Omi/HtrA2信号通路介导脓毒症脑病的作用及机制研究
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摘要
脓毒症的发病率和死亡率较高,神经系统是脓毒症时最早累及的器官。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。SAE的发病率为8%-70%,是ICU中最常见的脑病。并发SAE使脓毒症患者的死亡率明显增高。SAE病理生理机制较复杂,尚不明确,目前研究表明可能包括脑微血管内皮细胞功能障碍,血脑屏障(BBB)的破坏,脑局部炎症细胞的浸润,炎症介质的参与,脑灌注的下降,脑微血管调节障碍,星形胶质细胞和神经元功能障碍,神经递质紊乱,线粒体功能障碍,细胞凋亡,氧化应激和钙失调等。由此可见,SAE有多种病理生理机制参与,目前缺乏针对其病理生理机制的治疗和预防手段。
有研究证实SAE时存在线粒体功能障碍,脓毒症除了破坏线粒体膜电位,影响线粒体呼吸链功能,引起神经细胞能量供应不足,还介导神经细胞凋亡。现大量研究表明Omi/HtrA2是一种寡聚丝氨酸蛋白酶,在多种病理情况(心脏缺血/再灌注损伤,顺铂引起的肾损伤,脑缺血/再灌注损伤等)下介导细胞凋亡起关键作用,并且多项研究证实Omi/HtrA2参与多种神经系统疾病的病理过程(如亨廷顿氏病,脑缺血/再灌注及癫痫持续状态等)。本研究通过建立SAE大鼠模型,研究SAE大鼠海马组织氧化应激损伤及神经细胞凋亡,研究SAE对Omi/HtrA2信号通路(Omi/HtrA2线粒体内外表达及XIAP.caspase-3,9裂解产物、蛋白酶PARP裂解产物的胞浆内表达)的影响及应用Omi/HtrA2蛋白酶特异性抑制剂,Ucf-101对SAE干预,由此深入研究Omi/HtrA2信号通路介导SAE的作用及机制,为SAE提供新的治疗靶点。
第一部分脓毒症脑病模型的建立
目的：脓毒症相关性脑病(SAE)是ICU中最常见的脑病,使得脓毒症患者的死亡率明显增高,目前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,迫切需要大量动物基础研究。本部分选用金标准模型——盲肠结扎穿刺法(CLP)建立脓毒症模型,并通过评估大鼠行为学试验及神经反射评分,结合海马病理学改变,确定脓毒症脑病模型成功建立,以便于下一步实验研究。
方法：取SD大鼠,建立CLP模型,用4%水合氯醛(1ml/100g)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号针刺穿刺盲肠一次,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)。术后24小时,处死大鼠,取大鼠海马组织行苏木素-伊红(hematoxylin and eosin, H&E)染色观察脓毒症海马病理学改变(n=6)。另外相同分组(n=16),术后每天给予皮下注射生理盐水及头孢曲松,并允许正常进食及饮水,我们观察了实验大鼠7d生存率及大鼠体重的变化,行神经反射评分、新物体认知试验及Morris水迷宫试验行为学评估。
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；CLP组大鼠存活率明显下降,第3天为43.75%,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；海马H&E染色病理学检查提示CLP组大鼠可见明显神经元固缩、深染,细胞结构不清,细胞周围空隙明显增大,胶质细胞周围间隙扩大,空泡样改变,血管周围间隙扩大,以海马齿状回(dentate gyrus, DG)区最为显著,CA3区最轻。行为学试验(Morris水迷宫试验及新物体认知试验)均提示CLP大鼠学习记忆功能受损。
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,为后续SAE机制研究做好基础。海马组织病理改变提示海马损伤可能与SAE的发生有关。
第二部分Omi/HtrA2蛋白酶特异性抑制剂ucf-101对脓毒症脑病的保护作用
目的：大量研究提示SAE与海马神经细胞凋亡相关,而Omi/HtrA2是介导细胞凋亡的关键促凋亡蛋白,研究表明Omi/HtrA2蛋白酶特异性抑制剂ucf-101在多种病理情况下能抑制细胞凋亡,改善神经功能。本研究观察ucf-101对大鼠SAE的神经保护作用。
方法：实验动物随机分为四组,分别为假手术组(Sham)、假手术+Ucf-101组(Sham+Ucf-101)、CLP组(CLP)、CLP+Ucf-101组。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)。手术操作前30分钟、术后每日腹腔注射Ucf-101(10umol/kg)或生理盐水(10mL/kg)。实验24小时处死大鼠,取大鼠海马组织检测神经细胞的凋亡情况及病理学改变(n=6),检测海马组织氧化应激指标,包括谷胱甘肽(GSH),丙二醛(MDA),髓过氧化物酶(MPO)的含量及过氧化氢酶(CAT)的活性,检测海马组织炎症因子肿瘤坏死因子(TNF)-α及白介素(IL)-6水平(n=6)。另外相同分组(n=16),术后每天给予皮下注射生理盐水及头孢曲松,术后每日腹腔注射Ucf-101或生理盐水,并允许正常进食及饮水,我们观察了实验大鼠7d生存率及大鼠体重的变化,行神经反射评分、新物体认知试验及Morris水迷宫试验行为学评估。
