Ameliorative potential of phloridzin in type 2 diabetes-induced memory deficits in rats.

Diabetes associated oxidative stress and impaired cholinergic neurotransmission causes cognitive deficits. Although phloridzin shows antioxidant- and insulin sensitizing-activities, its ameliorative potential in diabetes-induced memory dysfunction remains unexplored. In the present study, type 2 diabetes (T2D) was induced by streptozotocin (35 mg/kg, intraperitoneal) in rats on ad libitum high-fat diet. Diabetic animals were treated orally with phloridzin (10 and 20 mg/kg) for four weeks. Memory functions were evaluated by passive avoidance test (PAT) and novel object recognition (NOR) test. Brains of rats were subjected to biochemical analysis of glutathione (GSH), brain-derived neurotrophic factor (BDNF), malonaldehyde (MDA) and acetylcholinesterase (AChE). Role of cholinergic system in the effects of phloridzin was evaluated by scopolamine pre-treatment in behavioral studies. While diabetic rats showed a significant decrease in step through latency in PAT, and exploration time and discrimination index in NOR test; a substantial increase in all parameters was observed following phloridzin treatment. Phloridzin reversed abnormal levels of GSH, BDNF, MDA and AChE in the brain of diabetic animals. Moreover, in silico molecular docking study revealed that phloridzin acts as a potent agonist at M1 receptor as compared to acetylcholine. Viewed collectively, reversal of T2D-induced memory impairment by phloridzin might be attributed to upregulation of neurotrophic factors, reduced oxidative stress and increased cholinergic signaling in the brain. Therefore, phloridzin may be a promising molecule in the management of cognitive impairment comorbid with T2D.

DHA modulates MANF and TREM2 abundance, enhances neurogenesis, reduces infarct size, and improves neurological function after experimental ischemic stroke.

AIMS: Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a secretory neurotrophic factor protein that promotes repair after neuronal injury. The microglia cell surface receptor (triggering receptor expressed on myeloid cells-2; TREM2) regulates the production of pro- and antiinflammatory mediators after stroke. Here, we study MANF and TREM2 expression after middle cerebral artery occlusion (MCAo) and explore if docosahexaenoic acid (DHA) treatment exerts a potentiating effect. METHODS: We used 2 hours of the MCAo model in rats and intravenously administered DHA or vehicle at 3 hours after the onset of MCAo. Neurobehavioral assessment was performed on days 1, 3, 7, and 14; MANF and TREM2 expression was measured by immunohistochemistry and Western blotting. RESULTS: MANF was upregulated in neurons and astrocytes on days 1, 7, and 14, and TREM2 was expressed on macrophages in the ischemic penumbra and dentate gyrus (DG) on days 7 and 14. DHA improved neurobehavioral recovery, attenuated infarct size on days 7 and 14, increased MANF and decreased TREM2 expression in ischemic core, penumbra, DG, and enhanced neurogenesis on Day 14. CONCLUSION: MANF and TREM2 protein abundance is robustly increased after MCAo, and DHA treatment potentiated MANF abundance, decreased TREM2 expression, improved neurobehavioral recovery, reduced infarction, and provided enhanced neuroprotection.

Effect of retinoic acid on midkine gene expression in rat anterior pituitary cells.

Retinoic acid (RA) is converted from retinal by retinaldehyde dehydrogenases (RALDHs) and is an essential signaling molecule in embryonic and adult tissue. We previously reported that RALDH1 was produced in the rat anterior pituitary gland and hypothesized that RA was generated in the gland. Midkine (MK) is an RA-inducible growth factor, and MK production in the rat anterior pituitary gland was recently reported. However, the mechanism that regulates gene expression of MK in the pituitary gland has not been determined. To investigate regulation of MK production in the anterior pituitary gland, we analyzed changes in MK mRNA in cultured rat anterior pituitary cells. We identified MK-expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for RALDH1. MK mRNA was expressed in RALDH1-producing cells in the anterior pituitary gland. Using isolated anterior pituitary cells of rats, we examined the effect of RA on gene expression of MK. Quantitative real-time PCR revealed that 72 h exposure to a concentration of 10-6 M of retinal and all-trans retinoic acid increased MK mRNA levels by about 2-fold. Moreover, the stimulatory effect of all-trans retinoic acid was mimicked by the RA receptor agonist Am80. This is the first report to show that RA is important in regulating MK expression in rat anterior pituitary gland.

