DFO调节海马增龄性铁沉积对老年大鼠术后认知功能的影响及机制研究

发布时间：2018-05-04 13:58

本文选题：术后认知功能障碍 + 海马　；参考：《第三军医大学》2015年硕士论文

【摘要】：背景与目的：术后认知功能障碍(Postoperative cognitive dysfunction, POCD)是一种常见的术后并发症。据流行病学资料,其发病率在接受心脏手术的病人中高达20-79%,在接受非心脏手术的病人中也达到了4.1-22.3%。POCD与高死亡率、住院时间延长、对医疗资源的大量消耗以及生活质量下降等不良事件密切相关。POCD主要在老年病人中发病较多,一般认为老龄是导致POCD发病最为重要的危险因素。尽管POCD的发病机制和病理生理变化尚未完全明确,但是有证据表明了该疾病与其他神经退行性疾病的密切关系。铁在脑内的增龄性沉积是神经退行性疾病的共同病理特征之一。过量的铁可导致氧化应激损伤和小胶质细胞过度激活,从而对神经细胞造成损伤。铁螫合剂去铁胺(Deferoxamine,DFO)已被用于多种脑内铁过载疾病和神经退行性疾病的动物模型研究,并发现了其良好的应用前景。尽管铁沉积在神经退行性疾病中发挥了重要作用,并且铁螯合剂的应用为预防和治疗带来了曙光,但是铁沉积在POCD发生发展中的潜在机制还缺乏深入研究,DFO对POCD的作用效应和作用机制也尚未阐明。因此,本研究拟通过动物实验揭示海马内铁沉积影响术后认知功能的原因,探讨DFO是否能改善术后认知功能及其潜在的作用机制。方法：1.通过电感耦合等离子体质谱(Inductively coupled plasma-mass spectrometry, ICP-MS)检测组织内总铁含量。Western blot检测ferritin表达量。以此评估成年和老年大鼠海马铁沉积的情况,并观察剖腹探查术对海马内铁沉积的影响。2.用Morris水迷宫(Morris water maze,MWM)评价海马内铁沉积对大鼠术后认知功能的影响。3.采用术前腹腔注射DFO的方法,观察该药物能否改善术后海马铁沉积以及对术后认知功能是否具有保护作用。4.通过免疫组化染色(Immunohistochemical staining, IHC)和real-time PCR检测海马内铁转运相关蛋白的表达、用试剂盒检测氧化应激和抗氧化应激指标、IHC检测小胶质细胞活化标记物OX-42表达、Western blot检测凋亡相关蛋白的表达和采用原位末端标记法(Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling, TUNEL)检测凋亡细胞数量,以阐明术后海马铁沉积加重的潜在原因及DFO改善术后认知功能的作用机制。结果：1.与成年大鼠相比,老年大鼠海马组织总铁含量和ferritin表达量显著升高并在术后进一步加重。MWM测试发现,老年大鼠术后潜伏期、游泳距离较成年大鼠明显增加,而目标象限停留时间百分比、中心区域停留时间百分比、平台穿越次数较成年大鼠明显减少。2.术前连续6天给予DFO可显著减少成年及老年大鼠术后海马组织总铁含量及ferritin表达量,并且在老年大鼠中减少的程度明显大于成年大鼠。MWM测试发现,DFO可减少老年大鼠术后潜伏期和游泳距离,增加目标象限停留时间百分比、中心区域停留时间百分比和平台区域穿越次数；但是在成年大鼠中上述指标无明显改善。3.老年大鼠术后TfR mRNA及蛋白表达明显减少,DMT1、hepcidin的mRNA及蛋白表达均显著增加,Fpnl虽然mRNA表达增加但蛋白表达急剧下降。术前给予DFO后虽使TfR表达增加,但可明显抑制DMT1和hepcidin的增加,防止Fpnl减少。4.DFO可抑制术后海马ROS及MDA的产生,保护总SOD活性；显著减少海马OX-42的表达；抑制Caspase-3的表达及活化,抑制促凋亡蛋白Bax的表达,增加抗凋亡蛋白Bcl-2的表达并提高Bcl-2/Bax比值,还可显著减少术后海马TUNEL阳性细胞的数量。结论：1.大鼠海马组织中存在增龄性铁沉积,手术刺激可使铁沉积加重并损害术后认知功能。提示海马内铁沉积可能与术后认知功能下降相关。2.铁螯合剂DFO可能有助于降低海马内铁沉积,并改善老年大鼠术后认知功能,但对成年大鼠作用不明显。3.老年大鼠术后海马铁转运蛋白表达改变使得细胞内铁外流减少,同时伴有细胞外的铁内流增加,可能是术后海马铁沉积增加的原因之一。4.DFO通过螫合作用和对铁转运相关蛋白的调控缓解了术后海马内铁沉积；同时对海马内氧化应激和小胶质细胞激活的抑制,减少了细胞凋亡的发生,从而改善了术后认知功能。5.DFO对海马内铁沉积的缓解和对术后认知功能的改善为预防和治疗POCD提供了新的策略。
[Abstract]:Background and purpose: postoperative cognitive dysfunction (Postoperative cognitive dysfunction, POCD) is a common postoperative complication. According to epidemiological data, the incidence is as high as 20-79% in patients undergoing cardiac surgery. In patients receiving non cardiac surgery, the incidence of 4.1-22.3%.POCD and high mortality is also achieved, and the time of hospitalization is prolonged. The large consumption of medical resources and the decline of adverse events, such as the decline of the quality of life, are closely related to.POCD mainly in the elderly patients. It is generally believed that aging is the most important risk factor for the pathogenesis of POCD. Although the pathogenesis and pathophysiological changes of POCD are not completely clear, there is evidence that the disease and other nerves have been shown. The close relationship of degenerative disease. AGE deposition in the brain is one of the common pathological features of neurodegenerative diseases. Excessive iron can cause oxidative stress and microglia to excessively activate and cause damage to nerve cells. Iron sting mixture (Deferoxamine, DFO) has been used in a variety of brain iron overload diseases and The animal model of neurodegenerative disease has been studied and its good prospects are found. Although iron deposition plays an important role in neurodegenerative diseases, and the application of iron chelating mixture has brought dawn to prevention and treatment, the potential mechanism of iron deposition in the development of POCD is still lack of in-depth study and the effect of DFO on POCD The effects and mechanisms of action have not yet been elucidated. Therefore, this study is intended to reveal the effects of iron deposition in the hippocampus on cognitive function in the hippocampus and to explore whether DFO can improve postoperative cognitive function and its potential mechanisms. Methods: 1. through inductively coupled plasma mass spectrometry (Inductively coupled plasma-mass spectrometry, ICP) -MS) detection of total iron content in the tissue.Western blot detection of ferritin expression. In order to evaluate the iron deposition in hippocampus of adult and old rats, and the effect of exploratory laparotomy on the iron deposition in the