结果：CLP组大鼠存活率明显下降,24h神经反射评分明显下降,学习记忆功能损害(行为学试验),Ucf-101台疗能明显提高大鼠生存率,明显提高大鼠术后24h神经反射评分,明显改善大鼠的学习记忆功能。CLP组大鼠海马区可见明显神经元固缩、深染,细胞结构不清及大量细胞凋亡,Ucf-101治疗能明显改善CLP导致的海马区病理损害及细胞凋亡。CLP组大鼠海马区TNF-α、IL-6水平明显升高,存在明显氧化应激损伤(MDA水平及MPO浓度明显增高,GSH水平和CAT活性明显下降),Ucf-101疗能明显改善海马区炎症反应,减轻氧化应激损伤。
结论：脓毒症可引起大鼠海马区炎症、氧化应激损害及细胞凋亡,导致大鼠认知记忆功能障碍,这提示脓毒症脑病发病与海马区神经细胞受损有关。Omi/HtrA2蛋白酶特异性抑制剂Ucf-101能明显减少海马区神经细胞凋亡,改善炎症和氧化应激损害,保护大鼠认知记忆功能。因此推测Ucf-101对SAE有神经保护作用,Omi/HtrA2可能是SAE治疗的新靶点。
第三部分Omi/HtrA2信号通路介导脓毒症脑病的机制研究
目的：Omi/HtrA2参与多种神经系统疾病的病理过程,本研究旨在观察SAE时Omi/HtrA2信号通路(Omi/HtrA2线粒体内外表达及XIAP、caspase-3、9裂解产物、蛋白酶PARP裂解产物胞浆内表达)的改变,并观察Ucf-101干预对Omi/HtrA2信号通路的影响,由此深入研究Omi/HtrA2信号通路介导SAE的作用及机制。
方法：实验动物随机分为四组(n=6),分别为假手术组(Sham)、假手术+Ucf-101组(Sham+Ucf-101)、CLP组(CLP)、CLP+Ucf-101组。手术操作前30分钟腹腔注射Ucf-101(10umol/kg)或生理盐水(10mL/kg)。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)。24小时处死大鼠,取大鼠海马组织,并分离线粒体和胞浆,用Western blot法分别检测Omi/HtrA2线粒体及胞浆中表达,并检测胞浆中XIAP、Caspase-3.9裂解产物、蛋白酶PARP裂解产物和cytochrome c表达。
结果：CLP组Omi/HtrA2从线粒体向胞浆内转移,胞浆内Omi/HtrA2蛋白表达明显升高,胞浆内XIAP表达明显下调,Caspase-3、9裂解产物及蛋白酶PARP裂解产物表达明显升高,Ucf-101处理可抑制Omi/HtrA2的转移,阻止XIAP的降解；明显减少胞浆内Caspase-3、9裂解产物及蛋白酶PARP裂解产物的表达；本研究也发现CLP组cytochrome c从线粒体释放至胞浆,而Ucf-101无法阻止cytochrome c的释放。
结论：本研究证实脓毒症可能导致Omi/HtrA2由海马神经细胞线粒体向胞浆内转移,通过与XIAP相结合,并降解XIAP,激活下游的细胞凋亡通路。可见Omi/HtrA2信号通路参与介导SAE的发病,Ucf-101通过干预此信号通路对SAE起神经保护作用。
The morbidity and mortality of sepsis is very high. Sepsis-associated encephalopathy(SAE) is diffuse brain dysfunction caused by sepsis. The nervous system is one of the earliest organs involved in sepsis. SAE incidence rate is8%-70%, and it is the most common encephalopathy in intensive care unit (ICU). The mortality of SAE is very high, but the pathophysiological mechanism is not clear, the current studies have found that brain microvascular endothelial cell dysfunction, blood-brain barrier (BBB) damage, inflammatory cell infiltration, inflammatory mediators, decreased cerebral perfusion, brain microvascular dysregulation, astrocytes and neurons dysfunction, neurotransmitters disorders, mitochondrial dysfunction, apoptosis, oxidative stress and calcium disorders are involved in SAE pathogenesis. And there are no effective therapies for SAE.
