Subarachnoid hemorrhage (SAH) is a devastating form of stroke, which poses a series of intractable challenges to clinical practice. Imbalance of mitochondrial homeostasis has been thought to be the crucial pathomechanism in early brain injury (EBI) cascade after SAH. Irisin, a protein related to metabolism and mitochondrial homeostasis, has been reported to play pivotal roles in post-stroke neuroprotection. However, whether this myokine can exert neuroprotection effects after SAH remains unknown. In the present study, we explored the protective effects of irisin and the underlying mechanisms related to mitochondrial biogenesis in a SAH animal model. Endovascular perforation was used to induce SAH, and recombinant irisin was administered intracerebroventricularly. Neurobehavioral assessments, TdT-UTP nick end labeling (TUNEL) staining, dihydroethidium (DHE) staining, immunofluorescence, western blot, and transmission electron microscopy (TEM) were performed for post-SAH assessments. We demonstrated that irisin treatment improved neurobehavioral scores, reduced neuronal apoptosis, and alleviated oxidative stress in EBI after SAH. More importantly, the administration of exogenous irisin conserved the mitochondrial morphology and promoted mitochondrial biogenesis. The protective effects of irisin were partially reversed by the mitochondrial uncoupling protein-2 (UCP-2) inhibitor. Taken together, irisin may have neuroprotective effects against SAH via improving the mitochondrial biogenesis, at least in part, through UCP-2 related targets.

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Neuroinflammation can be caused by various factors in early brain injury after subarachnoid hemorrhage (SAH). One of the most important features of this process is M1 microglial activation. In turn, the TLR4/NF-κB pathway plays an essential role in activating M1 phenotypic microglia. Biglycan, a small leucine-rich proteoglycan, functions as an endogenous ligand of TLR4 and TLR2 in macrophages. However, the underlying mechanisms associated with microglial activation in stroke pathogenesis are poorly understood. Here, we aimed to identify the role of biglycan in neuroinflammation following SAH. In our study, SAH was induced by endovascular perforation in young male C57BL/6J mice. Lentiviral vector was administered intracerebroventricularly to knock down Biglycan. Post-SAH assessments included neurobehavioral tests, immunofluorescence, western blot, qRT-PCR, Co-IP, flow cytometry, and ELISA. The biglycan level was markedly elevated following SAH in vivo. Of particularly note, knockdown of biglycan significantly improved neurological outcomes.TLR4 was bound with soluble biglycan in vitro. In addition, biglycan down-regulation suppressed the expression of phosphorylated-NF-κB p65 (p-NF-κB) and inducible nitric oxide synthase (iNOS), as well as the cytokine (TNF-α, IL-1β, and IL-6) production in vivo and in vitro. Moreover, we detected a decreased expression of CD16/32 and CD86, M1 markers when biglycan was inhibited in vitro. Our work suggests that biglycan can induce neuroinflammation by promoting M1 microglial activation at least in part through TLR4/NF-κB signaling pathway after experimental SAH. Targeting biglycan may be a promising strategy for the clinical management of SAH. K E Y W O R D Sbiglycan, early brain injury, microglia, neuroinflammation, subarachnoid hemorrhage, TLR4 | 369 XIE Et al.

Subarachnoid hemorrhage (SAH) is a life-threatening cerebrovascular disease with high rates of morbidity and mortality. Microglia, the resident immune cells of the central nervous system, are involved in initiating inflammatory response post-SAH through releasing a variety of inflammatory mediators. Regulation of neuroinflammation triggered by activated microglia has become a promising therapeutic strategy for SAH. Recent studies reported that bone marrow-derived mesenchymal stem cells (BM-MSCs) have therapeutic effects, resulting from the regulation of microglia activation and production of inflammatory cytokines post-SAH. However, the underlying molecular mechanisms of BM-MSCs in targeting microglia-mediated neuroinflammation after SAH are still unclear. In this study, we used murine microglia cell line BV2 treated with oxyhemoglobin (OxyHb) to mimic the SAH conditions in vitro. The results showed that BM-MSCs coculture modulated OxyHb-induced BV2 activation as well as polarization. We further implemented RNA-seq approaches to investigate differences in transcriptomes between OxyHb-stimulated BV2 cocultured with and without BM-MSCs. The RNA-seq results suggested that the levels of inflammatory genes were strongly altered when OxyHb-stimulated BV2 cells were cocultured with BM-MSCs. Moreover, we identified epigenetic regulators involved in the regulation of microglia-mediated inflammation by BM-MSCs. This study clarifies detailed transcriptomic mechanisms underlying the interaction between BM-MSCs and activated microglia and may lead to a new therapeutic strategy using stem cell therapy for SAH.

