亚甲蓝对创伤性颅脑损伤的神经保护作用及其机制研究

发布时间：2018-02-10 15:21

本文关键词： 创伤性脑损伤亚甲蓝神经元脑水肿神经功能缺损创伤性脑损伤亚甲蓝促炎细胞因子小胶质细胞自噬 mTOR/p70S6K　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：研究背景：创伤性脑损伤是一种严重危及人类健康的疾病,有着较高的发病率、致残率和死亡率。全世界每年约有1000多万人遭受各种类型和程度的颅脑损伤。创伤性颅脑损伤不仅导致患者死亡,即使有幸存活的患者也会在伤后遭受不同程度的并发症,包括各种神经功能缺损和运动功能的障碍等。最近数十年来,随着急诊抢救与重症监护设备、技术以及药物的进展,脑损伤患者的生存率得到了较大的改善。不过令人遗憾的是,到目前为止,仍然没有一种药物可以很好的防止颅脑损伤导致的各种长远期的并发症。一系列的研究已经表明,颅脑损伤的损害的严重程度不仅是和原发性创伤相关的,更多的是和继发性损伤密切相关,包括神经炎症反应、脑水肿和血脑屏障的破坏等等。继发性损伤可以从损伤开始后不久一直持续几天、几个月甚至几年,最终导致神经元的死亡或神经退行性疾病的发生。另一方面,原发性颅脑损伤在损伤当时就已经发生,在临床上是无法干预的。因此,目前脑损伤的研究都集中在如何减少继发性颅脑损伤。寻找合适的药物靶点及药物干预继发性颅脑损伤,是改善颅脑损伤患者预后的关键。亚甲蓝是一种噻吩嗪类化合物,具有自身氧化还原的特性。其在临床已经使用了120多年,既往主要用于治疗高铁血红蛋白血症、疟疾、氰化物中毒、心肺转流术和脓毒血症。近年来,亚甲蓝在中枢神经系统中的作用越来越引起大家的重视,亚甲蓝对于缺血性脑卒中、侧索性硬化、阿尔茨海默氏病、帕金森病、Leber视神经病变和其他神经退行性疾病等都具有潜在的治疗价值。由于颅脑损伤和其他类型的神经系统病变如缺血性脑卒中和退行性神经病变方面有不少共同的病理生理机制,因此,我们推测亚甲蓝在颅脑损伤中也具有同样的治疗价值。本研究拟采用ICR小鼠进行创伤性颅脑损伤的造模,探讨亚甲蓝对创伤性颅脑损伤的神经保护作用并进一步研究其可能的作用机制。第一部分亚甲蓝对创伤性颅脑损伤的神经保护作用研究目的：本研究采用ICR小鼠进行创伤性脑损伤造模,探讨亚甲蓝是否对创伤性颅脑损伤具有神经保护的作用。研究方法： (1)预实验确定亚甲蓝的最佳使用剂量：将小鼠分为5组,分别为假手术组、生理盐水处理组、低剂量亚甲蓝治疗组(0.lmg/kg)、中等剂量亚甲蓝治疗组(l.0mg/kg)和高剂量亚甲蓝治疗组(10mg/kg)。以脑损伤后24小时脑含水量和神经功能缺损评分为依据,确定亚甲蓝的最佳剂量。 (2)根据预实验结果,将小鼠分为假手术组、生理盐水处理组和亚甲蓝治疗组。结合文献资料,我们选择伤后24小时及72小时进行神经功能缺损评分、脑损伤体积、脑含水量和神经元数量的检测；在伤后14天进行神经功能缺损评分和脑损伤体积的测定。 (3)相关指标评估的方法：采用改良的神经功能缺损评分进行神经功能评估；采用干湿重量法测定脑含水量；采用免疫组化NeuN染色,用Image-Pro Plus软件检测神经元的数量；采用连续切片HE染色,用Image tool软件计算脑损伤体积。研究结果： (1)预实验结果表明：脑损伤后损伤侧大脑半球脑含水量及神经功能缺损较假手术组明显增高(P0.05)。与生理盐水处理组比较,采用l.Omg/kg亚甲蓝治疗后能显著降低伤后24小时的神经功能缺损评分和脑含水量(P0.05),亚甲蓝其他两个剂量与生理盐水组比较都无统计学差异(P0.05)。因此确定使用亚甲蓝1.0mg/kg为我们正式实验的剂量。 (2)脑含水量的检测结果表明,亚甲蓝可以显著降低ICR小鼠创伤性脑损伤后24小时损伤侧大脑半球的脑含水量(P0.05),而对侧大脑半球、脑干、小脑等部位的脑含水量无统计学差异(P0.05)。同时我们发现在伤后72小时两组小鼠在各个部位的脑含水量无统计学差异(P0.05)。 (3)神经元的免疫组化检测表明,采用亚甲蓝治疗的小鼠在伤后24小时及72小时损伤区的神经元数量要显著高于生理盐水处理组,两者存在着统计学的差异(P0.05)。 (4)神经功能缺损评分表明,与生理盐水处理组相比,采用亚甲蓝治疗能降低小鼠脑损伤后24小时和72小时的神经功能缺损评分,差异有统计学意义(P0.05)。而在脑损伤后14天,两组小鼠的神经功能缺失评分无显著差异(P0.05)。 (5)脑损伤体积检测结果表明,与生理盐水组比较,亚甲蓝治疗减少了脑损伤后24小时、72小时和14天脑损伤的体积(P0.05)。结论：亚甲蓝治疗可以降低创伤性脑损伤的神经功能缺损评分,降低创伤性脑损伤引起的脑水肿,减少脑损伤的体积,阻止创伤性脑损伤后神经元的死亡。第二部分亚甲蓝对创伤性颅脑损伤的神经保护作用机制探讨研究目的：探讨亚甲蓝对创伤性颅脑损伤神经保护作用的可能机制。研究方法： (1)我们将实验小鼠分为假手术组、生理盐水处理组和亚甲蓝治疗组。结合文献资料,在伤后24小时及72小时评估自噬激活情况、促炎细胞因子水平和小胶质细胞激活情况；在伤后14天评估小胶质细胞激活情况。 (2)相关指标评估的方法：免疫组化Beclin-1染色,Western Blot检测Beclin-1和LC3Ⅱ/Ⅰ来评估自噬激活情况；Western Blot来检测炎症因子IL-6、 ILlβ的表达量； Iba-1免疫组化染色来检测小胶质细胞激活情况。 (3) mTOR/p70S6K通路中相关蛋白的变化情况采用Western Blot检测。研究结果： (1)脑损伤后自噬显著激活；与生理盐水处理组比较,亚甲蓝治疗促进了创伤性脑损伤后24小时及72小时自噬的发生,表现为Beclin-1表达的上调和LC3Ⅱ/Ⅰ比值的增加(P0.05)。 (2)脑损伤后促炎细胞因子IL-6、IL1β的释放增加；与生理盐水处理组比较,亚甲蓝显著抑制了脑损伤后24小时及72小时IL-6、IL1β的表达(P0.05)。 (3)小胶质细胞在脑损伤后72小时和14天激活明显(P0.05)；与生理盐水处理组相比,亚甲蓝治疗抑制了创伤性脑损伤后72小时及14天小胶质细胞的激活(P0.05)。 (4)脑损伤后1nTOR/p70S6K通路激活,表现为磷酸化的mTOR和磷酸化的p70S6K表达增加(P0.05)。而与生理盐水处理组相比,亚甲蓝治疗后显著抑制了mTOR/p70S6K通路中磷酸化的mTOR及磷酸化的p70S6K蛋白的表达(P0.05),而对于总的mTOR及总的p70S6K蛋白的水平无明显的影响(P0.05)。结论：(1)亚甲蓝治疗可以通过减少创伤性脑损伤后促炎细胞因子的释放,抑制小胶质细胞的激活及促进自噬的发生起到神经保护作用。(2)结合文献资料和以上实验结果,我们认为亚甲蓝可能是通过抑制mTOR/p70S6K通路相关蛋白的激活,从而起到了抑制神经炎症反应和促进自噬的作用。
[Abstract]:Research background:
Traumatic brain injury is a serious threat to human health disease, with high morbidity, disability and mortality. There are about about 10000000 people suffering from traumatic brain injury and the degree of various types in the world each year. Not only leads to death in patients with traumatic brain injury, even survive patients will suffer different degrees of complications in trauma after including the deficit and motor function in various neurological disorders.
