ICH-induced Secondary Brain Injury Research Articles

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-1β. 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.

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

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. 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. 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. 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.

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.

Celastrol Alleviates Mitochondrial Oxidative Stress and Brain Injury After Intracerebral Hemorrhage by Promoting OPA1-Dependent Mitochondrial Fusion

Mitochondrial oxidative stress is one of the characteristics of secondary brain injury (SBI) after intracerebral hemorrhage (ICH), contributing largely to the apoptosis of neurons. Celastrol, a quinone methide triterpene that possesses antioxidant and mitochondrial protective properties, has emerged as a neuroprotective agent. However, the activity of celastrol has not been tested in ICH-induced SBI. In this study, we found that celastrol could effectively alleviate neurological function deficits and reduce brain oedema and neuronal apoptosis caused by ICH. Through electron microscopy, we found that celastrol could significantly attenuate mitochondrial morphology impairment. Therefore, we tested the regulatory proteins of mitochondrial dynamics and found that celastrol could reverse the downwards trend of OPA1 expression after ICH. In view of this, by culturing OPA1-deficient primary neurons and constructing neuron-specific OPA1 conditional knockout mice, we found that the protective effects of celastrol on mitochondrial morphology and function after ICH were counteracted in the absence of OPA1. Further experiments also showed that OPA1 is indispensable for the protective effects of celastrol on ICH-induced secondary brain injury. In summary, we have demonstrated that celastrol is a potential drug for the treatment of ICH and have revealed a novel mechanism by which celastrol exerts its antioxidant effects by promoting OPA1-mediated mitochondrial fusion.

Transient Receptor Potential Mucolipin-1 Participates in Intracerebral Hemorrhage-Induced Secondary Brain Injury by Inducing Neuroinflammation and Neuronal Cell Death.

Transient receptor potential mucolipin-1 (TRPML1) is the most abundantly and widely expressed channel protein in the TRP family. While numerous studies have been conducted involving many aspects of TRPML1, such as its role in cell biology, oncology, and neurodegenerative diseases, there are limited reports about what role it plays in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI). Here we examined the function of TRPML1 in ICH-induced SBI. The caudal arterial blood of rats was injected into the caudate nucleus of basal ganglia to establish an experimental ICH model. We observed that lentivirus downregulated the expression level of TRPML1 and chemical agonist promoted the enzyme activity of TRPML1. The results indicated that the protein levels of TRPML1 in brain tissues increased 24h after ICH. These results suggested that downregulated TRPML1 could significantly reduce inflammatory cytokines, and ICH induced the production of LDH and ROS. Furthermore, TRPML1 knockout relieved ICH-induced neuronal cell death and degeneration, and declines in learning and memory after ICH could be improved by downregulating the expression of TRPML1. In addition, chemical agonist-expressed TRPML1 showed the opposite effect and exacerbated SBI after ICH. In summary, this study demonstrated that TRPML1 contributed to brain injury after ICH, and downregulating TRPML1 could improve ICH-induced SBI, suggesting a potential target for ICH therapy.

Autophagy regulates inflammation in intracerebral hemorrhage: Enemy or friend?