Studies have confirmed that mitochondrial dysfunction is involved in SAE. Mitochondria membrane potential is destructed, Mitochondrial respiratory chain is influenced, Mitochondrial disfunction can cause cell energy supply shortage and induce cell apoptosis. The studies have demonstrated that Omi/HtrA2is a kind of oligomeric serine protease. It has been confirmed that Omi/HtrA2can regulate apoptosis in a variety of pathological conditions (cardiac ischemia/reperfusion injury, the renal dysfunction caused by cisplatin, cerebral ischemia/reperfusion injury, etc.), and several studies have confirmed that the Omi/HtrA2is involved in a variety of nervous system diseases (such as huntington's disease, cerebral ischemia/reperfusion and status epilepticus, etc.). In this study we would establish SAE rat models. We would be trying to study hippocampus cell apoptosis during sepsis, and Omi/HtrA2signaling pathways (Omi/HtrA2concentration in the mitochondria and cytoplasm, and XIAP concentration, cleaved Caspase-3, cleaved Caspase-9and cleaved PARP concentrations in the cytoplasm), and Omi/HtrA2protease inhibitor(ucf-101) neuroprotective effect. We would be trying to demonstrated Omi/HtrA2signaling pathway is invovled in SAE, It would help to discover a novel therapeutic target and new drug for SAE.
Part One Establishment of a rat model of sepsis associated encephalopathy
Objective:Sepsis associated encephalopathy (SAE) is the most common encephalopathy in intensive care unit (ICU). The mortality of SAE is very high, but the pathophysiological mechanism is not clear and no effective treatment has been confirmed. Ideal animal models are needed in basic research urgently. In this part, we selected the standard septic model of cecum ligation puncture (CLP) and septic encephalopathy model was considered to be set up successfully by evaluating the behavior trials, neurologic reflex scores and hippocampal pathology which can be used in the next experiments.
Methods:Animals were anesthetized with4%Chloral hydrate (1ml/100g, intraperitoneally). Under sterile surgical conditions, a2-cm abdominal incision was made along the ventral surface of the abdomen to expose the cecum, which was then ligated below the ileocecal junction with no bowel obstruction. The cecum was punctured once with an18-gauge needle, and the fecal contents were allowed to leak into the peritoneum by gently squeezing the cecum. The bowel was then returned to the abdomen and the abdominal cavity was closed. The sham-operated rats were submitted to laparotomy, and the cecum was manipulated but neither ligated nor punctured. All rats underwent surgical manipulation, received a subcutaneous injection of saline solution (3mL/100g body weight) plus antibiotics (ceftriaxone at30mg/kg) every6h for resuscitation. In the first experiment, blood pressure, heart rate and anus temperature were measured at baseline,6h,12h and24h after operation. At the same time, we measured blood lactic acid level. We also observed hippocampal pathology24h post-operation (n=6). In the second experiment, rats (n=16) were given normal saline and ceftriaxone every day and allowed normal eating and drinking. We would evaluate neurologic reflex scores, survival rate, new object recognition test (7day after operation) and Morris water maze test (4,7days after operation).
Results:The septic rats appeared crouching, pilomotor, hemiplegia or seizures. Significant decreased mean arterial pressure, increased heart rate, deteriorated neurological reflexes together with increased blood lactate levels suggested the successful induction of sepsis in the present study. Survival rate of the CLP group decreased obviously (3days,43.75%;4-7days,37.5%). Hippocampal H&E staining showed most neurons were shrunken and stained dark in the CLP group, significantly in dentate gyrus (DG) area. Behavior trials (Morris water maze test and new object recognition test) suggests that the CLP impaired learning and memory function.
