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.

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.

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.

Extracellular vesicle encapsulated Homer1a as novel nanotherapeutics against intracerebral hemorrhage in a mouse model.

Homer1a and A2 astrocytes are involved in the regulation of inflammation induced by intracerebral hemorrhage (ICH). However, there is no anticipated treatment strategy based on the anti-inflammatory effect of Homer1a and A2 astrocytes. Here, we successfully induced A2 astrocytes in vitro, and then we report an efficient method to prepare Homer1a+ EVs derived from A2 astrocytes which making it more stable, safe, and targetable to injured neurons. Homer1a+ EVs promotes the conversion of A1 to A2 astrocytes in ICH mice. Homer1a+ EVs inhibits activation and nuclear translocation of NF-κB, thereby regulating transcription of IL-17A in neurons. Homer1a+ EVs inhibits the RAGE/NF-κB/IL-17 signaling pathway and the binding ability of IL-17A: IL17-AR and RAGE: DIAPH1. In addition, Homer1a+ EVs ameliorates the pathology, behavior, and survival rate in GFAPCreHomer1fl/-Homer1a± and NestinCreRAGEfl/fl ICH mice. Our study provides a novel insight and potential for the clinical translation of Homer1a+ EVs in the treatment of ICH.

Age-related changes after intracerebral hemorrhage: a comparative proteomics analysis of perihematomal tissue.

The risk factors and causes of intracerebral hemorrhage (ICH) and the degree of functional recovery after ICH are distinct between young and elderly patients. The increasing incidence of ICH in young adults has become a concern; however, research on the molecules and pathways involved ICH in subjects of different ages is lacking. In this study, tandem mass tag (TMT)-based proteomics was utilized to examine the protein expression profiles of perihematomal tissue from young and aged mice 24 h after collagenase-induced ICH. Among the 5,129 quantified proteins, ICH induced 108 and 143 differentially expressed proteins (DEPs) in young and aged mice, respectively; specifically, there were 54 common DEPs, 54 unique DEPs in young mice and 89 unique DEPs in aged mice. In contrast, aging altered the expression of 58 proteins in the brain, resulting in 39 upregulated DEPs and 19 downregulated DEPs. Bioinformatics analysis indicated that ICH activated different proteins in complement pathways, coagulation cascades, the acute phase response, and the iron homeostasis signaling pathway in mice of both age groups. Protein-protein interaction (PPI) analysis and ingenuity pathway analysis (IPA) demonstrated that the unique DEPs in the young and aged mice were related to lipid metabolism and carbohydrate metabolism, respectively. Deeper paired-comparison analysis demonstrated that apolipoprotein M exhibited the most significant change in expression as a result of both aging and ICH. These results help illustrate age-related protein expression changes in the acute phase of ICH.

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.

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.

DMT1 ubiquitination by Nedd4 protects against ferroptosis after intracerebral hemorrhage.

OBJECTIVE: Neuronal precursor cells expressed developmentally down-regulated 4 (Nedd4) are believed to play a critical role in promoting the degradation of substrate proteins and are involved in numerous biological processes. However, the role of Nedd4 in intracerebral hemorrhage (ICH) remains unknown. This study aims to investigate the regulatory role of Nedd4 in the ICH model. METHODS: Male C57BL/6J mice were induced with ICH. Subsequently, the levels of glutathione peroxidase 4 (GPX4), malondialdehyde (MDA) concentration, iron content, mitochondrial morphology, as well as the expression of divalent metal transporter 1 (DMT1) and Nedd4 were assessed after ICH. Furthermore, the impact of Nedd4 overexpression was evaluated through analyses of hematoma area, ferroptosis, and neurobehavioral function. The mechanism underlying Nedd4-mediated degradation of DMT1 was elecidated using immunoprecipitation (IP) after ICH. RESULTS: Upon ICH, the level of DMT1 in the brain increased, but decreased when Nedd4 was overexpressed using Lentivirus, suggesting a negative correlation between Nedd4 and DMT1. Additionally, the degradation of DMT1 was inhibited after ICH. Furthermore, it was found that Nedd4 can interact with and ubiquitinate DMT1 at lysine residues 6, 69, and 277, facilitating the degradation of DMT1. Functional analysis indicated that overexpression of Nedd4 can alleviate ferroptosis and promote recovery following ICH. CONCLUSION: The results demonstrated that ferroptosis occurs via the Nedd4/DMT1 pathway during ICH, suggesting it potential as a valuable target to inhibit ferroptosis for the treatment of ICH.

