Immune-mediated disruption of the blood-brain barrier after intracerebral hemorrhage: Insights and potential therapeutic targets.

AIMS: Intracerebral hemorrhage (ICH) is a condition that arises due to the rupture of cerebral blood vessels, leading to the flow of blood into the brain tissue. One of the pathological alterations that occurs during an acute ICH is an impairment of the blood-brain barrier (BBB), which leads to severe perihematomal edema and an immune response. DISCUSSION: A complex interplay between the cells of the BBB, for example, pericytes, astrocytes, and brain endothelial cells, with resident and infiltrating immune cells, such as microglia, monocytes, neutrophils, T lymphocytes, and others accounts for both damaging and protective mechanisms at the BBB following ICH. However, the precise immunological influence of BBB disruption has yet to be richly ascertained, especially at various stages of ICH. CONCLUSION: This review summarizes the changes in different cell types and molecular components of the BBB associated with immune-inflammatory responses during ICH. Furthermore, it highlights promising immunoregulatory therapies to protect the integrity of the BBB after ICH. By offering a comprehensive understanding of the mechanisms behind BBB damage linked to cellular and molecular immunoinflammatory responses after ICH, this article aimed to accelerate the identification of potential therapeutic targets and expedite further translational research.

Examining Transcriptomic Alterations in Rat Models of Intracerebral Hemorrhage and Severe Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is a life-threatening condition associated with significant morbidity and mortality. This study investigates transcriptomic alterations in rodent models of ICH and severe ICH to shed light on the genetic pathways involved in hemorrhagic brain injury. We performed principal component analysis, revealing distinct principal component segments of normal rats compared to ICH and severe ICH rats. We employed heatmaps and volcano plots to identify differentially expressed genes and utilized bar plots and KEGG pathway analysis to elucidate the molecular pathways involved. We identified a multitude of differentially expressed genes in both the ICH and severe ICH models. Our results revealed 5679 common genes among the normal, ICH, and severe ICH groups in the upregulated genes group, and 1196 common genes in the downregulated genes, respectively. A volcano plot comparing these groups further highlighted common genes, including PDPN, TIMP1, SERPINE1, TUBB6, and CD44. These findings underscore the complex interplay of genes involved in inflammation, oxidative stress, and neuronal damage. Furthermore, pathway enrichment analysis uncovered key signaling pathways, including the TNF signaling pathway, protein processing in the endoplasmic reticulum, MAPK signaling pathway, and Fc gamma R-mediated phagocytosis, implicated in the pathogenesis of ICH.

Peripheral cytokine interleukin-10 alleviates perihematomal edema after intracerebral hemorrhage via interleukin-10 receptor/JAK1/STAT3 signaling.

AIMS: The extent of perihematomal edema following intracerebral hemorrhage (ICH) significantly impacts patient prognosis, and disruption of the blood-brain barrier (BBB) exacerbates perihematomal edema. However, the role of peripheral IL-10 in mitigating BBB disruption through pathways that link peripheral and central nervous system signals remains poorly understood. METHODS: Recombinant IL-10 was administered to ICH model mice via caudal vein injection, an IL-10-inhibiting adeno-associated virus and an IL-10 receptor knockout plasmid were delivered intraventricularly, and neurobehavioral deficits, perihematomal edema, BBB disruption, and the expression of JAK1 and STAT3 were evaluated. RESULTS: Our study demonstrated that the peripheral cytokine IL-10 mitigated BBB breakdown, perihematomal edema, and neurobehavioral deficits after ICH and that IL-10 deficiency reversed these effects, likely through the IL-10R/JAK1/STAT3 signaling pathway. CONCLUSIONS: Peripheral IL-10 has the potential to reduce BBB damage and perihematomal edema following ICH and improve patient prognosis.

Silencing CXCL16 alleviate neuroinflammation and M1 microglial polarization in mouse brain hemorrhage model and BV2 cell model through PI3K/AKT pathway.