hippocampus,.2. using Morris water maze (Morris water maze, MWM) to evaluate the effect of iron deposition on the cognitive function of rats in the hippocampus.3. before the operation. The method of intraperitoneal injection of DFO to observe whether the drug can improve the post operation hippocampal iron deposition and the protective effect of postoperative cognitive function on.4. through immunohistochemical staining (Immunohistochemical staining, IHC) and real-time PCR to detect the expression of iron transport related proteins in the hippocampus, and test the oxidative stress and antioxidant stress finger by the kit. IHC was used to detect the expression of OX-42 in microglia activation markers, Western blot was used to detect the expression of apoptosis related proteins and the number of apoptotic cells were detected by in situ terminal labeling (Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) in order to elucidate the potential cause of the aggravation of hippocampal iron deposition after operation and the improvement of the operation. Results: 1. compared with adult rats, the total iron content and ferritin expression in the hippocampus of the aged rats were significantly increased and the.MWM test was further aggravated after the operation. The incubation period of the aged rats was significantly increased in the incubation period than in the adult rats, while the percentage of the target quadrant residence time and the center area stayed in the aged rats. The total iron content and ferritin expression in hippocampus of adult and aged rats were significantly reduced by the time percentage of time compared with adult rats after.2. operation 6 days before operation, and the degree of decrease in old rats was significantly greater than that in adult rats. DFO could reduce the latency and the latency of postoperatively in old rats. The percentage of swimming distance, the percentage of stay in the target quadrant, the percentage of residence time in the central region and the number of regional crossing of the platform, but in the adult rats, the expression of TfR mRNA and protein in the.3. old rats decreased obviously, the mRNA and protein expression of DMT1, hepcidin increased significantly, and the expression of mRNA was increased in Fpnl. The expression of additive protein was sharply decreased. Although the expression of TfR increased before DFO, the increase of DMT1 and hepcidin was obviously inhibited, and Fpnl reduced.4.DFO could inhibit the production of ROS and MDA in the hippocampus, protect the total SOD activity, reduce the expression of OX-42 in hippocampus, inhibit the expression and activation of Caspase-3, and inhibit the expression of apoptotic protein Bax. Increase the expression of anti apoptotic protein Bcl-2 and increase the Bcl-2/Bax ratio, and significantly reduce the number of hippocampal TUNEL positive cells after operation. Conclusion: there is aging iron deposition in the hippocampus of 1. rats. The operation stimulation can aggravate the iron deposition and damage the postoperative cognitive function. It is suggested that the iron deposition in the hippocampus may be related to the decline of postoperative cognitive function. .2. iron chelating agent DFO may help to reduce the iron deposition in the hippocampus and improve the cognitive function of the aged rats, but the changes in the expression of iron transporter in the hippocampus of the adult rats are not obvious in the adult rats after operation, and the iron Exodus in the cells is reduced, and the increase of the iron internal flow outside the cells is also associated with the increase of the iron deposition in the hippocampus after the operation. One of the causes of.4.DFO can alleviate the iron deposition in the hippocampus after the chelation and the regulation of iron transport related proteins. Meanwhile, the inhibition of oxidative stress in the hippocampus and the inhibition of the activation of microglia reduces the occurrence of apoptosis, thus improving the remission of the postoperative cognitive function.5.DFO to the iron deposition in the hippocampus and the modification of postoperative cognitive function. It provides a new strategy for the prevention and treatment of POCD.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614

【参考文献】