Activating Transcription Factor 3 Diminishes Ischemic Cerebral Infarct and Behavioral Deficit by Downregulating Carboxyl-Terminal Modulator Protein.

Activating transcription factor 3 (ATF3) is a stress-induced transcription factor and a familiar neuronal marker for nerve injury. This factor has been shown to protect neurons from hypoxic insult in vitro by suppressing carboxyl-terminal modulator protein (CTMP) transcription, and indirectly activating the anti-apoptotic Akt/PKB cascade. Despite prior studies in vitro, whether this neuroprotective pathway also exists in the brain in vivo after ischemic insult remains to be determined. In the present study, we showed a rapid and marked induction of ATF3 mRNA throughout ischemia-reperfusion in a middle cerebral artery (MCA) occlusion model. Although the level of CTMP mRNA was quickly induced upon ischemia, its level showed only a mild increase after reperfusion. With the gain-of-function approach, both pre- and post-ischemic administration of Ad-ATF3 ameliorated brain infarct and neurological deficits. Whereas, with the loss-of-function approach, ATF3 knockout (KO) mice showed bigger infarct and worse functional outcome after ischemia. In addition, these congenital defects were rescued upon reintroducing ATF3 to the brain of KO mice. ATF3 overexpression led to a lower level of CTMP and a higher level of p-Akt(473) in the ischemic brain. On the contrary, ATF3 KO resulted in upregulation of CTMP and downregulation of p-Akt(473) instead. Furthermore, post-ischemic CTMP siRNA knockdown led to smaller infarct and better behaviors. CTMP siRNA knockdown increased the level of p-Akt(473), but did not alter the ATF3 level in the ischemic brain, upholding the ATF3→CTMP signal cascade. In summary, our proof-of-principle experiments support the existence of neuroprotective ATF3→CTMP signal cascade regulating the ischemic brain. Furthermore, these results suggest the therapeutic potential for both ATF3 overexpression and CTMP knockdown for stroke treatment.

Anti-Inflammatory Effects of Phytochemical Components of Clinacanthus nutans.

Recent studies on the ethnomedicinal use of Clinacanthus nutans suggest promising anti-inflammatory, anti-tumorigenic, and antiviral properties for this plant. Extraction of the leaves with polar and nonpolar solvents has yielded many C-glycosyl flavones, including schaftoside, isoorientin, orientin, isovitexin, and vitexin. Aside from studies with different extracts, there is increasing interest to understand the properties of these components, especially regarding their ability to exert anti-inflammatory effects on cells and tissues. A major focus for this review is to obtain information on the effects of C. nutans extracts and its phytochemical components on inflammatory signaling pathways in the peripheral and central nervous system. Particular emphasis is placed on their role to target the Toll-like receptor 4 (TLR4)-NF-kB pathway and pro-inflammatory cytokines, the antioxidant defense pathway involving nuclear factor erythroid-2-related factor 2 (NRF2) and heme oxygenase 1 (HO-1); and the phospholipase A2 (PLA2) pathway linking to cyclooxygenase-2 (COX-2) and production of eicosanoids. The ability to provide a better understanding of the molecular targets and mechanism of action of C. nutans extracts and their phytochemical components should encourage future studies to develop new therapeutic strategies for better use of this herb to combat inflammatory diseases.

Study of the antitumor mechanisms of apiole derivatives (AP-02) from Petroselinum crispum through induction of G0/G1 phase cell cycle arrest in human COLO 205 cancer cells.

