【作者】 王楠；

【导师】 徐忠信；

【作者基本信息】 吉林大学 ， 神经病学， 2020， 博士

【摘要】 慢性脑缺血(chronic cerebral hypoperfusion,CCH)是一种临床常见的脑缺血损伤,是血管性痴呆、阿尔茨海默症和Binswanger病等多种神经系统疾病或精神疾病发生发展过程中的病理因素。研究表明,长期脑血流灌注不足导致能量代谢障碍、氧化应激产生及炎性因子释放,神经细胞凋亡,进而造成了认知功能受损。研究表明,慢性脑缺血后,抑制氧化应激,促进血管新生,建立有效的侧支循环是改善全脑及局部脑组织低灌注,减轻神经功能损伤的有效方法。白藜芦醇是一类植物抗毒素,属于天然的多酚类,具有抗炎、抗氧化、抗糖尿病和心肌保护等一系列的保护作用。研究表明,白藜芦醇通过减轻氧化应激来保护氧化损伤的大脑神经,并对阿尔茨海默症小鼠的认知功能有一定的改善作用,但其是否对慢性脑缺血性认知功能损伤有所改善仍不明确。本研究拟建立CCH大鼠模型,给予白藜芦醇治疗,探索白藜芦醇对CCH大鼠认知功能的改善作用,分别从自噬流和血管再生两个层面探讨其作用机制。1.白藜芦醇改善慢性脑缺血性认知功能障碍目的:探讨白藜芦醇对慢性脑缺血认知功能障碍的保护作用。方法:SD大鼠随机分为假手术组(Sham组)、慢性脑低灌注模型组(CCH组)和白藜芦醇+慢性脑低灌注组(Res组);CCH组和Res组采用2VO法建立慢性脑缺血认知功能障碍大鼠模型,Res组于建模后每天灌胃白藜芦醇50 mg/kg,分别于3、6、9周采用Bederson神经功能缺损评分及Morris水迷宫检测各组大鼠学习记忆能力,HE评估病理组织学损伤,ELISA检测脑损伤标记物和氧化应激因子变化,TUNEL检测神经细胞凋亡情况,免疫荧光检测MAP2表达,观察神经元存活。结果:缺血发生3、6、9周,CCH组大鼠Bederson评分明显升高,潜伏期延长和游泳距离增加,与Sham组相比差异显著(P<0.05),说明大鼠学习记忆能力下降,白藜芦醇干预后认知功能明显改善,大鼠额叶皮层及海马区细胞结构和神经元形态得以修复,神经元变性坏死现象得到改善,脑损伤标记物降低,氧化应激损伤受到抑制;此外,Res组大鼠MAP2表达明显升高,海马区神经元细胞凋亡降低。结论:白藜芦醇可以减轻CCH大鼠脑损伤,抑制氧化应激损伤,降低神经元细胞凋亡,改善学习记忆能力。2.白藜芦醇通过PI3K/AKT/mTOR通路抑制自噬改善慢性脑缺血认知功能障碍目的:探索白藜芦醇对CCH大鼠自噬的影响,探讨PI3K/AKT/mTOR的调控机制。方法:免疫荧光和Western blot分别检测凋亡相关蛋白Bcl-2、Bax和cleaved Caspase3;自噬相关蛋白Beclin1和LC3II/I表达以及PI3K/AKT/mTOR通路相关蛋白表达。在此基础上建立白藜芦醇+慢性缺血+PI3K抑制剂LY294002组(PI3K组),Morris水迷宫检测各组大鼠学习记忆能力,HE染色观察脑组织损伤,ELISA检测氧化应激损伤,Western blot检测凋亡和自噬相关蛋白。结果:Res组促凋亡因子Bax和cleaved Caspase3表达明显降低,抑凋亡因子Bcl-2表达升高(P<0.05),自噬相关蛋白检测结果显示CCH组大鼠脑组自噬因子表达显著升高,存在过度自噬现象,当给予白藜芦醇治疗后Beclin1和LC3II/I表达明显降低(Vs.CCH,P<0.05);与此同时,白藜芦醇激活PI3K/AKT/mTOR信号通路,PI3K表达明显升高,同时AKT和mTOR磷酸化水平明显增加(Vs.CCH,P<0.05);将PI3K抑制剂LY294002和白藜芦醇一起给予CCH大鼠,实验结果显示PI3K组大鼠认知和空间记忆能力较差;大鼠额叶皮层和海马区的脑组织松弛,神经元损伤最为明显;氧化应激损伤未得到缓解,细胞凋亡率升高,促凋亡因子表达升高,过度自噬现象未能受到抑制。结论:白藜芦醇可以通过激活PI3K/AKT/mTOR信号通路抑制CCH大鼠自噬水平,改善CCH大鼠学习记忆能力。3.白藜芦醇激活Slit2/Robo4通路改善血管再生功能缓解慢性脑缺血性认知功能障碍目的:探索白藜芦醇对CCH大鼠血管再生功能的改善作用,探讨Slit/Robo通路的调控机制。方法:Western blot和q RT-PCR检测Slit/Robo通路相关蛋白Slit2、Robo4以及血管内皮相关因子ET-1、VEGF、SDF-1α和e NOS表达;建立白藜芦醇+慢性脑缺血+Robo阻断剂(SR组),免疫荧光检测MAP2评价血管损伤及修复情况;评估神经功能评分,水迷宫实验检测其认知功能。结果:通过q RT-PCR和Western blot法检测Slit2/Robo4通路相关蛋白Slit2/Robo4表达情况,结果显示,与Sham组相比,CCH组Slit2和Robo4表达升高,与CCH组相比,Res组Slit2和Robo4表达进一步升高(P<0.05);给予Robo阻断剂后,SR组MAP2表达降低。与CCH组相比,Res组e NOS、VEGF和SDF-1α表达升高,ET-1表达降低,组间比较具有统计学差异(P<0.05);加入Robo抑制剂后,Res对其相关因子的改变受到抑制,同时Robo抑制剂逆转白藜芦醇对神经功能评分的提升作用以及认知水平。结论:白藜芦醇可以激活Slit2/Robo4通路,改善血管内皮功能,改善CCH大鼠认知功能障碍。更多还原

【Abstract】 Chronic cerebral ischemia(CCH)is a common clinical cerebral ischemia injury,which is a pathological factor in the occurrence and development of vascular dementia,Alzheimer’s disease,Binswanger’s disease and other neurological diseases or mental diseases.According to reports,long-term cerebral blood perfusion deficiency leads to energy metabolism disorder,oxidative stress production,inflammatory factor release,neuronal apoptosis,and then causes cognitive impairment.The results show that,after chronic cerebral ischemia,inhibition of oxidative stress,promotion of angiogenesis and establishment of effective collateral circulation are effective methods to improve the global and local cerebral hypoperfusion and reduce the damage of nerve function.Resveratrol is a kind of plant antitoxin,which belongs to natural polyphenols.It has a series