Yanyong Liu

Melatonin Regulates the Viability and Differentiation of Rat Midbrain Neural Stem Cells

Abstract(1) Neurogenesis driven by neural stem cells (NSCs) is regulated by physiological and pathological factors. Melatonin (MT) has profound neurotrophic and neuroprotective effects. Hence, we studied the role of MT in regulating the viability and differentiation of NSCs derived from rat ventral midbrain. (2) NSCs were isolated from the rat ventral midbrain. The viability of NSCs was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-ulfophenyl)-2H-tetrazolium assay. The differentiation of NSCs was examined by analyzing the expression of the neural markers, MT receptors, brain derived neurotropic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) with semi-quantitative RT-PCR, immunofluorescence cytochemistry, and Western blot. (3) Our results showed that MT could promote the viability of NSCs. In addition, MT could significantly elevate the mRNA and protein levels of tyroxine hydroxylase (TH), a marker of dopaminergic neurons, and decrease the expression of the astrocytes maker glial fibrillary acidic protein (GFAP). MT also increased the production of BDNF and GDNF in the cultured NSCs. Meanwhile, we first found that two subtypes of MT receptors, MT1 and MT2, were expressed in the ventral midbrain NSCs. (4) These results demonstrated that MT could induce NSCs to differentiate into dopaminergic neurons and decrease astrocyte production. These findings also suggest that MT could offer a beneficial tool in guiding directional differentiation of NSCs.

Dynamic proteomic analysis of protein expression profiles in whole brain of Balb/C mice subjected to unpredictable chronic mild stress: implications for depressive disorders and future therapies.

The etiology and pathophysiology of depression remain unknown. Previous works were mostly performed on single observation time-point which might be insufficiently to reveal the molecular events changed during the disease development. Adult BALB/c mice were exposed to unpredictable chronic mild stress (UCMS) for different periods and differential 2D gel electrophoresis (DIGE) approach was employed to the brain tissue to explore the molecular disease signatures. Sustained elevation of corticosterone level was observed, suggesting the hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis when the mice were subjected to the stressful situation. The behavioral results indicated the depressive alterations of the mice exposing to UCMS. The altered proteins identified by proteomics showed that abnormal energy mobilization under stress condition was accompanied by overproduction of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Cytoskeleton protein and anti-oxidant enzymes were also changed by UCMS treatment. The results of biochemical and immunohistochemical assay confirmed the changes identified by DIGE analysis. These results indicated that the insufficiency of ATP synthesis, overwhelming ROS production and ER stress subsequently contributed to the cytoskeletal damage and inhibition to expression of some anti-oxidant proteins, which might ultimately bring functional neuron to apoptosis or death. Proteins whose expression is affected may provide tools for potential treatment strategies.

Protective effects of octacosanol on 6-hydroxydopamine-induced Parkinsonism in rats via regulation of ProNGF and NGF signaling.

AbstractAim:To investigate the protective effects of octacosanol in 6-hydroxydopamine-induced Parkinsonian rats and find whether octacosanol has effects on pro nerve growth factor (pro-NGF), NGF and the downstream effector proteins.Methods:Behavioral tests, enzymatic assay, tyrosine hydroxylase immunohistochemistry, TUNEL and Western blot were used to investigate the effects of octacosanol in this rat model of PD.Results:Oral administration of octacosanol (35–70 mg/kg, po for 14 d) significantly improved the behavioral impairments in rats induced by 6-OHDA and dose-dependently preserved the free radical scavenging capability of the striatum. Octacosanol treatment also effectively ameliorated morphological appearances of TH-positive neuronal cells in nigrostriatal systems and decreased the apoptotic cells induced by 6-OHDA in striatum. In addition, octacosanol strikingly blocked the 6-OHDA-induced increased expression of proNGF-p75NTR-sortilin death signaling complex and its downstream effector proteins. Meantime, octacosanol prevented the decreased levels of NGF, its receptors TrkA and p-Akt which together mediated the cell survival pathway.Conclusion:The findings implicated that the anti-parkinsonism effects afforded by octacosanol might be mediated by its neuro-microenvironment improving potency through retrieving the ratios of proNGF:NGF and the respective receptors p75NTR:TrkA in vivo. Due to its excellent tolerability and non-toxicity, octacosanol may be a promising agent for PD treatment.

