Notch信号通路介导脓毒症脑病的作用及机制研究
发布时间：2018-07-20 22:06
【摘要】：脓毒症脑病又称脓毒症相关性脑病(Sepsis-associated encephalopathy, SAE)是脓毒症引起的弥漫性脑功能障碍,其发病率为8%-70%。临床研究发现,SAE是ICU中最常见的脑病之一,并发SAE可导致脓毒症患者的死亡率明显增高。SAE病理生理机制较复杂,其发病机制尚不明确,但是大量的研究结果表明神经元凋亡是SAE发生及发展过程的重要表现之一。研究证实,在脓毒症大鼠模型中,SAE可导致大鼠长期的记忆损伤。其中,与空间记忆密切相关的海马等脑区的损伤程度尤为明显。 本研究以Notch信号通路为主要研究对象,探讨SAE发病机制。Notch信号通路是调控神经元凋亡的关键信号通路之一。最新研究表明,γ-分泌酶复合体和Notch1参与了一些神经变性疾病的发病过程,如阿尔茨海默病、缺血性脑卒中等。同时,Notch信号通路特异性抑制剂DAPT可有效抑制凋亡和炎症反应介导的脑缺血性损伤,其作用机制与Notch信号通路在PARP-1激活凋亡过程中起到调控作用有关。新近研究发现,PARP-1激活后通过自身分裂和核内凋亡相关因子,活化caspase通路,从而导致神经元的凋亡。 本研究以盲肠结扎穿刺法(CLP)建立大鼠的脓毒症模型,集中开展了Notch信号通路在SAE中的作用及其机制研究。研究表明,CLP后24小时,Notch受体细胞内区域(NICD)表达显著增加,大鼠海马组织炎症反应、神经元凋亡、神经元死亡等也明显增加。DAPT能明显减少海马组织神经元凋亡和死亡,以及TNF-αIL-lb、IL-6、PARP-1和Caspase-1的表达。同时,CLP引起NF-kB转核作用也可被DAPT明显抑制。 综上所述,Notch信号通路参与了脓毒症导致的脑损伤,其作用机制与调控NF-kB转核有关。研究结果可为SAE的治疗提供新的策略和药物靶标。 第一部分Notch信号通路在脓毒症脑病中的作用 目的：脓毒症相关性脑病(SAE)病理生理机制尚不明确,缺乏客观的诊断指标及有效的治疗和预防手段。研究发现γ-分泌酶复合体和Notch1参与了一些神经变性疾病的发病过程,γ-分泌酶抑制剂DAPT能抑制凋亡和炎症反应介导的脑缺血性损伤。本部分选用盲肠结扎穿刺法(CLP)建立脓毒症模型,观察Notch信号通路在脓毒症脑病中的作用及DAPT对脓毒症脑病的保护作用。 方法：取SD大鼠,建立CLP模型,戊巴比妥钠30mg/kg腹腔注射麻醉,腹部剃毛并用碘伏消毒,铺巾,沿腹中线剪开2cm腹部切口,暴露盲肠,在回盲瓣远端结扎盲肠,然后用18号针刺穿刺盲肠2次,轻轻挤压盲肠,使少量的肠内容物渗出,然后将盲肠返回到腹腔,关闭腹腔。假手术组(sham)大鼠予剖腹,并将盲肠外置1分钟,不予盲肠穿刺或结扎,然后将盲肠回纳至腹腔并关腹。每只手术操作的大鼠术后每6小时给予皮下注射生理盐水(3ml/100g)及头孢曲松(30mg/kg).实验于麻醉后直视下行股动脉切开置入24G输液管以测定血压、心率。记录术前、术后6h、12h和24h血压、心率和肛温,并在以上时间点经股动脉采血1ml测血乳酸水平(每个时间点,n=3)。59只大鼠随机分为四组：(a)假手术+生理盐水组(10ml/kg);(b)假手术+DAPT组(10μmol/kg):(c)CLP+生理盐水组(1ml/kg);(d) CLP+DAPT组(10μmol/kg)。每组分为2个亚组,第一亚组,24小时处死大鼠,断头取血,分离海马组织,-80℃保存,行NICD的Western-Blot蛋白检测。第二亚组,术后每6小时子皮下注射生理盐水(3ml/100g)及头孢曲松(30mg/kg),并允许自由进食及饮水。术后第7天存活大鼠检测行为学改变(新物体识别实验)。 结果：CLP组大鼠出现蜷缩少动、竖毛,个别大鼠出现偏瘫、癫痫发作表现；并出现平均动脉压明显下降,心率明显升高,血乳酸水平增高；体重明显下降；第3天为56.3%,第4-7天为37.5%；CLP组大鼠24h神经反射评分明显下降,并在第4天、第7天已逐渐恢复；新物体认知试验提示CLP大鼠学习记忆功能受损。在CLP后24h,NICD表达显著增加。Y-分泌酶抑制剂DAPT能降低CLP后NICD的表达,缓解认知功能损伤。 结论：CLP组大鼠完全模拟了脓毒症典型临床表现,神经反射评分及行为学检查提示大鼠脓毒症相关性脑病(SAE)模型制作成功；首次发现在CLP后24h,NICD表达显著增加,说明Notch信号通路可能参与了脓毒症导致的脑损伤；同时应用DAPT抑制Notch信号通路对脓毒症大鼠的记忆损伤有神经保护作用。 第二部分Notch信号通路参与脓毒症脑病的机制研究 目的：在本实验研究中,我们通过构建大鼠CLP脓毒症模型,主要探讨Notch信号通路参与脓毒症脑病的可能发病机制,以及γ-分泌酶抑制剂通过阻断Notch1信号通路对脓毒症脑病大鼠的干预作用。 