青藤碱在小鼠创伤性脑损伤中神经保护作用及其机制的研究
发布时间：2018-09-01 18:59
【摘要】：研究背景:创伤性脑损伤(traumatic brain injury,TBI)仍然是现代社会的主要公共健康问题,致残率和死亡率都很高。TBI分为原发性脑损伤和继发性脑损伤。原发性脑损伤是受伤当时立即发生,由外力直接造成的脑组织损伤。继发性脑损伤是指原发性脑损伤后一系列复杂的内源性病理生理过程被触发而造成神经细胞的损伤,又称为二次脑损伤。引起继发性脑损伤的主要病理生理过程主要包括氧化应激、谷氨酸兴奋毒性、炎性反应、离子平衡紊乱、血管渗透性增加、血脑屏障破坏等。其中氧化应激在继发性脑损伤中扮演重要角色。TBI后生成大量活性氧自由基和氮自由基,这些过量的自由基超过了体内抗氧化防御系统的降解能力,活性氧簇等有害毒物在体内大量堆积,导致脂质过氧化、蛋白质和DNA结构破坏,导致神经细胞损伤和死亡,因而造成神经功能障碍。核因子 E2 相关因子 2(nuclear factor erythroid 2-related factor 2,Nrf2)是机体调节氧化应激的关键因子。在生理情况下,Nrf2与Keap1和Cul3结合形成复合体,防止Nef2从胞浆进入胞核。Cul3是泛素连接酶,可以使Nrf2泛素化,从而被蛋白酶降解,使细胞内Nrf2处于一个平衡状态。在应激条件下,Keap1的空间构象发生改变,Nrf2从复合体中解离出来,未被降解的Nrf2进入细胞核,与Maf结合形成异二聚体激活ARE序列,从而启动Ⅱ相解毒和抗氧化基因等下游保护基因的转录。其中NQO-1和HO-1在对抗氧化应激和外来有害物质方面起重要作用。青藤碱是从中药青风藤中分离提纯获得的活性碱,临床上主要用于治疗类风湿性关节炎及系膜增生性肾小球肾炎。大量实验证明青藤碱具有抑制炎症反应、抑制免疫和细胞保护等多种药理作用。青藤碱在脑缺血、脑出血和神经退行性疾病等多种中枢神经系统疾病模型中表现出神经保护作用。然而青藤碱在TBI中的作用研究较少,相关机制也尚不清楚。因此,本实验通过建立小鼠TBI模型,探讨TBI后使用青藤碱是否起到神经保护作用及其分子机制。目的:本实验在雄性ICR小鼠TBI模型中,通过腹腔注射青藤碱后,探讨其神经保护作用及可能的分子机制。方法:1、实验模型建立及分组,选取健康成年雄性ICR小鼠,采用改良的Feeney法制作TBI模型。小鼠被随机分成4组:sham组、TBI组、TBI+溶剂组和TBI+SIN(30mg/kg)组;2、观察SIN对小鼠TBI后神经功能及脑水肿的影响;3、Western blot检测脑组织中凋亡相关蛋白Bcl-2和cleaved caspase-3的表达水平,用TUNEL检测脑组织切片中神经元凋亡;4、检测脑组织中丙二醛(malonaldehyde,MDA)含量和谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)、超氧化物歧化酶(superoxide dismutase,SOD)的活性;5、Western blot检测脑组织中NQO-1、HO-1、胞浆胞核Nrf2的表达,免疫荧光双染检测Nrf2的定位表达;6、实时定量PCR检测脑组织中NQO-1和HO-1的mRNA表达水平。结果:(1)与TBI组相比,青藤碱处理后明显降低了小鼠脑水含量(P0.001)和神经元凋亡指数(P0.001),明显提高了神经功能评分(1P0.01)。(2)青藤碱减轻了小鼠创伤性脑损伤后脑组织氧化应激,具体表现为青藤碱处理后明显降低了小鼠脑组织MDA水平(P0.001),SOD和GPx的活性明显提高(P0.001,P0.001)。(3)创伤性脑损伤后,Nrf2从细胞质转移至细胞核,而使用青藤碱后促进了这样转移,增加了 NQO-1和HO-1在蛋白和mRNA水平的表达。结论:我们的实验数据表明,在小鼠创伤性脑损伤模型中使用青藤碱可以降低氧化应激,减轻脑水肿,抑制神经元凋亡,改善小鼠神经功能评分,起到神经保护作用。青藤碱的神经保护作用可能是通过激活Nrf2-ARE通路获得的。
[Abstract]:Background: Traumatic brain injury (TBI) is still a major public health problem in modern society with high disability and mortality. TBI is divided into primary brain injury and secondary brain injury. After primary brain injury, a series of complex endogenous pathophysiological processes are triggered to cause neuronal damage, also known as secondary brain injury. Oxidative stress plays an important role in secondary brain injury. After TBI, a large number of reactive oxygen species (ROS) and nitrogen free radicals are produced. These excessive free radicals exceed the ability of the antioxidant defense system in vivo to degrade. Harmful toxic substances such as reactive oxygen species (ROS) accumulate in the body, leading to lipid peroxidation, protein and DNA structural damage and conduction. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key factor in regulating oxidative stress. In physiological conditions, Nrf2 binds to Keap1 and Cul3 to form a complex that prevents Nef2 from entering the nucleus from the cytoplasm. Under stress conditions, the spatial conformation of Keap1 changes, Nrf2 dissociates from the complex, Nrf2 enters the nucleus and binds with Maf to form a heterodimer to activate the ARE sequence, thereby initiating phase II detoxification and antioxidant genes. Among them, NQO-1 and HO-1 play an important role in antioxidant stress and exogenous harmful substances. Sinomenine is an active alkaloid isolated from the Chinese herbal medicine Sinomenine, which is mainly used in the treatment of rheumatoid arthritis and mesangial proliferative glomerulonephritis. Sinomenine has neuroprotective effects in many central nervous system diseases, such as cerebral ischemia, cerebral hemorrhage and neurodegenerative diseases. However, the role of sinomenine in TBI is seldom studied and the related mechanism is still unclear. AIM: To investigate the neuroprotective effects and possible molecular mechanisms of sinomenine in male ICR mice after intraperitoneal injection of sinomenine. The mice were randomly divided into four groups: sham group, TBI group, TBI + solvent group and TBI + SIN (30mg / kg) group; 2. To observe the effect of SIN on neurological function and brain edema after TBI in mice; 3. Western blot was used to detect the expression of apoptosis-related proteins Bcl-2 and cleaved caspase-3 in brain tissues, and TUNEL was used to detect neuronal apoptosis in brain sections. (5) Western blot was used to detect the expression of NQO-1, HO-1 and Nrf2 in brain tissue, immunofluorescence double staining was used to detect the localization of Nrf2. Results: (1) Compared with