BACKGROUND: Apiole was isolated from the leaves of various plants and vegetables and has been demonstrated to inhibit human colon cancer cell (COLO 205 cells) growth through induction of G0/G1 cell cycle arrest and apoptotic cell death. This study further explored the antitumor effects of apiole derivatives AP-02, 04, and 05 in COLO 205 cancer cells. METHODS: Human breast (MDA-MB-231, ZR75), lung (A549, PE089), colon (COLO 205, HT 29), and hepatocellular (Hep G2, Hep 3B) cancer cells were treated with apiole and its derivatives in a dose-dependent manner. Flow cytometry analysis was subsequently performed to determine the mechanism of AP-02-induced G0/G1 cell cycle arrest. The in vivo antitumor effect of AP-02 (1 and 5 mg/kg, administered twice per week) was examined by treating athymic nude mice bearing COLO 205 tumor xenografts. The molecular mechanisms of AP-02-induced antitumor effects were determined using western blot analysis. RESULTS: AP-02 was the most effective compound, especially for inhibition of COLO 205 colon cancer cell growth. The cytotoxicity of AP-02 in normal colon epithelial (FHC) cells was significantly lower than that in other normal cells derived from the breast, lung or liver. Flow cytometry analysis indicated that AP-02-induced G0/G1 cell cycle arrest in COLO 205 cells but not in HT 29 cells (< 5 µM for 24 h, **p < 0.01). Tumor growth volume was also significantly inhibited in AP-02 (> 1 mg/kg)-treated athymic nude mice bearing COLO 205 tumor xenografts compared to control mice (*p < 0.05). Furthermore, G0/G1 phase regulatory proteins (p53 and p21/Cip1) and an invasion suppressor protein (E-cadherin) were significantly upregulated, while cyclin D1 was significantly downregulated, in AP-02-treated tumor tissues compared to the control group (> 1 mg/kg, *p < 0.05). CONCLUSIONS: Our results provide in vitro and in vivo molecular evidence of AP-02-induced anti-proliferative effects on colon cancer, indicating that this compound might have potential clinical applications.

Histone deacetylases in stroke.

Stroke is the second leading cause of death and the leading cause of adult disability worldwide. Despite an impressive amount of neuroprotective agents that has been identified in experimental stroke, none of them proved efficient in clinical trials. There is a general consensus that an effective treatment requires the ability to interact with not one, but multiple pathophysiological cascades at different levels that induced by the insult - cocktail therapy. Luckily, recent progress in the field of epigenetics revealed that epigenetic modifications had influence on many known pathways involved in the complex course of ischemic disease development. The fact that epigenetic molecules, by altering transcriptional regulation, may simultaneously act on different levels of ischemic brain injury makes them promising candidates for clinical use. These modifications arise typically owing to deoxyribonucleic acid methylation and histone acetylation. The aim of this review is to give a comprehensive overview of current advances in stroke epigenetics, in particular, the physiological and pathological functions of the 11 classical histone deacetylases.

Interleukin 15 blockade protects the brain from cerebral ischemia-reperfusion injury.

Acute ischemic stroke is followed by a complex interplay between the brain and the immune system in which ischemia-reperfusion leads to a detrimental inflammatory response that causes brain injury. In the brain, IL-15 is expressed by astrocytes, neurons and microglia. Previous study showed that ischemia-reperfusion induces expression of IL-15 by astrocytes. Transgenic over-expression of IL-15 in astrocytes aggravates ischemia-reperfusion brain damage by increasing the levels and promoting the effector functions of CD8+ T and NK cells. Treatment of neonatal rats with IL-15 neutralizing antibody before hypoxia-ischemia induction reduces the infarct volume. However, as stroke-induced inflammatory responses differ between neonate and adult brain, the effects of IL-15 blockade on the injury and immune response arising from stroke in adult animals has remained unclear. In this study, we examined the effect of post-ischemia/reperfusion IL-15 blockade on the pathophysiology of cerebral ischemia-reperfusion in adult mice. Using a cerebral ischemia-reperfusion model, we compared infarct size and the infiltrating immune cells in the brain of wild type (WT) mice and Il15-/- mice lacking NK and memory CD8+ T cells. We also evaluated the effects of IL-15 neutralizing antibody treatment on brain infarct volume, motor function, and the status of brain-infiltrating immune cells in WT mice. Il15-/- mice show a smaller infarct volume and lower numbers of activated brain-infiltrating NK, CD8+ T, and CD4+ T cells compared to WT mice after cerebral ischemia-reperfusion. Post-ischemia/reperfusion IL-15 blockade reduces infarct size and improves motor and locomotor activity. Furthermore, IL-15 blockade reduces the effector function of NK, CD8+ T, and CD4+ T cells in the ischemia-reperfusion brain of WT mice. Ablation of IL-15 responses after cerebral ischemia-reperfusion ameliorates brain injury in adult mice. Therefore, targeting IL-15 is a potential effective therapy for ischemic stroke.

