Cyclopeptide moroidin inhibits vasculogenic mimicry formed by glioblastoma cells via regulating ß-catenin activation and EMT pathways.

Glioblastoma (GBM) is a highly vascularized malignant brain tumor with poor clinical outcomes. Vasculogenic mimicry (VM) formed by aggressive GBM cells is an alternative approach for tumor blood supply and contributes to the failure of anti-angiogenic therapy (AAT). However, there are still a lack of the effective drugs that target VM formation in GBM. In the present study, we evaluate the effects of a plant cyclopeptide moroidin on VM formed by GBM cells and explore its underlying molecular mechanisms. Moroidin evidently suppressed migration, tube formation and expression of α-SMA and metalloproteinase-9 in human GBM cell lines at sub-lethal concentrations. RNA sequencing data suggested the involvement of EMT pathway in the mechanism of moroidin. Exposing GBM cells to moroidin, the expression of EMT marker N-Cadherin and Vimentin decreased in a concentration-dependent manner. Moreover, moroidin significantly reduced the level of phosphorylated ERK and inhibited the activation ß-catenin. The plant cyclopeptide moroidin inhibited the VM formed by GBM cells by inhibiting ERK/ß-catenin-mediated EMT. Our study indicates a promising application of moroidin as an anti-VM agent to the treatment of GBM.

Moroidin, a Cyclopeptide from the Seeds of Celosia cristata That Induces Apoptosis in A549 Human Lung Cancer Cells.

Interference of microtubule dynamics with tubulin-targeted drugs is a validated approach for cancer chemotherapy. Moroidin (1) is an Urticaceae-type cyclopeptide having a potent inhibitory effect on purified tubulin polymerization. So far, moroidin has not been chemically synthesized, and its effect on cancer cells remains unknown. Herein, the cyclopeptide moroidin was isolated and identified from the seeds of Celosia cristata, and a revised assignment of its NMR data was presented. For the first time, moroidin (1) was demonstrated as having cytotoxic effects for several cancer cells, especially A549 lung cancer cells. The cellular evidence obtained showed that moroidin disrupts microtubule polymerization and decreases ß-tubulin protein levels, but is not as potent as colchicine. Molecular docking indicated that 1 has a high binding potential to the vinca alkaloid site on tubulin. Moreover, moroidin arrested A549 cells in the G2/M phase and induced cell apoptosis. The intrinsic mitochondrial pathway and AKT were involved in the moroidin-induced cell apoptosis. In addition, moroidin (1) inhibited the migration and invasion of A549 cells at sublethal concentrations.

Blended learning model via small private online course improves active learning and academic performance of embryology.

While blended learning has been growing in popularity in recent years, the effectiveness of this procedure remains controversial. In this report, we assess the effectiveness of blended learning of embryology within international medical students. The participants were international medical students taking embryology in the Bachelor of Medicine and Bachelor of Surgery program. The blended learning group (BLG) consisted of students (n = 43) in the 2018-2019 academic year, taught with blended learning model via a customized small private online course (SPOC). The control traditional teaching group (TTG) consisted students (n = 48) in the 2017-2018 academic year, taught with traditional teaching model. Academic performance, including mean scores and passing ratios on the final exam of two groups were compared and analyzed with a t-test. In addition, a questionnaire directed toward evaluating student's perceptions with the blended learning was administered to students in BLG. The majority of students in BLG actively participated in online self-study activities and discussion in face-to-face class sessions. The mean score and passing ratio were significantly greater than those of students in TTG (p < 0.01). Results from the questionnaire revealed that the majority of BLG students felt that this method was beneficial for their learning of human embryology. The blended learning model, that integrates SPOC with face-to-face class lectures proved a more effective means for the teaching of embryology than the traditional lecture-based teaching model. This blended learning method may serve as a feasible model that can be readily applied for use in other medical courses.

TIGAR mitigates atherosclerosis by promoting cholesterol efflux from macrophages.

