Background and Purpose
Oxidative stress and neuronal apoptosis play key roles in traumatic brain injury. We investigated the protective effects of astaxanthin against traumatic brain injury and its ...underlying mechanisms of action.
Experimental Approach
A weight‐drop model of traumatic brain injury in vivo and hydrogen peroxide exposure in vitro model were established. Brain oedema, behaviour tests, western blot, biochemical analysis, lesion volume, histopathological study and cell viability were performed.
Key Results
Astaxanthin significantly reduced oxidative insults on Days 1, 3 and 7 after traumatic brain injury. Neuronal apoptosis was also ameliorated on Day 3. Additionally, astaxanthin improved neurological functions up to 3 weeks after traumatic brain injury. Astaxanthin treatment dramatically enhanced the expression of peroxiredoxin 2 (Prx2), nuclear factor‐erythroid 2‐related factor 2 (NRF2/Nrf2) and sirtuin 1 (SIRT1), while it down‐regulated the phosphorylation of apoptosis signal‐regulating kinase 1 (ASK1) and p38. Inhibition of Prx2 by siRNA injection reversed the beneficial effects of astaxanthin against traumatic brain injury. Additionally, Nrf2 knockout prevented the neuroprotective effects of astaxanthin in traumatic brain injury. In contrast, overexpression of Prx2 in Nrf2 knockout mice attenuated the secondary brain injury after traumatic brain injury. Moreover, inhibiting SIRT1 by EX527 dramatically inhibited the neuroprotective effects of astaxanthin and suppressed SIRT1/Nrf2/Prx2/ASK1/p38 pathway both in vivo and in vitro.
Conclusion and Implications
Astaxanthin improved the neurological functions and protected the brain from injury after traumatic brain injury, primarily by reducing oxidative stress and neuronal death via SIRT1/Nrf2/Prx2/ASK1/p38 signalling pathway and might be a new candidate to ameliorate traumatic brain injury.
Aucubin (Au), an iridoid glycoside from natural plants, has antioxidative and anti-inflammatory bioactivities; however, its effects on a traumatic brain injury (TBI) model remain unknown. We explored ...the potential role of Au in an H
O
-induced oxidant damage in primary cortical neurons and weight-drop induced-TBI in a mouse model.
In vitro experiments, the various concentrations of Au (50 μg/ml, 100 μg/ml, or 200 μg/ml) were added in culture medium at 0 h and 6 h after neurons stimulated by H
O
(100 μM). After exposed for 12 h, neurons were collected for western blot (WB), immunofluorescence, and M29,79-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. In vivo experiments, Au (20 mg/kg or 40 mg/kg) was administrated intraperitoneally at 30 min, 12 h, 24 h, and 48 h after modeling. Brain water content, neurological deficits, and cognitive functions were measured at specific time, respectively. Cortical tissue around focal trauma was collected for WB, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, Nissl staining, quantitative real time polymerase chain reaction (q-PCR), immunofluorescence/immunohistochemistry, and enzyme linked immunosorbent assay (ELISA) at 72 h after TBI. RNA interference experiments were performed to determine the effects of nuclear factor erythroid-2 related factor 2 (Nrf2) on TBI mice with Au (40 mg/kg) treatment. Mice were intracerebroventricularly administrated with lentivirus at 72 h before TBI establishment. The cortex was obtained at 72 h after TBI and used for WB and q-PCR.
Au enhanced the translocation of Nrf2 into the nucleus, activated antioxidant enzymes, suppressed excessive generation of reactive oxygen species (ROS), and reduced cell apoptosis both in vitro and vivo experiments. In the mice model of TBI, Au markedly attenuated brain edema, histological damages, and improved neurological and cognitive deficits. Au significantly suppressed high mobility group box 1 (HMGB1)-mediated aseptic inflammation. Nrf2 knockdown in TBI mice blunted the antioxidant and anti-inflammatory neuroprotective effects of the Au.
Taken together, our data suggest that Au provides a neuroprotective effect in TBI mice model by inhibiting oxidative stress and inflammatory responses; the mechanisms involve triggering Nrf2-induced antioxidant system.
Background and Purpose
Dioscin has multiple biological activities and is beneficial for cardiovascular and cerebral vascular diseases. Here, we investigated the protective effects of dioscin against ...subarachnoid haemorrhage and the molecular mechanisms involved.
Experimental Approach
Dioscin was administered after subarachnoid haemorrhage induced in rats. MCC950, a potent selective nod‐like receptor pyrin domain‐containing 3 (NLRP3) inhibitor, was used to suppress NLRP3 and EX527 (selisistat) was used to inhibit sirtuin 1 (SIRT1).