Expression of DHA-Metabolizing Enzyme Alox15 is Regulated by Selective Histone Acetylation in Neuroblastoma Cells.

The omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) is enriched in neural membranes of the CNS, and recent studies have shown a role of DHA metabolism by 15-lipoxygenase-1 (Alox15) in prefrontal cortex resolvin D1 formation, hippocampo-prefrontal cortical long-term-potentiation, spatial working memory, and anti-nociception/anxiety. In this study, we elucidated epigenetic regulation of Alox15 via histone modifications in neuron-like cells. Treatment of undifferentiated SH-SY5Y human neuroblastoma cells with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate significantly increased Alox15 mRNA expression. Moreover, Alox15 expression was markedly upregulated by Class I HDAC inhibitors, MS-275 and depsipeptide. Co-treatment of undifferentiated SH-SY5Y cells with the p300 histone acetyltransferase (HAT) inhibitor C646 and TSA or sodium butyrate showed that p300 HAT inhibition modulated TSA or sodium butyrate-induced Alox15 upregulation. Differentiation of SH-SY5Y cells with retinoic acid resulted in increased neurite outgrowth and Alox15 mRNA expression, while co-treatment with the p300 HAT inhibitor C646 and retinoic acid modulated the increases, indicating a role of p300 HAT in differentiation-associated Alox15 upregulation. Increasing Alox15 expression was found in primary murine cortical neurons during development from 3 to 10 days-in-vitro, reaching high levels of expression by 10 days-in-vitro-when Alox15 was not further upregulated by HDAC inhibition. Together, results indicate regulation of Alox15 mRNA expression in neuroblastoma cells by histone modifications, and increasing Alox15 expression in differentiating neurons. It is possible that one of the environmental influences on the immature brain that can affect cognition and memory, may take the form of epigenetic effects on Alox15 and metabolites of DHA.

Astrocytic GAP43 Induced by the TLR4/NF-κB/STAT3 Axis Attenuates Astrogliosis-Mediated Microglial Activation and Neurotoxicity.

Growth-associated protein 43 (GAP43), a protein kinase C (PKC)-activated phosphoprotein, is often implicated in axonal plasticity and regeneration. In this study, we found that GAP43 can be induced by the endotoxin lipopolysaccharide (LPS) in rat brain astrocytes both in vivo and in vitro. The LPS-induced astrocytic GAP43 expression was mediated by Toll-like receptor 4 and nuclear factor-κB (NF-κB)- and interleukin-6/signal transducer and activator of transcription 3 (STAT3)-dependent transcriptional activation. The overexpression of the PKC phosphorylation-mimicking GAP43(S41D) (constitutive active GAP43) in astrocytes mimicked LPS-induced process arborization and elongation, while application of a NF-κB inhibitory peptide TAT-NBD or GAP43(S41A) (dominant-negative GAP43) or knockdown of GAP43 all inhibited astrogliosis responses. Moreover, GAP43 knockdown aggravated astrogliosis-induced microglial activation and expression of proinflammatory cytokines. We also show that astrogliosis-conditioned medium from GAP43 knock-down astrocytes inhibited GAP43 phosphorylation and axonal growth, and increased neuronal damage in cultured rat cortical neurons. These proneurotoxic effects of astrocytic GAP43 knockdown were accompanied by attenuated glutamate uptake and expression of the glutamate transporter excitatory amino acid transporter 2 (EAAT2) in LPS-treated astrocytes. The regulation of EAAT2 expression involves actin polymerization-dependent activation of the transcriptional coactivator megakaryoblastic leukemia 1 (MKL1), which targets the serum response elements in the promoter of rat Slc1a2 gene encoding EAAT2. In sum, the present study suggests that astrocytic GAP43 mediates glial plasticity during astrogliosis, and provides beneficial effects for neuronal plasticity and survival and attenuation of microglial activation. SIGNIFICANCE STATEMENT: Astrogliosis is a complex state in which injury-stimulated astrocytes exert both protective and harmful effects on neuronal survival and plasticity. In this study, we demonstrated for the first time that growth-associated protein 43 (GAP43), a well known growth cone protein that promotes axonal regeneration, can be induced in rat brain astrocytes by the proinflammatory endotoxin lipopolysaccharide via both nuclear factor-κB and signal transducer and activator of transcription 3-mediated transcriptional activation. Importantly, LPS-induced GAP43 mediates plastic changes of astrocytes while attenuating astrogliosis-induced microglial activation and neurotoxicity. Hence, astrocytic GAP43 upregulation may serve to indicate beneficial astrogliosis after CNS injury.