方法：随机将59只SD大鼠分为4组,分别为假手术组(Sham)、假手术+DAPT组(Sham+DAPT)、CLP组(CLP)、CLP+DAPT组,在CLP或假手术后24h,将大鼠处死,取出海马组织用于Westen blot法检测PARP-1、Caspase-1; ELISA法检测TNF-α IL-1b\IL-6水平。TUNEL法观察神经元凋亡,HE染色法观察神经元死亡。 结果：我们研究表明在实验动物中,比较于假手术组和假手术+DAPT组,在CLP组TNF-α IL-lb、IL-6水平显著增高,尽管在CLP+DAPT组增高受到抑制。通过HE染色和TUNEL染色法检测,评估海马组织神经细胞死亡和凋亡。在假手术组和假手术+DAPT组中几乎不能找到HE阳性的细胞。在CLP手术后,海马组织CA1区域神经细胞死亡明显增加。然而,与CLP组相比,HE染色阳性细胞在CLP+DAPT组明显减少。此外,TUNEL法检测神经细胞凋亡情况,在CLP组中TUNEL阳性细胞明显增加,但是在假手术组几乎不能检测到TUNEL阳性细胞。而神经细胞凋亡数目在CLP+DAPT组明显多于假手术组—RAPT组,与CLP组比较,前两组凋亡数目明显减少。 在脓毒症大鼠中,海马组织炎症反应、神经元凋亡、神经元死亡明显增加。DAPT能明显减少海马组织神经元凋亡和死亡,及其TNF-α、IL-1b、IL-6、PARP-1和Caspase-1的表达。 结论：证实了Notch信号通路通过激活PARP-1降解,导致caspase依赖性神经元凋亡,Y-分泌酶抑制剂DAPT抑制海马神经元PARP-1降解,抑制caspase依赖性神经元凋亡。DAPT通过抑制CLP脓毒症大鼠海马组织神经元凋亡和炎症反应改善脓毒症脑病。 第三部分Notch信号通路调控脓毒症脑病的分子机制研究：调控NF-kB激活 目的：NF-kB是在核内参与炎症反应的关键转录因子,亦广泛存在于神经系统的细胞中,调控神经细胞炎症反应及凋亡。研究显示,NF-kB信号途径在脓毒症神经系统炎症中也扮有重要角色。本实验部分采用CLP模型,研究脓毒症对神经细胞NF-kB信号通路的影响,并通过Notch特异性抑制剂DAPT干预,观察其对NF-kB信号通路的影响,由此深入研究Notch对脓毒症脑病NF-kB信号通路的调控作用。 方法：取SD大鼠,随机分为四组(n=6),分别为假手术组(Sham)、假手术+DAPT组(Sham+DAPT)、CLP组(CLP)、CLP+DAPT组。手术操作前30分钟腹腔注射DAPT(10μmo/kg)或生理盐水(10mL/kg).术后每6小时皮下注射生理盐水(3m1/100g)及头孢曲松(30mg/kg)复苏,24小时处死大鼠,取大鼠海马组织,液氮保存待测。用western blot和EMUSA分组检测NF-K蛋白表达水平。 结果：在Sham组动物中,核因子NF-kB在胞浆中表达,CLP使得NF-kB胞浆向胞核内转移。与Sham组相比,CLP组胞核内NF-kB蛋白含量明显升高,以p65最为显著。与Sham及Sham+DAPT组相比,CLP组胞核内NF-kB蛋白含量明显升高,但DAPT处理降低胞核内p65、p50及Re1B蛋白表达水平。与Sham组相比,CLP组磷酸化IKK-β表达明显升高,但DAPT处理可抑制CLP时磷酸化IKK-β表达。同样,DAPT处理可抑制p65磷酸化,其可能与IKK-β活性降低有关,相应的必然会导致p65DNA结合活性降低。 结论：本研究中我们证实CLP导致的脓毒症引起NF-kB由海马神经细胞胞浆向胞核内迁移,应用Notch信号通路特异性抑制剂DAPT抑制Notch靶基因表达,抑制NF-kB向胞核内迁移及其DNA结合活性。结合前文,NF-kB依赖的转录激活引起脓毒症海马神经细胞炎症反应及细胞凋亡,可能是导致脑病发生的原因之一。抑制Notch信号通路可能成为治疗脓毒症脑病的有效方法。
[Abstract]:Sepsis encephalopathy, also known as sepsis associated encephalopathy (Sepsis-associated encephalopathy, SAE), is a diffuse brain dysfunction caused by sepsis. The incidence of the disease is 8%-70%. clinical study. SAE is one of the most common encephalopathy in ICU. SAE can lead to a significant increase in mortality in patients