本文選題：吡咯烷二硫代氨基甲酸鹽 + 脂多糖��； 參考：《廣州醫(yī)科大學》2017年碩士論文

【摘要】：研究背景膿毒癥是指由感染引起的全身炎癥反應(yīng)綜合征(Systemic inflammatory response syndrome,SIRS),是機體和病原體相互作用所引起的抗炎和促炎反應(yīng)失衡的結(jié)果,其病原體可為細菌、病毒和真菌。疾病進一步加重可出現(xiàn)嚴重的膿毒癥、膿毒癥休克和多器官功能障礙(Multiple organ dysfunction syndrome,MODS)。膿毒癥相關(guān)性腦病(Sepsis associated encephalopathy,SAE)是膿毒癥患者常見的并發(fā)癥,膿毒癥發(fā)生時,中樞神經(jīng)系統(tǒng)(Central nervous system,CNS)被認為是最早遭受損傷的器官,其損傷可出現(xiàn)在多臟器功能不全之前,SAE具體的定義為膿毒癥患者在排除中樞神經(jīng)系統(tǒng)直接感染、結(jié)構(gòu)異�；蚱渌愋偷哪X病(如肺性腦病、肝性腦病)的情況下,所出現(xiàn)的彌漫性腦功能障礙[1]。目前膿毒癥的患病率是千分之三,在這些膿毒癥患者中約70%會出現(xiàn)SAE,SAE的發(fā)展和轉(zhuǎn)歸與患者的死亡率息息相關(guān)[2]。但是對于SAE的確切發(fā)病機制仍未明確,潛在的機制有血腦屏障的破壞、炎癥因子損傷、氧化應(yīng)激、小膠質(zhì)細胞的激活等[3]。在膿毒癥的患者中,由于自身免疫功能和耐受能力的下降,老年人已經(jīng)成為膿毒癥主要的患病群體,約58-65%的患者為老年人[4],他們的發(fā)病率和死亡率也較其他年齡段的患者有明顯的增高。此外,在美國膿毒癥已經(jīng)成為65歲以上老年人死亡的第十大原因[5]。Angus的研究結(jié)果顯示,85歲或以上的老年人在膿毒癥的發(fā)病率是青少年的100倍,而死亡率是青少年的3.8倍[4]。最近的一項研究評估了老年人在嚴重膿毒癥的長期死亡率,發(fā)現(xiàn)總的死亡率為55%,其中一年死亡率為30.6%,兩年死亡率為43%,這就意味著接近一半的老年患者會在出院后三年內(nèi)死亡,此外作者也指出膿毒癥愈后出現(xiàn)的癡呆與其長期死亡率息息相關(guān)[6]。SAE會導致認知功能急性和慢性的改變,其中最容易檢測到的是急性改變,表現(xiàn)為腦病和譫妄。然而,越來越多的研究證據(jù)表明在動物和人類中,SAE會導致長期的認知功能障礙。動物模型顯示,在SAE后,他們長期的行為、學習和記憶力都有明顯的變化[7]。海馬神經(jīng)元的損失被認為是長期記憶障礙的基礎(chǔ)。在細胞實驗中,脂多糖誘導的慢性炎癥模型γ-氨基丁酸相關(guān)活動增加,海馬神經(jīng)細胞膜的興奮性改變,這就進一步證明了SAE時海馬突觸功能出現(xiàn)了障礙。核轉(zhuǎn)錄因子κB(Nuclear transcriptrion factorκB,NF-κB)是廣泛存在于真核細胞的轉(zhuǎn)錄因子,調(diào)節(jié)著多種細胞的功能,如細胞免疫、細胞凋亡、細胞分化和增殖[8-11]。NF-κB轉(zhuǎn)錄調(diào)節(jié)的位點是在許多炎癥因子和免疫因子的啟動區(qū),因此對炎癥反應(yīng)的發(fā)展起著重要的作用。炎癥因子在腦組織中過度表達使神經(jīng)元存在的環(huán)境發(fā)生改變,使神經(jīng)元變性[12,13]。目前NF-κB的活性抑制劑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、糖皮質(zhì)激素和非甾體類抗炎藥物(水楊酸和阿司匹林)。研究表明PDTC是專一的NF-κB特異性抑制劑,能有效阻斷NF-κB的信號通路[14]。PDTC通過降低DNA與NF-κB的結(jié)合能力、增加I-κB的合成、阻止NF-κB的兩個亞基p50和p65轉(zhuǎn)移到細胞核等方式來發(fā)揮抑制作用[15]。因為NF-κB控制著免疫和炎癥的相關(guān)基因表達,而這些基因涉及到膿毒癥、哮喘、中毒等多種疾病的進展,與多種疾病的轉(zhuǎn)歸密切相關(guān),所以阻斷NF-κB的激活是在一個炎癥反應(yīng)的上游來對總體的環(huán)節(jié)進行控制,是預(yù)防各個器官發(fā)生功能障礙的有效途徑。