Conclusion:CLP models simulate the typical septic clinical manifestations, sepsis associated encephalopathy (SAE) model is established successfully by evaluating neurologic reflex scores, behavior trials and hippocampal pathology, which can be used in SAE mechanism study. Our study demonstrated hippocampus damage may be involved in SAE.
Part Two Neuro-protective effect of Omi/HtrA2protease inhibitor (Ucf-101) on sepsis associated encephalopathy
Objective:There are no effective treatments for SAE. It has been demonstrated that Omi/HtrA2protease inhibitors (Ucf-101) can improve a variety of pathological conditions (such as huntington's disease, cerebral ischemia/reperfusion and status epilepticus, etc.). We are trying to study Ucf-101neuro-protection of SAE.
Methods:The animals were categorized into four groups:(1) sham group plus normal saline (10mL/kg);(2) sham group plus Ucf-101(10umol/kg);(3) cecal ligation and puncture (CLP) plus normal saline (10mL/kg); and (4) CLP plus Ucf-101(10umol/kg). All rats underwent surgical manipulation, received a subcutaneous injection of saline solution (3mL/100g) plus antibiotics (ceftriaxone at30mg/kg) every6h for resuscitation. In the first experiment,24hours later, animals were sacrificed and their brains were harvested. Samples were used for analysis of inflammatory cytokine and oxidative stress (n=6), evaluation of apoptosis and pathologic changes (n=6). In the second experiment, All rats(n=16) received a intraperitoneal injection of saline solution (lOmL/kg) or Ucf-101(10umol/kg) every day, and was allowed eating and drinking normally, we would evaluate weight, neurologic reflex scores, survival rate, new object recognition test (7days after operation). Morris water maze test were also evaluated (4,7days after operation).
Results:CLP resulted in a poor survival rate, hippocampal oxidative injury, cell apoptosis and cognitive dysfunction as well as elevated TNF-a level and IL-6level, increased weight loss. Ucf-101could significantly inhibit cell apoptosis, reduce TNF-a and IL-6levels, MDA and MPO levels, elevated GSH level, slightly reverse CAT activities in the brain and attenuate this CLP effect on cognitive dysfunction. In addition, the survival rate and survival time was significantly improved by treatment with Ucf-101.
Conclusion:The present results demonstrated that sepsis can cause hippocampal inflammation, oxidative stress injury, apoptosis, and cognitive memory dysfunction. Ucf-101can significantly reduce the hippocampal cell apoptosis, inflammation and oxidative stress damage and improve cognitive function. So Ucf-101might have neuro-protective effect and Omi/HtrA2could be a new target for SAE.
Part Three Omi/HtrA2signaling pathways involved in sepsis associated encephalopathy
Objective:Omi/HtrA2is involved in a variety of nervous system disease.This study aims to study whether Omi/HtrA2signaling pathways were involved in SAE.
Methods:The animals were categorized into four groups (n=6):(1) sham group plus normal saline (10mL/kg);(2) sham group plus Ucf-101(10umol/kg);(3) cecal ligation and puncture (CLP) plus normal saline (10mL/kg); and (4) CLP plus Ucf-101(10umol/kg). All rats underwent surgical manipulation, received a subcutaneous injection of saline solution (3mL/100g) plus antibiotics (ceftriaxone at30mg/kg) every6h for resuscitation.24hours later, animals were sacrificed and their brains were harvested. Samples were used for analysis of Omi/HtrA2concentration in the mitochondria and cytoplasm, and XIAP concentration, cleaved Caspase-3, cleaved Caspase-9and cleaved PARP and cytochrome c concentrations in the cytoplasm.
Results:Omi/HtrA2is released into cytoplasm from mitochondria in the CLP group. XIAP expression decreased significantly, cleaved Caspase-3, cleaved Caspase-9and cleaved PARP concentration increased significantly. Ucf-101inhibited the translocation of Omi/HtrA2, reduced degradation of XIAP, and reduced cleaved Caspase-3, cleaved Caspase-9and cleaved PARP concentrations. This study also found that cytochrome c is released from mitochondria to the cytoplasm in CLP group and Ucf-101was unable to prevent the effect.