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.

Adjudin protects blood-brain barrier integrity and attenuates neuroinflammation following intracerebral hemorrhage in mice.

Secondary brain injury exacerbates neurological dysfunction and neural cell death following intracerebral hemorrhage (ICH), targeting the pathophysiological mechanism of the secondary brain injury holds promise for improving ICH outcomes. Adjudin, a potential male contraceptive, exhibits neuroprotective effects in brain injury disease models, yet its impact in the ICH model remains unknown. In this study, we investigated the effects of adjudin on brain injury in a mouse ICH model and explored its underlying mechanisms. ICH was induced in male C57BL/6 mice by injecting collagenase into the right striatum. Mice received adjudin treatment (50 mg/kg/day) for 3 days before euthanization and the perihematomal tissues were collected for further analysis. Adjudin significantly reduced hematoma volume and improved neurological function compared with the vehicle group. Western blot showed that Adjudin markedly decreased the expression of MMP-9 and increased the expression of tight junctions (TJs) proteins, Occludin and ZO-1, and adherens junctions (AJs) protein VE-cadherin. Adjudin also decreased the blood-brain barrier (BBB) permeability, as indicated by the reduced albumin and Evans Blue leakage, along with a decrease in brain water content. Immunofluorescence staining revealed that adjudin noticeably reduced the infiltration of neutrophil, activation of microglia/macrophages, and reactive astrogliosis, accompanied by an increase in CD206 positive microglia/macrophages which exhibit phagocytic characteristics. Adjudin concurrently decreased the generation of proinflammatory cytokines, such as TNF-α and IL-1ß. Additionally, adjudin increased the expression of aquaporin 4 (AQP4). Furthermore, adjudin reduced brain cell apoptosis, as evidenced by increased expression of anti-apoptotic protein Bcl-2, and decreased expression of apoptosis related proteins Bax, cleaved caspase-3 and fewer TUNEL positive cells. Our data suggest that adjudin protects against ICH-induced secondary brain injury and may serve as a potential neuroprotective agent for ICH treatment.

Electroacupuncture reduces oxidative stress response and improves secondary injury of intracerebral hemorrhage in rats by activating the peroxisome proliferator-activated receptor-γ/nuclear factor erythroid2-related factor 2/γ-glutamylcysteine synthetase pathway.

Intracerebral hemorrhage (ICH) is a severe stroke subtype. Secondary injury is a key factor leading to neurological deficits after ICH. Electroacupuncture (EA) can improve the neurological function after ICH, however, its internal mechanism is still unclear. The aim of this study is to investigate whether EA could ameliorate secondary injury after ICH through antioxidative stress and its potential regulatory mechanism. A rat model of ICH was established by injecting autologous blood into striatum. After the intervention of EA and EA combined with peroxisome proliferator-activated receptor-γ (PPARγ) blocker, Zea-longa scores, modified neurological severity scores and open field tests were used to evaluate the neurological function of the rats. Flow cytometry detected tissue reactive oxygen species (ROS) levels. Tissue tumor necrosis factor-α (TNF-α) levels were analyzed by enzyme-linked immunosorbent assays. The protein expressions of PPAR γ, nuclear factor erythroid2-related factor 2 (Nrf2) and γ-glutamylcysteine synthetase (γ-GCS) were detected by Western blot. Immunohistochemistry was used to observe the activation of microglia. The demyelination degree of axon myelin was observed by transmission electron microscope. Compared with the model group, EA intervention improved neurological function, decreased ROS and TNF-α levels, increased the protein expression of PPARγ, Nrf2 and γ-GCS, and reduced the activation of microglia, it also alleviated axonal myelin sheath damage. In addition, the neuroprotective effect of EA was partially attenuated by PPARγ blocker. EA ameliorated the neurological function of secondary injury after ICH in rats, possibly by activating the PPARγ/Nrf2/γ-GCS signaling pathway, reducing microglia activation, and inhibiting oxidative stress, thus alleviating the extent of axonal demyelination plays a role.

