【作者】 王飞；

【导师】 薛孟周；

【作者基本信息】 郑州大学 ， 康复医学与理疗学， 2022， 硕士

【摘要】 背景脑出血（Intracerebral Hemorrhage,ICH）具有较高的发病率和死亡率,是一种严重的神经系统疾病,其主要机制是由某种原因（如高血压、动脉瘤等）所致脑血管破裂引起的非外伤性脑实质内血肿形成。据报道,亚洲人ICH的年发病率约为60～80/10万人,急性期死亡率约40%。有关研究表明,给予重组激活因子VIIa可以促进血管损伤部位的止血功能,避免血肿进一步扩大,但尚未开发出有效减少发病后神经炎症的药物。此外,与脑出血后预后不良的相关因素有很多,其中炎症引起的血脑屏障（Blood-brain barrier,BBB）破坏是重要因素之一。1-磷酸鞘氨醇（Sphingosine 1-phosphate,S1P）通过与其受体1-磷酸鞘氨醇受体（Sphingosine 1-phosphate receptor,S1PR）结合,在不同细胞中发挥生物学效应（迁移、增殖、分化等方面）,近年来成为自体免疫系统疾病、神经系统疾病、炎症、肿瘤等多种疾病研究的热点之一。Ozanimod是最近开发的S1PR调节剂,国内外有关研究表明,该药物是S1PR1/5的选择性调节剂,对治疗神经变性相关疾病具有良好的生物学效应。基于这些发现,我们研究了Ozanimod对实验性脑出血小鼠模型的神经保护和血脑屏障的影响。Ozanimod作为新一代的S1PR激动剂。在啮齿类动物中,Ozanimod相关的研究证明该药物有着显著的神经保护作用,尤其是在中枢神经系统当中。该药物对于不同受体类型有着不同的选择性,主要针对1和5型S1PR,其中对1型受体的选择性是5型受体的数十倍,是其他2、3、4型受体的上万倍。这种选择性将3型受体激活相关的潜在安全问题最小化,因为其他S1PR亚型（尤其是3型受体）相关的不良事件有很多,包括高血压、黄斑水肿、肺毒性和肝毒性。此外,有报道称S1PR 1型在脑出血后的神经保护中发挥着重要作用。还有报道指出S1PR 5型具有不同种类神经细胞中优先表达的行为,尤其是少突胶质细胞。该药物在中枢神经系统中调节血脑屏障的内皮细胞（Endothelial cells,ECs）相关的紧密连接,这对维持和保护BBB的完整性至关重要。遗憾的是,尚未在脑出血动物模型中对受体S1PR1/5的同时调控进行研究。在本研究中,我们研究了口服S1PR1/5调节剂Ozanimod对脑出血动物模型的治疗效果。目的在本实验中,通过动物模型来模拟脑出血患者的病理改变,我们使用Ozanimod来探讨其在小鼠脑出血后的神经保护作用,从而为脑出血的临床治疗提供新思路。方法1.将购买于北京维通利华动物公司的126只成年C57BL/6雄性小鼠（6～8周左右）做随机分组,分别为:Sham组、ICH+Vehicle组、ICH+Ozanimod组。2.成年雄性C57BL/6小鼠在右侧纹状体部位注射VII型胶原酶诱导脑出血,Sham组小鼠注射等量的生理盐水。3.脑出血手术前后及脑出血1～3d分别进行神经行为实验,通过行为学评分判断造模成功。4.在术后3d时,对小鼠过量麻醉后取材进行相关的实验。5.实验相关的评价指标:每日记录实验小鼠的体重变化;Iba-1免疫组化染色评估小胶质细胞/巨噬细胞的激活情况;髓过氧化物酶（MPO）免疫组化染色观察中性粒细胞浸润情况;MRI和脑大体切片来评估血肿的大小;干湿重法评估脑水肿;TUNEL染色分析脑细胞死亡;Evans blue（EB）染色评估血脑屏障的完整性;Western blot技术评估紧密连接蛋白（ZO-1、Occludin）的表达水平。结果1.脑出血后神经功能损伤程度测评显示,Ozanimod组的神经功能损伤程度显著低于ICH+Vehicle组（p<0.01）。转角实验结果显示,与ICH+Vehicle组相比,Ozanimod组右转次数显著减少（p<0.001）。在实验过程中,我们测量了小鼠脑出血前后的体重变化。通过统计分析,我们发现药物干预后实验性脑出血小鼠的体重恢复显著（p<0.01）。2.脑出血后第三天通过计算血肿体积,Ozanimod组脑出血血肿体积明显低于ICH+Vehicle组（p<0.05）。因此,该药对实验性脑出血小鼠的血肿体积减少有显著作用。脑出血后3d的MRI评估结果显示,Ozanimod治疗有减轻脑水肿的趋势。使用传统的干湿重法测量脑水肿显示,Ozanimod组脑含水量在ICH后3d显著减少（p<0.01）。3.通过组织学分析,我们发现在Ozanimod组中血肿周围的激活态小胶质细胞（Iba-1）数量明显少于对照组（p<0.05）。而血肿周围的炎性细胞中性粒细胞也明显减少（p<0.001）。实验结果表示Ozanimod可减轻脑出血后的炎症浸润。4.Ozanimod降低脑出血后脑出血诱导的神经元死亡,TUNEL和Neu N双染色,结果显示Ozanimod处理后死亡神经元数量减少（p<0.01）,这与小鼠神经功能恢复的结果一致。5.Ozanimod在ICH后恢复血脑屏障完整性,Ozanimod通过增加受损脑组织中紧密连接蛋白ZO-1和Occludin来维持血脑屏障的完整性。通过免疫荧光染色,我们发现ICH+Ozanimod组ZO-1和Occludin的信号强度明显高于ICH+Vehicle组,这证明了Ozanimod治疗后血脑屏障的完整性明显改善(p_ZO-1<0.05;p_Occludin<0.01)。WB结果显示,ICH+Ozanimod组的ZO-1和Occludin蛋白的表达量明显高于ICH+Vehicle组(p_ZO-1<0.05;p_Occludin<0.05)。由此可见,Ozanimod处理后紧密连接蛋白表达明显改善。通过Evans blue渗透试验,我们发现Ozanimod干预后EB渗出比ICH+Vehicle组明显减少（p<0.001）。结论1.脑出血后,Ozanimod治疗可改善实验性脑出血后小鼠的神经行为缺陷并减轻小鼠的体重丢失,改善小鼠脑出血模型的神经炎症,降低胶原酶诱导脑出血模型的血肿体积,减轻小鼠大脑出血周围水肿,减少神经细胞死亡。2.脑出血后,Ozanimod降低了EB的通透性并稳定小鼠血脑屏障功能起到神经保护作用。更多还原

【Abstract】 BackgroundIntracerebral hemorrhage（ICH）is a serious neurological disease with high morbidity and mortality,and its main mechanism is the formation of non-traumatic intraparenchymal hematoma caused by rupture of intracerebral blood vessels for some reason（such as hypertension,aneurysm,etc.）.According to reports,the annual incidence of intracerebral hemorrhage（ICH）in Asians is about 60～80/100,000 people,and the mortality rate in the acute phase is about 40%.Relevant studies have shown that administration of recombinant activated factor VIIa can promote hemostasis at the site of vascular injury and avoid further expansion of hematoma,but no drug has been developed to effectively reduce post-onset neuroinflammation.In addition,there are many factors associated with poor prognosis after intracerebral hemorrhage,among which the destruction of the blood-brain barrier（BBB）caused by inflammation is one of the important factors.Sphingosine 1-phosphate（S1P）exerts biological effects in different cells