丙戊酸抑制大鼠脑损伤后局部炎症机制的研究
发布时间：2019-03-07 18:04
【摘要】：背景创伤性颅脑损伤(traumatic brain injury,TBI)在神经外科疾病中非常常见,颅脑损伤后启动一系列复杂的神经化学信号变化导致的病理事件包括神经元过度活跃,过多的谷氨酸释放,炎症,血脑屏障(blood-brain barrier,BBB)通透性增加和脑水肿。颅脑损伤后基因表达的改变和一系列神经功能损伤对患者的预后产生极大的影响,带来身体和经济的双重压力。目前,并没有确切的药物和治疗方法来应对对颅脑损伤后的继发损伤。目前研究的重点主要致力于颅脑损伤后对神经细胞的保护以及炎症反应对脑组织的损伤。丙戊酸(valproic acid,VPA)一种组蛋白去乙酰化酶抑制剂一直被用于临床,它的神经保护作用是多方面的。本研究利用大鼠脑损伤打击器建立闭合性大鼠脑损伤模型,并给予丙戊酸干预,通过对不同时间点的大鼠进行行为学评价及磁共振成像、乙酰化组蛋白H3、NF-κB表达的变化,探讨VPA的抗炎机制,为临床应用VPA治疗TBI提供可靠依据。目的通过观察VPA对TBI大鼠各时间点脑组织乙酰化组蛋白H3、NF-κB蛋白表达的影响,探讨丙戊酸对实验性脑损伤大鼠的减轻炎性反应的作用。方法按照随机的原则把75只雄性SD大鼠分为3组:假手术组(C组)、损伤组(TBI组)和丙戊酸治疗组(VPA组)。TBI和VPA组建立大鼠闭合性损伤模型,C组在相同部位仅切开头皮,缝合。VPA治疗组术后每天按300mg/kg腹腔注射VPA,至处死当天;单纯损伤组和假手术组在相同时间腹腔注射等体积的生理盐水。伤后各时间点对大鼠进行行为学评价。伤后6 h、24 h、48 h和72 h每组取5只处死取材,免疫荧光双标法在激光共聚焦显微镜下观察核因子κB(NF-κB)途径的激活状态,免疫组化检测乙酰化组蛋白H3含量,并在伤后72 h每组取一只大鼠腹腔注射水合氯醛,待麻醉完全后于北京国家纳米中心做磁共振成像。结果1.行为学评分结果:在给予VPA干预后,VPA组大鼠和TBI组大鼠相比损伤程度有所减轻,在伤后24 h内效果不明显,3组间差异无统计学意义(P0.05);VPA干预后7 d、14 d,VPA治疗组大鼠的运动、感觉及反射功能均优于TBI组,两组间差异有统计学意义(P0.05)。2.7.0T小动物超导磁共振成像显示:单纯损伤组和正常对照组相比损伤灶颜色接近红色,说明水肿程度较重,其中损伤灶中心为黄色,说明水肿程度最重,VPA治疗组和单纯损伤组相比损伤灶颜色接近蓝色,说明在应用VPA治疗后大鼠损伤脑组织水肿有所减轻。从图中还可以看出,VPA治疗组损伤灶脑组织体积比相同时间点单纯损伤组明显减小,说明VPA能减少脑损伤体积。3.免疫组织化学检测显示:3组大鼠乙酰化状态组蛋白H3表达水平差异均有统计学意义(P0.05),VPA组和C组相比无明显差异,TBI组明显低于C组(P0.05)。4.免疫荧光技术检测:C组大鼠脑组织切片NF-κB核阳性细胞各个时间点表达很少,甚至不表达,与C组相比,伤后6 h TBI组和VPA组大鼠脑组织切片NF-κB核阳性细胞数量高于C组,24 h的大鼠脑组织切片中NF-κB核阳性细胞数明显高于C组,48 h和72 h NF-κB核阳性细胞表达开始逐渐减少,但仍比C组表达高(P0.05);VPA治疗组NF-κB核阳性细胞各时间点表达量均低于同时间点TBI组(P0.05)。结论丙戊酸对大鼠脑损伤后的抗炎作用可能是通过抑制NF-κB途经的激活来实现的。
[Abstract]:Background Traumatic brain injury (TBI) is very common in neurosurgical diseases. The pathological events caused by a series of complex neurochemical signal changes after brain injury include hyperactive neurons, excessive glutamate release, inflammation, blood-brain barrier (blood-brain barrier), BBB) increased permeability and brain edema. The change of gene expression after head injury and a series of nerve function damage have a great effect on the prognosis of the patient, bringing the double pressure of the body and the economy. At present, there is no definite drug and treatment method to deal with the secondary injury after brain injury. The research focuses on the protection of nerve cells after brain injury and the damage of the inflammatory response to the brain tissue. Valproic acid (VPA), a histone deethanizing enzyme inhibitor, has been used in clinical and its neuroprotective effects are various. In this study, the model of brain injury of closed rats was established by using the rat brain injury killer, and the intervention of valproic acid was given, and the anti-inflammatory mechanism of VPA was discussed by the behavior evaluation of rats at different time points and the changes of the expression of the magnetic resonance imaging, the histone H3 and NF-EMAB. Provide reliable basis for clinical application of VPA in the treatment of TBI. Objective To study the effect of VPA on the expression of histone H3 and NF-B protein in the brain tissue of TBI rats, and to study the effect of valproic acid on the inflammatory response of rats with experimental brain injury. Methods 75 male SD rats were divided into three groups according to the random principle: sham operation group (group C), injury group (TBI group) and valproic acid treatment group (VPA group). The closed injury model of rats was established in the TBI and VPA groups. The VPA was injected intraperitoneally at 300 mg/ kg every day after the treatment of the VPA group, and the same time as that of the sham-operated group and the sham-operation group, the volume of saline was injected in the same time. The behavior of rats was evaluated at all time points after injury. At 6 h,24 h,48 h and 72 h after injury,5 rats were sacrificed, and