Huperzine A ameliorates neurological deficits after spontaneous subarachnoid hemorrhage through endothelial cell pyroptosis inhibition.

Spontaneous subarachnoid hemorrhage (SAH) is a kind of hemorrhagic stroke which causes neurological deficits in survivors. Huperzine A has a neuroprotective effect, but its role in SAH is unclear. Therefore, we explore the effect of Huperzine A on neurological deficits induced by SAH and the related mechanism. In this study, Evans blue assay, TUNEL staining, immunofluorescence, western blot analysis, and ELISA are conducted. We find that Huperzine A can improve neurological deficits and inhibit the apoptosis of nerve cells in SAH rats. Huperzine A treatment can improve the upregulation of brain water content, damage of blood-brain barrier, fibrinogen and matrix metalloprotein 9 expressions and the downregulation of ZO-1 and occludin expressions induced by SAH. Huperzine A inhibit the expressions of proteins involved in pyroptosis in endothelial cells in SAH rats. The increase in MDA content and decrease in SOD activity in SAH rats can be partly reversed by Huperzine A. The ROS inducer H 2O 2 can induce pyroptosis and inhibit the expressions of ZO-1 and occludin in endothelial cells, which can be blocked by Huperzine A. In addition, the increase in the entry of p65 into the nucleus in endothelial cells can be partly reversed by Huperzine A. Huperzine A may delay the damage of blood-brain barrier in SAH rats by inhibiting oxidative stress-mediated pyroptosis and tight junction protein expression downregulation through the NF-κB pathway. Overall, Huperzine A may have clinical value for treating SAH.

Brain metabolism response to intrahospital transfers in neurocritical ill patients and the impact of microdialysis probe location.

Intrahospital transfer (IHT), a routine in the management of neurocritical patients requiring imaging or interventions, might affect brain metabolism. Studies about IHT effects using microdialysis (MD) have produced conflicting results. In these studies, only the most damaged hemisphere was monitored, and those may not reflect the impact of IHT on overall brain metabolism, nor do they address differences between the hemispheres. Herein we aimed to quantify the effect of IHT on brain metabolism by monitoring both hemispheres with bilateral MD. In this study, 27 patients with severe brain injury (10 traumatic brain injury and 17 subarachnoid hemorrhage patients) were included, with a total of 67 IHT. Glucose, glycerol, pyruvate and lactate were measured by MD in both hemispheres for 10 h pre- and post-IHT. Alterations in metabolite levels after IHT were observed on both hemispheres; although these changes were more marked in hemisphere A (most damaged) than B (less damaged). Our results suggest that brain metabolism is altered after an IHT of neurocritical ill patients particularly but not limited to the damaged hemisphere. Bilateral monitorization may be more sensitive than unilateral monitorization for detecting metabolic disturbances not directly related to the course of the disease.

LPCAT3 exacerbates early brain injury and ferroptosis after subarachnoid hemorrhage in rats.

AIMS: Lysophosphatidylcholine acyltransferase 3 (LPCAT3) is known to play a pivotal role in lipid metabolism, but its role in the early brain injury (EBI) following subarachnoid hemorrhage (SAH) remains unclear. This study provides insights into LPCAT3 expression alterations and functional implications in EBI following SAH. METHODS: SAH models of adult male Sprague-Dawley (SD) rats were established by intravascular perforation. Lentivirus vectors were administered by intracerebroventricular injection (i.c.v.) to either induce LPCAT3 overexpression or knockdown 14 days before SAH induction. Western blot, immunofluorescence, Nissl staining, MDA detection, ROS detection, iron content detection, and short-term and long-term neurobehavioral tests were performed to investigate the effects of regulated-LPCAT3 after SAH. RESULTS: LPCAT3 levels were found to be significantly elevated in SAH. Suppression of LPCAT3 expression via shRNA improved oxidative stress, reduced brain edema, alleviated behavioral and cognitive deficits following SAH and decreased neuronal death, while upregulating LPCAT3 expression showed opposing effects. CONCLUSION: LPCAT3 is involved in SAH-induced EBI and associated with ferroptosis. Our findings provide a referential basis for potential therapeutic interventions aimed at alleviating EBI following SAH.

Maresin 1 Activates LGR6 to Alleviate Neuroinflammation via the CREB/JMJD3/IRF4 Pathway in a Rat Model of Subarachnoid Hemorrhage.

