Sirt1在脓毒血症继发的急性肺和肾损伤中的保护作用

发布时间：2018-08-22 11:04

【摘要】：脓毒血症是一种潜在的致命性的全身炎症反应，也称为系统性炎症反应综合症（SIRS），是一种由严重的感染引起的具有高发病率和死亡率的难治性疾病。脓毒血症同时也可以被认为是从感染到多器官功能损害综合症的一种过度。严重的脓毒血症可以引起衰竭型器官功能异常，临床观察发现脓毒血症患者发展成急性肺损伤（ALI）和急性肾损伤（AKI）的风险很高。目前脓毒血症中器官之间的损伤干扰及其机制的研究越来越受到重视，尤其是涉及到ALI和AKI之间关系的研究。ALI以及其严重模式-急性呼吸窘迫综合症（ARDS）常常导致多器官功能衰竭，临床上的死亡率大约为30%-50%。此外，作为一个天然的血液过滤系统，肾脏成为系统性炎症反应器官损伤的直接靶点。炎症引起的ALI及AKI在老年人中十分常见，通常死亡率较高，而目前研究发现Sirt1在老年人中表达下降。同时也有报道表明Sirt1在慢性阻塞性肺疾病（COPD）患者中的表达下降，所以我们推测Sirt1在炎症调节中起到一定的作用。我们分别用盲肠结扎穿刺手术（CLP）和脂多糖（LPS）引起的脓毒血症来研究Sirt1的对于肺脏和肾脏损伤的作用及其机制。 Sirtuin1（Sirt1）是Sirtuin家族的成员，是最初发现于酵母菌的一种脱乙酰酶。不同于经典的组蛋白脱乙酰途径，Sirt1发挥它的脱乙酰功能是通过对赖氨酸残基的脱乙酰作用来调节其它蛋白质的功能，例如核转录因子kB和p53等。也有越来越多的研究发现Sirt1在炎症、凋亡、抗应激、代谢、分化和老化中起到重要作用，而Sirt1在脓毒血症中作用的相关研究较少，机制尚不明确。在本研究中，我们利用Sirt1敲除鼠来研究其在脓毒血症引起的急性肺肾损伤中的作用，并进一步明确其调节作用机制。我们的实验证明Sirt1敲除鼠对脓毒血症模型引起的ALI和AKI更具有易感性。在实验的第一部分中，我们主要研究Sirt1对于脓毒血症继发的ALI的作用及其机制。实验中对Sirt1基因敲除及其同源性野生型小鼠进行CLP手术，成功造成小鼠脓毒血症模型。为了明确Sirt1对于肺损伤的作用及其机制，本部分实验从肺中性粒细胞的浸润情况、促炎症的释放和Sirt1调节机制三方面进行研究。为了了解中性粒细胞浸润情况，我们检测了CLP术后24小时肺组织匀浆中髓过氧化物酶（MPO）的活性。随后我们应用试剂盒检测造模后6小时和24小时后的血清中及肺组织中IL-6和TNF-α水平，结果显示Sirt1基因敲除明显增加MPO的活性，并且提高了促炎症因子的水平。为了进一步了解Sirt1调节肺内炎症的机制，我们应用western blot检测Sirt1调节的直接和间接靶点蛋白在肺内的表达。外源性和内源性感染因子进入到体内后，促进了NF-kB的活化，随后调节各种炎症介质的转录，最终导致不可逆的过度的炎症反应发生。我们的研究证明了Sirt1基因敲除鼠的肺内NF-kB的活化增强，同时其下游黏附分子-细胞间黏附分子-1（ICAM-1）和血管细胞黏附分子-1（VCAM-1）、细胞外调节激酶1/2（ERK1/2）和信号转导转录激活因子3（stat3）以及caspase3，caspase7的表达明显增加。以上实验结果证明Sirt1敲除通过触发过度的炎症以及相关炎症复合物途径而使肺损伤增强。在第二部分实验中，我们主要研究Sirt1在调节脓毒血症继发的肾损伤中的作用及其机制。我们应用LPS诱导小鼠建立脓毒血症模型。首先通过血清尿素氮（BUN）和尿液肾脏损伤分子（KIM-1）的检测显示在脓毒血症中肾脏功能明显受损。进一步的组织学检测发现脓毒血症也引起肾小管的损伤和炎症。肾脏的炎症与促炎症介质的产生增加与黏附分子的上调（例如ICAM-1和VCAM-1）有密切关系。这些早期的炎症反应在肾损伤的过程中引起炎症细胞的浸润，而过度的炎细胞浸润则可引起进一步的肾损伤。在炎症因子的调节过程中NF-kB调节各种炎症因子的转录，进而影响炎症反应进程，本部分研究发现Sirt1基因敲除导致NF-kB的活化，随后活化的NF-kB引起包括IL-6和TNF-α在内的细胞因子的增加，这些炎症因子的增加是AKI的明显标志。同时细胞因子也可以引起信号通路的活化，，例如Stat3和ERK1/2调节的级联反应等途径。以上实验证明Sirt1在脓毒血症引起的AKI中具有保护作用，并通过调节NF-kB的活化产生其保护机制。综上所述，Sirt1在脓毒血症引起的急性肺肾损伤中发挥保护作用。Sirt1发挥它的保护功能主要是通过对核转录因子的活化的调控，继而引发Stat3，ERK1/2、caspase等炎症通路的活化。因此我们的研究表明Sirt1是一个潜在的脓毒血症引起的肺肾损伤的治疗靶点。
[Abstract]:Sepsis is a potentially fatal systemic inflammatory response, also known as systemic inflammatory response syndrome (SIRS), a refractory disease with high morbidity and mortality from severe infections. Sepsis can also be considered an excess from infection to multiple organ dysfunction syndrome. Sepsis can cause organ dysfunction of failure type. Clinical observation shows that patients with sepsis have a high risk of developing acute lung injury (ALI) and acute kidney injury (AKI). Acute Respiratory Distress Syndrome (ARDS), a severe form of ARDS, often leads to multiple organ failure (MOF) with a clinical mortality rate of about 30% - 50%. In addition, as a natural blood filtration system, the kidney becomes a direct target for organ damage in systemic inflammatory reactors. The expression of Sirt1 was decreased in the elderly. It was also reported that the expression of Sirt1 was decreased in patients with chronic obstructive pulmonary disease (COPD), so we speculated that Sirt1 might play a role in the regulation of inflammation. We used cecum ligation puncture (CLP) and lipopolysaccharide (LPS) to induce sepsis, respectively. To study the effect and mechanism of Sirt1 on lung and kidney injury.
