【摘要】：膿毒癥腦病又稱膿毒癥相關(guān)性腦病(Sepsis-associated encephalopathy, SAE)是膿毒癥引起的彌漫性腦功能障礙,其發(fā)病率為8%-70%。臨床研究發(fā)現(xiàn),SAE是ICU中最常見的腦病之一,并發(fā)SAE可導(dǎo)致膿毒癥患者的死亡率明顯增高。SAE病理生理機(jī)制較復(fù)雜,其發(fā)病機(jī)制尚不明確,但是大量的研究結(jié)果表明神經(jīng)元凋亡是SAE發(fā)生及發(fā)展過程的重要表現(xiàn)之一。研究證實(shí),在膿毒癥大鼠模型中,SAE可導(dǎo)致大鼠長期的記憶損傷。其中,與空間記憶密切相關(guān)的海馬等腦區(qū)的損傷程度尤為明顯。 本研究以Notch信號(hào)通路為主要研究對(duì)象,探討SAE發(fā)病機(jī)制。Notch信號(hào)通路是調(diào)控神經(jīng)元凋亡的關(guān)鍵信號(hào)通路之一。最新研究表明,γ-分泌酶復(fù)合體和Notch1參與了一些神經(jīng)變性疾病的發(fā)病過程,如阿爾茨海默病、缺血性腦卒中等。同時(shí),Notch信號(hào)通路特異性抑制劑DAPT可有效抑制凋亡和炎癥反應(yīng)介導(dǎo)的腦缺血性損傷,其作用機(jī)制與Notch信號(hào)通路在PARP-1激活凋亡過程中起到調(diào)控作用有關(guān)。新近研究發(fā)現(xiàn),PARP-1激活后通過自身分裂和核內(nèi)凋亡相關(guān)因子,活化caspase通路,從而導(dǎo)致神經(jīng)元的凋亡。 本研究以盲腸結(jié)扎穿刺法(CLP)建立大鼠的膿毒癥模型,集中開展了Notch信號(hào)通路在SAE中的作用及其機(jī)制研究。研究表明,CLP后24小時(shí),Notch受體細(xì)胞內(nèi)區(qū)域(NICD)表達(dá)顯著增加,大鼠海馬組織炎癥反應(yīng)、神經(jīng)元凋亡、神經(jīng)元死亡等也明顯增加。DAPT能明顯減少海馬組織神經(jīng)元凋亡和死亡,以及TNF-αIL-lb、IL-6、PARP-1和Caspase-1的表達(dá)。同時(shí),CLP引起NF-kB轉(zhuǎn)核作用也可被DAPT明顯抑制。 綜上所述,Notch信號(hào)通路參與了膿毒癥導(dǎo)致的腦損傷,其作用機(jī)制與調(diào)控NF-kB轉(zhuǎn)核有關(guān)。研究結(jié)果可為SAE的治療提供新的策略和藥物靶標(biāo)。 第一部分Notch信號(hào)通路在膿毒癥腦病中的作用 目的：膿毒癥相關(guān)性腦病(SAE)病理生理機(jī)制尚不明確,缺乏客觀的診斷指標(biāo)及有效的治療和預(yù)防手段。研究發(fā)現(xiàn)γ-分泌酶復(fù)合體和Notch1參與了一些神經(jīng)變性疾病的發(fā)病過程,γ-分泌酶抑制劑DAPT能抑制凋亡和炎癥反應(yīng)介導(dǎo)的腦缺血性損傷。本部分選用盲腸結(jié)扎穿刺法(CLP)建立膿毒癥模型,觀察Notch信號(hào)通路在膿毒癥腦病中的作用及DAPT對(duì)膿毒癥腦病的保護(hù)作用。 方法：取SD大鼠,建立CLP模型,戊巴比妥鈉30mg/kg腹腔注射麻醉,腹部剃毛并用碘伏消毒,鋪巾,沿腹中線剪開2cm腹部切口,暴露盲腸,在回盲瓣遠(yuǎn)端結(jié)扎盲腸,然后用18號(hào)針刺穿刺盲腸2次,輕輕擠壓盲腸,使少量的腸內(nèi)容物滲出,然后將盲腸返回到腹腔,關(guān)閉腹腔。假手術(shù)組(sham)大鼠予剖腹,并將盲腸外置1分鐘,不予盲腸穿刺或結(jié)扎,然后將盲腸回納至腹腔并關(guān)腹。每只手術(shù)操作的大鼠術(shù)后每6小時(shí)給予皮下注射生理鹽水(3ml/100g)及頭孢曲松(30mg/kg).實(shí)驗(yàn)于麻醉后直視下行股動(dòng)脈切開置入24G輸液管以測(cè)定血壓、心率。記錄術(shù)前、術(shù)后6h、12h和24h血壓、心率和肛溫,并在以上時(shí)間點(diǎn)經(jīng)股動(dòng)脈采血1ml測(cè)血乳酸水平(每個(gè)時(shí)間點(diǎn),n=3)。