本文選題：肝X受體 + 自噬��； 參考：《第三軍醫(yī)大學(xué)》2014年博士論文

【摘要】：研究背景：復(fù)雜先天性心臟疾病的新生兒,在心臟手術(shù)后,常常會(huì)發(fā)生致死性術(shù)后并發(fā)癥,如低心輸出量綜合征。除了手術(shù)和體外循環(huán)創(chuàng)傷本身,體外循環(huán)下細(xì)胞損傷及破碎細(xì)胞炎癥因子釋放也可以激起全身炎癥反應(yīng)。此外,體外循環(huán)還與心臟術(shù)后血液中內(nèi)毒素水平的上升密切相關(guān)。血液總內(nèi)毒素的增加,可以導(dǎo)致新生小鼠血液中促炎細(xì)胞因子釋放,如TNF-α和IL-6。而循環(huán)中細(xì)胞因子水平的升高與心臟功能受損密切相關(guān)。大量體內(nèi)及體外研究結(jié)果表明,LXR是許多炎癥相關(guān)基因表達(dá)的調(diào)節(jié)因子。LXR表達(dá)于小鼠心肌中,并且在心肌損傷過(guò)程中起著保護(hù)作用。使用LXR激動(dòng)劑GW3965預(yù)處理可顯著緩解心肌缺血損傷小鼠的心臟缺血后左心舒張末壓的升高,并且進(jìn)一步改善缺血后所引起的左心室發(fā)展壓的降低。細(xì)胞實(shí)驗(yàn)表明,在LPS以及血管緊張素-Ⅱ誘導(dǎo)的心肌細(xì)胞炎癥過(guò)程中,LXR可以通過(guò)抑制NF-κB信號(hào)通路發(fā)揮抗炎作用。自噬是細(xì)胞內(nèi)自我消化的一個(gè)高度保守的過(guò)程,具有降解和回收利用長(zhǎng)壽蛋白質(zhì)以及細(xì)胞器的作用。已有研究表明,自噬在心肌缺血損傷的發(fā)病機(jī)制扮演著重要的角色。而且,在缺血引發(fā)炎癥損傷時(shí),自噬起著心肌保護(hù)作用。此外,LPS刺激可促進(jìn)乳鼠心肌細(xì)胞自噬,并且自噬通過(guò)抑制凋亡而發(fā)揮細(xì)胞保護(hù)的作用。前期研究結(jié)果表明,通過(guò)LXR激動(dòng)劑T0901317活化內(nèi)源性LXR配體,可以抑制乙醇誘導(dǎo)的小膠質(zhì)細(xì)胞活化,從而防止新生小鼠浦肯野細(xì)胞凋亡。因此,LXR對(duì)LPS誘導(dǎo)的心肌組織炎癥損傷的抗炎作用是否與自噬相關(guān)值得進(jìn)一步研究和探討。本課題分為L(zhǎng)XR激動(dòng)劑T0901317與新生小鼠心肌炎癥,以及T0901317與新生小鼠心肌自噬及凋亡兩個(gè)部分進(jìn)行研究。觀察T0901317對(duì)新生小鼠心肌炎性反應(yīng)的作用,并分別從顯微、超微和分子層面觀察在急性LPS刺激下T0901317對(duì)心肌自噬的作用。與此同時(shí),我們還進(jìn)一步探索了T0901317對(duì)心肌組織炎癥、自噬和凋亡影響的潛在機(jī)制。研究方法：1.T0901317與新生小鼠心肌炎癥：(1)建立LPS誘導(dǎo)的新生小鼠全身炎癥模型以及TO抗炎修復(fù)模型；(2)HE染色,計(jì)數(shù)心肌組織炎性細(xì)胞數(shù)；(3)熒光定量PCR檢測(cè)心肌組織IL-6及TNF-amRNA含量；(4)Western Blot檢測(cè)心肌組織NF-kB p65和磷酸化NF-kB p65、IκB-α和磷酸化IκB-α的蛋白水平。2.T0901317與新生小鼠心肌自噬以及凋亡：(1)Western Blot檢測(cè)LC3的表達(dá)；(2)免疫熒光染色含LC3斑點(diǎn)的自噬小體；(3)透射電鏡觀察自噬小體；(4)TUNEL檢測(cè)試劑盒檢測(cè)心肌組織凋亡細(xì)胞；(5)Western Blot檢測(cè)Akt和磷酸化Akt蛋白水平。結(jié)果：1.單次大劑量LPS腹腔注射可以誘導(dǎo)新生小鼠心肌的炎癥反應(yīng),表現(xiàn)為心肌組織明顯的炎性細(xì)胞浸潤(rùn),心肌組織促炎細(xì)胞因子的TNF-α、IL-6 mRNA表達(dá)水平分別上調(diào)2.26倍(p0.01)和1.48倍(p0.01)。2.經(jīng)過(guò)TO預(yù)處理的新生小鼠的心肌組織,在受到單次大劑量LPS腹腔注射后,表現(xiàn)出較輕的炎性細(xì)胞浸潤(rùn),心肌組織促炎細(xì)胞因子TNF-α、IL-6 mRNA的表達(dá)水平上升較未經(jīng)過(guò)TO預(yù)處理新生小鼠少,分別減少40%(p0.01)和36%(p0.05)。3.TO預(yù)處理的新生小鼠,在接受LPS刺激后,心肌組織內(nèi)NF-κB磷酸化水平較單純LPS刺激組進(jìn)一步降低56.6%(p0.01)。IκB-α降解程度同樣也進(jìn)一步降低28.2%(p0.01)。提示TO對(duì)新生小鼠心肌組織的抗炎作用與NF-κB信號(hào)通路有關(guān)。4.單次大劑量LPS腹腔注射后,新生小鼠心肌組織中LC3-Ⅱ/GAPDH比值增加3.32倍(p0.01),同時(shí)免疫熒光染色可見(jiàn)心肌細(xì)胞內(nèi)標(biāo)記有LC3的斑點(diǎn)數(shù)目增加5.34倍(p0.05)。5.經(jīng)過(guò)TO預(yù)處理的新生小鼠的心肌組織,在受到單次大劑量LPS腹腔注射后,相比單純LPS刺激的新生小鼠,LC3-Ⅱ/GAPDH比值進(jìn)一步增加1.15倍(p0.01),并且免疫熒光染色后LC3陽(yáng)性斑點(diǎn)數(shù)增加0.68倍(p0.05)。透射電鏡下可見(jiàn)同一視野下自噬小體以及自溶小體,進(jìn)一步確證自噬的發(fā)生。6.透射電鏡下單純LPS刺激的新生小鼠心肌組織內(nèi)可見(jiàn)凋亡小體。TUNEL-POD染色并計(jì)數(shù)TUNEL陽(yáng)性凋亡細(xì)胞,結(jié)果顯示LPS可增加新生小鼠心肌組織細(xì)胞凋亡(133.3±23.1 vs.67.6±4.7/105細(xì)胞核,p0.01),而經(jīng)過(guò)TO預(yù)處理的新生小鼠在受到LPS刺激后TUNEL陽(yáng)性細(xì)胞核數(shù)減少31%(p0.05)。提示LXR激動(dòng)劑TO在心肌組織中具有抗凋亡作用。7.免疫蛋白印跡技術(shù)結(jié)果發(fā)現(xiàn),急性LPS刺激可使心肌組織Akt磷酸化水平降低37%(p0.01,DMSO+LPS vs. DMSO+NaCl),而TO預(yù)處理的新生小鼠心肌內(nèi)Akt磷酸化水平進(jìn)一步降低27%(p 0.05, TO+LPS vs. DMSO+LPS)。結(jié)論：1.LXR激動(dòng)劑T0901317可以通過(guò)抑制NF-κB信號(hào)通路的活化,從而降低LPS腹腔注射所誘導(dǎo)的新生小鼠心肌組織炎癥反應(yīng)。2.LXR激動(dòng)劑T0901317可以強(qiáng)化LPS腹腔注射誘導(dǎo)的新生小鼠心肌組織自噬活性上升。并且還能降低單次LPS腹腔注射所致的新生小鼠心肌細(xì)胞凋亡。3.LXR激動(dòng)劑T0901317的抗炎作用,強(qiáng)化LPS刺激急性期自噬活性的作用以及抑制凋亡作用,可以用Akt磷酸化水平的變化解釋,但其詳細(xì)機(jī)制仍有待進(jìn)一步研究。