TBI group, Sinomenine treatment significantly decreased the brain water content (P 0.001) and neuron apoptosis index (P 0.001), significantly increased the neurological function score (1 P 0.01). (2) Sinomenine reduced oxidative stress in brain tissue after traumatic brain injury in mice. (3) After traumatic brain injury, Nrf2 transferred from cytoplasm to nucleus, and sinomenine promoted this metastasis, increasing the expression of NQO-1 and HO-1 in protein and mRNA levels. It is concluded that sinomenine can reduce oxidative stress, reduce brain edema, inhibit neuronal apoptosis, improve neurological function score and play a neuroprotective role in traumatic brain injury in mice. The neuroprotective effect of sinomenine may be achieved by activating Nrf2-ARE pathway.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15
[Abstract]:Background: Traumatic brain injury (TBI) is still a major public health problem in modern society with high disability and mortality. TBI is divided into primary brain injury and secondary brain injury. After primary brain injury, a series of complex endogenous pathophysiological processes are triggered to cause neuronal damage, also known as secondary brain injury. Oxidative stress plays an important role in secondary brain injury. After TBI, a large number of reactive oxygen species (ROS) and nitrogen free radicals are produced. These excessive free radicals exceed the ability of the antioxidant defense system in vivo to degrade. Harmful toxic substances such as reactive oxygen species (ROS) accumulate in the body, leading to lipid peroxidation, protein and DNA structural damage and conduction. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key factor in regulating oxidative stress. In physiological conditions, Nrf2 binds to Keap1 and Cul3 to form a complex that prevents Nef2 from entering the nucleus from the cytoplasm. Under stress conditions, the spatial conformation of Keap1 changes, Nrf2 dissociates from the complex, Nrf2 enters the nucleus and binds with Maf to form a heterodimer to activate the ARE sequence, thereby initiating phase II detoxification and antioxidant genes. Among them, NQO-1 and HO-1 play an important role in antioxidant stress and exogenous harmful substances. Sinomenine is an active alkaloid isolated from the Chinese herbal medicine Sinomenine, which is mainly used in the treatment of rheumatoid arthritis and mesangial proliferative glomerulonephritis. Sinomenine has neuroprotective effects in many central nervous system diseases, such as cerebral ischemia, cerebral hemorrhage and neurodegenerative diseases. However, the role of sinomenine in TBI is seldom studied and the related mechanism is still unclear. AIM: To investigate the neuroprotective effects and possible molecular mechanisms of sinomenine in male ICR mice after intraperitoneal injection of sinomenine. The mice were randomly divided into four groups: sham group, TBI group, TBI + solvent group and TBI + SIN (30mg / kg) group; 2. To observe the effect of SIN on neurological function and brain edema after TBI in mice; 3. Western blot was used to detect the expression of apoptosis-related proteins Bcl-2 and cleaved caspase-3 in brain tissues, and TUNEL was used to detect neuronal apoptosis in brain sections. (5) Western blot was used to detect the expression of NQO-1, HO-1 and Nrf2 in brain tissue, immunofluorescence double staining was used to detect the localization of Nrf2. Results: (1) Compared with TBI group, Sinomenine treatment significantly decreased the brain water content (P 0.001) and neuron apoptosis index (P 0.001), significantly increased the neurological function score (1 P 0.01). (2) Sinomenine reduced oxidative stress in brain tissue after traumatic brain injury in mice. (3) After traumatic brain injury, Nrf2 transferred from cytoplasm to nucleus, and sinomenine promoted this metastasis, increasing the expression of NQO-1 and HO-1 in protein and mRNA levels. It is concluded that sinomenine can reduce oxidative stress, reduce brain edema, inhibit neuronal apoptosis, improve neurological function score and play a neuroprotective role in traumatic brain injury in mice. The neuroprotective effect of sinomenine may be achieved by activating Nrf2-ARE pathway.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15
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