Ling-Yun Wu

Sirtuin 1 activation protects against early brain injury after experimental subarachnoid hemorrhage in rats

Increasing evidence indicates that sirtuin 1 (SIRT1) is implicated in a wide range of cellular functions, such as oxidative stress, inflammation and apoptosis. The aim of this study was to investigate the change of SIRT1 in the brain after subarachnoid hemorrhage (SAH) and its role on SAH-induced early brain injury (EBI). In the first set of experiments, rats were randomly divided into sham group and SAH groups at 2, 6, 12, 24, 48 and 72 h. The expression of SIRT1 was evaluated by western blot analysis, immunohistochemistry and immunofluorescence. In another set of experiments, SIRT1-specific inhibitor (sirtinol) and activator (activator 3) were exploited to study the role of SIRT1 in SAH-induced EBI. It showed that the protein level of SIRT1 was markedly elevated at the early stage of SAH and peaked at 24 h after SAH. The expression of SIRT1 could be observed in neurons and microglia, and the enhanced SIRT1 was mainly located in neurons after SAH. Administration of sirtinol inhibited the expression and activation of SIRT1 pathways after SAH, while activator 3 enhanced the expression and activation of SIRT1 pathways after SAH. In addition, inhibition of SIRT1 could exacerbate forkhead transcription factors of the O class-, nuclear factor-kappa B- and p53-induced oxidative damage, neuroinflammation and neuronal apoptosis, leading to aggravated brain injury after SAH. In contrast, activator 3 treatment could reduce forkhead transcription factors of the O class-, nuclear factor-kappa B-, and p53-induced oxidative damage, neuroinflammation and neuronal apoptosis to protect against EBI. These results suggest that SIRT1 plays an important role in neuroprotection against EBI after SAH by deacetylation and subsequent inhibition of forkhead transcription factors of the O class-, nuclear factor-kappa B-, and p53-induced oxidative, inflammatory and apoptotic pathways. SIRT1 might be a new promising molecular target for SAH.

Resveratrol Attenuates Acute Inflammatory Injury in Experimental Subarachnoid Hemorrhage in Rats via Inhibition of TLR4 Pathway

Toll-like receptor 4 (TLR4) has been proven to play a critical role in neuroinflammation and to represent an important therapeutic target following subarachnoid hemorrhage (SAH). Resveratrol (RSV), a natural occurring polyphenolic compound, has a powerful anti-inflammatory property. However, the underlying molecular mechanisms of RSV in protecting against early brain injury (EBI) after SAH remain obscure. The purpose of this study was to investigate the effects of RSV on the TLR4-related inflammatory signaling pathway and EBI in rats after SAH. A prechiasmatic cistern SAH model was used in our experiment. The expressions of TLR4, high-mobility group box 1 (HMGB1), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) were evaluated by Western blot and immunohistochemistry. The expressions of Iba-1 and pro-inflammatory cytokines in brain cortex were determined by Western blot, immunofluorescence staining, or enzyme-linked immunosorbent assay. Neural apoptosis, brain edema, and neurological function were further evaluated to investigate the development of EBI. We found that post-SAH treatment with RSV could markedly inhibit the expressions of TLR4, HMGB1, MyD88, and NF-κB. Meanwhile, RSV significantly reduced microglia activation, as well as inflammatory cytokines leading to the amelioration of neural apoptosis, brain edema, and neurological behavior impairment at 24 h after SAH. However, RSV treatment failed to alleviate brain edema and neurological deficits at 72 h after SAH. These results indicated that RSV treatment could alleviate EBI after SAH, at least in part, via inhibition of TLR4-mediated inflammatory signaling pathway.

Upregulation of miR-183 expression and its clinical significance in human brain glioma

Glioma is the most common type of primary malignant tumor in the central nervous system (CNS) with a high incidence and a high mortality rate, as well as an extremely low 5-year survival rate. As a class of small non-coding RNAs, microRNAs (miRNAs) may be closely involved in carcinogenesis and might also be connected with glioma diagnosis and prognosis. In this study, we aimed at investigating the expression level of microRNA-183 (miR-183) in 105 cases of glioma tissues of four World Health Organization (WHO) grades and 10 cases of normal brain tissues and its potential predictive and prognostic values in glioma. We found that the expression levels of miR-183 were significantly higher in glioma tissues than that in normal brain tissues, and also higher in high-grade gliomas (WHO grade III and IV) compared with low-grade gliomas (WHO grade I and II). The miR-183 expression level was classified as low or high according to the median value. High expression of miR-183 was found to significantly correlate with larger tumor size, higher WHO grade, and worse Karnofsky performance score (KPS). Kaplan–Meier survival analysis showed that patients with high miR-183 expression had worse overall survival (OS) and progression-free survival (PFS) than patients with low miR-183 expression. Moreover, univariate and multivariate analyses indicated that miR-183 expression level was an independent prognostic parameter of a patient’s OS and PFS. In conclusion, our study indicated that miR-183 was upregulated in glioma, and that it may be used as a potential biomarker of poor prognosis in patients with glioma.

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.

Sinomenine Provides Neuroprotection in Model of Traumatic Brain Injury via the Nrf2–ARE Pathway

The neuroprotective effect of sinomenine (SIN) has been demonstrated in several brain injury models. However, its role and molecular mechanism in traumatic brain injury (TBI) remain unknown. In this study, we investigated the neuroprotective effects of SIN in the weight-drop model of TBI in male ICR mice. Mice were randomly divided into the sham and TBI groups, SIN (10 mg/kg, 30 mg/kg and 50 mg/kg, administered intraperitoneally) or equal volume of vehicle was given at 30 min after TBI. Treatment with 30 mg/kg SIN significantly improved motor performance and alleviated cerebral edema. However, treatment with 10 mg/kg or 50 mg/kg SIN did not exhibit a better outcome. Therefore, we chose 30 mg/kg SIN for our subsequent experiments. SIN significantly increased the expression of Bcl-2 and decreased that of cleaved caspase-3, indicating that SIN is anti-apoptotic. This was confirmed by the observation that SIN-treated animals had fewer apoptotic neurons. Cortical malondialdehyde content, glutathione peroxidase (GPx) activity and superoxide dismutase (SOD) activity were restored in the group that received SIN. Furthermore, Western blot and immunofluorescence experiments showed that SIN enhanced the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus. SIN administration also significantly upregulated the expression of the downstream factors heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 at pre- and post-transcriptional levels. Together, these data demonstrate that SIN exerts a neuroprotective effect in a model of TBI, possibly by activating the Nrf2–antioxidant response element (ARE) pathway.

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

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.

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.

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

HIGHLIGHTSInhibition of LTB4 reduces neutrophil infiltration in the brain after SAH.Inhibition of LTB4 alleviates inflammation and oxidative stress after SAH.Inhibition of LTB4 alleviates early brain injury after SAH. ABSTRACT 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 24 h 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.

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.

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 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). www.fasebj.org