Conclusion:This study demonstrated that in sepsis Omi/HtrA2is released from mitochondria to cytoplasm, and it could degrade XIAP and activate the downstream of the apoptosis pathways. Omi/HtrA2mediated signal pathway is involved in sepsis associated encephalopathy, so it could be a new target for SAE.
有研究证实SAE时存在线粒体功能障碍,脓毒症除了破坏线粒体膜电位,影响线粒体呼吸链功能,引起神经细胞能量供应不足,还介导神经细胞凋亡。现大量研究表明Omi/HtrA2是一种寡聚丝氨酸蛋白酶,在多种病理情况(心脏缺血/再灌注损伤,顺铂引起的肾损伤,脑缺血/再灌注损伤等)下介导细胞凋亡起关键作用,并且多项研究证实Omi/HtrA2参与多种神经系统疾病的病理过程(如亨廷顿氏病,脑缺血/再灌注及癫痫持续状态等)。本研究通过建立SAE大鼠模型,研究SAE大鼠海马组织氧化应激损伤及神经细胞凋亡,研究SAE对Omi/HtrA2信号通路(Omi/HtrA2线粒体内外表达及XIAP.caspase-3,9裂解产物、蛋白酶PARP裂解产物的胞浆内表达)的影响及应用Omi/HtrA2蛋白酶特异性抑制剂,Ucf-101对SAE干预,由此深入研究Omi/HtrA2信号通路介导SAE的作用及机制,为SAE提供新的治疗靶点。
第一部分脓毒症脑病模型的建立
目的：脓毒症相关性脑病(SAE)是ICU中最常见的脑病,使得脓毒症患者的死亡率明显增高,目前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,迫切需要大量动物基础研究。本部分选用金标准模型——盲肠结扎穿刺法(CLP)建立脓毒症模型,并通过评估大鼠行为学试验及神经反射评分,结合海马病理学改变,确定脓毒症脑病模型成功建立,以便于下一步实验研究。
方法：取SD大鼠,建立CLP模型,用4%水合氯醛(1ml/100g)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号针刺穿刺盲肠一次,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)。术后24小时,处死大鼠,取大鼠海马组织行苏木素-伊红(hematoxylin and eosin, H&E)染色观察脓毒症海马病理学改变(n=6)。另外相同分组(n=16),术后每天给予皮下注射生理盐水及头孢曲松,并允许正常进食及饮水,我们观察了实验大鼠7d生存率及大鼠体重的变化,行神经反射评分、新物体认知试验及Morris水迷宫试验行为学评估。
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；CLP组大鼠存活率明显下降,第3天为43.75%,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；海马H&E染色病理学检查提示CLP组大鼠可见明显神经元固缩、深染,细胞结构不清,细胞周围空隙明显增大,胶质细胞周围间隙扩大,空泡样改变,血管周围间隙扩大,以海马齿状回(dentate gyrus, DG)区最为显著,CA3区最轻。行为学试验(Morris水迷宫试验及新物体认知试验)均提示CLP大鼠学习记忆功能受损。
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,为后续SAE机制研究做好基础。海马组织病理改变提示海马损伤可能与SAE的发生有关。
第二部分Omi/HtrA2蛋白酶特异性抑制剂ucf-101对脓毒症脑病的保护作用
目的：大量研究提示SAE与海马神经细胞凋亡相关,而Omi/HtrA2是介导细胞凋亡的关键促凋亡蛋白,研究表明Omi/HtrA2蛋白酶特异性抑制剂ucf-101在多种病理情况下能抑制细胞凋亡,改善神经功能。本研究观察ucf-101对大鼠SAE的神经保护作用。
方法：实验动物随机分为四组,分别为假手术组(Sham)、假手术+Ucf-101组(Sham+Ucf-101)、CLP组(CLP)、CLP+Ucf-101组。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)。手术操作前30分钟、术后每日腹腔注射Ucf-101(10umol/kg)或生理盐水(10mL/kg)。实验24小时处死大鼠,取大鼠海马组织检测神经细胞的凋亡情况及病理学改变(n=6),检测海马组织氧化应激指标,包括谷胱甘肽(GSH),丙二醛(MDA),髓过氧化物酶(MPO)的含量及过氧化氢酶(CAT)的活性,检测海马组织炎症因子肿瘤坏死因子(TNF)-α及白介素(IL)-6水平(n=6)。另外相同分组(n=16),术后每天给予皮下注射生理盐水及头孢曲松,术后每日腹腔注射Ucf-101或生理盐水,并允许正常进食及饮水,我们观察了实验大鼠7d生存率及大鼠体重的变化,行神经反射评分、新物体认知试验及Morris水迷宫试验行为学评估。
