【摘要】：研究背景:創(chuàng)傷性腦損傷(traumatic brain injury,TBI)仍然是現(xiàn)代社會的主要公共健康問題,致殘率和死亡率都很高。TBI分為原發(fā)性腦損傷和繼發(fā)性腦損傷。原發(fā)性腦損傷是受傷當(dāng)時立即發(fā)生,由外力直接造成的腦組織損傷。繼發(fā)性腦損傷是指原發(fā)性腦損傷后一系列復(fù)雜的內(nèi)源性病理生理過程被觸發(fā)而造成神經(jīng)細胞的損傷,又稱為二次腦損傷。引起繼發(fā)性腦損傷的主要病理生理過程主要包括氧化應(yīng)激、谷氨酸興奮毒性、炎性反應(yīng)、離子平衡紊亂、血管滲透性增加、血腦屏障破壞等。其中氧化應(yīng)激在繼發(fā)性腦損傷中扮演重要角色。TBI后生成大量活性氧自由基和氮自由基,這些過量的自由基超過了體內(nèi)抗氧化防御系統(tǒng)的降解能力,活性氧簇等有害毒物在體內(nèi)大量堆積,導(dǎo)致脂質(zhì)過氧化、蛋白質(zhì)和DNA結(jié)構(gòu)破壞,導(dǎo)致神經(jīng)細胞損傷和死亡,因而造成神經(jīng)功能障礙。核因子 E2 相關(guān)因子 2(nuclear factor erythroid 2-related factor 2,Nrf2)是機體調(diào)節(jié)氧化應(yīng)激的關(guān)鍵因子。在生理情況下,Nrf2與Keap1和Cul3結(jié)合形成復(fù)合體,防止Nef2從胞漿進入胞核。Cul3是泛素連接酶,可以使Nrf2泛素化,從而被蛋白酶降解,使細胞內(nèi)Nrf2處于一個平衡狀態(tài)。在應(yīng)激條件下,Keap1的空間構(gòu)象發(fā)生改變,Nrf2從復(fù)合體中解離出來,未被降解的Nrf2進入細胞核,與Maf結(jié)合形成異二聚體激活A(yù)RE序列,從而啟動Ⅱ相解毒和抗氧化基因等下游保護基因的轉(zhuǎn)錄。其中NQO-1和HO-1在對抗氧化應(yīng)激和外來有害物質(zhì)方面起重要作用。青藤堿是從中藥青風(fēng)藤中分離提純獲得的活性堿,臨床上主要用于治療類風(fēng)濕性關(guān)節(jié)炎及系膜增生性腎小球腎炎。大量實驗證明青藤堿具有抑制炎癥反應(yīng)、抑制免疫和細胞保護等多種藥理作用。青藤堿在腦缺血、腦出血和神經(jīng)退行性疾病等多種中樞神經(jīng)系統(tǒng)疾病模型中表現(xiàn)出神經(jīng)保護作用。然而青藤堿在TBI中的作用研究較少,相關(guān)機制也尚不清楚。因此,本實驗通過建立小鼠TBI模型,探討TBI后使用青藤堿是否起到神經(jīng)保護作用及其分子機制。目的:本實驗在雄性ICR小鼠TBI模型中,通過腹腔注射青藤堿后,探討其神經(jīng)保護作用及可能的分子機制。方法:1、實驗?zāi)Ｐ徒⒓胺纸M,選取健康成年雄性ICR小鼠,采用改良的Feeney法制作TBI模型。小鼠被隨機分成4組:sham組、TBI組、TBI+溶劑組和TBI+SIN(30mg/kg)組;2、觀察SIN對小鼠TBI后神經(jīng)功能及腦水腫的影響;3、Western blot檢測腦組織中凋亡相關(guān)蛋白Bcl-2和cleaved caspase-3的表達水平,用TUNEL檢測腦組織切片中神經(jīng)元凋亡;4、檢測腦組織中丙二醛(malonaldehyde,MDA)含量和谷胱甘肽過氧化物酶(glutathione peroxidase,GPx)、超氧化物歧化酶(superoxide dismutase,SOD)的活性;5、Western blot檢測腦組織中NQO-1、HO-1、胞漿胞核Nrf2的表達,免疫熒光雙染檢測Nrf2的定位表達;6、實時定量PCR檢測腦組織中NQO-1和HO-1的mRNA表達水平。結(jié)果:(1)與TBI組相比,青藤堿處理后明顯降低了小鼠腦水含量(P0.001)和神經(jīng)元凋亡指數(shù)(P0.001),明顯提高了神經(jīng)功能評分(1P0.01)。(2)青藤堿減輕了小鼠創(chuàng)傷性腦損傷后腦組織氧化應(yīng)激,具體表現(xiàn)為青藤堿處理后明顯降低了小鼠腦組織MDA水平(P0.001),SOD和GPx的活性明顯提高(P0.001,P0.001)。(3)創(chuàng)傷性腦損傷后,Nrf2從細胞質(zhì)轉(zhuǎn)移至細胞核,而使用青藤堿后促進了這樣轉(zhuǎn)移,增加了 NQO-1和HO-1在蛋白和mRNA水平的表達。結(jié)論:我們的實驗數(shù)據(jù)表明,在小鼠創(chuàng)傷性腦損傷模型中使用青藤堿可以降低氧化應(yīng)激,減輕腦水腫,抑制神經(jīng)元凋亡,改善小鼠神經(jīng)功能評分,起到神經(jīng)保護作用。青藤堿的神經(jīng)保護作用可能是通過激活Nrf2-ARE通路獲得的。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R651.15

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