GBE50 Attenuates Inflammatory Response by Inhibiting the p38 MAPK and NF- κ B Pathways in LPS-Stimulated Microglial Cells.

Overactivated microglia contribute to a variety of pathological conditions in the central nervous system. The major goal of the present study is to evaluate the potential suppressing effects of a new type of Ginko biloba extract, GBE50, on activated microglia which causes proinflammatory responses and to explore the underlying molecular mechanisms. Murine BV2 microglia cells, with or without pretreatmentof GBE50 at various concentrations, were activated by incubation with lipopolysaccharide (LPS). A series of biochemical and microscopic assays were performed to measure cell viability, cell morphology, release of tumor necrosis factor- α (TNF- α ) and interleukin-1 ß (IL-1 ß ), and signal transduction via the p38 MAPK and nuclear factor-kappa B (NF- κ B) p65 pathways. We found that GBE50 pretreatment suppressed LPS-induced morphological changes in BV2 cells. Moreover, GBE50 treatment significantly reduced the release of proinflammatory cytokines, TNF- α and IL-1 ß , and inhibited the associated signal transduction through the p38 MAPK and NF- κ B p65 pathways. These results demonstrated the anti-inflammatory effect of GBE50 on LPS-activated BV2 microglia cells, and indicated that GBE50 reduced the LPS-induced proinflammatory TNF- α and IL-1 ß release by inhibiting signal transduction through the NF- κ B p65 and p38 MAPK pathways. Our findings reveal, at least in part, the molecular basis underlying the anti-inflammatory effects of GBE50.

Azoxystrobin, a mitochondrial complex III Qo site inhibitor, exerts beneficial metabolic effects in vivo and in vitro.

BACKGROUND: Several anti-diabetes drugs exert beneficial effects against metabolic syndrome by inhibiting mitochondrial function. Although much progress has been made toward understanding the role of mitochondrial function inhibitors in treating metabolic diseases, the potential effects of these inhibitors on mitochondrial respiratory chain complex III remain unclear. METHODS: We investigated the metabolic effects of azoxystrobin (AZOX), a Qo inhibitor of complex III, in a high-fat diet-fed mouse model with insulin resistance in order to elucidate the mechanism by which AZOX improves glucose and lipid metabolism at the metabolic cellular level. RESULTS: Acute administration of AZOX in mice increased the respiratory exchange ratio. Chronic treatment with AZOX reduced body weight and significantly improved glucose tolerance and insulin sensitivity in high-fat diet-fed mice. AZOX treatment resulted in decreased triacylglycerol accumulation and down-regulated the expression of genes involved in liver lipogenesis. AZOX increased glucose uptake in L6 myotubes and 3T3-L1 adipocytes and inhibited de novo lipogenesis in HepG2 cells. The findings indicate that AZOX-mediated alterations to lipid and glucose metabolism may depend on AMP-activated protein kinase (AMPK) signaling. CONCLUSIONS: AZOX, a Qo inhibitor of mitochondrial respiratory complex III, exerts whole-body beneficial effects on the regulation of glucose and lipid homeostasis in high-fat diet-fed mice. GENERAL SIGNIFICANCE: These findings provide evidence that a Qo inhibitor of mitochondrial respiratory complex III could represent a novel approach for the treatment of obesity.

The important roles of RET, VEGFR2 and the RAF/MEK/ERK pathway in cancer treatment with sorafenib.

