ß2-Microglobulin exacerbates neuroinflammation, brain damage, and cognitive impairment after stroke in rats.

ß2-Microglobulin (ß2M), a component of the major histocompatibility complex class I molecule, is associated with aging-related cognitive impairment and Alzheimer's disease. Although upregulation of ß2M is considered to be highly related to ischemic stroke, the specific role and underlying mechanistic action of ß2M are poorly understood. In this study, we established a rat model of focal cerebral ischemia by occlusion of the middle cerebral artery. We found that ß2M levels in the cerebral spinal fluid, serum, and brain tissue were significantly increased in the acute period but gradually decreased during the recovery period. RNA interference was used to inhibit ß2M expression in the acute period of cerebral stroke. Tissue staining with 2,3,5-triphenyltetrazolium chloride and evaluation of cognitive function using the Morris water maze test demonstrated that decreased ß2M expression in the ischemic penumbra reduced infarct volume and alleviated cognitive deficits, respectively. Notably, glial cell, caspase-1 (p20), and Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation as well as production of the inflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor-α were also effectively inhibited by ß2M silencing. These findings suggest that ß2M participates in brain injury and cognitive impairment in a rat model of ischemic stroke through activation of neuroinflammation associated with the NLRP3 inflammasome.

Effects of STAT3 Inhibitor BP-1-102 on The Proliferation, Invasiveness, Apoptosis and Neurosphere Formation of Glioma Cells in Vitro.

Malignant glioma, especially glioblastoma (GBM), has historically been associated with a low survival rate. The hyperactivation of STAT3 played a key role in GBM initiation and resistance to therapy; thus, there is an urgent requirement for novel STAT3 inhibitors. BP-1-102 was recently reported as a biochemical inhibitor of STAT3, but its roles and mechanism in biological behavior of glioma cells were still unclear. In this study, the effects of BP-1-102 on proliferation, apoptosis, invasion and neurosphere formation of glioma cell were investigated. Our results indicated that BP-1-102 inhibited the proliferation of U251 and A172 cells, and their IC50 values were 10.51 and 8.534 µM, respectively. Furthermore, BP-1-102 inhibited the invasion and migration abilities of U251 and A172 cells by decreasing the expression of matrix metallopeptidase 9, and induced glioma cell apoptosis by decreasing the expression of B-cell lymphoma-2. BP-1-102 also inhibited the formation of neurosphere. Mechanically, BP-1-102 reduced the phosphorylation of STAT3 and the p-STAT3's nuclear translocation in glioma cells. Thus, this study herein provided a potential drug for glioma therapy.

The Potential Immunomodulatory Properties of Levornidazole Contribute to Improvement in Experimental Ulcerative Colitis.

The use of an antibiotic with immunomodulatory properties could be fascinating in treating multifactorial inflammatory conditions such as ulcerative colitis (UC). We report our investigations into the immunomodulatory properties of levornidazole, the S-enantiomer of ornidazole, which displayed a tremendous therapeutic potential in UC induced by dextran sodium sulfate (DSS). Levornidazole administration to DSS-colitic mice attenuated the intestinal inflammatory process, with an efficacy better than that shown by 5-amino salicylic acid. This was evidenced by decreased disease activity index, ameliorated macroscopic and microscopic colon damages, and reduced expression of inflammatory cytokines. Additionally, levornidazole displayed anti-inflammatory activity through Caveolin-1-dependent reducing IL-1ß and IL-18 secretion by macrophages contributing to its improvement of the intestinal inflammation, as confirmed in vitro and in vivo. In conclusion, these results pointed out that the immunomodulatory effects of levornidazole played a vital role in ameliorating the intestinal inflammatory process, which would be crucial for the translation of its use into clinical settings.

Author Correction: Transplanted miR-219-overexpressing oligodendrocyte precursor cells promoted remyelination and improved functional recovery in a chronic demyelinated model.

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Egr-1 and RNA POL II facilitate glioma cell GDNF transcription induced by histone hyperacetylation in promoter II.

