颅脑创伤(traumatic brain injury，TBI)是一类具有高死亡率和致残率的外伤类型，继发性损伤是决定TBI患者预后的关键因素。TBI后继发性神经元损伤机制错综复杂，且不同的机制之间相互影响和促进。同时，TBI后全身多种脏器均存在不同程度的继发性脏器功能损伤，同样影响TBI患者的预后。因此，目前的对抗单一损伤机制的治疗策略不能取得满意的效果，寻找一个可以同时拮抗多种继发性损伤机制的上游调控枢纽理论上应该具有更好的疗效。转录因子NF-E2 相关因子2 (nuclear factor E2-related factor 2，Nrf2)是近年来新发现的一种核转录因子，在不同的毒性攻击和疾病病理情况下，上调Nrf2信号转导通路都能够在多种细胞和组织中表现出保护作用，并同时发挥多重的保护作用。我们的前期工作已证实，Nrf2的激活与脑外伤具有相关性，其信号通路分别在脑、肺、肠道中激活，可能发挥保护作用。因此，本研究提出“Nrf2是调节脑外伤后多脏器多重病理生理机制的核心靶点”的假说，深入研究Nrf2在TBI中的作用及机制。 第一部分 绪论 本节旨在介绍相关研究背景，对Nrf2信号通路研究进展及Nrf2的全身性多脏器保护作用进行综述。 第二部分 Nrf2在小鼠创伤性脑损伤后继发性神经元损伤中的保护作用及机制研究 继发性神经元损伤机制非常复杂，氧化损伤、钙离子超载、炎症反应以及细胞凋亡等多种机制均参与TBI后继发性神经元损伤。因此寻找一个能够同时干预多种继发性损伤机制的靶点，可能成为TBI治疗策略的新方向。Nrf2是体内抑制氧化损伤、调节炎症反应以及代谢有害物质的关键分子，调控一系列细胞保护性蛋白的表达。我们的前期研究也证实了 TBI后脑组织中Nrf2-ARE通路被激活。因此，Nrf2有可能成为调节TBI后继发性神经元损伤中多重病理生理机制的核心靶点。本部分研究采用Hall自由落体法制成小鼠脑外伤模型，选择Nrf2(+/+) ICR小鼠和Nrf2(-/-) ICR小鼠为实验动物，在伤后24h分别对各创伤组和对照组小鼠进行神经功能评分；取损伤中心区脑皮层组织，干湿重比法测定脑组织含水量；原位末端标记法(TUNEL)检测细胞凋亡水平；EMSA法检测炎症信号通路上游调控因子NF-κB转录活性；免疫组织化学法检测炎症黏附分子ICAM-1分布；RT-PCR检测炎症细胞因子(TNF-α、IL-1β、IL-6)及抗氧化/解毒酶(NQO1、 GST-α1) mRNA表达水平；ELISA法检测炎症细胞因子(TNF-α、IL-1β、IL-6)蛋白含量；并作组间比较。研究发现，创伤组Nrf2(-/-)小鼠较创伤组Nrf2 (+/+)小鼠出现更为严重的神经功能缺失、脑水肿及细胞凋亡，同时伴TNF-α、 IL-1β、IL-6的上调及NQO1、GST-α1的缺失，表明Nrf2在小鼠创伤性脑损伤中的发挥保护性作用，这种保护作用可能通过调节炎症细胞因子表达及诱导抗氧化/解毒酶来实现；此外，进一步研究发现，创伤组Nrf2(-/-)小鼠较创伤组Nrf2(+/+)小鼠出现更为明显的NF-κB活化、ICAM-1表达及TNF-α、IL-1β、 IL-6的上调，表明Nrf2在小鼠创伤性脑损伤中通过抑制NF-κB炎症信号通路的活化发挥其抗炎作用。 第三部分 Nrf2在小鼠创伤性脑损伤后继发急性肺损伤中的保护作用及机制研究 急性肺损伤是TBI后的常见并发症，但是目前对其认识尚不充分。目前的观点是TBI后由于下丘脑—垂体—肾上腺轴及植物神经系统调节的异常，导致TBI后肺组织发生广泛的氧化损伤和炎症反应。我们的前期研究证实，TBI后肺组织中Nrf2-ARE通路被激活。而Nrf2是体内抑制氧化损伤和调节炎症反应的关键因子。因此我们推断，Nrf2有可能成为调节TBI后继发急性肺损伤中多重病理生理机制的核心靶点。本部分在实验性TBI模型中评估Nrf2对TBI后继发急性肺损伤的作用及相关机制。Nrf2(+/+)和Nrf2(-/-)小鼠各随机分为对照组和创伤组，在伤后24h留取肺组织标本，伊文思蓝渗透法检测肺毛细血管通透性；干湿重比法测定肺组织含水量；原位末端标记法(TUNEL)检测肺组织细胞凋亡水平； RT-PCR检测炎症细胞因子(TNF-α、IL-1β、IL-6)及抗氧化/解毒酶(NQO1、 GST-α1)mRNA表达水平；ELISA法检测炎症细胞因子(TNF-α、IL-1β、IL-6)蛋白含量；并作组间比较。研究发现，Nrf2敲除后，脑创伤小鼠出现更严重的肺组织水肿、通透性增加和细胞凋亡，并且出现更多的TNF-α、IL-1β、IL-6表达及NQO1、GST-α1缺失，表明Nrf2在小鼠创伤性脑损伤后继发急性肺损伤后通过调节炎症细胞因子表达及诱导抗氧化/解毒酶来发挥其保护性作用；进一步的研究中，通过EMSA法检测炎症信号通路上游调控因子NF-κB转录活性；免疫组织化学法检测炎症黏附分子ICAM-1分布；并作组间比较。我们发现，Nrf2在小鼠创伤性脑损伤后继发急性肺损伤中调节炎症反应的作用，亦通过抑制NF-κB炎症信号通路的活化而实现。 第四部分 Nrf2在小鼠创伤性脑损伤后继发性肠道功能损伤中的保护作用及机制研究 肠功能障碍是TBI的常见并发症，并对患者预后产生重要影响。本实验室前期研究发现，TBI后肠黏膜损伤不仅影响肠道自身功能，还造成远隔的组织或器官损伤，最终可能导致系统性炎症反应综合征和多脏器功能衰竭。而多种证据表明，炎症反应和氧化损伤广泛存在于这一病理生理过程中。我们的前期研究已经证实，TBI后肠组织中Nrf2-ARE通路被激活，并且可能发挥保护作用。因子，我们推断，Nrf2有可能成为调节TBI后继发急性肺损伤中多重病理生理机制的核心靶点。本部分在实验性TBI模型中评估Nrf2对TBI后继发肠黏膜损伤的作用及相关机制。Nrf2(+/+)和Nrf2(-/-)小鼠各随机分为对照组和创伤组，在伤后24h留取空场组织标本，光镜评价肠黏膜显微结构改变；电镜观察肠黏膜超微结构改变；乳果糖/甘露醇排泄比法检测肠黏膜屏障通透性；原位末端标记法(TUNEL)检测肠黏膜细胞凋亡水平；RT-PCR检测炎症细胞因子(TNF-α、IL-1β、 IL-6)及抗氧化/解毒酶(NQO1、GST-α1) mRNA表达水平；ELISA法检测炎症细胞因子(TNF-α、IL-1β、IL-6)蛋白含量；并作组间比较。研究发现，Nrf2敲除后，脑创伤小鼠出现更严重的肠黏膜结构损伤、屏障通透性增加和细胞凋亡，并且出现更多的TNF-α、IL-1β、IL-6表达及NQO1、GST-α1缺失，表明Nrf2在小鼠创伤性脑损伤后继发肠黏膜损伤后通过调节炎症细胞因子表达及诱导抗氧化/解毒酶来发挥其肠黏膜保护作用；进一步的研究中，通过EMSA法检测炎症信号通路上游调控因子NF-κB转录活性；免疫组织化学法检测炎症黏附分子ICAM-1分布；并作组间比较。我们发现，Nrf2在小鼠创伤性脑损伤后继发肠黏膜损伤中通过抑制NF-κB炎症信号通路的活化起到抗炎作用。 小结 本论文研究在我们前期实验工作的基础上，运用Nrf2基因敲除小鼠，深入研究了 Nrf2在小鼠创伤性脑损伤(TBI)后继发性神经元损伤、急性肺损伤和肠黏膜损伤中的保护性作用及相关机制。结果表明Nrf2对小鼠TBI后继发性神经元损伤、急性肺损伤和肠黏膜损伤均有明确的保护性作用，深入研究其保护机制，我们证实Nrf2通过内在性的抑制NF-κB炎症信号通路活化起到调节炎症反应、同时通过诱导抗氧化和解毒酶起到抵抗氧化损伤作用，从而发挥多重的保护作用。因此，我们得出结论：Nrf2是调节脑外伤后多脏器多重病理生理机制的核心靶点。因此，针对Nrf2为靶点的治疗，有可能具有“一个靶点，多重疗效”的作用，从而为TBI治疗策略提供一个全新的思路和方向。 关键词：创伤性脑损伤；Nrf2；炎症反应；氧化应激；急性肺损伤；肠粘膜损伤