Neuroinflammation and microglia polarization play pivotal roles in brain injury induced by intracerebral hemorrhage (ICH). Despite the well-established involvement of CXC motif chemokine ligand 16 (CXCL16) in regulating inflammatory responses across various diseases, its specific functions in the context of neuroinflammation and microglial polarization following ICH remain elusive. In this study, we investigated the impact of CXCL16 on neuroinflammation and microglia polarization using both mouse and cell models. Our findings revealed elevated CXCL16 expression in mice following ICH and in BV2 cells after lipopolysaccharide (LPS) stimulation. Specific silencing of CXCL16 using siRNA led to a reduction in the expression of neuroinflammatory factors, including IL-1ß and IL-6, as well as decreased expression of the M1 microglia marker iNOS. Simultaneously, it enhanced the expression of anti-inflammatory factors such as IL-10 and the M2 microglia marker Arg-1. These results were consistent across both mouse and cell models. Intriguingly, co-administration of the PI3K-specific agonist 740 Y-P with siRNA in LPS-stimulated cells reversed the effects of siRNA. In conclusion, silencing CXCL16 can positively alleviate neuroinflammation and M1 microglial polarization in BV2 inflammation models and ICH mice. Furthermore, in BV2 cells, this beneficial effect is mediated through the PI3K/Akt pathway. Inhibition of CXCL16 could be a novel approach for treating and diagnosing cerebral hemorrhage.

Altered brain expression and cerebrospinal fluid levels of TIMP4 in cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is a highly prevalent and progressive pathology, involving amyloid-ß (Aß) deposition in the cerebral blood vessel walls. CAA is associated with an increased risk for intracerebral hemorrhages (ICH). Insight into the molecular mechanisms associated with CAA pathology is urgently needed, to develop additional diagnostic tools to allow for reliable and early diagnosis of CAA and to obtain novel leads for the development of targeted therapies. Tissue inhibitor of matrix metalloproteinases 4 (TIMP4) is associated with cardiovascular functioning and disease and has been linked to vascular dementia. Using immunohistochemistry, we studied occipital brain tissue samples of 57 patients with CAA (39 without ICH and 18 with ICH) and 42 controls, and semi-quantitatively assessed expression levels of TIMP4. Patients with CAA had increased vascular expression of TIMP4 compared to controls (p < 0.001), and in these patients, TIMP4 expression correlated with CAA severity (τb = 0.38; p = 0.001). Moreover, TIMP4 expression was higher in CAA-ICH compared to CAA-non-ICH cases (p = 0.024). In a prospective cross-sectional study of 38 patients with CAA and 37 age- and sex-matched controls, we measured TIMP4 levels in cerebrospinal fluid (CSF) and serum using ELISA. Mean CSF levels of TIMP4 were decreased in patients with CAA compared to controls (3.36 ± 0.20 vs. 3.96 ± 0.22 ng/ml, p = 0.033), whereas median serum levels were increased in patients with CAA (4.51 ng/ml [IQR 3.75-5.29] vs 3.60 ng/ml [IQR 3.11-4.85], p-9.013). Moreover, mean CSF TIMP4 levels were lower in CAA patients who had experienced a symptomatic hemorrhage compared to CAA patients who did not (2.13 ± 0.24 vs. 3.57 ± 0.24 ng/ml, p = 0.007). CSF TIMP4 levels were associated with CSF levels of Aß40 (spearman r (rs) = 0.321, p = 0.009). In summary, we show that TIMP4 is highly associated with CAA and CAA-related ICH, which is reflected by higher levels in the cerebral vasculature and lower levels in CSF. With these findings we provide novel insights into the pathophysiology of CAA, and more specifically in CAA-associated ICH.

The silent information regulator 1 agonist SRT1720 reduces experimental intracerebral hemorrhagic brain injury by regulating the blood-brain barrier integrity.

Intracerebral hemorrhage (ICH) is a significant public health matter that has no effective treatment. ICH-induced destruction of the blood-brain barrier (BBB) leads to neurological deterioration. Astrocytic sonic hedgehog (SHH) alleviates brain injury by maintaining the integrity of the BBB after ICH. Silent information regulator 1 (SIRT1) is neuroprotective in several central nervous system diseases via BBB regulation. It is also a possible influential factor of the SHH signaling pathway. Nevertheless, the role of SIRT1 on BBB and the underlying pathological process associated with the SHH signaling pathway after ICH remain unclear. We established an intracerebral hemorrhagic mouse model by collagenase injection. SRT1720 (a selective agonist of SIRT1) was used to evaluate the effect of SIRT1 on BBB integrity after ICH. SIRT1 expression was reduced in the mouse brain after ICH. SRT1720 attenuated neurobehavioral impairments and brain edema of ICH mouse. After ICH induction, SRT1720 improved BBB integrity and tight junction expressions in the mouse brain. The SHH signaling pathway-related factors smoothened and glioma-associated oncogene homolog-1 were increased with the intervention of SRT1720, while cyclopamine (a specific inhibitor of the SHH signaling pathway) reversed these effects. These findings suggest that SIRT1 protects from ICH by altering BBB permeability and tight junction expression levels. This process is associated with the SHH signaling pathway, suggesting that SIRT1 may be a potential therapeutic target for ICH.