Neuroinflammation is an early event of brain injury after subarachnoid hemorrhage (SAH). Whether the macrophage mediators in resolving inflammation 1 (MaR1) is involved in SAH pathogenesis is unknown. In this study, 205 male Sprague-Dawley rats were subjected to SAH via endovascular perforation in the experimental and control groups. MaR1 was dosed intranasally at 1 h after SAH, with LGR6 siRNA and KG-501, GSK-J4 administered to determine the signaling pathway. Neurobehavioral, histological and biochemical data were obtained from the animal groups with designated treatments. The results showed: (i) The leucine-rich repeat containing G protein-coupled receptor 6 (LGR6) was decreased after SAH and reached to the lowest level at 24 h after SAH. Jumonji d3 (JMJD3) protein levels tended to increase and peaked at 24 h after SAH. LGR6 and JMJD3 expression were co-localized with microglia. (ii) MaR1 administration mitigated short-term neurological deficits, brain edema and long-term neurobehavioral performance after SAH, and attenuated microglial activation and neutrophil infiltration. (iii) Knockdown of LGR6, inhibition of CREB phosphorylation or JMJD3 activity abolished the anti-neuroinflammatory effect of MaR1 on the expression of CREB, CBP, JMJD3, IRF4, IRF5, IL-1ß, IL-6 and IL-10, thus prevented microglial activation and neutrophil infiltration. Together, the results show that MaR1 can activate LGR6 and affect CREB/JMJD3/IRF4 signaling to attenuate neuroinflammation after SAH, pointing to a potential pharmacological utility in this disorder.

Alpha-lipoic acid (ALA) ameliorates early brain injury after subarachnoid hemorrhage in Sprague-Dawley (SD) rats via inhibiting STING-NLRP3 inflammatory signaling.

Neuroinflammation is intimately associated with poor prognosis in patients with subarachnoid hemorrhage (SAH). Alpha-lipoic acid (ALA), a disulfide antioxidant, has been shown to be neuroprotective in an in vivo model of neurological injury; however, the role of ALA in SAH has never been evaluated. In this study, the Sprague-Dawley rats SAH model was induced by endovascular perforation method. ALA was transplanted intravenously into rats, and SR-717, a stimulator of interferon genes (STING) agonist, was injected intraperitoneally. The effects of ALA on early brain injury were assayed by neurological score, hematoxylin and eosin staining and Nissl staining. Immunohistochemistry staining and Western blotting were used to analyze various proteins. ALA significantly reduced STING- NLRP3 protein expression and decreased cell death, which in turn mitigated the neurobehavioral dysfunction following SAH. Furthermore, coadministration of ALA and SR-717 promoted STING-NLRP3 signaling pathway activation following SAH, which reversed the inhibitory effect of ALA on STING-NLRP3 protein activation and increased the neurological deficits. In conclusion, ALA may be a promising therapeutic strategy for alleviating early brain injury after SAH.

Neuroprotection via Carbon Monoxide Depends on the Circadian Regulation of CD36-Mediated Microglial Erythrophagocytosis in Hemorrhagic Stroke.

The molecular basis for circadian dependency in stroke due to subarachnoid hemorrhagic stroke (SAH) remains unclear. We reasoned that microglial erythrophagocytosis, crucial for SAH response, follows a circadian pattern involving carbon monoxide (CO) and CD36 surface expression. The microglial BV-2 cell line and primary microglia (PMG) under a clocked medium change were exposed to blood ± CO (250 ppm, 1 h) in vitro. Circadian dependency and the involvement of CD36 were analyzed in PMG isolated from control mice and CD36-/- mice and by RNA interference targeting Per-2. In vivo investigations, including phagocytosis, vasospasm, microglia activation and spatial memory, were conducted in an SAH model using control and CD36-/- mice at different zeitgeber times (ZT). In vitro, the surface expression of CD36 and its dependency on CO and phagocytosis occurred with changed circadian gene expression. CD36-/- PMG exhibited altered circadian gene expression, phagocytosis and impaired responsiveness to CO. In vivo, control mice with SAH demonstrated circadian dependency in microglia activation, erythrophagocytosis and CO-mediated protection at ZT2, in contrast to CD36-/- mice. Our study indicates that circadian rhythmicity modulates microglial activation and subsequent CD36-dependent phagocytosis. CO altered circadian-dependent neuroprotection and CD36 induction, determining the functional outcome in a hemorrhagic stroke model. This study emphasizes how circadian rhythmicity influences neuronal damage after neurovascular events.