BACKGROUND AND AIMS: TP53-induced glycolysis and apoptosis regulator (TIGAR) is now characterized as a fructose-2,6-bisphosphatase to reduce glycolysis and protect against oxidative stress. Recent studies have demonstrated that TIGAR is associated with cardiovascular disease. However, little is known about its role in atherosclerogenesis. In this study, we aimed to investigate the effect of TIGAR on atherosclerosis and explore the underlying molecular mechanism. METHODS: The Gene Expression Omnibus (GEO) datasets were used to analyze the differential expression of relative proteins. THP-1-derived macrophages were used as an in vitro model and apolipoprotein E-deficient (Apoe-/-) mice were used as an in vivo model. [3H] labeled cholesterol was used to assess the capacity of cholesterol efflux and reverse cholesterol transport (RCT). Both qPCR and Western blot were used to evaluate the mRNA and protein expression, respectively. Lentiviral vectors were used to disturb the expression of TIGAR in vitro and in vivo. Oil Red O, hematoxylin-eosin, and Masson staining were performed to evaluate atherosclerotic plaques in Apoe-/- mice fed a Western diet. Conventional assay kits were used to measure the levels of reactive oxygen species (ROS), plasma lipid profiles and 27-hydroxycholesterol (27-HC). RESULTS: Our results showed that TIGAR is increased upon the formation of macrophage foam cells and atherosclerosis. TIGAR knockdown markedly promoted lipid accumulation in macrophages. Silencing of TIGAR impaired cholesterol efflux and down-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 by interfering with liver X receptor α (LXRα) expression and activity, but did not influence cholesterol uptake by macrophages. Additionally, this inhibitory effect of TIGAR deficiency on cholesterol metabolism was mediated through the ROS/CYP27A1 pathway. In vivo experiments revealed that TIGAR deficiency decreased the levels of ABCA1 and ABCG1 in plaques and aorta and impaired the capacity of RCT, thereby leading to the progression of atherosclerosis in Apoe-/- mice. CONCLUSIONS: TIGAR mitigates the development of atherosclerosis by up-regulating ABCA1 and ABCG1 expression via the ROS/CYP27A1/LXRα pathway.

Smooth muscle-specific HuR knockout induces defective autophagy and atherosclerosis.

Human antigen R (HuR) is a widespread RNA-binding protein involved in homeostatic regulation and pathological processes in many diseases. Atherosclerosis is the leading cause of cardiovascular disease and acute cardiovascular events. However, the role of HuR in atherosclerosis remains unknown. In this study, mice with smooth muscle-specific HuR knockout (HuRSMKO) were generated to investigate the role of HuR in atherosclerosis. HuR expression was reduced in atherosclerotic plaques. As compared with controls, HuRSMKO mice showed increased plaque burden in the atherosclerotic model. Mechanically, HuR could bind to the mRNAs of adenosine 5'-monophosphate-activated protein kinase (AMPK) α1 and AMPKα2, thus increasing their stability and translation. HuR deficiency reduced p-AMPK and LC3II levels and increased p62 level, thereby resulting in defective autophagy. Finally, pharmacological AMPK activation induced autophagy and suppressed atherosclerosis in HuRSMKO mice. Our findings suggest that smooth muscle HuR has a protective effect against atherosclerosis by increasing AMPK-mediated autophagy.

Hepatic HuR protects against the pathogenesis of non-alcoholic fatty liver disease by targeting PTEN.

The liver plays an important role in lipid and glucose metabolism. Here, we show the role of human antigen R (HuR), an RNA regulator protein, in hepatocyte steatosis and glucose metabolism. We investigated the level of HuR in the liver of mice fed a normal chow diet (NCD) and a high-fat diet (HFD). HuR was downregulated in the livers of HFD-fed mice. Liver-specific HuR knockout (HuRLKO) mice showed exacerbated HFD-induced hepatic steatosis along with enhanced glucose tolerance as compared with control mice. Mechanistically, HuR could bind to the adenylate uridylate-rich elements of phosphatase and tensin homolog deleted on the chromosome 10 (PTEN) mRNA 3' untranslated region, resulting in the increased stability of Pten mRNA; genetic knockdown of HuR decreased the expression of PTEN. Finally, lentiviral overexpression of PTEN alleviated the development of hepatic steatosis in HuRLKO mice in vivo. Overall, HuR regulates lipid and glucose metabolism by targeting PTEN.