Corallocins A-C, Nerve Growth and Brain-Derived Neurotrophic Factor Inducing Metabolites from the Mushroom Hericium coralloides.

Three new natural products, corallocins A-C (1-3), along with two known compounds were isolated from the mushroom Hericium coralloides. Their benzofuranone and isoindolinone structures were elucidated by spectral methods. All corallocins induced nerve growth factor and/or brain-derived neurotrophic factor expression in human 1321N1 astrocytes. Furthermore, corallocin B showed antiproliferative activity against HUVEC and human cancer cell lines MCF-7 and KB-3-1.

Reducing the Risk of OHSS by GnRH Agonist Triggering.

CONTEXT: Overstimulation of follicle development in assisted reproductive technology cycles can lead to the development of life-threatening ovarian hyperstimulation syndrome (OHSS). There is evidence that administration of GnRH agonist as the trigger for final follicular maturation, instead of the usual human chorionic gonadotropin trigger, will reduce the risk of OHSS by shortening the duration of luteal stimulation, lowering estrogen levels by inducing luteolysis and reducing the secretion of vascular endothelial growth factor (VEGF). EVIDENCE ACQUISITION: The paper by Miller et al (1) in this month's issue of the Journal of Clinical Endocrinology and Metabolism (JCEM) demonstrates that GnRH agonist may directly reduce the activity of VEGF by stimulation of granulosa cell expression and secretion of pigment epithelial-derived factor (PEDF). EVIDENCE SYNTHESIS: The increased expression and secretion of PEDF in response to a bolus of GnRH agonist may antagonize the adverse effects of VEGF on ovarian vascular permeability and may contribute to luteolysis by reducing corpus luteum vascularity, thereby reducing the risk of OHSS. In addition, stimulation of PEDF may also be part of the protective mechanism of dopamine agonists used for prevention of OHSS. CONCLUSIONS: The new data presented by Miller et al (1) propose a likely mechanism for the reduced risk of OHSS following GnRH agonist triggering of follicle maturation in assisted reproductive technology cycles.

GnRH Agonist Triggering Modulates PEDF to VEGF Ratio Inversely to hCG in Granulosa Cells.

CONTEXT: GnRH agonist (GnRH-a) triggering is associated with a reduced risk of ovarian hyperstimulation syndrome (OHSS) compared with human chorionic gonadotropin (hCG) in assisted reproduction technology cycles. We have shown that ovarian pigment epithelium derived factor (PEDF), a potent antiangiogenic factor, counteracts vascular endothelial growth factor (VEGF) expression and that OHSS is correlated with hCG-induced impaired PEDF to VEGF ratio. OBJECTIVE: The objective of the study was to explore whether GnRH-a triggering could directly modulate PEDF/VEGF balance in granulosa cells. DESIGN: The design of the study was a mouse model and cultured granulosa cells. MAIN OUTCOME: Changes in PEDF and VEGF were measured by quantitative PCR and Western blot analysis. OHSS symptoms were recorded by changes in body weight and in peritoneal vascular leakage, quantified by the modified Miles vascular permeability assay. RESULTS: GnRH-a stimulation significantly increased PEDF and decreased VEGF mRNA and protein levels both in rat granulosa cell line and human primary granulosa cells in vitro. GnRH-a and hCG triggering inversely modulated PEDF mRNA and protein level in human granulosa cells in vivo. In the GnRH-a triggering mouse model, we showed similar increase in PEDF to VEGF ratio as in the in vitro results. OHSS-predisposed mice did not develop OHSS parameters after GnRH-a triggering, opposed to hCG-triggered mice. Finally, GnRH-a triggering of OHSS-predisposed mice significantly increased ovarian PEDF to VEGF ratio compared with hCG-triggered mice and control mice. CONCLUSIONS: GnRH-a triggering induces a direct effect on PEDF/VEGF balance in granulosa cells inversely to hCG. Our results suggest a novel elucidation to the GnRH-a triggering-mediated risk reduction of OHSS and may clarify the pros and cons of this triggering method.

