Sleep-wake disorder is one of the most common nonmotor symptoms of Parkinson's disease (PD). Melatonin has the potential to improve sleep-wake disorder, but its mechanism of action is still unclear. Our data showed that melatonin only improved the motor and sleep-wake behavior of a zebrafish PD model when melatonin receptor 1 was present. Thus, we explored the underlying mechanisms by applying a rotenone model. After the PD zebrafish model was induced by 10 nmol/L rotenone, the motor and sleep-wake behavior were assessed. In situ hybridization and real-time quantitative PCR were used to detect the expression of melatonin receptors and lipid-metabolism-related genes. In the PD model, we found abnormal lipid metabolism, which was reversed by melatonin. This may be one of the main pathways for improving PD sleep-wake disorder.
Animals ; Zebrafish ; Melatonin/pharmacology* ; Lipid Metabolism/drug effects* ; Disease Models, Animal ; Rotenone/pharmacology* ; Sleep Wake Disorders/metabolism* ; Parkinson Disease/metabolism* ; Motor Activity/drug effects* ; Sleep/drug effects*

OBJECTIVETo explore the protective effect of baicalin against rotenone-induced injury on PC12 cells, and the po-tential mechanism of action action was also explored.

METHODPC12 cells were injured by rotenone and were treated with different concentrations (0.1, 1, 10 μmol x L(-1)) of baicalin at the same time. Cell viability was analyzed by MTT, and morphology was observed by phase-contrast microscopy. The cell apoptosis was detected by flow cytometry by Annexin V-FITC/PI staining. The intracellular ROS level was determined by fluorescence microscope with DCF-DA staining. The expression of Bcl-2, Bax and Caspase-3 was analyzed by Western blot.

RESULTThe viability of PC12 cells exposure to rotenone for 24 hour was gradually decreased with dose escalating and 1.5 μmol x L was adopted to do the following experiment. Baicalin increased cell viability, improved cell morphology and decreased intracellular ROS level. Moreover, FACS indicated baicalin attenuated the apoptosis induced by rotenone significantly. Western blot showed that Bcl-2, Bax and Caspase-3 expression in rotenone-induced PC12 cells was reversed by baicalin.

CONCLUSIONThis study has demonstrated that baicalin protects PC12 cells against rotenone-induced apoptosis, at least in part, by scavenging excessive ROS and inhibiting the mitochondrion-dependent apoptotic pathway.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Survival ; drug effects ; Cytoprotection ; drug effects ; Flavonoids ; pharmacology ; Gene Expression Regulation ; drug effects ; Intracellular Space ; drug effects ; metabolism ; PC12 Cells ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism ; Rotenone ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo examine the effect of a selective inhibitor of 5-lipoxygenase (5-LOX) zileuton on microglia-mediated rotenone neurotoxicity.

METHODSThe supernatant from different concentrations of rotenone-stimulated mouse microglia BV2 cells was used as the conditioned media (CM) for PC12 cells. The viability of PC12 cells was determined by MTT assay and lactate dehydrogenase (LDH) release. Cell death was observed by LDH release and double fluorescence staining with Hoechst/propidiumiodide (PI). The effect of zileuton on microglia-mediated rotenone toxicity was evaluated by the above methods.

RESULTSRotenone at 1-10 nmol/L was nontoxic to PC12 cells directly. However, the CM from BV2 cells that were treated with rotenone (1-10 nmol/L) resulted in toxicity of PC12 cells. The BV2 CM which stimulated with rotenone (1-10 nmol/L) induced morphological changes, reduced cell viability, and increased LDH release and cell necrosis in PC12 cells. Pretreatment of BV2 cells with the 5-LOX inhibitor zileuton (0.01-1 μmol/L) protected PC12 cells from the microglia-mediated rotenone toxicity.

CONCLUSIONThe 5-LOX inhibitor zileuton effectively attenuates microglia-mediated rotenone toxicity in PC12 cells. These results suggest that 5-LOX pathway may be involved in neuronal death induced by microglial inflammation.