Myristica fragrans promotes ABCA1 expression and cholesterol efflux in THP-1-derived macrophages.

Myristica fragrans is a traditional herbal medicine and has been shown to alleviate the development of atherosclerosis. However, the anti-atherogenic mechanisms of M. fragrans are still to be addressed. In this study, we explored the effect of M. fragrans on lipid metabolism and inflammation and its mechanisms in THP-1-derived macrophages. The quantitative polymerase chain reaction and western blot analysis results showed that M. fragrans promotes cholesterol efflux from THP-1-derived macrophages and reduces intracellular total cholesterol, cholesterol ester, and free cholesterol contents in a dose- and a time-dependent manner. Further study found that liver X receptor alpha (LXRα) antagonist GGPP significantly blocked the upregulation of ABCA1 expression with M. fragrans treatment. In addition, chromatin immunoprecipitation assay confirmed that GATA binding protein 3 (GATA3) can bind to the LXRα promoter, and inhibition of GATA3 led to the downregulation of LXRα and ATP-binding cassette subfamily A member 1 expression. Furthermore, M. fragrans reduced lipid accumulation, followed by decreasing tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß and increasing IL-10 produced by THP-1-derived macrophages. Therefore, M. fragrans is identified as a valuable therapeutic medicine for atherosclerotic cardiovascular disease.

Maternal hyperglycemia disturbs neocortical neurogenesis via epigenetic regulation in C57BL/6J mice.

Offspring of mothers with hyperglycemia during pregnancy have a higher incidence of long-term neuropsychiatric disorders than offspring from a normal pregnancy, indicating that neocortical neurogenesis might be affected by maternal hyperglycemia. A paucity of study evaluating the effects of hyperglycemia on neocortical neurogenetic differentiation of neural stem cells, and the mechanism remains unclear. We sought to investigate the the roles and possible molecular mechanism of maternal hyperglycemia on neocortical neurogenetic differentiation of neural stem cells. We established a mouse model of a hyperglycemic pregnancy to study effects of intrauterine exposure to maternal hyperglycemia on neocortical neurogenesis. We observed morphological changes in the neocortex and detected the neurogenetic differentiation of neural stem cells in offspring affected by high glucose levels. We investigated the regulatory network between epigenetic modification and transcription factors in differentiated neural stem cells under hyperglycemic conditions. Maternal hyperglycemia disturbs neocortical lamination in some non-malformed offspring. Our results suggested that hyperglycemia altered the early-born neuron fate and the distribution of newborn neurons in deep layers by promoting the earlier differentiation of neural stem cells. Altered histone acetylation and its regulation on the transcription of proneural genes might be correlated to the disrupted differentiation of neural stem cells and altered distribution of newborn projection neurons in the neocortex. Our data raised the possibility that maternal hyperglycemia in pregnancy disturbs the laminar distribution of neocortical projection neurons in some non-malformed offspring via epigenetic regulation on neural stem cell differentiation and the birthdate of neocortical neurons.

Pregnancy-Associated Plasma Protein-A Accelerates Atherosclerosis by Regulating Reverse Cholesterol Transport and Inflammation.

