| BackgroundIschemic stroke,which accounts for approximately 87%of all stroke cases,is one of the most common cerebrovascular diseases.Ischemic stroke is one of the leading causes of neuronal death,neuroinflammatory response,and destruction of neurovascular units and can eventually lead to severe neurological disorders.Intravenous thrombolysis is currently the most effective treatment to restore cerebral blood flow.However,a narrow therapeutic window limits its clinical application,and reperfusion of blood flow to the ischemic brain tissue can further aggravate brain damage after cerebral ischemia,which is regarded as cerebral ischemia-reperfusion(I/R)injury.Numerous clinical studies and experiments have demonstrated that I/R injury triggers a vigorous immune and inflammatory response.And vigorous inflammatory response can aggravate the pathological damage of ischemic brain tissue.Importantly,adversely affect survival and patient long-term recovery.Therefore,blocking the inflammatory cascade following reperfusion is an ideal strategy to improve cerebral injury following ischemia-reperfusion events.Traditional Chinese medicine(TCM)has become popular because it has a definitive curative effect and few side effects.Many clinical studies have reported that TCM effectively improves the cerebral blood flow of patients,alleviate clinical symptoms,and reduces mortality.Dengzhan Shengmai capsule(DZSM)is a representative Chinese herbal compound of Yiqi Huoxue prescription.DZSM capsule is composed of Erigeron breviscapus,ginseng,ophiopogon,and schisandra.And it is widely used in the treatment of stroke,vascular dementia,cerebral infarction,and other cardiovascular and cerebrovascular diseases.Modern pharmacological investigations have shown that the DZSM can effectively reduce neuroinflammation after cerebral I/R injury.However,the mechanism of DZSM on neuroinflammation has not been elucidated.In this study,we first investigated the neuroprotective effect of DZSM against cerebral I/R injury.To investigate the characteristics of drug administration,RNAsequencing(RNA-seq)analysis was performed.Microglia are the main effector cells of neuroinflammation.To investigate the vital targets and pathways of the DZSM against ischemic stroke in microglia,single-cell RNA-sequencing(sc RNA-seq)was performed.And fully elucidating the mechanism of DZSM on neuroinflammation after cerebral I/R injury.These findings will provide more basic evidence for the clinical application of the DZSM against ischemic stroke.MethodsChapter One:Protective effect and RNA-seq analysis of DZSM against cerebral I/R injury.1.The protective effect of DZSM in middle cerebral artery occlusion(MCAO)rats.The rats were randomly divided into the sham,I/R,I/R+DZSM-L(0.1134 g/kg),I/R+DZSM-H(0.4536 g/kg),and I/R+Ginaton(20 mg/kg)groups.All the rats were dosed orally once per day for 5 days.On the fifth day after administration,the MCAO model was performed followed by 1.5 h ischemia and 24 h reperfusion.The protective effect of DZSM was assessed by infraction rate,neurological deficit scores,measurement of cerebral blood flow,hematoxylin and eosin(H&E)staining,and Nissl staining.2.To explore the characteristics of drug administration,RNA-seq analysis was performed.Brain tissue samples from the Sham group,I/R group,and I/R+DZSM-H group were used to perform RNA-seq.Differential expression analysis,cluster analysis,and enrichment analysis were performed.Chapter Two:Sc RNA-seq revealing vital targets of DZSM against ischemic stroke.1.Analysis of single-cell atlases of the rat brain of DZSM against ischemic stroke by scRNA-seq.Brain nucleus from the Sham group,I/R group,and I/R+DZSM-H group was extracted and sc RNA-seq was performed.Cell annotation,cell-type proportion analysis,analysis of differences between groups,and enrichment analysis were performed.2.The vital target of DZSM against ischemic stroke in microglia was revealed by sc RNA-seq.To explore vital targets,bioinformatics analysis of microglia based on the sc RNA-seq data was performed,such as GSEA analysis,differential expression analysis,cluster analysis,enrichment analysis,and Venn analysis.Immunofluorescence(IF)staining was used to detect the expression and localization of vital target.Chapter Three:The effect of MALT1 in cerebral I/R injury.1.The changes of MALT1 were observed in the MCAO rat model and oxygenglucose deprivation/reoxygenation(OGD/R)brain organoids model.Western blot was used to detect the levels of MALT1 in rat brain tissue(ischemia for 1.5 h and reperfusion for 3 h,6 h,12 h,and 24 h).ELISA kits were used to detect the expression of cytokines(TNF-α,IL-6,IL-1β,IL-10,and ARG1)in rat brain tissue after cerebral I/R injury.IF staining was used to detect the levels of MALT1 in brain organoids(oxygen sugar deprivation 8 h,12 h,and reoxygenation 24 h).2.The protective effect of MI-2 in