Attenuation of neuronal ferroptosis in intracerebral hemorrhage by inhibiting HDAC1/2: Microglial heterogenization via the Nrf2/HO1 pathway.

AIM: The class I histone deacetylases (HDACs) implicate in microglial heterogenization and neuroinflammation following Intracerebral hemorrhage (ICH). Ferroptosis has also been reported in the ICH model. However, the relationship between HDAC1/2's role in microglial heterogenization and neuronal ferroptosis remains unclear. METHODS: In both in vivo and in vitro models of ICH, we used Romidepsin (FK228), a selective HDAC1/2 inhibitor, to investigate its effects on microglial heterogenization and neuronal ferroptosis. In the in vitro ICH model using Hemin, a transwell system was utilized to examine how microglia-driven inflammation and ICH-triggered neuronal ferroptosis interact. Immunostaining, Western blotting and RT-qPCR were used to evaluate the microglial heterogenization and neuronal ferroptosis. Microglial heterogenization, neuronal ferroptosis, and neurological dysfunctions were assessed in vivo ICH mice model performed by autologous blood injection. RESULTS: HDAC1/2 inhibition altered microglial heterogenization after ICH, as showing the reducing neuroinflammation and shifting microglia towards an anti-inflammatory phenotype by immunostaining and qPCR results. HDAC1/2 inhibition reduced ferroptosis, characterized by high ROS and low GPx4 expression in HT22 cells, and reduced iron and lipid deposition post-ICH in vivo. Additionally, the Nrf2/HO1 signaling pathway, especially acetyl-Nrf2, activated in the in vivo ICH model due to HDAC1/2 inhibition, plays a role in regulating microglial heterogenization. Furthermore, HDAC1/2 inhibition improved sensorimotor and histological outcomes post-ICH, offering a potential mechanism against ICH. CONCLUSION: Inhibition of HDAC1/2 reduces neuro-ferroptosis by modifying the heterogeneity of microglia via the Nrf2/HO1 pathway, with a particular focus on acetyl-Nrf2. Additionally, this inhibition aids in the faster removal of hematomas and lessens prolonged neurological impairments, indicating novel approach for treating ICH.

Buyang huanwu decoction promotes remyelination via miR-760-3p/GPR17 axis after intracerebral hemorrhage.

ETHNOPHARMACOLOGICAL RELEVANCE: The repairment of myelin sheaths is crucial for mitigating neurological impairments of intracerebral hemorrhage (ICH). However, the current research on remyelination processes in ICH remains limited. A representative traditional Chinese medicine, Buyang Huanwu decoction (BYHWD), shows a promising therapeutic strategy for ICH treatment. AIM OF THE STUDY: To investigate the pro-remyelination effects of BYHWD on ICH and explore the underlying mechanisms. MATERIALS AND METHODS: The collagenase-induced mice ICH model was created for investigation. BYHWD's protective effects were assessed by behavioral tests and histological staining. Transmission electron microscopy was used for displaying the structure of myelin sheaths. The remyelination and oligodendrocyte differentiation were evaluated by the expressions of myelin proteolipid protein (PLP), myelin basic protein (MBP), MBP/TAU, Olig2/CC1, and PDGFRα/proliferating cell nuclear antigen (PCNA) through RT-qPCR and immunofluorescence. Transcriptomics integrated with disease database analysis and experiments in vivo and in vitro revealed the microRNA-related underlying mechanisms. RESULTS: Here, we reported that BYHWD promoted the neurological function of ICH mice and improved remyelination by increasing PLP, MBP, and TAU, as well as restoring myelin structure. Besides, we showed that BYHWD promoted remyelination by boosting the differentiation of PDGFRα+ oligodendrocyte precursor cells into olig2+/CC1+ oligodendrocytes. Additionally, we demonstrated that the remyelination effects of BYHWD worked by inhibiting G protein-coupled receptor 17 (GPR17). miRNA sequencing integrated with miRNA database prediction screened potential miRNAs targeting GPR17. By applying immunofluorescence, RNA in situ hybridization and dual luciferase reporter gene assay, we confirmed that BYHWD suppressed GPR17 and improved remyelination by increasing miR-760-3p. CONCLUSIONS: BYHWD improves remyelination and neurological function in ICH mice by targeting miR-760-3p to inhibit GPR17. This study may shed light on the orchestration of remyelination mechanisms after ICH, thus providing novel insights for developing innovative prescriptions with brain-protective properties.