with sepsis, and.SAE's pathophysiological mechanism is complex. The pathogenesis is not clear, but a large number of research results show that neuronal apoptosis is one of the important manifestations of the occurrence and development of SAE. In the rat model of sepsis, SAE can lead to long-term memory damage in rats. Among them, the damage degree of the hippocampus, which is closely related to spatial memory, is particularly obvious.
In this study, the main research object of this study is the Notch signaling pathway. The.Notch signaling pathway in the pathogenesis of SAE is one of the key signaling pathways in the regulation of neuronal apoptosis. The latest research shows that the gamma secretase complex and Notch1 are involved in the pathogenesis of some neurodegenerative diseases, such as Alzheimer's disease, ischemic stroke, and Notch letter. The pathway specific inhibitor DAPT can effectively inhibit the cerebral ischemic injury mediated by apoptosis and inflammatory response, and its mechanism of action is related to the regulatory role of the Notch signaling pathway in the process of PARP-1 activation and apoptosis. Recent studies have found that the activation of the caspase pathway through the activation of the PARP-1 through its own division and the related factors in the nuclear decay, resulting in the activation of the caspase pathway. Apoptosis of neurons.
In this study, a rat sepsis model was established by cecal ligation (CLP), and the role and mechanism of Notch signaling pathway in SAE was concentrated. The study showed that the expression of Notch receptor area (NICD) in the Notch receptor increased significantly after 24 hours of CLP, and the hippocampal inflammatory response, neuron apoptosis, neuron death and so on increased.D obviously. APT can significantly reduce the apoptosis and death of hippocampal neurons, as well as the expression of TNF- alpha IL-lb, IL-6, PARP-1 and Caspase-1. At the same time, CLP induced NF-kB nucleation can also be significantly inhibited by DAPT.
