【作者】 黄海燕；

【导师】 曾春雨；

【作者基本信息】 重庆大学 ， 生物学， 2024， 硕士

【摘要】 脑缺血再灌注（ischemia reperfusion,I/R）损伤是指脑组织缺血后恢复血液再灌注,反而进一步加重脑组织损伤和功能障碍的现象。脑I/R损伤常继发于缺血性脑卒中患者进行溶栓治疗、血管内支架置入术、心肺脑复苏等术后。脑I/R损伤具有较高的发病率和死亡率,深入探究脑I/R损伤的发病机制与干预措施有助于为临床的诊疗工作提供新的治疗方案。I/R损伤所引起的氧化应激、细胞凋亡等是脑组织损伤的重要病理机制,在上述机制的作用下,进一步发生血脑屏障破坏、脑水肿及脑出血。因此,降低I/R过程中神经细胞的氧化应激水平以及细胞凋亡水平对脑组织损伤具有极其重要的意义。肠-脑信号是一种双向交流途径,可以调节并稳定机体的生理功能,包括食物摄入、免疫调节和睡眠等。其中胃肠激素对于大脑的调节作用已经得到明确证实,而在众多胃肠道激素中,胃泌素的表达量相对较高。胃泌素伴随进餐而分泌,是调节胃酸分泌的节律性激素之一。目前已有研究表明急性脑卒中患者空腹血清胃泌素升高,然而,胃泌素是否在脑I/R损伤中发挥作用尚未可知。因此,本文对胃泌素在脑I/R损伤中的作用进行了研究,并初步探讨其潜在机制。主要研究内容和结果如下:（1）胃泌素对脑I/R损伤的保护作用为明确胃泌素在脑I/R损伤中的作用,采用线栓法构建C57BL/6J小鼠大脑中动脉阻塞再灌注（middle cerebral artery occlusion reperfusion,MCAO/R）模型。利用TTC染色检测梗死面积、神经功能缺损程度评分评价神经功能。实验结果表明,胃泌素治疗能够减少I/R小鼠的脑梗死面积,改善神经功能障碍。通过HE染色和尼氏染色观察脑组织的病理变化,TUNEL染色明确细胞凋亡情况。实验结果表明,胃泌素减轻了I/R小鼠的脑组织病理损伤,降低了脑组织细胞凋亡水平。（2）胃泌素对OGD/R模型中PC-12细胞的保护作用为验证胃泌素对神经细胞的保护作用,研究以大鼠肾上腺嗜铬细胞瘤PC-12细胞为研究对象,体外构建了氧糖剥夺再灌注（oxygen-glucose deprivation reperfusion,OGD/R）的模型。CCK-8检测显示,10^-9M为胃泌素处理PC-12细胞的适宜浓度,并且胃泌素对正常细胞活力无明显影响,但能够显著提高OGD/R处理后降低的细胞活力。使用10^-9M胃泌素处理细胞并检测培养基上清液中LDH的含量,发现胃泌素治疗可显著降低细胞培养基上清液中LDH的含量。通过DHE染色检测细胞中活性氧的情况,试剂盒测定细胞超氧化物歧化酶（superoxide dismutase,SOD）的活性和丙二醛（Malondialdehyde,MDA）的含量,TUNEL染色检测细胞凋亡情况,并测定caspase-3蛋白的活性。结果表明,胃泌素处理可显著降低OGD/R损伤后PC-12细胞中活性氧的水平,同时降低MDA的含量并增加抗氧化酶SOD的活性,降低细胞凋亡水平和促凋亡蛋白caspase-3的活性。（3）胃泌素激活PI3K/AKT/Bad信号通路介导对脑I/R损伤的保护作用上述研究发现,胃泌素可以通过减少细胞凋亡来发挥对脑I/R损伤的保护作用。胃泌素被报道可以激活PI3K的表达,而PI3K/AKT/Bad信号通路在抗凋亡方面具有重要作用。因此,为探究胃泌素是否可以通过PI3K/AKT/Bad信号通路发挥保护作用,本文利用Western blot实验检测了PI3K/AKT/Bad信号通路上相关蛋白的磷酸化表达量,结果表明,胃泌素能够增加该通路上PI3K、AKT和Bad的磷酸化。通过CCK-8检测渥曼青霉素和AKT抑制剂VIII处理后PC-12细胞的活力变化,结果表明,渥曼青霉素和AKT抑制剂VIII均能降低胃泌素对OGD/R模型中细胞的保护作用。提示胃泌素可能通过激活PI3K/AKT/Bad信号通路发挥对脑I/R损伤的保护作用。综上所述,本研究结果表明损伤后给予的胃泌素可能通过PI3K/AKT/Bad信号通路降低脑I/R损伤后的细胞凋亡水平,减少梗死面积,改善神经功能障碍,保护脑I/R损伤。本研究为胃泌素作为一种新型神经保护剂提供了新的线索。更多还原