AIM: To elucidate the roles of receptor tyrosine kinases RET and VEGFR2 and the RAF/MEK/ERK signaling cascade in cancer treatment with sorafenib. METHODS: The cell lines A549, HeLa, and HepG2 were tested. The enzyme activity was examined under cell-free conditions using 384-well microplate assays. Cell proliferation was evaluated using the Invitrogen Alarmar Blue assay. Gene expression was analyzed using the Invitrogen SYBR Green expression assays with a sequence detection system. Protein expression analysis was performed using Western blotting. RESULTS: Sorafenib potently suppressed the activities of cRAF, VEGFR2, and RET with IC(50) values of 20.9, 4 and 0.4 nmol/L, respectively. Sorafenib inhibited cRAF, VEGFR2, and RET via non-ATP-competitive, ATP-competitive and mixed-type modes, respectively. In contrast, sorafenib exerted only moderate cytotoxic effects on the proliferation of the 3 cell lines. The IC(50) values for inhibition of A549, HeLa, and HepG2 cells were 8572, 4163, and 8338 nmol/L, respectively. In the 3 cell lines, sorafenib suppressed the cell proliferation mainly by blocking the MEK/ERK downstream pathway at the posttranscriptional level, which in turn regulated related gene expression via a feed-back mechanism. CONCLUSION: This study provides novel evidence that protein kinases RET and VEGFR2 play crucial roles in cancer treatment with sorafenib.

Genetic informational RNA is not required for recombinant prion infectivity.

Whether a genetic informational nucleic acid is required for the infectivity of transmissible spongiform encephalopathies is central to the debate about the infectious agent. Here we report that an infectious prion formed with bacterially expressed recombinant prion protein plus synthetic polyriboadenylic acid and synthetic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol is competent to infect cultured cells and cause prion disease in wild-type mice. Our results show that genetic informational RNA is not required for recombinant prion infectivity.

Generating a prion with bacterially expressed recombinant prion protein.

The prion hypothesis posits that a misfolded form of prion protein (PrP) is responsible for the infectivity of prion disease. Using recombinant murine PrP purified from Escherichia coli, we created a recombinant prion with the attributes of the pathogenic PrP isoform: aggregated, protease-resistant, and self-perpetuating. After intracerebral injection of the recombinant prion, wild-type mice developed neurological signs in approximately 130 days and reached the terminal stage of disease in approximately 150 days. Characterization of diseased mice revealed classic neuropathology of prion disease, the presence of protease-resistant PrP, and the capability of serially transmitting the disease; these findings confirmed that the mice succumbed to prion disease. Thus, as postulated by the prion hypothesis, the infectivity in mammalian prion disease results from an altered conformation of PrP.

[The expression pattern and possible function of microRNA-34a in neurons].

Using Northern blotting, ISH and FISH, we demonstrated miR-34a expresses with aging in the brain of rhesus monkey and rat. Correspondingly, the same trend was found in the primary cultures of cortical neurons. Moreover, we found that induction of miR-34a led to neuronal apoptosis by targeting BCL-2. Our study suggests that miR-34a may play critical roles in neuronal development and ageing.

Grafted neural stem cells migrate to substantia nigra and improve behavior in Parkinsonian rats.

Neural stem cell (NSC) transplantation has the potential to treat neurodegenerative diseases such as Parkinson's disease (PD). In this study, we investigated the effect of transplanted NSCs in a PD animal model. NSCs isolated from the subventricular zone (SVZ) of E14 rats were cultured in vitro to produce neurospheres, which were subsequently infected with recombinant adeno-associated virus (rAAV(2)) expressing enhanced green fluorescent protein (EGFP). The PD animal model was established by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) of Sprague-Dawley rats. Once the model was established, EGFP-expressing NSCs were transplanted into the substantia nigra pars compacta (SNc) or striatum of PD rats. We found that NSCs transplanted into either site significantly reduced apomorphine-induced circling behavior of PD rats. Pathological analysis revealed that the EGFP-expressing NSCs could be detected at both injection sites at 1, 2 and 4 months after transplantation. SNc transplanted cells dispersed within the SNc with a significant portion differentiated into tyrosine hydroxylase-positive neurons. Whereas cells transplanted into the striatum migrated ventrally and posteriorly towards the SNc. These results suggest that the 6-OHDA damaged brain area attracts grafted NSCs, which migrated from the striatum and survived for a long time in SNc, resulting in behavioral improvement of PD rats.

[Chronic administration of CRF makes depression like changes in rats].