Animals ; Cell Death ; drug effects ; Cells, Cultured ; Hydroxyurea ; analogs & derivatives ; pharmacology ; Lipoxygenase Inhibitors ; pharmacology ; Mice ; Microglia ; cytology ; PC12 Cells ; Rats ; Rotenone ; toxicity

OBJECTIVETo investigate the neuroprotective potential of lycopene on oxidative stress and neurobehavioral abnormalities in rotenone induced Parkinson' disease (PD).

METHODSForty adult C57BL/6 mice were randomly divided into four groups (n = 10): control, lycopene (10 mg/kg body weight, orally), rotenone (3 mg/kg bw, intraperitoneally) and rotenone plus lycopene, which were sacrificed for 5 weeks. The spectrophotometry was used to determine the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and the content of malondialdehyde (MDA) in substantia nigra and right striatum. At the same time, the number of tyrosine hydroxylase (TH), alpha-synuclein (alpha-SYN) and microtubule-associated protein 3 light chain (LC3-B) positive neurons were estimated by immunohistochemistry. We also examined neurobehavioral abnormalities by WT-200 water maze.

RESULTSRotenone administration increased the MDA levels and significantly decreased the activities of SOD, GSH-Px and CAT. However, lycopene administration to the rotenone treated animals increased the activities of SOD, GSH-Px and CAT when compared to rotenone treated animals in substantia nigra and right striatum. The cognitive and motor deficits in rotenone administered animals, which were reversed on lycopene treatment. Along with this, the number of TH decreased, alpha-SYN increased and LC3-B positive neurons increased in rotenone administered animals, which were reversed on lycopene treatment.

CONCLUSIONCollectively, these observations provide an evidence for beneficial effect of lycopene supplementation in rotenone-induced PD and suggest therapeutic potential in neurodegenerative diseases involving accentuated oxidative stress.

Animals ; Behavior, Animal ; Brain ; drug effects ; Carotenoids ; pharmacology ; Disease Models, Animal ; Dopamine ; metabolism ; Male ; Malondialdehyde ; metabolism ; Mice ; Mice, Inbred C57BL ; Neurons ; metabolism ; Oxidative Stress ; drug effects ; Parkinson Disease ; metabolism ; Rotenone ; pharmacology ; Superoxide Dismutase ; metabolism

OBJECTIVETo observe the effects of deguelin on the apoptosis and proliferation of human esophageal cancer cell Ec-109, and to explore its possible mechanisms.

METHODSHuman esophageal cancer cells Ec-109 were in vitro cultured. They were divided into the blank control group, and 5, 10, 20, and 40 nmol/L deguelin groups. The inhibition on the proliferation was detected at 24, 48, and 72 h using CCK-8 assay. The early apoptosis rate at 24 h was detected by flow cytometry. The expressions of apoptosis-related proteins Bcl-2 and Bax were detected at 24 and 48 h respectively.

RESULTSCompared with the blank control group at the same point, the growth inhibition rate in all deguelin groups increased at 24, 48, and 72 h, showing statistical difference (P <0.05). The early apoptosis rate was 4.37% +/- 0.35%, 6.71% +/-0.14%, 15.62% +/- 0.21%, and 19.78% +/- 0.15% in 5, 10, 20, and 40 nmol/L deguelin groups, respectively, showing statistical difference when compared with that of the blank control group (1.10% +/- 0.08%, P < 0.05). Compared with the blank control group, Bcl-2 protein expression obviously decreased, and Bax protein expression obviously increased in 10, 20, and 40 nmol/L deguelin groups, showing statistical difference (P <0.05). The aforesaid indices were in time- and dose-dependent manners.

CONCLUSIONDeguelin showed obvious effects on inhibiting the proliferation of Ec-109 cells and promoting their apoptosis, which was correlated with up-regulating Bax protein expression and down-regulating Bcl-2 protein expression.

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Esophageal Neoplasms ; pathology ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rotenone ; analogs & derivatives ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo study the inhibitory effect of Akt inhibitor deguelin on PC-3 human prostate cancer cell lines and its possible mechanism.