MCAO rats.The rats were randomly divided into the sham,I/R,and I/R+MI-2(10 mg/kg)groups.All the rats were administered once a day by intraperitoneal injection for 5 days.On the fifth day after administration,the MCAO model was performed followed by 1.5 h ischemia and 24 h reperfusion.The protective effect was assessed by infraction rate,neurological deficit scores,measurement of cerebral blood flow,H&E staining,and Nissl staining.ELISA kits were used to detect the effect of MI-2 on the expression of cytokines(TNF-α,IL-6,IL-1 β,IL-10,and ARG1)in rat brain tissue.3.The protective effect of si-MALT1 in MCAO rats.The rats were randomly divided into si-NC(1μg/μL)and si-MALT1(1 μg/μL)groups.The siRNA was injected intracerebroventricularly on Day 1 and Day 3,and on the fifth day,the MCAO model was performed followed by 1.5 h ischemia and 24 h reperfusion.The protective effect was assessed by infraction rate,neurological deficit scores,H&E staining,and Nissl staining.ELISA kits were used to detect the effect of si-MALT1 on the expression of cytokines(TNF-α,IL-6,IL-1β,IL-10,and ARG1)in rat brain tissue.4.The effect of MI-2 on inflammation was evaluated in LPS-induced BV2 microglial cells.The cells were divided into the Control group,LPS group,and I/R+MI-2(0.01 μM、1 μM、2 μM)groups.IF staining was used to detect the level of MALT1 in BV2 microglial cells.ELISA kits were used to detect the effect of MI-2 on the expression of cytokines(TNF-α,IL-6,IL-1β,IL-10,and ARG1)in BV2 microglial cells.5.To explore the mechanism of MALT1 in cerebral I/R injury,RNA-seq analysis was performed.Brain tissue samples from the si-NC group and the si-MALT1 group were used to perform RNA-seq.To identify the key pathway,differential expression analysis,cluster analysis,enrichment analysis,and MCODE analysis were performed.Western blot was used to detect the expression of key proteins of the pathway.Chapter Four:The effect of DZSM and its active components on the expression of MALT1 and neuroinflammation.1.The effect of DZSM on the expression of MALT1 and the neuroinflammation in MCAO rats.The rats were randomly divided into five groups:the Sham,I/R,I/R+DZSM-L(0.1134 g/kg),I/R+DZSM-H(0.4536 g/kg),and I/R+Ginaton(20 mg/kg).On the fifth day after administration,the MCAO model was performed followed by 1.5 h ischemia and 24 h reperfusion.Western blot was used to detect the effect of DZSM on the expression of MALT1 in MCAO rat brain tissue.ELISA kits were used to detect the effect of DZSM on the expression of cytokines(TNF-α,IL-6,IL-1 β)in MCAO rat serum and the expression of cytokines(IL-6、IL-1β、TNF-α、ICAM-1、MMP9、IL-10、ARG1)in MCAO brain tissue.IF staining was used to detect the effect of DZSM on levels of MMP9,ICAM-1,and IBA-1 in MCAO rat brain tissue.2.The effect of DZSM on the expression of MALT1 and inflammation was evaluated in LPS-induced BV2 microglial cells.The cells were divided into the Control group,LPS group,and DZSM administration(15.625 μg/mL,31.25 μg/mL,62.5 μg/mL)groups.IF staining was used to detect the effect of DZSM on the level of MALT1 in BV2 microglial cells.ELISA kits were used to detect the effect of DZSM on the expression of cytokines(TNF-α,IL-6,IL-1 β,IL-10,and ARG1).3.The effect of scutellarin and chlorogenic acid on the expression of MALT1 and inflammation was evaluated in LPS-induced BV2 microglial cells.The cells were divided into the Control group,LPS group,and scutellarin and chlorogenic acid administration(0.1 μM,1 μM,2 μM)groups.IF staining was used to detect the effect of scutellarin and chlorogenic acid on the level of MALT1 in BV2 cells.ELISA kits were used to detect the effect of DZSM on the expression of cytokines(TNF-α,IL-6,IL-1β,IL-10,and ARGl).ResultsChapter One:Protective effect and RNA-seq analysis of DZSM against cerebral I/R injury.1.DZSM significantly alleviated cerebral I/R injury in MCAO rats.Administration of DZSM significantly reduced the infarction rate,and neurological deficit scores and ameliorated the reduction of rCBF.And alleviated the neuronal damage,such as increased neuronal density level and Nissl bodies density level in the cortex and CA1 and CA3 regions of the MCAO rat hippocampus.2.The inflammatory response was identified as the key biological process of DZSM against cerebral I/R injury.A total of 1483(Differental expressed genes)DEGs were in the I/R group and a total of 573 DEGs were in the I/R+DZSM group.In addition,hierarchical clustering of RNA-seq demonstrated that the I/R+DZSM group and the sham group had similar gene expression profiles.The enrichment analysis of DEGs in the I/R+DZSM group was significantly enriched in biological processes and signaling pathways related to inflammation,such as immune response,inflammatory response,TNF signaling pathway,NF-kappa B signaling pathway,etc.Chapter Two:Sc RNA-seq revealing vital targets of DZSM against ischemic