To sum up, Notch signaling pathway is involved in the brain damage caused by sepsis, and its mechanism is related to the regulation of NF-kB transnuclear. The results can provide new strategies and drug targets for the treatment of SAE.
Part 1 the role of Notch signaling pathway in sepsis encephalopathy
Objective: the pathophysiological mechanism of sepsis related encephalopathy (SAE) is not clear, lack of objective diagnosis and effective treatment and prevention. The study found that gamma secretase complex and Notch1 are involved in the pathogenesis of some neurodegenerative diseases. Gamma secretase inhibitor DAPT can inhibit the cerebral ischemia mediated by apoptosis and inflammatory reaction. In this part, the cecum ligation puncture method (CLP) was used to establish a sepsis model, to observe the role of Notch signaling pathway in sepsis encephalopathy and the protective effect of DAPT on sepsis encephalopathy.
Methods: taking SD rats, establishing CLP model, intraperitoneal injection of pentobarbital sodium 30mg/kg, abdominal shaving and iodophor disinfection, paaving with Iodophor, cutting out 2cm abdominal incision along the midline of abdomen, exposing the cecum, ligation of the cecum on the distal ileocecal flap, then puncturing the cecum with number 18 needle for 2 times, gently pressing the cecum, so as to exudate a small amount of intestinal contents and then return cecum and return the cecum. The rats were returned to the abdominal cavity and closed the abdominal cavity. The sham operation group (sham) rats were given a caesarean section, and the cecum was placed for 1 minutes without cecum puncture or ligation. Then the cecum was returned to the abdominal cavity and closed to the abdomen. The rats were given subcutaneous injection of saline (3ml/100g) and ceftriaxone (30mg/kg) every 6 hours after the operation. 24G infusion tube was inserted to measure blood pressure and heart rate. 6h, 12h and 24h blood pressure, heart rate and Anal temperature were recorded before operation, and the blood lactate level (n=3) of 1ml was randomly divided into four groups: (a) sham operation + saline group (10ml/kg) and (b) sham +DAPT group (10 mu mol/kg): (b) Saline group (1ml/kg); (d) group CLP+DAPT (10 mu mol/kg). Each group was divided into 2 subgroups, the first subgroup, 24 hours of death rats, the broken head to take the blood, the separation of the hippocampus, the -80 centigrade, the NICD Western-Blot protein detection. Second subgroup, every 6 hours after the operation, injected physiological saline (3ml/100g) and ceftriaxone (30mg/kg), and allowed free feeding every 6 hours after the operation. After seventh days of operation, rats were examined for behavioral changes (new object recognition test).
Results: in the CLP group, the rats in the CLP group were curled up and less moving, the erect hair, a few rats appeared hemiplegic, epileptic seizures, and the average arterial pressure decreased obviously, the heart rate increased obviously, the blood lactate level increased, the weight decreased obviously, the third day was 56.3%, and the 4-7 day was 37.5%; the 24h nerve reflex score of the group CLP rats decreased significantly, and was fourth days, seventh days. It has been gradually recovered; new body cognition test suggests that the learning and memory function of CLP rats is impaired. After CLP, the expression of.Y- secretase inhibitor DAPT can reduce the expression of NICD after CLP and alleviate the impairment of cognitive function in 24h.
Conclusion: CLP rats completely simulated the typical clinical manifestations of sepsis. Neural reflex score and behavioral examination suggested that the rat model of sepsis related encephalopathy (SAE) was successfully made. The expression of 24h and NICD was significantly increased after CLP, indicating that Notch signaling pathway may be involved in the brain damage caused by sepsis; and DAPT inhibition was also used. Notch signaling pathway has a neuroprotective effect on memory impairment in septic rats.
The second part is the mechanism of Notch signaling pathway in sepsis encephalopathy.