BACKGROUND: Recent studies have suggested that pregnancy-associated plasma protein-A (PAPP-A) is involved in the pathogenesis of atherosclerosis. This study aim is to investigate the role and mechanisms of PAPP-A in reverse cholesterol transport (RCT) and inflammation during the development of atherosclerosis. Methods and Results: PAPP-A was silenced in apolipoprotein E (apoE-/-) mice with administration of PAPP-A shRNA. Oil Red O staining of the whole aorta root revealed that PAPP-A knockdown reduced lipid accumulation in aortas. Oil Red O, hematoxylin and eosin (HE) and Masson staining of aortic sinus further showed that PAPP-A knockdown alleviated the formation of atherosclerotic lesions. It was found that PAPP-A knockdown reduced the insulin-like growth factor 1 (IGF-1) levels and repressed the PI3K/Akt pathway in both aorta and peritoneal macrophages. The expression levels of LXRα, ABCA1, ABCG1, and SR-B1 were increased in the aorta and peritoneal macrophages from apoE-/-mice administered with PAPP-A shRNA. Furthermore, PAPP-A knockdown promoted RCT from macrophages to plasma, the liver, and feces in apoE-/-mice. In addition, PAPP-A knockdown elevated the expression and secretion of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), tumor necrosis factor-α, and interleukin-1ß through the nuclear factor kappa-B (NF-κB) pathway. CONCLUSIONS: The present study results suggest that PAPP-A promotes the development of atherosclerosis in apoE-/-mice through reducing RCT capacity and activating an inflammatory response.

Melatonin enhances atherosclerotic plaque stability by inducing prolyl-4-hydroxylase α1 expression.

OBJECTIVE: Melatonin, an endogenous neurohormone secreted predominately by the pineal gland, has a variety of physiological functions. However, its protective role in atherosclerosis is not clear. In this study, we sought to investigate the potential effects of melatonin in modulating atherosclerotic plaque stability in apolipoprotein E knockout (ApoE) mice. METHOD AND RESULTS: Smooth muscle cells were treated with melatonin, which significantly increased mRNA and protein levels of a key intracellular enzyme essential for collagen maturation and secretion, prolyl-4-hydroxylase α1 (P4Hα1). Mechanistically, melatonin increased Akt phosphorylation and transcriptional activation of specificity protein 1 (Sp1), which bound with the P4Hα1 promoter and then induced P4Hα1 expression. Pretreatment with either Akt inhibitor LY294002 or Sp1 inhibitor mithramycin A (MTM) could inhibit melatonin-induced P4Hα1 expression. Finally, atherosclerotic lesions were induced by placing a perivascular collar on the right common carotid artery of ApoE mice, which were received with or without different doses of melatonin or MTM. High-dose melatonin enhanced atherosclerotic plaque stability in ApoE mice in vivo by inducing the expression of P4Hα1, which was reversed by MTM. CONCLUSION: We propose that melatonin supplementation may provide a novel and promising approach to atherosclerosis treatment.

Nicotine induces endothelial dysfunction and promotes atherosclerosis via GTPCH1.

Smoking is a major preventable risk factor for atherosclerosis. However, the causative link between cigarette smoke and atherosclerosis remains to be established. The objective of this study is to characterize the role of GTP cyclohydrolase 1 (GTPCH1), the rate-limiting enzyme for de novo tetrahydrobiopterin (BH4) synthesis, in the smoking-accelerated atherosclerosis and the mechanism involved. In vitro, human umbilical vein endothelial cells were treated with nicotine, a major component of cigarette smoke, which reduced the mRNA and protein levels of GTPCH1 and led to endothelial dysfunction. GTPCH1 overexpression or sepiapterin could attenuate nicotine-reduced nitric oxide and -increased reactive oxygen species levels. Mechanistically, human antigen R (HuR) bound with the adenylateuridylate-rich elements of the GTPCH1 3' untranslated region and increased its stability; nicotine inhibited HuR translocation from the nucleus to cytosol, which downregulated GTPCH1. In vivo, nicotine induced endothelial dysfunction and promoted atherosclerosis in ApoE-/- mice, which were attenuated by GTPCH1 overexpression or BH4 supplement. Our findings may provide a novel and promising approach to atherosclerosis treatment.

Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells.