METHODSPC-3 human prostate cancer cells were cultured in deguelin at the concentrations of 10, 100, 500 and 1 000 nmol/L for 24, 48 and 72 hours, respectively. Then the inhibitory effect of deguelin on the proliferation of the PC-3 cells was determined by MTT assay and that on the cell cycle was detected by flow cytometry. The expression levels of MDM2 and GSK3beta mRNA were measured by RT-PCR and those of MDM2 and GSK3beta proteins by Western blot.

RESULTSAt 24, 48 and 72 hours, the inhibition rates of deguelin on the proliferation of the PC-3 prostate cancer cells were (91.10 +/- 3.75), (86.39 +/- 1.16) and (79.51 +/- 2.63)% at 10 nmol/L, (82.46 +/- 3.65), (76.84 +/- 0.97) and (69.69 +/- 2.30) % at 100 nmol/L, (81.46 +/- 0.41), (75.56 +/- 1.12) and (54.07 +/- 3.21)% at 500 nmol/L, and (66.77 +/- 2.82), (58.22 +/- 0.35) and (39.34 +/- 2.40)% at 1000 nmol/L, all with statistically significant differences from the control group (P < 0.01). Deguelin at 10, 100, 500 and 1 000 nmol/L increased the cell cycles blocked in the G0/G1 phase ([62.4 +/- 2.2], [63.6 +/- 1.1 ], [65.0 +/- 0.3] and [66.5 +/- 1.9]%, P < 0.01) and reduced the percentage of the S-phase cells ([14.7 +/- 2.4], [11.1 +/- 5.2], [5.8 +/- 1.1] and [7.0 +/- 0.6]%, P < 0.01). RT-PCR and Western blot showed markedly up-regulated expressions of GSK3 P3 a3beta down-regulated expressions of MDM2 mRNA and proteins in the PC-3 cells treated with deguelin.

CONCLUSIONAkt inhibitor deguelin can inhibit the proliferation of PC-3 human prostate cancer cells by affecting the down-stream signal molecules GSK3P3 and betaDM2 in the Akt pathway.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Male ; Prostatic Neoplasms ; metabolism ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Rotenone ; analogs & derivatives ; pharmacology

The activation of nuclear factor of activated T cells 5 (NFAT5), a well-known osmoprotective factor, can be induced by isotonic stimuli, such as activated Toll-like receptors (TLRs). It is unclear, however, how NFAT5 discriminates between isotonic and hypertonic stimuli. In this study we identified a novel context-dependent suppression of NFAT5 target gene expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) or a high salt (NaCl) concentration. Although LPS and NaCl both used NFAT5 as a core transcription factor, these stimuli mutually inhibited distinct sets of NFAT5 targets within the cells. Although reactive oxygen species (ROS) are essential for this inhibition, the source of ROS differed depending on the context: mitochondria for high salt and xanthine oxidase for TLRs. Specifically, the high salt-induced suppression of interleukin-6 (IL-6) production was mediated through the ROS-induced inhibition of NFAT5 binding to the IL-6 promoter. The context-dependent inhibition of NFAT5 target gene expression was also confirmed in mouse spleen and kidney tissues that were cotreated with LPS and high salt. Taken together, our data suggest that ROS function as molecular sensors to discriminate between TLR ligation and osmotic stimuli in RAW 264.7 macrophages, directing NFAT5 activity toward proinflammatory or hypertonic responses in a context-dependent manner.
Animals ; *Gene Expression Regulation/drug effects ; Interleukin-6/biosynthesis/genetics ; Lipopolysaccharides/pharmacology ; Macrophages/drug effects/metabolism ; Male ; Mannitol/pharmacology ; Mice ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Promoter Regions, Genetic/genetics ; Protein Binding/drug effects/genetics ; Reactive Oxygen Species/*metabolism ; Rotenone/pharmacology ; Sodium Chloride/pharmacology ; Toll-Like Receptors ; Transcription Factors/genetics/*metabolism