Sirtuin 1 (Sirt1), a member of the Sirtuin family, is a deacetylase first discovered in yeast. Unlike the classical histone deacetylation pathway, Sirt1 exerts its deacetylation function by deacetylating lysine residues to regulate other protein functions, such as nuclear transcription factors kB and p53. Sirt1 has been found to play an important role in inflammation, apoptosis, anti-stress, metabolism, differentiation and aging, while the role of Sirt1 in sepsis has not been well understood. In this study, we used Sirt1 knockout mice to study its role in acute lung and kidney injury induced by sepsis and to further clarify its regulation. Our experiments showed that Sirt1 knockout mice were more susceptible to ALI and AKI induced by sepsis.
In the first part of the experiment, we mainly studied the effect and mechanism of Sirt1 on ALI secondary to sepsis. In the experiment, we performed CLP on the wild type mice with Sirt1 gene knockout and its homology, and successfully induced sepsis model in mice. To understand the infiltration of neutrophils, we examined the activity of myeloperoxidase (MPO) in lung tissue homogenate 24 hours after CLP. Then we used kits to detect IL-6 in serum and lung tissue 6 hours and 24 hours after CLP. In order to further understand the mechanism of Sirt1 regulating pulmonary inflammation, we used Western blot to detect the expression of Sirt1-regulated direct and indirect target proteins in the lung. Our study demonstrated that the activation of NF-kB in the lungs of Sirt1 knockout mice was enhanced, and the downstream adhesion molecule-intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were fine. The expression of extracellular regulated kinase 1/2 (ERK1/2), signal transduction activator 3 (stat3), caspase 3, and caspase 7 were significantly increased. The above results demonstrated that Sirt1 knockout enhanced lung injury by triggering excessive inflammation and related inflammatory complex pathways.
In the second part of the experiment, we mainly studied the role and mechanism of Sirt1 in regulating renal injury secondary to sepsis. We established sepsis model in LPS-induced mice. First, the detection of BUN and KIM-1 in serum showed that renal function was significantly impaired in sepsis. Histological examination revealed that sepsis also caused renal tubular injury and inflammation. Increased inflammation and the production of pro-inflammatory mediators were closely related to the up-regulation of adhesion molecules (such as ICAM-1 and VCAM-1). These early inflammatory reactions caused infiltration of inflammatory cells during renal injury, whereas excessive infiltration of inflammatory cells could be induced. NF-kB regulates the transcription of various inflammatory factors during the regulation of inflammatory factors, thereby affecting the process of inflammation. Sirt1 knockout leads to the activation of NF-kB, and subsequently activated NF-kB leads to the increase of cytokines including IL-6 and TNF-alpha, which is the result of AKI. Significant markers. Cytokines can also activate signaling pathways, such as the cascade of Stat3 and ERK1/2. These experiments have shown that Sirt1 has a protective effect on AKI induced by sepsis and produces its protective mechanism by regulating the activation of NF-kB.
To sum up, Sirt1 plays a protective role in acute lung and kidney injury induced by sepsis. Sirt1 exerts its protective function mainly by regulating the activation of nuclear transcription factors, which in turn leads to the activation of inflammatory pathways such as Stat3, ERK1/2 and caspase. Therapeutic targets for injury.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R459.7

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