Machine learning algorithms integrate bulk and single-cell RNA data to unveil oxidative stress following intracerebral hemorrhage.

BACKGROUND: Increased oxidative stress (OS) activity following intracerebral hemorrhage (ICH) had significantly impacting patient prognosis. Identifying optimal genes associated with OS could enhance the understanding of OS after ICH. METHODS: We employed single-cell RNA sequencing (scRNA-seq) to investigate the heterogeneity of OS across various cellular tiers following ICH, aiming to acquire biological insights into ICH. We utilized AUCell, Ucell, singscore, ssgsea, and AddModuleScore algorithms, along with correlation analysis, to identify hub genes influencing high OS post-ICH. Furthermore, we employed four machine learning algorithms, eXtreme Gradient Boosting, Boruta, Random Forest, and Least Absolute Shrinkage and Selection Operator, to identify the optimal feature genes. To validate the accuracy of our analysis, we conducted validation in ICH animal experiments. RESULTS: After analyzing the scRNA-seq dataset using various algorithms, we found that OS activity exhibited heterogeneity across different cellular layers following ICH, with particularly heightened activity observed in monocytes. Further integration of bulk data and machine learning algorithms revealed that ANXA2 and COTL1 were closely associated with high OS after ICH. Our animal experiments demonstrated an increase in OS expression post-ICH. Additionally, the protein expression of ANXA2 and COTL1 was significantly elevated and co-localized with microglia. Pearson correlation coefficient analysis revealed a significant correlation between ANXA2 and OS, indicating strong consistency (r = 0.84, p < 0.05). Similar results were observed for COTL1 and OS (r = 0.69, p < 0.05). CONCLUSIONS: Following ICH, ANXA2 and COTL1 might penetrate the brain via monocytes, localize within microglia, and enhance OS activity. This might help us better understand OS after ICH.

Toll-Like Receptors Mediate Opposing Dendritic Cell Effects on Treg/Th17 Balance in Mice With Intracerebral Hemorrhage.

BACKGROUND: Dendritic cells (DCs) regulate the immune response associated with T lymphocytes, but their role in stroke remains unclear. In this study, we investigated the causal relationship between DCs and T-cell response in intracerebral hemorrhage (ICH) by focusing on TLRs (toll-like receptors) that may modulate the function of DCs. METHODS: We studied the effects of TLR4, TLR2, and TLR9 on DC-mediated T-cell response and the outcomes of ICH using male C57BL/6 and CD11c-DTx (diphtheria toxin) receptor mice. We administered specific agents intraperitoneally or orally and evaluated the results using flow cytometry, real-time polymerase chain reaction, Western blotting, immunofluorescence staining, histopathology, and behavioral tests. RESULTS: TLR4 and TLR2 activation induces DC maturation and reduces the ratio of regulatory T to T-helper 17 cells in the brain and periphery after ICH. When either of these receptors is activated, it can worsen neuroinflammation and exacerbate ICH outcomes. TLR9 also promotes DC maturation, stabilizing the number of DCs, particularly conventional DCs. TLR9 has the opposite effects on regulatory T/T-helper 17 balance, neuroinflammation, and ICH outcomes compared with TLR4 and TLR2. Upon stimulation, TLR4 and TLR9 may achieve these effects through the p38-MAPK (p38-mitogen-activated protein kinase)/MyD88 (myeloid differentiation primary response gene 88) and indoleamine 2,3-dioxygenase 1 (IDO1)/GCN2 (general control nonderepressible 2) signaling pathways, respectively. DCs act as intermediaries for TLR-mediated T-cell response. CONCLUSIONS: TLR-mediated opposing effects of DCs on T-cell response may provide novel strategies to treat ICH.

Jingfang granules protects against intracerebral hemorrhage by inhibiting neuroinflammation and protecting blood-brain barrier damage.

Intracerebral hemorrhage (ICH) can induce intensive oxidative stress, neuroinflammation, and brain cell apoptosis. However, conventional methods for ICH treatment have many disadvantages. There is an urgent need for alternative, effective therapies with minimal side effects. Pharmacodynamics experiment, molecular docking, network pharmacology, and metabolomics were adopted to investigate the treatment and its mechanism of Jingfang Granules (JFG) in ICH. In this study, we investigated the therapeutic effects of JFG on ICH using behavioral, brain water content and Magnetic resonance imaging experiments. However, the key active component and targets of JFG remain unknown. Here we verified that JFG was beneficial to improve brain injury after ICH. A network pharmacology analysis revealed that the anti-inflammatory effect of JFG is predominantly mediated by its activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway through Luteolin, (+)-Anomalin and Phaseol and their targeting of AKT1, tumor necrosis factorα (TNF-α), and interleukin-1ß (IL-1ß). Molecular docking analyses revealed an average affinity of -8.633 kcal/mol, indicating a binding strength of less than -5 kcal/mol. Metabolomic analysis showed that JFG exerted its therapeutic effect on ICH by regulating metabolic pathways, such as the metabolism of taurine and hypotaurine, biosynthesis of valine, leucine, and isoleucine. In conclusion, we demonstrated that JFG attenuated neuroinflammation and BBB injury subsequent to ICH by activating the PI3K/Akt signaling pathway.

