电刺激迷走神经对急性脑缺血再灌注损伤的影响及机制研究

发布时间：2018-07-01 17:53

本文选题：脑缺血再灌注 + VNS　；参考：《重庆医科大学》2015年博士论文

【摘要】：研究背景与目的目前急性脑卒中已成为中国人致死性疾病的首位原因,及时恢复半暗带脑血流是最直接有效的治疗方式。但早期脑缺血再灌注(ischemia/reperfusion, I/R)后容易诱导产生大量氧自由基、瀑布式炎症反应、坏死和凋亡等级联反应,多种因素间互相作用,造成二次脑损伤。因此,成功发展作用于脑卒中多种分子机制的治疗方式是迫切需要的。1997年美国食品和药品监督局(Food and Drug Administration,FDA)推荐VNS (vagus nerve stimulation, VNS)应用于部分发作性难治性癫痫患者,随后多个国家相继批准。近年来,国外研究发现VNS对急性脑缺血再灌注大鼠具有神经保护作用,但其机制不明确。本研究拟通过VNS对急性脑缺血再灌注后氧化应激、凋亡、炎症反应的影响及可能分子机制进行深入探讨,为其临床应用提供一定理论依据。研究方法第一部分：采用颅内注射antagmiR210抑制内源性miR210表达,线栓法制备右侧大脑中动脉栓塞／再灌注(Middle cerebral artery occlusion/reperfusion, MCAO/R)模型,梗塞30min后给予VNS干预。MCAO手术及电刺激过程监测心率、尾动脉血压、血气以及右侧大脑中动脉供血区脑血流变化。再灌注24h时,荧光定量PCR检测各组miR210表达。采用Bederson 5分制评估再灌注24h时神经体征损伤、测定脑梗死体积；(Tdt-mediated dutp nick end labeling, TUNEL)原位凋亡检测技术检测缺血侧皮层神经元凋亡数量；ELISA检测缺血侧脑组织氧化应激反应水平(Superoxide Dismutase, SOD)、(Maleic Dialdehyde assay, MDA)、(Glutathione, GSH)。Western blot技术检测各组缺血侧皮层p-Akt和caspase-3活性蛋白表达情况,免疫荧光染色测各组缺血皮层caspase-3阳性细胞表达。第二部分：采用颅内注射PPARyRNAi技术抑制PPARy (Peroxisome proliferator -activated receptor y, PPARy)表达,构建右侧大脑中动脉栓塞/再灌注模型,梗塞30min后VNS干预,荧光定量PCR和Western blot技术检测各组缺血侧皮层PPARy基因和蛋白表达；采用Ludmila Belayev 12分制评估神经功能缺失和脑梗死体积;HE染色显示各组缺血侧脑组织的病理变化；免疫荧光双标检测缺血侧皮层小胶质细胞和星形胶质细胞表面a7乙酰胆碱能受体(a7nicotinic acetylcholine receptor, a7nAchR)表达；ELISA检测缺血侧脑组织肿瘤坏死因子a(Tumor necrosis factor α, TNF-α)、白介素-1β(Interleukin 1β, IL-1β)含量水平。研究结果第一部分：(1)实验过程中,VNS对大鼠心率(Heart rate, HR)、血压(Blood pressure, BP)、血气及右侧大脑中动脉供血区脑血流无明显影响(p0.05)；(2)与缺血组(I/R)相比,VNS进一步增加缺血诱导的miR210表达(p0.05),改善脑缺血再灌注急性期神经功能症状缺失和减少脑梗死体积,减少缺血侧皮层神经元凋亡数量(p0.05),沉默miR210后,进一步加重缺血导致的脑组织损伤同时也减少VNS神经保护作用(p0.05),提示miR210参与VNS的神经保护效应。(3)与假手术组(sham I/R)相比,缺血后导致大鼠缺血侧皮层SOD活性下降、GSH含量减少和MAD含量增加；与缺血组(I/R)相比,VNS明显抑制缺血侧脑组织氧化应激水平(p0.05)；沉默miR210后,进一步加重再灌注诱导的氧化应激反应(p0.05),同时削弱VNS抗氧化应激效应(p0.05)。(4)与假手术组(sham I/R)相比,缺血再灌注后缺血侧脑组织p-Akt含量减少(p0.05),VNS干预后逆转缺血抑制p-Akt表达的效应(p0.05),并且p-Akt蛋白变化与antagomiR210干扰无明显关联性(p0.05)。(5)与假手术组(sham I/R)相比,缺血组(I/R)缺血侧皮层caspase 3活性蛋白表达明显增加(p0.05)；沉默miR210后,进一步增加缺血侧脑组织caspase3活性(p0.05),而VNS后可抑制缺血侧皮层caspase 3活性蛋白表达(p0.05)；沉默miR210后,明显减弱VNS抑制凋亡反应效应(p0.05),各组免疫荧光caspase 3活性细胞变化趋势同蛋白表达基本一致,提示miR210参与VNS对氧化应激和凋亡的调控过程。第二部分：(1)荧光定量PCR和Western blot法检测颅内注射PPARyRNAi干预后各组PPARy表达示：与缺血+阴性病毒对照组(I/R+ LV-control)相比,缺血+电刺激+阴性病毒对照组(I/R+VNS+LV-control)中PPARγ基因和蛋白表达明显上调(p0.05)；PPARyRNAi干扰后,缺血+干扰组(I/R+LV-shPPARr)和缺血+电刺激+干扰组(I/R+VNS+LV-shPPARr)两组基因蛋白表达明显下降(p0.05)。(2)再灌注24h时,VNS改善神经功能症状缺失,减少脑梗死体积,减轻脑组织病理损伤,抑制缺血侧皮层炎症因子(TNF-α、IL-1β)水平(p0.05)；PPARyRNAi干扰后,VNS保护效应明显下降,炎症因子水平增加(p0.05),提示PPAR γ参与VNS对炎症反应的抑制过程。(3)免疫荧光显示VNS同时可以激活缺血侧皮层小胶质细胞和星形胶质细胞表面a7nAchR,调控神经免疫细胞形态和功能,发挥抗炎作用。结论(1)VNS改善急性脑缺血再灌注大鼠神经功能症状缺失和减少脑梗死体积。(2)VNS减少急性脑缺血再灌注大鼠缺血半暗带神经元凋亡数量。(3)VNS减少急性脑缺血再灌注大鼠缺血半暗带氧化应激和凋亡反应。(4)电刺激迷走神经减少急性脑缺血再灌注大鼠缺血半暗带炎症反应。(5)miR210表达活动影响氧化应激和凋亡反应,参与了电刺激迷走神经对急性脑缺血再灌注大鼠脑保护作用。PPARy表达活动调节脑缺血再灌注后炎症反应参与了电刺激迷走神经对急性脑缺血再灌注大鼠的脑保护作用。
[Abstract]:Background and objective acute cerebral apoplexy has become the first cause of fatal disease in China. It is the most direct and effective way to restore the cerebral blood flow in the semi dark zone in time. However, early cerebral ischemia reperfusion (ischemia/reperfusion, I/R) easily induces a large number of oxygen free radicals, waterfall inflammation, necrosis and apoptosis. A variety of factors interact with each other to cause two brain damage. Therefore, the successful development of a multiple molecular mechanism for stroke is an urgent need for the.1997 and Drug Administration (FDA) and the recommendation of the VNS (vagus nerve stimulation, VNS) for partial seizures of intractable epilepsy. In recent years, foreign studies have found that VNS has a neuroprotective effect on acute cerebral ischemia reperfusion rats, but its mechanism is not