Protective Effect of Ergothioneine Against Stroke in Rodent Models.

Ergothioneine (ET) is a naturally occurring antioxidant and cytoprotective agent that is synthesized by fungi and certain bacteria. Recent studies have shown a beneficial effect of ET on neurological functions, including cognition and animal models of depression. The aim of this study is to elucidate a possible effect of ET in rodent models of stroke. Post-ischemic intracerebroventricular (i.c.v.) infusion of ET significantly reduced brain infarct volume by as early as 1 day after infusion in rats, as shown by triphenyltetrazolium chloride (TTC) assay. There was a dose-dependent increase in protection, from 50 to 200 ng of ET infusion. These results suggest that ET could have a protective effect on CNS neurons. We next elucidated the effect of systemic ET on brain infarct volume in mice after stroke. Daily i.p. injection of 35 mg/kg ET (the first dose being administered 3 h after stroke) had no significant effect on infarct volume. However, daily i.p. injections of 70 mg/kg, 100 mg/kg, 125 mg/kg and 150 mg/kg ET, with the first dose administered 3 h after stroke, significantly decreased infarct volume at 7 days after vessel occlusion in mice. In order to elucidate at what time interval during the 7 days there could be effective protection, a second set of experiments was carried out in mice, using one of the effective loading protocols, i.e. 125 mg/kg i.p. ET but the brains were analyzed at 1, 4 and 7 days post-stroke by MRI. We found that ET was already protective against neuronal injury and decreased the size of the brain infarct from as early as 1 day post-stroke. Behavioral experiments carried out on a third set of mice (using 125 mg/kg i.p. ET) showed that this was accompanied by significant improvements in certain behaviors (pole test) at 1 day after stroke. Together, results of this study indicate that i.c.v. and systemic ET are effective in reducing brain infarct volume after stroke in rodent models.

COVID-19, Blood Lipid Changes, and Thrombosis.

Although there is increasing evidence that oxidative stress and inflammation induced by COVID-19 may contribute to increased risk and severity of thromboses, the underlying mechanism(s) remain to be understood. The purpose of this review is to highlight the role of blood lipids in association with thrombosis events observed in COVID-19 patients. Among different types of phospholipases A2 that target cell membrane phospholipids, there is increasing focus on the inflammatory secretory phospholipase A2 IIA (sPLA2-IIA), which is associated with the severity of COVID-19. Analysis indicates increased sPLA2-IIA levels together with eicosanoids in the sera of COVID patients. sPLA2 could metabolise phospholipids in platelets, erythrocytes, and endothelial cells to produce arachidonic acid (ARA) and lysophospholipids. Arachidonic acid in platelets is metabolised to prostaglandin H2 and thromboxane A2, known for their pro-coagulation and vasoconstrictive properties. Lysophospholipids, such as lysophosphatidylcholine, could be metabolised by autotaxin (ATX) and further converted to lysophosphatidic acid (LPA). Increased ATX has been found in the serum of patients with COVID-19, and LPA has recently been found to induce NETosis, a clotting mechanism triggered by the release of extracellular fibres from neutrophils and a key feature of the COVID-19 hypercoagulable state. PLA2 could also catalyse the formation of platelet activating factor (PAF) from membrane ether phospholipids. Many of the above lipid mediators are increased in the blood of patients with COVID-19. Together, findings from analyses of blood lipids in COVID-19 patients suggest an important role for metabolites of sPLA2-IIA in COVID-19-associated coagulopathy (CAC).

Clinacanthus nutans Mitigates Neuronal Death and Reduces Ischemic Brain Injury: Role of NF-κB-driven IL-1ß Transcription.