Intracerebral hemorrhage-induced brain injury in mice: The role of peroxiredoxin 2-Toll-like receptor 4 inflammatory axis.

BACKGROUND: Peroxiredoxin 2 (Prx2), an intracellular protein that regulates redox reactions, released from red blood cells is involved in inflammatory brain injury after intracerebral hemorrhage (ICH). Toll-like receptor 4 (TLR4) may be crucial in this process. This study investigated the role of the Prx2-TLR4 inflammatory axis in brain injury following experimental ICH in mice. METHODS: First, C57BL/6 mice received an intracaudate injection of autologous arterial blood or saline and their brains were harvested on day 1 to measure Prx2 levels. Second, mice received an intracaudate injection of either recombinant mouse Prx2 or saline. Third, the mice were co-injected with autologous arterial blood and conoidin A, a Prx2 inhibitor, or vehicle. Fourth, the mice received a Prx2 injection and were treated with TAK-242, a TLR4 antagonist, or saline (intraperitoneally). Behavioral tests, magnetic resonance imaging, western blot, immunohistochemistry/immunofluorescence staining, and RNA sequencing (RNA-seq) were performed. RESULTS: Brain Prx2 levels were elevated after autologous arterial blood injection. Intracaudate injection of Prx2 caused brain swelling, microglial activation, neutrophil infiltration, neuronal death, and neurological deficits. Co-injection of conoidin A attenuated autologous arterial blood-induced brain injury. TLR4 was expressed on the surface of microglia/macrophages and neutrophils and participated in Prx2-induced inflammation. TAK-242 treatment attenuated Prx2-induced inflammation and neurological deficits. CONCLUSIONS: Prx2 can cause brain injury following ICH through the TLR4 pathway, revealing the Prx2-TLR4 inflammatory axis as a potential therapeutic target.

ERBB1 alleviates secondary brain injury induced by experimental intracerebral hemorrhage in rats by modulating neuronal death via PLC-γ/PKC pathway.

AIMS: Intracerebral hemorrhage (ICH) is a disease with high rates of disability and mortality. The role of epidermal growth factor receptor 1 (ERBB1) in ICH was elucidated in this study. METHODS: ICH model was constructed by injecting autologous arterial blood into the right basal ganglia. The protein level of ERBB1 was detected by western blot analysis. To up- and downregulation of ERBB1 in rats, intraventricular injection of a lentivirus overexpression vector of ERBB1 and AG1478 (a specific inhibitor of ERBB1) was used. The cell apoptosis, neuronal loss, and pro-inflammatory cytokines were assessed by TUNEL, Nissl staining, and ELISA. Meanwhile, behavioral cognitive impairment of ICH rats was evaluated after ERBB1-targeted interventions. RESULTS: ERBB1 increased significantly in brain tissue of ICH rats. Overexpression of ERBB1 remarkably reduced cell apoptosis and neuronal loss induced by ICH, as well as pro-inflammatory cytokines and oxidative stress. Meanwhile, the behavioral and cognitive impairment of ICH rats were alleviated after upregulation of ERBB1; however, the secondary brain injury (SBI) was aggravated by AG1478 treatment. Furthermore, the upregulation of PLC-γ and PKC in ICH rats was reversed by AG1478 treatment. CONCLUSIONS: ERBB1 can improve SBI and has a neuroprotective effect in experimental ICH rats via PLC-γ/PKC pathway.