Bromodomain containing 4 (BRD4), a member of the bromodomain and extra-terminal family, has become a promising drug target for numerous types of cancer. BRD4 has been reported to be deregulated in gliomas; however, the precise molecular pathways regulated by BRD4 remained elusive. In the present study, BRD4 expression was silenced in the glioma cell line U251 and the results demonstrated that BRD4 knockdown attenuated cell proliferation and promoted cell apoptosis. A genome-wide analysis of BRD4-regulated transcripts in U251 cells was performed using microarray to reveal the possible molecular mechanism. A total of 3,529 differentially expressed genes were identified; 1,648 of these genes were upregulated and 1,881 were downregulated. The results of the gene ontology analysis revealed that these genes were mainly involved in membrane organization, mitotic cell cycle, cell division and DNA replication. Pathway analysis revealed that the pathways altered following BRD4 knockdown included multiple cellular processes, such as cell cycle and apoptosis. Candidate genes were identified through global signal transduction network analysis and were validated using reverse transcription-quantitative polymerase chain reaction and western blot analyses. The results demonstrated that BRD4 knockdown decreased the expression of KRAS proto-oncogene GTPase (KRAS). Downregulated KRAS expression in U251 cells restrained cell proliferation and promoted cell apoptosis, suggesting that the effect of BRD4 on glioma cells might occur through the Ras pathway. In conclusion, the present results confirmed the role of BRD4 in glioma and provided information for further exploration of the molecular mechanism of BRD4 in glioma development and progression.

Heterotrimeric G Stimulatory Protein α Subunit Is Required for Intestinal Smooth Muscle Contraction in Mice.

BACKGROUND & AIMS: The α subunit of the heterotrimeric G stimulatory protein (Gsa), encoded by the guanine nucleotide binding protein, α-stimulating gene (Gnas, in mice), is expressed ubiquitously and mediates receptor-stimulated production of cyclic adenosine monophosphate and activation of the protein kinase A signaling pathway. We investigated the roles of Gsa in vivo in smooth muscle cells of mice. METHODS: We performed studies of mice with Cre recombinase-mediated disruption of Gnas in smooth muscle cells (GsaSMKO and SM22-CreERT2, induced in adult mice by tamoxifen). Intestinal tissues were collected for histologic, biochemical, molecular, cell biology, and physiology analyses. Intestinal function was assessed in mice using the whole-gut transit time test. We compared gene expression patterns of intestinal smooth muscle from mice with vs without disruption of Gnas. Biopsy specimens from ileum of patients with chronic intestinal pseudo-obstruction and age-matched control biopsies were analyzed by immunohistochemistry. RESULTS: Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene (Foxf1) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo-obstruction, compared with tissues from controls. CONCLUSIONS: Gsa is required for intestinal smooth muscle contraction in mice, and its levels are reduced in ileum biopsies of patients with chronic intestinal pseudo-obstruction. Mice with disruption of Gnas might be used to study human chronic intestinal pseudo-obstruction.

Melatonin prevents neural tube defects in the offspring of diabetic pregnancy.

Melatonin, an endogenous neurohormone secreted by the pineal gland, has a variety of physiological functions and neuroprotective effects. However, its protective role on the neural tube defects (NTDs) was not very clear. The aim of this study was to investigate the effects of melatonin on the incidence of NTDs (including anencephaly, encephalocele, and spina bifida) of offspring from diabetic pregnant mice as well as its underlying mechanisms. Pregnant mice were given 10 mg/kg melatonin by daily i.p. injection from embryonic day (E) 0.5 until being killed on E11.5. Here, we showed that melatonin decreased the NTDs (especially exencephaly) rate of embryos exposed to maternal diabetes. Melatonin stimulated proliferation of neural stem cells (NSCs) under hyperglycemic condition through the extracellular regulated protein kinases (ERK) pathway. Furthermore, as a direct free radical scavenger, melatonin decreased apoptosis of NSCs exposed to hyperglycemia. In the light of these findings, it suggests that melatonin supplementation may play an important role in the prevention of neural malformations in diabetic pregnancy.