Intracerebral hemorrhage (ICH) is the second-largest stroke subtype and has a high mortality and disability rate. Secondary brain injury (SBI) is delayed after ICH. The main contributors to SBI are inflammation, oxidative stress, and excitotoxicity. Harmful substances from blood and hemolysis, such as hemoglobin, thrombin, and iron, induce SBI. When cells suffer stress, a critical protective mechanism called "autophagy" help to maintain the homeostasis of damaged cells, remove harmful substances or damaged organelles, and recycle them. Autophagy plays a critical role in the pathology of ICH, and its function remains controversial. Several lines of evidence demonstrate a pro-survival role for autophagy in ICH by facilitating the removal of damaged proteins and organelles. However, many studies have found that heme and iron can aggravate SBI by enhancing autophagy. Autophagy and inflammation are essential culprits in the progression of brain injury. It is a fascinating hypothesis that autophagy regulates inflammation in ICH-induced SBI. Autophagy could degrade and clear pro-IL-1β and apoptosis-associated speck-like protein containing a CARD (ASC) to antagonize NLRP3-mediated inflammation. In addition, mitophagy can remove endogenous activators of inflammasomes, such as reactive oxygen species (ROS), inflammatory components, and cytokines, in damaged mitochondria. However, many studies support the idea that autophagy activates microglia and aggravates microglial inflammation via the toll-like receptor 4 (TLR4) pathway. In addition, autophagy can promote ICH-induced SBI through inflammasome-dependent NLRP6-mediated inflammation. Moreover, some resident cells in the brain are involved in autophagy in regulating inflammation after ICH. Some compounds or therapeutic targets that regulate inflammation by autophagy may represent promising candidates for the treatment of ICH-induced SBI. In conclusion, the mutual regulation of autophagy and inflammation in ICH is worth exploring. The control of inflammation by autophagy will hopefully prove to be an essential treatment target for ICH.

The role of Tenascin C in intracerebral hemorrhage-induced secondary brain injury in rats via induction of neuronal cell death and neuroinflammation

BackgroundSpontaneous intracerebral hemorrhage (ICH) is a major cause of stroke that causes high rates of disability and mortality in adults. Tenascin C (TNC) protein, one of the matricellular proteins associated with platelet-derived growth factor receptor (PDGFR) activation, has been reported to induce neuronal apoptosis. However, the role and underlying mechanisms of TNC in ICH-induced secondary brain injury (SBI) have not yet been fully explained. The main purpose of this study was to explore the role of TNC and its potential mechanisms in ICH. MethodsAn ICH model was established by injecting autologous blood into the right basal ganglia in male Sprague Dawley (SD) rats, and imatinib, an inhibitor of PDGFR, was used to inhibit the release of TNC. ResultsWe found that TNC protein was significantly increased in the brain tissues after ICH and expressed in both neurons and microglia. We also found that the TNC level was elevated in the cerebrospinal fluid (CSF) after ICH. Additionally, we observed that the infiltration of activated microglia and the release of TNFα and IL-1β induced by ICH were decreased after inhibition of the protein levels of TNC and cleaved-TNC by a chemical inhibitor (imatinib). Furthermore, imatinib improved neuronal cell death and neurobehavioral abnormalities induced by ICH. ConclusionIn summary, our study revealed that TNC protein plays an important role in ICH-induced SBI, and inhibition of TNC could alleviate ICH-induced neuroinflammation, neuronal cell death, and neurobehaviour. Therefore, TNC may be a potential therapeutic target for ICH-induced SBI.

BMAL1 attenuates intracerebral hemorrhage-induced secondary brain injury in rats by regulating the Nrf2 signaling pathway.

BackgroundIntracerebral hemorrhage (ICH) is a severe cerebrovascular disease with high morbidity and mortality rates. Oxidative stress and inflammation are important pathological mechanisms of secondary brain injury (SBI) after ICH. Brain and muscle Arnt-like protein 1 (BMAL1), which forms the core component of the circadian clock, was previously shown to be involved in many diseases and to participate in oxidative stress and inflammatory responses. However, the role of BMAL1 in SBI following ICH is unknown. In addition, treatments targeting miR-155 and its downstream signaling pathway may exert a beneficial effect on SBI after ICH, while miR-155 may regulate Bmal1 mRNA stability and translation. Nevertheless, researchers have not clearly determined whetheantagomir-155 upregulates BMAL1 expression and subsequently attenuates ICH-induced brain injury in rats.MethodsAfter establishing an ICH rat model by injecting autologous blood, the time course of changes in levels of the BMAL1 protein after ICH was analyzed. Subsequently, this study was designed to investigate the potential role and mechanisms of BMAL1 in SBI following ICH using lentiviral overexpression and antagomir-155 treatments.ResultsBMAL1 protein levels were significantly decreased in the ICH group compared to the sham group. Genetic overexpression of BMAL1 alleviated oxidative stress, inflammation, brain edema, blood-brain barrier injury, neuronal death, and neurological dysfunction induced by ICH. On the other hand, we observed that inhibiting miRNA-155 using antagomir-155 promoted the expression of BMAL1 and further activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway to attenuate brain injury after ICH.ConclusionsThese results reveal that BMAL1 serves as a neuroprotective agent in ICH and upregulation of BMAL1 attenuates ICH-induced SBI. Therefore, BMAL1 may be a promising therapeutic target for SBI following ICH.