[Abstract]:Background: newborns with complex congenital heart disease often have fatal postoperative complications after cardiac surgery, such as low cardiac output syndrome. In addition to surgery and cardiopulmonary bypass wound itself, cell injury under extracorporeal circulation and the release of inflammatory factors in broken cells can also stimulate systemic inflammatory response. In addition, cardiopulmonary bypass is also used. The increase in total endotoxin in blood can lead to the release of proinflammatory cytokines in the blood of newborn mice, such as TNF- alpha and IL-6., and the elevated levels of cytokines in the circulation are closely related to the impairment of heart function. A large number of in vivo and in vitro studies have shown that LXR is a number of inflammatory phases. The regulatory factor.LXR is expressed in the myocardium of mice and plays a protective role in myocardial injury. The use of LXR agonist GW3965 preconditioning can significantly alleviate the increase of left ventricular end diastolic pressure after myocardial ischemia in mice, and further improve the decrease of the left ventricular development pressure caused by the absence of blood. Cell experiments showed that in the process of LPS and angiotensin - II induced cardiomyocyte inflammation, LXR can play an anti-inflammatory role by inhibiting the NF- kappa B signaling pathway. Autophagy is a highly conserved process of intracellular self digestion, which degrades and recycles long life proteins to the organelles. Autophagy has been shown to be in the process of autophagy. The pathogenesis of myocardial ischemia plays an important role. Moreover, autophagy plays a protective role in myocardial protection when it is induced by ischemia. In addition, LPS stimulation can promote autophagy in neonatal rat cardiomyocytes and autophagy plays a role in cell protection by inhibiting apoptosis. The previous research results showed that the activation of the LXR agonist T0901317 was within the activity. The source of LXR ligands can inhibit the activation of ethanol induced microglia and prevent the apoptosis of newborn mice. Therefore, it is worth further study and discussion whether the anti-inflammatory effects of LXR on LPS induced myocardial tissue inflammation and autophagy are worth further study and discussion. This topic is divided into LXR irritation agent T0901317 and neonatal myocarditis, and T09 01317 to study the two parts of autophagy and apoptosis in neonatal mice. Observe the effect of T0901317 on the myocarditis of newborn mice and observe the effect of T0901317 on autophagy at the microscopic, ultrastructural and molecular level under the acute LPS stimulation. At the same time, we also explore T0901317 to myocardium inflammation and autophagy. And the potential mechanism of apoptosis effect. Research methods: 1.T0901317 and neonatal myocarditis: (1) establish LPS induced systemic inflammation model and TO anti-inflammatory repair model in neonatal mice; (2) HE staining, count the number of inflammatory cells in myocardial tissue; (3) fluorescence quantitative PCR detection of cardiac muscle tissue IL-6 and TNF-amRNA content; (4) Western Blot detection heart The protein levels of NF-kB p65 and phosphorylated NF-kB p65, I kappa B- alpha and phosphorylated I kappa B- a were associated with autophagy and apoptosis in neonatal mice: (1) Western Blot detected the expression of LC3; (2) immunofluorescence staining autophagic bodies containing speckled autophagic bodies; (3) transmission electron