结果：CLP组大鼠存活率明显下降,24h神经反射评分明显下降,学习记忆功能损害(行为学试验),Ucf-101台疗能明显提高大鼠生存率,明显提高大鼠术后24h神经反射评分,明显改善大鼠的学习记忆功能。CLP组大鼠海马区可见明显神经元固缩、深染,细胞结构不清及大量细胞凋亡,Ucf-101治疗能明显改善CLP导致的海马区病理损害及细胞凋亡。CLP组大鼠海马区TNF-α、IL-6水平明显升高,存在明显氧化应激损伤(MDA水平及MPO浓度明显增高,GSH水平和CAT活性明显下降),Ucf-101疗能明显改善海马区炎症反应,减轻氧化应激损伤。
结论：脓毒症可引起大鼠海马区炎症、氧化应激损害及细胞凋亡,导致大鼠认知记忆功能障碍,这提示脓毒症脑病发病与海马区神经细胞受损有关。Omi/HtrA2蛋白酶特异性抑制剂Ucf-101能明显减少海马区神经细胞凋亡,改善炎症和氧化应激损害,保护大鼠认知记忆功能。因此推测Ucf-101对SAE有神经保护作用,Omi/HtrA2可能是SAE治疗的新靶点。
第三部分Omi/HtrA2信号通路介导脓毒症脑病的机制研究
目的：Omi/HtrA2参与多种神经系统疾病的病理过程,本研究旨在观察SAE时Omi/HtrA2信号通路(Omi/HtrA2线粒体内外表达及XIAP、caspase-3、9裂解产物、蛋白酶PARP裂解产物胞浆内表达)的改变,并观察Ucf-101干预对Omi/HtrA2信号通路的影响,由此深入研究Omi/HtrA2信号通路介导SAE的作用及机制。
方法：实验动物随机分为四组(n=6),分别为假手术组(Sham)、假手术+Ucf-101组(Sham+Ucf-101)、CLP组(CLP)、CLP+Ucf-101组。手术操作前30分钟腹腔注射Ucf-101(10umol/kg)或生理盐水(10mL/kg)。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)。24小时处死大鼠,取大鼠海马组织,并分离线粒体和胞浆,用Western blot法分别检测Omi/HtrA2线粒体及胞浆中表达,并检测胞浆中XIAP、Caspase-3.9裂解产物、蛋白酶PARP裂解产物和cytochrome c表达。
结果：CLP组Omi/HtrA2从线粒体向胞浆内转移,胞浆内Omi/HtrA2蛋白表达明显升高,胞浆内XIAP表达明显下调,Caspase-3、9裂解产物及蛋白酶PARP裂解产物表达明显升高,Ucf-101处理可抑制Omi/HtrA2的转移,阻止XIAP的降解；明显减少胞浆内Caspase-3、9裂解产物及蛋白酶PARP裂解产物的表达；本研究也发现CLP组cytochrome c从线粒体释放至胞浆,而Ucf-101无法阻止cytochrome c的释放。
结论：本研究证实脓毒症可能导致Omi/HtrA2由海马神经细胞线粒体向胞浆内转移,通过与XIAP相结合,并降解XIAP,激活下游的细胞凋亡通路。可见Omi/HtrA2信号通路参与介导SAE的发病,Ucf-101通过干预此信号通路对SAE起神经保护作用。
The morbidity and mortality of sepsis is very high. Sepsis-associated encephalopathy(SAE) is diffuse brain dysfunction caused by sepsis. The nervous system is one of the earliest organs involved in sepsis. SAE incidence rate is8%-70%, and it is the most common encephalopathy in intensive care unit (ICU). The mortality of SAE is very high, but the pathophysiological mechanism is not clear, the current studies have found that brain microvascular endothelial cell dysfunction, blood-brain barrier (BBB) damage, inflammatory cell infiltration, inflammatory mediators, decreased cerebral perfusion, brain microvascular dysregulation, astrocytes and neurons dysfunction, neurotransmitters disorders, mitochondrial dysfunction, apoptosis, oxidative stress and calcium disorders are involved in SAE pathogenesis. And there are no effective therapies for SAE.