Objective: in this experimental study, by constructing a rat CLP sepsis model, we mainly explore the possible pathogenesis of Notch signaling pathway involved in sepsis encephalopathy and the intervention effect of gamma secretase inhibitor on sepsis encephalopathy in rats by blocking the Notch1 signaling pathway.
Methods: 59 SD rats were randomly divided into 4 groups, which were sham operation group (Sham), sham operation +DAPT group (Sham+DAPT), CLP group (CLP), CLP+DAPT group. The rats were executed in CLP or after sham operation. The rats were killed and hippocampus tissue was used for Westen blot method to detect PARP-1. Neuron death was observed by color method.
Results: our study showed that in the experimental animals, the levels of TNF- alpha IL-lb, IL-6 in the CLP group were significantly higher than those in the sham operation group and the sham operation group (+DAPT group), although the increase in the CLP+DAPT group was inhibited. The neuronal cell death and apoptosis in the hippocampus were evaluated by HE staining and TUNEL staining. In the sham operation group and the sham operation group, the number of neurons in the sham operation group and the sham operation +DAPT group were in a few cases. HE positive cells could not be found. After CLP operation, the neuronal cell death in the CA1 region of the hippocampus increased significantly. However, compared with the CLP group, the HE staining positive cells were significantly reduced in the CLP+DAPT group. Furthermore, the TUNEL method was used to detect the apoptosis of the neurons, and the TUNEL positive cells in the CLP group were significantly increased, but they were almost undetected in the sham operation group. The number of apoptotic neurons in the CLP+DAPT group was significantly higher than that in the sham operated group RAPT group. Compared with the CLP group, the number of apoptotic cells in the first two groups was significantly reduced.
In the rats with sepsis, the inflammatory response of the hippocampus, the apoptosis of neurons, and the increase of neuron death obviously.DAPT can significantly reduce the apoptosis and death of hippocampal neurons, and the expression of TNF- alpha, IL-1b, IL-6, PARP-1 and Caspase-1.
Conclusion: it is confirmed that the Notch signaling pathway activates PARP-1 degradation and induces apoptosis of caspase dependent neurons. Y- secretase inhibitor DAPT inhibits the degradation of PARP-1 in hippocampal neurons, and inhibits the caspase dependent neuronal apoptosis.DAPT to improve the sepsis encephalopathy by inhibiting the apoptosis and inflammatory response of the hippocampal neurons in the rats with CLP sepsis.
The third part of Notch signaling pathway regulates the molecular mechanism of sepsis encephalopathy: regulation of NF-kB activation.
Objective: NF-kB is a key transcription factor that participates in the inflammatory response in the nucleus and is widely used in the cells of the nervous system to regulate the inflammatory response and apoptosis of the nerve cells. The study shows that the NF-kB signal pathway also plays an important role in the inflammation of the sepsis nervous system. In this experiment, the CLP model is used to study the NF-k of the sepsis to the nerve cells. The effects of B signaling pathway and the effect of Notch specific inhibitor DAPT on the NF-kB signaling pathway were observed, and the regulatory role of Notch on the NF-kB signaling pathway in sepsis encephalopathy was investigated.
Methods: SD rats were randomly divided into four groups (n=6), which were sham operation group (Sham), sham operation +DAPT group (Sham+DAPT), CLP group (CLP), CLP+DAPT group. 30 minutes before operation, DAPT (10 mu mo/kg) or physiological saline (10mL/kg) were intraperitoneally injected into the operation, and every 6 hours after operation, the physiological saline (3m1/100g) and ceftriaxone were resuscitation for 24 hours. Rats were harvested and stored in liquid nitrogen for preservation. The expression level of NF-K protein was detected by Western blot and EMUSA.
Results: in the Sham group, the nuclear factor NF-kB was expressed in the cytoplasm, and CLP made the NF-kB cytoplasm transfer into the nucleus. Compared with the Sham group, the content of NF-kB protein in the CLP group increased significantly, and the p65 was the most significant. Protein expression level. Compared with group Sham, the expression of phosphorylated IKK- beta in CLP group was significantly increased, but DAPT treatment inhibited the expression of phosphorylated IKK- beta in CLP. Similarly, DAPT treatment inhibited p65 phosphorylation, which may be related to the decrease of IKK- beta activity, which will inevitably lead to the decrease of p65DNA binding activity.