Striatoids A-F, Cyathane Diterpenoids with Neurotrophic Activity from Cultures of the Fungus Cyathus striatus.

Six new highly oxygenated polycyclic cyathane-xylosides, named striatoids A-F (1-6), were isolated from the cultures of the basidiomycete Cyathus striatus. Their structures were established by comprehensive spectroscopic analysis including 2D NMR (HMBC, HSQC, ROESY, (1)H-(1)H-COSY) and HRESIMS experiments. Compounds 2 and 3 possess an unusual 15,4'-ether ring system. The isolated compounds dose-dependently enhanced nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma (PC-12) cells.

Growth factors/chemokines in diabetic vitreous and aqueous alter the function of bone marrow-derived progenitor (CD34⁺) cells in humans.

Ocular ischemic microenvironment plays a critical role in the progression of diabetic retinopathy (DR). In this study, we investigated the effect of vitreous and aqueous obtained from proliferative DR patients on the function of CD34⁺ cells derived from healthy humans. Human CD34⁺ cells were incubated with vitreous or aqueous of subjects with PDR. After incubation, cell migration of CD34⁺ was evaluated with CXCL12. Intracellular levels of nitric oxide (NO) were measured with DAF-FM. Tube formation assay was used to evaluate the effect of treated CD34⁺ cells on in vitro angiogenesis. Angiogenic protein array and mass spectrometry (MS) were performed to ascertain the factors secreted by healthy nondiabetic CD34⁺ cells exposed to diabetic vitreous or aqueous. PDR vitreous/aqueous reduced migration of CD34⁺ cells (672.45 ± 42.1/736.75 ± 101.7 AFU; P < 0.01) and attenuated intracellular NO levels (182 ± 1.4/184.5 ± 6.3 AFU, P = 0.002). Pretreatment with PDR vitreous suppressed tube formation of human retinal endothelial cells (64 ± 1.6 vs. 80 ± 2.5). CD34⁺ exposed to PDR vitreous resulted in the increased expression of CXCL4 and serpin F1, whereas CD34⁺ exposed to PDR aqueous showed increased expression of CXCL4, serpin F1, and endothelin-1 (ET-1). MS analysis of CD34⁺ (exposed to PDR vitreous) expressed J56 gene segment, isoform 2 of SPARC-related modular calcium-binding protein 2, isoform 1 of uncharacterized protein c1 orf167, integrin α-M, and 40s ribosomal protein s21. Exposure of healthy nondiabetic CD34⁺ cells to PDR vitreous and aqueous resulted in decreased migration, reduced generation of NO, and altered paracrine secretory function. Our results suggest that the contribution of CD34⁺ cells to the aberrant neovascularization observed in PDR is driven more by the proangiogenic effects of the retinal cells rather than the influence of the vitreous.

A proteomics approach to identify the differential protein level in cardiac muscle of diabetic rat.