 Traumatic brain injury (TBI) is a major cause of death and disability worldwide, secondary brain injury plays a pivotal role in the outcome of patients suffering from TBI. The mechanisms underlying secondary brain injury are known to be complex, moreover, close interactions exist these pathological processes. In addition, TBI-induced secondary multiple organ injury also plays an important role in the outcome of patients suffering TBI. Therefore, it appears that the present therapeutic strategy aimed at single target has been proved to be inefficacy in clinical trials, and a therapeutic strategy aimed at multiple targets may be desirable. The transcription factor NF-E2-Related Factor 2 (Nrf2) is a basic leucine zipper redox sensitive transcription factor which has been reported to be a pleiotropic regulator in cell survival mechanisms. It induces expression and up-regulation of cytoprotective and antioxidant/detoxifying genes that attenuate tissue injury. In our previous study, we have demonstrated the activation of Nrf2 in the brain, the lung and the gut. In this study, we hypothesized that Nrf2 would play a pivotal role in protecting against TBI-induced secondary injury. Part Ⅰ: Introduction In this part, the background of the study is introduced, including reviews of the proceedings on the study of the Nrf2 signaling pathway, and the Nrf2-conferred multi-organ protection phenomenon. Part Ⅱ: Role of Nrf2 in protection against TBI-induced secondary brain injury in mice The mechanisms underlying secondary brain injury have been proved to be complex, including oxidative stress, calcium overload, inflammatory response, and apoptosis. A target that can interrupt multiple mechanisms underlying secondary brain injury may represent a promising new therapeutic approach for TBI. Nrf2 is the key regulator in mediating a series of cytoprotective proteins, which have been reported to reduce oxidative stress, inflammatory damage and the accumulation of toxic metabolites. Our previous study has also demonstrated the activation of Nrf2 signal ing pathway in the brain after TBI. Therefore, we hypothesized that Nrf2 would play a pivotal role in protecting against TBI-induced secondary brain injury. Wildtype Nrf2 (+/+) and Nrf2 (-/-) deficient mice were subjected to Hall' s weight-drop impact head injury. At 24h after TBI, neurological status was evaluated. We also measured brain edema by wet/dry method; Cortical apoptosis by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNED method; Nuclear factor kappa B (NF-κB) activity by electrophoretic mobility shift assay (EMSA); Expression of intercellular adhesion molecule-1 (ICAM-1) by immunohistochemistry ; mRNA expression of inflammatory cytokines (TNF-α 、 IL-1β、 IL-6 ) and antioxidant/detoxifying enzymes (NQO1、 GST-α1 ) by reverse transcriptase-polymerase chain reaction (RT-PCR). Protein expression of inflammatory cytokines ( TNF-α 、 