microscopy to observe autophagic corpuscles; (4) detection kit for detection of myocardial tissue Apoptotic cells, (5) Western Blot detection of Akt and phosphorylated Akt protein levels. Results: 1. a single large dose of LPS intraperitoneal injection can induce myocardial inflammation in newborn mice, manifested as obvious inflammatory cell infiltration in myocardial tissue, TNF- a of myocardium proinflammatory cytokines, and IL-6 mRNA expression level up to 2.26 times (P0.01) and 1.48 respectively. The myocardial tissue of P0.01.2., which was pretreated with TO, showed mild inflammatory cell infiltration after a single large dose of LPS intraperitoneal injection. The expression level of TNF- alpha and IL-6 mRNA in myocardial tissue increased less than that of non TO pretreated mice, and decreased by 40% (P0.01) and 36% (P0.05).3.TO pretreatment, respectively. In neonatal mice, the level of phosphorylation of NF- kappa B in myocardial tissue was further reduced by 56.6% (P0.01).I kappa B- degradation level by 28.2% (P0.01). The anti inflammatory effect of TO on neonatal mouse myocardium was related to NF- kappa B signaling pathway, which was associated with a single large dose of.4.. The ratio of LC3- II /GAPDH in rat myocardial tissue increased by 3.32 times (P0.01), and immunofluorescence staining showed that the number of spots marked with LC3 in cardiac myocytes increased by 5.34 times (P0.05).5. through TO pretreated neonatal mice. After a single large dose of LPS intraperitoneal injection, the LC3- II /GAPDH ratio compared to the pure LPS stimulated mice was compared. The value of LC3 positive spots increased by 1.15 times (P0.01), and the number of LC3 positive spots increased after immunofluorescence staining (P0.05). Under transmission electron microscopy, autophagosomes and autophagosomes were found under the same vision. Further confirmation of autophagy was confirmed by.6. transmission electron microscopy. The number of TUNEL positive apoptotic cells showed that LPS could increase the apoptosis of myocardium in neonatal mice (133.3 + 23.1 vs.67.6 + 4.7/105 nuclei, P0.01), while the number of TUNEL positive nuclei decreased by 31% (P0.05) after TO pretreatment in neonatal mice, suggesting that LXR irritation agent TO in myocardial tissue is immune to apoptosis. The results of Western blotting showed that acute LPS stimulation could reduce the level of Akt phosphorylation in myocardial tissue by 37% (P0.01, DMSO+LPS vs. DMSO+NaCl), while the level of Akt phosphorylation in the myocardium of newborn mice treated with TO was further reduced by 27% (P 0.05, TO+LPS vs. DMSO+LPS). Thus reducing the inflammatory reaction of neonatal mice induced by LPS intraperitoneal injection,.2.LXR agonist T0901317 can enhance the increase of autophagic activity in the myocardium of neonatal mice induced by intraperitoneal injection of LPS, and can also reduce the anti-inflammatory effect of.3.LXR agonist T0901317 of neonatal mouse cardiomyocyte apoptosis induced by single LPS intraperitoneal injection. The effect of enhanced LPS stimulation on autophagic activity and the inhibition of apoptosis can be explained by the changes in the level of Akt phosphorylation, but the detailed mechanism remains to be further studied.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R726.5

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