Fraxinellone ameliorates intracerebral hemorrhage-induced secondary brain injury by regulating Krüppel-like transcription factor 2 expression in rats

Damage to the blood–brain barrier (BBB) is an important factor leading to intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI). Krüppel-like transcription factor 2 (KLF2) plays an important role in the maintenance of the BBB. This study aims to detect the changes of KLF2 after ICH and evaluate the potential effects of fraxinellone on ICH-induced SBI and its correlation with KLF2. An ICH model was established by injecting autologous blood into the right basal ganglia of Sprague-Dawley (SD) rats. First, after ICH induction, the protein levels of KLF2 were reduced. Then, we found that the decrease of KLF2 protein levels induced by ICH could be effectively reversed with the treatment of fraxinellone in vascular endothelial cells. Furthermore, fraxinellone treatment effectively alleviated brain edema, decreased the levels of TNF-α and IL-1β, and improved neuronal cell degeneration induced by ICH. Meanwhile, fraxinellone ameliorated neurobehavioral disorders, motor and sensory impairments, and neurobehavioral disorders and memory loss caused by ICH. Collectively, these findings reveal that KLF2 may be a potential target for fraxinellone to exert neuroprotective effects after ICH, and fraxinellone could be a potential therapeutic agent for SBI after ICH.

Loss of MIC60 Aggravates Neuronal Death by Inducing Mitochondrial Dysfunction in a Rat Model of Intracerebral Hemorrhage.

Mitochondrial damage has been reported to be a critical factor for secondary brain injury (SBI) induced by intracerebral hemorrhage (ICH). MIC60 is a key element of the mitochondrial contact site and cristae junction organizing system (MICOS), which takes a principal part in maintaining mitochondrial structure and function. The role of MIC60 and its underlying mechanisms in ICH-induced SBI are not clear, which will be investigated in this present study. To establish and emulate ICH model in vivo and in vitro, autologous blood was injected into the right basal ganglia of Sprague-Dawley (SD) rats; and primary-cultured cortical neurons were treated by oxygen hemoglobin (OxyHb). First, after ICH induction, mitochondria were damaged and exhibited mitochondrial crista-structure remodeling, and MIC60 protein levels were reduced. Furthermore, MIC60 overexpression reduced ICH-induced neuronal death both in vivo and in vitro. In addition, MIC60 upregulation reduced ICH-induced cerebral edema, neurobehavioral impairment, and cognitive dysfunction; by contrast, MIC60 knockdown had the opposite effect. Additionally, in primary-cultured neurons, MIC60 overexpression could reverse ICH-induced neuronal cell death and apoptosis, mitochondrial membrane potential collapse, and decrease of mitophagy, indicating that MIC60 overexpression can maintain the integrity of mitochondrial structures. Moreover, loss of MIC60 is after ICH-induced reduction in PINK1 levels and mislocalization of Parkin in primary-cultured neurons. Taken together, our findings suggest that MIC60 plays an important role in ICH-induced SBI and may represent a promising target for ICH therapy.