IL-1β 、 IL-6 ) by enzyme linked immunosorbent assay (ELISA). Mice lacking Nrf2 function have significantly increased neurological deficit, brain edema and cortical apoptosis after TBI, Increased mRNA expression of TNF-α , IL-1β and IL-6, and decreased expression of NQ01 and GST were also observed in the brain samples of Nrf2 (-/-) mice as compared with Nrf2 (+/+) mice. These results suggest that Nrf2 plays an important role in protecting TBI-induced secondary brain injury, possibly by regulating of inflammatory cytokines and inducing of antioxidant and detoxifying enzymes. Moreover, Nrf2 (-/-) mice were shown to have more NF- κB activation, inflammatory cytokines production and ICAM-1 expression in brain after TBI compared with their wild-type Nrf2 (+/+) counterparts. The results suggest that Nrf2 plays an important protective role in limiting the cerebral inflammatory response may through modulating the proinflammatory NF-κB signaling pathway after TBI. Part Ⅲ: Role of Nrf2 in protection against TBI-induced acute lung injury in mice Acute lung injury (ALI) is a frequent but poorly understood complication of TBI. The prevailing theory has been that the dysregulation of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system resulting from the TBI may initiate the stimulation of inflammatory response and oxidative damage in the lung. Our previous study has also demonstrated the activation of Nrf2 signaling pathway in the lung after TBI. The present study investigated the role of Nrf2 in the regulation of TBI-induced ALI. Wildtype Nrf2 (+/+) and Nrf2 (-/-) deficient mice were subjected to Hall' s weight-drop impact head injury. Pulmonary capillary permeability (PCP), wet/dry weight ratio, apoptosis, NF-κB activity, ICAM-1 expression, inflammatory cytokines and antioxidant/detoxifying enzymes were measured at 24 h after TBI. Mice lacking Nrf2 were found to be more susceptible to TBI-induced ALI, as characterized by the higher increase in PCP, wet/dry weight, ratio and alveolar cells apoptosis after TBI. This exacerbation of lung injury in Nrf2 deficient mice was associated with increased pulmonary mRNA expression of inflammatory cytokines such as TNF-α , IL-1β and IL-6; and with decreased pulmonary mRNA expression antioxidant and detoxifying enzymes including NQ01 and GST-α1- as compared with their Nrf2 (+/+) counterparts after TBI. These results suggest that Nrf2 reduces TBI-induced acute lung injury, possibly by decreasing pulmonary inflammation and inducing antioxidant and detoxifying enzymes. Moreover, Nrf2 (-/-) mice were shown to have more increase in the levels of TNF-α , IL-1β , IL-6, ICAM-1 and their mediator NF-κB. The results suggest that Nrf2 plays an important protective role in attenuating pulmonary inflammatory response may through modulating the proinf1ammatory NF-κB signaling pathway after TBI. Part Ⅳ: Role of Nrf2 in protection against TBI-induced acute intestinal mucosal injury in mice Intestinal dysfunction occurs frequently in patients with TBI. The development of acute intestinal mucosal injury is also associated with the outcome