BACKGROUND: Cardiovascular proteomics investigation reveals the characterization and elucidation of the novel therapeutic targets and strategies to prevent the development of heart failure associated diabetic complication by using 2DE and MS. METHODS: The experimental animals were made diabetic with a single intraperitoneal injection of alloxan (150 mg/kg of bw). Albino rats were randomly divided into four individual groups: Group-I control (n=6), group-II alloxan-induced diabetic rats, untreated (n=6), group-III (n=6) and group-IV (n=6) alloxan-induced diabetic rats were treated with aqueous and ethanolic extracts of Cynodon dactylon for 15 days, respectively. Animals were euthanized to collect the heart tissues and blood samples. 2DE sample preparation, gel running and staining (n=6: each groups) were performed at the same time to avoid variation. The result of six gel images from each group were analyzed and evaluated as one match set with 2D software (P<0.05). RESULTS: The above experiment revealed two up-regulated proteins in group-II i.e. NTF4 and ETFB. CONCLUSIONS: NTF4 is a neuro-protective agent for neuro-degenerative diseases. It will prevent diabetic secondary complications, such as diabetic polyneuropathy and cardiomyopathy. ETFB is active in the mitochondria, the energy-producing centres in cells. It is clear from the experiment that because of up-regulation of ETFB more energy is availabile and the electron transfer for heart during diabetes is possible, what leads to reduce the oxidative stress and free-radical formation. The up-regulated proteins reduced CVD that occurred just before overt hyperglycaemia due to administration of C. dactylon. This approach established the preliminary reference map for decoding cellular mechanisms linked between pathogenesis CVD and diabetes.

Towards non invasive nerve growth factor therapies for Alzheimer's disease.

In the past thirty years, nerve growth factor (NGF) has received much attention for its potential role as a therapeutic agent for Alzheimer's disease (AD) due to its neurotrophic activities on basal forebrain cholinergic neurons. This attention has been renewed by recent findings that provide new causal links between defects in NGF signaling, transport or processing to the activation of the amyloidogenic route and, more generally, to AD neurodegeneration. Thus, the concept of therapeutic administration of human recombinant NGF in AD patients has a strong rationale, being further validated by recent and ongoing clinical trials with a gene-therapy approach. However, the widespread clinical application of gene or cell-therapy strategies for the delivery of NGF to AD patients seems unpractical, and it would be more advantageous to have non-invasive methods, that should also limit the adverse effects of NGF in activating nociceptive responses. This review will describe: 1) the data from preclinical and clinical studies underlying the rationale of NGF as a potential therapeutic agent for AD; and 2) the alternative strategies to reach adequate concentrations of NGF in relevant brain areas while preventing the onset of adverse effects.

Effects of Amphotericin B on the expression of neurotoxic and neurotrophic factors in cultured microglia.

Amphotericin B (AmB) is a polyene antibiotic and reported to have therapeutic effects on prion diseases, in which the microglial activation has been suggested to play important roles by proliferating and producing various factors such as nitric oxide, proinflammatory cytokines, and so on. However, the therapeutic mechanism of AmB on prion diseases remains elusive. In the present study, we investigated the effects of AmB on cellular functions of rat primary cultured microglia. We found that AmB, similarly as lipopolysaccharide (LPS), could activate microglia to produce nitric oxide via inducible nitric oxide synthase. Both AmB and LPS also induced mRNA expressions of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in microglia. AmB also changed the expression levels of neurotrophic factors mRNAs. AmB and LPS significantly down-regulated the level of ciliary neurotrophic factor mRNA. However, AmB, but not LPS, significantly up-regulated the level of glial cell-line derived neurotrophic factor mRNA in microglia. In addition, brain-derived neurotrophic factor mRNA expression level was tending upward by treatment with AmB, but not with LPS. Taken together, these results suggest that AmB regulates the microglial activation in different manner from LPS and that microglia may participate in the therapeutic effects of AmB on prion diseases by controlling the expression and production of such mediators.

Hypoxia-activated microglial mediators of neuronal survival are differentially regulated by tetracyclines.