Conclusion: in this study, we confirm that CLP induced sepsis causes NF-kB to migrate from the cytoplasm of hippocampal cells to the nucleus of the hippocampal cells. The Notch signaling pathway specific inhibitor DAPT inhibits the Notch target gene expression and inhibits the migration of NF-kB into the nucleus and its DNA binding activity. In the previous article, the transcription activation of NF-kB dependence causes the sepsis hippocampus God. Cell inflammation and apoptosis may be one of the causes of encephalopathy. Inhibition of Notch signaling pathway may be an effective method for the treatment of septic encephalopathy.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R459.7
本文编号：2134942
[Abstract]:Sepsis encephalopathy, also known as sepsis associated encephalopathy (Sepsis-associated encephalopathy, SAE), is a diffuse brain dysfunction caused by sepsis. The incidence of the disease is 8%-70%. clinical study. SAE is one of the most common encephalopathy in ICU. SAE can lead to a significant increase in mortality in patients with sepsis, and.SAE's pathophysiological mechanism is complex. The pathogenesis is not clear, but a large number of research results show that neuronal apoptosis is one of the important manifestations of the occurrence and development of SAE. In the rat model of sepsis, SAE can lead to long-term memory damage in rats. Among them, the damage degree of the hippocampus, which is closely related to spatial memory, is particularly obvious.
In this study, the main research object of this study is the Notch signaling pathway. The.Notch signaling pathway in the pathogenesis of SAE is one of the key signaling pathways in the regulation of neuronal apoptosis. The latest research shows that the gamma secretase complex and Notch1 are involved in the pathogenesis of some neurodegenerative diseases, such as Alzheimer's disease, ischemic stroke, and Notch letter. The pathway specific inhibitor DAPT can effectively inhibit the cerebral ischemic injury mediated by apoptosis and inflammatory response, and its mechanism of action is related to the regulatory role of the Notch signaling pathway in the process of PARP-1 activation and apoptosis. Recent studies have found that the activation of the caspase pathway through the activation of the PARP-1 through its own division and the related factors in the nuclear decay, resulting in the activation of the caspase pathway. Apoptosis of neurons.
In this study, a rat sepsis model was established by cecal ligation (CLP), and the role and mechanism of Notch signaling pathway in SAE was concentrated. The study showed that the expression of Notch receptor area (NICD) in the Notch receptor increased significantly after 24 hours of CLP, and the hippocampal inflammatory response, neuron apoptosis, neuron death and so on increased.D obviously. APT can significantly reduce the apoptosis and death of hippocampal neurons, as well as the expression of TNF- alpha IL-lb, IL-6, PARP-1 and Caspase-1. At the same time, CLP induced NF-kB nucleation can also be significantly inhibited by DAPT.
To sum up, Notch signaling pathway is involved in the brain damage caused by sepsis, and its mechanism is related to the regulation of NF-kB transnuclear. The results can provide new strategies and drug targets for the treatment of SAE.
Part 1 the role of Notch signaling pathway in sepsis encephalopathy
Objective: the pathophysiological mechanism of sepsis related encephalopathy (SAE) is not clear, lack of objective diagnosis and effective treatment and prevention. The study found that gamma secretase complex and Notch1 are involved in the pathogenesis of some neurodegenerative diseases. Gamma secretase inhibitor DAPT can inhibit the cerebral ischemia mediated by apoptosis and inflammatory reaction. In this part, the cecum ligation puncture method (CLP) was used to establish a sepsis model, to observe the role of Notch signaling pathway in sepsis encephalopathy and the protective effect of DAPT on sepsis encephalopathy.