Normobaric hyperoxia alleviates complement C3-mediated synaptic pruning and brain injury after intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a common cerebrovascular disease, and the complement cascade exacerbates brain injury after ICH. As the most abundant component of the complement system, complement component 3 (C3) plays essential roles in all three complement pathways. However, the effects of C3 on neurological impairment and brain injury in ICH patients and the related mechanism have not been fully elucidated. Normobaric hyperoxia (NBO) is regarded as a treatment for ICH patients, and recent clinical studies also have confirmed the neuroprotective role of NBO against acute ICH-mediated brain damage, but the underlying mechanism still remains elusive. AIMS: In the present study, we investigated the effects of complement C3 on NBO-treated ICH patients and model mice, and the underlying mechanism of NBO therapy in ICH-mediated brain injury. RESULTS: Hemorrhagic injury resulted in the high plasma C3 levels in ICH patients, and the plasma C3 levels were closely related to hemorrhagic severity and clinical outcomes after ICH. BO treatment alleviated neurologic impairments and rescued the hemorrhagic-induced increase in plasma C3 levels in ICH patients and model mice. Moreover, the results indicated that NBO exerted its protective effects of on brain injury after ICH by downregulating the expression of C3 in microglia and alleviating microglia-mediated synaptic pruning. CONCLUSIONS: Our results revealed that NBO exerts its neuroprotective effects by reducing C3-mediated synaptic pruning, which suggested that NBO therapy could be used for the clinical treatment of ICH.

Microglial uptake of hADSCs-Exo mitigates neuroinflammation in ICH.

Intracerebral hemorrhage (ICH) is associated with secondary neuroinflammation, leading to severe central nervous system damage. Exosomes derived from human adipose-derived mesenchymal stem cells (hADSCs-Exo) have shown potential therapeutic effects in regulating inflammatory responses in ICH. This study aims to investigate the role of hADSCs-Exo in ICH and its underlying mechanism involving miRNA-mediated regulation of formyl peptide receptor 1 (FPR1). Flow cytometry was used to identify hADSCs and extract exosomes. Transmission electron microscopy and Western blot were performed to confirm the characteristics of the exosomes. In vitro experiments were conducted to explore the uptake of hADSCs-Exo by microglia cells and their impact on inflammatory responses. In vivo, an ICH mouse model was established, and the therapeutic effects of hADSCs-Exo were evaluated through neurological function scoring, histological staining, and immunofluorescence. Bioinformatics tools and experimental validation were employed to identify miRNAs targeting FPR1. hADSCs-Exo were efficiently taken up by microglia cells and exhibited anti-inflammatory effects by suppressing the release of inflammatory factors and promoting M1 to M2 transition. In the ICH mouse model, hADSCs-Exo significantly improved neurological function, reduced hemorrhage volume, decreased neuronal apoptosis, and regulated microglia polarization. miR-342-3p was identified as a potential regulator of FPR1 involved in the neuroprotective effects of hADSCs-Exo in ICH. hADSCs-Exo alleviate neuroinflammation in ICH through miR-342-3p-dependent targeting of FPR1, providing a new therapeutic strategy for ICH.

Nrdp1-mediated Macrophage Phenotypic Regulation Promotes Functional Recovery in Mice with Mild Neurological Impairment after Intracerebral Hemorrhage.