Edaravone dexborneol regulates γ-aminobutyric acid transaminase in rats with acute intracerebral hemorrhage.

OBJECTIVES: Edaravone dexborneol is neuroprotective against ischemic stroke, with free radical-scavenging and anti-inflammatory effects, but its effects in hemorrhagic stroke remain unclear. We evaluated whether edaravone dexborneol has a neuroprotective effect in intracerebral hemorrhage, and its underlying mechanisms. MATERIALS AND METHODS: Bioinformatics were used to predict the pathway of action of edaravone dexborneol. An intracerebral hemorrhage model was established using type IV collagenase in edaravone dexborneol, intracerebral hemorrhage, Sham, adeno-associated virus + edaravone dexborneol, and adeno-associated virus + intracerebral hemorrhage groups. The modified Neurological Severity Score was used to evaluate neurological function in rats. Brain water content was measured using the dry-wet weight method. Tumor necrosis factor-α, interleukin-1ß, inducible nitric oxide synthase, and γ-aminobutyric acid levels were determined by enzyme-linked immunosorbent assay. The expression levels of neurofilament light chain and γ-aminobutyric acid transaminase were determined by western blot. Nissl staining was used to examine neuronal morphology. Cognitive behavior was evaluated using a small-animal treadmill. RESULTS: Edaravone dexborneol alleviated neurological defects, improved cognitive function, and reduced cerebral edema, neuronal degeneration, and necrosis in rats with cerebral hemorrhage. The expression levels of neurofilament light chain, tumor necrosis factor-α, interleukin-1ß, inducible nitric oxide synthase, and γ-aminobutyric acid were decreased, while γ-aminobutyric acid transaminase expression was up-regulated. CONCLUSIONS: Edaravone dexborneol regulates γ-aminobutyric acid content by acting on the γ-aminobutyric acid transaminase signaling pathway, thus alleviating oxidative stress, neuroinflammation, neuronal degeneration, and death caused by excitatory toxic injury of neurons after intracerebral hemorrhage.

The impact of experimental diabetes on intracerebral haemorrhage. A preclinical study.

Although diabetes mellitus negatively affects post-ischaemic stroke injury and recovery, its impact on intracerebral haemorrhage (ICH) remains uncertain. This study aimed to investigate the effect of experimental diabetes (ED) on ICH-induced injury and neurological impairment. Sprague-Dawley rats were induced with ED 2 weeks before ICH induction. Animals were randomly assigned to four groups: 1)Healthy; 2)ICH; 3)ED; 4)ED-ICH. ICH and ED-ICH groups showed similar functional assessment. The ED-ICH group exhibited significantly lower haemorrhage volume compared with the ICH group, except at 1 mo. The oedema/ICH volume ratio and cistern displacement ratio were significantly higher in the ED-ICH group. Vascular markers revealed greater expression of α-SMA in the ED groups (ED and ED-ICH) compared with ICH. Conversely, the ICH groups (ED-ICH and ICH) exhibited higher levels of VEGF compared to the healthy and ED groups. An assessment of myelin tract integrity showed an increase in fractional anisotropy in the ED and ED-ICH groups compared with ICH. The ED group showed higher cryomyelin expression than the ED-ICH and ICH groups. Additionally, the ED groups (ED and ED-ICH) displayed higher expression of MOG and Olig-2 than ICH. As for inflammation, MCP-1 levels were significantly lower in the ED-ICH groups compared with the ICH group. Notably, ED did not aggravate the neurological outcome; however, it results in greater ICH-related brain oedema, greater brain structure displacement and lower haemorrhage volume. ED influences the cerebral vascularisation with an increase in vascular thickness, limits the inflammatory response and attenuates the deleterious effect of ICH on white matter integrity.

TAK1 inhibition mitigates intracerebral hemorrhage-induced brain injury through reduction of oxidative stress and neuronal pyroptosis via the NRF2 signaling pathway.

