Analysis Of The Pathogenesis Of Hypertensive Intracerebral Hemorrhage By ScRNA-seq Of White Blood Cells

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 angiotensinⅡ(AngⅡ;1000 ng/kg per min)via osmotic pumps and treatment with L-NAME(100mg/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.