Methods: taking SD rats, establishing CLP model, intraperitoneal injection of pentobarbital sodium 30mg/kg, abdominal shaving and iodophor disinfection, paaving with Iodophor, cutting out 2cm abdominal incision along the midline of abdomen, exposing the cecum, ligation of the cecum on the distal ileocecal flap, then puncturing the cecum with number 18 needle for 2 times, gently pressing the cecum, so as to exudate a small amount of intestinal contents and then return cecum and return the cecum. The rats were returned to the abdominal cavity and closed the abdominal cavity. The sham operation group (sham) rats were given a caesarean section, and the cecum was placed for 1 minutes without cecum puncture or ligation. Then the cecum was returned to the abdominal cavity and closed to the abdomen. The rats were given subcutaneous injection of saline (3ml/100g) and ceftriaxone (30mg/kg) every 6 hours after the operation. 24G infusion tube was inserted to measure blood pressure and heart rate. 6h, 12h and 24h blood pressure, heart rate and Anal temperature were recorded before operation, and the blood lactate level (n=3) of 1ml was randomly divided into four groups: (a) sham operation + saline group (10ml/kg) and (b) sham +DAPT group (10 mu mol/kg): (b) Saline group (1ml/kg); (d) group CLP+DAPT (10 mu mol/kg). Each group was divided into 2 subgroups, the first subgroup, 24 hours of death rats, the broken head to take the blood, the separation of the hippocampus, the -80 centigrade, the NICD Western-Blot protein detection. Second subgroup, every 6 hours after the operation, injected physiological saline (3ml/100g) and ceftriaxone (30mg/kg), and allowed free feeding every 6 hours after the operation. After seventh days of operation, rats were examined for behavioral changes (new object recognition test).
Results: in the CLP group, the rats in the CLP group were curled up and less moving, the erect hair, a few rats appeared hemiplegic, epileptic seizures, and the average arterial pressure decreased obviously, the heart rate increased obviously, the blood lactate level increased, the weight decreased obviously, the third day was 56.3%, and the 4-7 day was 37.5%; the 24h nerve reflex score of the group CLP rats decreased significantly, and was fourth days, seventh days. It has been gradually recovered; new body cognition test suggests that the learning and memory function of CLP rats is impaired. After CLP, the expression of.Y- secretase inhibitor DAPT can reduce the expression of NICD after CLP and alleviate the impairment of cognitive function in 24h.
Conclusion: CLP rats completely simulated the typical clinical manifestations of sepsis. Neural reflex score and behavioral examination suggested that the rat model of sepsis related encephalopathy (SAE) was successfully made. The expression of 24h and NICD was significantly increased after CLP, indicating that Notch signaling pathway may be involved in the brain damage caused by sepsis; and DAPT inhibition was also used. Notch signaling pathway has a neuroprotective effect on memory impairment in septic rats.
The second part is the mechanism of Notch signaling pathway in sepsis encephalopathy.
Objective: in this experimental study, by constructing a rat CLP sepsis model, we mainly explore the possible pathogenesis of Notch signaling pathway involved in sepsis encephalopathy and the intervention effect of gamma secretase inhibitor on sepsis encephalopathy in rats by blocking the Notch1 signaling pathway.
Methods: 59 SD rats were randomly divided into 4 groups, which were sham operation group (Sham), sham operation +DAPT group (Sham+DAPT), CLP group (CLP), CLP+DAPT group. The rats were executed in CLP or after sham operation. The rats were killed and hippocampus tissue was used for Westen blot method to detect PARP-1. Neuron death was observed by color method.
Results: our study showed that in the experimental animals, the levels of TNF- alpha IL-lb, IL-6 in the CLP group were significantly higher than those in the sham operation group and the sham operation group (+DAPT group), although the increase in the CLP+DAPT group was inhibited. The neuronal cell death and apoptosis in the hippocampus were evaluated by HE staining and TUNEL staining. In the sham operation group and the sham operation group, the number of neurons in the sham operation group and the sham operation +DAPT group were in a few cases. HE positive cells could not be found. After CLP operation, the neuronal cell death in the CA1 region of the hippocampus increased significantly. However, compared with the CLP group, the HE staining positive cells were significantly reduced in the CLP+DAPT group. Furthermore, the TUNEL method was used to detect the apoptosis of the neurons, and the TUNEL positive cells in the CLP group were significantly increased, but they were almost undetected in the sham operation group. The number of apoptotic neurons in the CLP+DAPT group was significantly higher than that in the sham operated group RAPT group. Compared with the CLP group, the number of apoptotic cells in the first two groups was significantly reduced.