通過抑制NF-κB的活化,PDTC能降低粘附因子和中性粒細胞的表達,減少炎癥細胞的滲出與聚集,可有效減少炎癥部位所釋放的對組織有破壞作用的中性水解酶、過氧化物酶和酸性水解酶,從而減輕對人體的傷害[16]。因此,本研究旨在探討多次小劑量LPS對不同年齡小鼠認知功能的影響,以及PDTC的干預(yù)效應(yīng),為SAE的治療和預(yù)后提供依據(jù),同時也為PDTC在SAE中的保護作用提供新的研究方向。研究目的1、觀測脂多糖(Lipopolysaccharide,LPS)對不同年齡小鼠認知功能的影響2、探討吡咯烷二硫代氨基甲酸(Pyrrolidine dithiocarbamate,PDTC)對中樞炎癥老年小鼠的干預(yù)效應(yīng)研究方法1、青年雌性SPF級C57BL/6小鼠30只,6~8周齡,體重15-20g;老年雌性SPF級C57BL/小鼠60只,10~12月齡,體重20~30g。2、采用隨機數(shù)字表法,將其分為6組(n=15);青年對照組(Y組)、青年LPS組(YL組)、老年對照組(O組)、老年P(guān)DTC對照組(P組)、老年LPS組(OL組)、老年P(guān)DTC治療組(L+P組)。3、YL組和OL組腹腔注射LPS 250 ug/kg,1次/d,連續(xù)7d;L+P組每天于LPS注射前30min腹腔注射PDTC 50 mg/kg,1次/d,連續(xù)7d。4、末次給藥后2h取血漿及海馬組織,采用ELISA法測定血漿和海馬中TNF-α、IL-1β、IL-6含量,同時對O組和OL進行肺組織的取材,行HE染色觀察肺組織的病理改變。其余于末次給藥24h后進行行為學檢測,包括Morris水迷宮、曠場實驗和高架十字實驗,行為學結(jié)束后取腦組織行免疫組化和Western blot。結(jié)果1、肺組織HE染色結(jié)果顯示,O組肺泡結(jié)構(gòu)完整,肺泡腔無滲出、肺泡間隔無增寬、肺間質(zhì)無炎癥細胞浸潤。與O組比較,OL組肺組織未見明顯的病理學差異;2、與Y組比較,YL組在開臂時間百分比降低、進入開臂和閉臂次數(shù)減少,中央?yún)^(qū)活動時間降低(P0.05);3、與O組比較,OL組開臂時間百分比降低、進入開臂和閉臂次數(shù)減少,中央?yún)^(qū)活動時間降低,逃避潛伏期增加、原平臺所在象限停留時間減少、穿越平臺次數(shù)減少,血漿和海馬中TNF-α、IL-1β、IL-6含量增高(P0.05);4、與OL組比較,L+P組開臂時間百分比升高、進入開臂和閉臂次數(shù)增加,中央?yún)^(qū)活動時間增加,逃避潛伏期降低、原平臺所在象限停留時間增加,血漿和海馬中TNF-α、IL-1β、IL-6含量降低(P0.05);5、在免疫組化中,與O組比較,OL組在海馬的小膠質(zhì)細胞激活標記物IBA-1、Tau p T205和Aβ1 42的表達量明顯增加;與OL組比較,L+P組在海馬的小膠質(zhì)細胞激活標記物IBA-1、Tau p T205和Aβ1 42的表達量明顯降低;6、在Western blot中,與O組比較,OL組在海馬Tau p S396、Tau p T205、NF-κB P65和Aβ1 42的表達量明顯增加(P0.05);與OL組比較,L+P組在海馬Tau p S396、Tau p T205、NF-κB P65和Aβ1 42的表達量明顯降低(P0.05)。結(jié)論1、多次小劑量的LPS不能誘發(fā)青年小鼠認知功能障礙,卻能誘發(fā)老年小鼠出現(xiàn)認知功能障礙。2、應(yīng)用NF-κB特異性抑制劑PDTC干預(yù)LPS誘導的中樞炎癥老年小鼠,能明顯減少其外周和中樞炎癥因子TNF-α、IL-1β、IL-6的產(chǎn)生,減少小膠質(zhì)細胞的激活、Aβ1 42的沉積以及Tau過度的磷酸化,改善認知功能障礙。
[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 . 鑲烘場鑵旀棤娓楀嚭,鑲烘場闂撮殧鏃犲瀹，

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