Studies have confirmed that mitochondrial dysfunction is involved in SAE. Mitochondria membrane potential is destructed, Mitochondrial respiratory chain is influenced, Mitochondrial disfunction can cause cell energy supply shortage and induce cell apoptosis. The studies have demonstrated that Omi/HtrA2is a kind of oligomeric serine protease. It has been confirmed that Omi/HtrA2can regulate apoptosis in a variety of pathological conditions (cardiac ischemia/reperfusion injury, the renal dysfunction caused by cisplatin, cerebral ischemia/reperfusion injury, etc.), and several studies have confirmed that the Omi/HtrA2is involved in a variety of nervous system diseases (such as huntington's disease, cerebral ischemia/reperfusion and status epilepticus, etc.). In this study we would establish SAE rat models. We would be trying to study hippocampus cell apoptosis during sepsis, and Omi/HtrA2signaling pathways (Omi/HtrA2concentration in the mitochondria and cytoplasm, and XIAP concentration, cleaved Caspase-3, cleaved Caspase-9and cleaved PARP concentrations in the cytoplasm), and Omi/HtrA2protease inhibitor(ucf-101) neuroprotective effect. We would be trying to demonstrated Omi/HtrA2signaling pathway is invovled in SAE, It would help to discover a novel therapeutic target and new drug for SAE.
Part One Establishment of a rat model of sepsis associated encephalopathy
Objective:Sepsis associated encephalopathy (SAE) is the most common encephalopathy in intensive care unit (ICU). The mortality of SAE is very high, but the pathophysiological mechanism is not clear and no effective treatment has been confirmed. Ideal animal models are needed in basic research urgently. In this part, we selected the standard septic model of cecum ligation puncture (CLP) and septic encephalopathy model was considered to be set up successfully by evaluating the behavior trials, neurologic reflex scores and hippocampal pathology which can be used in the next experiments.
Methods:Animals were anesthetized with4%Chloral hydrate (1ml/100g, intraperitoneally). Under sterile surgical conditions, a2-cm abdominal incision was made along the ventral surface of the abdomen to expose the cecum, which was then ligated below the ileocecal junction with no bowel obstruction. The cecum was punctured once with an18-gauge needle, and the fecal contents were allowed to leak into the peritoneum by gently squeezing the cecum. The bowel was then returned to the abdomen and the abdominal cavity was closed. The sham-operated rats were submitted to laparotomy, and the cecum was manipulated but neither ligated nor punctured. All rats underwent surgical manipulation, received a subcutaneous injection of saline solution (3mL/100g body weight) plus antibiotics (ceftriaxone at30mg/kg) every6h for resuscitation. In the first experiment, blood pressure, heart rate and anus temperature were measured at baseline,6h,12h and24h after operation. At the same time, we measured blood lactic acid level. We also observed hippocampal pathology24h post-operation (n=6). In the second experiment, rats (n=16) were given normal saline and ceftriaxone every day and allowed normal eating and drinking. We would evaluate neurologic reflex scores, survival rate, new object recognition test (7day after operation) and Morris water maze test (4,7days after operation).
Results:The septic rats appeared crouching, pilomotor, hemiplegia or seizures. Significant decreased mean arterial pressure, increased heart rate, deteriorated neurological reflexes together with increased blood lactate levels suggested the successful induction of sepsis in the present study. Survival rate of the CLP group decreased obviously (3days,43.75%;4-7days,37.5%). Hippocampal H&E staining showed most neurons were shrunken and stained dark in the CLP group, significantly in dentate gyrus (DG) area. Behavior trials (Morris water maze test and new object recognition test) suggests that the CLP impaired learning and memory function.
Conclusion:CLP models simulate the typical septic clinical manifestations, sepsis associated encephalopathy (SAE) model is established successfully by evaluating neurologic reflex scores, behavior trials and hippocampal pathology, which can be used in SAE mechanism study. Our study demonstrated hippocampus damage may be involved in SAE.