Key Results
In vivo, dioscin inhibited acute inflammatory response, oxidative damage, neurological impairment and neural cell degeneration after subarachnoid haemorrhage along with dramatically suppressing NLRP3 inflammasome activation. While pretreatment with MCC950 reduced the inflammatory response and improved neurological outcomes it did not lessen ROS production. However, giving dioscin after MCC950 reduced acute brain damage and ROS production. Dioscin increased SIRT1 expression after subarachnoid haemorrhage, whereas EX527 abolished the up‐regulation of SIRT1 induced by dioscin and offset the inhibitory effects of dioscin on NLRP3 inflammasome activation. EX527 pretreatment also reversed the neuroprotective effects of dioscin against subarachnoid haemorrhage. Similarly, in vitro, dioscin dose‐dependently suppressed inflammatory response, oxidative damage and neuronal degeneration and improved cell viability in neurons and microglia co‐culture system. These effects were associated with inhibition of the NLRP3 inflammasome and stimulation of SIRT1 signalling, which could be inhibited by EX527 pretreatment.
Conclusion and Implications
Dioscin provides protection against subarachnoid haemorrhage via the suppression of NLRP3 inflammasome activation through SIRT1‐dependent pathway. Dioscin may be a new candidate to ameliorate early brain injury after subarachnoid haemorrhage.
Subarachnoid haemorrhage (SAH) has a high rate of disability and mortality. Extremely damaging molecules, including adenosine triphosphate (ATP), are released from extravasated red blood cells and ...nerve cells, which activate microglia and induce sterile tissue injury and organ dysfunction. P2X purinoceptor 7 (P2X7) is one of the most important purine receptors on the microglial surface and is involved in the proinflammatory activation of microglia. While P2X7 can also affect microglial phagocytosis, the mechanism is not clear. Here, we demonstrated that microglial phagocytosis is progressively impaired under continued BzATP exposure and P2X7 activation. Furthermore, we found that P2X7 activation leads to increased intracellular Ca2+ levels and activates Calcineurin, which dephosphorylates dynamin‐related protein 1 (DRP1) S637. The dephosphorylation of DRP1 at S637 leads to increased mitochondrial fission and decreased mitochondrial function, which may be responsible for the decreased microglial phagocytosis. Finally, we pharmacologically inhibited P2X7 activation in mice, which resulted in rescue of mitochondrial function and decreased microglial proliferation, but improved phagocytosis after SAH. Our study confirmed that P2X7 activation after SAH leads to the impairment of microglial phagocytosis through mitochondrial fission and verified that P2X7 inhibition restores microglial phagocytosis both in vitro and in vivo.
In the present study, we found that the expression of microglial P2X7 increased after SAH and that the continued activation of P2X7 resulted in a gradual decrease in phagocytosis, while specifically blocking P2X7 activation reversed the impairment in phagocytosis. We then clarified that the impairment in phagocytosis was as a result of excessive mitochondrial fission, which was mediated by Ca2+/Calcineurin/DRP1, and that preventing excessive mitochondrial fission could also alleviate phagocytic dysfunction. Our study deepens the understanding of microglial function after SAH and provides evidence for further regulation.
Abstract
Background
Early brain injury (EBI) has been thought to be a key factor affecting the prognosis of subarachnoid hemorrhage (SAH). Many pathologies are involved in EBI, with inflammation and ...neuronal death being crucial to this process. Resolvin D1 (RvD1) has shown superior anti-inflammatory properties by interacting with lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) in various diseases. However, it remains not well described about its role in the central nervous system (CNS). Thus, the goal of the present study was to elucidate the potential functions of the RvD1-ALX/FPR2 interaction in the brain after SAH.
Methods
We used an in vivo model of endovascular perforation and an in vitro model of hemoglobin (Hb) exposure as SAH models in the current study. RvD1 was used at a concentration of 25 nM in our experiments. Western blotting, quantitative polymerase chain reaction (qPCR), immunofluorescence, and other chemical-based assays were performed to assess the cellular localizations and time course fluctuations in ALX/FPR2 expression, evaluate the effects of RvD1 on Hb-induced primary microglial activation and neuronal damage, and confirm the role of ALX/FPR2 in the function of RvD1.
Results
ALX/FPR2 was expressed on both microglia and neurons, but not astrocytes. RvD1 exerted a good inhibitory effect in the microglial pro-inflammatory response induced by Hb, possibly by regulating the IRAK1/TRAF6/NF-κB or MAPK signaling pathways. RvD1 could also potentially attenuate Hb-induced neuronal oxidative damage and apoptosis. Finally, the mRNA expression of IRAK1/TRAF6 in microglia and GPx1/bcl-xL in neurons was reversed by the ALX/FPR2-specific antagonist Trp-Arg-Trp-Trp-Trp-Trp-NH2 (WRW4), indicating that ALX/FPR2 could mediate the neuroprotective effects of RvD1.