Introduction: Intracerebral hemorrhage (ICH) often triggers oxidative stress through reactive oxygen species (ROS). Transforming growth factor-ß-activated kinase 1 (TAK1) plays a pivotal role in regulating oxidative stress and inflammation across various diseases. 5Z-7-Oxozeaenol (OZ), a specific inhibitor of TAK1, has exhibited therapeutic effects in various conditions. However, the impact of OZ following ICH and its underlying molecular mechanisms remain elusive. This study aimed to explore the possible role of OZ in ICH and its underlying mechanisms by inhibiting oxidative stress-mediated pyroptosis. Methods: Adult male Sprague-Dawley rats were subjected to an ICH model, followed by treatment with OZ. Neurobehavioral function, blood-brain barrier integrity, neuronal pyroptosis, and oxidative stress markers were assessed using various techniques including behavioral tests, immunofluorescence staining, western blotting, transmission electron microscopy, and biochemical assays. Results: Our study revealed that OZ administration significantly inhibited phosphorylated TAK1 expression post-ICH. Furthermore, TAK1 blockade by OZ attenuated blood-brain barrier (BBB) disruption, neuroinflammation, and oxidative damage while enhancing neurobehavioral function. Mechanistically, OZ administration markedly reduced ROS production and oxidative stress by facilitating nuclear factor-erythroid 2-related factor 2 (NRF2) nuclear translocation. This was accompanied by a subsequent suppression of the NOD-like receptor protein 3 (NLRP3) activation-mediated inflammatory cascade and neuronal pyroptosis. Discussion: Our findings highlight that OZ alleviates brain injury and oxidative stress-mediated pyroptosis via the NRF2 pathway. Inhibition of TAK1 emerges as a promising approach for managing ICH.

Menaquinone-4 Alleviates Neurological Deficits Associated with Intracerebral Hemorrhage by Preserving Corticospinal Tract in Mice.

Menaquinone-4 (MK-4) is an isoform of vitamin K2 that has been shown to exert various biological actions besides its functions in blood coagulation and bone metabolism. Here we examined the effect of MK-4 on a mouse model of intracerebral hemorrhage (ICH). Daily oral administration of 200 mg/kg MK-4 starting from 3 h after induction of ICH by intrastriatal collagenase injection significantly ameliorated neurological deficits. Unexpectedly, MK-4 produced no significant effects on various histopathological parameters, including the decrease of remaining neurons and the increase of infiltrating neutrophils within the hematoma, the increased accumulation of activated microglia/macrophages and astrocytes around the hematoma, as well as the injury volume and brain swelling by hematoma formation. In addition, ICH-induced increases in nitrosative/oxidative stress reflected by changes in the immunoreactivities against nitrotyrosine and heme oxygenase-1 as well as the contents of malondialdehyde and glutathione were not significantly affected by MK-4. In contrast, MK-4 alleviated axon tract injury in the internal capsule as revealed by neurofilament-H immunofluorescence. Enhanced preservation of the corticospinal tract by MK-4 was also confirmed by retrograde labeling of neurons in the primary motor cortex innervating the spinal cord. These results suggest that MK-4 produces therapeutic effect on ICH by protecting structural integrity of the corticospinal tract.

Peroxiredoxin II exerts neuroprotective effects by inhibiting endoplasmic reticulum stress and oxidative stress-induced neuronal pyroptosis.

BACKGROUND: Intracerebral hemorrhage (ICH) is a critical neurological condition with few treatment options, where secondary immune responses and specific cell death forms, like pyroptosis, worsen brain damage. Pyroptosis involves gasdermin-mediated membrane pores, increasing inflammation and neural harm, with the NLRP3/Caspase-1/GSDMD pathway being central to this process. Peroxiredoxin II (Prx II), recognized for its mitochondrial protection and reactive oxygen species (ROS) scavenging abilities, appears as a promising neuronal pyroptosis modulator. However, its exact role and action mechanisms need clearer definition. This research aims to explore Prx II impact on neuronal pyroptosis and elucidate its mechanisms, especially regarding endoplasmic reticulum (ER) stress and oxidative stress-induced neuronal damage modulation. METHODS AND RESULTS: Utilizing MTT assays, Microscopy, Hoechst/PI staining, Western blotting, and immunofluorescence, we found Prx II effectively reduces LPS/ATP-induced pyroptosis and neuroinflammation in HT22 hippocampal neuronal cells. Our results indicate Prx II's neuroprotective actions are mediated through PI3K/AKT activation and ER stress pathway inhibition, diminishing mitochondrial dysfunction and decreasing neuronal pyroptosis through the ROS/MAPK/NF-κB pathway. These findings highlight Prx II potential therapeutic value in improving intracerebral hemorrhage outcomes by lessening secondary brain injury via critical signaling pathway modulation involved in neuronal pyroptosis. CONCLUSIONS: Our study not only underlines Prx II importance in neuroprotection but also opens new therapeutic intervention avenues in intracerebral hemorrhage, stressing the complex interplay between redox regulation, ER stress, and mitochondrial dynamics in neuroinflammation and cell death management.

miR-4685-3p Alleviates Human Brain Microvascular Endothelial Cells Injury by Regulating MMP9.