Metabolic Insight Into the Neuroprotective Effect of Tao-He-Cheng-Qi (THCQ) Decoction on ICH Rats Using Untargeted Metabolomics

Tao-He-Cheng-Qi decoction (THCQ) is an effective traditional Chinese medicine used to treat intracerebral hemorrhage (ICH). This study was performed to investigate the possible neuroprotective effect of THCQ decoction on secondary brain damage in rats with intracerebral hemorrhage and to elucidate the potential mechanism based on a metabolomics approach. Sprague-Dawley (SD) rats were randomly divided into five groups: the sham group, collagenase-induced ICH model group, THCQ low-dose (THCQ-L)-treated group, THCQ moderate-dose (THCQ-M)-treated group and THCQ high-dose (THCQ-H)-treated group. Following 3 days of treatment, behavioral changes and histopathological lesions in the brain were estimated. Untargeted metabolomics analysis with multivariate statistics was performed by using ultrahigh-performance liquid chromatography–mass spectrometry (UPLC-Q-Exactive Orbitrap MS). THCQ treatment at two dosages (5.64 and 11.27 g/kg·d) remarkably improved behavior (p < 0.05), brain water content (BMC) and hemorheology (p < 0.05) and improved brain nerve tissue pathology and inflammatory infiltration in ICH rats. Moreover, a metabolomic analysis demonstrated that the serum metabolic profiles of ICH patients were significantly different between the sham group and the ICH-induced model group. Twenty-seven biomarkers were identified that potentially predict the clinical benefits of THCQ decoction. Of these, 4 biomarkers were found to be THCQ-H group-specific, while others were shared between two clusters. These metabolites are mainly involved in amino acid metabolism and glutamate-mediated cell excitotoxicity, lipid metabolism-mediated oxidative stress, and mitochondrial dysfunction caused by energy metabolism disorders. In addition, a correlation analysis showed that the behavioral scores, brain water content and hemorheology were correlated with levels of serum metabolites derived from amino acid and lipid metabolism. In conclusion, the results indicate that THCQ decoction significantly attenuates ICH-induced secondary brain injury, which could be mediated by improving metabolic disorders in cerebral hemorrhage rats.

Neurovascular Units and Neural-Glia Networks in Intracerebral Hemorrhage: from Mechanisms to Translation.

Intracerebral hemorrhage (ICH), the most lethal type of stroke, often leads to poor outcomes in the clinic. Due to the complex mechanisms and cell-cell crosstalk during ICH, the neurovascular unit (NVU) was proposed to serve as a promising therapeutic target for ICH research. This review aims to summarize the development of pathophysiological shifts in the NVU and neural-glia networks after ICH. In addition, potential targets for ICH therapy are discussed in this review. Beyond cerebral blood flow, the NVU also plays an important role in protecting neurons, maintaining central nervous system (CNS) homeostasis, coordinating neuronal activity among supporting cells, forming and maintaining the blood-brain barrier (BBB), and regulating neuroimmune responses. During ICH, NVU dysfunction is induced, along with neuronal cell death, microglia and astrocyte activation, endothelial cell (EC) and tight junction (TJ) protein damage, and BBB disruption. In addition, it has been shown that certain targets and candidates can improve ICH-induced secondary brain injury based on an NVU and neural-glia framework. Moreover, therapeutic approaches and strategies for ICH are discussed.

Proteomic Analysis of Perihematoma Tissue from Patients with Intracerebral Hemorrhage Using iTRAQ-Based Quantitative Proteomics.

Intracerebral hemorrhage is a complicated disorder with limited proven prognostic and therapeutic targets and elusive mechanisms. With proteomic methods, we aimed to explore the global protein expression profile of perihematomal tissue from ICH patients and identify potential pathophysiological pathways and protein markers. Using iTRAQ-labeling quantitative proteomics technology, four ICH brain sample and four non-ICH brain samples were analyzed. Among the 3740 quantifiable proteins, 884 were dysregulated in the patients compared to those in the controls (p < 0.05). After bioinformatics analysis, the differentially expressed proteins were found to be mostly involved in hemostatic processes, nutrient metabolism signaling pathways, and antioxidation pathways. Moreover, fibronectin 1 was revealed to be at the center of the protein-protein interaction networks. In summary, the potential pathways and brain protein markers that could potentially be used to predict the prognosis of ICH were obtained from the altered proteomic profile of perihematomal tissue. Thus, these data may yield novel insights into the mechanisms of ICH-induced secondary brain injury.