The specific mechanisms for epigenetic regulation of gene transcription remain to be elucidated. We previously demonstrated that hyperacetylation of histone H3K9 in promoter II of glioma cells promotes high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene. This hyperacetylation significantly enhanced Egr-1 binding and increased the recruitment of RNA polymerase II (RNA POL II) to that region (P < 0.05). Egr-1 expression was abnormally increased in C6 glioma cells. Further overexpression of Egr-1 significantly increased Egr-1 binding to GDNF promoter II, while increasing RNA POL II recruitment, thus increasing GDNF transcription (P < 0.01). When the acetylation of H3K9 in the Egr-1 binding site was significantly reduced by the histone acetyltransferase (HAT) inhibitor curcumin, binding of Egr-1 to GDNF promoter II, RNA POL II recruitment, and GDNF mRNA expression were significantly downregulated (P < 0.01). Moreover, curcumin attenuated the effects of Egr-1 overexpression on Egr-1 binding, RNA POL II recruitment, and GDNF transcription (P < 0.01). Egr-1 and RNA POL II co-existed in the nucleus of C6 glioma cells, with overlapping regions, but they were not bound to each other. In conclusion, highly expressed Egr-1 may be involved in the recruitment of RNA POL II in GDNF promoter II in a non-binding manner, and thereby involved in regulating GDNF transcription in high-grade glioma cells. This regulation is dependent on histone hyperacetylation in GDNF promoter II.

Transplanted miR-219-overexpressing oligodendrocyte precursor cells promoted remyelination and improved functional recovery in a chronic demyelinated model.

Oligodendrocyte precursor cells (OPCs) have the ability to repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. Recent evidence suggests that miR-219 helps regulate the differentiation of OPCs into oligodendrocytes. We performed oligodendrocyte differentiation studies using miR-219-overexpressing mouse embryonic stem cells (miR219-mESCs). The self-renewal and multiple differentiation properties of miR219-mESCs were analyzed by the expression of the stage-specific cell markers Nanog, Oct4, nestin, musashi1, GFAP, Tuj1 and O4. MiR-219 accelerated the differentiation of mESC-derived neural precursor cells (NPCs) into OPCs. We further transplanted OPCs derived from miR219-mESCs (miR219-OPCs) into cuprizone-induced chronically demyelinated mice to observe remyelination, which resulted in well-contained oligodendrocyte grafts that migrated along the corpus callosum and matured to express myelin basic protein (MBP). Ultrastructural studies further confirmed the presence of new myelin sheaths. Improved cognitive function in these mice was confirmed by behavioral tests. Importantly, the transplanted miR219-OPCs induced the proliferation of endogenous NPCs. In conclusion, these data demonstrate that miR-219 rapidly transforms mESCs into oligodendrocyte lineage cells and that the transplantation of miR219-OPCs not only promotes remyelination and improves cognitive function but also enhances the proliferation of host endogenous NPCs following chronic demyelination. These results support the potential of a therapeutic role for miR-219 in demyelinating diseases.

GluR3B Ab's induced oligodendrocyte precursor cells excitotoxicity via mitochondrial dysfunction.

Studies have indicated that glutamate receptor subunit 3 peptide B antibodies (GluR3B Ab's) by directing against a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype glutamate receptors (AMPARs) subunit 3 (GluR3B) was involved in the hippocampal neuron damage in the pathogenesis of epilepsy. Glutamate accumulation is critical for oligodendrocyte precursors (OPCs) excitotoxic injury. However, remarkably little is known about whether GluR3B Ab's causes OPCs excitotoxicity, and the underlying mechanisms remain unclear. In this study, we found that the survival rate of OPCs decreased, apoptosis increased and the release of LDH increased with GluR3B Ab's treatment. GluR3B Ab's enhanced the level of intracellular Ca2+ and reactive oxygen species (ROS), caused mitochondrial potential collapse measured by JC-1 and promoted mitochondrial cytochrome C release. AMPARs antagonist NBQX reversed OPCs apoptosis caused by GluR3B Ab's. Taken together, these data suggests that AMPAR was involved in GluR3B Ab's-induced OPCs toxicity by mitochondrial dysfunction. The study revealed a new mechanism for OPCs excitotoxicity in many central nervous system diseases such as epilepsy.

An Epigenetic Mechanism of High Gdnf Transcription in Glioma Cells Revealed by Specific Sequence Methylation.