In the rats with sepsis, the inflammatory response of the hippocampus, the apoptosis of neurons, and the increase of neuron death obviously.DAPT can significantly reduce the apoptosis and death of hippocampal neurons, and the expression of TNF- alpha, IL-1b, IL-6, PARP-1 and Caspase-1.
Conclusion: it is confirmed that the Notch signaling pathway activates PARP-1 degradation and induces apoptosis of caspase dependent neurons. Y- secretase inhibitor DAPT inhibits the degradation of PARP-1 in hippocampal neurons, and inhibits the caspase dependent neuronal apoptosis.DAPT to improve the sepsis encephalopathy by inhibiting the apoptosis and inflammatory response of the hippocampal neurons in the rats with CLP sepsis.
The third part of Notch signaling pathway regulates the molecular mechanism of sepsis encephalopathy: regulation of NF-kB activation.
Objective: NF-kB is a key transcription factor that participates in the inflammatory response in the nucleus and is widely used in the cells of the nervous system to regulate the inflammatory response and apoptosis of the nerve cells. The study shows that the NF-kB signal pathway also plays an important role in the inflammation of the sepsis nervous system. In this experiment, the CLP model is used to study the NF-k of the sepsis to the nerve cells. The effects of B signaling pathway and the effect of Notch specific inhibitor DAPT on the NF-kB signaling pathway were observed, and the regulatory role of Notch on the NF-kB signaling pathway in sepsis encephalopathy was investigated.
Methods: SD rats were randomly divided into four groups (n=6), which were sham operation group (Sham), sham operation +DAPT group (Sham+DAPT), CLP group (CLP), CLP+DAPT group. 30 minutes before operation, DAPT (10 mu mo/kg) or physiological saline (10mL/kg) were intraperitoneally injected into the operation, and every 6 hours after operation, the physiological saline (3m1/100g) and ceftriaxone were resuscitation for 24 hours. Rats were harvested and stored in liquid nitrogen for preservation. The expression level of NF-K protein was detected by Western blot and EMUSA.
Results: in the Sham group, the nuclear factor NF-kB was expressed in the cytoplasm, and CLP made the NF-kB cytoplasm transfer into the nucleus. Compared with the Sham group, the content of NF-kB protein in the CLP group increased significantly, and the p65 was the most significant. Protein expression level. Compared with group Sham, the expression of phosphorylated IKK- beta in CLP group was significantly increased, but DAPT treatment inhibited the expression of phosphorylated IKK- beta in CLP. Similarly, DAPT treatment inhibited p65 phosphorylation, which may be related to the decrease of IKK- beta activity, which will inevitably lead to the decrease of p65DNA binding activity.
Conclusion: in this study, we confirm that CLP induced sepsis causes NF-kB to migrate from the cytoplasm of hippocampal cells to the nucleus of the hippocampal cells. The Notch signaling pathway specific inhibitor DAPT inhibits the Notch target gene expression and inhibits the migration of NF-kB into the nucleus and its DNA binding activity. In the previous article, the transcription activation of NF-kB dependence causes the sepsis hippocampus God. Cell inflammation and apoptosis may be one of the causes of encephalopathy. Inhibition of Notch signaling pathway may be an effective method for the treatment of septic encephalopathy.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R459.7
【参考文献】
相关期刊论文 前2条
1 潘长福;阮琼芳;沈晓黎;涂伟;娄远蕾;匡助才;赖贤良;汪泱;邓志锋;;脑外伤后小鼠海马区notch信号的表达变化[J];实验与检验医学;2010年04期
2 汤耀卿;李磊;;脓毒症动物模型制作方略及应用[J];中华实验外科杂志;2006年12期
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