OBJECTIVE: Cerebral microbleeds (CMBs) are punctate hemorrhagic lesions within the brain parenchyma and are a classic manifestation of cerebral small vessel disease (CSVD). The primary objective of this study is to investigate the potential role of miR-4685-3p and underlying mechanisms by which miR-4685-3p modulates matrix metalloproteinase-9 (MMP9) in cerebral microvascular endothelial cell injury. METHODS: We employed high-throughput sequencing to screen for differentially expressed miRNAs in the peripheral blood of patients with CMBs and healthy controls. Employing lipopolysaccharide (LPS) to induce cellular damage, we aim to establish a model of human brain microvascular endothelial cells (hCMEC/D3) injury. We also had cells transfected with miR-4685-3p mimic and MMP9 overexpression plasmid. We utilized quantitative polymerase chain reaction (qPCR) to assess the expression levels of miR-4685-3p and performed Western blot analysis to examine MMP9 expression levels in the cells. We employed the CCK-8 assay, TUNEL assay, and tube formation assay to evaluate cellular viability, apoptotic rates, and angiogenic capabilities. Furthermore, dual-luciferase reporter assay analysis was conducted to confirm the relationship between miR-4685-3p and MMP9. RESULTS: The sequencing results indicated a downregulation of miR-4685-3p in the peripheral blood of patients with CMBs. Within the context of LPS-induced injury to hCMEC/D3 cells, miR-4685-3p exhibits reduced expression, whereas MMP9 expression levels are elevated. The elevation of miR-4685-3p expression levels attenuates LPS-induced cellular apoptosis and enhances the viability and tube-forming capacity of hCMEC/D3 cells. Concomitant transfection with MMP9 overexpression constructs effectively reversed the detrimental effects of LPS on hCMEC/D3 cell integrity. We further confirmed that miR-4685-3p overexpression directly targets MMP9, leading to negative regulation of MMP9 expression. CONCLUSION: Upregulating miR-4685-3p, which targets the MMP9 axis, mitigated LPS-induced cerebral microvascular endothelial cell injury, potentially playing a protective role in the progression of CMBs.

Synergistic promotion of angiogenesis after intracerebral hemorrhage by ginsenoside Rh2 and chrysophanol in rats.

BACKGROUND: Intracerebral hemorrhage (ICH) is a debilitating condition characterized by the rupture of cerebral blood vessels, resulting in profound neurological deficits. A significant challenge in the treatment of ICH lies in the brain's limited capacity to regenerate damaged blood vessels. This study explores the potential synergistic effects of Ginsenoside Rh2 and Chrysophanol in promoting angiogenesis following ICH in a rat model. METHODS: Network pharmacology was employed to predict the potential targets and pathways of Ginsenoside Rh2 and Chrysophanol for ICH treatment. Molecular docking was utilized to assess the binding affinity between these compounds and their respective targets. Experimental ICH was induced in male Sprague-Dawley rats through stereotactic injection of type VII collagenase into the right caudate putamen (CPu). The study encompassed various methodologies, including administration protocols, assessments of neurological function, magnetic resonance imaging, histological examination, observation of brain tissue ultrastructure, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence staining, Western blot analysis, and statistical analyses. RESULTS: Network pharmacology analysis indicated that Ginsenoside Rh2 and Chrysophanol may exert their therapeutic effects in ICH by promoting angiogenesis. Results from animal experiments revealed that rats treated with Ginsenoside Rh2 and Chrysophanol exhibited significantly improved neurological function, reduced hematoma volume, and diminished pathological injury compared to the Model group. Immunofluorescence analysis demonstrated enhanced expression of vascular endothelial growth factor receptor 2 (VEGFR2) and CD31, signifying augmented angiogenesis in the peri-hematomal region following combination therapy. Importantly, the addition of a VEGFR2 inhibitor reversed the increased expression of VEGFR2 and CD31. Furthermore, Western blot analysis revealed upregulated expression of angiogenesis-related factors, including VEGFR2, SRC, AKT1, MAPK1, and MAPK14, in the combination therapy group, but this effect was abrogated upon VEGFR2 inhibitor administration. CONCLUSION: The synergistic effect of Ginsenoside Rh2 and Chrysophanol demonstrated a notable protective impact on ICH injury in rats, specifically attributed to their facilitation of angiogenesis. Consequently, this research offers a foundation for the utilization of Ginsenosides Rh2 and Chrysophanol in medical settings and offers direction for the advancement of novel pharmaceuticals for the clinical management of ICH.