the activation state of the nuclear factor B (NF-B) pathway was observed under the laser confocal microscope, and the content of the histone H3 was detected by immunohistochemistry. At 72 hours after injury, a rat was injected with water-chloral hydrate in each group, and the magnetic resonance imaging was performed in the national nano-center of Beijing after the anesthesia. Results 1. Results: After the VPA intervention, the degree of injury of the rats in the VPA group and the TBI group was reduced, the effect was not obvious in 24 hours after the injury, the difference between the groups was not significant (P0.05), and the rats in the treatment group of the VPA group were treated with the VPA intervention for 7 days,14 days, and the VPA treatment group. The sensory and reflective function was superior to that of the TBI group, and the difference between the two groups was statistically significant (P0.05). The degree of edema was the most, and the color of the lesion in the treatment group of the VPA and the simple injury group was close to the blue, indicating that the edema of the brain tissue of the rats after the treatment with VPA was reduced. It can also be seen from the figure that the volume of brain tissue in the damage range of the VPA treatment group is significantly reduced than that of the same time point, indicating that the VPA can reduce the volume of the brain injury. The results showed that there was no significant difference in the level of histone H3 in group 3 rats (P0.05), and there was no significant difference in the group of VPA and Group C, and the group of TBI was lower than that of group C (P0.05). The results showed that the expression of NF-B-B-positive cells in the brain tissue of the C-group was little or not even expressed, compared with that of the C group. The number of NF-and B-positive cells in the brain tissue of the rats at 6 h TBI and VPA was higher than that of the C group, and the number of NF-and B-positive cells in the brain tissue of the rats in 24 h was significantly higher than that of the C group, and the expression of NF-B-B-positive cells in the brain tissue of group C,48 h and 72 h was gradually decreased. However, the expression of NF-B-B-positive cells in the treatment group of VPA was lower than that of the TBI group at the same time point (P0.05). Conclusion The anti-inflammatory effect of valproic acid on rat brain injury may be achieved by inhibiting the activation of NF-B.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15
本文编号：2436330
[Abstract]:Background Traumatic brain injury (TBI) is very common in neurosurgical diseases. The pathological events caused by a series of complex neurochemical signal changes after brain injury include hyperactive neurons, excessive glutamate release, inflammation, blood-brain barrier (blood-brain barrier), BBB) increased permeability and brain edema. The change of gene expression after head injury and a series of nerve function damage have a great effect on the prognosis of the patient, bringing the double pressure of the body and the economy. At present, there is no definite drug and treatment method to deal with the secondary injury after brain injury. The research focuses on the protection of nerve cells after brain injury and the damage of the inflammatory response to the brain tissue. Valproic acid (VPA), a histone deethanizing enzyme inhibitor, has been used in clinical and its neuroprotective effects are various. In this study, the model of brain injury of closed rats was established by using the rat brain injury killer, and the intervention of valproic acid was given, and the anti-inflammatory mechanism of VPA was discussed by the behavior evaluation of rats