Investigation of the protective effects of piceatannol on experimental subarachnoid hemorrhage in rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is one of the most prevalent brain injuries in humans which has poor prognosis and high mortality rates. Due to several medical or surgical treatment methods, a gold standard method doesn't exist for SAH treatment. Piceatannol (PCN), a natural analog of resveratrol, was reported to reduce inflammation and apoptosis promising a wide range of therapeutic alternatives. In this study, we aimed to investigate the effects of PCN in an experimental SAH model. The alleviating effects of PCN in the hippocampus in an experimental SAH model were investigated for the first time. METHODS AND RESULTS: In this study, 27 Wistar Albino male rats (200-300 g; 7-8 week) were used. Animals were divided into three groups; SHAM, SAH, and SAH + PCN. SAH model was created with 120 µl of autologous arterial tail blood to prechiasmatic cisterna. 30 mg/kg PCN was administered intraperitoneally at 1st h after SAH. Neurological evaluation was performed with Garcia's score. RT-PCR was performed for gene expression levels in the hippocampus. Pyknosis, edema, and apoptosis were evaluated by H&E and TUNEL staining. Our results indicated that PCN administration reduced apoptosis (P < 0.01), cellular edema, and pyknosis (P < 0.05) in the hippocampus after SAH. Moreover, PCN treatment significantly decreased the expression levels of TNF-α (P < 0.01), IL-6 (P < 0.05), NF-κB (P < 0.05), and Bax (P < 0.05) in the hippocampus. CONCLUSIONS: Our results demonstrated that PCN might be a potential therapeutic adjuvant agent for the treatment of early brain injury (EBI) following SAH. Further studies are required to clarify the underlying mechanisms and treatment options of SAH.

Recombinant soluble form of receptor for advanced glycation end products ameliorates microcirculation impairment and neuroinflammation after subarachnoid hemorrhage.

Impaired cerebral microcirculation after subarachnoid hemorrhage (SAH) has been shown to be related to delayed ischemic neurological deficits (DIND). We previously demonstrated the involvement of the receptor for advanced glycation end products (RAGE) in the pathogenesis of SAH related neuronal death. In the present study, we aimed to investigate the therapeutic effects of a recombinant soluble form of RAGE (sRAGE) on microcirculation impairment following SAH. Intrathecal injection of autologous blood in rats, mixed primary astrocyte and microglia cultures exposed to hemolysates and endothelial cells â€‹(ECs) from human brain microvascular exposed to glia-conditioned medium or SAH patient's CSF were used as experimental SAH models in vivo and in vitro. The results indicated that intrathecal administration of recombinant sRAGE significantly ameliorated the vasoconstriction of cortical arterioles and associated perfusion impairment, brain edema, reduced cell death, endothelial dysfunction, and improved motor performance at 24 and 48 â€‹h after SAH induction in rats. The in vitro results further showed that recombinant sRAGE significantly reduced astrocyte swelling and microglia activation, in parallel with decreased mRNA expression levels of pro-inflammatory cytokines including interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in vitro. Moreover, the in vitro model of SAH-induced p-eNOS and eNOS suppression, along with stress fiber formation in brain microvascular ECs, was effectively reversed by sRAGE treatment and led to a decrease in cleaved-caspase 3 expression. In summary, recombinant sRAGE effectively lessened microcirculation impairment and vascular injury after SAH via the mechanism of anti-inflammation, which may provide a potential therapeutic strategy for SAH.

Inhibiting RIPK1-driven neuroinflammation and neuronal apoptosis mitigates brain injury following experimental subarachnoid hemorrhage.