Neuroinflammation has been shown to exacerbate ischemic brain injury, and is considered as a prime target for the development of stroke therapies. Clinacanthus nutans Lindau (C. nutans) is widely used in traditional medicine for treating insect bites, viral infection and cancer, due largely to its anti-oxidative and anti-inflammatory properties. Recently, we reported that an ethanol extract from the leaf of C. nutans could protect the brain against ischemia-triggered neuronal death and infarction. In order to further understand the molecular mechanism(s) for its beneficial effects, two experimental paradigms, namely, in vitro primary cortical neurons subjected to oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery (MCA) occlusion, were used to dissect the anti-inflammatory effects of C. nutans extract. Using promoter assays, immunofluorescence staining, and loss-of-function (siRNA) approaches, we demonstrated that transient OGD led to marked induction of IL-1ß, IL-6 and TNFα, while pretreatment with C. nutans suppressed production of inflammatory cytokines in primary neurons. C. nutans inhibited IL-1ß transcription via preventing NF-κB/p65 nuclear translocation, and siRNA knockdown of either p65 or IL-1ß mitigated OGD-mediated neuronal death. Correspondingly, post-ischemic treatment of C. nutans attenuated IκBα degradation and decreased IL-1ß, IL-6 and TNFα production in the ischemic brain. Furthermore, IL-1ß siRNA post-ischemic treatment reduced cerebral infarct, thus mimicking the beneficial effects of C. nutans. In summary, our findings demonstrated the ability for C. nutans to suppress NF-κB nuclear translocation and inhibit IL-1ß transcription in ischemic models. Results further suggest the possibility for using C. nutans to prevent and treat stroke patients.

Galectin-7 downregulation in lesional keratinocytes contributes to enhanced IL-17A signaling and skin pathology in psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by inflammatory cell infiltration, as well as hyperproliferation of keratinocytes in skin lesions, and is considered a metabolic syndrome. We found that the expression of galectin-7 is reduced in skin lesions of patients with psoriasis. IL-17A and TNF-α, 2 cytokines intimately involved in the development of psoriatic lesions, suppressed galectin-7 expression in human primary keratinocytes (HEKn cells) and the immortalized human keratinocyte cell line HaCaT. A galectin-7 knockdown in these cells elevated the production of IL-6 and IL-8 and enhanced ERK signaling when the cells were stimulated with IL-17A. Galectin-7 attenuated IL-17A-induced production of inflammatory mediators by keratinocytes via the microRNA-146a/ERK pathway. Moreover, galectin-7-deficient mice showed enhanced epidermal hyperplasia and skin inflammation in response to intradermal IL-23 injection. We identified fluvastatin as an inducer of galectin-7 expression by connectivity map analysis, confirmed this effect in keratinocytes, and demonstrated that fluvastatin attenuated IL-6 and IL-8 production induced by IL-17A. Thus, we validate a role of galectin-7 in the pathogenesis of psoriasis, in both epidermal hyperplasia and keratinocyte-mediated inflammatory responses, and formulate a rationale for the use of statins in the treatment of psoriasis.

Ligand-activated peroxisome proliferator-activated receptor-gamma protects against ischemic cerebral infarction and neuronal apoptosis by 14-3-3 epsilon upregulation.

BACKGROUND: Thiazolidinediones have been reported to protect against ischemia-reperfusion injury. Their protective actions are considered to be peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-dependent; however, it is unclear how PPAR-gamma activation confers resistance to ischemia-reperfusion injury. METHODS AND RESULTS: We evaluated the effects of rosiglitazone or PPAR-gamma overexpression on cerebral infarction in a rat model and investigated the antiapoptotic actions in the N2-A neuroblastoma cell model. Rosiglitazone or PPAR-gamma overexpression significantly reduced infarct volume. The protective effect was abrogated by PPAR-gamma small interfering RNA. In mice with knock-in of a PPAR-gamma dominant-negative mutant, infarct volume was enhanced. Proteomic analysis revealed that brain 14-3-3epsilon was highly upregulated in rats treated with rosiglitazone. Upregulation of 14-3-3epsilon was abrogated by PPAR-gamma small interfering RNA or antagonist. Promoter analysis and chromatin immunoprecipitation revealed that rosiglitazone induced PPAR-gamma binding to specific regulatory elements on the 14-3-3epsilon promoter and thereby increased 14-3-3epsilon transcription. 14-3-3epsilon Small interfering RNA abrogated the antiapoptotic actions of rosiglitazone or PPAR-gamma overexpression, whereas 14-3-3epsilon recombinant proteins rescued brain tissues and N2-A cells from ischemia-induced damage and apoptosis. Elevated 14-3-3epsilon enhanced binding of phosphorylated Bad and protected mitochondrial membrane potential. CONCLUSIONS: Ligand-activated PPAR-gamma confers resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3epsilon transcription. 14-3-3epsilon Upregulation enhances sequestration of phosphorylated Bad and thereby suppresses apoptosis.