Inhibition of lysophosphatidic acid receptor 1 attenuates neuroinflammation via PGE2/EP2/NOX2 signalling and improves the outcome of intracerebral haemorrhage in mice

Lysophosphatidic acid receptor 1 (LPA1) plays a critical role in proinflammatory processes in the central nervous system by modulating microglia activation. The aim of this study was to explore the anti-inflammatory effects and neurological function improvement of LPA1 inhibition after intracerebral haemorrhage (ICH) in mice and to determine whether prostaglandin E2 (PGE2), E-type prostaglandin receptor 2 (EP2), and NADPH oxidase 2 (NOX2) signalling are involved in LPA1-mediated neuroinflammation. ICH was induced in CD1 mice by autologous whole blood injection. AM966, a selective LPA1 antagonist, was administered by oral gavage 1 h and 12 h after ICH. The LPA1 endogenous ligand, LPA was administered to verify the effect of LPA1 activation. To elucidate potential inflammatory mechanisms of LPA1, the selective EP2 activator butaprost was administered by intracerebroventricular injection with either AM966 or LPA1 CRISPR knockout (KO). Water content of the brain, neurobehavior, immunofluorescence staining, and western blot were performed. After ICH, EP2 was expressed in microglia whereas LPA1 was expressed in microglia, neurons, and astrocytes, which peaked after 24 h. AM966 inhibition of LPA1 improved neurologic function, reduced brain oedema, and suppressed perihematomal inflammatory cells after ICH. LPA administration aggravated neurological deficits after ICH. AM966 treatment and LPA1 CRISPR KO both decreased the expressions of PGE2, EP2, NOX2, NF-κB, TNF-α, IL-6, and IL-1β expressions after ICH, which was reversed by butaprost. This study demonstrated that inhibition of LPA1 attenuated neuroinflammation caused by ICH via PGE2/EP2/NOX2 signalling pathway in mice, which consequently improved neurobehavioral functions and alleviated brain oedema. LPA1 may be a promising therapeutic target to attenuate ICH-induced secondary brain injury.

RAB7L1 Participates in Secondary Brain Injury Induced by Experimental Intracerebral Hemorrhage in Rats.

RAB7, a member of RAS oncogene family-like 1 (RAB7L1), is a GTPase belonging to the Rab family and acts as an upstream regulator to regulate the kinase activity of leucine-rich repeat kinase 2 (LRRK2). Although LRRK2 has been shown to aggravate secondary brain injury (SBI) after intracerebral hemorrhage (ICH), it is unknown whether RAB7L1 is also involved in this process. The purpose of the present study was to investigate the role of RAB7L1 in ICH-induced SBI in vivo. Autologous blood was injected into adult male Sprague-Dawley rats to induce an ICH model in vivo. The results showed that the protein levels of RAB7L1 increased after ICH. Overexpression of RAB7L1 induced neuronal apoptosis and damage, as demonstrated by TUNEL-positive and FJB-positive cells, and exacerbated ICH-induced learning and cognitive dysfunctions; in contrast, downregulation of RAB7L1 via RNA interference yielded comparatively opposite changes in these parameters. In summary, this study demonstrates that RAB7L1 promotes SBI after ICH and may represent a potential target for ICH therapy.

Activated WNK3 induced by intracerebral hemorrhage deteriorates brain injury maybe via WNK3/SPAK/NKCC1 pathway

