group. After the successful modeling, the cardiac function indexes such as left ventricular ejection fraction (EF%), short axis shortening rate (FS%) and left ventricular end diastolic diameter (LVEDD) were measured by animal echocardiography. Myocardial cells were isolated and measured under different conditions. Cell contractile force, calcium transient curve and active oxygen level. H-E staining was used to observe the changes in myocardial tissue structure. The number of CD45+ inflammatory cells infiltrated in myocardial slices was detected by immunohistochemistry. The plasma Tn I and TNF- alpha were measured by enzyme antibody sandwich (ELISA), ATP, MDA content and SERCA2a ATP enzyme activity of myocardial cells / tissues. Western blot and Real time PC R method were used to detect the expression of 4-HNE, SERCA2a, NCX, Ry R2. In vitro cell test, LPS made the cell contraction amplitude and calcium transient peak significantly decreased, after the contraction time and calcium transient attenuation time significantly prolonged, and RSV can obviously correct the myocardial cell mechanical function and calcium release abnormal.2. plasma Tn I and myocardial cleaved Caspase 3 expression in LPS no increase in mice. The plasma TNF- alpha is in LPS. In the mice, the CD45+ inflammatory cell infiltration in the myocardium was significantly increased, and the expression level of TNF- alpha in the myocardium was not significantly different between the mice in each group,.RSV did not reduce the level of plasma TNF- a, did not reduce the number of CD45+ inflammatory cells in the myocardium, and there was no difference between the NCX, Ry R2 and IP3R expression levels of myocardium in each group. The myocardial SER was no difference between each group. The myocardium SER was no difference between each group. The expression of CA2a in the mice treated with LPS decreased and the activity decreased, while RSV therapy could increase the expression of SERCA2a, improve the activity of.3.LPS to increase the monomer-PLB and pentamer-PLB, which is associated with the redox disorder caused by LPS, up regulating the content of 4-HNE and MDA in the myocardium and down regulation of the expression of Nrf-2 and downstream proteins. In vitro cell experiments further found that RSV could act as a ROS scavenger, a similar effect of N- acetyl cysteine, reduce the ROS level of cardiac myocytes, restore the inhibited SERCA2a function, and promote the transformation of PLB from monomer to pentamer. conclusion: 1.RSV can treat SIC.2.RSV SIC mechanism and promote PLB from monomers. Transformation to five polymer, and then up-regulated the mechanism of myocardial SERCA2a function related to.3.RSV regulation of PLB conformation changes and activation of Nrf-2 to regulate cell oxidation and reduction. Innovation: 1.RSV promotes PLB from monopolymer to five polymer by activating Nrf-2 to restore the inhibited SERC A2a function, and the therapeutic effect of SIC is expected to be resveratrol. Alcohol (or the direct agonist of Nrf-2) is used to provide theoretical basis for the clinical prevention and treatment of SIC. To sum up, this study is the first to clarify a new mechanism for the treatment of SIC by RSV: RSV regulates the redox state of intracellular redox by activating Nrf-2 and downstream genes, promoting the transformation of PLB from monomer to pentamer, and restoring the inhibited SERCA2a function.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R515.3;R542.2

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