RIPK1, a receptor-interacting serine/threonine protein kinase, plays a crucial role in maintaining cellular and tissue homeostasis by integrating inflammatory responses and cell death signaling pathways including apoptosis and necroptosis, which have been implicated in diverse physiological and pathological processes. Suppression of RIPK1 activation is a promising strategy for restraining the pathological progression of many human diseases. Neuroinflammation and neuronal apoptosis are two pivotal factors in the pathogenesis of brain injury following subarachnoid hemorrhage (SAH). In this study, we established in vivo and in vitro models of SAH to investigate the activation of RIPK1 kinase in both microglia and neurons. We observed the correlation between RIPK1 kinase activity and microglia-mediated inflammation as well as neuronal apoptosis. We then investigated whether inhibition of RIPK1 could alleviate neuroinflammation and neuronal apoptosis following SAH, thereby reducing brain edema and ameliorating neurobehavioral deficits. Additionally, the underlying mechanisms were also explored. Our research findings revealed the activation of RIPK1 kinase in both microglia and neurons following SAH, as marked by the phosphorylation of RIPK1 at serine 166. The upregulation of p-RIPK1(S166) resulted in a significant augmentation of inflammatory cytokines and chemokines, including TNF-α, IL-6, IL-1α, CCL2, and CCL5, as well as neuronal apoptosis. The activation of RIPK1 in microglia and neurons following SAH could be effectively suppressed by administration of Nec-1 s, a specific inhibitor of RIPK1. Consequently, inhibition of RIPK1 resulted in a downregulation of inflammatory cytokines and chemokines and attenuation of neuronal apoptosis after SAH in vitro. Furthermore, the administration of Nec-1 s effectively mitigated neuroinflammation, neuronal apoptosis, brain edema, and neurobehavioral deficits in mice following SAH. Our findings suggest that inhibiting RIPK1 kinase represents a promising therapeutic strategy for mitigating brain injury after SAH by attenuating RIPK1-driven neuroinflammation and neuronal apoptosis.

Nrf-2 modulates excitability of hippocampal neurons by regulating ferroptosis and neuroinflammation after subarachnoid hemorrhage in rats.

Excitability of hippocampal neurons in subarachnoid hemorrhage (SAH) rats has not been well studied. The rat SAH model was applied in this study to explore the role of nuclear factor E2-related factor (Nrf-2) in the early brain injury of SAH. The neural excitability of CA1 pyramidal cells (PCs) in SAH rats was evaluated by using electrophysiology experiments. Ferroptosis and neuroinflammation were measured by ELISA, transmission electron microscopy and western blotting. Our results indicated that SAH induced neurological deficits, brain edema, ferroptosis, neuroinflammation and neural excitability in rats. Ferrostatin-1 treatment significantly decreased the expression and distribution of IL-1ß, IL-6, IL-10, TGF-ß and TNF-α. Inhibiting ferroptosis by ferrostatin-1 can attenuate neural excitability, neurological deficits, brain edema and neuroinflammation in SAH rats. Inhibiting the expression of Nrf-2 significantly increased the neural excitability and the levels of IL-1ß, IL-6, IL-10, TGF-ß and TNF-α in Fer-1-treated SAH rats. Taken together, inhibiting the Nrf-2 induces early brain injury, brain edema and the inflammatory response with increasing of neural excitability in Fer-1-treated SAH rats. These results have indicated that inhibiting ferroptosis, neuroinflammation and neural excitability attenuates early brain injury after SAH by regulating the Nrf-2.

Anti-Apoptotic Effects of AMPA Receptor Antagonist Perampanel in Early Brain Injury After Subarachnoid Hemorrhage in Mice.

This study was aimed to investigate if acute neuronal apoptosis is induced by activation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors (AMPARs) and inhibited by a clinically available selective AMPAR antagonist and antiepileptic drug perampanel (PER) in subarachnoid hemorrhage (SAH), and if the mechanisms include upregulation of an inflammation-related matricellular protein periostin. Sham-operated and endovascular perforation SAH mice randomly received an administration of 3 mg/kg PER or the vehicle intraperitoneally. Post-SAH neurological impairments and increased caspase-dependent neuronal apoptosis were associated with activation of AMPAR subunits GluA1 and GluA2, and upregulation of periostin and proinflammatory cytokines interleukins-1ß and -6, all of which were suppressed by PER. PER also inhibited post-SAH convulsion-unrelated increases in the total spectral power on video electroencephalogram (EEG) monitoring. Intracerebroventricularly injected recombinant periostin blocked PER's anti-apoptotic effects on neurons. An intracerebroventricular injection of a selective agonist for GluA1 and GluA2 aggravated neurological impairment, neuronal apoptosis as well as periostin upregulation, but did not increase the EEG total spectral power after SAH. A higher dosage (10 mg/kg) of PER had even more anti-apoptotic effects compared with 3 mg/kg PER. Thus, this study first showed that AMPAR activation causes post-SAH neuronal apoptosis at least partly via periostin upregulation. A clinically available AMPAR antagonist PER appears to be neuroprotective against post-SAH early brain injury through the anti-inflammatory and anti-apoptotic effects, independent of the antiepileptic action, and deserves further study.

Deferoxamine Mitigates Ferroptosis and Inflammation in Hippocampal Neurons After Subarachnoid Hemorrhage by Activating the Nrf2/TXNRD1 Axis.

