Low-toxic, fluorescent labeled and size-controlled graphene oxide quantum dots@polystyrene nanospheres as reference material for quantitative determination and in vivo tracing.

Polystyrene (PS) is selected as a representative nanoplastic and persistent pollutant for its difficult degradation and wide application. The environmental risk assessment of PS is obstructed by the toxic dye-based fluorescent PS, which false positives could be induced by the leakage of dye. For high biocompatibility, low toxicity, hydrophilicity, good water dispersibility, strong fluorescent stability, graphene oxide quantum dots (o-CQDs) are selected and embedded into PS microspheres, i.e., o-CQDs@PS, by microemulsion polymerization and denoted as CPS. Meanwhile, the sizes of CPS, e.g., 100, 150, and 200 nm, could be controlled by optimizing the type and number of water-soluble initiators. The anti-interference, low toxicity, and in vivo fluorescent tracing of CPS are proven by the coexistence of metals (including Fe2+, Fe3+, K+, Ba2+, Al3+, Zn2+, Mg2+, Ca2+, and Na+) on the fluorescence intensity of CPS, the growth of Chlorella pyrenoidosa and Artemia cysts as aquatic phytoplankton and zooplankton cultured with CPS, and the transfer of CPS from water into brine shrimp. In the concentration range of 0.1-100 mg/L, CPS can be quantitatively determined, which is suitable for coastal water and wastewater treatment plants. Therefore, CPS with standard size is suitable as reference material of PS.

Isoliquiritigenin mitigates oxidative damage after subarachnoid hemorrhage in vivo and in vitro by regulating Nrf2-dependent Signaling Pathway via Targeting of SIRT1.

BACKGROUND: Oxidative stress is a crucial factor leading to subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). Isoliquiritigenin has been verified as a powerful anti-oxidant in a variety of diseases models and can activate sirtuin 1 and nuclear factor-erythroid 2-related factor 2 (Nrf2) pathways. However, the effects of isoliquiritigenin against EBI after SAH and the underlying mechanisms remain elusive. PURPOSE: The primary goal of this study is to verify the therapeutic effects of isoliquiritigenin on EBI after SAH and the possible molecular mechanisms. STUDY DESIGN: A prechiasmatic cistern SAH model in rats and a hemoglobin incubation SAH model in primary neurons were established. Isoliquiritigenin was administered after SAH induction. EX527 was employed to inhibit sirtuin 1 activation and ML385 was used to suppress Nrf2 signaling. METHODS: In our study, neurological scores, brain edema, biochemical estimation, western blotting, and histopathological study were performed to explore the therapeutic action of isoliquiritigenin against SAH. RESULTS: Our data revealed that isoliquiritigenin significantly mitigated oxidative damage after SAH as evidenced by decreased reactive oxygen species overproduction and enhanced intrinsic anti-oxidative system. Concomitant with the reduced oxidative insults, isoliquiritigenin improved neurological function and reduced neuronal death in the early period after SAH. Additionally, isoliquiritigenin administration significantly enhanced Nrf2 and sirtuin 1 expressions. Inhibition of Nrf2 by ML385 reversed the anti-oxidative and neuroprotective effects of isoliquiritigenin against SAH. Moreover, inhibiting sirtuin 1 by EX527 pretreatment suppressed isoliquiritigenin-induced Nrf2-dependent pathway and abated the cerebroprotective effects of isoliquiritigenin. In primary cortical neurons, isoliquiritigenin treatment also ameliorated oxidative insults and repressed neuronal degeneration. The beneficial aspects of isoliquiritigenin were attributed to the promotion of sirtuin 1 and Nrf2 signaling pathways and were counteracted by EX527. CONCLUSION: Our findings suggest that isoliquiritigenin exerts cerebroprotective effects against SAH-induced oxidative insults by modulating the Nrf2-mediated anti-oxidant signaling in part through sirtuin 1 activation. Isoliquiritigenin might be a new potential drug candidate for SAH.

Luteolin Confers Cerebroprotection after Subarachnoid Hemorrhage by Suppression of NLPR3 Inflammasome Activation through Nrf2-Dependent Pathway.

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.

Peroxiredoxin 1/2 protects brain against H2O2-induced apoptosis after subarachnoid hemorrhage.

Recent studies suggest that peroxiredoxin1/2 (Prx1/2) may be involved in the pathophysiology of postischemic 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.

Recombinant milk fat globule-EGF factor-8 reduces apoptosis via integrin ß3/FAK/PI3K/AKT signaling pathway in rats after traumatic brain injury.