clear. This study intends to explore the effects and possible molecular mechanisms of oxidative stress, apoptosis, inflammatory response and the possible molecular mechanism of acute cerebral ischemia reperfusion in order to provide the clinical application of VNS. Part 1: the first part of the study: using intracranial injection of antagmiR210 to inhibit endogenous miR210 expression, and to prepare the right middle cerebral artery embolism / reperfusion (Middle cerebral artery occlusion/reperfusion, MCAO/R) model by the thread thrombus method. After the infarct 30min, the heart rate and the tail artery were monitored by the VNS dry pre.MCAO operation and the electrical stimulation process. Blood pressure, blood gas and the cerebral blood flow in the right middle cerebral artery supply area. MiR210 expression in each group was detected by fluorescence quantitative PCR at 24h. Bederson 5 fraction was used to evaluate the neurological damage, and the volume of cerebral infarction was measured; (Tdt-mediated dUTP nick end labeling, TUNEL) in situ apoptosis detection technique for the detection of ischemic cortex God The number of apoptotic cells, the level of oxidative stress response (Superoxide Dismutase, SOD), (Maleic Dialdehyde assay, MDA), (Glutathione, GSH).Western blot techniques were detected by ELISA, and the expression of active protein in the ischemic cortex of each group was detected by Glutathione, GSH.Western blot, and the immunofluorescence staining was used to detect the positive cells of ischemic cortex The second part: the second part: using the intracranial injection of PPARyRNAi technique to inhibit the expression of PPARy (Peroxisome proliferator -activated receptor y, PPARy), construct the right middle cerebral artery embolization / reperfusion model, the VNS intervention after the infarct 30min, and the fluorescence quantitative PCR and Western techniques to detect the gene and protein expression of the ischemic side cortex. The Ludmila Belayev 12 score was used to evaluate the loss of nerve function and the volume of cerebral infarction; HE staining showed the pathological changes of the ischemic brain tissue in each group; the expression of A7 acetylcholinergic receptor (a7nicotinic acetylcholine receptor, a7nAchR) on the surface of ischemic lateral cortex and astrocytes was detected by double labeling immunofluorescence; ELISA detection was absent. The level of tumor necrosis factor A (Tumor necrosis factor A, TNF- alpha) and interleukin -1 beta (Interleukin 1 beta, IL-1 beta) in the cerebral tissue of the blood side. Results the first part: (1) in the experimental process, VNS has no obvious effect on the heart rate (Heart rate, HR), blood pressure, blood gas and the blood flow of the right middle cerebral artery in the rat. (2) compared with the ischemic group (I/R), VNS further increased the miR210 expression (P0.05) induced by ischemia, improved the loss of nerve function symptoms and reduced the volume of cerebral infarction in the acute phase of cerebral ischemia reperfusion, reduced the number of neuronal apoptosis in the ischemic lateral cortex (P0.05). After silent miR210, the brain tissue damage caused by ischemia was further aggravated and the VNS God was also reduced. The protective effect (P0.05) showed that miR210 was involved in the neuroprotective effect of VNS. (3) compared with the sham operation group (sham I/R), the ischemic side cortex SOD activity decreased, the GSH content decreased and the MAD content increased. Compared with the ischemic group (I/R), VNS significantly inhibited the level of oxidative stress in the ischemic side brain tissue (P0.05); after silencing