Ferroptosis is a distinct peroxidation-driven form of cell death tightly involved in subarachnoid hemorrhage (SAH). This study delved into the mechanism of deferoxamine (DFO, an iron chelator) in SAH-induced ferroptosis and inflammation. SAH mouse models were established by endovascular perforation method and injected intraperitoneally with DFO, or intraventricularly injected with the Nrf2 pathway inhibitor ML385 before SAH, followed by detection of neurological function, blood-brain barrier (BBB) permeability, and brain water content. Apoptotic level of hippocampal neurons, symbolic changes of ferroptosis, and levels of pro-inflammatory cytokines were assessed using TUNEL staining, Western blotting, colorimetry, and ELISA. The localization and expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) were detected. HT22 cells were exposed to Hemin as in vitro SAH models and treated with FIN56 to induce ferroptosis, followed by evaluation of the effects of DFO on FIN56-treated HT22 cells. The regulation of Nrf2 in thioredoxin reductase 1 (TXNRD1) was analyzed by co-immunoprecipitation and Western blotting. Moreover, HT22 cells were treated with DFO and ML385 to identify the role of DFO in the Nrf2/TXNRD1 axis. DFO extenuated brain injury, and ferroptosis and inflammation in hippocampal neurons of SAH mice. Nrf2 localized at the CA1 region of hippocampal neurons, and DFO stimulated nuclear translocation of Nrf2 protein in hippocampal neurons of SAH mice. Additionally, DFO inhibited ferroptosis and inflammatory responses in FIN56-induced HT22 cells. Nrf2 positively regulated TXNRD1 protein expression. Indeed, DFO alleviated FIN56-induced ferroptosis and inflammation via activation of the Nrf2/TXNRD1 axis. DFO alleviated neurological deficits, BBB disruption, brain edema, and brain injury in mice after SAH by inhibiting hippocampal neuron ferroptosis via the Nrf2/TXNRD1 axis. DFO ameliorates SAH-induced ferroptosis and inflammatory responses in hippocampal neurons by activating the Nrf2/TXNRD1 axis.

Menaquinone-4 attenuates ferroptosis by upregulating DHODH through activation of SIRT1 after subarachnoid hemorrhage.

BACKGROUND: Menaquinone-4(MK-4), the isoform of vitamin K2 in the brain, exerts neuroprotective effects against a variety of central nervous system disorders. This study aimed to demonstrate the anti-ferroptosis effects of MK-4 in neurons after SAH. METHODS: A subarachnoid hemorrhage (SAH) model was prepared by endovascular perforation in mice. In vitro hemoglobin stimulation of primary cortical neurons mimicked SAH. MK-4, Brequinar (BQR, DHODH inhibitor), and Selisistat (SEL, SIRT1 inhibitor) were administered, respectively. Subsequently, WB, immunofluorescence was used to determine protein expression and localization, and transmission electron microscopy was used to observe neuronal mitochondrial structure while other indicators of ferroptosis were measured. RESULTS: MK-4 treatment significantly upregulated the protein levels of DHODH; decreased GSH, PTGS2, NOX1, ROS, and restored mitochondrial membrane potential. Meanwhile, MK-4 upregulated the expression of SIRT1 and promoted its entry into the nucleus. BQR or SEL partially abolished the protective effect of MK-4 on, neurologic function, and ferroptosis. CONCLUSIONS: Taken together, our results suggest that MK-4 attenuates ferroptosis after SAH by upregulating DHODH through the activation of SIRT1.

Neuronal pyroptosis mediated by STAT3 in early brain injury after subarachnoid hemorrhage.