Melatonin antagonizes hypoxia-mediated glioblastoma cell migration and invasion via inhibition of HIF-1α.

Hypoxia is a crucial factor in tumor aggressiveness and resistance to therapy, especially in glioblastoma. Our previous results have shown that melatonin exerts antimigratory and anti-invasive action in glioblastoma cells under normoxia. However, the effect of melatonin on migration and invasion of glioblastoma cells under hypoxic condition remains poorly understood. Here, we show that melatonin strongly reduced hypoxia-mediated invasion and migration of U251 and U87 glioblastoma cells. In addition, we found that melatonin significantly blocked HIF-1α protein expression and suppressed the expression of downstream target genes, matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF). Furthermore, melatonin destabilized hypoxia-induced HIF-1α protein via its antioxidant activity against ROS produced by glioblastoma cells in response to hypoxia. Along with this, HIF-1α silencing by small interfering RNA markedly inhibited glioblastoma cell migration and invasion, and this appeared to be associated with MMP-2 and VEGF under hypoxia. Taken together, our findings suggest that melatonin suppresses hypoxia-induced glioblastoma cell migration and invasion via inhibition of HIF-1α. Considering the fact that overexpression of the HIF-1α protein is often detected in glioblastoma multiforme, melatonin may prove to be a potent therapeutic agent for this tumor.

Saturated fatty acids activate microglia via Toll-like receptor 4/NF-κB signalling.

Diets rich in SFA have been implicated in Alzheimer's disease (AD). There is strong evidence to suggest that microglial activation augments the progression of AD. However, it remains uncertain whether SFA can initiate microglial activation and whether this response can cause neuronal death. Using the BV-2 microglial cell line and primary microglial culture, we showed that palmitic acid (PA) and stearic acid (SA) could activate microglia, as assessed by reactive morphological changes and significantly increased secretion of pro-inflammatory cytokines, NO and reactive oxygen species, which trigger primary neuronal death. In addition, the mRNA level of these pro-inflammatory mediators determined by RT-PCR was also increased by PA and SA. We further investigated the intracellular signalling mechanism underlying the release of pro-inflammatory mediators from PA-activated microglial cells. The present results showed that PA activated the phosphorylation and nuclear translocation of the p65 subunit of NF-κB. Furthermore, pyrrolidine dithiocarbamate, a NF-κB inhibitor, attenuated the production of pro-inflammatory mediators except for IL-6 in PA-stimulated microglia. Administration of anti-Toll-like receptor (TLR)4-neutralising antibody repressed PA-induced NF-κB activation and pro-inflammatory mediator production. In conclusion, the present in vitro study demonstrates that SFA could activate microglia and stimulate the TLR4/NF-κB pathway to trigger the production of pro-inflammatory mediators, which may contribute to neuronal death.

Expression profile of embryonic stem cell-associated genes Oct4, Sox2 and Nanog in human gliomas.

AIMS: To investigate whether Oct4, Sox2 and Nanog, three core regulatory factors maintaining pluripotency and self-renewal of embryonic stem cells (ESCs), are coexpressed in human gliomas, and whether their expression might be linked to carcinogenesis and the formation of cancer stem cells (CSCs). METHODS AND RESULTS: Forty cases of human glioma were examined. The expression of Oct4, Sox2 and Nanog was analysed by immunohistochemistry, reverse transcription polymerase chain reaction and western blot. We found a positive correlation between the expression levels of Oct4, Sox2 and Nanog and tumour malignancy. Immunohistochemistry showed that Oct4 and Nanog were expressed in both the nuclei and the cytoplasm of glioma cells, whereas Sox2 was expressed only in the nuclei. Double immunofluorescence staining revealed that a majority of Oct4-positive cells coexpressed Sox2 and Nanog. More than 50% of Oct4-positive cells coexpressed the putative CSC markers CD133 and Nestin. Moreover, some cells exhibited Oct4 and Nanog immunoexpression in the cytoplasm, but the frequency of positive cells did not correlate with tumour malignancy. CONCLUSIONS: The present findings suggest that ESC-associated pathways are activated in human gliomas and that these may be involved in glioma progression, a role that is distinct from that in ESCs.

