背景和目的：脑卒中（cerebral stroke）为一种急性脑血管病，是全世界第二大死亡原因，也是造成老年人长期残疾的重要原因 ^[1]。出血性脑卒中是常见的卒中类型，约占整个脑卒中的15%，在所有脑卒中疾病中的死亡率最高。高血压脑出血大概占所有脑出血的70%，同时，高血压也是目前已知诱发脑出血的最大危险因素。中国是世界上脑出血负担最重的国家，脑出血发病率和死亡率约为全世界水平的两倍。然而，目前高血压脑出血的血管病变机制十分不明晰。因此，较为系统地研究高血压脑出血的发病机制，是亟待解决的重要科学问题，也是中国特色的重大需求。单细胞测序可以在单个细胞水平上进行多组学的高通量测序研究，反映单个细胞的基因表达状态以及基因结构等信息，揭示细胞之间的异质性，为深入研究疾病的发生与发展机制，以及其诊疗提供了新的研究方法。本研究通过单细胞测序手段寻找高血压脑出血相关的血细胞中独特的差异基因、细胞类型或亚类型，为高血压脑出血的早期诊断提供依据，为高血压脑出血血管病变防治提供新的线索或靶点。
方法：本研究将8月龄的野生型C57BL/6 雄性小鼠（28-34g）通过皮下埋血管紧张素II（1000 ng/kg/min）的微量渗透泵并同时配合饮水中添加L-NAME（100 mg/kg/天）的形式构建自发性的高血压脑出血模型。在模型给予后每日三次观察小鼠的行为学特征来评估脑出血临床症状^[2]，中风症状出现后对小鼠血液样本进行单细胞转录组测序(single-cell RNA sequencing, scRNA-seq)处理: 分别收取对照组、高血压组、模型三天组和脑出血组的外周血液样本，裂解红细胞，将得到的全部血液白细胞进行单细胞转录组测序和生物信息学分析研究。通过聚类及差异基因分析等，绘制疾病状态下血细胞图谱，找出与高血压脑出血病变相关的血液中特殊的细胞类型，通过进一步的分类和注释，获得这些异质性的细胞中新的细胞亚型C8 cluster，通过IPA分析转录调控因子筛选对关键的亚型细胞C8 cluster具有调控作用的转录因子NRF2，以及与脑血管破裂密切相关的调控通路。通过给小鼠体内注射NRF2激活剂CDDO-EA (2 mg/kg) 探究对C8 cluster的影响，通过H&E染色，MRI检测多种手段观察出血点个数和出血量大小，从而研究其对高血压脑出血的作用。
结果：我们对14个小鼠外周血白细胞样本进行单细胞转录组测序，共捕获12万个白细胞，并注释出六大类型细胞，包括粒细胞，T细胞、B细胞、单核细胞、自然杀伤细胞以及红细胞。我们根据这六种细胞各自存在异质性又分为21个不同的细胞亚群。有部分细胞亚群在不同的疾病进展时期存在数量比例上的差异。与正常小鼠相比，高血压脑出血模型后的小鼠外周血白细胞中，粒细胞显著增多而淋巴细胞大量减少，这可能导致机体免疫功能紊乱，加重出血和死亡风险。高血压脑出血样本中Adam8、 Mmp9、 Adam19、Ctsd等蛋白水解酶基因均较正常组显著升高，提示中性粒细胞增多后可能通过大量分泌蛋白水解酶，分解蛋白质以及胶原，破坏血管壁结构，进而引发了高血压脑出血。我们发现了一群独特的粒细胞亚型C8 cluster，该亚型在高血压脑出血模型建模后持续显著增多，在造模后第3天数量最多。这提示C8的增多可能与高血压脑出血的发生密切相关，而非脑出血后的伴随现象。同时我们发现C8与其他粒细胞相比，具有较为独特的差异表达基因，高表达Gp5、Itga2b等基因。为进一步明确C8在高血压脑出血血管病变中的作用，我们通过IPA分析C8的差异基因获得C8 cluster转录调控因子NRF2。通过在小鼠体内给予NRF2激活剂CDDO-EA干预C8数量探究其对高血压脑出血表型的影响。结果表明，CDDO-EA可以通过降低C8 cluster 的数量降低小鼠高血压脑出血的发病率、减少脑出血体积、脑出血灶数量，保护脑出血的发生发展。
结论： 自发性高血压脑出血模型刺激后，小鼠外周血中粒细胞显著增多同时淋巴细胞大量减少，且高血压脑出血模型组分泌的蛋白水解酶显著增加。中性粒细胞中的Gp5⁺Itga2b⁺细胞亚群，即C8 cluster在高血压脑出血的进展中起到了重要的作用，减少血液中C8 cluster数量可以减少高血压脑出血的发生。
 

~Background and Objective：Stroke is an acute cerebrovascular disease, which is the second leading cause of death and the main cause of long-term disability of the elderly worldwide ^[1]. Hemorrhagic stroke is a common subtype of stroke, accounting for about 15% of all stroke, and its mortality is the highest among all stroke diseases. Hypertensive cerebral hemorrhage accounts for about 70% of the total cerebral hemorrhage. Besides, hypertension is also the most known risk factor for cerebral hemorrhage. The incidence rate and mortality of cerebral hemorrhage are two times that of the global level, which makes the burden of cerebral hemorrhage there become the heaviest all over the world. However, the vascular pathological mechanism of hypertensive intracerebral hemorrhage is still unclear. Therefore, a more systematic study of the pathogenesis of hypertensive intracerebral hemorrhage is an important scientific problem to be solved, and it is also a major demand with Chinese characteristics. Single-cell RNA sequencing can analyze multiomics at the level of single cell, reveal the gene structure and gene expression status and reflect the heterogeneity between cells. It provides a new research idea for further study on disease occurrence, development mechanism, diagnosis and treatment. The purpose of this study is to find out the unique differential genes, cell types or subtypes in the blood cells of hypertensive intracerebral hemorrhage by single-cell RNA sequencing, which can provide evidences for the early diagnosis of hypertensive intracerebral hemorrhage, and provide new targets for the treatment and prevention of hypertensive intracerebral hemorrhage.