Conclusions
The results of the present study indicated that the RvD1-ALX/FPR2 interaction could potentially play dual roles in the CNS, as inhibiting Hb promoted microglial pro-inflammatory polarization and ameliorating Hb induced neuronal oxidant damage and death. These results shed light on a good therapeutic target (ALX/FPR2) and a potential effective drug (RvD1) for the treatment of SAH and other inflammation-associated brain diseases.
Microglia are resident immune cells in the central nervous system and central to the innate immune system. Excessive activation of microglia after subarachnoid haemorrhage (SAH) contributes greatly ...to early brain injury, which is responsible for poor outcomes. Dehydroepiandrosterone (DHEA), a steroid hormone enriched in the brain, has recently been found to regulate microglial activation. The purpose of this study was to address the role of DHEA in SAH.
We used in vivo models of endovascular perforation and in vitro models of haemoglobin exposure to illustrate the effects of DHEA on microglia in SAH.
In experimental SAH mice, exogenous DHEA administration increased DHEA levels in the brain and modulated microglial activation. Ameliorated neuronal damage and improved neurological outcomes were also observed in the SAH mice pretreated with DHEA, suggesting neuronal protective effects of DHEA. In cultured microglia, DHEA elevated the mRNA and protein levels of Jumonji d3 (JMJD3, histone 3 demethylase) after haemoglobin exposure, downregulated the H3K27me3 level, and inhibited the transcription of proinflammatory genes. The devastating proinflammatory microglia-mediated effects on primary neurons were also attenuated by DHEA; however, specific inhibition of JMJD3 abolished the protective effects of DHEA. We next verified that DHEA-induced JMJD3 expression, at least in part, through the tropomyosin-related kinase A (TrkA)/Akt signalling pathway.
DHEA has a neuroprotective effect after SAH. Moreover, DHEA increases microglial JMJD3 expression to regulate proinflammatory/anti-inflammatory microglial activation after haemoglobin exposure, thereby suppressing inflammation.
Inflammatory response plays an important role in the pathogenesis of secondary damage after traumatic brain injury (TBI). The inflammasome is a multiprotein complex involved in innate immunity and a ...number of studies have suggested that the inflammasome plays a critical role in a host inflammatory signaling. Nucleotide-binding domain, leucine-rich repeat, pyrin domain containing 3 (NLRP3) is a key component of the NLRP3-inflammasome, which also includes apoptotic speck-containing protein (ASC) with a cysteine protease (caspase) -activating recruitment domain and pro-caspase1. Activation of the NLRP3-inflammasome causes the processing and release of the interleukin 1 beta (IL-1β) and interleukin 18 (IL-18). Based on this, we hypothesized that the NLRP3-inflammasome could participate in the inflammatory response following TBI. However, the expression of NLRP3-inflammasome in cerebral cortex after TBI is not well known. Rats were randomly divided into control, sham and TBI groups (including 6 h, 1 day, 3 day and 7 day sub-group). TBI model was induced, and animals were sacrificed at each time point respectively. The expression of NLRP3-inflammasome was measured by quantitative real-time polymerase chain reaction, western blot and immunohistochemistry respectively. Immunofluorescent double labeling was performed to identify the cell types of NLRP3-inflammasome’s expression. Moreover, enzyme linked immunosorbent assay was used to detect the alterations of IL-1β and IL-18 at each time point post-injury. The results showed that, TBI could induce assembly of NLRP3-inflammasome complex, increased expression of ASC, activation of caspase1, and processing of IL-1β and IL-18. These results suggested that NLRP3-inflammasome might play an important role in the inflammation induced by TBI and could be a target for TBI therapy.
Intracranial hypertension (IH) is a medical or surgical emergency that can be the common ending of various neurological disorders, such as traumatic brain injury, cerebral vascular diseases and brain ...tumors. However, the molecular mechanisms underlying IH-induced neuronal apoptosis have not been fully determined, and the treatments are symptomatic, insufficient and complicated by side-effects. In this study, a cellular model induced by compressed gas treatment in primary cultured rat cortical neurons was performed to mimic IH-induced neuronal injury in vitro. We found that compression induced cytotoxicity and apoptosis in cortical neurons in a dose- and time-dependent manner. Compression resulted in oxidative stress, which could be prevented by the ROS scavenger N-acetylcysteine (NAC). Compression produced mitochondrial oxidative stress, ATP loss and mitochondrial fragmentation. The results of western blot showed that compression differently regulated the expression of mitochondrial dynamic proteins, and the Drp1 inhibitor mdivi-1 partially reversed the compression-induced cytotoxicity. Compression significantly increased the expression of ER stress-associated factors in a time-dependent manner. The results of calcium imaging showed that compression induced intracellular calcium overload via promoting ER calcium release. Furthermore, the results using inhibitors of each signaling pathway demonstrated that ROS mediated the compression-induced ER stress and mitochondrial dysfunction in cortical neurons. In conclusion, our results demonstrated that compression induced apoptosis in primary cultured cortical neurons, which was associated with ROS mediated ER stress and mitochondrial dysfunction. Pharmacological compounds or agents targeting mitochondrial dysfunction and ER stress associated oxidative stress might be ideal candidates for the treatment of IH-related neurological diseases.