Downregulation of GRIM-19 promotes growth and migration of human glioma cells.

It has become increasingly clear that there are notable parallels between normal development and tumorigenesis. Glioma is a classic model that links between tumorigenesis and development. We evaluated the expression of GRIM-19, a novel gene essential for normal development, in various grades of gliomas and several human glioma cell lines. We showed that GRIM-19 mRNA and protein expression were markedly lower in gliomas than in control brain tissues and negatively correlated with the malignancy of gliomas. Downregulation of GRIM-19 in glioma cells significantly enhanced cell proliferation and migration, whereas overexpression of GRIM-19 showed the opposite effects. We also showed that the activation of signal transducer and activator of transcription 3 (STAT3) and the expression of many STAT3-dependent genes were regulated by the expression of GRIM-19. In addition, GRIM-19 exerted its role probably through the non-STAT3 signaling pathway. Collectively, our data suggest that most gliomas expressed GRIM-19 at low levels, which may play a major role in tumorigenesis in the brain.

Roles of activated astrocyte in neural stem cell proliferation and differentiation.

Recent studies demonstrated that the molecules secreted from astrocytes play important roles in the cell fate determination of neural stem cells (NSCs). However, the exact molecules involved and its possible mechanisms in the process remain largely unknown. In this study, astrocyte-conditioned medium (ACM) obtained from astrocytes unstimulated or stimulated by lipopolysaccharide was prepared to treat NSCs. The results showed that both the proliferation and differentiation of NSCs treated with stimulated ACMs were significantly increased compared with those treated with unstimulated ACM. Interleukin-6 (IL-6) antibody neutralization of the ACMs decreased NSC proliferation and astrogliogenesis, while NSC neurogenesis was increased. In contrast, recombinant IL-6 cytokine increased NSC proliferation and astrogliogenesis, but decreased neurogenesis. Furthermore, the expression of phosphorylated signal transducer and activator of transcription 3 (p-stat3) protein as well as serial of basic helix-loop-helix transcription factors (bHLH) mRNA in NSCs exposed to stimulated ACMs significantly increased, respectively. The expression levels of p-stat3 protein and bHLH mRNA of NSCs were significantly altered after adding anti-IL-6 antibody or recombinant IL-6, respectively. The data suggest that IL-6 secreted from activated astrocytes participates in ACM-induced proliferation and differentiation of NSCs via the phosphorylation of stat3 signals and the expression of bHLH transcription factors.

Anti-inflammatory effects of fluoxetine in lipopolysaccharide(LPS)-stimulated microglial cells.

Recent evidence has suggested that microglial activation plays an important role in the pathogenesis of depression. Activated microglia can secrete various pro-inflammatory cytokines and neurotoxic mediators, which may contribute to the development and maintenance of depression. Thus, inhibition of microglial activation may have a therapeutic benefit in the treatment of depression. In the present study, using BV2 microglial cell line and primary microglial culture, we investigated if fluoxetine, the most widely used antidepressant, can inhibit microglia activation. Our results showed that fluoxetine significantly inhibited lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α), interleukin- 6 (IL-6) and nitric oxide (NO). By RT-PCR, the mRNA level of these pro-inflammatory cytokines and iNOS was also attenuated by fluoxetine. We further investigated the intracellular signaling mechanism regulating the production of pro-inflammatory cytokines and NO from LPS-activated microglia. The results showed that fluoxetine inhibited IκB-a degradation, phosphorylation and nuclear translocation of the p65 subunit of NF-κB, and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in the LPS-stimulated microglia. Taken together, our results suggest that the therapeutic effects of fluoxetine are partially mediated by modulating microglial activation.