Rosiglitazone and PPAR-gamma overexpression protect mitochondrial membrane potential and prevent apoptosis by upregulating anti-apoptotic Bcl-2 family proteins.

To determine the involvement of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in cytoprotection, we subjected N2-A cells to oxygen-glucose deprivation followed by reoxygenation (H-R). Following H-R insults, H(2)O(2) production was increased while cell viability declined, which was accompanied by loss of mitochondrial membrane potential (MMP), cytochrome c release, caspases 9 and 3 activation, poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis. Rosiglitazone up to 5 microM protected cell viability, normalized MMP, and prevented apoptotic signals. The protective effect of rosiglitazone was abrogated by GW9662, a PPAR-gamma antagonist, or a specific PPAR-gamma small interference RNA (siRNA) but not a control scRNA. PPAR-gamma overexpression alone was effective in maintaining MMP and preventing apoptosis and its protective effect was also abrogated by PPAR-gamma siRNA or GW9662. To elucidate the mechanism by which PPAR-gamma protects MMP and prevents apoptosis, we analyzed Bcl-2, Bcl-xl, and phosphorylated Bad (p-Bad). H-R suppressed them. Rosiglitazone or PPAR-gamma overexpression restored them via PPAR-gamma. Rosiglitazone or PPAR-gamma overexpression preserved phosphorylated Akt and 3-phosphoinositide-dependent kinase-1 (PDK-1) in a PPAR-gamma dependent manner. These results indicate that ligand-activated PPAR-gamma protects N2-A cells against H-R damage by enhancing Bcl-2/Bcl-xl and maintaining p-Bad via preservation of p-Akt.

In vivo cerebromicrovasculatural visualization using 3D DeltaR2-based microscopy of magnetic resonance angiography (3DDeltaR2-mMRA).

This study proposed a novel methodology for depicting cerebral small vessels including veins, arterioles, and venules, called 3DDeltaR(2)-mMRA (three-dimensional, steady-state DeltaR(2)-based, and flow-independent microscopic magnetic resonance angiography). The DeltaR(2) map calculated by a fast spin-echo imaging technique before and after the injection of an iron-oxide contrast agent was used to delineate the relative cerebral blood volume, primarily to microvasculature. The proposed 3DDeltaR(2)-mMRA method, which employs 3D reconstruction techniques, can simultaneously provide high-resolution 3D information on the cerebral anatomy, in vivo microvascular architecture, and hemodynamic response, which can be used to evaluate pathological microvascular changes over time in cerebromicrovascular disease. Since spin-echo-based DeltaR(2) imaging was applied, the inflow effects, susceptibility artifacts, and the overestimation of vessel size in brain were reduced. A well-defined three-vessel occlusion model in the rat was performed to evaluate the capability of the proposed method in evaluating alterations to the microvasculature.

The apple polyphenol phloretin inhibits breast cancer cell migration and proliferation via inhibition of signals by type 2 glucose transporter.