For studying the role of CRF in the process of depression, chronic intra-cerebral ventricular administration of CRF in normal rats for 21 days were performed and compared with the depression model of chronic unpredictable miled stress (CUMS) in rats. The Open-field Test and Morris water Maze were employed to test the ability of locomotion and learning and memory. HPLC-UV, RT-PCR were employed to analyze the level of blood serum cortisol and the expression of CRF as well as its receptors (R1 and R2) of CUMS models. After chronic stress, the locomotion activity and spatial learning and memory ability decreased obviously, while the level of serum cortisol increased evidently, and the CRF and its receptor-1 mRNA levels were higher compared with those in normal rats. The rats with chronic administration of CRF also consistently decreased the weight gain, locomotion activity and the ability of spatial learning and memory as the CUMS model. This work demonstrates that CRF plays a very important role in the depression genesis and development, sustained elevation of CRF induced by stress may be the chief factor for depression.

[The change of learning and memory ability in the rat model of depression].

The present study was performed to explore learning and memory ability in the depression models of rats. The chronic unpredictable stress and olfactory bulbectomy model of rats were adopted. Open-field test was used to detect the locomotion activity and HPLC-UV was employed to analyze the level of blood serum cortisol. The method of Morris water Maze was used to measure learning and memory ability and the results of LTP and LTD in hippocampus CA1 were recorded to observe the synaptic plasticity of hippocampus neurons. The results showed that compared with control group, the locomotion activity and learning ability for two models decreased extremely, while there was no apparent difference in the feedback of memory. Meanwhile, the synaptic plasticity of hippocampus neurons for two models decreased significantly and the level of serum cortisol increased evidently. These results suggested that both methods employed to build the models could cause rats depression and learning inhibition, but do no effects on the feedback of memory.

[Effects of traditional Chinese herbal medicine on the neurobehavioral manifestations and the activity of dopamine D2 receptor in corpora striatum of rats with levodopa-induced dyskinesias].

OBJECTIVE: To explore the effect of traditional Chinese herbal medicine (TCM) for nourishing liver and kidney, clearing meridians and removing toxic substances, on the neurobehavioral manifestations and the activity of the dopamine D2 receptor in rat with levodopa-induced dyskinesias (LID). METHODS: The rat model of Parkinson's disease (PD) was established by injecting 6-hydroxydopamine (6-OHDA) into right substantia nigra of brain, then, the model of LID in rat was produced by injecting levodopa (LD) and benserazide for 4 weeks. The rats were divided into normal control group, 4-week LD treated group, 4-week LD plus TCM treated group, 8-week LD treated group, and 8-week LD plus TCM treated group, and the effect of the TCM on neurobehavioral manifestations was observed. The radioligand binding assay (RLBA) and Scatchard drawing were used to measure the maximal binding capacity of receptor (Bmax) and equilibrium dissociation constant (KD) of the dopamine D2 receptor in corpora striatum. RESULTS: Compared with the 4-week LD treated group and 8-week LD treated group, TCM could decrease abnormal involuntary movement scores of the rats with LID; the RLBA revealed that the dopamine D2 receptor Bmax significantly increased (P<0.05, P<0.01) and the KD significantly decreased (P<0.05). CONCLUSION: TCM can improve the activity of the dopamine D2 receptor and relieve the symptoms of LID.

[Knockdown and overexpression of miR-219 lead to embryonic defects in zebrafish development].

MicroRNAs (miRNAs) are important post-transcriptional modulators of gene expression, which have been implicated in many physiological and pathological processes. In this study, we found that miR-219 expressed since early segmentation stages (16s) in spinal cord and mid-, hindbrain in zebrafish using Northern blotting and whole mounts ISH technologies. Moreover, knockdown or overexpression of miR-219 led to specific embryonic defects. Furthermore, TUNEL assay showed that overexpression of miR-219 induced significant cell apoptosis in the head and tail of zebrafish. Our study suggested that miR-219 may play an important role in zebrafish embryonic development.

[Expression of galanin and galanin receptors in neurogenesis regions of adult mouse brain and effect of galanin on the neural stem cell's differentiation].