In recent decades, with the emergency and intensive care equipment, technology and drug development, the survival rate of patients with brain injury has been greatly improved. But unfortunately, so far, there is not a drug can prevent brain injury caused by long-term complications as well. A series of research have shown that the severity of craniocerebral injury and damage is not only the primary injury related, is more closely related and secondary injury, including nerve inflammation, brain edema and blood brain barrier damage. Following the injury from injury shortly after continued for several days, or even months in recent years, resulting in the death of neurons or neurodegenerative diseases. On the other hand, primary craniocerebral injury in the injury had occurred, in the clinic is not intervention. Because of this, the brain The research of injury is focused on how to reduce secondary craniocerebral injury. Finding the right drug targets and drugs to intervene secondary craniocerebral injury is the key to improve the prognosis of patients with craniocerebral injury.
Methylene blue is a phenothiazine compounds, has the characteristics of self oxidation reduction. It has been used for 120 years in the clinical history, mainly used for the treatment of methemoglobinemia, malaria, cyanide poisoning, cardiopulmonary bypass and sepsis. In recent years, methylene blue and function in the central nervous system more attention, methylene blue for ischemic stroke, side simply sclerosis, Alzheimer's disease, Parkinson's disease, has the potential therapeutic value of Leber optic neuropathy and other neurodegenerative diseases. Due to disease of the central nervous system and other types of brain injury such as ischemic stroke and neurodegenerative diseases are many common pathophysiological mechanisms, therefore, we speculate that methylene blue has the same therapeutic value in traumatic brain injury.
The purpose of this study is to establish a model of traumatic brain injury in ICR mice, and to explore the neuroprotective effect of methylene blue on traumatic brain injury and to further study its possible mechanism.
The neuroprotective effect of methylene blue on traumatic brain injury in the first part
The purpose of the study is:
In this study, ICR mice were used to make a model of traumatic brain injury and to explore the effect of methylene blue on the neuroprotective effect of traumatic brain injury.
Research methods:
(1) methylene blue dosage determined pre experiment: mice were divided into 5 groups, namely sham operated group, normal saline group, low dose of methylene blue treatment group (0.lmg/kg), medium dose of methylene blue (l.0mg/kg) treatment group and high-dose treatment group of methylene blue (10mg/kg) based on the water and nerve function. 24 hours after brain injury with cerebral impairment score as the basis, to determine the optimal dose of methylene blue.
(2) according to the results of the experiment, the mice were divided into sham operation group, saline treatment group and methylene blue group. Combined with the literature, we selected 24 hours after injury and 72 hours of neurological deficit score, brain injury volume, detection of brain water content and the number of neurons in the neural function defect determination; score and brain injury Volume 14 days after injury.
(3) method evaluation: the neural function defect score on the modified neurological function assessment; brain water content was determined by dry wet weight method; by using immunohistochemical NeuN staining, with the number of Image-Pro Plus software to detect neurons; using serial sections of HE staining, calculate the brain injury volume using Image tool software.
The results of the study:
(1) the results of pre experiments show that: after brain injury, ipsilateral hemisphere brain water content and neurological function were significantly higher than those in the sham operation group (P0.05). With the saline treated group compared with l.Omg/kg methylene blue after treatment can significantly reduce the injury of nerve function defect score and 24 hours of brain water content (P0.05), methylene blue, the other two dose compared with normal saline group had no significant difference (P0.05). It was determined using methylene blue 1.0mg/kg as our formal experimental dose.