Human triple-negative breast cancer (TNBC) is the most aggressive and poorly understood subclass of breast cancer. Glucose transporters (GLUTs) are required for glucose uptake in malignant cancer cells and are ideal targets for cancer therapy. To determine whether the inhibition of GLUTs could be used in TNBC cell therapy, the apple polyphenol phloretin (Ph) was used as a specific antagonist of GLUT2 protein function in human TNBC cells. Interestingly, we found that Ph (10-150 µM, for 24 h) inhibited cell growth and arrested the cell cycle in MDA-MB-231 cells in a p53 mutant-dependent manner, which was confirmed by pre-treatment of the cells with a p53-specific dominant-negative expression vector. We also found that Ph treatment (10-150 µM, for 24 h) significantly decreased the migratory activity of the MDA-MB-231 cells through the inhibition of paxillin/FAK, Src, and alpha smooth muscle actin (α-sMA) and through the activation of E-cadherin. Furthermore, the anti-tumorigenic effect of Ph (10, 50 mg/kg or DMSO twice a week for six weeks) was demonstrated in vivo using BALB/c nude mice bearing MDA-MB-231 tumor xenografts. A decrease in N-cadherin, vimentin and an increase in p53, p21 and E-cadherin were detected in the tumor tissues. In conclusion, inhibition of GLUT2 by the apple polyphenol Ph could potentially suppress TNBC tumor cell growth and metastasis.

Clinacanthus nutans Mitigates Neuronal Apoptosis and Ischemic Brain Damage Through Augmenting the C/EBPß-Driven PPAR-γ Transcription.

Clinacanthus nutans Lindau (C. nutans) is a traditional herbal medicine widely used in Asian countries for treating a number of remedies including snake and insect bites, skin rashes, viral infections, and cancer. However, the underlying molecular mechanisms for its action and whether C. nutans can offer protection on stroke damage in brain remain largely unknown. In the present study, we demonstrated protective effects of C. nutans extract to ameliorate neuronal apoptotic death in the oxygen-glucose deprivation model and to reduce infarction and mitigate functional deficits in the middle cerebral artery occlusion model, either administered before or after hypoxic/ischemic insult. Using pharmacological antagonist and siRNA knockdown approaches, we demonstrated ability for C. nutans extract to protect neurons and ameliorate ischemic injury through promoting the anti-apoptotic activity of peroxisome proliferator-activated receptor-gamma (PPAR-γ), a stress-induced transcription factor. Reporter and chromatin immunoprecipitation promoter analysis further revealed C. nutans extract to selectively increase CCAAT/enhancer binding protein (C/EBP)ß binding to specific C/EBP binding site (-332~-325) on the PPAR-γ promoter to augment its transcription. In summary, we report a novel transcriptional activation involving C/EBPß upregulation of PPAR-γ expression to suppress ischemic neuronal apoptosis and brain infarct. Recognition of C. nutans to enhance the C/EBPߠ→ PPAR-γ neuroprotective signaling pathway paves a new way for future drug development for prevention and treatment of ischemic stroke and other neurodegenerative diseases.

15d-prostaglandin J2 protects brain from ischemia-reperfusion injury.

OBJECTIVE: Brain expresses abundant lipocalin-type prostaglandin (PG) D2 (PGD2) synthase but the role of PGD2 and its metabolite, 15-deoxy-Delta(12,14) PGJ2 (15d-PGJ2) in brain protection is unclear. The aim of this study is to assess the effect of 15d-PGJ2 on neuroprotection. METHODS AND RESULTS: Adenoviral transfer of cyclooxygenase-1 (Adv-COX-1) was used to amplify the production of 15d-PGJ2 in ischemic cortex in a rat focal infarction model. Cortical 15d-PGJ2 in Adv-COX-1-treated rats was increased by 3-fold over control, which was correlated with reduced infarct volume and activated caspase 3, and increased peroxisome proliferator activated receptor-gamma (PPARgamma) and heme oxygenase-1 (HO-1). Intraventricular infusion of 15d-PGJ2 resulted in reduction of infarct volume, which was abrogated by a PPARgamma inhibitor. Rosiglitazone infusion had a similar effect. 15d-PGJ2 and rosiglitazone at low concentrations suppressed H2O2-induced rat or human neuronal apoptosis and necrosis and induced PPARgamma and HO-1 expression. The anti-apoptotic effect was abrogated by PPARgamma inhibition. CONCLUSIONS: 15d-PGJ2 suppressed ischemic brain infarction and neuronal apoptosis and necrosis in a PPARgamma dependent manner. 15d-PGJ2 may play a role in controlling acute brain damage induced by ischemia-reperfusion.