by binding to its receptor,Sphingosine1-phosphate receptor（S1PR）,and has become an autologous in recent years.It is one of the hot spots in the research of various diseases such as immune system diseases,nervous system diseases,inflammation and tumors.Ozanimod is a recently developed S1PR modulator.Relevant studies at home and abroad have shown that the drug is a selective modulator of S1PR 1/5 receptors and has a good biological effect on the treatment of neurodegenerative diseases.Based on these findings,we investigated the effects of Ozanimod on neuroprotection and blood-brain barrier in a mouse model of experimental intracerebral hemorrhage.Ozanimod as a new generation of sphingosine-1-phosphate receptor agonists.In rodents,studies related to Ozanimod have demonstrated that the drug has a significant neuroprotective effect,especially in the central nervous system.The drug has different selectivity for different receptor types,mainly targeting 1 and 5 sphingosine1-phosphate receptors,among which the selectivity for type 1 receptor is dozens of times that of type 5 receptor,and the other 2,3,4 receptors tens of thousands of times.This selectivity minimizes potential safety concerns related to type 3 receptor activation,as adverse events associated with other sphingosine 1-phosphate receptor subtypes（especially type 3 receptors）are numerous,including hypertension,macular edema,pulmonary toxicity and hepatotoxicity.In addition,sphingosine-1-phosphate receptor type 1 has been reported to play an important role in neuroprotection after intracerebral hemorrhage.It has also been reported that sphingosine 1-phosphate receptor type 5 has a behavior that is preferentially expressed in different types of neural cells,especially oligodendrocytes.The drug regulates ECs-associated tight junctions of the blood-brain barrier in the central nervous system,which are critical for maintaining and protecting the integrity of the BBB.Unfortunately,the simultaneous regulation of receptors S1PR1/5 has not been studied in animal models of intracerebral hemorrhage.In the present study,we investigated the therapeutic effect of the oral S1PR1/5 modulator Ozanimod in an animal model of intracerebral hemorrhage.ObjectiveIn this project,we used animal models to simulate the pathological changes of clinical human intracerebral hemorrhage,and we used Ozanimod to explore its neuroprotective effect after intracerebral hemorrhage in mice,so as to provide new ideas for the clinical treatment of intracerebral hemorrhage.Materials and Methods1.126 adult C57BL/6 male mice（about 6～8 weeks）purchased from Beijing Vital River Animal Co.,Ltd.were randomly divided into:Sham group,ICH+Vehicle group,and ICH+Ozanimod group.2.Adult male C57BL/6 mice were injected with collagenase type VII in the striatum to induce intracerebral hemorrhage,and mice in the sham-operated group were injected with the same volume of normal saline.3.Neurobehavioral tests were performed before and after intracerebral hemorrhage surgery and from 1 to 3 days,and the success of modeling was judged by behavioral score.4.At the 3rd day after operation,the mice were collected after excessive anesthesia.5.Experiment-related evaluation methods:Iba-1 immunostaining to evaluate the activation of microglia/macrophages;myeloperoxidase（MPO）immunostaining to observe neutrophil infiltration;MRI and gross sections to evaluate The size of the hematoma;the dry-wet method to determine the cerebral edema after intracerebral hemorrhage;use the TUNEL kit provided by Sigma（St.Louis,MO,USA）to analyze the nerve cell death;Evans blue（EB）staining to evaluate the integrity of