Methods: In this study, C57BL/6 male mice aged 8 months were treated with angiotensin II (AngII; 1000 ng/kg per min) via osmotic pumps and treatment with L-NAME (100 mg/kg per day) in drinking water which was started on the same day as the implantation of osmotic pumps. Thus, a spontaneous hypertensive cerebral hemorrhage model was established.  After the model was given, clinical signs of stroke especially behavioral characteristics were assessed by daily neurological examinations three times per day to evaluate the clinical symptoms of cerebral hemorrhage ^[2]. After the appearance of stroke symptoms, the blood samples of mice were collected to perform single-cell RNA-sequencing. Peripheral blood samples of the control group, hypertension group, model three-day group and mice with intracerebral hemorrhage were collected to lyse red blood cells, and all white blood cells obtained were analyzed by single-cell RNA-sequencing and bioinformatics. After clustering and differential gene analysis, we constructed an atlas of the blood cell under the disease state, and found out the special cell types in the blood related to hypertensive intracerebral hemorrhage, next, further classification and annotation were carried out to obtain the new cell subtype in these heterogeneous cells and C8 cluster was determined. We used IPA to analyze the transcriptional regulatory factors and obtained NRF2, the transcriptional regulatory factor affecting the key subtypes C8 cluster, as well as the regulatory pathways closely related to cerebral vascular rupture. We injected NRF2 activator CDDO-EA (2 mg/kg) into mice to explore the effect on C8 cluster. H&E staining and MRI were used to observe the number and size of lesions, and then to study its effect on hypertensive intracerebral hemorrhage, which was significantly different from other granulocytes,
Results: We constructed an atlas of >120,000 single-blood cell transcriptomes from 14 mouse peripheral blood leukocytes and identified six cell types. It mainly includes granulocytes, T cells, B cells, monocytes, natural killer cells and red blood cells. We divided these six cell types into 21 different cell subclusters according to their heterogeneity. There are differences in the number and proportion of some subclusters in different stages of disease progression. Compared with control mice, the number of granulocytes increased significantly and the number of lymphocytes decreased significantly in mice with hypertensive intracerebral hemorrhage, which leads immune dysfunction and increases the risk of hemorrhage and death. The protease of mice with hypertensive intracerebral hemorrhage was highly expressed than that of the control group, such as Adam8、 Mmp9、 Adam19、Ctsd，which indicating that the increase of neutrophils may promote the destruction of vascular by secreting a large number of proteases and then lead to cleaving protein and collagen, destroying the structure of vascular wall, and eventually lead to the occurrence of hypertensive intracerebral hemorrhage. We found a unique group of granulocyte subsets C8 cluster. The number of this subtype increased significantly after the establishment of hypertensive intracerebral hemorrhage model, and reached the maximum on the third day after the establishment of the model. This suggests that the increase of C8 may be closely related to the occurrence of hypertensive intracerebral hemorrhage, rather than the accompanying phenomenon after intracerebral hemorrhage. We found that C8 had significantly different genes from other subclusters, C8 cluster highly expressed Gp5 and Itga2b genes. To further clarify the effect of C8 in hypertensive cerebral hemorrhage vascular lesions, we obtained the transcriptional regulator NRF2 of C8 cluster by IPA analysis. In order to intervene the number of C8 and explore its impact on the phenotype of hypertensive intracerebral hemorrhage, mice were injected with NRF2 activator CDDO-EA intraperitoneally. The results show that C8 cluster was decreased after injection of CDDO-EA and then reduced the incidence of hypertensive cerebral hemorrhage in mice, reduced the volume of cerebral hemorrhage and the number of cerebral hemorrhage focus, and protected the mice from cerebral hemorrhage.
Conclusion: The number of the peripheral blood granulocytes increased significantly, while the number of lymphocytes decreased, and the secretion of the protease was increased significantly after stimulation of spontaneous hypertensive intracerebral hemorrhage model. The Gp5 ⁺Itga2b⁺subcluster caused C8 cluster in neutrophils plays an important role in the incidence of hypertensive intracerebral hemorrhage. Reducing the number of C8 clusters in blood can reduce the incidence of hypertensive cerebral hemorrhage.