of protective effects,such as anti-inflammatory,anti-oxidation,anti-diabetes,myocardial protection and so on.It has been proved that resveratrol can protect the brain nerves from oxidative damage by reducing oxidative stress and improve the cognitive function of mice with Alzheimer’s disease,but whether it can improve the cognitive function of chronic cerebral ischemia is still unclear.The aim of this study is to establish a CCH rat model and to explore the effect of resveratrol on the cognitive function of CCH rats.1.Resveratrol improves chronic cerebral ischemic cognitive dysfunctionObjective: To explore the protective effects of resveratrol on chronic cerebral ischemic cognitive dysfunction.Methods: SD rats were randomly divided into sham operation group(Sham group),chronic cerebral hypoperfusion model group(CCH group),resveratrol + chronic cerebral hypoperfusion group(Res group);2CH method was used in CCH group and Res group.A rat model of chronic cerebral ischemic cognitive dysfunction was established.The Res group was orally administered with resveratrol 50 mg/kg daily after modeling.Bederson neurological deficit score and Morris water maze were used at 3,6,and 9 weeks,respectively.The learning and memory abilities of rats in each group,HE evaluation of histopathological damage,ELISA detection of brain injury markers and changes of oxidative stress factors,TUNEL detection of neuronal apoptosis,immunofluorescence detection of MAP2 expression,observation of neuron survival,microvascular Density and angiogenesis.Results: At 3,6,and 9 weeks after the onset of ischemia,the Bederson score of the rats in the CCH group was significantly increased,the incubation period was prolonged,and the swimming distance was increased.Compared with the Sham group,the differences were significant(P <0.05),indicating that the learning and memory abilities of the rats were decreased.After resveratrol intervention,the cognitive function was significantly improved,the cell structure and neuron morphology of the frontal cortex and hippocampus of rats were repaired,the neuronal degeneration and necrosis phenomenon was improved,the brain injury markers were reduced,and oxidative stress injury was suppressed;In addition,the expression of MAP2 in the Res group was significantly increased,and the apoptosis of neurons in the hippocampus was reduced.Conclusion: Resveratrol can reduce brain damage,inhibit oxidative stress injury,reduce neuronal cell apoptosis,and improve learning and memory ability in CCH rats.2.Resveratrol inhibits autophagy through PI3K/AKT/mTOR pathway to improve chronic cerebral ischemic cognitive dysfunctionObjective: To explore the effect of resveratrol on autophagy in CCH rats,and to explore the regulatory mechanism of PI3K/AKT/mTOR.Methods: Immunofluorescence and Western blot were used to detect apoptosis-related proteins Bcl-2,Bax and cleaved Caspase3,expressions of autophagy-related proteins Beclin1,LC3II/I,and PI3K/AKT/mTOR pathway-related proteins.On this basis,a resveratrol + chronic ischemia + PI3 K inhibitor LY294002 group(PI3K group)was established.Morris water maze was used to detect the