| Background and objectsStroke is currently the second leading cause of death after cardiovascular disease and also the major cause of long-term disability,including hemorrhagic stroke and ischemic stroke,of which ischemic stroke accounts for 70%of strokes.It is mainly caused by occlusion of large cerebral blood vessels leading to the stop of blood supply,causing hypoxia and nutrient deficiency of nerve cells,leading to primary irreversible damage and secondary damage of cells,with high morbidity,high disability rate and high mortality.Many researches have shown that neuroinflammation is an important pathogenic mechanism and pathological process of ischemic stroke,and the interaction of central nervous immunity and peripheral circulating neuroinflammation leads to brain tissue injury after stroke.However,because the current research on neuroinflammation is still unclear,many targeted drugs for neuroinflammation are limited to preclinical studies,especially peripheral nerve immunity after stroke.Therefore,it is particularly important to further understand the development of neuroinflammation after stroke,especially the relationship between peripheral circulatory neuroinflammation and central nerve center,which is the basis of targeted neuroimmunology drugs after stroke.In conclusion,this study focused on the role and mechanism of neural immunity in ischemic stroke,built a mouse model of cerebral ischemia reperfusion injury,and performed single-cell RNA-sequencing of 10×Genomics on peripheral blood of mice at different time points(sham,1 day,7day,14 day)to dynamically analyze the changes of peripheral blood immune cells of mice after ischemic stroke.To identify possible cell subsets and key molecules associated with stroke,and to conduct targeted interventions for these subsets and molecules.In addition,we also conducted single-cell transcriptome sequencing on central immune cells after stroke to explore the heterogeneity of central T cells,so as to better understand the dynamic changes of neuroinflammation after stroke.This study is expected to reveal the dynamic changes of peripheral immune cells and central interaction after stroke,further understand the occurrence and development of neuroinflammation after stroke,and provide a new approach and idea for targeted therapy after stroke.Materials and Methods1.Cerebral ischemia reperfusion injury(I/R)model construction:Mouse models with 1 day,3day,7day and 14day cerebral ischemia reperfusion injury were constructed,neurological function scores and death assessment were performed,then orbital blood was taken and cerebral tissue of ischemic hemisphere of mice with corresponding injury days was collected,and peripheral blood mononuclear cells were extracted or preserved at-80 degrees immediately.2.Peripheral blood mononuclear cells(PBMC)extraction:Different peripheral blood immune cells were isolated according to their different densities in Percoll gradient centrifuge for single-cell sequencing analysis.3.Peripheral blood single cell sequencing:Detecting the dynamic changes of peripheral blood immune cells by single cell sequencing on sham at different time points after cerebral ischemic injury,1 day(acute stage),7 day(subacute stage),and 14day(chronic stage),then cell cluster and molecular clustering function analysis were performed,and qPCR,WB,in situ hybridization and ELISA were used for verification.4.Detection of brain injury indicators:TTC staining was used to detect cerebral infarction size 3 days after cerebral ischemia-reperfusion injury,Longa score,and neurological function scores of mice at different time after ischemia reperfusion(sham,1 day,3day,7day,14 day)were detected by rod-climbing experiment and rod-rotating experiment.The cross maze and mine tests examined anxiety and memory function in normal mice.5.Evaluation of neuroinflammation:Flow cytometry was used to detect the amount of CD45+cells in brain tissue 3 days after cerebral ischemia-reperfusion injury in mice to determine the degree of infiltration of peripheral inflammatory cells.The expression levels of IL-1,IL-6 and TNF in brain tissue and peripheral blood 3 days after cerebral I/R were detected by ELISA kit to assess the degree of inflammatory factor infiltration in peripheral blood and stroke.In addition,TUNEL staining was performed to detect neuronal apoptosis 3 days after cerebral ischemia reperfusion injury.6.Assessment of BBB damage:Green fluorescent protein dye was injected through caudal vein and CD31 immunofluorescence co-staining was used to detect the degree of dye leakage to assess the extent of BBB damage.7.Protein interaction:The interaction between CTSS and JAM protein was detected by CO-IP and WB experiments;To prepare for the next protease cutting experiment.8.Protein cutting experiment in vitro:The enzyme digestion degree of CTSS protein on JAM-A,JAM-B and JAM-C recombinant protein was detected under different PH conditions(PH4.5,PH7.2 and PH9.8),and the cutting bands were presented by silver staining experiment.Results1.Peripheral blood single cell sequencing(RNA-SEQ)analysis suggested the dynamic changes