The underlying mechanism of deguelin regulating the cell cycle in human Burkitt's lymphoma cell line Raji cells in vitro, and the cytotoxicity of deguelin to Raji cells and human peripheral blood monocular cells (PBMCs) were investigated. The effects of deguelin on the growth of Raji cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was detected through Hoechst 33258 staining. The effect of deguelin on the cell cycle of Raji cells was studied by a propidium iodide method. The expression levels of cyclin D1, P21 and pRb were examined by using Western blotting. The results showed that the proliferation of Raji cells was inhibited in the deguelin-treated group, with a 24-h IC(50) value of 21.61 nmol/L and a 36-h IC(50) value of 17.07 nmol/L. Proliferation in Raji cells was inhibited significantly by deguelin, while little change was observed in PBMCs. Deguelin induced G(2)/M arrest in Raji cells. The expression of cyclin D1, P21 and pRb was dramatically down-regulated by deguelin in a dose-dependent manner. It was concluded that deguelin could inhibit the proliferation of Raji cells by arresting the cells at G(2)/M phase and inducing the cell apoptosis. Moreover, deguelin selectively induced apoptosis of Raji cells with low toxicity to PBMCs. The antitumor effects of deguelin were related to the down-regulated expression of cyclin D1, P21 and pRb proteins.
Cell Cycle ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Humans ; Rotenone ; analogs & derivatives ; pharmacology

OBJECTIVETo determine whether 5-lipoxygenase (5-LOX) is involved in rotenone-induced injury in PC12 cells, which is a cell model of Parkinson disease.

METHODSAfter rotenone treatment for various durations, cell viability was determined by colorimetric MTT reduction assay, and 5-LOX translocation was detected by immunocytochemistry. The effect of 5-LOX inhibitor zileuton was also investigated.

RESULTRotenone (0.3-30 μmol/L) induced PC12 cell injury, and zileuton (3-100 μmol/L) attenuated this injury. Rotenone also time-and concentration-dependently induced 5-LOX translocation into the nuclear envelope, and zileuton (1-30 μmo/L) significantly inhibited rotenone-induced 5-LOX translocation.

CONCLUSION5-LOX is involved in rotenone-induced injury in PC12 cells, and 5-LOX inhibitor zileuton can reduce rotenone-induced 5-LOX activation and cell injury.

Animals ; Arachidonate 5-Lipoxygenase ; metabolism ; physiology ; Cell Survival ; drug effects ; Hydroxyurea ; analogs & derivatives ; pharmacology ; Lipoxygenase Inhibitors ; pharmacology ; PC12 Cells ; Rats ; Rotenone ; pharmacology

OBJECTIVETo prepare and identify a polyclonal antibody (pAb) against (mouse) cysteinyl leukotriene receptor 1 (CysLT(1)) and to investigate the changes of CysLT(1) receptor expression in BV2 microglial cells after rotenone treatment.

METHODSRabbits were immunized with KLH-coupled CysLT(1) peptide to prepare the pAb. The titer of the pAb in rabbit plasma was detected by ELISA method, and the specificity of the pAb was tested by antigen blockade. After BV2 cells were treated with rotenone (0.01-1 μmol/L) for 24 h, the expression of CysLT(1) was determined by immunostaining, Western blotting and RT-PCR.

RESULTThe pAb showed a titer of 1/32728, and was not cross-reacted with antigens of CysLT(2) receptor and GPR17. Immunostaining, Western blotting and RT-PCR analysis showed the expression of CysLT(1) receptor in BV2 microglia. Rotenone at 1μmol/L significantly induced an increased expression of CysLT(1) receptor.

CONCLUSIONThe prepared CysLT(1) receptor polyclonal antibody has a high titer and high specificity to meet testing requirements of Western blotting and immunostaining; CysLT(1) is associated with rotenone-induced injury of BV2 microglial cells.

Animals ; Cells, Cultured ; Male ; Mice ; Microglia ; drug effects ; metabolism ; pathology ; Rabbits ; Receptors, Leukotriene ; immunology ; metabolism ; Rotenone ; pharmacology