Neuregulin receptor degradation protein 1 (Nrdp1) is a ring finger E3 ubiquitin ligase involved in some inflammation through ubiquitination, including macrophage polarization following cerebral hemorrhage. However, there is limited understanding regarding the mechanisms through which Nrdp1 modulates macrophage polarization and the potential impact of this modulation on neurological function. Using stereotactic injection and adenoviral transfection techniques, the corresponding animal models were constructed through injecting adenovirus, saline, or blood into the mouse striatum at different periods of time in this research. The alteration in the ratio of various M1/M2 phenotype-associated markers (e.g., CD86, CD206, IL-6, IL-10, etc.) was evaluated through immunohistochemistry, immunofluorescence, western blotting, and elisa assays. Additionally, neurological function scores and behavioral tests were utilized to evaluate changes in neurological function in mice after cerebral hemorrhage. Our results show that overexpression of Nrdp1 promotes the expression of a variety of M2 macrophage-associated markers and enhance transcriptional activity of arginase-1 (Arg1) protein through ubiquitination for early regulation M2 macrophage polarization. Additionally, Nrdp1 promotes hematoma absorption, increases IL-10 expression, inhibits inducible nitric oxide synthase (iNOS), IL-6, and TNF-α production, alleviates neurological impairment and brain edema, and accelerates functional recovery. These findings suggest that modulating macrophage polarization through Nrdp1 could be a therapeutic strategy for neurofunctional impairment in cerebral hemorrhage.

Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP-Activated Exchange Protein-1.

Mitochondrial dysfunction contributes to the development of secondary brain injury (SBI) following intracerebral hemorrhage (ICH) and represents a promising therapeutic target. Celastrol, the primary active component of Tripterygium wilfordii, is a natural product that exhibits mitochondrial and neuronal protection in various cell types. This study aims to investigate the neuroprotective effects of celastrol against ICH-induced SBI and explore its underlying mechanisms. Celastrol improves neurobehavioral and cognitive abilities in mice with autologous blood-induced ICH, reduces neuronal death in vivo and in vitro, and promotes mitochondrial function recovery in neurons. Single-cell nuclear sequencing reveals that the cyclic adenosine monophosphate (cAMP)/cAMP-activated exchange protein-1 (EPAC-1) signaling pathways are impacted by celastrol. Celastrol binds to cNMP (a domain of EPAC-1) to inhibit its interaction with voltage-dependent anion-selective channel protein 1 (VDAC1) and blocks the opening of mitochondrial permeability transition pores. After neuron-specific knockout of EPAC1, the neuroprotective effects of celastrol are diminished. In summary, this study demonstrates that celastrol, through its interaction with EPAC-1, ameliorates mitochondrial dysfunction in neurons, thus potentially improving SBI induced by ICH. These findings suggest that targeting EPAC-1 with celastrol can be a promising therapeutic approach for treating ICH-induced SBI.

Txnrd2 Attenuates Early Brain Injury by Inhibition of Oxidative Stress and Endoplasmic Reticulum Stress via Trx2/Prx3 Pathway after Intracerebral Hemorrhage in Rats.

Thioredoxin-reductase 2 (Txnrd2) belongs to the thioredoxin-reductase family of selenoproteins and is a key antioxidant enzyme in mammalian cells to regulate redox homeostasis. Here, we reported that Txnrd2 exerted a major influence in brain damage caused by Intracerebral hemorrhage (ICH) by suppressing endoplasmic reticulum (ER) stress oxidative stress and via Trx2/Prx3 pathway. Furthermore, we demonstrated that pharmacological selenium (Se) rescued the brain damage after ICH by enhancing Txnrd2 expression. Primarily, expression and localization of Txnrd2, Trx2 and Prx3 were determined in collagenase IV-induced ICH model. Txnrd2 was then knocked down using siRNA interference in rats which were found to develop more severe encephaledema and neurological deficits. Mechanistically, we observed that loss of Txnrd2 leads to increased lipid peroxidation levels and ER stress protein expression in neurons and astrocytes. Additionally, it was revealed that Se effectively restored the expression of Txnrd2 in brain and inhibited both the activity of ER stress protein activity and the generation of reactive oxygen species (ROS) by promoting Trx2/Prx3 kilter when administrating sodium selenite in lateral ventricle. This study shed light on the effect of Txnrd2 in regulating oxidative stress and ER stress via Trx2/Prx3 pathway upon ICH and its promising potential as an ICH therapeutic target.