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 ß3 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-ß3/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-ß3/FAK/PI3K/AKT pathway-related components. Administration of integrin-ß3 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-ß3/FAK/PI3K/AKT signaling pathway, highlighting rhMFG-E8 as a potentially promising therapeutic strategy for TBI patients.

Peroxiredoxin 2 activates microglia by interacting with Toll-like receptor 4 after subarachnoid hemorrhage.

BACKGROUND: Peroxiredoxin (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. METHODS: We 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. RESULTS: Prx2 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. CONCLUSIONS: Extracellular 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.

Inhibition of leukotriene B4 synthesis protects against early brain injury possibly via reducing the neutrophil-generated inflammatory response and oxidative stress after subarachnoid hemorrhage in rats.

Leukotriene B4 (LTB4) is a highly potent neutrophil chemoattractant and neutrophils induces inflammatory response and oxidative stress when they recruit to and infiltrate in the injuried/inflamed site, such as the brain parenchyma after aneurysmal subarachnoid hemorrhage (SAH). This study is to investigate the potential effects of inhibition of LTB4 synthesis on neutrophil recruitment, inflammatory response and oxidative stress, as well as early brain injury (EBI) in rats after SAH. A pre-chiasmatic cistern SAH model of rats was used in this experiment. SC 57461A was used to inhibit LTB4 synthesis via intracerebroventricular injection. The brain tissues of temporal lobe after SAH were analyzed. Neuronal injury, brain edema and neurological function were evaluated to investigate the development of EBI. We found that inhibition of LTB4 synthesis after SAH could reduce the level of myeloperoxidase, alleviate the inflammatory response and oxidative stress, and reduce neuronal death in the brain parenchyma, and ameliorate brain edema and neurological behavior impairment at 24h after SAH. These results suggest that inhibition of LTB4 synthesis might alleviate EBI after SAH possibly via reducing the neutrophil-generated inflammatory response and oxidative stress.

Roles of Pannexin-1 Channels in Inflammatory Response through the TLRs/NF-Kappa B Signaling Pathway Following Experimental Subarachnoid Hemorrhage in Rats.

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.

RETRACTED: Downregulation of miR-204 expression correlates with poor clinical outcome of glioma patients.

Glioma is the most common type of malignant neoplasm in the central nervous system, with high incidence and mortality rate. MicroRNAs, as a class of small noncoding RNAs, play an important role in carcinogenesis and correlate with glioma diagnosis and prognosis. In this study, we investigated the microRNA-204 (miR-204) concentration in glioma tissues and its relation to the expression of ezrin and bcl-2 mRNA, as well as its potential predictive and prognostic values in glioma. The concentrations of miR-204 were significantly lower in glioma tissues than in nontumor brain tissues and also were lower in high-grade than in low-grade gliomas (World Health Organization grades III and IV versus grades I and II). The miR-204 concentration was inversely correlated with the ezrin and bcl-2 concentrations. The miR-204 concentration was classified as high or low according to the median value, and low miR-204 correlated with higher World Health Organization grade, larger tumor, and worse Karnofsky performance score. Kaplan-Meier survival analysis demonstrated that patients with low miR-204 expression had shorter progression-free survival and overall survival than patients with high miR-204 expression. In addition, univariate and multivariate analyses showed that miR-204 expression was an independent prognostic feature of overall survival and progression-free survival. In conclusion, our study indicates that miR-204 is downregulated in glioma and may be a biomarker of poor prognosis in patients with this cancer.

Expression and cell distribution of leukotriene B4 receptor 1 in the rat brain cortex after experimental subarachnoid hemorrhage.

Convincing evidence supports that nuclear factor kappa B (NF-κB)-meditated inflammation contributes to the adverse prognosis of aneurysmal subarachnoid hemorrhage (SAH), and pathologic neutrophil accumulation after SAH in the brain parenchyma enhances the inflammatory process. Leukotriene B4 (LTB4) is a highly potent lipid chemoattractant of neutrophils, and its biological effects are mediated primarily through the high-affinity LTB4 receptor 1 (BLT1). It is verified that NF-κB-dependent BLT1 mediates LTB4 signaling and LTB4 stimulates NF-κB-dependent inflammation via BLT1. This study aimed to determine the expression and cell distribution of BLT1 in the brain cortex after SAH and investigate the potential relationship between protein expressions of BLT1 and NF-κB. Male Sprague-Dawley rats were randomly assigned into sham group and SAH groups at 6h, 12h and on day 1, day 2 and day 3 (n=6 for each subgroup). SAH groups suffered experimental SAH by injecting 0.3ml autologous blood into the prechiasmatic cistern. BLT1 expression was measured by real-time PCR, western blot, immunohistochemistry and immunofluorescence. Nuclear expression of p65 protein, the major subunit of NF-κB, was also detected by western blot. Our data showed that the expression levels of BLT1 and nuclear p65 protein were both markedly increased after SAH. Moreover, there was a significant positive correlation between BLT1 and nuclear p65 protein expressions in the same specific time course. Double immunofluorescence staining showed that BLT1 were mainly expressed in neurons, microglia and endothelial cells rather than astrocytes after SAH. These results suggest that BLT1 may participate in the NF-κB-mediated inflammatory response after SAH, and there might be important implications for further studies using specific BLT1 antagonists to attenuate the NF-κB-mediated inflammation after SAH.

