本文選題：脂多糖 + 心力衰竭　； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：研究背景:敗血癥心肌病(SIC)嚴重影響敗血癥預(yù)后。目前對SIC缺乏有效的治療手段,尋找SIC的防治方法至關(guān)重要。研究證實,心肌肌漿網(wǎng)鈣三磷酸腺苷酶(SERCA2a)功能下調(diào)是SIC發(fā)生的重要原因,但其下調(diào)的機制沒有闡明。受磷蛋白(PLB)是調(diào)控SERCA2a功能的重要蛋白,在機體內(nèi)有兩種存在形式,單聚體monomer和五聚體pentamer。單聚體能直接與SERCA2a結(jié)合,抑制SERCA2a的Ca2+攝取功能,而五聚體被認為是PLB在體內(nèi)的儲存形式,不具有生物功能和活性[6]。上述兩種PLB存在形式在體內(nèi)保持動態(tài)平衡。然而,目前還沒有研究指出敗血癥是否能影響心肌PLB的存在形式進而下調(diào)SERCA2a功能。白藜蘆醇(RSV)對多種心血管疾病中有治療作用。我們的預(yù)實驗發(fā)現(xiàn)RSV能預(yù)防SIC的發(fā)生。在這基礎(chǔ)上,我們擬研究RSV是否對SIC有治療作用,并探討其機制是否與糾正SERCA2a-PLB異常有關(guān)。研究目的:1.RSV是否能治療SIC。2.RSV治療SIC的機制是否與糾正SERCA2a-PLB有關(guān)。3.RSV調(diào)控SERCA2a-PLB的機制。研究方法:采用腹腔注射(i.p.)大腸桿菌脂多糖(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周齡)分為4組,依次是對照組,RSV組,LPS組和LPS+RSV組。建模成功后,用動物心臟超聲機測量小鼠心功能指標如:左室射血分數(shù)(EF%)、短軸縮短率(FS%)和左室舒張末期內(nèi)徑(LVEDD)等。并分離心肌細胞,測量不同條件下心肌細胞收縮力、鈣瞬變曲線和活性氧水平。應(yīng)用H-E染色觀察心肌組織結(jié)構(gòu)是否改變,使用免疫組織化學(xué)法檢測心肌切片CD45+炎癥細胞浸潤數(shù)量。應(yīng)用酶-抗體夾心法(ELISA)測量血漿Tn I和TNF-α、心肌ATP、MDA含量和心肌細胞/組織SERCA2a ATP酶活性。應(yīng)用Western blot和或Real time PC R法檢測心肌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)表達情況。研究結(jié)果:1.LPS使EF%、FS%下降50%以上,伴有LVEDD顯著增大,而在LPS注射后2小時用RSV治療能顯著糾正上述異常。離體細胞實驗,LPS使細胞收縮幅度和鈣瞬變峰值顯著下降,收縮后恢復(fù)時間和鈣瞬變衰減時間顯著延長,而RSV能明顯糾正心肌細胞機械功能和鈣釋放異常。2.血漿Tn I和心肌cleaved Caspase 3表達在LPS處理的小鼠中沒有升高。血漿TNF-α在LPS處理的小鼠中顯著升高,伴有心肌組織CD45+炎性細胞浸潤明顯增加,而心肌組織TNF-α表達水平在各組小鼠之間沒有顯著差異。RSV不降低血漿TNF-α水平,不減少心肌組織CD45+炎性細胞數(shù)量,心肌NCX、Ry R2和IP3R表達水平在各組之間無差異。心肌SERCA2a在LPS處理的小鼠中表達下降,活性降低,而RSV治療可以上調(diào)SERCA2a表達,改善活性。3.LPS使心肌monomer-PLB增多,pentamer-PLB減少,這與LPS引起的氧化還原紊亂有關(guān)。LPS上調(diào)心肌4-HNE和MDA的含量,下調(diào)Nrf-2及下游蛋白(CAT和NQO-1)的表達,RSV治療能明顯減輕上述異常。離體細胞實驗進一步發(fā)現(xiàn):RSV能起到和一種ROS清除劑,N-乙酰半胱氨酸相似的作用,減少心肌細胞ROS水平,恢復(fù)受到抑制的SERCA2a功能,促進PLB從monomer轉(zhuǎn)變?yōu)閜entamer。結(jié)論:1.RSV能治療SIC。2.RSV治療SIC的機制與促進PLB從單聚體向五聚體轉(zhuǎn)變,進而上調(diào)心肌SERCA2a功能有關(guān)。3.RSV調(diào)控PLB構(gòu)象改變的機制與激活Nrf-2,調(diào)控細胞氧化還原有關(guān)。創(chuàng)新點:1.RSV通過激活Nrf-2,促進PLB從單聚體向五聚體轉(zhuǎn)變,恢復(fù)受抑制的SERC A2a功能,對SIC起到治療作用。2.本研究有望為白藜蘆醇(或Nrf-2的直接激動劑)應(yīng)用于臨床防治SIC提供理論依據(jù)。綜上所述,本研究首次闡明了RSV治療SIC的一種新的機制:RSV通過激活Nrf-2及下游基因,調(diào)節(jié)細胞內(nèi)氧化還原狀態(tài),促進心肌PLB從monomer向pentamer轉(zhuǎn)變,恢復(fù)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R515.3;R542.2

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