Salvianolic acid A inhibits ferroptosis and protects against intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a common cerebral vascular disease with high incidence, disability, and mortality. Ferroptosis is a regulated type of iron-dependent, non-apoptotic programmed cell death. There is increasing evidence that ferroptosis may lead to neuronal damage mediated by hemorrhagic stroke mediated neuronal damage. Salvianolic acid A (SAA) is a natural bioactive polyphenol compound extracted from salvia miltiorrhiza, which has anti-inflammatory, antioxidant, and antifibrosis activities. SAA is reported to be an iron chelator that inhibits lipid peroxidation and provides neuroprotective effects. However, whether SAA improves neuronal ferroptosis mediated by hemorrhagic stroke remains unclear. The study aims to evaluate the therapeutic effect of SAA on Ferroptosis mediated by Intracerebral hemorrhage and explore its potential mechanisms. We constructed in vivo and in vitro models of intracerebral hemorrhage in rats. Multiple methods were used to analyze the inhibitory effect of SAA on ferroptosis in both in vivo and in vitro models of intracerebral hemorrhage in rats. Then, network pharmacology is used to identify potential targets and mechanisms for SAA treatment of ICH. The SAA target ICH network combines SAA and ICH targets with protein-protein interactions (PPIs). Find the specific mechanism of SAA acting on ferroptosis through molecular docking and functional enrichment analysis. In rats, SAA (10 mg/kg in vivo and 50 µM in vitro, p < 0.05) alleviated dyskinesia and brain injury in the ICH model by inhibiting ferroptosis (p < 0.05). The molecular docking results and functional enrichment analyses suggested that AKT (V-akt murine thymoma viral oncogene homolog) could mediate the effect of SAA. NRF2 (Nuclear factor erythroid 2-related factor 2) was a potential target of SAA. Our further experiments showed that salvianolic acid A enhanced the Akt /GSK-3ß/Nrf2 signaling pathway activation in vivo and in vitro. At the same time, SAA significantly expanded the expression of GPX4, XCT proteins, and the nuclear expression of Nrf2, while the AKT inhibitor SH-6 and the Nrf2 inhibitor ML385 could reduce them to some extent. Therefore, SAA effectively ameliorated ICH-mediated neuronal ferroptosis. Meanwhile, one of the critical mechanisms of SAA inhibiting ferroptosis was activating the Akt/GSK-3ß/Nrf2 signaling pathway.

MMP-9 release into collateral blood vessels before endovascular thrombectomy to assess the risk of major intracerebral haemorrhages and poor outcome for acute ischaemic stroke: a proof-of-concept study.

BACKGROUND: Matrix metalloproteinases (MMPs) are implied in blood-brain barrier degradation and haemorrhagic transformation following ischaemic stroke, but their local relevance in the hyperacute disease phase is unknown. We aimed to examine ultra-early MMP-9 and MMP-2 release into collateral blood vessels, and to assess its prognostic value before therapeutic recanalisation by endovascular thrombectomy (EVT). METHODS: We report a cross-sectional proof-of-concept study including patients undergoing EVT for large-vessel ischaemic stroke at the University Hospital Würzburg, Germany. We obtained liquid biopsies from the collateral circulation before recanalisation, and systemic control samples. Laboratory workup included quantification of MMP-9 and MMP-2 plasma concentrations by cytometric bead array, immunohistochemical analyses of cellular MMP-9 and MMP-2 expression, and detection of proteolytic activity by gelatine zymography. The clinical impact of MMP concentrations was assessed by stratification according to intracranial haemorrhagic lesions on postinterventional computed tomography (Heidelberg Bleeding Classification, HBC) and early functional outcome (modified Rankin Scale, mRS). We used multivariable logistic regression, receiver-operating-characteristic (ROC) curves, and fixed-level estimates of test accuracy measures to study the prognostic value of MMP-9 concentrations. FINDINGS: Between August 3, 2018, and September 16, 2021, 264 matched samples from 132 patients (86 [65.2%] women, 46 [34.8%] men, aged 40-94 years) were obtained. Median (interquartile range, IQR) MMP-9 (279.7 [IQR 126.4-569.6] vs 441 [IQR 223.4-731.5] ng/ml, p < 0.0001) but not MMP-2 concentrations were increased within collateral blood vessels. The median MMP-9 expression level of invading neutrophils was elevated (fluorescence intensity, arbitrary unit: 2276 [IQR 1007-5086] vs 3078 [IQR 1108-7963], p = 0.0018). Gelatine zymography experiments indicated the locally confined proteolytic activity of MMP-9 but not of MMP-2. Pretherapeutic MMP-9 release into stroke-affected brain regions predicted the degree of intracerebral haemorrhages and clinical stroke severity after recanalisation, and independently increased the odds of space-occupying parenchymal haematomas (HBC1c-3a) by 1.54 times, and the odds of severe disability or death (mRS ≥5 at hospital discharge) by 2.33 times per 1000 ng/ml increase. Excessive concentrations of MMP-9 indicated impending parenchymal haematomas and severe disability or death with high specificity. INTERPRETATION: Measurement of MMP-9 within collateral blood vessels is feasible and identifies patients with stroke at risk of major intracerebral haemorrhages and poor outcome before therapeutic recanalisation by EVT, thereby providing evidence of the concept validity of ultra-early local stroke biomarkers. FUNDING: This work was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) and the Interdisciplinary Centre for Clinical Research (IZKF) at the University of Würzburg.