Gene expression profile of amyloid beta protein-injected mouse model for Alzheimer disease

AbstractAim:To investigate the gene expression profile changes in the cerebral cortex of mice injected icv with amyloid beta-protein (Aβ) fragment 25-35 using cDNA microarray.Methods:Balb/c mice were randomly divided into a control group and Aβ-treated group. The Morris water maze test was performed to detect the peffect of Aβ-injection on the learning and memory of mice. Atlas Mouse 1.2 Expression Arrays containing 1176 genes were used to investigate the gene expression pattern of each group.Results:The gene expression profiles showed that 19 genes including TBX1, NF-κB, AP-1/c-Jun, cadherin, integrin, erb-B2, and FGFR1 were up-regulated after 2 weeks of icv administration of Aβ; while 12 genes were down-regulated, including NGF, glucose phosphate isomerase 1, AT motif binding factor 1, Na+/K+-ATPase, and Akt.Conclusion:The results provide important leads for pursuing a more complete understanding of the molecular events of Aβ-injection into mice with Alzheimer disease.

Enhanced in vivo antitumor efficacy of dual-functional peptide-modified docetaxel nanoparticles through tumor targeting and Hsp90 inhibition

Although conventional anticancer drugs exhibit excellent efficacy, serious adverse effects and/or even toxicity have occurred due to their nonselectivity. Moreover, active targeting approaches have not consistently led to successful outcomes. Ligands that simultaneously possess targeting capability and exert a strong influence on intracellular signaling cascades may be expected to improve the therapeutic efficacy of active targeting nanoparticulate carriers. In this study, we screened a targeting peptide, LPLTPLP, which specifically bound to non-small cell lung cancer (NSCLC) specimens in vitro. Surprisingly, this peptide inhibited the expression of Hsp90 and induced apoptosis by preventing autophagy in A549 cells treated with docetaxel. The results suggested that this peptide might be used as a promising dual-functional ligand for cancer treatment. Based on these findings, we designed and developed a novel active targeting delivery system by modifying docetaxel nanoparticles (DNP) with the dual-functional ligand LPLTPLP. We consistently demonstrated that the cellular uptake of nanoparticles (NPs) was significantly enhanced in vitro. Furthermore, the targeting NPs exhibited significantly improved antitumor efficacy and biodistribution compared with nontargeting nanodrug and free docetaxel. These findings demonstrate the feasibility of dual-functional NPs for efficient anticancer therapy.

Active targeting docetaxel-PLA nanoparticles eradicate circulating lung cancer stem-like cells and inhibit liver metastasis.

Lung cancer is the major cause of cancer related lethality worldwide, and metastasis to distant organs is the pivotal cause of death for the vast majority of lung cancer patients. Accumulated evidence indicates that lung cancer stem-like cells (CSLCs) play important roles in metastagenesis, and these circulating CSLCs may be important targets to inhibit the subsequent metastasis. The present study was aimed at establishing CSLC-targeting polylactic acid (PLA) encapsulated docetaxel nanoparticles for antimetastatic therapy. Cyclic binding peptides were screened on CSLCs in vitro and the peptide CVKTPAQSC exhibiting high specific binding ability to pulmonary adenocarcinoma tissue was subsequently conjugated to the nanoparticles loaded with docetaxel (NDTX). Antimetastatic effect of CSLC-targeting nanoparticles loaded with docetaxel (TNDTX) was evaluated in a nude mouse model of liver metastasis. Results showed that, in the absence of targeting peptide, NDTX hardly exhibited any antimetastatic effect. However, TNDTX treatment significantly decreased the metastatic tumor area in the nude mouse liver. Histopathological and serological results also confirmed the antimetastatic efficacy of TNDTX. To our knowledge, this is the first report on establishing a CSLC-based strategy for lung cancer metastatic treatment, and we hope this will offer a potential therapeutic approach for management of metastatic lung cancer.

The neuroprotective effects of phytoestrogen α-zearalanol on β-amyloid-induced toxicity in differentiated PC-12 cells

Although favorable effects of estrogen replacement therapy on Alzheimers disease on postmenopausal women have been recognized, an associated increased incidence of uterine and breast tumors has jeopardized the clinical use of estrogen. Phytoestrogen α-zearalanol (α-ZAL) is a reductive product of the Gibberella zeae metabolite and abundant in plants and vegetables, which has been shown to protect cell injury with low side-effects on uterine and breast. This study was designed to evaluate the neuroprotective effects of α-ZAL, on the cultured differentiated PC-12 cells, while 17β-estradiol (17β-E2) has been used as an estrogen positive control. Following a 24 h exposure of the cells to amyloid β-peptide fragment 25-35 (Aβ₂₅₋₃₅), a significant reduction in cell survival and activities of total superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as increased of malondialdehyde (MDA) were observed. However, preincubation of the cells with α-ZAL or 17β-E2 prior to Aβ₂₅₋₃₅ exposure elevated the cell survival and SOD and GSH-Px activities, and decreased the level of MDA. In addition, Aβ₂₅₋₃₅ caused a significant cell apoptosis and increased apoptotic rate, accompanied by decreasing of bcl-2 expression and increasing bax, caspase-3 expression, pretreatment of the cells with α-ZAL or 17β-E2 ameliorated these changes induced by Aβ₂₅₋₃₅. Taken together, these data indicated that the phytoestrogen α-ZAL may effectively antagonize Aβ₂₅₋₃₅-induced cell toxicity by attenuating oxidative stress and apoptotic cell death, in a manner similar to 17β-E2. Our results suggested that α-ZAL can be used as a potential substitute of 17β-E2 in postmenopausal women for Alzheimers disease prevention.