Glioma cells express high levels of GDNF. When investigating its transcriptional regulation mechanism, we observed increased or decreased methylation of different cis-acting elements in the gdnf promoter II. However, it is difficult to determine the contributions of methylation changes of each cis-acting element to the abnormally high transcription of gdnf gene. To elucidate the contributions of methylation changes of specific cis-acting elements to the regulation of gdnf transcription, we combined gene site-directed mutation, molecular cloning, and dual luciferase assay to develop the "specific sequence methylation followed by plasmid recircularization" method to alter methylation levels of specific cis-acting elements in the gdnf promoter in living cells and assess gene transcriptional activity. This method successfully introduced artificial changes in the methylation of different cis-acting elements in the gdnf promoter II. Moreover, compared with unmethylated gdnf promoter II, both silencer II hypermethylation plus enhancer II unmethylation and hypermethylation of the entire promoter II (containing enhancer II and silencer II) significantly enhanced gdnf transcriptional activity (P < 0.05), and no significant difference was noted between these two hypermethylation patterns (P > 0.05). Enhancer II hypermethylation plus silencer II unmethylation did not significantly affect gene transcription (P > 0.05). Furthermore, we found significantly increased DNA methylation in the silencer II of the gdnf gene in high-grade astroglioma cells with abnormally high gdnf gene expression (P < 0.01). The absence of silencer II significantly increased gdnf promoter II activity in U251 cells (P < 0.01). In conclusion, our specific sequence methylation followed by plasmid recircularization method successfully altered the methylation levels of a specific cis-acting element in a gene promoter in living cells. This method allows in-depth investigation of the impact of methylation changes of different cis-acting elements in the same promoter on gene transcriptional activity. Our findings provide preliminary evidence that silencer II hypermethylation in the gdnf promoter II may underlie high gene transcription in high-grade glioma cells.

Hyperacetylation of histone H3K9 involved in the promotion of abnormally high transcription of the gdnf gene in glioma cells.

The mechanism underlying abnormally high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene in glioma cells is not clear. In this study, to assess histone H3K9 acetylation levels in promoters I and II of the gdnf gene in normal human brain tissue, low- and high-grade glioma tissues, normal rat astrocytes, and rat C6 glioblastoma cells, we employed chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR), real-time PCR, and a pGL3 dual fluorescence reporter system. We also investigated the influence of treatment with curcumin, a histone acetyltransferase inhibitor, and trichostatin A (TSA), a deacetylase inhibitor, on promoter acetylation and activity and messenger RNA (mRNA) expression level of the gdnf gene in C6 cells. Compared to normal brain tissue, H3K9 acetylation in promoters I and II of the gdnf gene increased significantly in high-grade glioma tissues but not in low-grade glioma tissues. Moreover, H3K9 promoter acetylation level of the gdnf gene in C6 cells was also remarkably higher than in normal astrocytes. In C6 cells, curcumin markedly decreased promoter II acetylation and activity and GDNF mRNA expression. Conversely, all three measurements were significantly increased following TSA treatment. Our results suggest that histone H3K9 hyperacetylation in promoter II of the gdnf gene might be one of the reasons for its abnormal high transcription in glioma cells.

Quercetin attenuates cell apoptosis in focal cerebral ischemia rat brain via activation of BDNF-TrkB-PI3K/Akt signaling pathway.

Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF-TrkB-PI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF-TrkB-PI3K/Akt signaling pathway.

[Effects of quercetin on the learning and memory ability of neonatal rats with hypoxic-ischemic brain damage].

OBJECTIVE: To study the effects of quercetin, a flavonoid, on the learning and memory ability of 3-day-old neonatal rats with hypoxic-ischemic brain white matter damage (WMD). METHODS: Sixty 3-day-old Sprague-Dawley rats were randomly divided into four groups: control, WMD model,and quercetin treatment groups (20 and 40 mg/kg). There were 15 rats in each group. Rats in the WMD model and the two quercetin treatment groups were subjected to right common carotid artery ligation followed by 2 hrs of exposure to 8% O2 to induce periventricular white matter injury. After the operation quercetin was administered daily in the two quercetin treatment groups for 6 weeks. Six weeks later, Morris water maze and open-field tests were carried out to test memory and learning ability as well as behavior and cognition. RESULTS: From the second day of training, escape latency in the Morris water maze test was more prolonged in the WMD model group than in the control group (P<0.01). The escape latency in the two quercetin treatment groups was shortened significantly compared with the WMD model group (P<0.05). The WMD model group crossed the original platform fewer times compared with the control and quercetin treatment groups (P<0.05). The open-field test indicated that the number of rearings increased and time spent in the centre was extended in the WMD model group compared with the control group. Compared with the WMD model group, the number of rearings was significantly reduced (P<0.05) and time spent in the centre was significantly shortened in the quercetin treatment groups (P<0.05). CONCLUSIONS: Quercetin treatment can improve memory and learning ability as well as cognitive ability in neonates with WMD, suggesting that quercetin protects against WMD resulting from hypoxia-ischemia.