BackgroundPeroxiredoxin (Prx) protein family have been reported as important damage-associated molecular patterns (DAMPs) in ischemic stroke. Since peroxiredoxin 2 (Prx2) is the third most abundant protein in erythrocytes and the second most protein in the cerebrospinal fluid in traumatic brain injury and subarachnoid hemorrhage (SAH) patients, we assessed the role of extracellular Prx2 in the context of SAH.MethodsWe introduced a co-culture system of primary neurons and microglia. Prx2 was added to culture medium with oxyhemoglobin (OxyHb) to mimic SAH in vitro. Neuronal cell viability was assessed by lactate dehydrogenase (LDH) assay, and neuronal apoptosis was determined by TUNEL staining. Inflammatory factors in culture medium were measured by ELISA, and their mRNA levels in microglia were determined by qPCR. Toll-like receptor 4 knockout (TLR4-KO) mice were used to provide TLR4-KO microglia; ST-2825 was used to inhibit MyD88, and pyrrolidine dithiocarbamate (PDTC) was used to inhibit NF-κB. Related cellular signals were analyzed by Western blot. Furthermore, we detected the level of Prx2 in aneurysmal SAH patients’ cerebrospinal fluids (CSF) and compared its relationship with Hunt-Hess grades.ResultsPrx2 interacted with TLR4 on microglia after SAH and then activated microglia through TLR4/MyD88/NF-κB signaling pathway. Pro-inflammatory factors were expressed and released, eventually caused neuronal apoptosis. The levels of Prx2 in SAH patients positively correlated with Hunt-Hess grades.ConclusionsExtracellular Prx2 in CSF after SAH is a DAMP which resulted in microglial activation via TLR4/MyD88/NF-κB pathway and then neuronal apoptosis. Prx2 in patients’ CSF may be a potential indicator of brain injury and prognosis.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1118-4) contains supplementary material, which is available to authorized users.

Accumulating evidence suggests neuronal apoptosis has the potential to lead to more harmful effects in the pathological processes following traumatic brain injury (TBI). Previous studies have established that milk fat globule-EGF factor-8 (MFG-E8) provides neuroprotection through modulation of inflammation, oxidative stress, and especially apoptosis in cerebral ischemia and neurodegenerative disease. However, the effects of MFG-E8 on neuronal apoptosis in TBI have not yet been investigated. Therefore, we explored the role of MFG-E8 on anti-apoptosis and its potential mechanism following TBI. In the first set of experiments, adult male Sprague–Dawley (SD) rats were randomly divided into Sham and TBI groups that were each further divided into five groups representing different time points (6 h, 24 h, 72 h, and 7 days) (n = 9 each). Western blotting, quantitative real-time PCR, and immunofluorescence staining were performed to identify the expression and cellular localization of MFG-E8. In the second set of experiments, four groups were randomly assigned: Sham group, TBI + Vehicle group, and TBI + rhMFG-E8 (1 and 3 µg) (n = 15). Recombinant human MFGE8 (rhMFG-E8) was administrated as two concentrations through intracerebroventricular (i.c.v.) injection at 1 h after TBI induction. Brain water content, neurological severity score, western blotting, and immunofluorescence staining were measured at 24 and 72 h following TBI. In the final set of experiments, MFG-E8 siRNA (500 pmol/3 µl), integrin β3 siRNA (500 pmol/3 µl), and PI3K inhibitor LY294002 (5 and 20 µM) were injected i.c.v. and thereafter rats exposed to TBI. Western blotting, immunofluorescence staining, brain water content, neurological severity score, and Fluoro-Jade C (FJC) staining were used to investigate the effect of the integrin-β3/FAK/PI3K/AKT signaling pathway on MFG-E8-mediated anti-apoptosis after TBI. The expression of MFG-E8 was mainly located in microglial cells and increased to peak at 24 h after TBI. Treatment with rhMFG-E8 (3 µg) markedly decreased brain water content, improved neurological deficits, and reduced neuronal apoptosis at 24 and 72 h after TBI. rhMFG-E8 significantly enhanced the expression of integrin-β3/FAK/PI3K/AKT pathway-related components. Administration of integrin-β3 siRNA and LY294002 (5 and 20 µM) abolished the effect of rhMFG-E8 on anti-apoptosis and neuroprotection after TBI. This study demonstrated for the first time that rhMFG-E8 inhibits neuronal apoptosis and offers neuroprotection. This is suggested to occur through the modulation of the integrin-β3/FAK/PI3K/AKT signaling pathway, highlighting rhMFG-E8 as a potentially promising therapeutic strategy for TBI patients.

