脂多糖对不同年龄小鼠认知功能的影响及吡咯烷二硫代氨基甲酸的干预效应
发布时间：2018-04-20 14:46
本文选题：吡咯烷二硫代氨基甲酸盐 + 脂多糖 ； 参考：《广州医科大学》2017年硕士论文
【摘要】：研究背景脓毒症是指由感染引起的全身炎症反应综合征(Systemic inflammatory response syndrome,SIRS),是机体和病原体相互作用所引起的抗炎和促炎反应失衡的结果,其病原体可为细菌、病毒和真菌。疾病进一步加重可出现严重的脓毒症、脓毒症休克和多器官功能障碍(Multiple organ dysfunction syndrome,MODS)。脓毒症相关性脑病(Sepsis associated encephalopathy,SAE)是脓毒症患者常见的并发症,脓毒症发生时,中枢神经系统(Central nervous system,CNS)被认为是最早遭受损伤的器官,其损伤可出现在多脏器功能不全之前,SAE具体的定义为脓毒症患者在排除中枢神经系统直接感染、结构异常或其他类型的脑病(如肺性脑病、肝性脑病)的情况下,所出现的弥漫性脑功能障碍[1]。目前脓毒症的患病率是千分之三,在这些脓毒症患者中约70%会出现SAE,SAE的发展和转归与患者的死亡率息息相关[2]。但是对于SAE的确切发病机制仍未明确,潜在的机制有血脑屏障的破坏、炎症因子损伤、氧化应激、小胶质细胞的激活等[3]。在脓毒症的患者中,由于自身免疫功能和耐受能力的下降,老年人已经成为脓毒症主要的患病群体,约58-65%的患者为老年人[4],他们的发病率和死亡率也较其他年龄段的患者有明显的增高。此外,在美国脓毒症已经成为65岁以上老年人死亡的第十大原因[5]。Angus的研究结果显示,85岁或以上的老年人在脓毒症的发病率是青少年的100倍,而死亡率是青少年的3.8倍[4]。最近的一项研究评估了老年人在严重脓毒症的长期死亡率,发现总的死亡率为55%,其中一年死亡率为30.6%,两年死亡率为43%,这就意味着接近一半的老年患者会在出院后三年内死亡,此外作者也指出脓毒症愈后出现的痴呆与其长期死亡率息息相关[6]。SAE会导致认知功能急性和慢性的改变,其中最容易检测到的是急性改变,表现为脑病和谵妄。然而,越来越多的研究证据表明在动物和人类中,SAE会导致长期的认知功能障碍。动物模型显示,在SAE后,他们长期的行为、学习和记忆力都有明显的变化[7]。海马神经元的损失被认为是长期记忆障碍的基础。在细胞实验中,脂多糖诱导的慢性炎症模型γ-氨基丁酸相关活动增加,海马神经细胞膜的兴奋性改变,这就进一步证明了SAE时海马突触功能出现了障碍。核转录因子κB(Nuclear transcriptrion factorκB,NF-κB)是广泛存在于真核细胞的转录因子,调节着多种细胞的功能,如细胞免疫、细胞凋亡、细胞分化和增殖[8-11]。NF-κB转录调节的位点是在许多炎症因子和免疫因子的启动区,因此对炎症反应的发展起着重要的作用。炎症因子在脑组织中过度表达使神经元存在的环境发生改变,使神经元变性[12,13]。目前NF-κB的活性抑制剂主要有吡咯烷二硫代氨基甲酸(Pyrrolidine dithiocarbamate,PDTC)、抗炎因子IL-10、一氧化氮、糖皮质激素和非甾体类抗炎药物(水杨酸和阿司匹林)。研究表明PDTC是专一的NF-κB特异性抑制剂,能有效阻断NF-κB的信号通路[14]。PDTC通过降低DNA与NF-κB的结合能力、增加I-κB的合成、阻止NF-κB的两个亚基p50和p65转移到细胞核等方式来发挥抑制作用[15]。因为NF-κB控制着免疫和炎症的相关基因表达,而这些基因涉及到脓毒症、哮喘、中毒等多种疾病的进展,与多种疾病的转归密切相关,所以阻断NF-κB的激活是在一个炎症反应的上游来对总体的环节进行控制,是预防各个器官发生功能障碍的有效途径。通过抑制NF-κB的活化,PDTC能降低粘附因子和中性粒细胞的表达,减少炎症细胞的渗出与聚集,可有效减少炎症部位所释放的对组织有破坏作用的中性水解酶、过氧化物酶和酸性水解酶,从而减轻对人体的伤害[16]。因此,本研究旨在探讨多次小剂量LPS对不同年龄小鼠认知功能的影响,以及PDTC的干预效应,为SAE的治疗和预后提供依据,同时也为PDTC在SAE中的保护作用提供新的研究方向。研究目的1、观测脂多糖(Lipopolysaccharide,LPS)对不同年龄小鼠认知功能的影响2、探讨吡咯烷二硫代氨基甲酸(Pyrrolidine dithiocarbamate,PDTC)对中枢炎症老年小鼠的干预效应研究方法1、青年雌性SPF级C57BL/6小鼠30只,6~8周龄,体重15-20g;老年雌性SPF级C57BL/小鼠60只,10~12月龄,体重20~30g。2、采用随机数字表法,将其分为6组(n=15);青年对照组(Y组)、青年LPS组(YL组)、老年对照组(O组)、老年PDTC对照组(P组)、老年LPS组(OL组)、老年PDTC治疗组(L+P组)。3、YL组和OL组腹腔注射LPS 250 ug/kg,1次/d,连续7d;L+P组每天于LPS注射前30min腹腔注射PDTC 50 mg/kg,1次/d,连续7d。4、末次给药后2h取血浆及海马组织,采用ELISA法测定血浆和海马中TNF-α、IL-1β、IL-6含量,同时对O组和OL进行肺组织的取材,行HE染色观察肺组织的病理改变。其余于末次给药24h后进行行为学检测,包括Morris水迷宫、旷场实验和高架十字实验,行为学结束后取脑组织行免疫组化和Western blot。结果1、肺组织HE染色结果显示,O组肺泡结构完整,肺泡腔无渗出、肺泡间隔无增宽、肺间质无炎症细胞浸润。与O组比较,OL组肺组织未见明显的病理学差异;2、与Y组比较,YL组在开臂时间百分比降低、进入开臂和闭臂次数减少,中央区活动时间降低(P0.05);3、与O组比较,OL组开臂时间百分比降低、进入开臂和闭臂次数减少,中央区活动时间降低,逃避潜伏期增加、原平台所在象限停留时间减少、穿越平台次数减少,血浆和海马中TNF-α、IL-1β、IL-6含量增高(P0.05);4、与OL组比较,L+P组开臂时间百分比升高、进入开臂和闭臂次数增加,中央区活动时间增加,逃避潜伏期降低、原平台所在象限停留时间增加,血浆和海马中TNF-α、IL-1β、IL-6含量降低(P0.05);5、在免疫组化中,与O组比较,OL组在海马的小胶质细胞激活标记物IBA-1、Tau p T205和Aβ1 42的表达量明显增加;与OL组比较,L+P组在海马的小胶质细胞激活标记物IBA-1、Tau p T205和Aβ1 42的表达量明显降低;6、在Western blot中,与O组比较,OL组在海马Tau p S396、Tau p T205、NF-κB P65和Aβ1 42的表达量明显增加(P0.05);与OL组比较,L+P组在海马Tau p S396、Tau p T205、NF-κB P65和Aβ1 42的表达量明显降低(P0.05)。结论1、多次小剂量的LPS不能诱发青年小鼠认知功能障碍,却能诱发老年小鼠出现认知功能障碍。2、应用NF-κB特异性抑制剂PDTC干预LPS诱导的中枢炎症老年小鼠,能明显减少其外周和中枢炎症因子TNF-α、IL-1β、IL-6的产生,减少小胶质细胞的激活、Aβ1 42的沉积以及Tau过度的磷酸化,改善认知功能障碍。