of peripheral blood immune cells at different time points in mice after I/R:tSNE analysis of cerebral ischemia-reperfusion injury(sham,1 day,7 days,14 days)divided peripheral immune cells into 28 cell subsets.The 28 cell subsets were divided into 4 groups according to their expression changes(Group 1,Group 2,Group 3,Group 4),with Group 2 representing a continuous decrease in the whole process.Group 3 represented an increase in the acute stage,but a decrease in the subacute stage and chronic stage.Group 4 represented decreased expression in the acute phase and increased expression in the subacute and chronic phases.Among them,the expression of mononuclear cell related Group 1(2,9,10,18,24,26 subgroups)continued to increase after cerebral ischemia reperfusion injury.2.Peripheral blood single cell sequencing(SCNA-SEQ)revealed a subpopulation of cathepsin S positive monocytes continuously elevated after I/R in mice.Pseudotime analysis suggests that subgroup 2,10 May play a key role in the activation and regulation of mononuclear macrophages.GO analysis suggests that subgroup 2 is mainly involved in toll-like receptor pathway related functions,while subgroup 10 is mainly associated with inflammatory response,protease cleavage,collagen binding and other functions.A key molecule cathepsin S associated with protease cleavage was identified in monocyte subpopulation 10 by Gene functional analysis.3.Central CD3+single-cell transcriptome sequencing revealed the heterogeneity of T cells in chronic post-stroke phase:Central T cells were divided into 15 subgroups by UMAP dimension reduction analysis,each subgroup was named and described by immune cell classical marker,and subgroup 0(CD8-teff-Cc15)and subgroup 2(CD4-Th1-like-lfng)were differentially expressed at 7 and 14 days according to GO analysis and heat map.Subgroup 3(CD8-Trm-Cd69)and 6(gdT-Tcrg-V4).4.Expression of Cathepsin S was significantly elevated after I/R in mice,mainly in mononuclear macrophages/microglia cells:ELISA kit showed significantly elevated expression of cathepsin S in peripheral blood of mice after I/R.WB and qPCR showed elevated expression of cathepsin S in cerebral tissue of ischemic hemisphere after I/R.In situ hybridization,cathepsin S was expressed in monocytes/microglia cells.In addition,cathepsin S was also significant increase in peripheral blood of patients 24 hours after stroke,according to the Gene Expression Omnibus database.5.Knockout cathepsin S and intraperitoneal injection of cathepsin S inhibitors in mice can play a protective effect on cerebral ischemia-reperfusion injury:cathepsin S has been associated with protease cutting and inflammation,and thus cathepsin S was found to reduce cerebral infarct size and neurological function score after I/R in CTSS-KO mice(Longa score,rod climbing experiment,rod rolling experiment).cathepsin S was found to reduce the damage of blood-brain barrier(BBB)by injecting cathepsin S into caudal vein.Reduce the infiltration of peripheral immune cells into the center and the expression of inflammatory factors in brain tissue.intraperitoneal injection of cathepsin S inhibitors can also reduce damage to cerebral infarction size,neural function score and blood-brain barrier after cerebral ischemia-reperfusion injury in mice.6.Cathepsin S has caused injury to the BBB in mice after I/R,mainly due to its enzymatic action on vascular JAM protein:Since functional cluster analysis of cathepsin S has indicated inflammation and protease cleavage,and protein interaction software prediction analysis has found that cathepsin S may have an interaction relationship with JAM proteins.We demonstrated the interaction of cathepsin S with JAM proteins using CO-IP experiments.Therefore,using in vitro enzyme digestion experiments,we found that CTSS can enzymatically cut JAM protein at different PH conditions.ConclusionAfter cerebral ischemia-reperfusion injury,peripheral inflammation and central immunity play an important role together.Peripheral blood single cell sequencing at different time points after cerebral ischemia-reperfusion in mice suggests that peripheral blood immune cells have dynamic changes at different time points,and the subgroup 10 associated with monocyte function continues to increase in the acute and chronic stages.cathepsin S,a key molecule associated with protease cleavage,was highly expressed in mononuclear macrophages/microglia after cerebral ischemia-reperfusion injury.cathepsin S knockdown and intraperitoneal injection of cathepsin S inhibitors have significant protective effects on cerebral ischemia reperfusion injury in mice,mainly due to the inhibiting involvement of cathepsin S in inflammation and enzymatic inhibition of BBB tight protein JAM.In addition,the central CD3+single-cell transcriptome sequencing analysis revealed the heterogeneity of T cells after stroke,the central T cells were divided into 15 subgroups,and the differential expression of cluster 0(CD8-teff-Cc15)、cluster 2(CD4-Th1-like-lfng)、cluster 3(CD8-Trm-Cd69)and 6(gdTTcrg-V4)was found at 7 and 14 days. |
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