A growing body of evidence demonstrates that Akt may serve as a therapeutic target for treatment of early brain injury following subarachnoid hemorrhage (SAH). The purpose of the current study was to evaluate the neuroprotective effect of Akt specific activator SC79 in an experimental rat model of SAH. SAH was induced by injecting 300 μL of blood into the prechiasmatic cistern. Intracerebroventricular (ICV) injection of SC79 (30 min post-SAH) induced the p-Akt (Ser473) expression in a dose-dependent manner. A single ICV dose treatment of SC79 (100 μg/rat) significantly increased the expression of Bcl-2 and p-GSK-3β (Ser9), decreased the protein levels of Bax, cytoplasm cytochrome c, and cleaved caspase-3, indicating the antiapoptotic effect of SC79. As a result, the number of apoptotic cells was reduced 24 h post SAH. Moreover, SC79 treatment alleviated SAH-induced oxidative stress, restored mitochondrial morphology, and improved neurological deficits. Strikingly, treatment of SC79 provided a beneficial outcome against neurologic deficit with a therapeutic window of at least 4 h post SAH by ICV injection and 30 min post SAH by intraperitoneal injection. Collectively, SC79 exerts its neuroprotective effect likely through the dual activities of antioxidation and antiapoptosis. These data provide a basic platform to consider SC79 as a novel therapeutic agent for treatment of SAH.

Background: Accumulating evidence suggests that neuroinflammation plays a critical role in early brain injury after subarachnoid hemorrhage (SAH). Pannexin-1 channels, as a member of gap junction proteins located on the plasma membrane, releases ATP, ions, second messengers, neurotransmitters, and molecules up to 1 kD into the extracellular space, when activated. Previous studies identified that the opening of Pannexin-1 channels is essential for cellular migration, apoptosis and especially inflammation, but its effects on inflammatory response in SAH model have not been explored yet.Methods: Adult male Sprague-Dawley rats were divided into six groups: sham group (n = 20), SAH group (n = 20), SAH + LV-Scramble-ShRNA group (n = 20), SAH + LV-ShRNA-Panx1 group (n = 20), SAH + LV-NC group (n = 20), and SAH + LV-Panx1-EGFP group (n = 20). The rat SAH model was induced by injection of 0.3 ml fresh arterial, non-heparinized blood into the prechiasmatic cistern in 20 s. In SAH + LV-ShRNA-Panx1 group and SAH + LV-Panx1-EGFP group, lentivirus was administered via intracerebroventricular injection (i.c.v.) at 72 h before the induction of SAH. The Quantitative real-time polymerase chain reaction, electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, immunofluorescence staining, and western blotting were performed to explore the potential interactive mechanism between Pannexin-1 channels and TLR2/TLR4/NF-κB-mediated signaling pathway. Cognitive and memory changes were investigated by the Morris water maze test.Results: Administration with LV-ShRNA-Panx1 markedly decreased the expression levels of TLR2/4/NF-κB pathway-related agents in the brain cortex and significantly ameliorated neurological cognitive and memory deficits in this SAH model. On the contrary, administration of LV-Panx1-EGFP elevated the expressions of TLR2/4/NF-κB pathway-related agents, which correlated with augmented neuronal apoptosis.Conclusion: Pannexin-1 channels may contribute to inflammatory response and neurobehavioral dysfunction through the TLR2/TLR4/NF-κB-mediated pathway signaling after SAH, suggesting a potential role of Pannexin-1 channels could be a potential therapeutic target for the treatment of SAH.