BackgroundIntracerebral hemorrhage (ICH) is the common brain diseases in middle-aged and elderly people, with high disability and/or mortality rate, and is a serious public health concern. Both WNK3 kinase and the WNK3/SPAK/NKCC1 signaling pathway play an integral role in maintaining normal cell homeostasis. However, their role and underlying mechanisms in ICH-induced secondary brain injury (SBI) have yet to be elucidated. MethodsWe established an ICH model using male Sprague-Dawley (SD) rats by injecting autologous arterial blood into the unilateral basal ganglia. To establish ICH model in vitro, oxyhemoglobin (OxyHb; 20 μM) and neurons were cultured for 6 h at 37 °C, 5% CO2 atmosphere. To investigate the role of WNK3 and the WNK3/SPAK/NKCC1 signaling pathway in SBI, after genetic interventions, rotation and water maze test, brain edema and neuroinflammation were detected, and terminal-deoxynucleoitidyl transferase mediated dUTP nick end labeling (TUNEL), Fluoro-Jade C (FJC), and Nissl staining were performed. ResultsOur data showed that WNK3 expression in brain tissue were upregulated after ICH induction. In addition, silencing of WNK3 reduced neuronal apoptosis, and inflammatory responses in rats that underwent ICH. Inhibition of WNK3 expression reduced the damaged blood-brain barrier (BBB), alleviated the impaired degree of cerebral edema, and improved disruptive neurobehavioral cognition caused by ICH. Moreover, overexpression of WNK3 had the opposite effects. Finally, WNK3/SPAK/NKCC1 signaling pathway may be involved in the above-mentioned processes. ConclusionsIn conclusion, our findings showed that WNK3 and WNK3/SPAK/NKCC1 signaling pathway play a vital biological function in ICH-induced SBI. Depletion of WNK3 attenuated brain injury after ICH both in vivo and in vitro. Thus, WNK3 and WNK3/SPAK/NKCC1 signaling pathway are potential targets for treating SBI after ICH.

Miro1 Regulates Neuronal Mitochondrial Transport and Distribution to Alleviate Neuronal Damage in Secondary Brain Injury After Intracerebral Hemorrhage in Rats.

Intracerebral hemorrhage (ICH) is a primary cause of death and disability in adults worldwide. Secondary brain injury (SBI) induced by ICH can lead to impaired mitochondrial function, which ultimately contributes to apoptosis and necrosis. Mitochondrial Rho GTPase 1 (Miro1) is a key regulator of mitochondrial movement and motor protein binding. Although Miro1 has been demonstrated to be implicated in various types of central nervous system damage, its potential effect on ICH-induced SBI has not been studied in detail. Hence, in the present new study, we explored the effect of Miro1 on SBI in vivo and in vitro. Self-body heart blood was injected into the right basal ganglia of the rat brain in vivo. Meanwhile, our in vitro model of ICH was based on the stimulation of oxygen hemoglobin (OxyHb) to neurons. Then, Miro1 was overexpressed both in the brains of rats after ICH in vivo and in OxyHb-treated cultured neurons in vitro. Miro1 overexpression in vivo reduced several pathological indexes such as brain edema, neurobehavioral impairment, and neuronal death. Immunofluorescent staining in vitro showed that overexpression of Miro1 ameliorated neuronal damage via facilitation of mitochondrial transport and distribution. JC-1 staining indicated that overexpression of Miro1 reduced the collapse of mitochondrial membrane potential and enhanced mitochondrial mass. Additionally, live-dead cellular staining and flow cytometry analysis revealed that Miro1 overexpression in cultured neurons reduced both necrotic and apoptotic rates. In contrast, inhibition of Miro1 expression yielded opposite effects to those of Miro1 overexpression. Above all, the upregulation of Miro1 significantly alleviated pathological symptoms on SBI in vivo and in vitro.

Luteolin alleviates neuroinflammation via downregulating the TLR4/TRAF6/NF-κB pathway after intracerebral hemorrhage

The activation of microglia and inflammatory responses is essential for the process of intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI). In this study, we investigated the effects of luteolin on ICH-induced SBI and the potential mechanisms. Autologous blood was injected to establish the ICH model in vivo, and oxyhemoglobin (OxyHb) was used to mimic the ICH model in vitro. We found that the administration of luteolin significantly improved motor and sensory impairments and inhibited neuronal cell degeneration in vivo. In the in vitro study, the decrease of the neuronal cell viability induced by activated microglia was alleviated by luteolin treatment. Furthermore, by antagonizing the activation of the Toll-like receptor 4 (TLR4)/TNF receptor-associated factor 6 (TRAF6)/nuclear transcription factor-κB (NF-κB) signaling pathway, the ICH-induced elevation of cytokine release was decreased after treatment with luteolin, which was confirmed both in vivo and in vitro. Additionally, we found that luteolin engaged with TRAF6 and inhibited the ubiquitination of TRAF6. Taken together, our findings demonstrate the neuroprotective effects of luteolin after ICH and the potential mechanisms, which suggest that luteolin is a potential therapeutic candidate for ICH treatment.