【Abstract】 Cerebral ischemia reperfusion(I/R)injury refers to the phenomenon of cerebral ischemia after the restoration of blood reperfusion,but further aggravates brain tissue injury and dysfunction.Cerebral I/R injury which has a high morbidity and mortality,is often secondary to patients with ischemic stroke after thrombolytic therapy,endovascular stent implantation,cardiopulmonary resuscitation,etc.The in-depth research on the pathogenesis and intervention of cerebral I/R injury is helpful to provide a new treatment plan for clinical diagnosis and treatment.Oxidative stress and apoptosis induced by I/R injury are important pathological mechanisms of brain tissue injury.Under the action of these mechanisms,blood-brain barrier destruction,cerebral edema and cerebral hemorrhage further occur.Therefore,reducing the level of oxidative stress and apoptosis of nerve cells during I/R is of great significance for brain tissue injury.Gut-brain signaling is a two-way communication pathway that regulates and stabilizes physiological functions,including food intake,immune regulation,and sleep.The regulating effect of gastrointestinal hormones on the brain has been clearly confirmed,and the expression of gastrin is relatively high among many gastrointestinal hormones.Gastrin is secreted with meals and is one of the rhythmical hormones that regulate the secretion of gastric acid.At present,studies have shown that fasting serum gastrin is elevated in patients with acute stroke.However,whether gastrin plays a role in cerebral I/R injury remains unclear.Therefore,the role of gastrin in cerebral I/R injury was studied in this paper,and its potential mechanism was preliminarily discussed.The main research contents and results are as follows:(1)Protective effect of gastrin on cerebral I/R injuryTo study the function of gastrin in cerebral I/R injury,line plug method is used to construct C57BL/6J mice middle cerebral artery occlusion reperfusion(MCAO/R)model.Infarct size and neurological impairment are measured by TTC staining.The results show that gastrin therapy could reduce cerebral infarction size and improve neurological dysfunction in mice cerebral I/R injury.The pathological changes of the brain tissues are observed by HE and Nissl staining.Cell apoptosis is determined by TUNEL staining.The experimental results indicate that gastrin alleviates the pathological injury of brain tissue and inhibits apoptosis in mice cerebra with I/R injury.(2)Protective effect of gastrin on PC-12 cells in OGD/R modelTo verify the effect of gastrin on nerve cells,a model of oxygen-glucose deprivation reperfusion(OGD/R)is established in vitro,taking PC-12 cells of rat adrenal pheochromocytoma as the research object.CCK-8 detection showed that gastrin treated PC-12 cells with an appropriate concentration of 10-9M had no obvious effect on cell viability,but significantly improved the decreased cell viability after OGD/R treatment.LDH content in the supernatant of the cell culture medium,treated with 10-9M gastrin,is detected.It is found that gastrin treatment significantly reduces LDH content in the supernatant.Active oxygen species in cells are measured by DHE staining,superoxide dismutase(SOD)activity and Malondialdehyde(MDA)content are measured by kit,cell apoptosis is measured by TUNEL staining and the activity of Caspase-3 protein is also determined.The results show that gastrin treatment significantly decline the level of reactive oxygen species in PC-12 cells after OGD/R injury,decrease the content of MDA,increase the activity of antioxidant enzyme SOD,reduce the level of apoptosis and lower the activity of proapoptotic protein Caspase-3.(3)The activation of PI3K/AKT/Bad signaling pathway by gastrin mediates the protective effect against cerebral I/R injuryThe above studies suggest that gastrin can play a protective role against cerebral I/R damage by reducing apoptosis.Gastrin has been reported to activate the expression of PI3K,and the PI3K/AKT/Bad signaling pathway plays an important role in anti-apoptosis.Therefore,to explore whether the protective function of gastrin on cerebral I/R injury is through PI3K/AKT/Bad signaling pathway,western blot assay is used to detect the phosphorylated expression levels of related proteins in PI3K/AKT/Bad signaling pathway.The results show that gastrin increase the phosphorylation of PI3K,AKT and Bad.The activity changes of PC-12 cells after treatment with Wortmannin and AKT inhibitor VIII were detected by CCK-8.The results showed that both Wortmannin and AKT inhibitor VIII could reduce the protective effect of gastrin on OGD/R cells.It is suggested that gastrin can play a protective role in cerebral I/R injury by activating PI3K/AKT/Bad signaling pathway.In conclusion,the results of this study suggest that gastrin administered after injury can reduce the level of oxidative stress and apoptosis after cerebral I/R injury through PI3K/AKT/Bad signaling pathway,reduce infarct size,improve neurological dysfunction and protect cerebral I/R injury.This study provides a new clue for gastrin as a novel neuroprotective agent.更多还原