亚铁离子对星形胶质细胞的损伤作用及香芹酚在实验性脑出血中的神经保护作用

发布时间：2018-11-12 13:07

【摘要】：相关研究显示,亚铁离子对神经上皮细胞具有毒性损伤作用。但亚铁离子是否可以通过改变星形胶质细胞内水通道蛋白4(AQP4)的表达而对后者造成毒性损伤作用,目前尚不清楚。亦有文献报道香芹酚在中枢神经系统疾病中具有重要的神经保护作用。但到目前为止,对于香芹酚在出血性脑卒中中是否同样具有神经保护的作用,尚缺乏相关研究。本研究通过亚铁离子诱导的原代培养星形胶质细胞损伤模型以及胶原酶诱导的小鼠脑出血模型,探讨亚铁离子的毒性损伤机制以及香芹酚的神经保护作用。我们利用亚铁离子诱导的原代培养星形胶质细胞损伤模型,行Cell Counting Kit8细胞活力检测,验证亚铁离子对星形胶质细胞的损伤作用；行real time RT-PCR和Western Blot检测亚铁离子作用后AQP4表达的变化；行Western Blot检测亚铁离子对丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)通路中信号蛋白表达的影响；在阻断亚铁离子的氧化应激反应后,检测亚铁离子对AQP4表达的影响。我们利用亚铁离子诱导的星形胶质细胞损伤模型,验证香芹酚对星形胶质细胞的保护作用。我们利用胶原酶诱导的小鼠脑出血模型,对行为学指标、脑水肿程度、血脑屏障(BBB)破坏程度、AQP4 mRNA表达程度以及AQP4蛋白表达程度进行测定和评估,验证香芹酚的神经保护作用。研究结果显示,在体外培养的原代星形胶质细胞中,给予亚铁离子刺激后,星形胶质细胞的死亡随着亚铁离子浓度的增高而增多、随着亚铁离子作用时间的延长而增多；同时,星形胶质细胞中AQP4基因及蛋白的表达量均增高；磷酸化p38、磷酸化ERK以及磷酸化JNK的表达量亦有上调；给予抗氧化剂阻断亚铁离子引起的氧化应激反应后,星形胶质细胞中AQP4蛋白的表达量降低。在体外培养的原代星形胶质细胞中,同时给予亚铁离子及香芹酚作用后,发现星形胶质细胞的死亡有减少趋势；同时,星形胶质细胞中AQP4蛋白的表达量减少。在胶原酶注射诱导小鼠脑出血模型中,给予香芹酚治疗后,小鼠的神经功能得到改善；其损伤侧脑组织的含水量及BBB功能障碍明显减轻；同时,损伤侧脑组织的AQP4基因及蛋白表达量均下调。综上所述,亚铁离子对原代培养的星形胶质细胞具有明显的损伤作用；其损伤作用可能是通过氧化应激反应激活MAPK通路导致星形胶质细胞AQP4的表达上调而引起。香芹酚对亚铁离子损伤后的星形胶质细胞有一定的保护作用；其作用机制可能是通过抑制AQP4的过量表达。香芹酚对小鼠脑出血后的神经功能有一定的保护作用；其机制可能是通过抑制AQP4的过量表达、减轻血管源性脑水肿而产生保护作用。
[Abstract]:Related studies have shown that ferrous ions have toxic effects on neuroepithelial cells. However, it is not clear whether ferrous ions can cause toxic damage to astrocytes by changing the expression of aquaporin 4 (AQP4) in astrocytes. It has also been reported that carvonol plays an important neuroprotective role in central nervous system diseases. So far, however, there has been little research on whether carvonol has the same neuroprotective effect in hemorrhagic stroke. In this study, the injury model of primary cultured astrocytes induced by ferrous ions and the model of intracerebral hemorrhage induced by collagenase were used to investigate the mechanism of the toxicity of ferrous ions and the neuroprotective effect of carvingol. We used the primary cultured astrocyte injury model induced by ferrous ions to detect the viability of Cell Counting Kit8 cells to verify the damage effect of ferrous ions on astrocytes. Real time RT-PCR and Western Blot were used to detect the changes of AQP4 expression after the action of ferrous ions, and Western Blot was used to detect the effect of ferrous ions on the expression of signal proteins in the mitogen-activated protein kinase (mitogen-activated protein kinases, MAPKs) pathway. After blocking the oxidative stress reaction of ferrous ions, the effect of ferrous ions on the expression of AQP4 was detected. We used the model of astrocyte injury induced by ferrous ions to verify the protective effect of carvingol on astrocytes. Using collagenase-induced cerebral hemorrhage model in mice, we measured and evaluated the behavioral indexes, brain edema, (BBB) damage of blood-brain barrier, AQP4 mRNA expression and AQP4 protein expression. To verify the neuroprotective effect of carvingol. The results showed that in primary astrocytes cultured in vitro, the death of astrocytes increased with the increase of the concentration of ferrous ions, and increased with the prolongation of the action time of ferrous ions. At the same time, the expression of AQP4 gene and protein were increased in astrocytes, and the expression of phosphorylated p38, phosphorylated ERK and phosphorylated JNK were also up-regulated. The expression of AQP4 protein in astrocytes decreased after antioxidants blocked the oxidative stress induced by ferrous ions. In the primary astrocytes cultured in vitro, the death of astrocytes decreased and the expression of AQP4 protein in astrocytes decreased after the treatment of ferrous ions and carvingol. In the model of intracerebral hemorrhage induced by collagenase injection, the nerve function of the mice was improved after the treatment of carvingol, and the water content and BBB dysfunction of the injured brain tissue were obviously alleviated. At the same time, the expression of AQP4 gene and protein were down-regulated. In conclusion, ferrous ions have obvious damage on primary cultured astrocytes, which may be due to the up-regulation of AQP4 expression in astrocytes through the activation of MAPK pathway by oxidative stress. Carvingol has a protective effect on astrocytes injured by ferrous ions, and its mechanism may be by inhibiting the overexpression of AQP4. Carvonol has a protective effect on the neurological function of mice after intracerebral hemorrhage, and its mechanism may be to reduce the vasogenic brain edema by inhibiting the overexpression of AQP4.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.34

【共引文献】