Luteolin Exerts Neuroprotection via Modulation of the p62/Keap1/Nrf2 Pathway in Intracerebral Hemorrhage

Upregulation of neuronal oxidative stress is involved in the progression of secondary brain injury (SBI) following intracerebral hemorrhage (ICH). In this study, we investigated the potential effects and underlying mechanisms of luteolin on ICH-induced SBI. Autologous blood and oxyhemoglobin (OxyHb) were used to establish in vivo and in vitro models of ICH, respectively. Luteolin treatment effectively alleviated brain edema and ameliorated neurobehavioral dysfunction and memory loss in vivo. Also, in vivo, we found that luteolin promoted the activation of the sequestosome 1 (p62)/kelch‐like enoyl-coenzyme A hydratase (ECH)‐associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by enhancing autophagy and increasing the translocation of Nrf2 to the nucleus. Meanwhile, luteolin inhibited the ubiquitination of Nrf2 and increased the expression levels of downstream antioxidant proteins, such as heme oxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide phosphate (NADPH): quinine oxidoreductase 1 (NQO1). This effect of luteolin was also confirmed in vitro, which was reversed by the autophagy inhibitor, chloroquine (CQ). Additionally, we found that luteolin inhibited the production of neuronal mitochondrial superoxides (MitoSOX) and alleviated neuronal mitochondrial injury in vitro, as indicated via tetrachloro-tetraethylbenzimidazol carbocyanine-iodide (JC-1) staining and MitoSOX staining. Taken together, our findings demonstrate that luteolin enhances autophagy and anti-oxidative processes in both in vivo and in vitro models of ICH, and that activation of the p62-Keap1-Nrf2 pathway, is involved in such luteolin-induced neuroprotection. Hence, luteolin may represent a promising candidate for the treatment of ICH-induced SBI.

Ghrelin attenuates secondary brain injury following intracerebral hemorrhage by inhibiting NLRP3 inflammasome activation and promoting Nrf2/ARE signaling pathway in mice

Ghrelin, a brain-gut peptide, has been proven to exert neuroprotection in different kinds of neurological diseases; however, its role and the potential molecular mechanisms in secondary brain injury (SBI) after intracerebral hemorrhage (ICH) are still unknown. In this study, we investigate whether treatment with ghrelin may attenuate SBI in a murine ICH model, and if so, whether the neuroprotective effects are due to the inhibition of nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation and promotion of nuclear factor-E2-related factor 2 (Nrf2)/antioxidative response element (ARE) signaling pathway. Stereotactically intrastriatal infusion of autologous blood was performed to mimic ICH. Ghrelin was given intraperitoneally immediately following ICH and again 1 h later. Results showed that ghrelin attenuated neurobehavioral deficits, brain edema, hematoma volume, and perihematomal cell death post-ICH. Ghrelin inhibited the NLRP3 inflammasome activation and subsequently suppressed the neuroinflammatory response as evidenced by reduced microglia activation, neutrophil infiltration, and pro-inflammatory mediators release after ICH. Additionally, ghrelin alleviated ICH-induced oxidative stress according to the chemiluminescence of luminol and lucigenin, malondialdehyde (MDA) content, and total superoxide dismutase (SOD) activity assays. These changes were accompanied by upregulation of Nrf2 expression, Nrf2 nuclear accumulation, and enhanced Nrf2 DNA binding activity, as well as by increased expressions of Nrf2 downstream target antioxidative genes, including NAD(P)H quinine oxidoreductase-1 (NQO1), glutathione cysteine ligase regulatory subunit (GCLC), and glutathione cysteine ligase modulatory subunit (GCLM). Together, our data suggested that ghrelin protected against ICH-induced SBI by inhibiting NLRP3 inflammasome activation and promoting Nrf2/ARE signaling pathway.