TPPU对大鼠脑缺血再灌注损伤血脑屏障的保护作用
发布时间：2018-10-31 17:36
【摘要】：目的:用线栓法复制大鼠脑缺血/再灌注(I/R)损伤模型,以细胞色素P450表氧化酶CYP-EETs信号通路为调控靶点,研究可溶性表氧化物水解酶抑制剂(sEHi)TPPU对脑缺血/再灌注损伤所致血脑屏障(BBB)破坏的作用,探索脑缺血治疗新靶点。方法:通过线栓法建立大鼠MCAO脑缺血/再灌注模型;大鼠随机分为5组,sham组、MCAO组、TPPU(1.0mg/kg)组、TPPU(0.3mg/kg)组、TPPU(0.1mg/kg)组;激光多普勒血流仪及激光散斑评估模型成功与否,TTC法检测MCAO梗死体积,伊文氏蓝法检测血脑屏障通透性,采用免疫荧光染色、western blot等方法检测MCAO及TPPU干预后血脑屏障相关蛋白含量的变化,干湿重法评估脑水肿程度,Garcia18分制行为学评分法评估神经功能缺损及恢复情况。结果:线栓法t-MCAO模型成功率高、死亡率低,激光多普勒血流仪能够全程动态反映MCA(middle cerebral artery大脑中动脉)区域脑组织的血流变化,有助于判定MCA是否形成有效梗阻;激光散斑成像能够直观显示感兴趣区血管的血流变化,缺点是损伤大且不能连续不间断的监测;MCAO后大鼠均出现神经功能缺损症状,与sham组比较有显著统计学差异(P0.01);MCAO大鼠血脑屏障通透性增高,梗死侧脑组织可见大量伊文思蓝渗漏,进而继发血管性脑水肿;TTC染色直观反映出梗死面积,与神经功能缺损严重程度相符合;与MCAO组比较,可溶性环氧化物水解酶抑制剂TPPU可以减小脑皮质梗死体积(P0.05),而对纹状体梗死作用不明显(P0.05);TPPU能够减轻MCAO后血脑屏障紧密连接(tight junction,TJ)蛋白的丢失(P0.05),有助于维持血脑屏障完整性,减少伊文思蓝向脑组织渗漏(P0.001),进而减轻血管性脑水肿(P0.05);TPPU能够改善MCAO大鼠脑梗死后神经功能缺损症状,各剂量组与对照组比较均有统计学意义(P0.01);上述获益随TPPU剂量增大而增加,呈剂量依赖性。结论:可溶性环氧化物水解酶抑制剂TPPU能够保护MCAO大鼠血脑屏障完整性,维持其屏障功能,减轻脑水肿,减小脑梗死体积,减轻神经功能缺损症状;上述作用具有剂量依赖性。
[Abstract]:Aim: to establish a rat model of cerebral ischemia / reperfusion (I / R) injury with thread embolization, and to regulate the cytochrome P450 epioxygenase CYP-EETs signaling pathway. To study the effect of soluble surface oxide hydrolase inhibitor (sEHi) TPPU) on the damage of blood-brain barrier (BBB) induced by cerebral ischemia / reperfusion injury, and to explore a new target for the treatment of cerebral ischemia. Methods: MCAO cerebral ischemia / reperfusion model was established by thread occlusion in rats. Rats were randomly divided into 5 groups: sham group, MCAO group (, TPPU (1.0mg/kg) group (, TPPU (0.3mg/kg) group (, TPPU (0.1mg/kg) group; TTC method was used to measure the infarct volume of MCAO, Yi Wen's blue method was used to detect the permeability of blood-brain barrier, and immunofluorescence staining was used. Western blot and other methods were used to detect the changes of blood-brain barrier related proteins after intervention of MCAO and TPPU. Brain edema was evaluated by dry and wet weight method and neurological functional defect and recovery was evaluated by Garcia18 score. Results: the success rate of t-MCAO model was high and the mortality was low. Laser Doppler Flowmeter could dynamically reflect the changes of cerebral blood flow in the middle cerebral artery (MCA) region of MCA (middle cerebral artery. It was helpful to judge whether MCA was an effective obstruction. Laser speckle imaging can visualize the changes of blood flow in the area of interest, but the disadvantages are that the blood flow changes in the region of interest can not be continuously monitored without continuous monitoring. After MCAO, there is a significant difference between the two groups (P0.01). In MCAO rats, the blood-brain barrier permeability was increased, a large number of Evans blue leakage was observed in the infarcted brain tissue, and then vascular cerebral edema was found, and TTC staining reflected the infarct size directly, which was consistent with the severity of nerve function defect. Compared with MCAO group, soluble epoxide hydrolase inhibitor (TPPU) could decrease the volume of cerebral cortex infarction (P0.05), but had no obvious effect on striatum infarction (P0.05). TPPU can reduce the loss of Blood-brain Barrier tight junction (tight junction,TJ) protein after MCAO (P0.05), which can help to maintain the integrity of BBB and reduce the leakage of Evans blue to brain tissue (P0.001). Then the vascular brain edema was alleviated (P0.05). TPPU could improve the symptoms of neurological deficit after cerebral infarction in MCAO rats, which was significantly higher in each dose group than that in the control group (P0.01), and the above benefits increased with the increase of TPPU dose in a dose-dependent manner. Conclusion: soluble epoxide hydrolase inhibitor (TPPU) can protect the integrity of blood-brain barrier, maintain its barrier function, reduce cerebral edema, reduce cerebral infarction volume and alleviate neurological deficit symptoms in MCAO rats, and these effects are dose-dependent.