Gossypium herbaceam Extracts Attenuate Ibotenic Acid-induced Excitotoxicity in Rat Hippocampus

Excitotoxicity is one of the most extensively studied processes of neuronal death and plays an important role in Alzheimers disease. In the present study, the protective effects of Gossypium herbaceam extracts (GHE) on learning and memory impairment induced by excitatory neurotoxin ibotenic acid were examined in vivo using Morris water maze. Furthermore, neuroprotective effects of GHE were investigated with methods of immunohistochemistry and biochemistry. Our data showed that oral administration with GHE at the doses of 35, 70 and 140 mg/kg exerted an improved effect on the learning and memory impairment in rats induced by intracerebral injection of ibotenic acid. To confirm the precise mechanism of memory improvement by presence of GHE, we further investigated the potential protection on the hippocampus. Our findings suggest that GHE afforded a beneficial inhibition on pro-apoptosis proteins expression following ibotenic acid. Additionally, calcium pump activity and calbindin-D28k expression were dramatically increased after GHE treatment, implicating that the modulation of calcium homeostasis could be involved in the mechanism underlying neuroprotection of GHE against ibotenic acid-induced excitotoxicity. These data suggested that GHE could be a potential agent for preventing or retarding the development or progression of Alzheimers disease.

NASAL ADMINISTRATION OF CHOLERA TOXIN B SUBUNIT-NERVE GROWTH FACTOR IMPROVES THE SPACE LEARNING AND MEMORY ABILITIES IN β-AMYLOID PROTEIN25-35-INDUCED AMNESIC MICE

Nerve growth factor (NGF) is a potential drug for Alzheimers disease treatment, but delivering NGF to the brain is difficult. To increase the content of NGF in brain, we prepared cholera toxin B subunit (CB) -NGF by the improved sodium metaperiodate method and compared its pharmacodynamics with NGF. In vitro, CB-NGF, as well as NGF, could promote neurite outgrowth and increase choline acetyltransferase activities. But the time window of TrkA phosphorylation induced by CB-NGF and NGF was different. In vivo, nasal administration of CB-NGF could increase the stay time and partially improve abilities of space learning and memory in amnesic mice, and protected the cholinergic neurons in basal forebrain against Abeta(25-35). CB-NGF treatment has better curative effects than NGF in Alzheimers disease model mice.

Neuroprotective effects of 5-(4-hydroxy-3-dimethoxybenzylidene)-thiazolidinone in MPTP induced Parkinsonism model in mice

Parkinsons disease (PD) is a neurological disorder characterized by degeneration of nigrostriatal dopaminergic (DAergic) system. Present treatment targeting to DAergic system solely ameliorated the symptoms but failed to retard the DAergic neuron degeneration, therefore new therapeutic methods aiming at preventing or delaying the neurodegenerative process are urgently needed. In the present study, we found that 5-(4-hydroxy-3-dimethoxybenzylidene)-2-thioxo-4-thiazolidinone (RD-1), a compound derived from rhodanine, protected DAergicneurons from neurotoxicity of MPTP/MPP(+). Firstly, RD-1 significantly improved the locomotor ability in the MPTP mice model, and elevated the tyrosine hydroxylase (TH) positive cell numbers in substantianigra pars compacta (SNpc) and the integrated optical density (IOD) of TH-positive nerve fibers in striatum respectively. Since mitochondrial dysfunction plays an important role in pathogenesis of PD, thereby we investigated the molecular mechanisms of RD-1 against MPTP/MPP(+) neurotoxicity, focusing on its effects on the mitochondrial dysfunction. Immunoblotting analysis showed that RD-1 significantly elevated the Parkin and Miro2 expression levels in acute MPTP treated mice, and improved mitochondrial membrane potential and ATP synthesis in MPP(+)-treated Neuro-2a cells. Moreover, RD-1attenuated impaired mitochondrial transport and vesicle release dysfunction evoked by MPP(+) cytotoxicity in cultured primary mesencephalic neurons. Taken together, these results indicate that improving the mitochondrial dysfunction may be a good choice to delay the neurodegenerative progression commonly associated with PD.