The neuropeptid galanin is widely expressed in the central nervous system and has a diverse range of physiological effects including food intaking, arousal/sleep, nociception and reproduction. In this study, expression of galanin and galanin receptors (GalR1 and GalR2) mRNA were identified not only in the neurogenisis regions including subventricular zone (SVZ), rostral migratory stream (RMS) and dentate gyrus (DG) of adult mice but also in the SVZ-derived neural stem cell (NSC) culture. Here, we also showed that the addition of galanin and GalR2-specific agonist Gal2-11 to wild-type or GALKO NSCs under differentiation condition significantly promote the neuritogenesis and increase the length of neurites on the betaIII-tubulin positive cells. This effect could be reduced by treatment of the galanin antagonist M35. These results indicate that galanin and its receptors might regulate neurite extension in differentiating neural stem cells and even participate in the development of the nervous system.

Differential effect of galanin on proliferation of PC12 and B104 cells.

The effect of galanin (GAL) on neural cell proliferation was studied using PC12 and B104 cells. Reverse transcription-PCR was used to determine the expression of GAL and GAL receptors and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay of cell viability was employed to detect the effects of GAL on cell proliferation. These studies revealed firstly that PC12 cells express mRNAs encoding all three GAL receptors (GalR1-3) but not GAL mRNA, whereas B104 cells express GAL, GalR2 and GalR3 mRNAs, but not GalR1 mRNA; and secondly that GAL inhibited the proliferation of PC12 cells, but in contrast significantly activated the proliferation of B104 cells. Moreover, these effects of GAL were blocked by M35, a nonselective, chimera peptide antagonist of GAL receptors. These data suggest that GAL can alter neural cell proliferation via GAL receptor activation, and that different GAL receptors and/or cellular complements of receptors produce different net effects via activation of different signaling pathways.

[The expression of galanin and galanin receptor-2 in the brain of chronic stress model of depression].

The expression of galanin and galanin receptor-2 in hippocampus and dorsal raphe nucleus of depression model was studied. The chronic stress rat model was adopted as the modal of depression. Open-field test was used to observe the transformation of their behavior and HPLC-EC was employed to analysis the level of blood serum cortisol. The method of in situ hybridization was used for testing the expression of Galanin and galanin receptor-2 in hippocampus and dorsal raphe nucleus and the method of RT-PCR was used to further analysis of the expression. The results showed that the locomotion activity decreased extremely after chronic stress, but the level of serum cortisol increased evidently. The expression of Galanin and its receptor-2 in hippocampus and dorsal raphe nucleus increased obviously. The higher expression for galanin and galanin receptor-2 in some brain area suggested that galanin probably takes part in the modulation of the function of neurons during the stress process.

[Different effect of galanin on the proliferation of PC12 and B104 cells].

The effect of galanin (GAL) on neural proliferation was studied in this article using PC12 and B104 cells. RT-PCR was used to determine the expression of galanin and its receptors in both cells. MTT analysis method was employed to detect the effects of galanin and the agonist, antagonist of galanin receptors on the proliferation of both cells. Results showed that PC12 cells expressed mRNAs for all the three galanin receptors (GalRs) but not for galanin, while B104 cells expressed galanin, GalR2, GalR3 except GalR1. In addition, galanin and two receptor agonists, GAL-11 and GAL2-11, could inhibit the proliferation of PC12 cells but activated the proliferation of B104 cells significantly. Moreover, the influences could be blocked by M35, a nonspecific antagonist of galanin receptors. It suggested that GAL can affect cell proliferation via GalRs, but the different galanin receptors might mediate different cell functions.

[Galanin increases the survival rate of hippocampal cells injured by H2O2 in vitro].

The method of primary hippocampal nerve cell culture was used to study the injury effect of H2O2 and the protective effect of galanin (GAL) and GAL receptor agonists. Result demonstrated that H2O2 has obvious dose relative toxicity to hippocampal cells in vitro. GAL and GAL's nonselective agonist GAL1-11, GalR2's selective agonist GAL2-11 can increase the survival rate of hippocampal cells suffered form H2O2. All the protective effects can be blocked by nonselective antagonist M35. The result indicates that GAL can protect hippocampal cells from oxidative injury in vitro, which is most probably mediated by GalR2.