the blood-brain barrier;The expression levels of tight junction proteins（ZO-1,Occludin）were detected by Western blot;the weight changes of experimental mice were recorded every day.Results1.The evaluation of the degree of neurological damage after intracerebral hemorrhage showed that the degree of neurological damage in the Ozanimod group was significantly lower than that in the ICH+Vehicle group（p<0.01）.The results of the turning angle test showed that compared with the ICH+Vehicle group,the number of right turns in the Ozanimod group was significantly reduced（p<0.001）.During the experiment,we measured changes in body weight in mice before and after intracerebral hemorrhage.Through statistical analysis,we found that the weight recovery of experimental intracerebral hemorrhage mice after drug intervention was significant（p<0.01）.2.By calculating the hematoma volume on the third day after intracerebral hemorrhage,the hematoma volume in the Ozanimod group was significantly lower than that in the ICH+Vehicle group（p<0.05）.Therefore,the drug has a significant effect on the reduction of hematoma volume in mice with experimental intracerebral hemorrhage.MRI assessment results 3 days after intracerebral hemorrhage showed that Ozanimod treatment tended to reduce brain edema.Measurement of brain edema using traditional wet and dry weight method showed that the brain water content in Ozanimod group was significantly decreased 3 days after ICH（p<0.01）.3.Through histological analysis,we found that the number of activated microglia（Iba-1）around the hematoma in the Ozanimod group was significantly less than that in the control group（p<0.05）.The inflammatory cells and neutrophils around the hematoma were also significantly reduced（p<0.001）.The experimental results show that Ozanimod can reduce the inflammatory infiltration after intracerebral hemorrhage.4.Ozanimod reduces neuronal death induced by intracerebral hemorrhage.Double staining of TUNEL and Neu N showed that the number of dead neurons decreased after Ozanimod treatment（p<0.01）,which was consistent with the results of neurological recovery in mice.5.Ozanimod restores BBB integrity after ICH.Ozanimod maintains BBB integrity by increasing the tight junction proteins ZO-1 and Occludin in damaged brain tissue.By immunofluorescence staining,we found that the signal intensities of ZO-1 and Occludin in the ICH+Ozanimod group were significantly higher than those in the ICH+Vehicle group,which proved that the integrity of the blood-brain barrier was significantly improved after Ozanimod treatment(p_ZO-1<0.05;p_Occludin<0.01).WB results showed that the expression levels of ZO-1 and Occludin proteins in the ICH+Ozanimod group were significantly higher than those in the ICH+Vehicle group(p_ZO-1<0.05;p_Occludin<0.05).It can be seen that the expression of tight junction protein was significantly improved after Ozanimod treatment.Through Evans blue penetration test,we found that EB exudation was significantly reduced after Ozanimod intervention compared with ICH+Vehicle group（p<0.001）.Conclusions1.After intracerebral hemorrhage,Ozanimod treatment can ameliorate neurobehavioral deficits and reduce weight loss in mice after experimental intracerebral hemorrhage,improve neuroinflammation in a mouse intracerebral hemorrhage model,and reduce hematoma volume in a collagenase-induced intracerebral hemorrhage model,attenuates edema around intracerebral hemorrhage and reduces neuronal cell death in mice.2.After intracerebral hemorrhage,Ozanimod reduced the permeability of EB and stabilized the blood-brain barrier function in mice to play a neuroprotective role.更多还原