59只大鼠隨機(jī)分為四組：(a)假手術(shù)+生理鹽水組(10ml/kg);(b)假手術(shù)+DAPT組(10μmol/kg):(c)CLP+生理鹽水組(1ml/kg);(d) CLP+DAPT組(10μmol/kg)。每組分為2個(gè)亞組,第一亞組,24小時(shí)處死大鼠,斷頭取血,分離海馬組織,-80℃保存,行NICD的Western-Blot蛋白檢測(cè)。第二亞組,術(shù)后每6小時(shí)子皮下注射生理鹽水(3ml/100g)及頭孢曲松(30mg/kg),并允許自由進(jìn)食及飲水。術(shù)后第7天存活大鼠檢測(cè)行為學(xué)改變(新物體識(shí)別實(shí)驗(yàn))。 結(jié)果：CLP組大鼠出現(xiàn)蜷縮少動(dòng)、豎毛,個(gè)別大鼠出現(xiàn)偏癱、癲癇發(fā)作表現(xiàn)；并出現(xiàn)平均動(dòng)脈壓明顯下降,心率明顯升高,血乳酸水平增高；體重明顯下降；第3天為56.3%,第4-7天為37.5%；CLP組大鼠24h神經(jīng)反射評(píng)分明顯下降,并在第4天、第7天已逐漸恢復(fù)；新物體認(rèn)知試驗(yàn)提示CLP大鼠學(xué)習(xí)記憶功能受損。在CLP后24h,NICD表達(dá)顯著增加。Y-分泌酶抑制劑DAPT能降低CLP后NICD的表達(dá),緩解認(rèn)知功能損傷。 結(jié)論：CLP組大鼠完全模擬了膿毒癥典型臨床表現(xiàn),神經(jīng)反射評(píng)分及行為學(xué)檢查提示大鼠膿毒癥相關(guān)性腦病(SAE)模型制作成功；首次發(fā)現(xiàn)在CLP后24h,NICD表達(dá)顯著增加,說明Notch信號(hào)通路可能參與了膿毒癥導(dǎo)致的腦損傷；同時(shí)應(yīng)用DAPT抑制Notch信號(hào)通路對(duì)膿毒癥大鼠的記憶損傷有神經(jīng)保護(hù)作用。 第二部分Notch信號(hào)通路參與膿毒癥腦病的機(jī)制研究 目的：在本實(shí)驗(yàn)研究中,我們通過構(gòu)建大鼠CLP膿毒癥模型,主要探討Notch信號(hào)通路參與膿毒癥腦病的可能發(fā)病機(jī)制,以及γ-分泌酶抑制劑通過阻斷Notch1信號(hào)通路對(duì)膿毒癥腦病大鼠的干預(yù)作用。 方法：隨機(jī)將59只SD大鼠分為4組,分別為假手術(shù)組(Sham)、假手術(shù)+DAPT組(Sham+DAPT)、CLP組(CLP)、CLP+DAPT組,在CLP或假手術(shù)后24h,將大鼠處死,取出海馬組織用于Westen blot法檢測(cè)PARP-1、Caspase-1; ELISA法檢測(cè)TNF-α IL-1b\IL-6水平。TUNEL法觀察神經(jīng)元凋亡,HE染色法觀察神經(jīng)元死亡。 結(jié)果：我們研究表明在實(shí)驗(yàn)動(dòng)物中,比較于假手術(shù)組和假手術(shù)+DAPT組,在CLP組TNF-α IL-lb、IL-6水平顯著增高,盡管在CLP+DAPT組增高受到抑制。通過HE染色和TUNEL染色法檢測(cè),評(píng)估海馬組織神經(jīng)細(xì)胞死亡和凋亡。在假手術(shù)組和假手術(shù)+DAPT組中幾乎不能找到HE陽性的細(xì)胞。在CLP手術(shù)后,海馬組織CA1區(qū)域神經(jīng)細(xì)胞死亡明顯增加。然而,與CLP組相比,HE染色陽性細(xì)胞在CLP+DAPT組明顯減少。此外,TUNEL法檢測(cè)神經(jīng)細(xì)胞凋亡情況,在CLP組中TUNEL陽性細(xì)胞明顯增加,但是在假手術(shù)組幾乎不能檢測(cè)到TUNEL陽性細(xì)胞。而神經(jīng)細(xì)胞凋亡數(shù)目在CLP+DAPT組明顯多于假手術(shù)組—RAPT組,與CLP組比較,前兩組凋亡數(shù)目明顯減少。 在膿毒癥大鼠中,海馬組織炎癥反應(yīng)、神經(jīng)元凋亡、神經(jīng)元死亡明顯增加。DAPT能明顯減少海馬組織神經(jīng)元凋亡和死亡,及其TNF-α、IL-1b、IL-6、PARP-1和Caspase-1的表達(dá)。 