【学位授予单位】：湖北医药学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3
[Abstract]:Aim: to establish a rat model of cerebral ischemia / reperfusion (I / R) injury with thread embolization, and to regulate the cytochrome P450 epioxygenase CYP-EETs signaling pathway. To study the effect of soluble surface oxide hydrolase inhibitor (sEHi) TPPU) on the damage of blood-brain barrier (BBB) induced by cerebral ischemia / reperfusion injury, and to explore a new target for the treatment of cerebral ischemia. Methods: MCAO cerebral ischemia / reperfusion model was established by thread occlusion in rats. Rats were randomly divided into 5 groups: sham group, MCAO group (, TPPU (1.0mg/kg) group (, TPPU (0.3mg/kg) group (, TPPU (0.1mg/kg) group; TTC method was used to measure the infarct volume of MCAO, Yi Wen's blue method was used to detect the permeability of blood-brain barrier, and immunofluorescence staining was used. Western blot and other methods were used to detect the changes of blood-brain barrier related proteins after intervention of MCAO and TPPU. Brain edema was evaluated by dry and wet weight method and neurological functional defect and recovery was evaluated by Garcia18 score. Results: the success rate of t-MCAO model was high and the mortality was low. Laser Doppler Flowmeter could dynamically reflect the changes of cerebral blood flow in the middle cerebral artery (MCA) region of MCA (middle cerebral artery. It was helpful to judge whether MCA was an effective obstruction. Laser speckle imaging can visualize the changes of blood flow in the area of interest, but the disadvantages are that the blood flow changes in the region of interest can not be continuously monitored without continuous monitoring. After MCAO, there is a significant difference between the two groups (P0.01). In MCAO rats, the blood-brain barrier permeability was increased, a large number of Evans blue leakage was observed in the infarcted brain tissue, and then vascular cerebral edema was found, and TTC staining reflected the infarct size directly, which was consistent with the severity of nerve function defect. Compared with MCAO group, soluble epoxide hydrolase inhibitor (TPPU) could decrease the volume of cerebral cortex infarction (P0.05), but had no obvious effect on striatum infarction (P0.05). TPPU can reduce the loss of Blood-brain Barrier tight junction (tight junction,TJ) protein after MCAO (P0.05), which can help to maintain the integrity of BBB and reduce the leakage of Evans blue to brain tissue (P0.001). Then the vascular brain edema was alleviated (P0.05). TPPU could improve the symptoms of neurological deficit after cerebral infarction in MCAO rats, which was significantly higher in each dose group than that in the control group (P0.01), and the above benefits increased with the increase of TPPU dose in a dose-dependent manner. Conclusion: soluble epoxide hydrolase inhibitor (TPPU) can protect the integrity of blood-brain barrier, maintain its barrier function, reduce cerebral edema, reduce cerebral infarction volume and alleviate neurological deficit symptoms in MCAO rats, and these effects are dose-dependent.
【学位授予单位】：湖北医药学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3
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