of patients suffering from TBI. This pathologic course may not only influence the intestinal mucosa itself, but also impair the remote tissue and organs, leading to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). Several pieces of evidence have shown that inflammation and oxidative stress are involved in the progression of TBI-induced intestinal mucosal injury. Our previous study has also demonstrated the activation of Nrf2 signaling pathway in the gut after TBI. The present study investigated the role of Nrf2 in the regulation of TBI-induced acute intestinal mucosal injury. Wildtype Nrf2 (+/+) and Nrf2 (-/-) deficient mice were subjected to Hall' s weight.-drop impact head injury. Intestinal mucosal morphological changes, plasma endotoxin, intestinal permeability, apoptosis, inflammatory cytokines and antioxidant/detoxifying enzymes were measured at 24 h after TBI. Nrf2 deficient mice were found to be more susceptible to TBI-induced acute intestinal mucosal injury, as characterized by the higher increase in gut structure damage, plasma endotoxin, intestinal permeability and apoptosis after TBI. This exacerbation of intestinal mucosal injury in Nrf2 deficient mice was associated with increased intestinal mRNA expression of inflammatory cytokines such as TNF-α , IL-1β and IL-6, and with decreased intestinal mRNA expression levels of antioxidant and detoxifying enzymes including NQ01 and GST-α1- as compared with their wildtype Nrf2 (+/+) counterparts after TBI. Our data suggests that Nrf2 plays an important role in protecting TBI-induced intestinal mucosal injury, possibly by regulating of inflammatory cytokines arid inducing of anti oxidant and detoxifying enzymes. Moreover, Intestinal levels of NF-κB, pro-inflammatory cytokines and ICAM-1 in Nrf2 (-/-) deficient mice were significantly higher compared with Nrf2 (+/+) mice at 24 h after TBI. The results suggest that Nrf2 plays an important protective role in attenuating intestinal inflammatory response may through modulating the proinflammatory NF-κB signaling pathway after TBI. Summary Based on the previous researches of us, the current study focused on the precise role of Nrf2 in the regulation of TBI-induced secondary brain injury, acute lung injury, and acute intestinal mucosal injury. The protective roles of Nrf2 in regulation of TBI-induced secondary brain injury, acutc lung injury and acute intestinal mucosal injury have been demonstrated. Furthermore, we have also demonstrated that Nrf2 plays an important role in attenuating inflammatory response through modulating the proinfiammatory NF-κB signaling pathway, and plays an important role in attenuating oxidative stress through inducing of antioxidant and detoxifying enzymes. In sum, we reported here for the first time that Nrf2 plays a pivotal role in protecting against TBI-induced secondary injury. These findings raise the possibility that Nrf2 may become a new therapeutic target for the treatment of TBI. Key words: traumatic brain injury; Nrf2; inflammatory response; oxidative stress; acute lung injury; acute intestinal mucosal injury.