Neuroinflammation has been reported to be associated with white matter injury (WMI) after subarachnoid hemorrhage (SAH). As the main resident immune cells of the brain, microglia can be activated into proinflammatory and anti‐inflammatory phenotypes. Toll‐like receptor 4 (TLR4), expressed on the surface of the microglia, plays a key role in microglial inflammation. However, the relationship between TLR4, microglial polarization, and WMI following SAH remains unclear. In this study, a total of 121 male adult C57BL/6 wild‐type (WT) mice, 20 WT mice at postnatal day 1 (P1), and 41 male adult TLR4 gene knockout (TLR4−/−) mice were used to investigate the potential role of TLR4‐induced microglial polarization in early WMI after SAH by radiological, histological, microstructural, transcriptional, and cytological evidence. The results indicated that microglial inflammation was associated with myelin loss and axon damage, shown as a decrease in myelin basic protein (MBP), as well as increase in degraded myelin basic protein (dMBP) and amyloid precursor protein (APP). Gene knockout of TLR4 revised microglial polarization toward the anti‐inflammatory phenotype and protected the white matter at an early phase after SAH (24 h), as shown through reduction of toxic metabolites, preservation of myelin, reductions in APP accumulation, reductions in white matter T2 hyperintensity, and increases in FA values. Cocultures of microglia and oligodendrocytes, the cells responsible for myelin production and maintenance, were established to further elucidate the relationship between microglial polarization and WMI. In vitro, TLR4 inhibition decreased the expression of microglial MyD88 and phosphorylated NF‐κB, thereby inhibiting M1 polarization and mitigating inflammation. Decrease in TLR4 in the microglia increased preservation of neighboring oligodendrocytes. In conclusion, microglial inflammation has dual effects on early WMI after experimental SAH. Future explorations on more clinically relevant methods for modulating neuroinflammation are warranted to combat stroke with both WMI and gray matter destruction.

BackgroundMicroglial necroptosis exacerbates neurodegenerative diseases, central nervous system injury and demonstrates a pro-in ammatory process, but its contribution to subarachnoid hemorrhage (SAH) is poorly characterized. BCL-2 homologous antagonist-killer protein (Bak1), a critical regulatory molecule of endogenous apoptosis, can be involved in the pathological process of necroptosis by regulating mitochondrial permeability. Despite its possible signi cance in microglia necroptosis-mediated in ammation, few studies have investigated Bak1's role in microglia necroptosis regulation. MethodsIn vivo, an experimental subarachnoid hemorrhage in mice identi ed microglia necroptosis and adenoassociated virus targeted interference with microglia Bak1 to determine its effect on neuroin ammation. In vitro knockout of Bak1 in BV2 microglia identi ed its regulatory role in microglia necroptosis and neuroin ammation, and RNA-Seq clari ed the potential downstream regulatory mechanisms. ResultsMicroglia undergo necroptosis after subarachnoid hemorrhage in vivo and vitro. Knocked-down of Bak1 by adeno-associated virus attenuates microglial necroptosis, alleviates neuroin ammation, and improves neurological function after SAH. Increasing Bak1 expression and mediating BV2 microglia proin ammatory phenotype transformation, exacerbating oxidative stress and neuroin ammation.Abrogating Bak1 reduces necroptosis by downregulating the expression of phosphorylated pseudokinase mixed lineage kinase domain-like protein (p-MLKL), then downregulating pro-in ammatory phenotype gene expression. RNA-Seq shows that disrupting BV2 Bak1 downregulates multiple immune and in ammatory pathways and ameliorates cell injury by elevating Thrombospondin 1 (THBS1) expression. ConclusionsOur nding demonstrated that deletion of bak1 alleviates microglial necroptosis, neuroin ammation and improved neurological functions after SAH, which was, at least in part, mediated by activation of THBS1 signaling pathway. Bak1 may be a potential therapeutic strategy for the management of SAH.

In recent years, wastewater treatment to remove tetracycline hydrochloride (TCH), has received much attention in water treatment problems. ZIF-67/C 3 N 4 composite adsorbent, a nanosheet structured material stacked with MOFs, was prepared by in situ growth method, which has high adsorption activity for tetracycline hydrochloride in wastewater. Comparing the effect of monomeric and composite adsorbents, Z 6 C 2 had the best adsorption effect (206 mg•g -1 ), which was 77.6% higher than that of ZIF-67 (116 mg•g -1 ) and 10.8 times higher than that of C 3 N 4 (19 mg•g -1 ).The structure of ZIF-67 stacked on C 3 N 4 nanosheets has an excellent specific surface area and number of active sites, as well as π-π interactions, electrostatic interactions and hydrogen bonding interactions between the adsorbent and TCH, which combine to enhance the adsorption performance. The adsorption process is accompanied by a combination of chemisorption, mass transport and internal diffusion rate-limiting. The adsorbent exhibits good stability and adsorption capacity, which may be suitable for efficient and low-cost water purification.

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