LXR激动剂TO901317对LPS诱导的新生小鼠心肌炎症和自噬的影响

发布时间：2018-05-03 04:07

本文选题：肝X受体 + 自噬　；参考：《第三军医大学》2014年博士论文

【摘要】：研究背景：复杂先天性心脏疾病的新生儿,在心脏手术后,常常会发生致死性术后并发症,如低心输出量综合征。除了手术和体外循环创伤本身,体外循环下细胞损伤及破碎细胞炎症因子释放也可以激起全身炎症反应。此外,体外循环还与心脏术后血液中内毒素水平的上升密切相关。血液总内毒素的增加,可以导致新生小鼠血液中促炎细胞因子释放,如TNF-α和IL-6。而循环中细胞因子水平的升高与心脏功能受损密切相关。大量体内及体外研究结果表明,LXR是许多炎症相关基因表达的调节因子。LXR表达于小鼠心肌中,并且在心肌损伤过程中起着保护作用。使用LXR激动剂GW3965预处理可显著缓解心肌缺血损伤小鼠的心脏缺血后左心舒张末压的升高,并且进一步改善缺血后所引起的左心室发展压的降低。细胞实验表明,在LPS以及血管紧张素-Ⅱ诱导的心肌细胞炎症过程中,LXR可以通过抑制NF-κB信号通路发挥抗炎作用。自噬是细胞内自我消化的一个高度保守的过程,具有降解和回收利用长寿蛋白质以及细胞器的作用。已有研究表明,自噬在心肌缺血损伤的发病机制扮演着重要的角色。而且,在缺血引发炎症损伤时,自噬起着心肌保护作用。此外,LPS刺激可促进乳鼠心肌细胞自噬,并且自噬通过抑制凋亡而发挥细胞保护的作用。前期研究结果表明,通过LXR激动剂T0901317活化内源性LXR配体,可以抑制乙醇诱导的小胶质细胞活化,从而防止新生小鼠浦肯野细胞凋亡。因此,LXR对LPS诱导的心肌组织炎症损伤的抗炎作用是否与自噬相关值得进一步研究和探讨。本课题分为LXR激动剂T0901317与新生小鼠心肌炎症,以及T0901317与新生小鼠心肌自噬及凋亡两个部分进行研究。观察T0901317对新生小鼠心肌炎性反应的作用,并分别从显微、超微和分子层面观察在急性LPS刺激下T0901317对心肌自噬的作用。与此同时,我们还进一步探索了T0901317对心肌组织炎症、自噬和凋亡影响的潜在机制。研究方法：1.T0901317与新生小鼠心肌炎症：(1)建立LPS诱导的新生小鼠全身炎症模型以及TO抗炎修复模型；(2)HE染色,计数心肌组织炎性细胞数；(3)荧光定量PCR检测心肌组织IL-6及TNF-amRNA含量；(4)Western Blot检测心肌组织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与新生小鼠心肌自噬以及凋亡：(1)Western Blot检测LC3的表达；(2)免疫荧光染色含LC3斑点的自噬小体；(3)透射电镜观察自噬小体；(4)TUNEL检测试剂盒检测心肌组织凋亡细胞；(5)Western Blot检测Akt和磷酸化Akt蛋白水平。结果：1.单次大剂量LPS腹腔注射可以诱导新生小鼠心肌的炎症反应,表现为心肌组织明显的炎性细胞浸润,心肌组织促炎细胞因子的TNF-α、IL-6 mRNA表达水平分别上调2.26倍(p0.01)和1.48倍(p0.01)。2.经过TO预处理的新生小鼠的心肌组织,在受到单次大剂量LPS腹腔注射后,表现出较轻的炎性细胞浸润,心肌组织促炎细胞因子TNF-α、IL-6 mRNA的表达水平上升较未经过TO预处理新生小鼠少,分别减少40%(p0.01)和36%(p0.05)。3.TO预处理的新生小鼠,在接受LPS刺激后,心肌组织内NF-κB磷酸化水平较单纯LPS刺激组进一步降低56.6%(p0.01)。IκB-α降解程度同样也进一步降低28.2%(p0.01)。提示TO对新生小鼠心肌组织的抗炎作用与NF-κB信号通路有关。4.单次大剂量LPS腹腔注射后,新生小鼠心肌组织中LC3-Ⅱ/GAPDH比值增加3.32倍(p0.01),同时免疫荧光染色可见心肌细胞内标记有LC3的斑点数目增加5.34倍(p0.05)。5.经过TO预处理的新生小鼠的心肌组织,在受到单次大剂量LPS腹腔注射后,相比单纯LPS刺激的新生小鼠,LC3-Ⅱ/GAPDH比值进一步增加1.15倍(p0.01),并且免疫荧光染色后LC3阳性斑点数增加0.68倍(p0.05)。透射电镜下可见同一视野下自噬小体以及自溶小体,进一步确证自噬的发生。6.透射电镜下单纯LPS刺激的新生小鼠心肌组织内可见凋亡小体。TUNEL-POD染色并计数TUNEL阳性凋亡细胞,结果显示LPS可增加新生小鼠心肌组织细胞凋亡(133.3±23.1 vs.67.6±4.7/105细胞核,p0.01),而经过TO预处理的新生小鼠在受到LPS刺激后TUNEL阳性细胞核数减少31%(p0.05)。提示LXR激动剂TO在心肌组织中具有抗凋亡作用。7.免疫蛋白印迹技术结果发现,急性LPS刺激可使心肌组织Akt磷酸化水平降低37%(p0.01,DMSO+LPS vs. DMSO+NaCl),而TO预处理的新生小鼠心肌内Akt磷酸化水平进一步降低27%(p 0.05, TO+LPS vs. DMSO+LPS)。结论：1.LXR激动剂T0901317可以通过抑制NF-κB信号通路的活化,从而降低LPS腹腔注射所诱导的新生小鼠心肌组织炎症反应。2.LXR激动剂T0901317可以强化LPS腹腔注射诱导的新生小鼠心肌组织自噬活性上升。并且还能降低单次LPS腹腔注射所致的新生小鼠心肌细胞凋亡。3.LXR激动剂T0901317的抗炎作用,强化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磷酸化水平的变化解释,但其详细机制仍有待进一步研究。
[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.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R726.5

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