[The migration and differentiation of subventricular zone neural stem cells transplanted into the adult striatum].

Adeno-associated virus vectors (type2) containing the marker gene--green fluorescent protein (AAV2-GFP) were used to transduce subventricular zone neural stem cells (NSCs). NSCs labelled by gene product--GFP were transplanted into adult Sprague-Dawley rat striatum. The animals were allowed to survive for 45 days, 90 days and 120 days before they were perfused. All the fixed brains were serially sectioned in the saggital plane,at the sickness of 30 microm with a cryostat microtome. These results showed that, at all stages, GFP labelled NSCs were seen in the injection sites,and a lot of them dispersed in the host brains. GFP labelled NSCs showed directional migration 45 days following transplantation. 120 days after transplantation, a group of GFP labelled NSCs migrated dorsally and posteriorly, reached corpus callosum; another group of transplanted NSCs migrated ventrally and posteriorly,reached substantia nigra, moreover,a number of these cells migrated through or over the substantia nigra and reached the more ventral boundary of the substantia nigra. The migrating NSCs were in chains, GFP labelled cells in substantia nigra were beta-tubulin III immunoreactive.

Galanin inhibits the proliferation of glial olfactory ensheathing cells.

The effect of galanin (GAL) on neural proliferation was studied in this article using olfactory ensheathing cells (OECs). OECs were isolated from newborn rat olfactory bulb and cultured in vitro. RT-PCR was used to determine the expression of GAL and its receptors in these cells. MTT analysis and LDH assay were used to detect the effects of GAL and the agonist, antagonist of GAL receptors on the proliferation of OECs. Results show that OECs express mRNAs for GAL and GAL receptor2 (GalR2) but not for the two other GAL receptors, GalR1 and GalR3. In addition, GAL and two receptor agonists, GAL1-11 and GAL2-11, can inhibit the proliferation of OECs significantly, but cause no cytotoxicity in the OECs population. Moreover, the influence can be blocked by M35, a nonspecific antagonist of GAL receptors. It is suggested that GAL is an inhibitory factor in regulating OECs proliferation.

[The expression of galanin and galanin receptors in olfactory ensheathing cells and effects of galanin on cells proliferation].

Olfactory ensheathing cells (OECs) were isolated from newborn rat olfactory bulb and cultured in vitro. RT-PCR was used to determine the expression of galanin and it's receptors in these cells. MTT analysis was used to detect the effects of galanin and agonist, antagonist of galanin receptors on the proliferation of OECs. Results show that OECs express mRNAs for galanin and GalR2 but not for two another receptors, GalR1 and GalR3. In addition, galanin and two receptor agonists, GAL1-11 and GAL2-11, can significantly inhibit the proliferation of OECs. But the influence can be blocked by M35, nonspecific antagonist of galanin receptors.

Galanin in neuro(glio)genesis: expression of galanin and receptors by progenitor cells in vivo and in vitro and effects of galanin on neurosphere proliferation.

Considerable recent evidence suggests that in addition to its neuromodulatory role, galanin, like several other neuropeptides, also plays an important trophic role during development and after adult neural injury. Studies in our laboratory have identified high levels of galanin and galanin receptor expression in the subventricular zone, rostral migratory stream, subgranular zone of dentate gyrus and the medial corpus callosum--which include the main sites for continuing cell proliferation in both adult and developing rat brain. Galanin expression was also strongly and transiently induced in oligodendrocyte progenitor cells (OPCs) throughout the neocortex and corpus callosum by a benign physiological stimulus, cortical spreading depression (CSD). SD-like depolarization also occurs in peri-infarction areas following cerebral ischemia and is associated with proliferation of OPCs and transiently increased galanin expression. Together, these data suggest a putative role for galanin in regulating progenitor or 'stem cell' proliferation, migration and/or differentiation. Cultured adult and embryonic stem cells or 'neurospheres' express galanin and galanin receptor mRNA and preliminary studies suggest that sub-acute galanin treatment of cultured neurospheres decreases cell proliferation/survival, possibly by effects on the rate of apoptosis via GalR2 receptors.