(2) the detection results of cerebral water content showed that methylene blue can significantly reduce the amount of water for 24 hours the ipsilateral cerebral hemisphere of brain after traumatic brain injury in mice with ICR (P0.05), and the contralateral cerebral hemisphere, brainstem, cerebellum and other parts of the brain water content had no significant difference (P0.05). At the same time we found in in various parts of the brain injury after 72 hours in two groups were no significant difference in water content (P0.05).
(3) immunohistochemistry showed that the number of neurons in the injured area treated by methylene blue was significantly higher than that in saline group at 24 hours and 72 hours after injury, and there was a significant difference between them (P0.05).
(4) the neurological deficit scores showed that compared with normal saline group, using methylene blue treatment can reduce brain injury in mice after nerve function defect in 24 hours and 72 hours of score, the difference was statistically significant (P0.05). The brain injury after 14 days, there was no significant difference between the scores of neurological deficits in two groups of mice the (P0.05).
(5) the results of brain damage volume test showed that methylene blue therapy reduced the volume of brain damage (P0.05) at 24 hours, 72 hours and 14 days after brain damage compared with the saline group.
Conclusion:
Methylene blue treatment can reduce neurological function after traumatic brain injury caused by traumatic brain injury, decrease brain edema and reduce the volume of brain damage, prevent traumatic neuronal death after brain injury. The second part of the research of methylene blue on traumatic brain injury of the nerve protection mechanism
Objective: To investigate the possible mechanism of methylene blue on the neuroprotective effect of traumatic brain injury.
Research methods:
We (1) mice were randomly divided into sham operation group, saline treatment group and methylene blue group. According to the data, in 24 hours and 72 hours to assess the activation of autophagy after injury, levels of proinflammatory cytokines and activation of microglia activation; on the 14 day after injury assessment of microglia.
(2) related indicators evaluation methods: immunohistochemistry Beclin-1 staining, Western Blot detection Beclin-1 and LC3 II / I to evaluate autophagy activation; Western Blot to detect inflammatory factor IL-6, ILl beta expression; Iba-1 immunohistochemical staining to detect microglia activation.
(3) the changes of related proteins in the mTOR/p70S6K pathway were detected by Western Blot.
The results of the study:
(1) autophagy was significantly activated after brain injury. Compared with saline group, methylene blue treatment promoted the occurrence of autophagy at 24 hours and 72 hours after traumatic brain injury, which was manifested by the up regulation of Beclin-1 expression and the increase of LC3 II / I ratio (P0.05).
(2) the release of proinflammatory cytokines IL-6 and IL1 beta increased after brain injury. Compared with saline group, methylene blue significantly inhibited the expression of IL-6 and IL1 IL1 at 24 hours and 72 hours after brain injury.
(3) microglia activated significantly in 72 hours and 14 days after brain injury (P0.05). Compared with saline group, methylene blue treatment inhibited microglial activation (P0.05) at 72 hours and 14 days after traumatic brain injury.
(4) the activation of 1nTOR/p70S6K after brain injury, manifested as mTOR phosphorylation and phosphorylation of the increased expression of p70S6K (P0.05). Compared with the normal saline group, methylene blue treatment significantly inhibited the phosphorylation of mTOR and phosphorylation of mTOR/p70S6K pathway of p70S6K protein expression (P0.05), and for the total mTOR and total p70S6K protein levels had no significant effect (P0.05).
Conclusion: (1) methylene blue treatment can reduce traumatic brain injury after the release of proinflammatory cytokines and inhibit the activation of microglia and promote autophagy plays a neuroprotective role. (2) combined with literature data and experimental results, we believe that the methylene blue probably by inhibiting activation of mTOR/p70S6K pathway protein, so as to inhibit nerve inflammation and promote autophagy.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R651.15

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