Neuroinflammation induced by early brain injury (EBI) seriously affects the prognosis of patients after subarachnoid hemorrhage (SAH). Pyroptosis can aggravate inflammatory injury by promoting the secretion of inflammatory cytokines. Meanwhile, STAT3 plays a critical role in the inflammatory response of EBI after SAH. However, whether it plays a pyroptotic role in SAH is mainly unknown. This study aimed to explore the mechanism of STAT3 in pyroptosis in EBI after SAH. C57BL/6J mice were used to establish the SAH model. Brain tissues were collected at different time points for q-RT-PCR and western blot to detect the expression level of STAT3. After intracerebroventricular injection of STAT3 inhibitor S3I-201, they were divided into sham, SAH, SAH + Vehicle, and SAH + S3I-201. Then, the SAH grade, cerebral edema content, blood-brain barrier (BBB) damage, and neurological scores of mice in each group were detected. qRT-PCR and western blot were used to detect related genes and proteins, and enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of IL-18 and IL-1ß. Immunofluorescence staining was used to observe the expression level of proteins. At the same time, S3I-201 was added to the primary neuron cells of the culture medium containing OxyHb to simulate the in vitro experiment, and the relevant indicators consistent with the in vivo experiment were detected. The expression of STAT3 was upregulated after SAH. Inhibition of STAT3 with S3I-201 attenuated neurological deficits, cerebral edema, and BBB damage after SAH. In addition, S3I-201 can also reduce the expression of pyroptosis-related inflammasomes such as GSDMD, NLRP3, Caspase 1, and AIM2 after SAH and the neurological damage caused by IL-18 and IL-1ß. Further studies have shown that STAT3 regulates pyroptosis by promoting the nuclear translocation of NF-κB p65. Our finding demonstrated that STAT3 regulates neuronal pyroptosis in EBI after SAH. Inhibition of STAT3 may be a potential target to attenuate the damage that triggers neuroinflammation after SAH.

Wu-zhu-yu Decoction reduces early brain injury following subarachnoid hemorrhage in vivo and in vitro by activating the Nrf2 antioxidant system via SIRT6 targeting.

ETHNOPHARMACOLOGICAL RELEVANCE: Early brain damage (EBI) following subarachnoid hemorrhage (SAH) is a long-lasting condition with a high occurrence. However, treatment options are restricted. Wu-zhu-yu Decoction (WZYD) can treat headaches and vomiting, which are similar to the early symptoms of subarachnoid hemorrhage (SAH). However, it is yet unknown if WZYD can reduce EBI following SAH and its underlying mechanisms. AIM OF THE STUDY: This study aimed to investigate whether WZYD protects against EBI following SAH by inhibiting oxidative stress through activating nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling via Sirtuin 6 (SIRT6)-mediated histone H3 lysine 56 (H3K56) deacetylation. MATERIALS AND METHODS: In the current investigation, the principal components of WZYD were identified using high-performance liquid chromatography-diode array detection (HPLC-DAD). The SAH model in rats using the internal carotid artery plug puncture approach and the SAH model in primary neurons using hemoglobin incubation were developed. WZYD with different doses (137 mg kg-1, 274 mg kg-1, 548 mg kg-1) and the positive drug-Nimodipine (40 mg kg-1) were intragastrically administered in SAH model rats, respectively. The PC12 cells were cultured with corresponding medicated for 24h. In our investigation, neurological scores, brain water content, Evans blue leakage, Nissl staining, TUNEL staining, oxidative stress, expression of apoptosis-related proteins, and Nrf2/HO-1 signaling were evaluated. The interaction between SIRT6 and Nrf2 was detected by co-immunoprecipitation. SIRT6 knockdown was used to confirm its role in WZYD's neuroprotection. RESULTS: The WZYD treatment dramatically reduced cerebral hemorrhage and edema, and enhanced neurological results in EBI following SAH rats. WZYD administration inhibited neuronal apoptosis via reducing the expression levels of Cleaved cysteinyl aspartate specific proteinase-3(Cleaved Caspase-3), cysteinyl aspartate specific proteinase-3(caspase-3), and Bcl-2, Associated X Protein (Bax) and increasing the expression of B-cell lymphoma-2(Bal2). It also decreased reactive oxygen species and malondialdehyde levels and increased Nrf2 and HO-1 expression in the rat brain after SAH. In vitro, WZYD attenuated hemoglobin-induced cytotoxicity, oxidative stress and apoptosis in primary neurons. Mechanistically, WZYD enhanced SIRT6 expression and H3K56 deacetylation, activated Nrf2/HO-1 signaling, and promoted the interaction between SIRT6 and Nrf2. Knockdown of SIRT6 abolished WZYD-induced neuroprotection. CONCLUSIONS: WZYD attenuates EBI after SAH by activating Nrf2/HO-1 signaling through SIRT6-mediated H3K56 deacetylation, suggesting its therapeutic potential for SAH treatment.

Aloe-emodin from Sanhua Decoction inhibits neuroinflammation by regulating microglia polarization after subarachnoid hemorrhage.