[Abstract]:background sepsis refers to systemic inflammatory response syndrome ( s ) caused by infection , which is the result of the anti - inflammatory and pro - inflammatory response imbalance caused by the interaction of organism and pathogen , which can be bacteria , viruses and fungi . Severe sepsis , septic shock , and multiple organ dysfunction syndrome ( MODS ) can be further aggravated by disease . Septic associated encephalopathy ( SAE ) is a common complication in sepsis . In the event of sepsis , the central nervous system ( CNS ) is considered to be the first organ to be injured , and its injury may occur before multiple organ dysfunction , which is specifically defined as sepsis patients with diffuse brain dysfunction in the case of direct infection , structural abnormalities , or other types of encephalopathy ( e.g . , pulmonary encephalopathy , hepatic encephalopathy ) in the central nervous system . At present , the prevalence of sepsis is three , and about 70 % of these sepsis patients will have SAEs , and the development and outcome of SAEs is closely related to the mortality of the patient . However , the exact pathogenesis of SAE is still not clear . Potential mechanisms include destruction of blood brain barrier , inflammatory factor injury , oxidative stress , activation of microglial cells , and so on . In patients with sepsis , the elderly have become a major group of sepsis due to the decline in their own immune function and tolerance , and about 58 - 65 % of the patients are elderly patients , and their morbidity and mortality are also significantly higher than those in other age groups . In addition , the number of deaths in the United States has become the tenth largest cause of death in older persons over 65 years of age . The results of the study showed that the incidence of sepsis in the aged 85 or older was 100 times that of adolescents , while the mortality rate was 3.8 times that of adolescents . A recent study assessed the long - term mortality of older persons in severe sepsis , with a total mortality rate of 55 per cent , with a one - year mortality rate of 36.6 per cent and a two - year mortality rate of 43 per cent , which means that nearly half of older patients died within three years after discharge , and the authors also noted that the more developed dementia was associated with its long - term mortality . SAEs can lead to acute and chronic changes in cognitive function , most easily detected are acute changes , manifested as encephalopathy and Delirium . However , an increasing number of studies have shown that SAEs can lead to long - term cognitive impairment in animals and humans . Animal models show that , after SAE , they have a significant change in their long - term behavior , learning and memory . NF - 魏B is a specific inhibitor of NF - 魏B , which can effectively block the signal pathway of NF - 魏B . NF - 魏B is a specific inhibitor of NF - 魏B , which can effectively block the signaling pathway of NF - 魏B . PDTC can inhibit the expression of NF - 魏B by decreasing the binding ability of DNA and NF - 魏B , and inhibiting the transfer of two subunits of NF - 魏B to the nucleus and so on . NF - 魏B controls the expression of related genes of immune and inflammation , and these genes relate to the progression of various diseases such as sepsis , asthma and poisoning , and are closely related to the prognosis of various diseases . Therefore , blocking NF - 魏B activation is an effective way to prevent the dysfunction of various organs . By inhibiting NF - 魏B activation , PDTC can reduce the expression of adhesion factor and neutrophils , reduce exudation and aggregation of inflammatory cells , and reduce the damage to human body . The effects of multiple doses of LPS on cognitive function in aged mice were investigated . The effects of PDTC on cognitive function in aged mice were studied . 鑲烘场鑵旀棤娓楀嚭,鑲烘场闂撮殧鏃犲瀹，
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