結(jié)論：證實(shí)了Notch信號(hào)通路通過激活PARP-1降解,導(dǎo)致caspase依賴性神經(jīng)元凋亡,Y-分泌酶抑制劑DAPT抑制海馬神經(jīng)元PARP-1降解,抑制caspase依賴性神經(jīng)元凋亡。DAPT通過抑制CLP膿毒癥大鼠海馬組織神經(jīng)元凋亡和炎癥反應(yīng)改善膿毒癥腦病。 第三部分Notch信號(hào)通路調(diào)控膿毒癥腦病的分子機(jī)制研究：調(diào)控NF-kB激活 目的：NF-kB是在核內(nèi)參與炎癥反應(yīng)的關(guān)鍵轉(zhuǎn)錄因子,亦廣泛存在于神經(jīng)系統(tǒng)的細(xì)胞中,調(diào)控神經(jīng)細(xì)胞炎癥反應(yīng)及凋亡。研究顯示,NF-kB信號(hào)途徑在膿毒癥神經(jīng)系統(tǒng)炎癥中也扮有重要角色。本實(shí)驗(yàn)部分采用CLP模型,研究膿毒癥對(duì)神經(jīng)細(xì)胞NF-kB信號(hào)通路的影響,并通過Notch特異性抑制劑DAPT干預(yù),觀察其對(duì)NF-kB信號(hào)通路的影響,由此深入研究Notch對(duì)膿毒癥腦病NF-kB信號(hào)通路的調(diào)控作用。 方法：取SD大鼠,隨機(jī)分為四組(n=6),分別為假手術(shù)組(Sham)、假手術(shù)+DAPT組(Sham+DAPT)、CLP組(CLP)、CLP+DAPT組。手術(shù)操作前30分鐘腹腔注射DAPT(10μmo/kg)或生理鹽水(10mL/kg).術(shù)后每6小時(shí)皮下注射生理鹽水(3m1/100g)及頭孢曲松(30mg/kg)復(fù)蘇,24小時(shí)處死大鼠,取大鼠海馬組織,液氮保存待測(cè)。用western blot和EMUSA分組檢測(cè)NF-K蛋白表達(dá)水平。 結(jié)果：在Sham組動(dòng)物中,核因子NF-kB在胞漿中表達(dá),CLP使得NF-kB胞漿向胞核內(nèi)轉(zhuǎn)移。與Sham組相比,CLP組胞核內(nèi)NF-kB蛋白含量明顯升高,以p65最為顯著。與Sham及Sham+DAPT組相比,CLP組胞核內(nèi)NF-kB蛋白含量明顯升高,但DAPT處理降低胞核內(nèi)p65、p50及Re1B蛋白表達(dá)水平。與Sham組相比,CLP組磷酸化IKK-β表達(dá)明顯升高,但DAPT處理可抑制CLP時(shí)磷酸化IKK-β表達(dá)。同樣,DAPT處理可抑制p65磷酸化,其可能與IKK-β活性降低有關(guān),相應(yīng)的必然會(huì)導(dǎo)致p65DNA結(jié)合活性降低。 結(jié)論：本研究中我們證實(shí)CLP導(dǎo)致的膿毒癥引起NF-kB由海馬神經(jīng)細(xì)胞胞漿向胞核內(nèi)遷移,應(yīng)用Notch信號(hào)通路特異性抑制劑DAPT抑制Notch靶基因表達(dá),抑制NF-kB向胞核內(nèi)遷移及其DNA結(jié)合活性。結(jié)合前文,NF-kB依賴的轉(zhuǎn)錄激活引起膿毒癥海馬神經(jīng)細(xì)胞炎癥反應(yīng)及細(xì)胞凋亡,可能是導(dǎo)致腦病發(fā)生的原因之一。抑制Notch信號(hào)通路可能成為治療膿毒癥腦病的有效方法。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R459.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 潘長福;阮瓊芳;沈曉黎;涂偉;婁遠(yuǎn)蕾;匡助才;賴賢良;汪泱;鄧志鋒;;腦外傷后小鼠海馬區(qū)notch信號(hào)的表達(dá)變化[J];實(shí)驗(yàn)與檢驗(yàn)醫(yī)學(xué);2010年04期

2 湯耀卿;李磊;;膿毒癥動(dòng)物模型制作方略及應(yīng)用[J];中華實(shí)驗(yàn)外科雜志;2006年12期

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