ETHNOPHARMACOLOGICAL RELEVANCE: Subarachnoid hemorrhage (SAH) triggers a cascade of events that lead to early brain injury (EBI), which contributes to poor outcomes and appears within 3 days after SAH initiation. EBI involves multiple process including neuronal death, blood-brain barrier (BBB) injury and inflammation response. Microglia are cluster of immune cells originating in the brain which respond to SAH by changing their states and releasing inflammatory molecules through various signaling pathways. M0, M1, M2 are three states of microglia represent resting state, promoting inflammation state, and anti-inflammation state respectively, which can be modulated by pharmacological strategies. AIM OF THE STUDY: After identified potential active ingredients and targets of Sanhua Decoction (SHD) for SAH, we selected aloe-emodin (AE) as a potential ingredient modulating microglia activation states. MATERIALS AND METHODS: Molecular mechanisms, targets and pathways of SHD were reveal by network pharmacology technique. The effects of AE on SAH were evaluated in vivo by assessing neurological deficits, neuronal apoptosis and BBB integrity in a mouse SAH model. Furthermore, BV-2 cells were used to examine the effects of AE on microglial polarization. The influence of AE on microglia transformation was measured by Iba-1, TNF-α, CD68, Arg-1 and CD206 staining. The signal pathways of neuronal apoptosis and microglia polarization was measured by Western blot. RESULTS: Network pharmacology identified potential active ingredients and targets of SHD for SAH. And AE is one of the active ingredients. We also confirmed that AE via NF-κB and PKA/CREB pathway inhibited the microglia activation and promoted transformation from M1 phenotype to M2 at EBI stage after SAH. CONCLUSIONS: AE, as one ingredient of SHD, can alleviate the inflammatory response and protecting neurons from SAH-induced injury. AE has potential value for treating SAH-induced nerve injury and is expected to be applied in clinical practice.

Role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in autophagy activation following subarachnoid hemorrhage.

BACKGROUND: Early brain injury (EBI) refers to a severe brain injury that occurs within hours to days after subarachnoid hemorrhage (SAH). Neuronal damage in EBI is considered a key factor leading to poor prognosis. Currently, our understanding of the mechanisms of neuronal damage, such as neuronal autophagy, is still incomplete. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in metabolism and plays an important role in autophagy. Based on this, this study will further explore the regulation of autophagy by GAPDH after SAH, which may provide a new treatment strategy for improving the prognosis of SAH patients. METHODS: The rat SAH model was established by endovascular puncturing, and the trend of autophagy in hippocampal neurons at different time points was discussed. Additionally, an in vitro SAH model was created using the oxygenated hemoglobin and hippocampal neuronal HT22 cell line. Through siRNA and overexpression adenovirus techniques, we further investigated the relationship between the key enzyme GAPDH and autophagy in the in vitro SAH model. RESULTS: We observed significant neuronal damage in the hippocampus 24 h after SAH, and the proteomics showed significant enrichment of autophagy-related pathways at this time point. Further studies showed that the expression of LC3 and Beclin1 peaked at 24 h, and the nuclear translocation of GAPDH occurred simultaneously with SAH-induced neuronal autophagy. Our in vitro SAH model confirmed the role of GAPDH in regulating the level of autophagy in HT22 cells. Knockdown of GAPDH significantly reduced the level of autophagy, while overexpression of GAPDH increased the level of autophagy. CONCLUSION: This study shows the trend of autophagy in hippocampal neurons after SAH, and reveals the regulatory role of GAPDH in SAH-induced autophagy. However, further studies are needed to reveal the exact mechanism of GAPDH in the nuclear translocation regulation of autophagy and validate in animal models.

Osteopontin modulates microglial activation states and attenuates inflammatory responses after subarachnoid hemorrhage in rats.