learning and memory abilities of rats in each group.HE staining was used to observe brain tissue damage and ELISA was used to detect oxidative stress injury.Western blot was used to detect apoptosis and autophagy related proteins.Results: The expressions of pro-apoptotic factors Bax and cleaved Caspase3 in the Res group were significantly reduced,and the expression of anti-apoptotic factors Bcl-2 was increased(P <0.05).The results of autophagy-related protein detection showed that the autophagy factor expression in the brain group of the CCH group was significant.The expression of Beclin1 and LC3II/I was significantly reduced after resveratrol treatment(Vs.CCH,P <0.05);meanwhile,resveratrol activated PI3K/AKT/mTOR in the signal pathway,PI3 K expression was significantly increased,and AKT and mTOR phosphorylation levels were significantly increased(Vs.CCH,P <0.05).PI3 K inhibitor LY294002 was given to CCH rats together with resveratrol.As a result,rats in the PI3 K group recognized The cognitive and spatial memory ability is poor;the brain tissue of the rat’s frontal cortex and hippocampus relaxes,and neuronal damage is most obvious;oxidative stress injury is not alleviated,the apoptosis rate increases,and the expression of pro-apoptotic factors increases Excessive autophagy has not been suppressed.Conclusion: Resveratrol can inhibit the autophagy level of CCH rats by improving the PI3K/AKT/mTOR signaling pathway and improve the learning and memory ability of CCH rats.3.Resveratrol activates Slit2/Robo4 pathway to improve angiogenesis and relieve chronic cerebral ischemic cognitive dysfunctionObjective: To explore the effect of resveratrol on angiogenesis in CCH rats,and to explore the mechanism of Slit / Robo pathway regulation.Methods: Western blot and q RT-PCR were used to detect the expressions of Slit2,Robo4 related proteins Slit2,Robo4 and vascular endothelial related factors ET-1,VEGF,SDF-1α and e NOS;establish resveratrol + chronic cerebral ischemia + Robo resistance Decoction(SR group),immunofluorescence detection of MAP2 expression to evaluate vascular injury and repair;assessment of neural function score,water maze test to detect cognitive function.Results: The expression of Slit2 / Robo4 related proteins of Slit2 / Robo4 pathway was detected by PCR and Western blot.The results showed that compared with the Sham group,the expression of Slit2 and Robo4 in the CCH group was increased.Compared with the CCH group,the expression of Slit2 and Robo4 in the Res group was further increased.Increased(P <0.05);After the Robo blocker was administered,MAP2 expression decreased in the SR group.Compared with the CCH group,the expression of e NOS,VEGF,and SDF-1α in the Res group was increased,and the expression of ET-1 was decreased.There was a statistical difference between the groups(P <0.05).After adding the Robo inhibitor,Res related factors at the same time,Robo inhibitors reversed the effect of resveratrol on neurological score and cognitive level.Conclusion: Resveratrol can activate the Slit2 / Robo4 pathway,improve vascular endothelial function,and improve cognitive dysfunction in CCH rats.更多还原