Direct Conversion of Human Fibroblasts into Neural Progenitors Using Transcription Factors Enriched in Human ESC-Derived Neural Progenitors.

Early human embryonic stem cell (hESC)-derived neural populations consist of various embryonic neural progenitors (ENPs) with broad neural developmental propensity. Here, we sought to directly convert human somatic cells into ENP-like phenotypes using hESC-ENP-enriched neural transcription factors (TFs). We demonstrated that induced ENP could be efficiently converted from human fibroblasts using two TF combinations. The iENPs exhibit cellular and molecular characteristics resembling hESC-ENPs and can give rise to astrocytes, oligodendrocytes, and functional neuronal subtypes of the central and peripheral nervous system. Nevertheless, our analyses further revealed that these two iENP populations differ in terms of their proliferation ability and neuronal propensity. Finally, we demonstrated that the iENPs can be induced from fibroblasts from patients with Huntington's disease and Alzheimer's disease, and the diseased iENPs and their neuronal derivatives recapitulated the hallmark pathological features of the diseases. Collectively, our results point toward a promising strategy for generating iENPs from somatic cells for disease modeling and future clinical intervention.

Promoter region methylation and reduced expression of thrombospondin-1 after oxygen-glucose deprivation in murine cerebral endothelial cells.

Angiogenesis is induced in response to ischemia. Thrombospondin-1 (TSP-1) is a potent angiostatic factor. Silencing of TSP-1 expression may contribute to the postischemic angiogenesis. Upregulation of TSP-1, in contrast, may terminate the postischemic angiogenesis. A possible mechanism that silences TSP-1 expression is the DNA methylation of its promoter region. DNA methylation has been reported following cerebral ischemia. The present study aimed to explore whether methylation of the promoter region of TSP-1 regulates its expression after oxygen-glucose deprivation (OGD) in murine cerebral endothelial cells (CECs) in vitro. Sublethal OGD increased the extent of methylation of the promoter region of TSP-1 with a concurrent decrease in TSP-1 mRNA and protein expression in CECs. After reoxygenation, demethylation of the TSP-1 promoter region led to the restoration of TSP-1 mRNA and protein expression. The extent of methylation of the promoter region of TSP-1 was inversely correlated with the extent of TSP-1 gene expression at mRNA and protein levels after OGD. Oxygen-glucose deprivation-induced reduction in the TSP-1 mRNA level was not accompanied by a change in mRNA stability. These findings raise the possibility that OGD downregulation of TSP-1 expression is at least in part due to methylation of its promoter region.

Induction of prostacyclin/PGI2 synthase expression after cerebral ischemia-reperfusion.

Prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet aggregation and leukocyte activation, is crucial in vascular diseases such as stroke. Prostacyclin synthase (PGIS) is the key enzyme for PGI2 synthesis. Although expression of PGIS was noted in the brain, its role in ischemic insult remains unclear. Here we reported the temporal and spatial expression of PGIS mRNA and protein after 60-min transient ischemia. Northern blot and in situ hybridization revealed a delayed increase of PGIS mRNA in the ischemic cortex at 24- to 72-h after ischemia; PGIS was detected mainly in the ipsilateral penumbra area, pyriform cortex, hippocampus, and leptomeninges. Western blot and immunohistochemical analysis revealed that PGIS proteins were expressed temporally and spatially similar to PGIS mRNA. PGIS was heavily colocalized with PECAM-1 to endothelial cells at the leptomeninges, large and small vessels, and localized to neuronal cells, largely at the penumbra area. A substantial amount of PGIS was also detected in the macrophage and glial cells. To evaluate its role against ischemic infarct, we overexpressed PGIS by adenoviral gene transfer. When infused 72 h before ischemia (- 72 h), Adv-PGIS reduced infarct volume by approximately 50%. However, it had no effect on infarct volume when infused immediately after ischemia (0 h). Eicosanoid analysis revealed selective elevation of PGI2 at - 72 h while PGI2 and TXB2 were both elevated at 0 h, altering the PGI2/thromboxane A2 (TXA2) ratio from 10 to 4. These findings indicate that PGIS protects the brain by enhancing PGI2 synthesis and creating a favorable PGI2/TXA2 ratio.