AIMS: Osteopontin (OPN) has demonstrated neuroprotective effects in various stroke models. Its role in neuroinflammation after brain injury remains to be elucidated. This study aims to clarify the effect of OPN on neuroinflammation, particularly on the functional states of microglia after subarachnoid hemorrhage (SAH). METHODS: 77 rats were randomly divided into the following groups: Sham, SAH 24 h, SAH + rOPN, SAH + Vehicle (PBS), SAH + OPN siRNA, and SAH + Scr siRNA, SAH + rOPN+Fib-14 and SAH + rOPN+DMSO. Modified Garcia and beam balance tests were used to evaluate neurobehavioral outcomes. Semi-quantitative immunofluorescence staining was performed to measure expression of myeloperoxidase (MPO) and microglia activation state markers CD16, CD206 after SAH and recombinant OPN treatment. The quantification of microglia activation and functional markers CD16, CD206, TNF-α and IL-10 were further evaluated using Western-blotting. RESULTS: Nasal administration of rOPN improved neurological dysfunction, attenuated neutrophil infiltration, and decreased expression of phenotypic and functional markers of pro-inflammatory microglia CD16 and TNF-α. It also promoted an anti-inflammatory microglial state, as evidenced by increased expression of CD206 and IL-10. Furthermore, after blocking the phosphorylation of FAK signaling, the effects of rOPN on microglial activation states were partially reversed. The downstream pathways of STAT3 and NF-κB also exhibited consistent changes, suggesting the involvement of the STAT3 and NF-κB pathways in OPN's modulation of microglial activation via integrin-FAK signaling. CONCLUSION: OPN attenuates inflammatory responses after SAH by promoting an anti-inflammatory microglial state, potentially mediated through the integrin-FAK-STAT3 and NF-κB signaling pathways.

Perampanel attenuates oxidative stress and pyroptosis following subarachnoid hemorrhage via the SIRT3/FOXO3α pathway.

Subarachnoid hemorrhage (SAH) occurs most commonly after rupture of an aneurysm, resulting in high disability and mortality due to the absence of effective therapy. Its subsequent stage, early brain injury (EBI), promotes the sustainable development of injury in the brain and ultimately leads to poor prognosis. As a new antiepileptic drug, the effect of perampanel on EBI after SAH is unknown. Pyroptosis, a process of inflammatory programmed cell death, has been confirmed in most studies to play a substantial role in aggravating SAH-post EBI. Similarly, oxidative stress is closely involved in neuronal pyroptosis and the pathophysiological mechanism of SAH-post EBI, leading to a devastating outcome for SAH patients. Nonetheless, no studies have been conducted to determine whether perampanel reduces pyroptosis and oxidative stress in the context of SAH-induced EBI. Rat SAH model via endovascular perforation was constructed in this study, to assess the neuroprotective effect of perampanel on SAH-post EBI, and to clarify the possible molecular mechanism. By means of the neurological score, brain edema detection, FJB staining, immunofluorescence, WB, ELISA, and ROS assay, we found that perampanel can improve neuroscores and reduce brain edema and neuronal degeneration at 24 h after SAH; we also found that perampanel reduced oxidative stress, neuronal pyroptosis, and inhibition of the SIRT3-FOXO3α pathway at 24 h after SAH. When 3-TYP, an inhibitor of SIRT3, was administered, the effects of perampanel on the SIRT3-FOXO3a pathway, antioxidant stress, and neuronal pyroptosis were reversed. Taken together, our data indicate that perampanel attenuates oxidative stress and pyroptosis following subarachnoid hemorrhage via the SIRT3/FOXO3α pathway. This study highlights the application value of perampanel in subarachnoid hemorrhage and lays a foundation for clinical research and later transformation of perampanel in SAH.

Protective mechanisms of tea polyphenols regulating the PI3K/Akt pathway on early brain injury after subarachnoid hemorrhage in rats.

In recent years, numerous studies have demonstrated that tea polyphenols (TPPs) can exert neuroprotective effects through the regulation of the PI3K/Akt pathway. The objective of this work was to verify whether TPPs could protect against early brain injury in rats after subarachnoid hemorrhage (SAH) by modulating the PI3K/Akt pathway. A total of 150 rats were randomly rolled into control (C), TPP, and SAH groups. The TPP and SAH groups underwent endovascular perforation to induce SAH, while C group received only endovascular needle puncture and saline injection. Brain water content, Evans Blue (EB) extravasation assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, Western blot, and RT-PCR analyses were performed. Relative to SAH group, TPP treatment considerably improved neurological function scores following SAH, reduced brain edema, cortical neuronal apoptosis, and blood-brain barrier damage. Levels of aquaporin-4 (AQP4) and apoptosis-related protein Bax were considerably lower in the TPP group than in SAH group. Conversely, levels of anti-apoptotic protein Bcl-2 and tight junction protein Zona occludens 1 (ZO-1) were considerably higher in the TPP group. Furthermore, TPP treatment was found to activate the PI3K/Akt signaling. TPPs can mitigate early brain injury caused by SAH in rats by reducing AQP4 levels, alleviating cortical damage, and attenuating neuronal apoptosis. These findings elucidate the protective mechanisms of TPPs against early brain injury following SAH through the regulation of the PI3K/Akt signaling.