stroke.1.Construction of single-cell atlases of the rat brain of DZSM against ischemic stroke by scRNA-seq.Dimensionality reduction followed by clustering analysis of the longitudinal scRNAseq data identified 23 distinct clusters.And 8 cell types were identified throughmarker genes,mainly including astrocyte,endothelial cell,ependymocyte,neuron,microglia,neuroendocrine cell,oligodendrocyte,oligodendrocyte precursor.The results of cell-type proportion analysis and analysis of differences between groups showed that microglia changed significantly,and the proportion of microglia increased after MCAO injury but decreased after DZSM treatment.Differential gene expression analysis showed that 546 genes were up-regulated and 342 genes were down-regulated in microglia after cerebral I/R injury,442 genes were up-regulated and 384 genes were downregulated after DZSM treatment.The top 50 DEGs of microglia were significantly enriched in immune response and defense response.2.MALT1 was identified as a potential target of microglia in the protection of DZSM against cerebral I/R injury.The results of the GSEA enrichment analysis of the I/R group showed that the proinflammatory-related NF-κB signaling pathway was upregulated in the I/R group.Compared with the I/R group,a total of 888 DEGs in the I/R group,and 826 differential genes in the I/R+DZSM group.Enrichment analysis of DEGs of microglia in the I/R+DZSM group significantly enriched in the inflammatory response and immune response.Venn analysis showed that there are 201 DEGs in common between the up-regulated I/R vs down-IR+DZSM group.The top 50 down-regulated DEGs of the I/R+DZSM group were shown in the heat map.And MALT1 was identified as a potential vital target in microglia of the DZSM against cerebral I/R injury.Sc RNA-seq results showed that the expression of MALT1 in microglia was significantly increased in the I/R group,and the expression of MALT1 in microglia was significantly decreased after DZSM treatment.However,MALT1 did not have statistically significant expression differences between other cell types.IF staining results showed that the expression of MALT1 in microglia was significantly increased in the I/R group,and the expression of MALT1 in the I/R+DZSM group was significantly decreased in microglia,but there was no statistical significance in neurons and astrocytes.Chapter Three:The effect of MALT1 in cerebral I/R injury.1-MALT1 showd a trend of sustained high expression over time in MCAO rats and OGD/R injured brain organoids.Western blot results showed that the expression of MALT1 was significantly increased at ischemia 1.5 h followed by 3 h,6 h,12 h,and 24 h reperfusion,and MALT1 showed a sustained high expression trend with the prolongation of reperfusion time.In addition,ELISA results showed that the expression of IL-6,TNF-α,and IL-1β in the rat brain tissue was significantly increased,and the expression of IL-10 and ARG1 were significantly decreased at 1.5 h followed by 24 h reperfusion.IF staining results showed that the expression of MALT1 was significantly increased at 8 h and 12 h after oxygen-glucose deprivation,followed by 24 h reoxygenation.And the expression of MALT1 was significantly increased with the prolongation of oxygen-glucose deprivation.2.MI-2 significantly alleviated cerebral I/R injury in MCAO rats.MI-2 significantly reduced the infarction rate and neurological deficit scores and ameliorated the reduction in rCBF.And alleviated the neuronal damage,such as increased neuronal density level in the cortex and CA1 and CA3 regions of the rat hippocampus and Nissl bodies density level in the cortex and CA1 region of the MCAO rat hippocampus.In addition,ELISA results showed that MI-2 significantly reduced the expression of IL-6,TNF-α,and IL-1β in rat brain tissue,and significantly increased the expression of IL-10 and ARG1.Western blot results showed that MI-2 significantly decreased the expression of MALT1 and CYLD-Ct,and significantly increased the expression of CYLD in MCAO rat brain tissue.It is further proved that MI-2 plays a protective role in cerebral I/R injury by changing the expression of protease and protease activity of MALT 1.3.MI-2 significantly decreased the expression of MALT1 and inhibited inflammation response in LPS-induced BV2 microglial cells.IF staining results showed that MI-2 significantly reduced the expression of MALT1 in LPS-induced BV2 microglial cells.In addition,ELISA results showed that MI-2 significantly decreased the expression of NO,IL-6,TNF-α,and IL-1β but significantly increased the expression of IL-10 and ARG1 in LPS-induced BV2 microglial cells,suggesting a protective effect of MI-2 on inflammation.4.si-MALT1 significantly reduced cerebral I/R injury in MCAO rats.Western blot results showed that the expression of MALT1 and CYLD-ct were significantly reduced,and the expression of CYLD was significantly