miR210, further further Aggravated reperfusion induced oxidative stress response (P0.05) and weakened VNS antioxidant stress effect (P0.05). (4) compared with the sham group (sham I/R), the ischemic side cerebral tissue p-Akt content decreased (P0.05) after ischemia-reperfusion, and VNS intervention reversed the inhibitory effect of ischemia on the inhibitory effect of p-Akt (P0.05), and p-Akt protein changes and antagomiR210 interference were not clear. Significant correlation (P0.05). (5) compared with the sham group (sham I/R), the expression of caspase 3 active protein in the ischemic side cortex of ischemic group (I/R) increased significantly (P0.05). After silence miR210, Caspase3 activity (P0.05) was further increased in ischemic side brain tissue, while VNS could inhibit the expression of caspase 3 active protein (P0.05) in the ischemic side cortex after VNS. Weak VNS inhibits apoptosis response effect (P0.05), and the change trend of immunofluorescent caspase 3 active cells is basically the same as protein expression, suggesting that miR210 participates in the regulatory process of VNS on oxidative stress and apoptosis. The second part: (1) fluorescence quantitative PCR and Western blot method for the detection of PPARy expression in each group after the intervention of intracranial injection PPARyRNAi: and ischemia + Compared with the negative virus control group (I/R+ LV-control), the PPAR gamma gene and protein expression in the ischemic + electrical stimulation + negative virus control group (I/R+VNS+LV-control) increased significantly (P0.05). After PPARyRNAi interference, the expression of gene protein in the two groups of ischemic + interference group (I/R+LV-shPPARr) and ischemia + Electrical stimulation + interference group (I/R+VNS+LV-shPPARr) decreased significantly (P0.05). (2) when reperfusion of 24h, VNS improved the lack of neurological symptoms, reduced the volume of cerebral infarction, reduced the pathological damage of brain tissue, and inhibited the level of inflammatory factors (TNF-, IL-1 beta) in the ischemic lateral cortex (P0.05). After PPARyRNAi interference, the protective effect of VNS was significantly decreased, and the level of inflammatory factors increased (P0.05), suggesting that PPAR gamma participates in the inhibition of the inflammatory reaction of VNS. (3) immunofluorescence showed that VNS could activate the a7nAchR of the microglia and astrocytes on the ischemic side of the ischemic cortex, regulate the morphology and function of the neurocyte and play an anti-inflammatory effect. Conclusion (1) VNS can improve the lack of neurological symptoms and reduce the volume of cerebral infarction in the rats with acute cerebral ischemia reperfusion. (2) VNS reduces the acute cerebral ischemia reperfusion The number of apoptosis in the rat ischemic penumbra neurons. (3) VNS reduces the oxidative stress and apoptosis response in the ischemic penumbra of rats with acute cerebral ischemia reperfusion. (4) electric stimulation of the vagus nerve to reduce the inflammatory response in the ischemic penumbra of rats with acute cerebral ischemia reperfusion. (5) miR210 expression activity affects oxidative stress and apoptosis response, and participates in the electrical stimulation of the vagus. The protective effect of.PPARy on acute cerebral ischemia reperfusion rats regulates the inflammatory response after cerebral ischemia reperfusion and the protective effect of electric stimulation of the vagus nerve on the rats with acute cerebral ischemia reperfusion.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R743.3

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