Elevated cerebrospinal fluid levels of thrombospondin-1 correlate with adverse clinical outcome in patients with aneurysmal subarachnoid hemorrhage.

BACKGROUND: Thrombospondin-1 (TSP-1) is a homotrimeric glycoprotein which modulates a wide range of biological functions. Elevated level of TSP-1 in plasma was reported to be correlated with intracerebral hemorrhage. Our study was designed to investigate the relationship between cerebrospinal fluid (CSF) TSP-1 levels and clinical outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH). METHODS: CSF TSP-1 levels were measured in 31 aSAH patients on days 1-3, days 5-7 and days 8-10 after aSAH onset using enzyme-linked immunosorbent assay. Patients were under a close follow-up until death or completion of three months after aSAH. Binary logistic regression analyses were performed to determine independent risk factors for the clinical outcomes. RESULTS: TSP-1 levels peaked on days 1-3 after aSAH, kept up high on days 5-7 and remained elevated until days 8-10 (p<0.05). Significant elevation of CSF TSP-1 levels were found in patients both with and without vasospasm. Modified Rankin Scale at 3months after aSAH showed a significant correlation with CSF TSP-1 levels on days 1-3 and days 5-7 (both p<0.01). Binary logistic regression analysis showed that higher TSP-1 level on days 1-3 (p<0.05) and on days 5-7 (p<0.05) was a predictive marker of cerebrovasospasm and poor outcome of patient with aSAH. CONCLUSIONS: Upregulation of TSP-1 may involve in the pathological process of aSAH and might be a risk factor of future adverse prognosis of aSAH.

The effect of subarachnoid erythrocyte lysate on brain injury: a preliminary study.

Abundant erythrocytes remain and lyse partially in the subarachnoid space after severe subarachnoid haemorrhage (SAH). But the effect of subarachnoid erythrocyte lysate on brain injury is still not completely clear. In this study, autologous erythrocytes (the non-lysate group) and their lysate (the lysate group) were injected separately into the cistern magna of rabbits to induce a model of experimental SAH, although the control group received isotonic sodium chloride solution instead of erythrocyte solution. Results showed that vasospasm of the basilar artery was observed at 72 h after experimental SAH, but there was no significant difference between the non-lysate group and the lysate group. Brain injury was more severe in the lysate group than in the non-lysate group. Meanwhile, the levels of peroxiredoxin 2 (Prx2), IL-6 and TNF-α in brain cortex and in CSF were significantly higher in the lysate group than those in the non-lysate group. These results demonstrated that brain injury was more likely to be caused by erythrocyte lysate than by intact erythrocytes in subarachnoid space, and inflammation response positively correlated with Prx2 expression might be involved in mechanism of brain injury after SAH.

Inhibition of myeloid differentiation factor 88(MyD88) by ST2825 provides neuroprotection after experimental traumatic brain injury in mice.

Myeloid differentiation factor 88(MyD88) is an endogenous adaptor protein that plays an important role in coordinating intracellular inflammatory responses induced by agonists of the Toll-like receptor and interleukin-1 receptor families. MyD88 has been reported to be essential for neuronal death in animal models and may represent a therapeutic target for pharmacologic inhibition following traumatic brain injury (TBI). The purpose of the current study was to investigate the neuroprotective effect of MyD88 specific inhibitor ST2825 in an experimental mouse model of TBI. Intracerebroventricular (ICV) injection of high concentration (20µg/µL) ST2825 (15min post TBI) attenuated the development of TBI in mice, markedly improved neurological function and reduced brain edema. Decreased neural apoptosis and increased neuronal survival were also observed. Biochemically, the high concentration of ST2825 significantly reduced the levels of MyD88, further decreased TAK1, p-TAK1, nuclear p65 and increased IκB-α. Additionally, ST2825 significantly reduced the levels of Iba-1 and inflammatory factors TNF-α and IL-1ß. These data provide an experimental rationale for evaluation of MyD88 as a drug target and highlight the potential therapeutic implications of ST2825 in TBI.