The tetracycline derivatives minocycline (MINO) and doxycycline (DOXY) have been shown to be neuroprotective in in vivo and in vitro models of stroke. This neuroprotection is thought to be due to the suppression of microglial activation. However, the specific molecular parameters in microglia of the tetracyclines' effect are not understood. We subjected cultured rat microglial and neuronal cells to in vitro hypoxia and examined the effects of MINO and DOXY pre-treatments. Our data showed that MINO and DOXY protect against hypoxia-induced neuronal death by a mechanism dependent on regulation of microglial factors, but likely unrelated to regulation of microglial proliferation/viability. Both MINO and DOXY suppressed the hypoxic activation of ED-1, a marker for microglial activation. Morphological analyses of hypoxic microglia using the microglial marker Iba1 revealed that treatment with MINO and DOXY caused a higher percentage of microglia to remain in a non-activated state. MINO suppressed the hypoxic upregulation of pro-inflammatory agents nitric oxide (NO), interleukin-1 beta (IL-1beta), and tumor necrosis factor alpha (TNF-alpha), while DOXY down-regulated only NO and IL-1beta. In contrast, the hypoxic activation of pro-survival/neuroprotective microglial proteins, such as brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), were unaffected by tetracycline treatments. Taken together, these results suggest that MINO and DOXY may provide neuroprotection against stroke by selectively down-regulating microglial toxic factors while maintaining functional pro-survival factors.

How the brain repairs itself: new therapeutic strategies in inflammatory and degenerative CNS disorders.

In the early 20th century, seminal work by Tello and Cajal showed that the CNS has the ability to regenerate itself after injury. In the most recent years, this pivotal observation has been rejuvenated by detailed in vitro and in vivo evidence supporting the idea of an innate self-maintenance programme to sustain brain homoeostasis and repair. These observations support the idea that chronic inflammatory and degenerative disorders of the brain might result from defective repair mechanisms rather than uncontrollable pathogenetic events. Investigation of the molecular and cellular events sustaining intrinsic brain-repair mechanisms and a better understanding of why they fail over time in chronic disorders might, therefore, provide an attractive conceptual framework within which to develop new and efficacious therapies for neurological diseases.

GDNF and somatostatin in sensory neurones.

Somatostatin (SOM) is a regulatory peptide produced throughout the central nervous system and in most major peripheral organs. In humans, the SOM stable analogue octreotide (OCT) is used to treat opioid-resistant pain. In animal models, SOM and OCT are analgesics and SOM released from the peripheral endings of sensory neurones exerts anti-inflammatory actions. The expression of SOM in sensory neurones can be modulated by the trophic factor glial-cell-line-derived neurotrophic factor (GDNF). Recent data show that GDNF modulates activity-induced release of endogenous SOM from both central and peripheral terminals of adult sensory neurones. This novel mechanism deserves exploration as a potential new therapeutic strategy to control two major features of inflammation: pain and leukocyte recruitment.

Alzheimer's therapeutics: neurotrophin small molecule mimetics.

A substantial portion of neuronal populations undergoing degeneration in Alzheimer's and other neurodegenerative disorders express neurotrophin receptors. Neurotrophin small molecule mimetics constitute candidate compounds that might be useful in preventing or delaying loss of neuronal function, neural networks or neuronal death in neurodegenerative states. We are testing the hypothesis that pharmacophores based on a combination of the crystal structures of neurotrophins and structure-activity relationships of active neurotrophin peptidomimetics can be used to screen small molecule libraries to identify non-peptide small molecules with neurotrophin agonist or antagonist activity. In preliminary screens using pharmacophores based on two nerve growth factor (NGF) loop domains, a number of small molecules have been identified that display neurotrophic activity using in vitro bioassays. Current studies are focused on determining whether these small molecules function via neurotrophin receptors and whether they activate neurotrophin signaling cascades. Assessment of structure-activity relationships between active and inactive small molecules will allow modification of pharmacophores and provide a basis for the iterative process if identifying compounds with increased potency and efficacy. A collection of such compounds will provide a basis for synthesis of compounds with targeted pharmacological properties.

Development of pharmacological agents for targeting neurotrophins and their receptors.

Neurotrophins comprise a family of protein growth factors that control the survival, growth, and/or differentiation of neurons and several other cell populations derived from the neuroectoderm. Neurotrophins and their receptors are important targets for the therapy of human disease, with potential applications ranging from the treatment of chronic or acute neurodegeneration to pain and cancer. Neurotrophins have been used clinically but are poor pharmacological agents. Consequently, approaches to develop pharmacological agents that target neurotrophins, their receptors or neurotrophin signaling pathways have been attempted.