•Compression induces mitochondrial dysfunction and oxidative stress in neurons.•Compression regulates mitochondrial dynamics and ER stress in neurons.•Compression regulates ER Ca2+ homeostasis in cortical neurons.•ROS mediates compression-induced ER stress and mitochondrial dysfunction.
Delayed neuronal death is associated with neurological deficits and mortality after traumatic brain injury (TBI), where post-synaptic density (PSD) proteins are thought to play key roles. The ...immediate-early gene (IEG) coded protein Arc is a brain-specific PSD protein that controls synaptic plasticity and learning behaviors. In this study, we investigated the expression and biological function of Arc in neuronal death after TBI in an in vitro model mimicked by traumatic neuronal injury (TNI) in cortical neurons. TNI caused a temporal increase of Arc expression at 3 and 6 h. Knockdown of Arc expression using small interfering RNA (Si-Arc-3) promoted TNI-induced cytotoxicity and apoptosis. The results of western blot showed that Si-Arc-3 transfection further enhanced the activation of endoplasmic reticulum (ER) stress-associated factors, including glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and caspase-12 after TNI. In addition, knockdown of Arc significantly increased expression of (receptor-interacting protein kinase 1) RIP1 and the number of necroptotic cells, which were apparently prevented by necrostatin-1 (Nec-1). The results of immunostaining and western blot showed that knockdown of Arc activated the metabotropic glutamate receptor 1 (mGluR1) and intracellular Ca
release in neurons. Mechanistically, the Si-Arc-3-induced activation of ER stress-associated factors, RIP1 expression, apoptosis, and necroptosis were partially reversed by the mGluR1 antagonist AIDA. In summary, our data suggest that silence of Arc expression aggravates neuronal death after TNI by promoting apoptosis and necroptosis. These data support for the first time that Arc may represent a novel candidate for therapies against TBI.
Translocation of high-mobility group box 1 (HMGB1) from nucleus could trigger inflammation. Extracellular HMGB1 up-regulates inflammatory response in sepsis as a late mediator. However, little was ...known about its role in subarachnoid hemorrhage-inducible inflammation, especially in the early stage. This study aims to identify whether HMGB1 translocation occurred early after SAH and also to clarify the potential role of HMGB1 in brain injury following SAH.
Sprague-Dawley (SD) rats were randomly divided into sham group and SAH groups at 2 h, 12 h and on day 1, day 2. SAH groups suffered experimental subarachnoid hemorrhage by injection of 0.3 ml autoblood into the pre-chiasmatic cistern. Rats injected by recombinant HMGB1(rHMGB1) solution were divided into four groups according to different time points. Cultured neurons were assigned into control group and four hemoglobin (Hb) incubated groups. Mixed glial cells were cultured and stimulated in medium from neurons incubated by Hb. HMGB1 expression is measured by western blot analysis, real-time polymerase chain reaction (PCR), immunohistochemistry and immunofluorescence. Downstream nuclear factor kappa B (NF-κB) subunit P65 and inflammatory factor Interleukin 1β (IL-1β) were measured by western blot and real-time PCR, respectively. Brain injury was evaluated by cleaved caspase-3 staining.
Our results demonstrated HMGB1 translocation occurred as early as 2 h after experimental SAH with mRNA and protein level increased. Immunohistochemistry and immunofluorescence results indicated cytosolic HMGB1 was mainly located in neurons while translocated HMGB1 could also be found in some microglia. After subarachnoid injection of rHMGB1, NF-κB, downstream inflammatory response and cleaved caspase-3 were up-regulated in the cortex compared to the saline control group. In-vitro, after Hb incubation, HMGB1 was also rapidly released from neurons to medium. Incubation with medium from neurons up-regulated IL-1β in mixed glial cells. This effect could be inhibited by HMGB1 specific inhibitor glycyrrhizic acid (GA) treatment.
HMGB1 was released from neurons early after SAH onset and might trigger inflammation as an upstream inflammatory mediator. Extracellular HMGB1 contributed to the brain injury after SAH. These results might have important implications during the administration of specific HMGB1 antagonists early in order to prevent or reduce inflammatory response following SAH.
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