Recent studies suggest that peroxiredoxin1/2 (Prx1/2) may be involved in the pathophysiology of post‐ischemic inflammatory responses in the brain. In this study, we assessed the distribution and function of Prx1/2 in mice after experimental subarachnoid hemorrhage (SAH). We investigated the distribution of Prx1/2 in the brains of mice both in vivo and in vitro using immunofluorescence staining. The expression of Prx1/2 after SAH was determined by Western blot. Adenanthin was used to inhibit Prx1/2 function, and Prx1/2 overexpression was achieved by injecting adeno‐associated virus. Oxidative stress and neuronal apoptosis were assessed both in vivo and in vitro. The neurologic function, inflammatory response, and related cellular signals were analyzed. The results showed that Prx1 was mainly expressed in astrocytes, and Prx2 was abundant in neurons. The expression of Prx1/2 was elevated after SAH, and their expression levels peaked before proinflammatory cytokines. Inhibiting Prx1/2 promoted neuronal apoptosis by increasing the hydrogen peroxide (H2O2) levels via the apoptosis signal‐regulating kinase 1/p38 pathway. By contrast, overexpression of Prx1/2 attenuated oxidative stress and neuronal apoptosis after SAH. Thus, early expression of Prx1/2 may protect the brain from oxidative damage after SAH and may provide a novel target for treating SAH.—Lu, Y., Zhang, X.‐S., Zhou, X.‐M., Gao, Y.‐Y., Chen, C.‐L., Liu, J.‐P., Ye, Z.‐N., Zhang, Z.‐H., Wu, L.‐Y., Li, W., Hang, C.‐H. Peroxiredoxin 1/2 protects brain against H2O2‐induced apoptosis after subarachnoid hemorrhage. FASEB J. 33, 3051–3062 (2019). http://www.fasebj.org

BackgroundSubarachnoid hemorrhage (SAH) is a devastating neurological injury with high morbidity and mortality that is mainly caused by early brain injury (EBI). Progranulin (PGRN) is known to be involved in various biological functions, such as anti-inflammation and tissue repair. This study aimed to investigate the change of PGRN in the brain after SAH and its role on EBI.MethodsThe levels of PGRN, myeloperoxidase (MPO), interleukin1β (IL-1β), and tumor necrosis factor-α (TNF-α) were detected in the cerebrospinal fluid (CSF) from SAH patients by enzyme-linked immunosorbent assay (ELISA). In addition, PGRN levels were also detected in the cerebral cortex after experimental SAH in rats by western blotting and immunohistochemistry (IHC). Recombinant human PGRN (r-PGRN) or an equal volume of phosphate-buffered saline (PBS) was administrated at 30 min after SAH. All rats were subsequently sacrificed at 24 h after SAH. Neurological score and brain water content were assessed. For mechanistic studies, the changes of MPO, matrix metalloproteinase-9 (MMP-9), zonula occludens 1 (ZO-1), Bcl-2, and cleaved caspase-3 were examined by western blotting and the levels of pro-inflammatory cytokines (IL-1β and TNF-α) were determined by ELISA. In addition, neuronal apoptosis and blood brain barrier (BBB) permeability were examined.ResultsThe levels of PGRN significantly decreased, and the levels of MPO, IL-1β, and TNF-α were markedly elevated in the CSF from SAH patients. In rats, PGRN levels in the brain also decreased after SAH. Administration of r-PGRN decreased brain water content and improved neurological scores at 24 h after SAH. These changes were associated with marked reductions in MPO, MMP-9, and proinflammation cytokine levels, as well as increased levels of Bcl-2 and ZO-1. In addition, neuronal apoptosis and BBB permeability were alleviated by r-PGRN.ConclusionsThese results indicate that the levels of PGRN decreased after SAH and that r-PGRN alleviates EBI after SAH possibly via inhibition of neutrophil recruitment, providing a new target for the treatment of SAH.

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Luteolin (LUT) possesses multiple biologic functions and has beneficial effects for cardiovascular and cerebral vascular diseases. Here, we investigated the protective effects of LUT against subarachnoid hemorrhage (SAH) and the involvement of underlying molecular mechanisms. In a rat model of SAH, LUT significantly inhibited SAH-induced neuroinflammation as evidenced by reduced microglia activation, decreased neutrophil infiltration, and suppressed proinflammatory cytokine release. In addition, LUT markedly ameliorated SAH-induced oxidative damage and restored the endogenous antioxidant systems. Concomitant with the suppressed oxidative stress and neuroinflammation, LUT significantly improved neurologic function and reduced neuronal cell death after SAH. Mechanistically, LUT treatment significantly enhanced the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), while it downregulated nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation. Inhibition of Nrf2 by ML385 dramatically abrogated LUT-induced Nrf2 activation and NLRP3 suppression and reversed the beneficial effects of LUT against SAH. In neurons and microglia coculture system, LUT also mitigated oxidative stress, inflammatory response, and neuronal degeneration. These beneficial effects were associated with activation of the Nrf2 and inhibitory effects on NLRP3 inflammasome and were reversed by ML385 treatment. Taken together, this present study reveals that LUT confers protection against SAH by inhibiting NLRP3 inflammasome signaling pathway, which may be modulated by Nrf2 activation.