Part Two Neuro-protective effect of Omi/HtrA2protease inhibitor (Ucf-101) on sepsis associated encephalopathy
Objective:There are no effective treatments for SAE. It has been demonstrated that Omi/HtrA2protease inhibitors (Ucf-101) can improve a variety of pathological conditions (such as huntington's disease, cerebral ischemia/reperfusion and status epilepticus, etc.). We are trying to study Ucf-101neuro-protection of SAE.
Methods:The animals were categorized into four groups:(1) sham group plus normal saline (10mL/kg);(2) sham group plus Ucf-101(10umol/kg);(3) cecal ligation and puncture (CLP) plus normal saline (10mL/kg); and (4) CLP plus Ucf-101(10umol/kg). All rats underwent surgical manipulation, received a subcutaneous injection of saline solution (3mL/100g) plus antibiotics (ceftriaxone at30mg/kg) every6h for resuscitation. In the first experiment,24hours later, animals were sacrificed and their brains were harvested. Samples were used for analysis of inflammatory cytokine and oxidative stress (n=6), evaluation of apoptosis and pathologic changes (n=6). In the second experiment, All rats(n=16) received a intraperitoneal injection of saline solution (lOmL/kg) or Ucf-101(10umol/kg) every day, and was allowed eating and drinking normally, we would evaluate weight, neurologic reflex scores, survival rate, new object recognition test (7days after operation). Morris water maze test were also evaluated (4,7days after operation).
Results:CLP resulted in a poor survival rate, hippocampal oxidative injury, cell apoptosis and cognitive dysfunction as well as elevated TNF-a level and IL-6level, increased weight loss. Ucf-101could significantly inhibit cell apoptosis, reduce TNF-a and IL-6levels, MDA and MPO levels, elevated GSH level, slightly reverse CAT activities in the brain and attenuate this CLP effect on cognitive dysfunction. In addition, the survival rate and survival time was significantly improved by treatment with Ucf-101.
Conclusion:The present results demonstrated that sepsis can cause hippocampal inflammation, oxidative stress injury, apoptosis, and cognitive memory dysfunction. Ucf-101can significantly reduce the hippocampal cell apoptosis, inflammation and oxidative stress damage and improve cognitive function. So Ucf-101might have neuro-protective effect and Omi/HtrA2could be a new target for SAE.
Part Three Omi/HtrA2signaling pathways involved in sepsis associated encephalopathy
Objective:Omi/HtrA2is involved in a variety of nervous system disease.This study aims to study whether Omi/HtrA2signaling pathways were involved in SAE.
Methods:The animals were categorized into four groups (n=6):(1) sham group plus normal saline (10mL/kg);(2) sham group plus Ucf-101(10umol/kg);(3) cecal ligation and puncture (CLP) plus normal saline (10mL/kg); and (4) CLP plus Ucf-101(10umol/kg). All rats underwent surgical manipulation, received a subcutaneous injection of saline solution (3mL/100g) plus antibiotics (ceftriaxone at30mg/kg) every6h for resuscitation.24hours later, animals were sacrificed and their brains were harvested. Samples were used for analysis of Omi/HtrA2concentration in the mitochondria and cytoplasm, and XIAP concentration, cleaved Caspase-3, cleaved Caspase-9and cleaved PARP and cytochrome c concentrations in the cytoplasm.
Results:Omi/HtrA2is released into cytoplasm from mitochondria in the CLP group. XIAP expression decreased significantly, cleaved Caspase-3, cleaved Caspase-9and cleaved PARP concentration increased significantly. Ucf-101inhibited the translocation of Omi/HtrA2, reduced degradation of XIAP, and reduced cleaved Caspase-3, cleaved Caspase-9and cleaved PARP concentrations. This study also found that cytochrome c is released from mitochondria to the cytoplasm in CLP group and Ucf-101was unable to prevent the effect.
Conclusion:This study demonstrated that in sepsis Omi/HtrA2is released from mitochondria to cytoplasm, and it could degrade XIAP and activate the downstream of the apoptosis pathways. Omi/HtrA2mediated signal pathway is involved in sepsis associated encephalopathy, so it could be a new target for SAE.
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