increased in MCAO rats.And si-MALT1 significantly reduced the infarction rate and neurological deficit scores and alleviated the neuronal damage,such as increased neuronal density level and Nissl bodies density level in the cortex and CA1 and CA3 regions of the rat hippocampus.In addition,ELISA results showed that si-MALT1 significantly decreased the expression of IL-6,TNF-α,and IL-1β in rat brain tissue,but significantly increased the expression of IL-10 and ARG1.5.si-MALT1 inhibited the NF-κB signaling pathway in MCAO rats,RNA-seq results showed that there were 1033 DEGs in the si-MALT1 group.Enrichment analysis of DEGs in the si-MALT1 group significantly enriched in immune response inflammatory response,NF-kappa B signaling pathway,and TNF signaling pathway.MCODE analysis results showed that a total of 24 modules were identified,and the top-ranked module was identified as the main candidate module.Enrichment analysis results showed that the up-regulated DEGs were significantly enriched in biological processes such as the regulation of cytokine activity,and the down-regulated DEGs were significantly enriched in immune response and inflammatory response.KEGG enrichment analysis showed that DEGs were significantly enriched in the HIF-1 signaling pathway,NF-kappa B signaling pathway,and TNF signaling pathway.Combined with the GSEA analysis of the sc RNA-seq,the NF-κB signaling pathway was upregulated after MCAO surgery,and the data of RNA-seq confirmed the importance of the pathway once again.Western blot results showed that the expression of RELB and IKB-α was significantly increased,and the expression of pp65/p65 was significantly decreased in the si-MALT1 group in MCAO rat brain tissue.Chapter Four:The effect of DZSM and its active components on the expression of MALT1 and neuroinflammation.1.DZSM significantly reduced the expression of MALT1 and inhibited neuroinflammation in MCAO rats.Western blot and IF staining results showed that the expression of MALT1 in MCAO rats was significantly reduced in the I/R+DZSM group compared with the I/R group.At the same time,ELISA results showed that DZSM significantly decreased the expression of TNF-α,IL-1β,and IL-6 in serum and TNF-α,IL-1β,ICAM-1,and MMP9 in the brain,but significantly increased the expression of IL-10 and ARG1.2.DZSM significantly decreased the expression of MALT1 and inhibits inflammation response in LPS-induced BV2 microglial cells.IF staining results showed that the expression of MALT1 was significantly reduced in the I/R+DZSM group.In addition,ELISA results showed that DZSM significantly decreased the expression of NO,IL-6,TNF-α,and IL-1β but significantly increased the expression of IL-10 and ARG1,suggesting a protective effect of DZSM on inflammation.3.Scutellarin and chlorogenic acid significantly decreased the expression of MALT1 and inhibited inflammation response in LPS-induced BV2 microglial cells.IF staining results showed that scutellarin and chlorogenic acid significantly reduced the expression of MALT1 in LPS-induced BV2 microglial cells.In addition,ELISA results showed that scutellarin and chlorogenic acid significantly reduced the expression of NO,IL-6,TNF-α,and IL-1β expression but significantly increased the expression of IL-10 and ARG1,suggesting a protective effect for scutellarin and chlorogenic acid on the inflammatory response.Conclusion1.DZSM has a protective effect against cerebral I/R injury in MCAO rats,and inflammatory response is one of the key biological process of DZSM against cerebral I/R injury.2.MALT1 may be a vital target that regulates the activation of microglia to mediate neuroinflammation.In addition,MI-2 and si-MALT1 can inhibit cerebral I/R injury and neuroinflammation,and inhibit the NF-κB signaling pathway.3.DZSM can reduce neuroinflammation after cerebral I/R injury by inhibiting the expression of MALT1,and scutellarin and chlorogenic acid may be the active ingredients of DZSM in cerebral I/R injury treatment.Innovation1.Based on sc RNA-seq,we found that MALT1 is a potential target of microglia against cerebral I/R injury.The role and potential mechanism of MALT1 in cerebral I/R injury were verified.Our findings provide a new potential therapeutic target and strategy for the prevention and treatment of ischemic stroke2.To investigate the mechanism of the protective effect of DZSM on cerebral I/R injury,our study based on the evaluation of drug efficacy,combined with histopathological changes,deeply studied the changes of effector cells and screened the signaling pathways regulated by vital targets.We found that DZSM may serve a protective role in cerebral I/R injury and reduce the neuroinflammation by inhibiting the expression of MALT1,which provides more basic evidence for the clinical application of the DZSM against ischemic stroke. |
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