Activating transcription factor 6 alleviates secondary brain injury by increasing cystathionine γ-lyase expression in a rat model of intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) comprises primary and secondary injuries, the latter of which induces increased inflammation and apoptosis and is more severe. Activating transcription factor 6 (ATF6) is a type-II transmembrane protein in the endoplasmic reticulum (ER). ATF6 target genes could improve ER homeostasis, which contributes to cryoprotection. Hence, we predict that ATF6 will have a protective effect on brain tissue after ICH. METHOD: The ICH rat model was generated through autologous blood injection into the right basal ganglia, the expression of ATF6 after ICH was determined by WB and IF. The expression of ATF6 was effectively controlled by means of intervention, and a series of measures was used to detect cell death, neuroinflammation, brain edema, blood-brain barrier and other indicators after ICH. Finally, the effects on long-term neural function of rats were measured by behavioral means. RESULT: ATF6 was significantly increased in the ICH-induced brain tissues. Further, ATF6 was found to modulate the expression of cystathionine γ-lyase (CTH) after ICH. Upregulation of ATF6 attenuated neuronal apoptosis and inflammation in ICH rats, along with mitigation of ICH-induced brain edema, blood-brain barrier deterioration, and cognitive behavior defects. Conversely, ATF6 genetic knockdown induced effects counter to those aforementioned. CONCLUSIONS: This study thereby emphasizes the crucial role of ATF6 in secondary brain injury in response to ICH, indicating that ATF6 upregulation may potentially ameliorate ICH-induced secondary brain injury. Consequently, ATF6 could serve as a promising therapeutic target to alleviate clinical ICH-induced secondary brain injuries.

Egr1 promotes Nlrc4-dependent neuronal pyroptosis through phlda1 in an in-vitro model of intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a fatal brain injury, but the current treatments for it are inadequate to reduce the severity of secondary brain injury. Our study aims to explore the molecular mechanism of Egr1 and Phlda1 in regulating hemin-induced neuronal pyroptosis, and hope to provide novel therapeutic targets for ICH treatment. Mouse hippocampal neuron cells treated with hemin were used to simulate an in-vitro ICH model. Using qRT-PCR and western blot to evaluate mRNA and protein concentrations. MTT assay was utilized to assess cell viability. LDH levels were determined by lactate Dehydrogenase Activity Assay Kit. IL-1ß and IL-18 levels were examined by ELISA. The interaction of Egr1 and Phlda1 promoter was evaluated using chromatin immunoprecipitation and dual-luciferase reporter assays. Egr1 and Phlda1 were both upregulated in HT22 cells following hemin treatment. Hemin treatment caused a significant reduction in HT22 cell viability, an increase in Nlrc4 and HT22 cell pyroptosis, and heightened inflammation. However, knocking down Egr1 neutralized hemin-induced effects on HT22 cells. Egr1 bound to the promoter of Phlda1 and transcriptionally activated Phlda1. Silencing Phlda1 significantly reduced Nlrc4-dependent neuronal pyroptosis. Conversely, overexpressing Phlda1 mitigated the inhibitory effects of Egr1 knockdown on Nlrc4 and neuronal pyroptosis during ICH. Egr1 enhanced neuronal pyroptosis mediated by Nlrc4 under ICH via transcriptionally activating Phlda1.