at different time points and the changes of the expression of the magnetic resonance imaging, the histone H3 and NF-EMAB. Provide reliable basis for clinical application of VPA in the treatment of TBI. Objective To study the effect of VPA on the expression of histone H3 and NF-B protein in the brain tissue of TBI rats, and to study the effect of valproic acid on the inflammatory response of rats with experimental brain injury. Methods 75 male SD rats were divided into three groups according to the random principle: sham operation group (group C), injury group (TBI group) and valproic acid treatment group (VPA group). The closed injury model of rats was established in the TBI and VPA groups. The VPA was injected intraperitoneally at 300 mg/ kg every day after the treatment of the VPA group, and the same time as that of the sham-operated group and the sham-operation group, the volume of saline was injected in the same time. The behavior of rats was evaluated at all time points after injury. At 6 h,24 h,48 h and 72 h after injury,5 rats were sacrificed, and the activation state of the nuclear factor B (NF-B) pathway was observed under the laser confocal microscope, and the content of the histone H3 was detected by immunohistochemistry. At 72 hours after injury, a rat was injected with water-chloral hydrate in each group, and the magnetic resonance imaging was performed in the national nano-center of Beijing after the anesthesia. Results 1. Results: After the VPA intervention, the degree of injury of the rats in the VPA group and the TBI group was reduced, the effect was not obvious in 24 hours after the injury, the difference between the groups was not significant (P0.05), and the rats in the treatment group of the VPA group were treated with the VPA intervention for 7 days,14 days, and the VPA treatment group. The sensory and reflective function was superior to that of the TBI group, and the difference between the two groups was statistically significant (P0.05). The degree of edema was the most, and the color of the lesion in the treatment group of the VPA and the simple injury group was close to the blue, indicating that the edema of the brain tissue of the rats after the treatment with VPA was reduced. It can also be seen from the figure that the volume of brain tissue in the damage range of the VPA treatment group is significantly reduced than that of the same time point, indicating that the VPA can reduce the volume of the brain injury. The results showed that there was no significant difference in the level of histone H3 in group 3 rats (P0.05), and there was no significant difference in the group of VPA and Group C, and the group of TBI was lower than that of group C (P0.05). The results showed that the expression of NF-B-B-positive cells in the brain tissue of the C-group was little or not even expressed, compared with that of the C group. The number of NF-and B-positive cells in the brain tissue of the rats at 6 h TBI and VPA was higher than that of the C group, and the number of NF-and B-positive cells in the brain tissue of the rats in 24 h was significantly higher than that of the C group, and the expression of NF-B-B-positive cells in the brain tissue of group C,48 h and 72 h was gradually decreased. However, the expression of NF-B-B-positive cells in the treatment group of VPA was lower than that of the TBI group at the same time point (P0.05). Conclusion The anti-inflammatory effect of valproic acid on rat brain injury may be achieved by inhibiting the activation of NF-B.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15
【参考文献】
相关期刊论文 前1条
1 张亚峰;王骏飞;蒋青;;NF-κB信号转导途径与炎症性疾病[J];国际免疫学杂志;2007年05期
，本文编号：2436330
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